Scientific Bibliography

Nanobiology and Cancer Nanotechnology
Nanotechnology Devices and Nanomaterials
Nanotechnology-Enabled Therapeutics Development and Delivery
Cancer Diagnostics and Biosensors
Nanotechnologies in Advanced Imaging
Environment, Health and Safety

Nanotechnology - Enabled Therapeutics Development and Delivery

2007   2006   2005   2004   2003   2002

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2007

Carbon nanotube-enhanced thermal destruction of cancer cells in a noninvasive radiofrequency field.
Gannon CJ, Cherukuri P, Yakobson BI, Cognet L, Kanzius JS, Kittrell C, Weisman RB, Pasquali M, Schmidt HK, Smalley RE, Curley SA.
Eur J Pharm Sci. 2007 Nov;32(3):159-68.
[ expand abstract ]

BACKGROUND.: Single-walled carbon nanotubes (SWNTs) have remarkable physicochemical properties that may have several medical applications. The authors have discovered a novel property of SWNTs-heat release in a radiofrequency (RF) field-that they hypothesized may be used to produce thermal cytotoxicity in malignant cells. METHODS.: Functionalized, water-soluble SWNTs were exposed to a noninvasive, 13.56-megahertz RF field, and heating characteristics were measured with infrared thermography. Three human cancer cell lines were incubated with various concentrations of SWNTs and then treated in the RF field. Cytotoxicity was measured by fluorescence-activated cell sorting. Hepatic VX2 tumors in rabbits were injected with SWNTs or with control solutions and were treated in the RF field. Tumors were harvested 48 hours later to assess viability. RESULTS.: The RF field induced efficient heating of aqueous suspensions of SWNTs. This phenomenon was used to produce a noninvasive, selective, and SWNT concentration-dependent thermal destruction in vitro of human cancer cells that contained internalized SWNTs. Direct intratumoral injection of SWNTs in vivo followed by immediate RF field treatment was tolerated well by rabbits bearing hepatic VX2 tumors. At 48 hours, all SWNT-treated tumors demonstrated complete necrosis, whereas control tumors that were treated with RF without SWNTs remained completely viable. Tumors that were injected with SWNTs but were not treated with RF also were viable. CONCLUSIONS.: The current results suggested that SWNTs targeted to cancer cells may allow noninvasive RF field treatments to produce lethal thermal injury to the malignant cells. Now, the authors are developing SWNTs coupled with cancer cell-targeting agents to enhance SWNT uptake by cancer cells while limiting uptake by normal cells.

Enhanced cytotoxicity of monoclonal anticancer antibody 2C5-modified doxorubicin-loaded PEGylated liposomes against various tumor cell lines.
Elbayoumi TA, Torchilin VP.
Eur J Pharm Sci. 2007 Nov;32(3):159-68.
[ expand abstract ]

Doxorubicin-loaded long-circulating liposomes (Doxil, ALZA Corp.) were additionally modified with the nucleosome-specific monoclonal antibody 2C5 (mAb 2C5) recognizing a broad variety of tumor cells via the tumor cell surface-bound nucleosomes. These mAb 2C5-modified PEGylated liposomes demonstrated 3-8-fold increase in the in vitro binding and internalization by multiple cancer cell lines of diverse origins (murine LLC, 4T1, C26 and human BT-20, MCF-7, and PC3), as shown by flow cytometry (FACS) and epi and confocal microscopy. As a result, mAb 2C5-modified Doxil demonstrated significantly higher cytotoxicity towards various cancer cells, including those resistant to doxorubicin, than all control preparations. The specific internalization of the mAb 2C5-Doxil into cytosol, along with the nuclear localization of their drug load, inside the target cancer cells were mainly responsible the superior anticancer activity. The IC50 values of mAb 2C5-Doxil with various murine and human cancer cells were 5-8-fold lower than those of control doxorubicin-loaded liposomes, Doxil or Doxil modified with a nonspecific IgG.

Polymer-caged lipsomes: a pH-responsive delivery system with high stability.
Lee SM, Chen H, Dettmer CM, O'Halloran TV, Nguyen ST.
J Am Chem Soc. 2007 Dec 12;129(49):15096-7.
[ expand abstract ]

Polymer-incorporated liposomes were prepared from preformed liposomes and a cholesterol-functionalized poly(acrylic acid) additive via a simple drop-in procedure. These modified liposomes possess surface-active carboxylate groups that can be cross-linked with telechelic 2,2'-(ethylenedioxy)bis(ethylamine) linkers, resulting in polymer-caged liposomes (PCLs) that are highly stable and have tunable pH-sensitive responses.

Microfluidic self-assembly of tumor spheroids for anticancer drug discovery.
Wu LY, Di Carlo D, Lee LP.
Biomed Microdevices. 2007 Oct 27; [Epub ahead of print].
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Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of in vivo tumors. Moreover, continuous dynamic perfusion allows the establishment of physiologically relevant drug profiles to exposed spheroids. Here we present a physiologically inspired design allowing microfluidic self-assembly of spheroids, formation of uniform spheroid arrays, and characterizations of spheroid dynamics all in one platform. Our microfluidic device is based on hydrodynamic trapping of cancer cells in controlled geometries and the formation of spheroids is enhanced by maintaining compact groups of the trapped cells due to continuous perfusion. It was found that spheroid formation speed (average of 7 h) and size uniformity increased with increased flow rate (up to 10 mul min(-1)). A large amount of tumor spheroids (7,500 spheroids per square centimeter) with a narrow size distribution (10 +/- 1 cells per spheroid) can be formed in the device to provide a good platform for anticancer drug assays.

A Cellular Trojan Horse for Delivery of Therapeutic Nanoparticles into Tumors.
Choi MR, Stanton-Maxey KJ, Stanley JK, Levin CS, Bardhan R, Akin D, Badve S, Sturgis J, Robinson JP, Bashir R, Halas NJ, Clare SE.
Nano Lett. 2007 Dec 12;7(12):3759-3765.
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Destruction of hypoxic regions within tumors, virtually inaccessible to cancer therapies, may well prevent malignant progression. The tumor's recruitment of monocytes into these regions may be exploited for nanoparticle-based delivery. Monocytes containing therapeutic nanoparticles could serve as "Trojan Horses" for nanoparticle transport into these tumor regions. Here we report the demonstration of several key steps toward this therapeutic strategy: phagocytosis of Au nanoshells, and photoinduced cell death of monocytes/macrophages as isolates and within tumor spheroids.

Temperature-sensitive poly(N-(2-hydroxypropyl)methacrylamide mono/dilactate)-coated liposomes for triggered contents release.
Paasonen L, Romberg B, Storm G, Yliperttula M, Urtti A, Hennink WE.
Bioconjug Chem. 2007 Nov-Dec;18(6):2131-6.
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We prepared thermosensitive poly( N-(2-hydroxypropyl)methacrylamide mono/dilactate) (pHPMA mono/dilactate) polymer and studied temperature-triggered contents release from polymer-coated liposomes. HPMA mono/dilactate polymer was synthesized with a cholesterol anchor suitable for incorporation in the liposomal bilayers and with a cloud point (CP) temperature of the polymer slightly above normal body temperature (42 degrees C). Dynamic light scattering (DLS) measurements showed that whereas the size of noncoated liposomes remained stable upon raising the temperature from 25 to 46 degrees C, polymer-coated liposomes aggregated around 43 degrees C. Also, noncoated liposomes loaded with calcein showed hardly any leakage of the fluorescent marker when heated to 46 degrees C. However, polymer-coated liposomes showed a high degree of temperature-triggered calcein release above the CP of the polymer. Likely, liposome aggregation and bilayer destabilization are triggered because of the precipitation of the thermosensitive polymer above its CP onto the liposomal bilayers, followed by permeabilization of the liposomal membrane. This study demonstrates that liposomes surface-modified with HPMA mono/dilactate copolymer are attractive systems for achieving temperature-triggered contents release.

Multifunctional poly(d,l-lactide-co-glycolide)/montmorillonite (PLGA/MMT) nanoparticles decorated by Trastuzumab for targeted chemotherapy of breast cancer.
Sun B, Ranganathan B, Feng SS.
Biomaterials. 2008 Feb;29(4):475-86.
[ expand abstract ]

This paper continued our earlier work on the poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles (PLGA/MMT NPs), which were further decorated by human epidermal growth factor receptor-2 (HER2) antibody Trastuzumab for targeted breast cancer chemotherapy with paclitaxel as a model anticancer drug. Such a NP system is multifunctional, which formulates anticancer drugs with no harmful adjuvant, reduces the side effects of the formulated anticancer drug, promotes synergistic therapeutic effects, and achieves targeted delivery of the therapy. The paclitaxel-loaded PLGA/MMT NPs were prepared by a modified solvent extraction/evaporation technique, which were then decorated with Trastuzumab. The effects of the surface decoration on particle size and size distribution, surface morphology, drug encapsulation efficiency, as well as the drug release kinetics, were investigated. The NP formulation exhibited a biphasic drug release with a moderate initial burst followed by a sustained release profile. The surface decoration speeded the drug release. Surface chemistry analysis was conducted by X-ray photoelectron spectroscopy (XPS), which confirmed the presence of Trastuzumab on the NP surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the stability of the antibody in the NP preparation process. Internalization of the coumarin-6-loaded PLGA/MMT NPs with or without the antibody decoration by both of Caco-2 colon adeno carcinoma cells and SK-BR-3 breast cancer cells was visualized by confocal laser scanning microscopy and quantitatively analyzed, which shows that the antibody decoration achieved significantly higher cellular uptake of the NPs. The results of in vitro cytotoxicity experiment on SK-BR-3 cells further proved the targeting effects of the antibody decoration. Judged by IC50 after 24h culture, the therapeutic effects of the drug formulated in the NPs with surface decoration could be 12.74 times higher than that of the bare NPs and 13.11 times higher than Taxol((R)).

Tumor-selective vessel occlusions by platelets after vascular targeting chemotherapy using paclitaxel encapsulated in cationic liposomes.
Strieth S, Nussbaum CF, Eichhorn ME, Fuhrmann M, Teifel M, Michaelis U, Berghaus A, Dellian M.
Int J Cancer. 2008 Jan 15;122(2):452-60.
[ expand abstract ]

Paclitaxel encapsulated in cationic liposomes (EndoTAG-1) significantly impairs tumor growth by a significant reduction of functional tumor microcirculation and induction of endothelial cell apoptosis. The aim of the study was to analyze whether platelet activation within the tumor microcirculation contributes to the antivascular effects of vascular targeting chemotherapy using EndoTAG-1. In vitro, FACS analysis revealed a significant activation of platelets upon treatment with EndoTAG-1. In vivo, using A-Mel-3 tumors in Syrian Golden hamsters equipped with dorsal skinfold chamber preparations, the contribution of platelets to the antivascular effects of EndoTAG-1 was evaluated by fluorescence and laser-scanning microscopy. Immediately after a single treatment with EndoTAG-1 or cationic liposomes devoid of paclitaxel, an increase of platelet adherence in tumor microvessels was observed. This was accompanied by an acute impairment of the microcirculation within the treated tumors leading to reduced tumor perfusion. After repetitive therapy, an increase of platelet adherence and subsequent tumor microvessel occlusions occurred only after treatment with EndoTAG-1. Comparing to "tumor free" normal tissue controls these microthromboses were tumor selective. Significantly disbalancing the coagulation system within tumors by targeted induction of microthromboses within the tumor microcirculation appears to be an important mechanism of EndoTAG-1 therapy.

Oligonucleotide Loading Determines Cellular Uptake of DNA-Modified Gold Nanoparticles.
Giljohann DA, Seferos DS, Patel PC, Millstone JE, Rosi NL, Mirkin CA.
Nano Lett. 2007 Dec;7(12):3818-21.
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The cellular internalization of oligonucleotide-modified nanoparticles is investigated. Uptake is dependent on the density of the oligonucleotide loading on the surface of the particles, where higher densities lead to greater uptake. Densely functionalized nanoparticles adsorb a large number of proteins on the nanoparticle surface. Nanoparticle uptake is greatest where a large number of proteins are associated with the particle.

Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging.
Derek W. Bartlett, Helen Su, Isabel J. Hildebrandt, Wolfgang A. Weber, Mark E. Davis.
Proc Natl Acad Sci U S A. 2007 September 25; 104(39): 15549–15554.
[ expand abstract ]

Targeted delivery represents a promising approach for the development of safer and more effective therapeutics for oncology applications. Although macromolecules accumulate nonspecifically in tumors through the enhanced permeability and retention (EPR) effect, previous studies using nanoparticles to deliver chemotherapeutics or siRNA demonstrated that attachment of cell-specific targeting ligands to the surface of nanoparticles leads to enhanced potency relative to nontargeted formulations. Here, we use positron emission tomography (PET) and bioluminescent imaging to quantify the in vivo biodistribution and function of nanoparticles formed with cyclodextrin-containing polycations and siRNA. Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the 5' end of the siRNA molecules allows labeling with 64Cu for PET imaging. Bioluminescent imaging of mice bearing luciferase-expressing Neuro2A s.c. tumors before and after PET imaging enables correlation of functional efficacy with biodistribution data. Although both nontargeted and transferrin-targeted siRNA nanoparticles exhibit similar biodistribution and tumor localization by PET, transferrin-targeted siRNA nanoparticles reduce tumor luciferase activity by ˜50% relative to nontargeted siRNA nanoparticles 1 d after injection. Compartmental modeling is used to show that the primary advantage of targeted nanoparticles is associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization. Optimization of internalization may therefore be key for the development of effective nanoparticle-based targeted therapeutics.

Active nanodiamond hydrogels for chemotherapeutic delivery.
Huang H, Pierstorff E, Osawa E, Ho D.
Nano Lett. 2007 Nov;7(11):3305-14.
[ expand abstract ]

Nanodiamond materials can serve as highly versatile platforms for the controlled functionalization and delivery of a wide spectrum of therapeutic elements. In this work, doxorubicin hydrochloride (DOX), an apoptosis-inducing drug widely used in chemotherapy, was successfully applied toward the functionalization of nanodiamond materials (NDs, 2-8 nm) and introduced toward murine macrophages as well as human colorectal carcinoma cells with preserved efficacy. The adsorption of DOX onto the NDs and its reversible release were achieved by regulating Cl- ion concentration, and the NDs were found to be able to efficiently ferry the drug inside living cells. Comprehensive bioassays were performed to assess and confirm the innate biocompatibility of the NDs, via real-time quantitative polymerase chain reaction (RT-PCR), and electrophoretic DNA fragmentation as well as MTT analysis confirmed the functional apoptosis-inducing mechanisms driven by the DOX-functionalized NDs. We extended the applicability of the DOX-ND composites toward a translational context, where MTT assays were performed on the HT-29 colon cancer cell line to assess DOX-ND induced cell death and ND-mediated chemotherapeutic sequestering for potential slow/sustained released capabilities. These and other medically relevant capabilities enabled by the NDs forge its strong potential as a therapeutically significant nanomaterial.

Quantum dot-aptamer conjugates for synchronous cancer imaging, therapy, and sensing of drug delivery based on bi-fluorescence resonance energy transfer.
Bagalkot V, Zhang L, Levy-Nissenbaum E, Jon S, Kantoff PW, Langer R, Farokhzad OC.
Nano Lett. 2007 Oct;7(10):3065-70.
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We report a novel quantum dot (QD)-aptamer(Apt)-doxorubicin (Dox) conjugate [QD-Apt(Dox)] as a targeted cancer imaging, therapy, and sensing system. By functionalizing the surface of fluorescent QD with the A10 RNA aptamer, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA), we developed a targeted QD imaging system (QD-Apt) that is capable of differential uptake and imaging of prostate cancer cells that express the PSMA protein. The intercalation of Dox, a widely used antineoplastic anthracycline drug with fluorescent properties, in the double-stranded stem of the A10 aptamer results in a targeted QD-Apt(Dox) conjugate with reversible self-quenching properties based on a Bi-FRET mechanism. A donor-acceptor model fluorescence resonance energy transfer (FRET) between QD and Dox and a donor-quencher model FRET between Dox and aptamer result when Dox intercalated within the A10 aptamer. This simple multifunctional nanoparticle system can deliver Dox to the targeted prostate cancer cells and sense the delivery of Dox by activating the fluorescence of QD, which concurrently images the cancer cells. We demonstrate the specificity and sensitivity of this nanoparticle conjugate as a cancer imaging, therapy and sensing system in vitro.

Apoptotic epidermal growth factor (EGF)-conjugated block copolymer micelles as a nanotechnology platform for targeted combination therapy.
Lee H, Hu M, Reilly RM, Allen C.
Mol Pharm. 2007 Sep-Oct;4(5):769-81.
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The overexpression of epidermal growth factor receptor (EGFR) in human epithelial cancers has been associated with aggressive disease, poor patient prognosis, and a high incidence of metastases. In the present study, block copolymer micelles are conjugated with epidermal growth factor (EGF), which acts as both a targeting ligand for the drug carrier and an apoptotic factor against EGFR-overexpressing cancers. Drug-free EGF-conjugated micelles are shown to result in cell-cycle arrest at the G 1 phase and subsequent induction of cell-type-specific apoptosis in EGFR-overexpressing breast cancer cells as demonstrated by flow cytometric analysis. EGF delivered as EGF-conjugated micelles was found to be 13-fold more potent than free EGF; the IC 50 was decreased from 0.98 +/- 0.1 nM for free EGF to 0.076 +/- 0.01 nM for EGF micelles. The apoptotic micelles, however, are non-antiproliferative to cells expressing a low level of EGFR, suggesting that the apoptotic micelles have minimal or no toxicity against normal healthy tissues. Ellipticine, a chemotherapeutic agent, was loaded into the EGF-micelles after it had been shown, using the combination index-isobologram equation, to act synergistically with EGF. A 10-fold increase in EGF content in the ellipticine-loaded micelles lowered the IC 50 of ellipticine in EGFR-overexpressing breast cancer cells by more than 18-fold. The EGF-micelles have the potential to be further pursued as a versatile nanotechnology platform for targeted delivery of a wide range of chemotherapeutic agents as a combination therapy for the treatment of EGFR-overexpressing cancers.

Synthetic nano-LDL with paclitaxel oleate as a targeted drug delivery vehicle for glioblastoma multiforme.
Nikanjam M, Gibbs AR, Hunt CA, Budinger TF, Forte TM.
J Control Release. 2007 Dec 20;124(3):163-71.
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The low density lipoprotein (LDL) receptor has been shown to be upregulated in GBM tumor cells in vitro and is therefore a potential molecular target for the delivery of therapeutic agents. A synthetic nano-LDL (nLDL) particle was developed as a drug delivery vehicle targeted to GBM cells by incorporating a lipophilic prodrug, paclitaxel oleate, into the particle. Nano-LDL containing paclitaxel oleate (nLDL-PO) was constructed by combining a synthetic peptide containing a lipid binding motif and the LDL receptor (LDLR) binding domain of apolipoprotein B-100 with a lipid emulsion consisting of phosphatidyl choline, triolein, and paclitaxel oleate. Paclitaxel oleate incorporated into the core of the lipid particle. nLDL-PO cell survival in GBM cell lines was found to be time, concentration, and cell line dependent. Cell killing was observed with short drug incubations and exhibited saturation at 6 h. nLDL-PO cell survival improved in the presence of the LDL receptor inhibitor, suramin, demonstrating that the drug was delivered via the LDL receptor. Collectively, these data strongly suggest that the synthetic nano-LDLs can incorporate lipophilic drugs and are capable of killing GBM cells. nLDL-PO has the potential to serve as a selective drug delivery vehicle for targeting GBM tumors via the LDL receptor.

Radiosensitization of paclitaxel, etanidazole and paclitaxel+etanidazole nanoparticles on hypoxic human tumor cells in vitro.
Jin C, Bai L, Wu H, Tian F, Guo G.
Biomaterials. 2007 Sep;28(25):3724-30.
[ expand abstract ]

Paclitaxel and etanidazole are hypoxic radiosensitizers that exhibit cytotoxic action at different mechanisms. The poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel, etanidazole and paclitaxel+etanidazole were prepared by o/w and w/o/w emulsification-solvent evaporation method. The morphology of the nanoparticles was investigated by scanning electron microscope (SEM). The drug encapsulation efficiency (EE) and release profile in vitro were measured by high-performance liquid chromatography (HPLC). The cellular uptake of nanoparticles for the human breast carcinoma cells (MCF-7) and the human carcinoma cervicis cells (HeLa) was evaluated by transmission electronic microscopy and fluorescence microscopy. Cell viability was determined by the ability of single cell to form colonies in vitro. The prepared nanoparticles were spherical shape with size between 80 and 150 nm. The EE was higher for paclitaxel and lower for etanidazole. The drug release was controlled over time. The cellular uptake of nanoparticles was observed. Co-culture of the two tumor cell lines with drug-loaded nanoparticles demonstrated that released drug effectively sensitized hypoxic tumor cells to radiation. The radiosensitization of paclitaxel+etanidazole nanoparticles was more significant than that of single drug-loaded nanoparticles.

Enhanced retention of the alpha-particle-emitting daughters of Actinium-225 by liposome carriers.
Sofou S, Kappel BJ, Jaggi JS, McDevitt MR, Scheinberg DA, Sgouros G.
Bioconjug Chem. 2007 Nov-Dec;18(6):2061-7.
[ expand abstract ]

Targeted alpha-particle emitters hold great promise as therapeutics for micrometastatic disease. Because of their high energy deposition and short range, tumor targeted alpha-particles can result in high cancer-cell killing with minimal normal-tissue irradiation. Actinium-225 is a potential generator for alpha-particle therapy: it decays with a 10-day half-life and generates three alpha-particle-emitting daughters. Retention of (225)Ac daughters at the target increases efficacy; escape and distribution throughout the body increases toxicity. During circulation, molecular carriers conjugated to (225)Ac cannot retain any of the daughters. We previously proposed liposomal encapsulation of (225)Ac to retain the daughters, whose retention was shown to be liposome-size dependent. However, daughter retention was lower than expected: 22% of theoretical maximum decreasing to 14%, partially due to the binding of (225)Ac to the phospholipid membrane. In this study, Multivesicular liposomes (MUVELs) composed of different phospholipids were developed to increase daughter retention. MUVELs are large liposomes with entrapped smaller lipid-vesicles containing (225)Ac. PEGylated MUVELs stably retained over time 98% of encapsulated (225)Ac. Retention of (213)Bi, the last daughter, was 31% of the theoretical maximum retention of (213)Bi for the liposome sizes studied. MUVELs were conjugated to an anti-HER2/neu antibody (immunolabeled MUVELs) and were evaluated in vitro with SKOV3-NMP2 ovarian cancer cells, exhibiting significant cellular internalization (83%). This work demonstrates that immunolabeled MUVELs might be able to deliver higher fractions of generated alpha-particles per targeted (225)Ac compared to the relative fractions of alpha-particles delivered by (225)Ac-labeled molecular carriers.

Anti-CD166 single chain antibody-mediated intracellular delivery of liposomal drugs to prostate cancer cells.
Roth A, Drummond DC, Conrad F, Hayes ME, Kirpotin DB, Benz CC, Marks JD, Liu B.
Mol Cancer Ther. 2007 Oct;6(10):2737-46.
[ expand abstract ]

Targeted delivery of small-molecule drugs has the potential to enhance selective killing of tumor cells. We have identified previously an internalizing single chain [single chain variable fragment (scFv)] antibody that targets prostate cancer cells and identified the target antigen as CD166. We report here the development of immunoliposomes using this anti-CD166 scFv (H3). We studied the effects of a panel of intracellularly delivered, anti-CD166 immunoliposomal small-molecule drugs on prostate cancer cells. Immunoliposomal formulations of topotecan, vinorelbine, and doxorubicin each showed efficient and targeted uptake by three prostate cancer cell lines (Du-145, PC3, and LNCaP). H3-immunoliposomal topotecan was the most effective in cytotoxicity assays on all three tumor cell lines, showing improved cytotoxic activity compared with nontargeted liposomal topotecan. Other drugs such as liposomal doxorubicin were highly effective against LNCaP but not PC3 or Du-145 cells, despite efficient intracellular delivery. Post-internalization events thus modulate the overall efficacy of intracellularly delivered liposomal drugs, contributing in some cases to the lower than expected activity in a cell line-dependent manner. Further studies on intracellular tracking of endocytosed liposomal drugs will help identify and overcome the barriers limiting the potency of liposomal drugs.

Gold Nanorods mediate tumor cell death by compromising membrane integrity.
L. Tong, Y. Zhao, T. B. Huff, M. N. Hansen, A. Wei, J.X. Cheng.
Adv Materials. 2007;19(20):3136 – 3141.
[ expand abstract ]

No abstract available (Citation link)

Bio-nano-informatics: an integrated information management system for personalized oncology.
Stokes TH, Phan J, Quo CF, Nie S, Wang MD.
Conf Proc IEEE Eng Med Biol Soc. 2006;1:3325-8.
[ expand abstract ]

The Emory-Georgia Tech Nanotechnology Center for Personalized and Predictive Oncology is one of the eight national centers funded by the National Cancer Institute (NCI). Its overall goal is to combine nanotechnology and biocomputing with clinical oncology for personalized detection, diagnosis and treatment of human cancer. Within this large-scale and multifaceted center, a key challenge is how to integrate and manage data and resources. Here we have developed an "intelligent" information system for data management, interpretation, and for translation of new results to clinical applications.

Multifunctional poly(d,l-lactide-co-glycolide)/montmorillonite (PLGA/MMT) nanoparticles decorated by Trastuzumab for targeted chemotherapy of breast cancer.
Sun B, Ranganathan B, Feng SS.
Biomaterials. 2008 Feb;29(4):475-86.
[ expand abstract ]

This paper continued our earlier work on the poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles (PLGA/MMT NPs), which were further decorated by human epidermal growth factor receptor-2 (HER2) antibody Trastuzumab for targeted breast cancer chemotherapy with paclitaxel as a model anticancer drug. Such a NP system is multifunctional, which formulates anticancer drugs with no harmful adjuvant, reduces the side effects of the formulated anticancer drug, promotes synergistic therapeutic effects, and achieves targeted delivery of the therapy. The paclitaxel-loaded PLGA/MMT NPs were prepared by a modified solvent extraction/evaporation technique, which were then decorated with Trastuzumab. The effects of the surface decoration on particle size and size distribution, surface morphology, drug encapsulation efficiency, as well as the drug release kinetics, were investigated. The NP formulation exhibited a biphasic drug release with a moderate initial burst followed by a sustained release profile. The surface decoration speeded the drug release. Surface chemistry analysis was conducted by X-ray photoelectron spectroscopy (XPS), which confirmed the presence of Trastuzumab on the NP surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the stability of the antibody in the NP preparation process. Internalization of the coumarin-6-loaded PLGA/MMT NPs with or without the antibody decoration by both of Caco-2 colon adeno carcinoma cells and SK-BR-3 breast cancer cells was visualized by confocal laser scanning microscopy and quantitatively analyzed, which shows that the antibody decoration achieved significantly higher cellular uptake of the NPs. The results of in vitro cytotoxicity experiment on SK-BR-3 cells further proved the targeting effects of the antibody decoration. Judged by IC50 after 24h culture, the therapeutic effects of the drug formulated in the NPs with surface decoration could be 12.74 times higher than that of the bare NPs and 13.11 times higher than Taxol((R)).

Organically modified silica nanoparticles with covalently incorporated photosensitizer for photodynamic therapy of cancer.
Ohulchanskyy TY, Roy I, Goswami LN, Chen Y, Bergey EJ, Pandey RK, Oseroff AR, Prasad PN.
Nano Lett. 2007 Sep;7(9):2835-42.
[ expand abstract ]

We report a novel nanoformulation of a photosensitizer (PS), for photodynamic therapy (PDT) of cancer, where the PS molecules are covalently incorporated into organically modified silica (ORMOSIL) nanoparticles. We found that the covalently incorporated PS molecules retained their spectroscopic and functional properties and could robustly generate cytotoxic singlet oxygen molecules upon photoirradiation. The synthesized nanoparticles are of ultralow size ( approximately 20 nm) and are highly monodispersed and stable in aqueous suspension. The advantage offered by this covalently linked nanofabrication is that the drug is not released during systemic circulation, which is often a problem with physical encapsulation. These nanoparticles are also avidly uptaken by tumor cells in vitro and demonstrate phototoxic action, thereby highlighting their potential in diagnosis and PDT of cancer.

Tumour cell toxicity of intracellular hyperthermia mediated by magnetic nanoparticles.
Wilhelm C, Fortin JP, Gazeau F.
J Nanosci Nanotechnol. 2007 Aug;7(8):2933-7.
[ expand abstract ]

Intracellular hyperthermia is a process by which malignant cells can be selectively killed by heat generated by nanomediators located inside the cell. Here we show that maghemite anionic nanoparticles are efficiently captured by human prostatic tumor cells (PC3) and concentrate within intracellular vesicles. When submitted to an alternative magnetic field, maghemite nanocrystals generate heat from the cell inside, inducing a temperature elevation of eight degree in a loose pellet of 20 million magnetically labeled cells. We demonstrate that this heating modality was as lethal as external waterbath heating. A one hour AC magnetic field (700 kHz-31 mT) exposure of the magnetically labeled cells killed 44% of the cells. Interestingly, more than 80% of the cells were killed after being submitted twice to the magnetic field. Finally, when magnetic cells coexist with non magnetic ones, the same proportions of cells were damaged for both populations, after magnetic field exposure. These findings pave the way for an efficient cell killing mediated by intracellular magnetic hyperthermia.

Dendrimer-modified magnetic nanoparticles enhance efficiency of gene delivery system.
Pan B, Cui D, Sheng Y, Ozkan C, Gao F, He R, Li Q, Xu P, Huang T.
Cancer Res. 2007 Sep 1;67(17):8156-63.
[ expand abstract ]

Magnetic nanoparticles (MNP) with a diameter of 8 nm were modified with different generations of polyamidoamine (PAMAM) dendrimers and mixed with antisense survivin oligodeoxynucleotide (asODN). The MNP then formed asODN-dendrimer-MNP composites, which we incubated with human tumor cell lines such as human breast cancer MCF-7, MDA-MB-435, and liver cancer HepG2 and then analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, quantitative reverse transcription-PCR, Western blotting, laser confocal microscopy, and high-resolution transmission electron microscopy. Results showed that the asODN-dendrimer-MNP composites were successfully synthesized, can enter into tumor cells within 15 min, caused marked down-regulation of the survivin gene and protein, and inhibited cell growth in dose- and time-dependent means. No.5 generation of asODN-dendrimer-MNP composites exhibits the highest efficiency for cellular transfection and inhibition. These results show that PAMAM dendrimer-modified MNPs may be a good gene delivery system and have potential applications in cancer therapy and molecular imaging diagnosis.

Synthesis and characterization of PAMAM dendrimer-based multifunctional nanodevices for targeting alphavbeta3 integrins.
Lesniak WG, Kariapper MS, Nair BM, Tan W, Hutson A, Balogh LP, Khan MK.
Bioconjug Chem. 2007 Jul-Aug;18(4):1148-54.
[ expand abstract ]

We have synthesized a stable and clinically relevant nanodevice (cRGD-BT-ND; ND for short) that exhibits superior binding to the biologic target alphavbeta3 integrins, when either compared to the same free cRGD peptide or to the biotinylated nanodevice without covalently attached peptides (BT-ND). Selective targeting of alphavbeta3 integrins was achieved by coupling cyclic cRGD peptides to the nanodevice (ND) surface, while biotin groups (BT) were used for amplified detection of bound cRGD-BT-ND by anti-biotin antibody or avidin linked to horseradish peroxidase after binding. The synthesis involved the following steps: the amino-terminated ethylenediamine core generation 5 poly(amidoamine) (PAMAM_E5.NH2) dendrimer was first partially acetylated and then biotinylated, and residual primary amine termini were converted to succinamic acid groups (SAH), some of which finally were conjugated with cRGD peptide residues through the amino group of the lysine side chain. The starting material and all derivatives were extensively characterized by polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC), potentiometric acid-base titration, MALDI-TOF, and NMR. Cytotoxicity of all dendrimer derivatives was examined in B16F10 melanoma cell cultures using the XTT colorimetric assay for cellular viability. Binding of nanodevices to the biological target was determined using plates coated with human alphavbeta3 integrin and alphavbeta3 receptor expressing human dermal microvascular endothelial cells (HDMECs). The PAMAM_E5.(NHAc)72(NHBT)8(NHSAH)35(NHSA-cR GD)4 nanodevice is nontoxic within physiologic concentration ranges and specifically binds to the alphavbeta3 integrins, apparently much stronger than the cyclic cRGD peptide itself.

Paclitaxel-functionalized gold nanoparticles.
Gibson JD, Khanal BP, Zubarev ER.
J Am Chem Soc. 2007 Sep 19;129(37):11653-61.
[ expand abstract ]

Here we describe the first example of 2 nm gold nanoparticles (Au NPs) covalently functionalized with a chemotherapeutic drug, paclitaxel. The synthetic strategy involves the attachment of a flexible hexaethylene glycol linker at the C-7 position of paclitaxel followed by coupling of the resulting linear analogue to phenol-terminated gold nanocrystals. The reaction proceeds under mild esterification conditions and yields the product with a high molecular weight, while exhibiting an extremely low polydispersity index (1.02, relative to linear polystyrene standards). TGA analysis of the hybrid nanoparticles reveals the content of the covalently attached organic shell as nearly 67% by weight, which corresponds to approximately 70 molecules of paclitaxel per 1 nanoparticle. The presence of a paclitaxel shell with a high grafting density renders the product soluble in organic solvents and allows for detailed (1)H NMR analysis and, therefore, definitive confirmation of its chemical structure. High-resolution TEM was employed for direct visualization of the inorganic core of hybrid nanoparticles, which were found to retain their average size, shape, and high crystallinity after multiple synthetic steps and purifications. The interparticle distance substantially increases after the attachment of paclitaxel as revealed by low-magnification TEM, suggesting the presence of a larger organic shell. The method described here demonstrates that organic molecules with exceedingly complex structures can be covalently attached to gold nanocrystals in a controlled manner and fully characterized by traditional analytical techniques. In addition, this approach gives a rare opportunity to prepare hybrid particles with a well-defined amount of drug and offers a new alternative for the design of nanosized drug-delivery systems.

Differential tumor cell targeting of anti-HER2 (Herceptin) and anti-CD20 (Mabthera) coupled nanoparticles.
Cirstoiu-Hapca A, Bossy-Nobs L, Buchegger F, Gurny R, Delie F.
Int J Pharm. 2007 Mar 1;331(2):190-6.
[ expand abstract ]

Two types of antibody-labeled nanoparticles (mAb-NPs) were prepared with the aim to achieve specific tumor targeting. Anti-HER2 and anti-CD20 monoclonal antibodies (mAb) were used as model ligands. Small poly(dl-lactic acid) nanoparticles (PLA NPs) with a mean size of about 170 nm were prepared by the salting out method. Thereafter, the coating of PLA NPs with mAbs was performed in two steps. First, thiol groups (-SH) were introduced on the surface of PLA-NPs by a two-step carbodiimide reaction. The number of -SH groups on the surface of NPs increased from 150 to 400 mmol-SH/mol PLA when cystamine concentrations of 25-1518 mol cystamine/mol PLA were used during the thiolation reaction. In the second step, covalent coupling of antibodies to thiolated NPs (NPs-SH) was obtained via a bifunctional cross-linker, m-maleimidobenzoyl-N-hydroxy-sulfosuccinimide ester (sulfo-MBS). For both mAbs anti-HER2 and anti-CD20, respectively, the number of -SH functions on the NPs had no influence on the amount of mAb coupled to the NPs. Approximately, 295 anti-HER2 and 557 anti-CD20 molecules, respectively, were covalently coupled per nanoparticle. The NPs size after the coupling reactions was about 250 nm. The specific interaction between tumor cells and mAb-NPs was determined by confocal microscopy using two cell lines: SKOV-3 human ovarian cancer cells (overexpressing HER2) and Daudi lymphoma cells (overexpressing CD20). The results showed the selective targeting of mAb-NPs to tumor cells overexpressing the specific antigen. While anti-CD20 labeled NPs (anti-CD20 NPs) bound to and remained at the cellular surface, anti-HER2 labeled NPs (anti-HER2 NPs) were efficiently internalized. The mAb-NPs represent a promising approach to improve the efficacy of NPs in active targeting for cancer therapy while the choice of the antibody-target system defines the fate of the mAb-NPs after their binding to the cells.

Synthesis and antibody conjugation of magnetic nanoparticles with improved specific power absorption rates for alternating magnetic field cancer therapy
Grüttnera C, Müllera K, Tellera J, Westphala F, Foremanb A, Ivkovb R.
J Magn Magn Mater. 2007 Apr; 311(1):181-186.
[ expand abstract ]

Bionized nanoferrite (BNF) particles with high specific power absorption rates were synthesized in the size range of 20–100 nm by high-pressure homogenization for targeted cancer therapy with alternating magnetic fields. Several strategies were used to conjugate antibodies to the BNF particles. These strategies were compared using an immunoassay to find optimal conditions to reach a high immunoreactivity of the final antibody–particle conjugate.

Paclitaxel and ceramide co-administration in biodegradable polymeric nanoparticulate delivery system to overcome drug resistance in ovarian cancer.
Devalapally H, Duan Z, Seiden MV, Amiji MM.
Int J Cancer. 2007 Oct 15;121(8):1830-8.
[ expand abstract ]

The objective of this study was to overcome drug resistance upon systemic administration of combination paclitaxel (PTX) and the apoptotic signaling molecule C(6)-ceramide (CER) in biodegradable poly(ethylene oxide)-modified poly(epsilon-caprolactone (PEO-PCL) nanoparticles. Subcutaneous sensitive (wild-type) and multidrug resistant (MDR-1 positive) SKOV-3 human ovarian adenocarcinoma xenografts were established in female Nu/Nu mice. PTX and CER were administered intravenously either as a single agent or in combination in aqueous solution and in PEO-PCL nanoparticles to the tumor-bearing mice. There was significant (p< 0.05) tumor growth suppression in both wild-type SKOV-3 and multidrug resistant SKOV-3(TR) models upon single dose co-administration of PTX (20 mg/kg) and CER (100 mg/kg) in nanoparticle formulations as compared to the individual agents and administration in aqueous solutions. For instance, in SKOV-3 wild-type model, more than 4.3-fold increase (p < 0.05) in tumor growth delay and 3.6-fold (p < 0.05) increase in tumor volume doubling time (DT) were observed with the combination treatment in nanoparticles as compared to untreated animals. Similarly, 3-fold increase (p < 0.05) in tumor growth delay and tumor volume DT was observed in SKOV-3(TR) model. Body weight changes and blood cells counts were used as measures of safety and, except for an increase in platelet counts (p < 0.05) in PTX + CER treated animals, there was no difference between various treatment strategies. The results of this study show that combination of PTX and CER in biodegradable polymeric nanoparticles can serve as a very effective therapeutic strategy to overcome drug resistance in ovarian cancer.

Immunonanoparticles--an effective tool to impair harmful proteolysis in invasive breast tumor cells.
Obermajer N, Kocbek P, Repnik U, Kuznik A, Cegnar M, Kristl J, Kos J.
FEBS J. 2007 Sep;274(17):4416-27.
[ expand abstract ]

Breast cancer cells exhibit excessive proteolysis, which is responsible for extensive extracellular matrix degradation, invasion and metastasis. Besides other proteases, lysosomal cysteine protease cathepsin B has been implicated in these processes and the impairment of its intracellular activity was suggested to reduce harmful proteolysis and hence diminish progression of breast tumors. Here, we present an effective system composed of poly(D,L-lactide-coglycolide) nanoparticles, a specific anti-cytokeratin monoclonal IgG and cystatin, a potent protease inhibitor, that can neutralize the excessive intracellular proteolytic activity as well as invasive potential of breast tumor cells. The delivery system distinguishes between breast and other cells due to the monoclonal antibody specifically recognizing cytokeratines on the membrane of breast tumor cells. Bound nanoparticles are rapidly internalized by means of endocytosis releasing the inhibitor cargo within the lysosomes. This enables intracellular cathepsin B proteolytic activity to be inhibited, reducing the invasive and metastatic potential of tumor cells without affecting proteolytic functions in normal cells and processes. This approach may be applied for treatment of breast and other tumors in which intracellular proteolytic activity is a part of the process of malignant progression.

Synthesis, liposomal preparation, and in vitro toxicity of two novel dodecaborate cluster lipids for boron neutron capture therapy.
Justus E, Awad D, Hohnholt M, Schaffran T, Edwards K, Karlsson G, Damian L, Gabel D.
Bioconjug Chem. 2007 Jul-Aug;18(4):1287-93.
[ expand abstract ]

A new class of lipids, containing the closo-dodecaborate cluster, has been synthesized. Two lipids, S-(N, N-(2-dimyristoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-14) and S-(N, N-(2-dipalmitoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-16) are described. Both of them have a double-tailed lipophilic part and a headgroup carrying two negative charges. Differential scanning calorimetry shows that B-6-14 and B-6-16 bilayers have main phase transition temperatures of 18.8 and 37.9 degrees C, respectively. Above the transition temperature of 18.8 degrees C, B-6-14 can form liposomal vesicles, representing the first boron-containing lipid with this capability. Upon cooling below the transition temperature, stiff bilayers are formed. When incorporated into liposomal formulations with equimolar amounts of distearoyl phosphatidylcholine (DSPC) and cholesterol, stable liposomes are obtained. The zeta-potential measurements indicate that both B-6-14- and B-6-16-containing vesicles are negatively charged, with the most negative potential described of any liposome so far. The liposomes are of high potential value as transporters of boron to tumor cells in treatments based on boron neutron capture therapy (BNCT). Liposomes prepared from B-6-14 were slightly less toxic in V79 Chinese hamster cells (IC50 5.6 mM) than unformulated Na2B12H11SH (IC50 3.9 mM), while liposomes prepared from B-6-16 were not toxic even at 30 mM.

Preparation, characterisation and maintenance of drug efficacy of doxorubicin-loaded human serum albumin (HSA) nanoparticles.
Dreis S, Rothweiler F, Michaelis M, Cinatl J Jr, Kreuter J, Langer K.
Int J Pharm. 2007 Aug 16;341(1-2):207-14.
[ expand abstract ]

Human serum albumin (HSA) nanoparticles represent promising drug carrier systems. Binding of cytostatics to HSA nanoparticles may diminish their toxicity, optimise their body distribution and/or may overcome multidrug resistance. In the present study, doxorubicin-loaded HSA nanoparticle preparations were prepared. Doxorubicin was loaded to the HSA nanoparticles either by adsorption to the nanoparticles' surfaces or by incorporation into the particle matrix. Both loading strategies resulted in HSA nanoparticles of a size range between 150nm and 500nm with a loading efficiency of 70-95%. The influence on cell viability of the resulting nanoparticles was investigated in two different neuroblastoma cell lines. The anti-cancer effects of the drug-loaded nanoparticles were increased in comparison to doxorubicin solution. Based on these result a standard protocol for the preparation of doxorubicin-loaded HSA nanoparticles for further antitumoural studies was established.

Anticancer activity of cisplatin-loaded PLGA-mPEG nanoparticles on LNCaP prostate cancer cells.
Gryparis EC, Hatziapostolou M, Papadimitriou E, Avgoustakis K.
Eur J Pharm Biopharm. 2007 Aug;67(1):1-8.
[ expand abstract ]

The in vitro anticancer activity of cisplatin-loaded PLGA-mPEG nanoparticles on human prostate cancer LNCaP cells was investigated. The uptake of the PLGA-mPEG nanoparticles by the LNCaP cells was also studied. Blank PLGA-mPEG nanoparticles exhibited low cytotoxicity, which increased with increasing PLGA/PEG ratio in the PLGA-mPEG copolymer used to prepare the nanoparticles, possibly due to the increased cell uptake observed with increasing PLGA/PEG ratio. PLGA-mPEG nanoparticles loaded with cisplatin exerted in vitro anticancer activity against LNCaP cells that was comparable to the activity of free (non-entrapped in nanoparticles) cisplatin. Little differences in the in vitro anticancer activity of the different nanoparticle compositions were found, which may result from the differences observed between the different nanoparticles compositions in the uptake by the LNCaP cells and in the leakage of cisplatin from the nanoparticles during incubation with the cells. Visual evidence of nanoparticles' uptake by the LNCaP cells was obtained with nanoparticles labeled with PLGA(4165)-PyrBu(274) or dextran-rhodamine B isothiocyanate using fluorescence microscopy. Moreover, in some cases fluorescence around or inside cell nuclei was observed, which, if verified by further studies, would indicate that PLGA-PEG nanoparticles might prove to be useful in site-specific delivery of drugs whose site of pharmacological activity is cell nucleus.

Development of gelatin nanoparticles with biotinylated EGF conjugation for lung cancer targeting.
Tseng CL, Wang TW, Dong GC, Yueh-Hsiu Wu S, Young TH, Shieh MJ, Lou PJ, Lin FH.
Biomaterials. 2007 Sep;28(27):3996-4005.
[ expand abstract ]

Since lung cancer is the most malignant cancer today, a specific drug-delivery system has been developed for superior outcome. In this study, gelatin nanoparticles (GPs) employed as native carriers were grafted with NeutrAvidin(FITC) on the particle's surface (GP-Av). Next, the biotinylated epithelial growth factor (EGF) molecules were conjugated with NeutrAvidin(FITC), forming a core-shell-like structure (GP-Av-bEGF) to achieve the enhancement of targeting efficiency. These nanoparticles were applied as an EGF receptor (EGFR)-seeking agent to detect lung adenocarcinoma. The results showed that the modification process had no significant influence on particle size (220 nm) and zeta potential (-9.3 mV). By the in vitro cell culture test, GP-Av-bEGF resulted in higher entrance efficiency on adenocarcinoma cells (A549) than that on normal lung cells (HFL1) because A549 possessed greater amounts of EGFR. We also found that uptake of GP-Av-bEGF by A549 cells was time and dose dependent. Confocal microscopy confirmed the cellular internalization of GP-Av-bEGF, and more fluorescent spots of GP-Av-bEGF nanoparticles were obviously observed as well as lysosomal entrapment in A549. Finally, the delivery was demonstrated by in vivo aerosol administration to cancerous lung of the SCID mice model, and specific accumulation in cancerous lung was confirmed by image quantification. The targeting ability of GP-Av-bEGF was proved in vitro and in vivo, which holds promise for further anti-cancer drug applications.

Simplified preparation via streptavidin of antisense oligomers/carriers nanoparticles showing improved cellular delivery in culture.
Wang Y, Nakamura K, Liu X, Kitamura N, Kubo A, Hnatowich DJ.
Bioconjug Chem. 2007 Jul-Aug;18(4):1338-43.
[ expand abstract ]

OBJECTIVE: Carriers are increasingly now viewed as helpful or even essential to improve cellular uptake in connection with antisense tumor targeting and other applications requiring transmembrane delivery of oligomers. Evaluation of many of the large number of available and potentially useful carriers is limited only by the complexities of preparing the oligomer/carriers by covalent conjugation. However, using streptavidin as a linker between biotinylated carriers and biotinylated antisense oligomers would require only simple mixing for preparation. The goal of this study was to evaluate the preparation and cell accumulation in culture of carrier/streptavidin nanoparticle of an antisense phosphorodiamide morpholino (MORF) oligomer. METHODS: The model carriers were cholesterol, a 10 mer HIV-tat peptide, and a 10 mer polyarginine, each having been reported elsewhere to improve cellular delivery of oligomers. The model antisense oligomer was the 25 mer MORF targeting the survivin mRNA. The accumulations of the antisense MORF/carrier nanoparticle were compared to the sense MORF/carrier, to the carrier-free nanoparticles, and to the naked antisense MORF in the survivin-expressing MCF-7 cells. The MORFs and peptides were purchased biotinylated, while the cholesterol was biotinylated in-house. In all cases, the 99mTc radiolabel was placed on the oligomers. Cell studies were performed at low nM concentration as required for antisense imaging applications and at 37 degrees C primarily in 1% FBS. RESULTS: Each radiolabeled oligomer/streptavidin/carrier nanoparticle was successfully prepared by careful mixing at a 1:1 molar ratio. As evidence of carrier participation, the radiolabeled MORF showed increased accumulation in cells when incubated as the nanoparticle compared to the carrier-free nanoparticle and by as much as a factor of 11. Accumulation of the antisense MORF/streptavidin/tat nanoparticle was significantly higher than the sense MORF/streptavidin/tat nanoparticle as evidence of specific antisense targeting.
CONCLUSIONS: The preparation of oligomer/carrier nanoparticles was greatly simplified over covalent conjugations by using streptavidin as a linker. Furthermore, our results suggest that the addition of streptavidin did not interfere with the cellular delivery function of the tat, polyarginine, or cholesterol carriers nor with the specific antisense mRNA binding function of the MORF oligomer.

Targeting cancer cells using PLGA nanoparticles surface modified with monoclonal antibody.
Kocbek P, Obermajer N, Cegnar M, Kos J, Kristl J.
J Control Release. 2007 Jul 16;120(1-2):18-26.
[ expand abstract ]

Targeting drugs to their sites of action is still a major challenge in pharmaceutical research. In this study, polylactic-co-glycolic acid (PLGA) immuno-nanoparticles were prepared for targeting invasive epithelial breast tumour cells. Monoclonal antibody (mAb) was used as a homing ligand and was attached to the nanoparticle surface either covalently or non-covalently. The presence of mAb on the nanoparticle surface, its stability and recognition properties were tested. Protein assay, surface plasmon resonance, flow cytometry and fluorescence-immunostaining confirmed the presence of mAb on nanoparticles in both cases. However, a binding assay using cell lysate revealed that the recognition properties were preserved only for nanoparticles with adsorbed mAb. These nanoparticles were more likely to be bound to the targeted cells than non-coated nanoparticles. Both types of nanoparticles entered the target MCF-10A neoT cells in mono-culture. In co-culture of MCF-10A neoT and Caco-2 cells immuno-nanoparticles were localized solely to MCF-10A neoT cells, whereas non-coated nanoparticles were distributed randomly. Immuno-nanoparticles entered only MCF-10A neoT cells, while non-coated nanoparticles were taken up by both cell types, indicating specific targeting of the immuno-nanoparticles. In conclusion, we demonstrate a method by which mAbs can be bound to nanoparticles without detriment to their targeting ability. Furthermore, the results show the effectiveness of the new carrier system for targeted delivery of small or large active substances into cells or tissues of interest.

Mesoporous silica nanoparticles as a delivery system for hydrophobic anticancer drugs.
Lu J, Liong M, Zink JI, Tamanoi F.
Small. 2007 Aug;3(8):1341-6.
[ expand abstract ]

No abstract available. (Citation link )

The drug loading, cytotoxicty and tumor vascular targeting characteristics of magnetite in magnetic drug targeting.
Dandamudi S, Campbell RB.
Biomaterials. 2007 Nov;28(31):4673-83.
[ expand abstract ]

Chemotherapy is a popular treatment approach against cancer but significant uptake of drugs by normal tissues is still a major limitation. Magnetic drug targeting (MDT) has been used to improve localized drug delivery to interstitial tumor targets. MDT is now being developed to improve drug delivery to tumor vessels. We thus seek to understand the role of magnetite (MAG-C) in drug loading, influence on cytotoxicity and vascular targeting characteristics. The inclusion of MAG-C at lower concentrations (0.5 mg/ml) in cationic liposomes did not alter the efficiency of loading etoposide, but at higher concentrations (2.5 mg/ml) incorporation decreased from 80+/-3.4% to 44+/-4.26%. MAG-C reduced the incorporation of dacarbazine. The incorporation was significantly lower compared to liposomal etoposide, both in the presence and absence of MAG-C. The incorporation efficiency of vinblastine sulfate in cationic liposomes was similar for low and relatively high MAG-C content; values for incorporation were 21+/-0.11 and 23+/-2, respectively. Polyethylene-glycol improved the efficiency of loading chemotherapeutic agents regardless of drug type. Additionally, cytotoxicity and tumor vascular targeting characteristics of liposome therapeutics were not influenced by MAG-C. The components used to prepare magnetic liposomes for MDT should be optimized for maximum therapeutic benefit.

Targeted quantum dot conjugates for siRNA delivery.
Derfus AM, Chen AA, Min DH, Ruoslahti E, Bhatia SN.
Bioconjug Chem. 2007 Sept-Oct; 18(5):1391-6.
[ expand abstract ]

Treatment of human diseases such as cancer generally involves the sequential use of diagnostic tools and therapeutic modalities. Multifunctional platforms combining therapeutic and diagnostic imaging functions in a single vehicle promise to change this paradigm. in particular, nanoparticle-based multifunctional platforms offer the potential to improve the pharmacokinetics of drug formulations, while providing attachment sites for diagnostic imaging and disease targeting features. We have applied these principles to the delivery of small interfering RNA (siRNA) therapeutics, where systemic delivery is hampered by rapid excretion and nontargeted tissue distribution. Using a PEGlyated quantum dot (QD) core as a scaffold, siRNA and tumor-homing peptides (F3) were conjugated to functional groups on the particle's surface. We found that the homing peptide was required for targeted internalization by tumor cells, and that siRNA cargo could be coattached without affecting the function of the peptide. Using an EGFP model system, the role of conjugation chemistry was investigated, with siRNA attached to the particle by disulfide cross-linkers showing greater silencing efficiency than when attached by a nonreducible thioether linkage. Since each particle contains a limited number of attachment sites, we further explored the tradeoff between number of F3 peptides and the number of siRNA per particle, leading to an optimized formulation. Delivery of these F3/siRNA-QDs to EGFP-transfected HeLa cells and release from their endosomal entrapment led to significant knockdown of EGFP signal. By designing the siRNA sequence against a therapeutic target (e.g., oncogene) instead of EGFP, this technology may be ultimately adapted to simultaneously treat and image metastatic cancer.

Multifunctional nanoparticles for combining ultrasonic tumor imaging and targeted chemotherapy.
Rapoport N, Gao Z, Kennedy A.
J Natl Cancer Inst. 2007 Jul 18;99(14):1095-106.
[ expand abstract ]

BACKGROUND: Drug delivery in polymeric micelles combined with tumor irradiation by ultrasound results in effective drug targeting, but this technique requires prior tumor imaging. A technology that combined ultrasound imaging with ultrasound-mediated nanoparticle-based targeted chemotherapy could therefore have important applications in cancer treatment. METHODS: Mixtures of drug-loaded polymeric micelles and perfluoropentane (PFP) nano/microbubbles stabilized by the same biodegradable block copolymer were prepared. Size distribution of nanoparticles was measured by dynamic light scattering. Cavitation activity (oscillation, growth, and collapse of microbubbles) under ultrasound was assessed based on the changes in micelle/microbubble volume ratios. The effect of the nano/microbubbles on the ultrasound-mediated cellular uptake of doxorubicin (Dox) in MDA MB231 breast tumors in vitro and in vivo (in mice bearing xenograft tumors) was determined by flow cytometry. Statistical tests were two-sided. RESULTS: Phase state and nanoparticle sizes were sensitive to the copolymer/perfluorocarbon volume ratio. At physiologic temperatures, nanodroplets converted into nano/microbubbles. Doxorubicin was localized in the microbubble walls formed by the block copolymer. Upon intravenous injection into mice, Dox-loaded micelles and nanobubbles extravasated selectively into the tumor interstitium, where the nanobubbles coalesced to produce microbubbles with a strong, durable ultrasound contrast. Doxorubicin was strongly retained in the microbubbles but released in response to therapeutic ultrasound. Microbubbles cavitated under the action of tumor-directed ultrasound, which enhanced intracellular Dox uptake by tumor cells in vitro to a statistically significant extent relative to that observed with unsonicated microbubbles (drug uptake ratio = 4.60; 95% confidence interval [CI] = 1.70 to 12.47; P = .017) and unsonicated micelles (drug uptake ratio = 7.97; 95% CI = 3.72 to 17.08; P = .0032) and resulted in tumor regression in the mouse model. CONCLUSIONS: Multifunctional nanoparticles that are tumor-targeted drug carriers, long-lasting ultrasound contrast agents, and enhancers of ultrasound-mediated drug delivery have been developed and deserve further exploration as cancer therapeutics.

211AtCl@US-tube nanocapsules: a new concept in radiotherapeutic-agent design
Hartman KB, Hamlin DK, Wilbur S, Wilson LJ.
Small. 3(9):1496–1499.
[ expand abstract ]

No abstract available (Citation link)

Near-infrared resonant nanoshells for combined optical imaging and photothermal cancer therapy.
Gobin AM, Lee MH, Halas NJ, James WD, Drezek RA, West JL.
Nano Lett. 2007 Jul;7(7):1929-34.
[ expand abstract ]

Metal nanoshells are core/shell nanoparticles that can be designed to either strongly absorb or scatter within the near-infrared (NIR) wavelength region ( approximately 650-950 nm). Nanoshells were designed that possess both absorption and scattering properties in the NIR to provide optical contrast for improved diagnostic imaging and, at higher light intensity, rapid heating for photothermal therapy. Using these in a mouse model, we have demonstrated dramatic contrast enhancement for optical coherence tomography (OCT) and effective photothermal ablation of tumors.

Functionalized fullerenes mediate photodynamic killing of cancer cells: Type I versus Type II photochemical mechanism.
Mroz P, Pawlak A, Satti M, Lee H, Wharton T, Gali H, Sarna T, Hamblin MR.
Free Radic Biol Med. 2007 Sep 1;43(5):711-9.
[ expand abstract ]

Photodynamic therapy (PDT) employs the combination of nontoxic photosensitizers (PS) and harmless visible light to generate reactive oxygen species (ROS) and kill cells. Most clinically studied PS are based on the tetrapyrrole structure of porphyrins, chlorines, and related molecules, but new nontetrapyrrole PS are being sought. Fullerenes are soccer-ball shaped molecules composed of 60 or 70 carbon atoms and have attracted interest in connection with the search for biomedical applications of nanotechnology. Fullerenes are biologically inert unless derivatized with functional groups, whereupon they become soluble and can act as PS. We have compared the photodynamic activity of six functionalized fullerenes with 1, 2, or 3 hydrophilic or 1, 2, or 3 cationic groups. The octanol-water partition coefficients were determined and the relative contributions of Type I photochemistry (photogeneration of superoxide in the presence of NADH) and Type II photochemistry (photogeneration of singlet oxygen) were studied by measurement of oxygen consumption, 1270-nm luminescence and EPR spin trapping of the superoxide product. We studied three mouse cancer cell lines: (J774, LLC, and CT26) incubated for 24 h with fullerenes and illuminated with white light. The order of effectiveness as PS was inversely proportional to the degree of substitution of the fullerene nucleus for both the neutral and the cationic series. The monopyrrolidinium fullerene was the most active PS against all cell lines and induced apoptosis 4-6 h after illumination. It produced diffuse intracellular fluorescence when dichlorodihydrofluorescein was added as an ROS probe, suggesting a Type I mechanism for phototoxicity. We conclude that certain functionalized fullerenes have potential as novel PDT agents and phototoxicity may be mediated both by superoxide and by singlet oxygen.

Intracellular heating of living cells through Néel relaxation of magnetic nanoparticles.
Fortin JP, Gazeau F, Wilhelm C.
Eur Biophys J. 2008 Feb;37(2):223-8.
[ expand abstract ]

Maghemite and cobalt ferrite anionic magnetic nanoparticles enter tumor cells and can be used as heat sources when exposed to a high-frequency magnetic field. Comparative studies of the two particles enable to unravel the magnetic heating mechanisms (Néel relaxation vs. Brown relaxation) responsible for the cellular temperature rise, and also to establish a simple model, adjusted to the experimental results, allowing to predict the intracellular heating efficiency of iron oxide nanoparticles. Hence, we are able to derive the best nanoparticle design for a given material with a view to intracellular hyperthermia-based applications.

Tumor targeting with antibody-functionalized, radiolabeled carbon nanotubes.
McDevitt MR, Chattopadhyay D, Kappel BJ, Jaggi JS, Schiffman SR, Antczak C, Njardarson JT, Brentjens R, Scheinberg DA.
J Nucl Med. 2007 Jul;48(7):1180-9
[ expand abstract ]

Single-walled carbon nanotubes (CNT) are mechanically robust graphene cylinders with a high aspect ratio that are comprised of sp(2)-bonded carbon atoms and possessing highly regular structures with defined periodicity. CNT exhibit unique mechanochemical properties that can be exploited for the development of novel drug delivery platforms. We hypothesized that novel prototype nanostructures consisting of biologics, radionuclides, fluorochromes, and CNT could be synthesized and designed to target tumor cells. METHODS: Tumor-targeting CNT constructs were synthesized from sidewall-functionalized, water-soluble CNT platforms by covalently attaching multiple copies of tumor-specific monoclonal antibodies, radiometal-ion chelates, and fluorescent probes. The constructs were characterized spectroscopically, chromatographically, and electrophoretically. The specific reactivity of these constructs was evaluated in vitro by flow cytometry and cell-based immunoreactivity assays and in vivo using biodistribution in a murine xenograft model of lymphoma. RESULTS: A soluble, reactive CNT platform was used as the starting point to build multifunctional constructs with appended antibody, metal-ion chelate, and fluorescent chromophore moieties to effect specific targeting, to carry and deliver a radiometal-ion, and to report location, respectively. These nanoconstructs were found to be specifically reactive with the human cancer cells they were designed to target in vivo in a model of disseminated human lymphoma and in vitro by flow cytometry and cell-based immunoreactivity assays versus appropriate controls. CONCLUSION: The key achievement in these studies was the selective targeting of tumor in vitro and in vivo by the use of specific antibodies appended to a soluble, nanoscale CNT construct. The ability to specifically target tumor with prototype-radiolabeled or fluorescent-labeled, antibody-appended CNT constructs was encouraging and suggested further investigation of CNT as a novel delivery platform.

Multiplex targeting, tracking, and imaging of apoptosis by fluorescent surface enhanced Raman spectroscopic dots.
Yu KN, Lee SM, Han JY, Park H, Woo MA, Noh MS, Hwang SK, Kwon JT, Jin H, Kim YK, Hergenrother PJ, Jeong DH, Lee YS,Cho MH.
Bioconjug Chem. 2007 Jul-Aug;18(4):1155-62.
[ expand abstract ]

We have developed multifunctional fluorescent surface enhanced Raman spectroscopic tagging material (F-SERS dots) composed of silver nanoparticle-embedded silica spheres with fluorescent organic dye and specific Raman labels for multiplex targeting, tracking, and imaging of cellular/molecular events in the living organism. In this study, F-SERS dots fabricated with specific target antibodies (BAX and BAD) were employed for the detection of apoptosis. The F-SERS dots did not show any particular toxicity in several cell lines. The F-SERS dots could monitor the apoptosis effectively and simultaneously through fluorescent images as well as Raman signals in both cells and tissues with high selectivity. Our results clearly demonstrate that F-SERS dots can be easily applicable to multiplex analysis of diverse cellular/molecular events important for maintaining cellular homeostasis.

Internalization of MWCNTs by microglia: possible application in immunotherapy of brain tumors.
Kateb B, Van Handel M, Zhang L, Bronikowski MJ,Manohara H, Badie B.
Neuroimage. 2007;37 Suppl 1:S9-17.
[ expand abstract ]

There is a pressing need for new therapeutic, diagnostic, and drug delivery approaches for treating brain cancers. Nanotechnology offers a new method for targeted brain cancer therapy and could play a major role in gene and drug delivery. The goals of our study were to visualize in vitro ingestion, cytotoxicity, and loading capacity of Multi-Walled Carbon Nanotubes (MWCNTs) in microglia. Furthermore, we investigated internalization differences between microglia and glioma cells. BV2 microglia and GL261 glioma cells were incubated with MWCNTs, which were synthesized through catalytic chemical vapor deposition technique. Real-time RT-PCR, cell proliferation analysis, siRNA and DNA loading, electron microscopy, and flow cytometry were performed. We demonstrated that MWCNTs do not result in proliferative or cytokine changes in vitro, are capable of carrying DNA and siRNA and are internalized at higher levels in phagocytic cells as compared to tumor cells. This study suggests MWCNTs could be used as a novel, non-toxic, and biodegradable nano-vehicles for targeted therapy in brain cancers. Further studies are needed to demonstrate the full capacity of MWCNTs as nanovectors.

Methylene blue-containing silica-coated magnetic particles: a potential magnetic carrier for photodynamic therapy.
Tada DB, Vono LL, Duarte EL, Itri R, Kiyohara PK, Baptista MS, Rossi LM.
Langmuir. 2007 Jul 17;23(15):8194-9.
[ expand abstract ]

We present the preparation and characterization of methylene blue-containing silica-coated magnetic particles. The entrapment of methylene blue (MB), a photodynamic therapy drug under study in our group, in the silica matrix took place during the growth of a silica layer over a magnetic core composed of magnetite nanoparticles. The resulting material was characterized by transmission electron microscopy (TEM), light scattering, and X-ray diffraction. It is composed of approximately 30 nm silica spheres containing magnetic particles of 11 +/- 2 nm and methylene blue entrapped in the silica matrix. The immobilized drug can generate singlet oxygen, which was detected by its characteristic phosphorescence decay curve in the near-infrared and by a chemical method using 1,3-diphenylisobenzofuran to trap singlet oxygen. The lifetime of singlet oxygen was determined to be 52 micros (in acetonitrile) and 3 micros (in water), with both values being in good agreement with those in the literature. The release of singlet oxygen (etaDelta) was affected by the encapsulation of MB in the silica matrix, which caused a reduction to 6% of the quantum yield of MB free in solution. The magnetization curve confirmed the superparamagnetic behavior with a reduced saturation magnetization in respect to uncoated magnetic nanoparticles, which is consistent with the presence of a diamagnetic component over the magnetite surface. The result is a single particle platform that combines therapy (photosensitizer) and diagnostic (MRI contrast agent) possibilities at the same time, as well as drug targeting.

Co-Delivery of Hydrophobic and Hydrophilic Drugs from Nanoparticle-Aptamer Bioconjugates.
Zhang L, Radovic-Moreno AF, Alexis F, Gu FX, Basto PA, Bagalkot V, Jon S, Langer RS, Farokhzad OC.
ChemMedChem. 2(9):1268 – 1271.
[ expand abstract ]

No abstract available. (Citation link)

Modulation of intracellular ceramide using polymeric nanoparticles to overcome multidrug resistance in cancer.
van Vlerken LE, Duan Z, Seiden MV, Amiji MM.
Cancer Res. 2007 May 15;67(10):4843-50.
[ expand abstract ]

Although multidrug resistance (MDR) is known to develop through a variety of molecular mechanisms within the tumor cell, many tend to converge toward the alteration of apoptotic signaling. The enzyme glucosylceramide synthase (GCS), responsible for bioactivation of the proapoptotic mediator ceramide to a nonfunctional moiety glucosylceramide, is overexpressed in many MDR tumor types and has been implicated in cell survival in the presence of chemotherapy. The purpose of this study was to investigate the therapeutic strategy of coadministering ceramide with paclitaxel, a commonly used chemotherapeutic agent, in an attempt to restore apoptotic signaling and overcome MDR in the human ovarian cancer cell line SKOV3. Poly(ethylene oxide)-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticles were used to encapsulate and deliver the therapeutic agents for enhanced efficacy. Results show that indeed the cotherapy eradicates the complete population of MDR cancer cells when they are treated at their IC(50) dose of paclitaxel. More interestingly, when the cotherapy was combined with the properties of nanoparticle drug delivery, the MDR cells can be resensitized to a dose of paclitaxel near the IC(50) of non-MDR (drug sensitive) cells, indicating a 100-fold increase in chemosensitization via this approach. Molecular analysis of activity verified the hypothesis that the efficacy of this therapeutic approach is indeed due to a restoration in apoptotic signaling, although the beneficial properties of PEO-PCL nanoparticle delivery seemed to enhance the therapeutic success even further, showing the promising potential for the clinical use of this therapeutic strategy to overcome MDR.

Dendrimer-entrapped gold nanoparticles as a platform for cancer-cell targeting and imaging.
Shi X, Wang S, Meshinchi S, Van Antwerp ME, Bi X, Lee I, Baker JR Jr.
Small. 2007 Jul;3(7):1245-52.
[ expand abstract ]

We present a general approach for the targeting and imaging of cancer cells using dendrimer-entrapped gold nanoparticles (Au DENPs). Au DENPs were found to be able to covalently link with targeting and imaging ligands for subsequent cancer-cell targeting and imaging. The Au DENPs linked with defined numbers of folic acid (FA) and fluorescein isothiocyanate (FI) molecules are water soluble, stable, and biocompatible. In vitro studies show that the FA- and FI-modified Au DENPs can specifically bind to KB cells (a human epithelial carcinoma cell line) that overexpress high-affinity folate receptors and they are internalized dominantly into lysosomes of target cells within 2 h. These findings demonstrate that Au DENPs may serve as a general platform for cancer imaging and therapeutics.

Morbidity and quality of life during thermotherapy using magnetic nanoparticles in locally recurrent prostate cancer: results of a prospective phase I trial.
Johannsen M, Gneveckow U, Taymoorian K, Thiesen B, Waldöfner N, Scholz R, Jung K, Jordan A, Wust P, Loening SA.
Int J Hyperthermia. 2007 May;23(3):315-23.
[ expand abstract ]

PURPOSE: To investigate the treatment-related morbidity and quality of life (QoL) during thermotherapy using superparamagnetic nanoparticles in patients with locally recurrent prostate cancer. MATERIALS AND METHODS: Ten patients with biopsy-proven locally recurrent prostate cancer following primary therapy with curative intent and no detectable metastases were entered on a prospective phase I trial. Endpoints were feasibility, toxicity and QoL. Following intraprostatic injection of a nanoparticle dispersion, six thermal therapy sessions of 60 min duration were delivered at weekly intervals using an alternating magnetic field. National Cancer Institute (NCI) common toxicity criteria (CTC) and the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 and QLQ-PR25 questionnaires were used to evaluate toxicity and QoL, respectively. In addition, prostate specific antigen (PSA) measurements were carried out. RESULTS: Maximum temperatures up to 55 degrees C were achieved in the prostates at 25-30% of the available magnetic field strength. Nanoparticle deposits were detectable in the prostates one year after thermal therapy. At a median follow-up of 17.5 months (3-24), no systemic toxicity was observed. Acute urinary retention occurred in four patients with previous history of urethral stricture. Treatment-related morbidity was moderate and QoL was only temporarily impaired. Prostate-specific antigen (PSA) declines were observed in eight patients. CONCLUSIONS: Interstitial heating using magnetic nanoparticles was feasible and well tolerated in patients with locally recurrent prostate cancer. Deposition of nanoparticles in the prostate was highly durable. Further refinement of the technique is necessary to allow application of higher magnetic field strengths.

A p53-derived apoptotic peptide derepresses p73 to cause tumor regression in vivo.
Bell, HS, Dufes C, O'Prey J, Crighton D, Bergamaschi D, Lu X, Schatzlein AG, Vousden KH, Ryan KM.
J Clin Invest. 2007;117(4):1008-18.
[ expand abstract ]

The tumor suppressor p53 is a potent inducer of tumor cell death, and strategies exist to exploit p53 for therapeutic gain. However, because about half of human cancers contain mutant p53, application of these strategies is restricted. p53 family members, in particular p73, are in many ways functional paralogs of p53, but are rarely mutated in cancer. Methods for specific activation of p73, however, remain to be elucidated. We describe here a minimal p53-derived apoptotic peptide that induced death in multiple cell types regardless of p53 status. While unable to activate gene expression directly, this peptide retained the capacity to bind iASPP - a common negative regulator of p53 family members. Concordantly, in p53-null cells, this peptide derepressed p73, causing p73-mediated gene activation and death. Moreover, systemic nanoparticle delivery of a transgene expressing this peptide caused tumor regression in vivo via p73. This study therefore heralds what we believe to be the first strategy to directly and selectively activate p73 therapeutically and may lead to the development of broadly applicable agents for the treatment of malignant disease.

Ceramic nanoparticles deliver genes to the spleen, trigger antitumor immune response. Nanosized bioceramic particles could function as efficient gene delivery vehicles with target specificity for the spleen.
Tan K, Cheang P, Ho IA, Lam PY, Hui KM.
Gene Ther. 2007 May;14(10):828-35.
[ expand abstract ]

We have compared the ability of several nanosized bioceramic particles including negatively charged silica (SiO(2)), neutrally charged hydroxyapatite (HA) and positively charged zirconia (ZrO(2)) nanoparticles as non-viral vectors for efficient in vivo gene delivery. A mixture of highly monodispersed aqueous suspension of HA or SiO(2) nanoparticles, coated with protamine sulfate (PS), complexed efficiently with plasmid DNA and significantly enhanced transgene expression in vitro. In comparison, ZrO(2) nanoparticles gave poor transfection efficiency under similar conditions tested. It was also determined that, under the same conditions, PS-SiO(2)-DNA, but not PS-HA-DNA-nanoplexes, were able to mediate efficient transgene expression in vitro in the presence of 50% serum. Intraperitoneal injections of PS-SiO(2)-luciferase DNA nanoplexes targeted the highest level of transgene expression in the spleen of recipient mice that lasted for more than 48 h. Injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes was able to mediate the production of Flt-3L in the sera of recipient mice. Simultaneously, the production of Flt-3L was accompanied by the stimulation of IL-2 and interferon-gamma (IFN-gamma). Most importantly, the injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes could mount potent anti-tumour specific immune responses that led to the subsequent regression of parental tumor cells containing the muc-1 determinant.

Solid lipid nanoparticles: could they help to improve the efficacy of pharmacologic treatments for brain tumors?
Brioschi A, Zenga F, Zara GP, Gasco MR, Ducati A, Mauro A.
Neurol Res. 2007 Apr;29(3):324-30.
[ expand abstract ]

OBJECTIVES: Brain malignant neoplasms are still characterized by poor prognosis due to their peculiar hallmarks that severely limit aggressive multimodal therapeutic approaches. The optimization of the intratumoral drug delivery, directed to achieve effective concentrations and to reduce systemic undesired toxicity, is one of the primary goals of the brain tumors therapeutic strategies. Different passive and active delivery carriers allowing to a better control of drug distribution, metabolism, and elimination after parenteral administration have been developed. In the present review we will describe general characteristics and evaluate the efficacy of Solid Lipid Nanoparticles (SLN) as carriers of different drugs in experimental brain malignant tumor therapy. METHODS: SLN vehiculating different illustrative types of antineoplastic agents (conventional cytotoxic drugs such as doxorubicin and paclitaxel, the prodrug Cholesteryl butyrate, and anti VEGF antisense oligonucleotides) have been tested in experimental animal models of cerebral gliomas. RESULTS: SLN proved to successfully vehiculate into the brain different types of cytotoxic and gene therapeutical agents (otherwise unable to pass through the Blood-Brain Barrier) and to induce effective anti-tumoral therapeutical response. DISCUSSION: Compared to other vehicules, SLN seem to offer more advantages (such as higher physical stability, greater protection from degradation and better release profile of incorporated drugs, good tolerability and possibility of site-specific targeting) and could be regarded as an effective carrier for chemotherapeutic drugs, gene therapeutical agents, and diagnostic tools in neuro-oncology.

Preparation and characterization of radioactive dirhenium decacarbonyl-loaded PLLA nanoparticles for radionuclide intra-tumoral therapy.
Hamoudeh M, Salim H, Barbos D, Paunoiu C, Fessi H.
Eur J Pharm Biopharm. 2007 Nov;67(3):597-611.
[ expand abstract ]

This study describes the development of biocompatible radioactive rhenium-loaded nanoparticles for radionuclide anti-cancer therapy. To achieve this goal, dirhenium decacarbonyl [Re2(CO)10] has been encapsulated in poly(L-lactide) based nanoparticles by an oil-in-water emulsion-solvent evaporation method. A 3(3) factorial design method was applied to investigate the influence of both the proceeding and formulation parameters including the stirring speed and the concentration of both the PLLA polymer and the poly(vinyl alcohol) stabiliser on both nanoparticles size and the Re2(CO)10 encapsulation efficacy. The factorial design results attributed a clear negative effect for the stirring speed and the stabiliser concentration on the nanoparticles size while the polymer concentration exhibited a positive one. Regarding the Re2(CO)10 encapsulation efficacy, higher values were obtained when higher polymer concentrations, lower stabiliser concentrations or slower stirring speeds were applied in the preparation. Different tests were thereafter performed to characterize the Re2(CO)10-loaded nanoparticles. The nanoparticles size, being experimentally controlled by the above mentioned parameters, ranged between 330 and 1500 nm and the maximum rhenium loading was 24% by nanoparticles weight as determined by atomic emission assays and neutron activation analysis. Furthermore, the rhenium distribution within nanoparticles has been shown to be homogeneous as confirmed by the energy dispersive X-ray spectrometry. DSC assays demonstrated that Re2(CO)10 was encapsulated in its crystalline initial state. Other experiments including FT-IR and NMR did not show interactions between PLLA and Re2(CO)10. To render them radioactive, these nanoparticles have been bombarded with a neutron flux of 1.45x10(13) n/cm2/s during 1 h. The SEM micrographs of nanoparticles after neutron bombardment showed that the nanoparticles remained spherical and separated but slightly misshaped. These applied neutron activation conditions yielded a specific activity of about 32.5 GBq per gram of nanoparticles. Preliminary estimations allow us to think that a sole injection of 50 mg of these activated nanoparticles into a brain tumor model (4.2 cm diameter) would deliver a tumor absorbed dose of up to 47 Gy. In conclusion, these dirhenium decacarbonyl-loaded nanoparticles represent a novel promising tool for radionuclide anti-cancer therapy.

Encapsulation of paclitaxel in macromolecular nanoshells.
Zahr AS, Pishko MV.
Biomacromolecules. 2007 Jun;8(6):2004-10.
[ expand abstract ]

An electrostatic layer-by-layer self-assembly technique was used to encapsulate solid core paclitaxel nanoparticles within a polymeric nanometer-scale shell. This approach provides a new strategy for the development of polymeric vehicles that control drug release and target diseased tissues and cells specific to the ailment, such as breast cancer. Core paclitaxel nanoparticles, 153 +/- 28 nm in diameter, were prepared using a modified nanoprecipitation technique. A nanoshell composed of multilayered polyelectrolytes, poly(allylamine hydrochloride) and poly(styrene-4-sulfonate) was assembled stepwise onto core charged drug nanoparticles. In vitro studies were performed to determine the anticancer activity of paclitaxel core-shell nanoparticles. Paclitaxel core-shell nanoparticles induced cell cycle arrest in the G2/M phase after 24 and 48 h of incubation with a human breast carcinoma cell line, MCF-7. Changes in MCF-7 cell morphology, fragmentation of the nucleus, and loss of cell-cell contacts indicated that the cells responded to paclitaxel core nanoparticles upon treatment for 24 and 48 h. Cells arrested in G2/M phase illustrated abnormal microtubule and actin cytoskeleton morphology. The core-shell drug nanoparticles fabricated using this procedure provide a new approach in the delivery of paclitaxel devoid of Cremophor EL, a solvent that causes adverse side effects in patients undergoing chemotherapy for treatment of metastasized mammary cancers.

Nanoparticulate delivery of suicide DNA to murine prostate and prostate tumors.
Peng W, Anderson DG, Bao Y, Padera RF Jr, Langer R, Sawicki JA.
Prostate. 2007 Jun 1;67(8):855-62.
[ expand abstract ]

BACKGROUND: Currently available treatments for benign prostatic hyperplasia (BPH) and localized prostate cancer are generally effective but are often attended by serious side effects that impact on the quality of life. In particular, most current therapies are non-specific, with surgery, radiation, and chemical ablation having the potential to cause damage to surrounding tissue. Here, we demonstrate the effectiveness of a prostate-specific, locally delivered gene therapy for the targeted killing of prostate cells. METHODS: Using a degradable, poly(beta-amino ester) polymer, poly(butane diol diacrylate co amino pentanol) (C32), we developed a nanoparticulate system to deliver a diphtheria toxin suicide gene (DT-A) driven by a prostate specific promoter to cells. These C32/DT-A nanoparticles were directly injected to the normal prostate and to prostate tumors in mice. RESULTS: Nearly 50% of normal prostates showed a significant reduction in size, attributable to cellular apoptosis, whereas injection with naked DT-A-encoding DNA had little effect. Significant apoptosis was also observed in C32/DT-A injected prostate tumors. Importantly, no damage to surrounding tissue was observed. CONCLUSIONS: These results suggest that local delivery of poly(beta-amino ester) polymer/ DT-A nanoparticles may have application in the treatment of BPH and prostate cancer.

Nanosized bioceramic particles could function as efficient gene delivery vehicles with target specificity for the spleen.
Tan K, Cheang P, Ho IA, Lam PY, Hui KM.
Gene Ther. 2007 May;14(10):828-35.
[ expand abstract ]

We have compared the ability of several nanosized bioceramic particles including negatively charged silica (SiO(2)), neutrally charged hydroxyapatite (HA) and positively charged zirconia (ZrO(2)) nanoparticles as non-viral vectors for efficient in vivo gene delivery. A mixture of highly monodispersed aqueous suspension of HA or SiO(2) nanoparticles, coated with protamine sulfate (PS), complexed efficiently with plasmid DNA and significantly enhanced transgene expression in vitro. In comparison, ZrO(2) nanoparticles gave poor transfection efficiency under similar conditions tested. It was also determined that, under the same conditions, PS-SiO(2)-DNA, but not PS-HA-DNA-nanoplexes, were able to mediate efficient transgene expression in vitro in the presence of 50% serum. Intraperitoneal injections of PS-SiO(2)-luciferase DNA nanoplexes targeted the highest level of transgene expression in the spleen of recipient mice that lasted for more than 48 h. Injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes was able to mediate the production of Flt-3L in the sera of recipient mice. Simultaneously, the production of Flt-3L was accompanied by the stimulation of IL-2 and interferon-gamma (IFN-gamma). Most importantly, the injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes could mount potent anti-tumour specific immune responses that led to the subsequent regression of parental tumor cells containing the muc-1 determinant.

Quantum dots are phagocytized by macrophages and colocalize with experimental gliomas.
Jackson H, Muhammad O, Daneshvar H, Nelms J, Popescu A, Vogelbaum MA, Bruchez M, Toms SA.
Neurosurgery. 2007 Mar;60(3):524-9; discussion 529-30.
[ expand abstract ]

OBJECTIVE: The identification of neoplastic tissue within normal brain during biopsy and tumor resection remains a problem in the operative management of gliomas. A variety of nanoparticles are phagocytized by macrophages in vivo. This feature may allow optical nanoparticles, such as quantum dots, to colocalize with brain tumors and serve as an optical aid in the surgical resection or biopsy of brain tumors. METHODS: Male Fisher rats (Charles River Labs, Wilmington, MA) were implanted intracranially with C6 gliosarcoma cell lines to establish tumors. Two weeks after the implantation of tumors, 705-nm emission Qdot ITK Amino(PEG) Quantum Dots (Quantum Dot Corp., Hayward, CA) were injected via the tail vein at doses of 3 to 17 nmol. The animals were sacrificed 24 hours after the injection of quantum dots and their tissues were examined. RESULTS: Quantum dots are avidly phagocytized by macrophages and are taken up by the liver, spleen, and lymph nodes. A dose-response relationship was noted. At low doses, the majority of the quantum dots are sequestered in the liver, spleen, and lymph nodes. At higher doses, increasing quantities of quantum dots are noted within the experimental brain tumors. Macrophages and microglia colocalize with glioma cells, carrying the quantum dot and thereby optically outlining the tumor. Excitation with blue or ultraviolet wavelengths stimulates the quantum dots, which give off a deep red fluorescence detectable with charge-coupled device cameras, optical spectroscopy units, and in dark-field fluorescence microscopy. CONCLUSION: Quantum dots are optical nanoparticles that, when delivered in nanomole doses, are phagocytized by the macrophages and microglia that infiltrate experimental gliomas. The optical signal may be detected, allowing for improved identification and visualization of tumors, potentially augmenting brain tumor biopsy and resection.

Folic acid-conjugated protein cages of a plant virus: a novel delivery platform for doxorubicin.
Ren Y, Wong SM, Lim LY.
Bioconjug Chem. 2007 May-Jun;18(3):836-43.
[ expand abstract ]

The protein cage of a plant virus may provide a template for monodispersed nanosized systems for drug delivery. Using the Hibiscus chlorotic ringspot virus (HCRSV) as a model plant virus, we have prepared nanosized protein cages (30 nm) capable of encapsulating the anticancer drug, doxorubicin. The technique utilized the simultaneous encapsulation of a polyprotic acid of mw 200 kDa to produce an encapsulation efficiency for doxorubicin of about 7.5%. Folic acid was conjugated onto the capsids to impart cancer-targeting capability. The resultant nanosized systems improved the uptake and cytotoxicity of doxorubicin in the ovarian cancer cells, OVCAR-3, with statistical significance. Plant virus capsids may therefore provide viable templates for targeted drug delivery in cancer chemotherapy.

Polymeric nanoparticle-encapsulated curcumin ("nanocurcumin"): a novel strategy for human cancer therapy.
Bisht S, Feldmann G, Soni S, Ravi R, Karikar C, Maitra A, Maitra A.
J Nanobiotechnology. 2007 Apr 17;5:3.
[ expand abstract ]

BACKGROUND: Curcumin, a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa), has potent anti-cancer properties as demonstrated in a plethora of human cancer cell line and animal carcinogenesis models. Nevertheless, widespread clinical application of this relatively efficacious agent in cancer and other diseases has been limited due to poor aqueous solubility, and consequently, minimal systemic bioavailability. Nanoparticle-based drug delivery approaches have the potential for rendering hydrophobic agents like curcumin dispersible in aqueous media, thus circumventing the pitfalls of poor solubility. RESULTS: We have synthesized polymeric nanoparticle encapsulated formulation of curcumin - nanocurcumin - utilizing the micellar aggregates of cross-linked and random copolymers of N-isopropylacrylamide (NIPAAM), with N-vinyl-2-pyrrolidone (VP) and poly(ethyleneglycol)monoacrylate (PEG-A). Physico-chemical characterization of the polymeric nanoparticles by dynamic laser light scattering and transmission electron microscopy confirms a narrow size distribution in the 50 nm range. Nanocurcumin, unlike free curcumin, is readily dispersed in aqueous media. Nanocurcumin demonstrates comparable in vitro therapeutic efficacy to free curcumin against a panel of human pancreatic cancer cell lines, as assessed by cell viability and clonogenicity assays in soft agar. Further, nanocurcumin's mechanisms of action on pancreatic cancer cells mirror that of free curcumin, including induction of cellular apoptosis, blockade of nuclear factor kappa B (NFkappaB) activation, and downregulation of steady state levels of multiple pro-inflammatory cytokines (IL-6, IL-8, and TNFalpha). CONCLUSION: Nanocurcumin provides an opportunity to expand the clinical repertoire of this efficacious agent by enabling ready aqueous dispersion. Future studies utilizing nanocurcumin are warranted in pre-clinical in vivo models of cancer and other diseases that might benefit from the effects of curcumin.

Purpose: To determine if pulsed-high intensity focused ultrasound (HIFU) could effectively serve as a source of hyperthermia with thermosensitive liposomes to enhance delivery and efficacy of doxorubicin in tumors.
Experimental Design: Comparisons in vitro and in vivo were carried out between non–thermosensitive liposomes (NTSL) and low temperature–sensitive liposomes (LTSL). Liposomes were incubated in vitro over a range of temperatures and durations, and the amount of doxorubicin released was measured. For in vivo experiments, liposomes and free doxorubicin were injected i.v. in mice followed by pulsed-HIFU exposures in s.c. murine adenocarcinoma tumors at 0 and 24 h after administration. Combinations of the exposures and drug formulations were evaluated for doxorubicin concentration and growth inhibition in the tumors.
Results: In vitro incubations simulating the pulsed-HIFU thermal dose (42°C for 2 min) triggered release of 50% of doxorubicin from the LTSLs; however, no detectable release from the NTSLs was observed. Similarly, in vivo experiments showed that pulsed-HIFU exposures combined with the LTSLs resulted in more rapid delivery of doxorubicin as well as significantly higher i.t. concentration when compared with LTSLs alone or NTSLs, with or without exposures. Combining the exposures with the LTSLs also significantly reduced tumor growth compared with all other groups.
Conclusions: Combining low-temperature heat-sensitive liposomes with noninvasive and nondestructive pulsed-HIFU exposures enhanced the delivery of doxorubicin and, consequently, its antitumor effects. This combination therapy could potentially produce viable clinical strategies for improved targeting and delivery of drugs for treatment of cancer and other diseases.

Antiangiogenic gene therapy with systemically administered sFlt-1 plasmid DNA in engineered gelatin-based nanovectors.
Kommareddy S, Amiji M.
Cancer Gene Ther. 2007 May;14(5):488-98.
[ expand abstract ]

This study examined the potential of engineered gelatin-based nanoparticulate vectors for systemic delivery of therapeutic genes to human solid tumor xenografts in vivo. Plasmid DNA encoding for the soluble form of the extracellular domain of vascular endothelial growth factor receptor-1 (VEGF-R1 or sFlt-1) was encapsulated in the control and poly(ethylene glycol) (PEG)-modified gelatin-based nanoparticles. When the plasmid DNA was delivered in PEG-modified thiolated gelatin nanoparticles, highest levels of sFlt-1 expression was observed in vitro in MDA-MB-435 human breast adenocarcinoma cell line. In addition, upon intravenous administration in female Nu/Nu mice bearing orthotopic MDA-MB-435 breast adenocarcinoma xenografts, efficient in vivo expression of sFlt-1 plasmid DNA was confirmed quantitatively by enzyme-linked immunosorbent assay and qualitatively by Western blot analysis. The expressed sFlt-1 was therapeutically active as shown by suppression of tumor growth and microvessel density measurements. The results of this study show that PEG-modified gelatin-based nanovectors can serve as a safe and effective systemically administered gene delivery vehicle for solid tumor.

Modulation of Intracellular Ceramide Using Polymeric Nanoparticles to Overcome Multidrug Resistance in Cancer
van Vlerken LE, Duan Z, Seiden MV, Amiji MM.
Cancer Res. May 15; 67 (10):4843-50.
[ expand abstract ]

Although multidrug resistance (MDR) is known to develop through a variety of molecular mechanisms within the tumor cell, many tend to converge toward the alteration of apoptotic signaling. The enzyme glucosylceramide synthase (GCS), responsible for bioactivation of the proapoptotic mediator ceramide to a nonfunctional moiety glucosylceramide, is overexpressed in many MDR tumor types and has been implicated in cell survival in the presence of chemotherapy. The purpose of this study was to investigate the therapeutic strategy of coadministering ceramide with paclitaxel, a commonly used chemotherapeutic agent, in an attempt to restore apoptotic signaling and overcome MDR in the human ovarian cancer cell line SKOV3. Poly(ethylene oxide)-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticles were used to encapsulate and deliver the therapeutic agents for enhanced efficacy. Results show that indeed the cotherapy eradicates the complete population of MDR cancer cells when they are treated at their IC50 dose of paclitaxel. More interestingly, when the cotherapy was combined with the properties of nanoparticle drug delivery, the MDR cells can be resensitized to a dose of paclitaxel near the IC50 of non-MDR (drug sensitive) cells, indicating a 100-fold increase in chemosensitization via this approach. Molecular analysis of activity verified the hypothesis that the efficacy of this therapeutic approach is indeed due to a restoration in apoptotic signaling, although the beneficial properties of PEO-PCL nanoparticle delivery seemed to enhance the therapeutic success even further, showing the promising potential for the clinical use of this therapeutic strategy to overcome MDR.

Ultrafine hydrogel nanoparticles: synthetic approach and therapeutic application in living cells.
Gao D, Xu H, Philbert MA, Kopelman R.
Angew Chem Int Ed Engl. 2007;46(13):2224-7.
[ expand abstract ]

No abstract available. (Citation link)

On the suitability of nanocrystalline ferrites as a magnetic carrier for drug delivery: functionalization, conjugation and drug release kinetics.
Rana S, Gallo A, Srivastava RS, Misra RD.
Acta Biomater. 2007 Mar;3(2):233-42.
[ expand abstract ]

Superparamagnetic nickel ferrite nanoparticles functionalized with polyvinyl alcohol, polyethylene oxide and polymethacrylic acid (PMAA) polymers and subsequently conjugated with doxorubicin anti-cancer drug are studied for their use as a magnetic carrier for drug delivery. Fourier transform infrared spectroscopy enabled examination of the ability of the nanoparticles to be functionalized with polymers and conjugated with doxorubicin drug. The functionalized polymer-coated nanocrystalline nickel ferrites retain the magnetic characteristics of non-functionalized nanocrystalline nickel ferrites (superparamagnetism, absence of hysteresis, remanence and coercivity at room temperature), encouraging their application as a magnetic carrier for drug delivery. The PMAA-coated nanoferrites are demonstrated as being a potentially superior magnetically targeted drug carrier based on FTIR results and drug release kinetics in the absence and presence of an external magnetic field.

Improvement of cancer-targeting therapy, using nanocarriers for intractable solid tumors by inhibition of TGF-Β signaling.
Kano MR, Bae Y, Iwata C, Morishita Y,Yashiro M, Oka M, Fujii T, Komuro A, Kiyono K, Kaminishi M, Hirakawa K, Ouchi Y, Nishiyama N, Kataoka K, Miyazono K.
Proc Natl Acad Sci U S A. 2007 Feb 27;104 (9):3460-65.
[ expand abstract ]

Transforming growth factor (TGF)-Β plays a pivotal role in regulation of progression of cancer through effects on tumor microenvironment as well as on cancer cells. TGF-Β inhibitors have recently been shown to prevent the growth and metastasis of certain cancers. However, there may be adverse effects caused by TGF-Β signaling inhibition, including the induction of cancers by the repression of TGF-Β-mediated growth inhibition. Here, we present an application of a short-acting, small-molecule TGF-Β type I receptor (TΒR-I) inhibitor at a low dose in treating several experimental intractable solid tumors, including pancreatic adenocarcinoma and diffuse-type gastric cancer, characterized by hypovascularity and thick fibrosis in tumor microenvironments. Low-dose TΒR-I inhibitor altered neither TGF-Β signaling in cancer cells nor the amount of fibrotic components. However, it decreased pericyte coverage of the endothelium without reducing endothelial area specifically in tumor neovasculature and promoted accumulation of macromolecules, including anticancer nanocarriers, in the tumors. Compared with the absence of TΒR-I inhibitor, anticancer nanocarriers exhibited potent growth-inhibitory effects on these cancers in the presence of TΒR-I inhibitor. The use of TΒR-I inhibitor combined with nanocarriers may thus be of significant clinical and practical importance in treating intractable solid cancers.

Immunological properties of engineered nanomaterials.
Dobrovolskaia M, McNeil SE.
Nature Nanotechnology. 2007 Jul 29
[ view article ]

Nanoparticles of poly(lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) copolymers for protein drug delivery.
Lee SH, Zhang Z, Feng SS.
Biomaterials. 2007 Apr;28(11):2041-50.
[ expand abstract ]

Nanoparticles (NPs) of poly(lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) copolymers with various PLA:TPGS component ratios were prepared by the double emulsion technique for protein drug formulation with bovine serum albumin (BSA) as a model protein. Influence of the PLA:TPGS component ratio and the BSA loading level on the drug encapsulation efficiency (EE) and in vitro drug release behavior was investigated. The PLA-TPGS NPs achieved 16.7% protein drug loading and 75.6% EE, which exhibited a biphasic pattern of controlled protein release with higher initial burst for those NPs of more TPGS content. Furthermore, the released proteins retained good structural integrity for at least 35 days at 37 degrees C as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD) spectroscopy. Compared with other biodegradable polymeric NPs such as poly(d,l-lactide-co-glycolide) (PLGA) NPs, PLA-TPGS NPs could provide the encapsulated proteins a milder environment. Confocal laser scanning microscopy (CLSM) observation demonstrated the intracellular uptake of the PLA-TPGS NPs by NIH-3T3 fibroblast cells and Caco-2 cancer cells. This research suggests that PLA-TPGS NPs could be of great potential for clinical formulation of proteins and peptides.

The Antioxidant Tempamine: In Vitro Antitumor and Neuroprotective Effects and Optimization of Liposomal Encapsulation and Release.
Wasserman V, Kizelsztein P, Garbuzenko O, Kohen R, Ovadia H, Tabakman R, Barenholz Y.
Langmuir. 2007 Feb 13;23(4):1937-1947.
[ expand abstract ]

The piperidine nitroxide tempamine (TMN) is a cell-permeable, stable radical having antioxidant, anticancer, and proapoptotic and/or pronecrotic activities, as was demonstrated by us in cell cultures. We also demonstrated synergism between TMN and doxorubicin in doxorubicin-sensitive and doxorubicin-resistant cell lines. Treatment of the C26 mouse colon carcinoma model in vivo also demonstrated synergism between TMN and doxorubicin in sterically stabilized liposomes (SSLs) containing TMN (SSL-TMN) and those containing doxorubicin. The above effects of TMN and SSL-TMN motivated us to develop and optimize the SSL-TMN formulation so that it will be able to reach the disease site with a sufficiently high TMN level and a release rate needed to achieve a therapeutic effect. Because TMN is an amphipathic weak base, it was remote loaded by an intraliposome high/extraliposome low transmembrane ammonium sulfate gradient. The kinetics and level of TMN loading were monitored by cyclic voltammetry (CV) and electron paramagnetic resonance (EPR); the latter also indicates TMN precipitation in the intraliposomal aqueous phase. The regeneration of the original CV and EPR signals by the ionophore nigericin indicates that TMN remained fully intact during loading and release. The cardinal role of the transmembrane ammonium ion gradient in the loading process was proven by the use of the selective ionophores nonactin (for NH4+) and nigericin (for H+). The anion of the ammonium salts affects loading stability and the rate of TMN release, both mediated through the TMN state of aggregation in the intraliposomal aqueous phase. The greater the TMN salt precipitation, the slower the TMN release rate. This was supported by measurement of osmolality, which is inversely related to TMN salt precipitate. Precipitation is in the order SO4-2 > Cl-1 > glucuronate-1. Liposome lipid composition, magnitude of the transmembrane ammonium ion gradient, and type of anion of the ammonium salt determine the amount of TMN loaded and its release rate.

Organically Modified Silica Nanoparticles Co-encapsulating Photosensitizing Drug and Aggregation-Enhanced Two-Photon Absorbing Fluorescent Dye Aggregates for Two-Photon Photodynamic Therapy.
Kim S, Ohulchanskyy TY, Pudavar HE, Pandey RK, Prasad PN.
J Am Chem Soc. 2007 Feb 9; [Epub ahead of print].
[ expand abstract ]

We report energy-transferring organically modified silica nanoparticles for two-photon photodynamic therapy. These nanoparticles co-encapsulate two-photon fluorescent dye nanoaggregates as an energy up-converting donor and a photosensitizing PDT drug as an acceptor. They combine two features: (i) aggregation-enhanced two-photon absorption and emission properties of a novel two-photon dye and (ii) nanoscopic fluorescence resonance energy transfer between this nanoaggregate and a photosensitizer, 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide. Stable aqueous dispersions of the co-encapsulating nanoparticles (diameter </= 30 nm) have been prepared in the nonpolar interior of micelles by coprecipitating an organically modified silica sol with the photosensitizer and an excess amount of the two-photon dye which forms fluorescent aggregates by phase separation from the particle matrix. Using a multidisciplinary nanophotonic approach, we show: (i) indirect excitation of the photosensitizer through efficient two-photon excited intraparticle energy transfer from the dye aggregates in the intracellular environment of tumor cells and (ii) generation of singlet oxygen and in vitro cytotoxic effect in tumor cells by photosensitization under two-photon irradiation.

siRNA Delivery into Human T Cells and Primary Cells with Carbon-Nanotube Transporters.
Liu Z, Winters M, Holodniy M, Dai H.
Angew Chem Int Ed Engl. 2007 Feb 9; [Epub ahead of print].
[ expand abstract ]

No abstract available

Bifunctional compounds for targeted hepatic gene delivery.
Kim KS, Lei Y, Stolz DB, Liu D.
Gene Ther. 2007 Feb 8; [Epub ahead of print].
[ expand abstract ]

A series of bifunctional compounds with galactosyl residues as targeting ligand for asialoglycoprotein receptors on hepatocytes and various dendrimers as the DNA-binding domain was synthesized. When mixed with plasmid DNA, these compounds self assembled into particles that exhibited high transfection activity both in vitro and in vivo. Optimal activity in liver cells was observed with compounds containing three galactosyl residues and 16 dendrimer arms. These results suggest that domain-based design is an effective strategy for development of a new generation of synthetic gene carriers.

Dendritic Molecular Transporters Provide Control of Delivery to Intracellular Compartments.
Huang K, Voss B, Kumar D, Hamm HE, Harth E.
Bioconjug Chem. 2007 Feb 7; [Epub ahead of print].
[ expand abstract ]

Novel biocompatible macromolecular vectors were developed that not only enable transport of bioactive cargo across the cell membrane but also control the delivery into defined intracellular compartments. This work describes the synthesis and design of two non-peptidic fluorescently labeled Newkome-type dendrimers, differentiated over a varied alkyl spacer with guanidine end moieties. The internalization of the fluorescein-labeled molecular transporter into mammalian cells showed strong subcellular localizations, evident with both live cells and fixed cells costained with DAPI, a nuclear stain. We observed that the subcellular distribution of these vectors varied significantly, as one of the vectors concentrates in the nucleus (FD-1) while the other (FD-2) concentrates in the cytosol. All experiments performed with NIH-3T3 fibroblasts and human microvascular endothelial cells (HMEC) showed similar results. The differential localization patterns of the two molecular transporters can be controlled through the variation of alkyl spacer length at the terminal generation of the dendrimer. Intracellular delivery of bioactive entities into specific subcellular locations, utilizing this practical approach, might overcome limitations in drug delivery and pioneer future technologies in drug transport.

Folate Receptor Targeted Delivery of Polyelectrolyte Complex Micelles Prepared from ODN-PEG-Folate Conjugate and Cationic Lipids.
Kim SH, Jeong JH, Mok H, Lee SH, Kim SW, Park TG.
Biotechnol Prog. 2007 Feb 2;23(1):232-237.
[ expand abstract ]

A polyelectrolyte complex micelle (PECM)-based delivery system for targeting folate (FOL) receptor overexpressing tumor cells is demonstrated using poly(ethylene glycol) (PEG)-conjugated oligonucleotide (ODN). The tumor targeting property was conferred to the PECM by tethering a folate moiety to the distal end of the PEG segment in an anti-sense green fluorescent protein (GFP) ODN-PEG conjugate. Nanoscale PECMs were spontaneously produced from ionic interactions between the ODN-PEG-FOL conjugate and a cationic lipid, lipofectamine (Lf). When treated with FOL receptor overexpressing cells (KB), the PCEMs caused a significant reduction in GFP expression in a dose-dependent manner. This effect was not observed in FOL receptor deficient cells (A549). The enhanced transfection of ODN-PEG-FOL/Lf PECMs to KB cells was caused by FOL receptor mediated endocytosis. The efficiency of target-specific gene suppression by ODN-PEG-FOL/Lf PECMs was maintained even in the presence of 10% fetal bovine serum in the transfection medium.

New Method for Delivering a Hydrophobic Drug for Photodynamic Therapy Using Pure Nanocrystal Form of the Drug.
Baba K, Pudavar HE, Roy I, Ohulchanskyy TY, Chen Y, Pandey RK, Prasad PN.
Mol Pharm. 2007 Feb 1; [Epub ahead of print].
[ expand abstract ]

A carrier-free method for delivery of a hydrophobic drug in its pure form, using nanocrystals (nanosized crystals), is proposed. To demonstrate this technique, nanocrystals of a hydrophobic photosensitizing anticancer drug, 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide (HPPH), have been synthesized using the reprecipitation method. The resulting drug nanocrystals were monodispersed and stable in aqueous dispersion, without the necessity of an additional stabilizer (surfactant). As shown by confocal microscopy, these pure drug nanocrystals were taken up by the cancer cells with high avidity. Though the fluorescence and photodynamic activity of the drug were substantially quenched in the form of nanocrystals in aqueous suspension, both these characteristics were recovered under in vitro and in vivo conditions. This recovery of drug activity and fluorescence is possibly due to the interaction of nanocrystals with serum albumin, resulting in conversion of the drug nanocrystals into the molecular form. This was confirmed by demonstrating similar recovery in presence of fetal bovine serum (FBS) or bovine serum albumin (BSA). Under similar treatment conditions, the HPPH in nanocrystal form or in 1% Tween-80/water formulation showed comparable in vitro and in vivo efficacy. Keywords: Nanocrystals; reprecipitation method; photosensitizers; photodynamic therapy; singlet oxygen; drug delivery.

Effect of coupling of albumin onto surface of PEG liposome on its in vivo disposition.
Furumoto K, Yokoe J, Ogawara K, Amano S, Takaguchi M, Higaki K, Kai T, Kimura T.
Int J Pharm. 2007 Feb 1;329(1-2):110-6.
[ expand abstract ]

To evaluate the effect of coupling of albumin onto the surface of poly(ethylene glycol)-modified liposome (PEG liposome) on the in vivo disposition of liposome, pharmacokinetics and tissue distribution were examined after intravenous administration of rat serum albumin-modified PEG (RSA/PEG) liposome into rats. RSA/PEG liposome showed longer blood-circulating property than PEG liposome and the hepatic clearance for RSA/PEG liposome was significantly smaller than that for PEG liposome. Single-pass liver perfusion experiments also showed that the hepatic disposition of RSA/PEG liposome was much less than that of PEG liposome and that pre-treatment of liver with trypsin did not significantly reduce the hepatic disposition of RSA/PEG liposome, suggesting that RSA/PEG liposome could avoid the hepatic uptake via the receptor-mediated endocytosis. To unravel the mechanism behind the less affinity of RSA/PEG liposome to the liver, serum proteins associated on their surface were quantitatively and qualitatively assessed. The results showed that the coupling of albumin onto PEG liposome significantly reduced the total amount of serum proteins associated onto the surface, and SDS-PAGE revealed that the decrease in the association with liposomes for several serum proteins, which might have opsonic activity. From these findings, introduction of serum albumin onto PEG liposome could be useful to develop a new nanoparticulate formulation with a better pharmacokinetic property.

Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery.
Khalil IA, Kogure K, Futaki S, Hama S, Akita H, Ueno M, Kishida H, Kudoh M, Mishina Y, Kataoka K, Yamada M, Harashima H.
Gene Ther. 2007 Feb 1; [Epub ahead of print].
[ expand abstract ]

This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirus-mediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies.

Effect of transferrin receptor-targeted liposomal doxorubicin in P-glycoprotein-mediated drug resistant tumor cells.
Kobayashi T, Ishida T, Okada Y, Ise S, Harashima H, Kiwada H.
Int J Pharm. 2007 Feb 1;329(1-2):94-102.
[ expand abstract ]

The over-expression of P-glycoprotein (P-gp) has been associated with the development of multidrug resistance (MDR) in cancer cells. In this study, we examined whether transferrin receptor (Tf-R) targeted liposomes can efficiently deliver encapsulated doxorubicin (DXR) into MDR cells (SBC-3/ADM) via Tf-R-mediated endocytosis thus overcoming MDR by by-passing P-gp-mediated drug efflux. We prepared four types of liposome, i.e. untargeted and Tf-R-targeted, made of either egg-PC/cholesterol or hydrogenated egg PC/cholesterol. Only with the targeted EPC-liposome we achieved significant delivery of encapsulated DXR and increased cytotoxicity of encapsulated DXR on the MDR cells (3.5-fold higher than free DXR). Confocal microscopy and an intracellular drug-accumulation assay indicated that the targeted liposomes efficiently delivered DXR into cells where it readily accumulated in the nucleus, in both drug-sensitive and MDR cells. These findings suggest that the targeted liposomes are rapidly internalized via Tf-R-mediated endocytosis followed by release of their contents into the cytoplasm. The rapid internalization and content release, most likely facilitated by the higher fluidity of the EPC-based liposomes, may explain why only targeted EPC-liposomes were able to prevent drug efflux by P-gp and to consequently circumvent MDR. Our results indicate that in order to achieve MDR circumvention by means of liposomal encapsulation of DXR the liposomes not only need to be targeted, but also to have the proper physicochemical properties for adequate release of the drug. Furthermore, these in vitro results suggest that Tf-R targeted EPC-liposomes are a potentially useful drug delivery system to circumvent P-gp-mediated MDR of tumors.

Oil-Encapsulating PEO-PPO-PEO/PEG Shell Cross-Linked Nanocapsules for Target-Specific Delivery of Paclitaxel.
Bae KH, Lee Y, Park TG.
Biomacromolecules. 2007 Feb;8(2):650-6.
[ expand abstract ]

PEO-PPO-PEO/PEG shell cross-linked nanocapsules encapsulating an oil phase in their nanoreservoir structure was developed as a target-specific carrier for a water-insoluble drug, paclitaxel. Oil-encapsulating PEO-PPO-PEO/PEG composite nanocapsules were synthesized by dissolving an oil (Lipiodol) and an amine-reactive PEO-PPO-PEO derivative in dichloromethane and subsequently dispersing in an aqueous solution containing amine-functionalized six-arm-branched poly(ethylene glycol) by ultrasonication. The resultant shell cross-linked nanocapsules had a unique core/shell architecture with an average size of 110.7 +/- 9.9 nm at 37 degrees C, as determined by dynamic light scattering and transmission electron microscopy. Paclitaxel could be effectively solubilized in the inner Lipiodol phase surrounded by a cross-linked PEO-PPO-PEO/PEG shell layer. The paclitaxel-loaded nanocapsules were further conjugated with folic acid to achieve folate receptor targeted delivery. Confocal microscopy and flow cytometric analysis revealed that folate-mediated targeting significantly enhanced the cellular uptake and apoptotic effect against folate receptor overexpressing cancer cells. The present study suggested that these novel nanomaterials encapsulating an oil reservoir could be potentially applied for cancer cell targeted delivery of various water-insoluble therapeutic and diagnostic agents.

Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery.
Cheng J, Teply BA, Sherifi I, Sung J, Luther G, Gu FX, Levy-Nissenbaum E, Radovic-Moreno AF, Langer R, Farokhzad OC.
Biomaterials. 2007 Feb;28(5):869-76.
[ expand abstract ]

Nanoparticle (NP) size has been shown to significantly affect the biodistribution of targeted and non-targeted NPs in an organ specific manner. Herein we have developed NPs from carboxy-terminated poly(d,L-lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG-COOH) polymer and studied the effects of altering the following formulation parameters on the size of NPs: (1) polymer concentration, (2) drug loading, (3) water miscibility of solvent, and (4) the ratio of water to solvent. We found that NP mean volumetric size correlates linearly with polymer concentration for NPs between 70 and 250 nm in diameter (linear coefficient=0.99 for NPs formulated with solvents studied). NPs with desirable size, drug loading, and polydispersity were conjugated to the A10 RNA aptamer (Apt) that binds to the prostate specific membrane antigen (PSMA), and NP and NP-Apt biodistribution was evaluated in a LNCaP (PSMA+) xenograft mouse model of prostate cancer. The surface functionalization of NPs with the A10 PSMA Apt significantly enhanced delivery of NPs to tumors vs. equivalent NPs lacking the A10 PSMA Apt (a 3.77-fold increase at 24h; NP-Apt 0.83%+/-0.21% vs. NP 0.22%+/-0.07% of injected dose per gram of tissue; mean+/-SD, n=4, p=0.002). The ability to control NP size together with targeted delivery may result in favorable biodistribution and development of clinically relevant targeted therapies.

The effect of paclitaxel-loaded nanoparticles with radiation on hypoxic MCF-7 cells.
Jin C, Wu H, Liu J, Bai L, Guo G.
J Clin Pharm Ther. 2007 Feb;32(1):41-7.
[ expand abstract ]

Background and Objective: The inability of radiotherapy to eradicate completely certain human tumours may be due to the presence of resistant hypoxic cells. Several studies have confirmed the radiosensitizing effect of paclitaxel, a microtubular inhibitor. The object of this study was to evaluate the physicochemical characteristics of paclitaxel-loaded nanoparticles, and determine the ability of the released paclitaxel to radiosensitize hypoxic human breast carcinoma cells (MCF-7) with respect to radiation dose. Methods: The poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel were prepared by o/w emulsification-solvent evaporation method. The morphology of the paclitaxel-loaded nanoparticles was investigated by scanning electron microscopy. The drug encapsulation efficiency (EE) and in vitro release profile were measured by high-performance liquid chromatography. Cell cycle was evaluated by flow cytometry. Cell viability was measured by the ability of single cells to form colonies in vitro. Results: The prepared nanoparticles were spherical with diameter between 200 and 800 nm. The EE was 85.5%. The drug release pattern was biphasic with a fast release rate followed by a slow one. Co-culture of human breast carcinoma cells (MCF-7) with paclitaxel-loaded nanoparticles demonstrated that released paclitaxel retained its bioactivity to block cells in the G2/M phase of the cell cycle and effectively sensitized hypoxic MCF-7 cells to radiation with radiosensitivity shown to be dependent of radiation dose at levels of dosages studied. The sensitizer enhancement ratio for paclitaxe-loaded nanoparticles at 10% survival is approximately 1.4. Conclusion: This work has demonstrated that paclitaxel can be effectively released from a biodegradable PLGA nanoparticle delivery system while maintaining potent combined cytotoxic and radiosensitizing abilities for hypoxic tumour cells.

Challenges to macromolecular drug delivery.
Juliano R.
Biochem Soc Trans. 2007 Feb;35(Pt 1):41-3.
[ expand abstract ]

The use of macromolecules, particularly monoclonal antibodies, as therapeutic agents has come to the forefront in recent years. The biodistribution and delivery issues for protein drugs are shared to a substantial degree with other emerging therapeutic approaches including pharmacologically active nucleic acids and nanoparticles. A generalized approach to these issues involves consideration of the multiple biological barriers that stand between the macromolecular drug or nanoparticle at its site of administration and its ultimate biological target. Considerations of size, stability, non-specific versus specific associations and potency versus toxicity all play a role. The creation of delivery approaches that combine high specificity for the target cell or tissue, high therapeutic payload and modest toxicity remains a challenge, although some very promising examples have emerged recently. A variety of sophisticated targeting strategies, based primarily on combinatorial library methods, when used in combination with new technologies to identify cell-surface receptor 'signatures' of specific tissues, will facilitate advances in targeted delivery of macromolecules and nanoparticles. The challenges to contemporary macromolecule drug delivery are complex, thus new research paradigms are emerging that combine the talents of physical and biological scientists to address this key issue for modern pharmacology and therapeutics.

Low-density lipoprotein receptor-mediated endocytosis of PEGylated nanoparticles in rat brain endothelial cells.
Kim HR, Gil S, Andrieux K, Nicolas V, Appel M, Chacun H, Desmaele D, Taran F, Georgin D, Couvreur P.
Cell Mol Life Sci. 2007 Feb;64(3):356-64.
[ expand abstract ]

Poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to diffuse through the blood-brain barrier after intravenous administration. However, the mechanism of transport of these nanoparticles into brain has not yet been clearly elucidated. The development of a model of rat brain endothelial cells (RBEC) in culture has allowed investigations into this mechanism. A study of the intracellular trafficking of nanoparticles by cell fractionation and confocal microscopy showed that nanoparticles are internalized by the endocytic pathway. Inhibition of the caveolae-mediated pathway by preincubation with filipin and nystatin did not modify the cellular uptake of the nanoparticles. In contrast, chlorpromazine and NaN(3) pretreatment, which interferes with clathrin and energy-dependent endocytosis, caused a significant decrease of nanoparticle internalization. Furthermore, cellular uptake experiments with nanoparticles preincubated with apolipoprotein E and blocking of low-density lipoprotein receptors (LDLR) clearly suggested that the LDLR-mediated pathway was involved in the endocytosis of PEGPHDCA nanoparticles by RBEC.

Biodistribution and pharmacokinetic analysis of long-circulating thiolated gelatin nanoparticles following systemic administration in breast cancer-bearing mice.
Kommareddy S, Amiji M.
J Pharm Sci. 2007 Feb;96(2):397-407.
[ expand abstract ]

The objective of the present study was to modify thiolated gelatin nanoparticles with poly(ethylene glycol) (PEG) chains and examine their long circulating and tumor-targeting properties in vivo in an orthotopic a human breast adenocarcinoma xenograft model. The crosslinked nanoparticle systems were characterized to have a size of 150-250 nm with rapid payload release properties in a highly reducing environment. Upon PEG modification, the nanoparticle size increased to 300-350 nm in diameter. The presence of PEG chains on the surface was confirmed by characterization with electron spectroscopy for chemical analysis. The in vivo long-circulating potential, biodistribution and passive tumor targeting of the controls, and PEG-modified thiolated gelatin nanoparticles were evaluated by injecting indium-111 ((111)In)-labeled nanoparticles into breast tumor (MDA-MB-435)-bearing nude mice. Upon modification with PEG, the nanoparticles were found to have longer circulation times, with the plasma and tumor half-lives of 15.3 and 37.8 h, respectively. The results also showed preferential localization of thiolated nanoparticles in the tumor mass. The resulting nanoparticulate systems with long circulation properties could be used to target encapsulated drugs and genes to tumors passively by utilizing the enhanced permeability and retention effect of the tumor vasculature.

The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing.
Liu X, Howard KA, Dong M, Andersen MO, Rahbek UL, Johnsen MG, Hansen OC, Besenbacher F, Kjems J.
Biomaterials. 2007 Feb;28(6):1280-8.
[ expand abstract ]

We have previously introduced the use of the biomaterial chitosan to form chitosan/siRNA nanoparticles for gene silencing protocols. This present study shows that the physicochemical properties (size, zeta potential, morphology and complex stability) and in vitro gene silencing of chitosan/siRNA nanoparticles are strongly dependent on chitosan molecular weight (Mw) and degree of deacetylation (DD). High Mw and DD chitosan resulted in the formation of discrete stable nanoparticles approximately 200 nm in size. Chitosan/siRNA formulations (N:P 50) prepared with low Mw (approximately 10 kDa) showed almost no knockdown of endogenous enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells, whereas those prepared from higher Mw (64.8-170 kDa) and DD (approximately 80%) showed greater gene silencing ranging between 45% and 65%. The highest gene silencing efficiency (80%) was achieved using chitosan/siRNA nanoparticles at N:P 150 using higher Mw (114 and 170 kDa) and DD (84%) that correlated with formation of stable nanoparticles of approximately 200 nm. In conclusion, this work confirms the application of chitosan as a non-viral carrier for siRNA and the importance of polymeric properties for the optimisation of gene silencing using chitosan/siRNA nanoparticles.

Paclitaxel nanoparticle inhibits growth of ovarian cancer xenografts and enhances lymphatic targeting.
Lu H, Li B, Kang Y, Jiang W, Huang Q, Chen Q, Li L, Xu C.
Cancer Chemother Pharmacol. 2007 Feb;59(2):175-81.
[ expand abstract ]

OBJECTIVES: Ovarian cancer has the highest mortality of all the gynecologic cancers. The antitumor agent paclitaxel has been proved to be efficient in the treatment of ovarian cancer. Our study is to develop a polymeric drug delivery system for paclitaxel and determine whether paclitaxel nanoparticle can inhibit growth of ovarian carcinoma xenografts in Fisher344 (F344) rats by intraperitoneal administration. The mechanism of paclitaxel nanoparticles in rats bearing ovarian cancer has been investigated in this study. METHODS: Synthesize paclitaxel loading nanoparticle (PLA) by ultrasonic emulsification; MTT analysis identified cytotoxic activity of paclitaxel nanoparticle in vitro; rat ovarian carcinoma cells were injected into the peritoneal cavity of F344 rats. The antitumor effect of paclitaxel nanoparticle in vivo has been evaluated by measuring tumor weight and ascite volume. At the end of the procedure rats were sacrificed; tumors were excised and processed for PCNA staining, tissue terminal deoxynucleotide transferase-mediated dUTP nick and labeling assay and RT-PCR to evaluate the proliferative and apoptotic changes and cancer transfer-related gene expression induced by PLA. Paclitaxel concentration in plasma, pelvic lymph nodes, liver, and heart were determined by high-performance liquid chromatography. RESULTS: Paclitaxel nanoparticle and PTX (Cremophor) showed equivalent cytotoxic activity in vitro. In rats implanted carcinoma cells, paclitaxel nanoparticles significantly reduced tumor weight and ascites volume, and induced apoptosis of tumor cells. PLA also inhibited cell proliferation and matrix metalloproteinase 9 mRNA expression. The paclitaxel concentration of pelvic lymph nodes in PLA treated animals was 20-fold higher than that of free PTX treated animals at 48 h after intraperitoneal administration. CONCLUSION: The intraperitoneal administration of paclitaxel nanoparticle can significantly inhibit the progression of ovarian carcinoma in peritoneal cavity of female F344 rat. The paclitaxel nanoparticle is safe and lymphatic targeting.

Nanostructured calcium phosphates (NanoCaPs) for non-viral gene delivery: influence of the synthesis parameters on transfection efficiency.
Olton D, Li J, Wilson ME, Rogers T, Close J, Huang L, Kumta PN, Sfeir C.
Biomaterials. 2007 Feb;28(6):1267-79.
[ expand abstract ]

Calcium phosphate (CaP) based approaches remain an attractive option for delivering plasmid DNA (pDNA) into cultured cells. However, despite their appeal, current synthesis methodologies typically yield lower, less consistent transfection efficiencies when compared to viral approaches. Therefore, we report here a novel method to consistently synthesize efficient, nano-sized, mono-dispersed CaP-pDNA particles; accomplished by optimizing both the stoichiometry (Ca/P ratio) of the CaP particles as well as the mode in which the calcium and phosphate precursor solutions are mixed. Our results indicate that calcium and phosphate precursors when mixed in a controlled and regulated manner reproducibly result in nano-sized particles that consistently yield higher transfection efficiencies when compared to particles synthesized via manual mixing (a two-fold increase was observed). Also, maximum transfection efficiencies in both HeLa and MC3T3-E1 cells lines were obtained when a Ca/P ratio between 100 and 300 was used. Particles synthesized within this optimum Ca/P ratio range were between 25 and 50 nm. Our data suggests that these maximized transfection efficiencies were obtained because these particles not only effectively condensed (70% efficient) but also efficiently bound (90% efficient) the pDNA. In addition, X-ray diffraction and Fourier transform infrared spectroscopy analyses confirmed that all of the synthesized CaP structures exhibited the hydroxyapatite phase.

Nanotechnology approaches for drug and small molecule delivery across the blood brain barrier.
Silva GA.
Surg Neurol. 2007 Feb;67(2):113-6.
[ expand abstract ]

Nanotechnology involves the design, synthesis, and characterization of materials and devices that have a functional organization in at least one dimension on the nanometer (ie, one billionth of a meter) scale. One area in which nanotechnology may have a significant clinical impact in neuroscience is the selective transport and delivery of drugs and other small molecules across the blood brain barrier that cannot cross otherwise. Using a variety of nanoparticles composed of different chemical compositions, different groups are exploring proof-of-concept approaches for the delivery of different antineoplastic drugs, oligonucleotides, genes, and magnetic resonance imaging contrast agents. This review discusses some of the main technical challenges associated with the development of nanotechnologies for delivery across the blood brain barrier and summarizes ongoing work.

Interaction of photosensitizers with liposomes containing unsaturated lipid.
Voszka I, Budai M, Szabo Z, Maillard P, Csik G, Grof P.
Chem Phys Lipids. 2007 Feb;145(2):63-71.
[ expand abstract ]

Small unilamellar liposomes were made of dipalmitoyl-phosphatidylcholine and dioleoyl-phosphatidylcholine, and photosensitized by a symmetrically or an asymmetrically substituted glycosilated tetraphenyl-porphyrin derivative. As differential scanning calorimetry and electron paramagnetic resonance spectroscopy (EPR) revealed these porphyrin derivatives were localized in different depth within the lipid bilayer. Both porphyrin derivatives were able to induce photoreaction and consequent structural changes in the membrane. 5-, 12-, or 16-doxyl stearic acid labeled lipid bilayers were applied and the efficiency of photoinduced reaction was followed by the decay of their EPR signal amplitude. Light dose-dependent destruction of nitroxide radical proved to be dependent on the position of spin label. In this process the porphyrin localized in closer connection with the double bond of unsaturated fatty acid was more effective. EPR signal decay was also dependent on the unsaturated fatty acid content of the liposome and the oxygen saturation of the solvent.

Examination of Nonendocytotic Bulk Transport of Nanoparticles Across Phospholipid Membranes.
Banerji SK, Hayes MA.
Langmuir. 2007 Jan 30; [Epub ahead of print].
[ expand abstract ]

Nonendocytotic transport is believed to play a role in the transmigration of particles less than 100 nm within biological systems. Determining the fundamental mechanism of this transport across cell membranes is essential if nanotechnology is to be utilized in general medical practice and may lead to methods of treating the deleterious internalization of ambient, possibly pollutant, nanoparticles. In order to gain a broader understanding of nonendocytotic transmembrane transport, it becomes essential to devise a method which allows the isolation of fundamental modes of transport such as passive Brownian diffusion through a membrane, as opposed to effusion-like transport of particles through transmembrane channels. The passive Brownian diffusion contribution was investigated using gold nanoparticles and mimetic biomembranes. Specifically, gold nanoparticle dispersions consisting of 7, 10, and 15 nm diameter particles were captured in giant unilamelar vesicles composed of phosphatidylcholine, phosphatidic acid, and cholesterol. Nonendocytotic transmembrane transport was modeled as the time derivative of the appearance of nanoparticles in the phosphate buffer outside the vesicles at 37 degrees C. The results show the transport rate to be zero; hence, a simple diffusive process of transmembrane transport is not supported.

Studies on the oridonin-loaded poly(D,L-lactic acid) nanoparticles in vitro and in vivo.
Xing J, Zhang D, Tan T.
Int J Biol Macromol. 2007 Jan 30;40(2):153-8.
[ expand abstract ]

The purpose of this paper was to investigate the possibility of developing a polymeric nanoparticle delivery system for ORI to increase its solubility, blood circulation time and tissue targeting. Oridonin-loaded poly(D,L-lactic acid) nanoparticles (ORI-PLA-NP) were prepared by the further modified spontaneous emulsion solvent diffusion (MSESD) method. Studies were carried out to characterize and evaluate the produced ORI-PLA-NP both in vitro and in vivo. The experimental results showed that the mean size of the nanoparticles were 137.3 nm, with 87.2% of the nanoparticles distributed between the range of 107 and 195 nm. The entrapment efficiency and actual drug loading of the nanoparticles were 91.88+/-1.83 and 2.32+/-0.05%, respectively. It was demonstrated by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD) that ORI existed in the form of amorphous in the nanoparticles. The in vitro release profile of ORI-PLA-NP could be expressed well by the Higuchi equation: Q=8.944t(1/2)+11.246. The results of pharmacokinetics demonstrated that being encapsulated in PLA nanoparticles was remarkably effective for ORI to prolong its blood circulation time. After the i.v. administration of ORI-PLA-NP, we could observe a stable and high concentration of ORI in liver, lung and spleen, while its distribution in heart and kidney decreased.

Cisplatin encapsulated in phosphatidylethanolamine liposomes enhances the in vitro cytotoxicity and in vivo intratumor drug accumulation against melanomas.
Hwang TL, Lee WR, Hua SC, Fang JY.
J Dermatol Sci. 2007 Jan 29; [Epub ahead of print].
[ expand abstract ]

BACKGROUND: Cisplatin is a potent anticancer drug for treating melanoma. OBJECTIVE: The aim of this study was to evaluate the possibility of using liposomes, for intratumoral distribution in a melanoma, composed of phosphatidylethanolamine (PE), for its cytotoxicity. METHOD: The in vitro drug release, in vitro cytotoxicity against melanoma, and in vivo residence time in the tumor of liposome-encapsulated cisplatin were investigated. The liposomes were prepared and characterized in terms of their morphology, size, zeta potential, and drug loading. RESULT: The size of the PE liposomes attained a level of approximately 100nm. The concentration of cisplatin encapsulated in PE liposomes was 50-70% dependent on the presence or absence of polyethylene glycol (PEG) derivatives. On the other hand, no or negligible cisplatin molecules were encapsulated in egg phosphatidylcholine (EPC) liposomes. PE liposomes had higher cytotoxicity than classic liposomes or free cisplatin. Images of confocal laser scanning microscopy confirmed the great potency of PE liposomes to deliver cisplatin into cells. The incorporation of PEG derivatives completely inhibited the proliferation of melanoma cells. With in vivo intratumoral administration, the cisplatin concentration in the tumor tissue was maintained at a high level for 72h after application of the PE liposomes. The PE liposomes delivered cisplatin into the tumor approximately 3.6 times more efficiently than the free drug. CONCLUSION: These results demonstrate that PE liposomes represent a potentially useful strategy for targeting cisplatin delivery into melanomas.

Lipid-based Nanoparticles for Nucleic Acid Delivery.
Li W, Szoka FC Jr.
Pharm Res. 2007 Jan 25; [Epub ahead of print].
[ expand abstract ]

Lipid-based colloidal particles have been extensively studied as systemic gene delivery carriers. The topic that we would like to emphasize is the formulation/assembly of lipid-based nanoparticles (NP) with diameter under 100 nm for delivering nucleic acid in vivo. NP are different from cationic lipid-nucleic acid complexes (lipoplexes) and are vesicles composed of lipids and encapsulated nucleic acids with a diameter less than 100 nm. The diameter of the NP is an important attribute to enable NP to overcome the various in vivo barriers for systemic gene delivery such as: the blood components, reticuloendothelial system (RES) uptake, tumor access, extracellular matrix components, and intracellular barriers. The major formulation factors that impact the diameter and encapsulation efficiency of DNA-containing NP include the lipid composition, nucleic acid to lipid ratio and formulation method. The particle assembly step is a critical one to make NP suitable for in vivo gene delivery. NP are often prepared using a dialysis method either from an aqueous-detergent or aqueous-organic solvent mixture. The resulting particles have diameters about 100 nm and nucleic acid encapsulation ratios are >80%. Additional components can then be added to the particle after it is formed. This ordered assembly strategy enables one to optimize the particle physico-chemical attributes to devise a biocompatible particle with increased gene transfer efficacy in vivo. The components included in the sequentially assembled NP include: poly(ethylene glycol) (PEG)-shielding to improve the particle pharmacokinetic behavior, a targeting ligand to facilitate the particle-cell recognition and in some case a bioresponsive lipid or pH-triggered polymer to enhance nucleic acid release and intracellular trafficking. A number of groups have observed that a PEG-shielded NP is a robust and modestly effective system for systemic gene or small interfering RNA (siRNA) delivery.

Employment of Cationic Solid-Lipid Nanoparticles as RNA Carriers.
Montana G, Bondi ML, Carrotta R, Picone P, Craparo EF, Biagio PL, Giammona G, Carlo MD.
Bioconjug Chem. 2007 Jan 25; [Epub ahead of print].
[ expand abstract ]

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. In this article, the suitability of cationically modified solid-lipid nanoparticles (SLN) as a nonviral vector for gene delivery was investigated, in order to obtain stable materials able to condense RNA. Cationic SLN were produced by microemulsion using Compritol ATO 888 as matrix lipid, Pluronic F68 as tenside, and dimethyldioctadecylammonium bromide (DDAB) as cationic lipid. The resulting particles were approximately 100 nm in size and showed a highly positive surface charge (+41 mV) in water. Size and shape were further characterized by scanning electron microscopy (SEM) measurements. Moreover, we utilized the sea urchin as a model system to test their applicability on a living organism. To evaluate cationic SLN ability to complex the in vitro transcribed Paracentrotus lividus bep3 RNA, we utilized both light scattering and gel mobility experiments, and protection by nuclease degradation was also investigated. By microinjection experiment, we demonstrated that the nanoparticles do not inference with the viability of the P. lividus embryo and the complex nanoparticles-bep3 permits movement of the RNA during its localization in the egg, suggesting that it could be a suitable system for gene delivery. Taken together, all these results indicate that the cationic SNL are a good RNA carrier for gene transfer system and the sea urchin a simple and versatile candidate to test biological properties of nanotechnology devices.

A Parallel Approach for Subwavelength Molecular Surgery Using Gene-Specific Positioned Metal Nanoparticles as Laser Light Antennas.
Csaki A, Garwe F, Steinbruck A, Maubach G, Festag G, Weise A, Riemann I, Konig K, Fritzsche W.
Nano Lett. 2007 Jan 24; [Epub ahead of print].
[ expand abstract ]

An optical technique for the parallel manipulation of nanoscale structures with molecular resolution is presented. Bioconjugated metal nanoparticles are thereby positioned at the location of interest, such as, e.g., certain DNA sequences along metaphase chromosomes, prior to pulsed laser light irradiation of the whole sample. The nanoparticles are designed to absorb the introduced energy highly efficiently, in that way acting as nanoantenna. As result of the interaction, structural changes of the sample with subwavelength dimensions and nanoscale precision are observed at the location of the particles. The process leading to the nanolocalized destruction is caused by particle ablation as well as thermal damage of the surrounding material.

Nanosized Paclitaxel Particles from Supercritical Carbon Dioxide Processing and Their Biological Evaluation.
Pathak P, Prasad GL, Meziani MJ, Joudeh AA, Sun YP.
Langmuir. 2007 Jan 23; [Epub ahead of print].
[ expand abstract ]

The rapid expansion of a supercritical solution into a liquid solvent (RESOLV) technique with benign supercritical carbon dioxide was applied to obtain aqueous suspended nanoparticles of the highly potent anticancer drug paclitaxel. The paclitaxel nanoparticles were protected from agglomeration by using a known nontoxic stabilization agent. The aqueous suspended paclitaxel nanoparticles of different average particle sizes were evaluated in vitro against human breast cancer cells. The results suggest that the nanosized paclitaxel particles are effective, with an antineoplastic activity comparable to that of the commercial paclitaxel formulation. The technique should be generally applicable to the processing of nanoparticles from other important drugs with aqueous solubility problems.

Coating of negatively charged liposomes by polylysine: Drug release study.
Volodkin D, Mohwald H, Voegel JC, Ball V.
J Control Release. 2007 Jan 22;117(1):111-20.
[ expand abstract ]

The present work describes surface coating of carboxyfluorescein(CF)-loaded liposomes with poly-l-lysine (PLL) and liposome membrane permeability. The vesicles were prepared from synthetic or natural lipids. Interaction between PLL and the liposomes leads to the formation of complexes - either single PLL-coated vesicles or vesicle aggregates. Formation of the complexes is strongly affected by PLL/lipid molar ratio and the molecular mass of the PLL chains. Liposome permeability depends strongly on the lipid phase state - vesicles in the solid state retained the entrapped dye for a long time, but continuous CF release was registered for "fluid" vesicles. Crossing the transition temperature leads to intensive dye leakage because of the appearance of leaky interfacial domains between the coexisting solid and liquid phases and also because of a reversible change in the vesicle size upon the solid-liquid state phase transition. PLL coverage does not cause permeabilization of "solid" liposomes, but increases the permeability of "fluid" vesicles. At the same time, the results of differential scanning calorimetry and vesicle fusion suggest that PLL adsorption occurs exclusively on the vesicular surface and that the lipidic organization is not significantly disturbed. Moreover, PLL does not prevent lipid exchange between vesicles induced by temperature change.

The use of fullerene substituted phenylalanine amino acid as a passport for peptides through cell membranes.
Yang J, Wang K, Driver J, Yang J, Barron AR.
Org Biomol Chem. 2007 Jan 21;5(2):260-6.
[ expand abstract ]

We report the formation of a fullerene-peptide conjugate via the incorporation of a fullerene substituted phenylalanine derivative, "Bucky amino acid" (Baa), to a cationic peptide, which acts as a passport for intracellular delivery, enabling transport of a range of sequences into HEK-293, HepG2, and neuroblastoma cells where the peptides in the absence of the fullerene amino acid cannot enter the cell. Delivery of the fullerene species to either the cytoplasm or nucleus of the cell is demonstrated. Fullerene peptides based on the nuclear localization sequence (NLS), H-Baa-Lys(FITC)-Lys-Lys-Arg-Lys-Val-OH, can actively cross over the cell membrane and accumulate significantly around the nucleus of HEK-293 and neuroblastoma cells, while H-Baa-Lys(FITC)-Lys8-OH accumulates in the cytoplasm. Cellular studies show that the uptake for the anionic peptide Baa-Lys(FITC)Glu4Gly3Ser-OH is greatly reduced in comparison with the cationic fullerene peptides of the same concentration. The hydrophobic nature of the fullerene assisting peptide transport is suggested by the effect of gamma-cyclodextrin (CD) in lowering the efficacy of transport. These data suggest that the incorporation of a fullerene-based amino acid provides a route for the intracellular delivery of peptides and as a consequence the creation of a new class of cell penetrating peptides.

Biomimetic amplification of nanoparticle homing to tumors.
Simberg D, Duza T, Park JH, Essler M, Pilch J, Zhang L, Derfus AM, Yang M, Hoffman RM, Bhatia S, Sailor MJ, Ruoslahti E.
Proc Natl Acad Sci USA. 2007 Jan 16;104(3):932-6.
[ expand abstract ]

Nanoparticle-based diagnostics and therapeutics hold great promise because multiple functions can be built into the particles. One such function is an ability to home to specific sites in the body. We describe here biomimetic particles that not only home to tumors, but also amplify their own homing. The system is based on a peptide that recognizes clotted plasma proteins and selectively homes to tumors, where it binds to vessel walls and tumor stroma. Iron oxide nanoparticles and liposomes coated with this tumor-homing peptide accumulate in tumor vessels, where they induce additional local clotting, thereby producing new binding sites for more particles. The system mimics platelets, which also circulate freely but accumulate at a diseased site and amplify their own accumulation at that site. The self-amplifying homing is a novel function for nanoparticles. The clotting-based amplification greatly enhances tumor imaging, and the addition of a drug carrier function to the particles is envisioned.

Aclarubicin-loaded cationic albumin-conjugated pegylated nanoparticle for glioma chemotherapy in rats.
Lu W, Wan J, Zhang Q, She Z, Jiang X.
Int J Cancer. 2007 Jan 15;120(2):420-31.
[ expand abstract ]

Traditional glioma chemotherapy with those second-line drugs such as anthracyclines usually failed because they are inaccessible to blood-brain barrier (BBB) in tumor. In our study, we incorporated aclarubicin (ACL) into cationic albumin-conjugated pegylated nanoparticle (CBSA-NP-ACL) to determine its therapeutic potential of rats with intracranially implanted C6 glioma cells. When labeled with fluorescent probe, 6-coumarin, CBSA-NP was shown to accumulate much more in tumor mass than nanoparticle without conjugated CBSA (NP) 1 hr post intravenous injection, as well as better retention after 24 hr. Tumor drug concentration of CBSA-NP-ACL displayed 2.6- and 3.3-fold higher than that of NP-ACL and ACL solution 1 hr post injection, while 2.7 and 6.6-fold higher after 24 hr, respectively. Moreover, using tumor microdialysis sampling, AUC(0-24 hr) of free drug amount in tumor interstitium delivered by CBSA-NP-ACL was about 2.0- and 2.7-fold higher than that of NP-ACL and ACL solutions, respectively. When the tumor rat model was subjected to 4 cycles of 2 mg/kg of ACL in different formulations, a significant increase of median survival time was found in the group of CBSA-NP-ACL compared with that of saline control animals, animals treated with NP-ACL and ACL solution. By terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, CBSA-NP-ACL can extensively make the tumor cell apoptosis. Histochemical evaluation by periodic acid Shiff staining and biochemical analysis depicted that the incorporation of ACL into CBSA-NP reduced its toxicity to liver, kidney and heart. Besides, CBSA-NP-ACL was not shown to open tight junction evaluated by BBB coculture. It was concluded that CBSA-NP-ACL could have a therapeutic potential for treatment of glioma.

Biomimetic amplification of nanoparticle homing to tumors.
Simberg D, Duza T, Park JH, Essler M, Pilch J, Zhang L, Derfus AM, Yang M, Hoffman RM, Bhatia S, Sailor MJ, Ruoslahti E.
Proc Natl Acad Sci USA. 2007 Jan 10; [Epub ahead of print].
[ expand abstract ]

Nanoparticle-based diagnostics and therapeutics hold great promise because multiple functions can be built into the particles. One such function is an ability to home to specific sites in the body. We describe here biomimetic particles that not only home to tumors, but also amplify their own homing. The system is based on a peptide that recognizes clotted plasma proteins and selectively homes to tumors, where it binds to vessel walls and tumor stroma. Iron oxide nanoparticles and liposomes coated with this tumor-homing peptide accumulate in tumor vessels, where they induce additional local clotting, thereby producing new binding sites for more particles. The system mimics platelets, which also circulate freely but accumulate at a diseased site and amplify their own accumulation at that site. The self-amplifying homing is a novel function for nanoparticles. The clotting-based amplification greatly enhances tumor imaging, and the addition of a drug carrier function to the particles is envisioned.

Hydrotropic Polymeric Micelles for Enhanced Paclitaxel Solubility: In Vitro and In Vivo Characterization.
Lee SC, Huh KM, Lee J, Cho YW, Galinsky RE, Park K.
Biomacromolecules. 2007 Jan 8;8(1):202-208.
[ expand abstract ]

The purpose of this investigation was to characterize the in vitro stability and in vivo disposition of paclitaxel in rats after solubilization of paclitaxel into hydrotropic polymeric micelles. The amphiphilic block copolymers consisted of a micellar shell-forming poly(ethylene glycol) (PEG) block and a core-forming poly(2-(4-vinylbenzyloxy)-N,N-diethylnicotinamide) (P(VBODENA)) block. N,N-Diethylnicotinamide (DENA) in the micellar inner core resulted in effective paclitaxel solubilization and stabilization. Solubilization of paclitaxel using polymeric micelles of poly(ethylene glycol)-b-P(d,l-lactide) (PEG-b-PLA) served as a control for the stability study. Up to 37.4 wt % paclitaxel could be loaded in PEG-b-P(VBODENA) micelles, whereas the maximum loading amount for PEG-b-PLA micelles was 27.6 wt %. Thermal analysis showed that paclitaxel in the polymeric micelles existed in the molecularly dispersed amorphous state even at loadings over 30 wt %. Paclitaxel-loaded hydrotropic polymeric micelles retained their stability in water for weeks, whereas paclitaxel-loaded PEG-b-PLA micelles precipitated in a few days. Hydrotropic polymer micelles were more effective than PEG-PLA micelle formulations in inhibiting the proliferation of human cancer cells. Paclitaxel in hydrotropic polymer micelles was administered orally (3.8 mg/kg), intravenously (2.5 mg/kg), or via the portal vein (2.5 mg/kg) to rats. The oral bioavailability was 12.4% of the intravenous administration. Our data suggest that polymeric micelles with a hydrotropic structure are superior as a carrier of paclitaxel due to a high solubilizing capacity combined with long-term stability, which has not been accomplished by other existing polymeric micelle systems.

Synthetic nano-low density lipoprotein as targeted drug delivery vehicle for glioblastoma multiforme.
Nikanjam M, Blakely EA, Bjornstad KA, Shu X, Budinger TF, Forte TM.
Int J Pharm. 2007 Jan 2;328(1):86-94.
[ expand abstract ]

The low density lipoprotein (LDL) receptor has been shown to be upregulated in GBM tumor cells and is therefore a potential molecular target for the delivery of therapeutic agents. A synthetic nano-LDL (nLDL) particle was developed and tested to determine its utility as a drug delivery vehicle targeted to GBM tumors. nLDL particles were constructed by combining a synthetic peptide containing a lipid binding motif and the LDL receptor (LDLR) binding domain of apolipoprotein B-100 with a lipid emulsion consisting of phosphatidyl choline, triolein, and cholesteryl oleate. Composition analysis, fast protein liquid chromatography, and electron microscopy revealed that nLDL was highly reproducible and intermediate in size between high density lipoprotein and LDL particles (10.5+/-2.8nm diameter). The binding and uptake of fluorescently labeled nLDL particles was assessed using fluorescence microscopy. Uptake of nLDL was time dependent, exhibiting saturation at approximately 3h, and concentration dependent, exhibiting saturation at concentrations greater than 5muM peptide. Using Lysotracker as a cellular marker, nLDL co-localized with lysosomes. nLDL binding was eliminated by blocking LDLRs with suramin and nLDL inhibited binding of plasma LDL to LDLRs. Collectively these data strongly suggest that the synthetic nano-LDLs described here are taken up by LDLR and can serve as a drug delivery vehicle for targeting GBM tumors via the LDLR.

Synthesis and characterization of chitosan-g-poly(ethylene glycol)-folate as a non-viral carrier for tumor-targeted gene delivery.
Chan P, Kurisawa M, Chung JE, Yang YY.
Biomaterials. 2007 Jan;28(3):540-9.
[ expand abstract ]

Poor water solubility and low transfection efficiency of chitosan are major drawbacks for its use as a gene delivery carrier. PEGylation can increase its solubility, and folate conjugation may improve gene transfection efficiency due to promoted uptake of folate receptor-bearing tumor cells. The aim of this study was to synthesize and characterize folate-poly(ethylene glycol)-grafted chitosan (FA-PEG-Chi) for targeted plasmid DNA delivery to tumor cells. Gel electrophoresis study showed strong DNA binding ability of modified chitosan. The pH(50) values, defined as the pH when the transmittance of a polymer solution at 600 nm has reached 50% of the original value, suggested that the water solubility of PEGylated chitosan had improved significantly. Regression analysis of pH(50) value as a function of substitution degree of PEG yielded an almost linear correlation for PEG-Chi and FA-PEG-Chi. The solubility of PEGylated chitosan decreased slightly by further conjugation of folic acid due to the relatively more hydrophobic nature of folic acid when compared to PEG. In addition, the chitosan-based DNA complexes did not induce remarkable cytotoxicity against HEK 293 cells. FA-PEG-Chi can be a promising gene carrier due to its solubility in physiological pH, efficiency in condensing DNA, low cytotoxicity and targeting ability.

Amine-containing core-shell nanoparticles as potential drug carriers for intracellular delivery.
Feng M, Li P.
J Biomed Mater Res A. 2007 Jan;80(1):184-93.
[ expand abstract ]

The present study aimed at exploring the use of amine-containing core-shell nanoparticles as potential drug carriers for intracellular delivery. Stable nanoparticles (100-200 nm in diameter) that consisted of poly(methyl methacrylate) (PMMA) cores with hydrophilic poly(ethyleneimine) (PEI) shells were synthesized and used to study their complexation with model drug, ibuprofen (IB), and release it under various electrolyte concentrations. The complexed IB/PEI-PMMA nanoparticles were characterized with FTIR, photon correlation spectroscopy, zeta-potential, and transmission electron microscopy (TEM). Results suggested that the PEI-PMMA nanoparticles could effectively complex with the IB via electrostatic interaction. The thick PEI shells (approximately 30 nm) significantly enhanced the drug loading capacity up to 23% (w/w) of the complexed nanopartricle. In vitro release of the drug from the complexed nanoparticles was sensitive to the ionic strength of the media. Study of cellular entry of fluorescently labeled IB/nanoparticle complexes using a confocal laser scanning microscopy demonstrated that the entry of the complexed nanoparticles strongly depended on the complexing ratio between IB and PEI-PMMA nanoparticles.

Conjugates of poly(DL-lactide-co-glycolide) on amino cyclodextrins and their nanoparticles as protein delivery system.
Gao H, Wang YN, Fan YG, Ma JB.
J Biomed Mater Res A. 2007 Jan;80(1):111-22.
[ expand abstract ]

Poly(DL-lactide-co-glycolide) (PLG) was chemically conjugated on two amino cyclodextrins, mono(6-(2-aminoethyl)amino-6-deoxy)-beta-cyclodextrin and ethylenediamino bridged bis(beta-cyclodextrin), to afford novel amphiphilic conjugates. Those conjugates were then characterized with infrared spectrometry (IR), proton nuclear magnetic resonance ((1)H NMR) and gel permeation chromatography (GPC). A repeat-nanoprecipitation (RP-NP) method was also developed to fabricate the nanoparticles of the conjugates with a water-soluble model protein, bovine serum albumin (BSA). At the end of RP-NP process, the availability of BSA was over 80% while the entrapment efficiency was 40-50% for each nanoprecipitation. The nanoparticles were rigid and spherical with diameters of 110-180 nm determined by transmission electron microscope (TEM), atomic force microscopy (AFM) and particle size analyzer. Nanoparticles possessed good steric stability during freeze-drying and resuspensions due to the existence of cyclodextrins corona. Interactions between BSA and the conjugates in the nanoparticles were then elucidated with IR experiments. About 25% BSA adsorbed on the surface of nanoparticles due to the interaction and was easy to release in the first day. The release of BSA from the nanoparticles was in three phases: a burst effect in the first day, a followed plateau in about a week, and a sustained release of the protein over 14 days. By changing the lactide/glycolide ratio, the degradation time of the conjugates and the release rate of BSA could be controlled. The loss of CDs content was faster than that of overall Mw during degradation since CDs formed outer corona of the nanoparticles. Both the novel biomaterials and the nanosphere fabrication technique contributed to the maintenance of protein structure.

Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid.
Hatakeyama H, Akita H, Kogure K, Oishi M, Nagasaki Y, Kihira Y, Ueno M, Kobayashi H, Kikuchi H, Harashima H.
Gene Ther. 2007 Jan;14(1):68-77.
[ expand abstract ]

For successful cancer gene therapy via intravenous (i.v.) administration, it is essential to optimize the stability of carriers in the systemic circulation and the cellular association after the accumulation of the carrier in tumor tissue. However, a dilemma exists regarding the use of poly(ethylene glycol) (PEG), which is useful for conferring stability in the systemic circulation, but is undesirable for the cellular uptake and the following processes. We report the development of a PEG-peptide-lipid ternary conjugate (PEG-Peptide-DOPE conjugate (PPD)). In this strategy, the PEG is removed from the carriers via cleavage by a matrix metalloproteinase (MMP), which is specifically expressed in tumor tissues. An in vitro study revealed that the PPD-modified gene carrier (Multifunctional Envelope-type Nano Device: MEND) exhibited pDNA expression activity that was dependent on the MMP expression level in the host cells. In vivo studies further revealed that the PPD was potent in stabilizing MEND in the systemic circulation and facilitating tumor accumulation. Moreover, the i.v. administration of PPD or PEG/PPD dually-modified MEND resulted in the stimulation of pDNA expression in tumor tissue, as compared with a conventional PEG-modified MEND. Thus, MEND modified with PPD is a promising device, which has the potential to make in vivo cancer gene therapy achievable.Gene Therapy (2007) 14, 68-77. doi:10.1038/sj.gt.3302843; published online 17 August 2006.

Dendrimer-based targeted delivery of an apoptotic sensor in cancer cells.
Myc A, Majoros IJ, Thomas TP, Baker JR Jr.
Biomacromolecules. 2007 Jan;8(1):13-8.
[ expand abstract ]

Our previous studies have demonstrated the applicability of poly(amidoamine) (PAMAM) dendrimers as a platform for the targeted delivery of chemotherapeutic drugs both in vitro and in vivo. To monitor the rate and extent of cell-killing caused by the delivered chemotherapeutic drug, we wished to analyze the degree of apoptosis in targeted cells on a real-time basis. As the apoptosis-regulating caspases are activated during the apoptotic process, several caspase-hydrolyzable, fluorescence resonance energy transfer (FRET)-based substrates have been marketed for the detection of apoptosis. However, the applicability of these agents is limited because of their nonspecificity and the consequent high background fluorescence in tissues. Here we show the synthesis, characterization, and in vitro targeting of an engineered PAMAM nanodevice in which folic acid (FA) is conjugated as the targeting molecule and a caspase-specific FRET-based agent (PhiPhiLux G1D2) is conjugated as the apoptosis-detecting agent. This conjugate specifically targets FA-receptor-positive, KB cells. In these cells, the apoptosis-inducing agent staurosporine caused a 5-fold increase in the cellular fluorescence. These results show, for the first time, the potential applicability of a targeted apoptosis-measuring nanodevice, which could be used for simultaneously monitoring the apoptotic potential of a delivered drug.

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2006

Polymer genomics: An insight into pharmacology and toxicology of nanomedicines.
Kabanov AV.
Adv Drug Deliv Rev. 2006 Dec 30;58(15):1597-621.
[ expand abstract ]

Synthetic polymers and nanomaterials display selective phenotypic effects in cells and in the body signal transduction mechanisms involved in inflammation, differentiation, proliferation, and apoptosis. When physically mixed or covalently conjugated with cytotoxic agents, bacterial DNA or antigens, polymers can drastically alter specific genetically controlled responses to these agents. These effects, in part, result from cooperative interactions of polymers and nanomaterials with plasma cell membranes and trafficking of polymers and nanomaterials to intracellular organelles. Cells and whole organism responses to these materials can be phenotype or genotype dependent. In selected cases, polymer agents can bypass limitations to biological responses imposed by the genotype, for example, phenotypic correction of immune response by polyelectrolytes. Overall, these effects are relatively benign as they do not result in cytotoxicity or major toxicities in the body. Collectively, however, these studies support the need for assessing pharmacogenomic effects of polymer materials to maximize clinical outcomes and understand the pharmacological and toxicological effects of polymer formulations of biological agents, i.e. polymer genomics.

Intraperitoneal Delivery of Liposomal siRNA for Therapy of Advanced Ovarian Cancer.
Landen CN, Merritt WM, Mangala LS, Sanguino AM, Bucana C, Lu C, Lin YG, Han LY, Kamat AA, Schmandt R, Coleman RL, Gershenson DM, Lopez-Berestein G, Sood AK.
Cancer Biol Ther. 2006 Dec 30;5(12) [Epub ahead of print].
[ expand abstract ]

Purpose: Intravenous (IV) delivery of siRNA incorporated into neutral liposomes allows efficient delivery to tumor tissue, and has therapeutic efficacy in preclinical proof-of-concept studies using EphA2-targeting siRNA. We sought to determine whether intraperitoneal (IP) delivery of these siRNA complexes was as effective at delivery and therapy as IV delivery. Experimental design: SiRNA was incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Alexa555-siRNA-DOPC was injected IP into nude mice bearing established ovarian tumors, and organs were collected for microscopic fluorescent examination. Subsequently, therapeutic efficacy of the IP versus IV routes was directly compared. Results: Alexa555-siRNA in DOPC liposomes injected IP was diffusely distributed into intraperitoneal ovarian tumors. Delivery was also seen deeply into the liver and kidney parenchyma, suggesting that the predominant means of distribution was through the vasculature, rather than direct diffusion from the peritoneal cavity. In mice with orthotopic ovarian tumors, treatment with combined paclitaxel and IP EphA2-targeting siRNA-DOPC reduced tumor growth by 48-81% compared to paclitaxel/control siRNA-DOPC IP (HeyA8: 0.34 g v 0.66 g; SKOV3ip1: 0.04 v 0.21, p < 0.01). This reduction was comparable to concurrently-treated mice with paclitaxel and EphA2 siRNA-DOPC injected IV, which showed a reduction in growth by 45-69% compared to paclitaxel/control siRNA-DOPC injected IV (HeyA8: 0.23g v. 0.42g; SKOV3ip1: 0.04 v. 0.13 g). Conclusions: IP injection of siRNA incorporated in DOPC allows intra-tumoral delivery and has therapeutic efficacy in orthotopic ovarian tumors. These findings may have therapeutic implications for siRNA-based strategies.

Arginine-conjugated polypropylenimine dendrimer as a non-toxic and efficient gene delivery carrier.
Kim TI, Baek JU, Zhe Bai C, Park JS.
Biomaterials. 2006 Dec 28; [Epub ahead of print].
[ expand abstract ]

We synthesized arginine-conjugated polypropylenimine dendrimer G2 (DAB-8), PPI2-R for gene delivery systems. Synthesized PPI2-R could retard plasmid DNA at a weight ratio of 4 completely and PPI2-R polyplexes showed a fluorescence of less than 10% over a charge ratio of 2 by PicoGreen reagent assay, suggesting its good DNA condensing ability. The size of PPI2-R polyplex was measured to about 200nm at a charge ratio of 150. PPI2-R displayed 80-90% cell viability at even a 150mug/mL concentration. Transfection efficiency of PPI2-R was found to be high comparable to that of PEI25kD and to be 8-214 times higher than that of unmodified PPI2 on HeLa and 293 cells. Moreover, PPI2-R showed 4 times higher transfection efficiency than PEI25kD, treating with 10mug pDNA because of its low cytotoxicity on HeLa cells. Finally, PPI2-R showed a transfection efficiency 2-3 times higher than PEI25kD on HUVECs, showing its potency as a gene delivery carrier for primary cells. These results demonstrate that arginine-conjugation of PPI2 is successful in developing a low toxic and highly transfection efficient gene delivery carrier.

Targeted and intracellular delivery of paclitaxel using multi-functional polymeric micelles.
Seow WY, Xue JM, Yang YY.
Biomaterials. 2006 Dec 18; [Epub ahead of print].
[ expand abstract ]

Natural paclitaxel (Taxol((R)) is an effective anti-cancer drug, although a critical disadvantage is its non-targeting nature. To address this issue, cholesterol-grafted poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-undecenoic acid) was synthesized with different starting monomer ratios via a free radical copolymerization route. Folate was subsequently attached to the hydrophilic segment of the polymer in order to target folate receptors-overexpressing cancer cells. The success of synthesis was confirmed with (1)H-NMR carried out in CDCl(3)/D(2)O. Using a membrane dialysis method, the polymer was then self-assembled into micelles whose hydrophobic cores could be utilized to encapsulate paclitaxel, an extremely hydrophobic compound. The polymer had a low CMC of approximately 20mg/L in water. Dynamic light scattering further showed that the sizes of blank micelles formed from the polymer were below 180nm at different pH values tested and approximately 220nm upon drug incorporation. More importantly, it was demonstrated that the micelles exhibited a useful pH-induced thermo-sensitivity, such that drug was released more rapidly at pH 5.0 (acidic endosomal/lysosomal environment) than at pH 7.4 (normal extracellular pH). In vitro cytotoxicity assays performed against KB cells then provided concluding evidences that the cellular uptake of micelles surface-functionalised with folate was indeed enhanced due to a receptor-assisted endocytosis process. This novel polymeric design thus has the potential to be a useful paclitaxel vehicle for the treatment of folate-receptor positive cancers.

Differential tumor cell targeting of anti-HER2 (Herceptin((R))) and anti-CD20 (Mabthera((R))) coupled nanoparticles.
Cirstoiu-Hapca A, Bossy-Nobs L, Buchegger F, Gurny R, Delie F.
Int J Pharm. 2006 Dec 15; [Epub ahead of print].
[ expand abstract ]

Two types of antibody-labeled nanoparticles (mAb-NPs) were prepared with the aim to achieve specific tumor targeting. Anti-HER2 and anti-CD20 monoclonal antibodies (mAb) were used as model ligands. Small poly(dl-lactic acid) nanoparticles (PLA NPs) with a mean size of about 170nm were prepared by the salting out method. Thereafter, the coating of PLA NPs with mAbs was performed in two steps. First, thiol groups (-SH) were introduced on the surface of PLA-NPs by a two-step carbodiimide reaction. The number of -SH groups on the surface of NPs increased from 150 to 400mmol-SH/molPLA when cystamine concentrations of 25-1518molcystamine/molPLA were used during the thiolation reaction. In the second step, covalent coupling of antibodies to thiolated NPs (NPs-SH) was obtained via a bifunctional cross-linker, m-maleimidobenzoyl-N-hydroxy-sulfosuccinimide ester (sulfo-MBS). For both mAbs anti-HER2 and anti-CD20, respectively, the number of -SH functions on the NPs had no influence on the amount of mAb coupled to the NPs. Approximately, 295 anti-HER2 and 557 anti-CD20 molecules, respectively, were covalently coupled per nanoparticle. The NPs size after the coupling reactions was about 250nm. The specific interaction between tumor cells and mAb-NPs was determined by confocal microscopy using two cell lines: SKOV-3 human ovarian cancer cells (overexpressing HER2) and Daudi lymphoma cells (overexpressing CD20). The results showed the selective targeting of mAb-NPs to tumor cells overexpressing the specific antigen. While anti-CD20 labeled NPs (anti-CD20 NPs) bound to and remained at the cellular surface, anti-HER2 labeled NPs (anti-HER2 NPs) were efficiently internalized. The mAb-NPs represent a promising approach to improve the efficacy of NPs in active targeting for cancer therapy while the choice of the antibody-target system defines the fate of the mAb-NPs after their binding to the cells.

HER2-specific T-cell immune responses in patients vaccinated with truncated HER2 protein complexed with nanogels of cholesteryl pullulan.
Kitano S, Kageyama S, Nagata Y, Miyahara Y, Hiasa A, Naota H, Okumura S, Imai H, Shiraishi T, Masuya M, Nishikawa M, Sunamoto J, Akiyoshi K, Kanematsu T, Scott AM, Murphy R, Hoffman EW, Old LJ, Shiku H.
Clin Cancer Res. 2006 Dec 15;12(24):7397-405.
[ expand abstract ]

PURPOSE: We developed a complex of tumor antigen protein with a novel nanoparticle antigen delivery system of cholesteryl pullulan (CHP). To target HER2 antigen, we prepared truncated HER2 protein 1-146 (146HER2) complexed with CHP, the CHP-HER2 vaccine. We designed a clinical study to assess the safety of the vaccine and HER2-specific T-cell immune responses measured by the newly developed enzyme-linked immunospot assay with mRNA-transduced phytohemagglutinin-stimulated CD4(+) T cells in HLA-A2402-positive patients with therapy-refractory HER2-expressing cancers. EXPERIMENTAL DESIGN: Nine patients with various types of solid tumors were enrolled. Each patient was s.c. vaccinated biweekly with 300 microg of CHP-HER2 vaccine for three times followed by booster doses. HER2-specific T-cell responses were evaluated by enzyme-linked immunospot assay by targeting autologous phytohemagglutinin-stimulated CD4(+) T cells transduced with 146HER2-encoding mRNA to cover both identified peptides and unknown epitopes for MHC class I and class II that might exist in the sequence of the vaccine protein. RESULTS: CHP-HER2 vaccine was well tolerated; the only adverse effect was grade 1 transient skin reaction at the sites of vaccination. HER2-specific CD8(+) and/or CD4(+) T-cell immune responses were detected in five patients who received four to eight vaccinations, among whom both T-cell responses were detected in these patients. In four patients with CD8(+) T-cell responses, two patients reacted to previously identified HER2(63-71) peptide and the other two reacted only to 146HER2 mRNA-transduced cells. CONCLUSIONS: CHP-HER2 vaccine was safe and induced HER2-specific CD8(+) and/or CD4(+) T-cell immune responses.

Dendrimer-encapsulated camptothecins: increased solubility, cellular uptake, and cellular retention affords enhanced anticancer activity in vitro.
Morgan MT, Nakanishi Y, Kroll DJ, Griset AP, Carnahan MA, Wathier M, Oberlies NH, Manikumar G, Wani MC, Grinstaff MW.
Cancer Res. 2006 Dec 15;66(24):11913-21.
[ expand abstract ]

A biocompatible polyester dendrimer composed of the natural metabolites, glycerol and succinic acid, is described for the encapsulation of the antitumor camptothecins, 10-hydroxycamptothecin and 7-butyl-10-aminocamptothecin. The cytotoxicity of the dendrimer-drug complex toward four different human cancer cell lines [human breast adenocarcinoma (MCF-7), colorectal adenocarcinoma (HT-29), non-small cell lung carcinoma (NCI-H460), and glioblastoma (SF-268)] is also reported, and low nmol/L IC(50) values are measured. Cellular uptake and efflux measurements in MCF-7 cells show an increase of 16-fold for cellular uptake and an increase in drug retention within the cell when using the dendrimer vehicle.

The Anionic Boron Cluster (B(12)H(11)SH)(2-) as a Means To Trigger Release of Liposome Contents.
Gabel D, Awad D, Schaffran T, Radovan D, Daraban D, Damian L, Winterhalter M, Karlsson G, Edwards K.
ChemMedChem. 2006 Dec 12; [Epub ahead of print].
[ expand abstract ]

No abstract available

Characterization of (Aminoethyl)chitin/DNA Nanoparticle for Gene Delivery.
Je JY, Cho YS, Kim SK.
Biomacromolecules. 2006 Dec 11;7(12):3448-3451.
[ expand abstract ]

Nonviral gene delivery systems have been increasingly proposed as a safer alternative to viral vehicles. In the present study, we synthesized water-soluble chitin by aminoalkylating onto chitin at the C-6 position, and its transfection efficiency was investigated. Aminoethyl-chitin (AEC) was complexed with DNA, and AEC/DNA nanoparticles were characterized. AEC/DNA nanoparticles showed good DNA binding ability, high protection of DNA from nuclease and serum, and low cytotoxicity. Mean particle size decreased from 367 to 290 nm and &zgr; potential increased from -4.58 to 22.87 mV when the AEC/DNA charge ratio (N/P) increased from 1.15 to 18.5. The transfection efficiency of AEC/DNA nanoparticles was investigated in a human embryonic kidney cell line (HEK293), and the results showed that AEC/DNA nanoparticles were much enhanced compare with naked DNA.

Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference.
Tan WB, Jiang S, Zhang Y.
Biomaterials. 2006 Dec 9; [Epub ahead of print].
[ expand abstract ]

Gene silencing using short interfering RNA (siRNA) is fast becoming an attractive approach to probe gene function in mammalian cells. Although there have been some success in the delivery of siRNA using various methods, tracking their delivery and monitoring their transfection efficiency prove to be hard without a suitable tracking agent. Therefore, a challenge lies with the design of an efficient and at the same time, self-tracking, transfection agent for RNA interference. In this paper, chitosan nanoparticles (NPs) with encapsulated quantum dots (QDs) were synthesized and used to deliver HER2/neu siRNA. Using such a construct, the delivery and transfection of the siRNA can be monitored by the presence of fluorescent QDs in the chitosan NPs. Targeted delivery of HER2 siRNA to HER2-overexpressing SKBR3 breast cancer cells was shown to be specific with chitosan/QD NP surface labeled with HER2 antibody targeting the HER2 receptors on SKBR3 cells. Gene-silencing effects of the conjugated siRNA was also established using the luciferase and HER2 ELISA assays. These self-tracking siRNA delivery NPs will also aid in the monitoring of future gene silencing studies in vivo.

Fluorocarbon nanoparticles as multifunctional drug delivery vehicles.
Dielectrophoresis microsystem with integrated flow cytometers for on-line monitoring of sorting efficiency.
Wang Z, Hansen O, Petersen PK, Rogeberg A, Kutter JP, Bang DD, Wolff A.
Electrophoresis. 2006 Dec 7;27(24):5081-5092 [Epub ahead of print].
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Dielectrophoresis (DEP) and flow cytometry are powerful technologies and widely applied in microfluidic systems for handling and measuring cells and particles. Here, we present a novel microchip with a DEP selective filter integrated with two microchip flow cytometers (FCs) for on-line monitoring of cell sorting processes. On the microchip, the DEP filter is integrated in a microfluidic channel network to sort yeast cells by positive DEP. The two FCs detection windows are set upstream and downstream of the DEP filter. When a cell passes through the detection windows, the light scattered by the cell is measured by integrated polymer optical elements (waveguide, lens, and fiber coupler). By comparing the cell counting rates measured by the two FCs, the collection efficiency of the DEP filter can be determined. The chips were used for quantitative determination of the effect of flow rate, applied voltage, conductivity of the sample, and frequency of the electric field on the sorting efficiency. A theoretical model for the capture efficiency was developed and a reasonable agreement with the experimental results observed. Viable and non-viable yeast cells showed different frequency dependencies and were sorted with high efficiency. At 2 MHz, more than 90% of the viable and less than 10% of the non-viable cells were captured on the DEP filter. The presented approach provides quantitative real-time data for sorting a large number of cells and will allow optimization of the conditions for, e.g., collecting cancer cells on a DEP filter while normal cells pass through the system. Furthermore, the microstructure is simple to fabricate and can easily be integrated with other microstructures for lab-on-a-chip applications.

A novel PEGylation of chitosan nanoparticle for gene delivery.
Zhang Y, Chen J, Zhang Y, Pan Y, Zhao J, Ren L, Liao M, Hu Z, Kong L, Wang J.
Biotechnol Appl Biochem. 2006 Dec 6; [Epub ahead of print].
[ expand abstract ]

Chitosan (CS) has emerged as a promising non-viral vector for gene delivery because of its capability to form complexes with plasmid DNA (pDNA) and enhance its transport across cellular membranes through endocytosis. Complexes of CS and pDNA may improve transfection efficiency; however, they are not capable of sustained DNA release and prolonging gene transfer. In order to achieve prolonged delivery of CS/DNA complexes, we had prepared CS nanoparticle (NP) and CS/DNA complexes; then, alpha-methoxy-omega-succinimidyl-polyethylene glycol (MSS-PEG) was conjugated to the surface of CS/DNA complexes using an active ester scheme; finally, the potential of PEGylation of CS NP as a non-viral gene delivery vector to transfer exogenous gene in vitro and in vivo were examined. Electrophoretic analysis suggested that CS NPs could protect the DNA from nuclease degradation. The pDNA carried by CS NPs could enter and express in HepG2 cells. However, its transfection efficiency was very low and the better dose of DNA transferred was 1.6microgram. The transfection activities of CS/DNA-PEG still remained and the better dose of pDNA transferred was 2.4microgram. It indicated that the transfection activity of PEGylation of complexes was improved efficiently. In vivo experiments also showed that CS/DNA-PEG complexes mediated higher gene expression in tissues than CS/DNA complexes did and the gene expression in tumor induced by CS/DNA-PEG complexes was highest of all. These results suggested that PEGylation of CS/DNA complexes have favorable properties for non-viral gene delivery in vitro or in vivo and have the potential to deliver therapeutic genes directly into hepatoma tissues.

Polyarginine segments in block copolypeptides drive both vesicular assembly and intracellular delivery.
Holowka EP, Sun VZ, Kamei DT, Deming TJ.
Nat Mater. 2006 Dec 3; [Epub ahead of print].
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Polymeric vesicles are a relatively new class of nanoscale self-assembled materials that show great promise as robust encapsulants. Compared with liposomes, use of polymeric building blocks for membrane formation allows increased stability, stimuli responsiveness and chemical diversity, which may prove advantageous for drug-delivery applications . A major drawback of most polymeric vesicles is the lack of biofunctionality, which restricts their ability to interact with cells and tissues. We have prepared vesicles composed of polyarginine and polyleucine segments that are stable in media, can entrap water soluble species, and can be processed to different sizes and prepared in large quantities. The remarkable feature of these materials is that the polyarginine segments both direct structure for vesicle formation and provide functionality for efficient intracellular delivery of the vesicles. This unique synergy between nanoscale self-assembly and inherent peptide functionality provides a new approach for design of multifunctional materials for drug delivery.

In vivo Radioprotection by the Fullerene Nanoparticle DF-1 as Assessed in a Zebrafish Model.
Daroczi B, Kari G, McAleer MF, Wolf JC, Rodeck U, Dicker AP.
Clin Cancer Res. 2006 Dec 1;12(23):7086-91.
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PURPOSE: We have previously shown that zebrafish (Danio rerio) embryos can be used as an in vivo model to validate modifiers of the radiation response. Here, we evaluated the radioprotective effect of the nanoparticle DF-1, a fullerene with antioxidant properties, in zebrafish embryos. EXPERIMENTAL DESIGN: Zebrafish embryos were exposed to different doses of ionizing radiation ranging from 20 to 80 Gy in the presence and absence of DF-1. Toxicity and radioprotective effects were assessed by monitoring overall survival and morphology as well as organ functions by employing assays to measure kidney excretory function and development of sensory nerve cells (neuromasts). Antioxidant properties of DF-1 were assessed in whole fish. RESULTS: DF-1 had no apparent adverse effects on normal zebrafish morphology or viability throughout the concentration range tested (1-1,000 mumol/L). Ionizing radiation (10-40 Gy) caused time-dependent and dose-dependent perturbations of normal zebrafish morphology and physiology, notably defective midline development resulting in dorsal curvature of the body axis ("curly-up"), neurotoxicity, impaired excretory function, and decreased survival of the exposed embryos. DF-1 (100 mumol/L) markedly attenuated overall and organ-specific radiation-induced toxicity when given within 3 hours before or up to 15 minutes after radiation exposure. By contrast, DF-1 afforded no protection when given 30 minutes after ionizing radiation. The degree of radioprotection provided by DF-1 was comparable with that provided by the Food and Drug Administration-approved radioprotector amifostine (4 mmol/L). Protection against radiation-associated toxicity using DF-1 in zebrafish embryos was associated with marked reduction of radiation-induced reactive oxygen species. CONCLUSION: The fullerene DF-1 protects zebrafish embryos against deleterious effects of ionizing radiation due, in part, to its antioxidant properties.

Nanomedicine: Developing smarter therapeutic and diagnostic modalities.
Farokhzad OC, Langer R.
Adv Drug Deliv Rev. 2006 Dec 1;58(14):1456-9.
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The early impact of nanotechnology on medicine is beginning to get realized, with novel nanoscale therapeutic and diagnostic modalities under development or in clinical practice today. In this commentary the field of "nanomedicine" is briefly reviewed form the perspective of where we were; where we are today; and where we are likely to go tomorrow.

Determination of the Minimum Temperature Required for Selective Photothermal Destruction of Cancer Cells Using Immunotargeted Gold Nanoparticles.
Huang X, Jain PK, El-Sayed IH, El-Sayed MA.
Photochem Photobiol. 2006 Dec 1; [Epub ahead of print].
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Laser photothermal therapy of cancer using gold nanoparticles immunotargeted to molecular markers on the cell surface has been shown to be an effective modality to selectively kill cancer cells at much lower laser powers than those needed for healthy cells. To elucidate the minimum light dosimetry required to induce cell death, photothermal destruction of two cancerous cell lines and a noncancerous cell line treated with anti-epidermal growth factor receptor (anti-EGFR) antibody conjugated gold nanoparticles is studied and a numerical heat transport model is used to estimate the local temperature rise within the cells as a result of the laser heating of the gold nanoparticles. It is found that cell samples with higher nanoparticle loading require a lower incident laser power to achieve a certain temperature rise. Numerically estimated temperatures of 70-80 degrees C achieved by heating the gold particles agree well with the measured threshold temperature for destruction of the cell lines by oven-heating and those measured in an earlier nanoshell method. Specific binding of anti-EGFR antibody to cancerous cells overexpressing EGFR selectively increases the gold nanoparticle loading within cancerous cells, thus allowing the cancerous cells to be destroyed at lower laser power thresholds needed for the noncancerous cells. In addition, photothermal therapy using gold nanoparticles requires lower laser power thresholds than that using conventional dyes due to the much higher absorption coefficient of the gold nanoparticles.

PEGylated lysine dendrimers for tumor-selective targeting after intravenous injection in tumor-bearing mice.
Okuda T, Kawakami S, Akimoto N, Niidome T, Yamashita F, Hashida M.
J Control Release. 2006 Dec 1;116(3):330-336.
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In this study, we synthesized a sixth generation lysine dendrimer (KG6) and two PEGylated derivatives thereof and evaluated their biodistribution characteristics in both normal and tumor-bearing mice. The intact KG6 showed a rapid clearance from the blood stream and non-specific accumulation in the liver and kidney. In contrast, the PEGylated derivatives showed a better retention in blood and low accumulativeness in organs dependent of the rate of PEGylation. In addition, PEGylated KG6 with a high modification rate was accumulated effectively in tumor tissue via the enhanced permeability and retention (EPR) effect. Moreover, we clarified that multiple administrations did not affect the biodistribution characteristics of a second dose of PEGylated KG6. PEGylated lysine dendrimer would be a useful material for a clinically applicable tumor-targeting carrier.

Simultaneous delivery of doxorubicin and GG918 (Elacridar) by new Polymer-Lipid Hybrid Nanoparticles (PLN) for enhanced treatment of multidrug-resistant breast cancer.
Wong HL, Bendayan R, Rauth AM, Wu XY.
J Control Release. 2006 Dec 1;116(3):275-84.
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Multidrug-resistant (MDR) cancer may be treated using combinations of encapsulated cytotoxic drugs and chemosensitizers. To optimize for the effectiveness of this combinational approach, novel polymer-lipid hybrid nanoparticle (PLN) formulations capable of delivering a cytotoxic drug, doxorubicin (Dox), a chemosensitizer, GG918, or their combination were prepared. Both acute and long-term anticancer activities of various combinations of Dox and GG918 in solution or PLN form were evaluated in a human MDR breast cancer cell line (MDA435/LCC6/MDR1) using trypan blue exclusion and clonogenic assays. Cellular Dox uptake and drug distribution within the cells were determined by fluoremetry and fluorescence microscopy. The results showed that the encapsulation efficiencies of Dox and GG918 in PLN were up to 89% and were not compromised by co-encapsulation of the two agents. Of various combinational treatment approaches, the Dox and GG918 co-encapsulated PLN formulation ((DG)n) demonstrated the greatest Dox uptake and anticancer activity to the MDR cells, while co-administration of two single-agent loaded PLN was least effective. Fluorescence microscopy indicated cellular internalization of (DG)n. These findings suggest that in addition to the total drug concentrations, the simultaneous delivery of Dox and GG918 to the same cellular location is critical in determining the therapeutic effectiveness of this anticancer drug-chemosensitizer combination.

In vitro assessment of transferrin-conjugated liposomes as drug delivery systems for inhalation therapy of lung cancer.
Anabousi S, Bakowsky U, Schneider M, Huwer H, Lehr CM, Ehrhardt C.
Eur J Pharm Sci. 2006 Dec;29(5):367-74.
[ expand abstract ]

Most human tumours over-express receptors for growth factors and peptide hormones, which are being increasingly studied as a means to selectively deliver cytotoxic agents. An example being the transferrin receptor (TfR, CD71). Here, we studied expression levels and location of TfR in different lung epithelial cell types (i.e., bronchial and alveolar epithelial cells) by flow-cytometry and confocal laser scanning microscopy (CLSM). Furthermore, we assessed uptake levels and cytotoxicity of transferrin (Tf)-conjugated liposomes in vitro. TfR was found to be expressed at a significantly higher level in bronchial epithelial cells compared with their alveolar counterparts. Cells of cancerous origin (i.e., A549 cell line) showed a higher TfR expression level than healthy alveolar epithelial type II cells in primary culture. CLSM revealed TfR to be located primarily at the basolateral aspect of cells, with the exception of cells undergoing mitotic proliferation, which also showed TfR at their apical membranes, due to their loss of cell polarity. Higher expression levels of TfR correlated well with enhanced uptake of Tf-liposomes and increased levels of cytotoxicity. Liposome uptake was temperature-dependent and inhibitable by excess free Tf. Tf-conjugated liposomes appear as good candidates for an approach to deliver cytostatic drugs to sites of lung cancer by inhalation.

Thermotropic phase behavior of DPPC liposome systems in the presence of the anti-cancer agent 'Ellipticine'.
Cavalcanti LP, Torriani IL.
Eur Biophys J. 2006 Dec;36(1):67-71.
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This letter presents our first results on the structural changes in DPPC multilamellar vesicles dispersed in water in the presence of the anti-cancer agent Ellipticine. The thermotropic phase transitions of the lamellar packing inside lipid vesicles were characterized in situ by small angle X ray diffraction. The results lead to the determination of a critical concentration value for drug loading on the vesicle system around 4% molar fraction of Ellipticine, an indication of the localization of the drug in the alkyl chains and the influence of the drug on the decreasing rate of the bilayer period after the main phase transition.

Inhibitory effect of the polyinosinic-polycytidylic acid/cationic liposome on the progression of murine B16F10 melanoma.
Fujimura T, Nakagawa S, Ohtani T, Ito Y, Aiba S.
Eur J Immunol. 2006 Dec;36(12):3371-80.
[ expand abstract ]

Cellular proteins, retinoic acid inducible gene-I and Toll-like receptor 3, sense dsRNA including polyinosinic-polycytidylic acid (PIC) to stimulate innate immune response. The local administration of PIC has been demonstrated to be effective in anti-tumor immunotherapy. However, the effects of PIC delivered cross the cell membrane have not yet been examined. To address this issue, we used a complex of PIC and cationic liposome (PIC liposome) and examined its anti-tumor effects in vitro and in vivo. PIC liposome could directly suppress the growth of B16F10 melanoma in vitro and repeated peritumoral injections of PIC liposome inhibited melanoma growth in a dose-dependent manner. This treatment induced tyrosinase-related protein-2 (TRP-2)-tetramer(+) CD8(+) cells in the lymph nodes. As the mechanism for its anti-tumor immune response, we showed that the intradermal injection of PIC liposome induced the maturation of dendritic cells (DC). Moreover, the intratumoral injection of immature DC after treatment with PIC liposome significantly increased the number of TRP-2-specific IFN-gamma-producing cells in the lymph nodes as well as spleen, which resulted in an augmentation of the anti-tumor immune response. These studies demonstrate the potential of peritumoral injection of PIC liposome as immunotherapy for malignant melanoma.

[Magnetically based enhancement of nanoparticle uptake in tumor cells: combination of magnetically induced cell labeling and magnetic heating.]
Kettering M, Winter J, Zeisberger M, Alexiou C, Bremer-Streck S, Bergemann C, Kaiser WA, Hilger I.
Rofo. 2006 Dec;178(12):1255-60.
[ expand abstract ]

PURPOSE: Magnetic nanoparticles (MNP) are known to be versatile tools in diagnostic and interventional radiology. The goal of the present study was to assess whether MNP can be selectively accumulated on human adenocarcinoma cells in vitro using an external magnetic field (magnetically induced cell labeling) and whether these labeled tumor cells can then be destroyed after being exposed to an alternating magnetic field (magnetically induced heating). In this context, a long-term goal is to combine these two developing methods to achieve an additive effect in tumor therapy. MATERIALS AND METHODS: BT-474 cells were incubated until confluence. Magnetic nanoparticles (0.32 mg Fe/ml culture medium) were then added and the flask was exposed to an external magnetic field gradient (magnetically induced cell labeling, 56 or 83 mT magnets) for 24 hours in order to label the tumor cells with nanoparticles. Cells without both MNP and magnetic labeling as well as cells with MNP incubation but without magnetic labeling served as controls. After MNP incubation, the magnetically labeled cells (5 x 10 (7) cells/ml) were exposed to an alternating magnetic field for 5.45 minutes (frequency 400 kHz, amplitude 24.6 kA/m). The combination effect of both magnetic labeling and magnetic heating was assessed by determining the temperature increase. The amount of MNP accumulated within the cells was determined by measuring the iron content via atomic absorption spectrometry. For statistical analysis mean values and standard deviations of temperature increases and iron contents were calculated and the differences were analyzed using the Student's t-test. RESULTS: A significant temperature increase (p < 0.01) during magnetic heating of 41.76 +/- 4.60 K was detected after magnetic labeling of the cells (5 x 10 (7) cells/ml, 83 mT) incubated with MNP. In comparison, the cells incubated with MNP but without magnetic labeling revealed a temperature increase of 32.03 +/- 3.33 K, naked cells of only 2.69 +/- 0.34 K. CONCLUSION: The results demonstrated the magnetically based enhancement of cellular uptake of nanoparticles by tumor cells, resulting in the intensification of the generated temperature increase during magnetic heating. Consequently, magnetic nanoparticles are shown to be valuable tools for the combination of magnetically based therapy modalities.

Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery.
Nishiyama N, Kataoka K.
Pharmacol Ther. 2006 Dec;112(3):630-48.
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Polymeric micelles, self-assemblies of block copolymers, are promising nanocarrier systems for drug and gene delivery. Until now, several micellar formulations of antitumor drugs have been intensively studied in preclinical and clinical trials, and their utility has been demonstrated. Even compared with long-circulating liposomes, polymeric micelles might have several advantages, such as controlled drug release, tissue-penetrating ability and reduced toxicity such as hand-foot syndrome and hypersensitivity reaction. Importantly, critical features of the polymeric micelles as drug carriers, including particle size, stability, and loading capacity and release kinetics of drugs, can be modulated by the structures and physicochemical properties of the constituent block copolymers. Also, nano-engineering of block copolymers might allow the preparation of polymeric micelles with integrated smart functions, such as specific-tissue targetability, as well as chemical or physical stimuli-sensitivity. Thus, polymeric micelles are nanotechnology-based carrier systems that might exert the activity of potent bioactive compounds in a site-directed manner, ensuring their effectiveness and safety in the clinical use.

Transition Metal-Mediated Liposomal Encapsulation of Irinotecan (CPT-11) Stabilizes the Drug in the Therapeutically Active Lactone Conformation.
Ramsay E, Alnajim J, Anantha M, Taggar A, Thomas A, Edwards K, Karlsson G, Webb M, Bally M.
Pharm Res. 2006 Dec;23(12):2799-808.
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PURPOSE: To determine whether entrapped transition metals could mediate the active encapsulation of the anticancer drug irinotecan into preformed liposomes. Further, to establish that metal complexation could stabilize liposomal irinotecan in the therapeutically active lactone conformation. MATERIALS AND METHODS: Irinotecan was added to preformed 1,2-distearoyl-sn-glycero-phosphocholine/cholesterol (DSPC/chol) liposomes prepared in CuSO(4), ZnSO(4), MnSO(4), or CoSO(4) solutions, and drug encapsulation was determined over time. The roles of the transmembrane pH gradient and internal pH were evaluated. TLC and HPLC were used to monitor drug stability and liposome morphology was assessed by cryo-TEM. RESULTS: Irinotecan was rapidly and efficiently loaded into preformed liposomes prepared in unbuffered ( approximately pH 3.5) 300 mM CuSO(4) or ZnSO(4). For Cu-containing liposomes, results suggested that irinotecan loading occurred when the interior pH and the exterior pH were matched; however, addition of nigericin to collapse any residual transmembrane pH gradient inhibited irinotecan loading. Greater than 90% of the encapsulated drug was in its active lactone form and cryo-TEM analysis indicated dark intravesicular electron-dense spots. CONCLUSION: Irinotecan is stably entrapped in the active lactone conformation within preformed copper-containing liposomes as a result of metal-drug complexation.

Fluorocarbon nanoparticles as multifunctional drug delivery vehicles.
Yu YB.
J Drug Target. 2006 Dec;14(10):663-9.
[ expand abstract ]

The history and current status of fluorocarbon nanoparticles in biomedicine is briefly reviewed. The deficiencies of current fluorocarbon nanoparticle formulations are highlighted. Strategies to remedy such deficiencies and to functionalize fluorocarbon nanoparticles are presented. Potential applications of fluorocarbon nanoparticles as multifunctional drug delivery vehicles are discussed. The strength of fluorocarbon nanoparticles as drug delivery vehicles is that they integrate drug delivery with non-invasive MR imaging so that the biodistribution of the pharmaceutical entity (drug+delivery vehicle) can be monitored in real time. This, in turn, permits the physician to adjust treatment plan for each patient based on his/her actual response to the ongoing treatment.

Comparing transfection efficiency and safety for antisense oligodeoxyribonucleotide between phospholipids-based microbubbles and liposomes.
Zhao YZ, Luo YK, Liang HD, Mei XG, Tang J, Lu CT, Zhang Y, Lin Q.
J Drug Target. 2006 Dec;14(10):687-93.
[ expand abstract ]

Objective: To compare transfection efficiency and safety for antisense oligodeoxynucleotides (AS-ODNs) between two type of phospholipids-based vectors.Methods: An AS-ODNs sequence HA824 combined with luciferase reporter plasmid was used. Under low intensity ultrasound (US), a breast cancer cell line SK-BR-3 was exposed to different concentration of microbubbles and liposomes. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by propidium iodide assay. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the inhibitory effect of HA824 on HER-2 expression at mRNA level. Atomic force microscopy (AFM) scanning techniques was employed to observe the change of membrane pore size.Results: AS-ODNs transfection efficiency showed an increasing tend with microbubble concentration, but not with liposome concentration. Maximum transfection efficiency with minimum cell viability was achieved under 2% microbubble concentration. Too strong sonoporation activity would enlarge membrane pores significantly and cause low cell viability.Conclusion: US-mediated AS-ODNs transfection enhanced by phospholipids-based microbubbles represents an effective and safe avenue.

Mathematical modeling and simulation of drug release from microspheres: Implications to drug delivery systems.
Arifin DY, Lee LY, Wang CH.
Adv Drug Deliv Rev. 2006 Nov 30;58(12-13):1274-325.
[ expand abstract ]

This article aims to provide a comprehensive review of existing mathematical models and simulations of drug release from polymeric microspheres and of drug transport in adjacent tissues. In drug delivery systems, mathematical modeling plays an important role in elucidating the important drug release mechanisms, thus facilitating the development of new pharmaceutical products by a systematic, rather than trial-and-error, approach. The mathematical models correspond to the known release mechanisms, which are classified as diffusion-, swelling-, and erosion-controlled systems. Various practical applications of these models which explain experimental data are illustrated. The effect of gamma-irradiation sterilization on drug release mechanism from erosion-controlled systems will be discussed. The application of existing models to nanoscale drug delivery systems specifically for hydrophobic and hydrophilic molecules is evaluated. The current development of drug transport modeling in tissues utilizing computational fluid dynamics (CFD) will also be described.

Bio-functional micelles self-assembled from a folate-conjugated block copolymer for targeted intracellular delivery of anticancer drugs.
Liu SQ, Wiradharma N, Gao SJ, Tong YW, Yang YY.
Biomaterials. 2006 Nov 30; [Epub ahead of print].
[ expand abstract ]

In this study, a block copolymer, poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-2-aminoethyl methacrylate)-b-poly(10-undecenoic acid) (P(NIPAAm-co-DMAAm-co-AMA)-b-PUA) was synthesized, and folic acid was conjugated to the hydrophilic block through the amine group in AMA. This polymer was self-assembled into micelles, which exhibited pH-induced temperature sensitivity. They were smaller in size, and possessed a better-defined core-shell structure as well as more stable hydrophobic core than the random copolymer P(NIPAAm-co-DMAAm-co-UA), and provided a shell with folate molecules. An anti-cancer drug, doxorubicin (DOX) was encapsulated into the micelles. The mean diameter of the blank and DOX-loaded micelles was less than 100nm. DOX release was pH-dependent, being faster at low pH (endosomes/lysosomes). Therefore, DOX was readily released from the micelles into the nucleus after being taken up. More importantly, IC50 of DOX-loaded micelles with folate against folate receptor-expressing 4T1 and KB cells was much lower than that of the DOX-loaded micelles without folate (3.8 vs. 7.6mg/L for 4T1 cells and 1.2 vs. 3.0mg/L for KB cells). In vivo experiments conducted in a 4T1 mouse breast cancer model demonstrated that DOX-loaded micelles had a longer blood circulation time than free DOX (t(1/2): 30min and 140min, respectively). In addition, the micelles delivered an increased amount of DOX to the tumor when compared to free DOX. These bio-functional micelles may make a promising carrier to transport anticancer drugs specifically to tumor cells and release the drug molecules inside the cells to the cytosols for improved chemotherapy.

A bacterial protein enhances the release and efficacy of liposomal cancer drugs.
Cheong I, Huang X, Bettegowda C, Diaz LA Jr, Kinzler KW, Zhou S, Vogelstein B.
Science. 2006 Nov 24;314(5803):1308-11.
[ expand abstract ]

Clostridium novyi-NT is an anaerobic bacterium that can infect hypoxic regions within experimental tumors. Because C. novyi-NT lyses red blood cells, we hypothesized that its membrane-disrupting properties could be exploited to enhance the release of liposome-encapsulated drugs within tumors. Here, we show that treatment of mice bearing large, established tumors with C. novyi-NT plus a single dose of liposomal doxorubicin often led to eradication of the tumors. The bacterial factor responsible for the enhanced drug release was identified as a previously unrecognized protein termed liposomase. This protein could potentially be incorporated into diverse experimental approaches for the specific delivery of chemotherapeutic agents to tumors.

Thermotherapy of Prostate Cancer Using Magnetic Nanoparticles: Feasibility, Imaging, and Three-Dimensional Temperature Distribution.
Johannsen M, Gneveckow U, Thiesen B, Taymoorian K, Cho CH, Waldofner N, Scholz R, Jordan A, Loening SA, Wust P.
Eur Urol. 2006 Nov 17; [Epub ahead of print].
[ expand abstract ]

OBJECTIVES: To investigate the feasibility of thermotherapy using biocompatible superparamagnetic nanoparticles in patients with locally recurrent prostate cancer and to evaluate an imaging-based approach for noninvasive calculations of the three-dimensional temperature distribution. METHODS: Ten patients with locally recurrent prostate cancer following primary therapy with curative intent were entered into a prospective phase 1 trial. The magnetic fluid was injected transperineally into the prostates according to a preplan. Patients received six thermal therapies of 60-min duration at weekly intervals using an alternating magnetic field applicator. A method of three-dimensional thermal analysis based on computed tomography (CT) of the prostates was developed and correlated with invasive and intraluminal temperature measurements. The sensitivity of nanoparticle detection by means of CT was investigated in phantoms. RESULTS: The median detection rate of iron oxide nanoparticles in tissue specimens using CT was 89.5% (range: 70-98%). Maximum temperatures up to 55 degrees C were achieved in the prostates. Median temperatures in 20%, 50%, and 90% of the prostates were 41.1 degrees C (range: 40.0-47.4 degrees C), 40.8 degrees C (range: 39.5-45.4 degrees C), and 40.1 degrees C (range: 38.8-43.4 degrees C), respectively. Median urethral and rectal temperatures were 40.5 degrees C (range: 38.4-43.6 degrees C) and 39.8 degrees C (range: 38.2-43.4 degrees C). The median thermal dose was 7.8 (range: 3.5-136.4) cumulative equivalent minutes at 43 degrees C in 90% of the prostates. CONCLUSION: The heating technique using magnetic nanoparticles was feasible. Hyperthermic to thermoablative temperatures were achieved in the prostates at 25% of the available magnetic field strength, indicating a significant potential for higher temperatures. A noninvasive thermometry method specific for this approach could be developed, which may be used for thermal dosimetry in future studies.

PLA/PLGA nanoparticles for sustained release of docetaxel.
Musumeci T, Ventura CA, Giannone I, Ruozi B, Montenegro L, Pignatello R, Puglisi G.
Int J Pharm. 2006 Nov 15;325(1-2):172-9.
[ expand abstract ]

This study investigates the potentiality of nanosphere colloidal suspensions as sustained release systems for intravenous administration of docetaxel (DTX). Nanospheres were prepared by solvent displacement method using polylactic acids (PLA) at different molecular weight and polylactic-co-glycolic (PLGA) as biodegradable matrices. The systems were characterized by light scattering analysis for their mean size, size distribution and zeta potential and by scanning electron microscopy (SEM) for surface morphology. The average diameters of the nanoparticles ranged from 100 to 200 nm. Negative zeta potential values were observed for all systems, particularly the nanospheres produced with the lowest molecular weight PLA showed a zeta potential value of -28mV. Differential scanning calorimetry analysis (DSC) suggested that DTX was molecularly dispersed in the polymeric matrices. A biphasic release of DTX was observed for all colloidal suspensions, after a burst effect in which about 50% (w/w) of the loaded drug was released a sustained release profile for about 10 days was observed. To evaluate the influence of the polymeric carrier on the interaction of DTX with biological membranes, we performed an in vitro study using lipid vesicles made of dipalmitoylphosphatidylcholine (DPPC) as a biomembrane model. DSC was used as a simple and not invasive technique of analysis. DTX produced a depression of DPPC pretransition peak, no variation of the main phase transition temperature and a significative increase of DeltaH value, showing a superficial penetration of the drug into DPPC bilayer. Kinetic experiments demonstrated that the release process of DTX form nanospheres is affected by the molecular weight of the employed polymers.

PEGylated Nanoparticles Based on a Polyaspartamide. Preparation, Physico-Chemical Characterization, and Intracellular Uptake.
Craparo EF, Cavallaro G, Bondi ML, Mandracchia D, Giammona G.
Biomacromolecules. 2006 Nov 13; 7(11): 3083-3092.
[ expand abstract ]

Nanoparticles with different surface PEGylation degree were prepared by using as starting material alpha,beta-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA). PHEA was functionalized with a PEG amino-derivative for obtaining PHEA-PEG(2000) copolymer. Both PHEA and PHEA-PEG(2000) were derivatized with methacrylic anhydride (MA) for obtaining poly(hydroxyethylaspartamide methacrylated) (PHM) and poly(hydroxyethylaspartamide methacrylated)-PEGylated (PHM-PEG(2000)), respectively. Nanoparticles were obtained by UV irradiation of an inverse microemulsion, using as internal phase an aqueous solution of PHM alone or of the PHM/PHM-PEG(2000) mixture at different weight ratio and as external phase a mixture of propylene carbonate and ethyl acetate. Obtained nanoparticles were characterized by FT-IR analysis, dimensional analysis, and TEM micrography. XPS analysis and zeta potential measurements demonstrated the presence of PEG onto the nanoparticle surface. Moreover, the partial degradation of nanoparticles in the presence of esterase as a function of time was demonstrated. Finally, nanoparticles did not possess any cytotoxic activity against K-562 cells and were able to escape from phagocytosis depending on the surface PEGylation degree.

Rapid and Precise Release from Nano-Tracted Poly(N-isopropylacrylamide) Hydrogels Containing Linear Poly(acrylic acid).
Asoh TA, Kaneko T, Matsusaki M, Akashi M.
Macromol Biosci. 2006 Nov 10;6(11):959-965 [Epub ahead of print].
[ expand abstract ]

We investigated the rapid and precise molecular release from hydrogels in response to dual stimuli. To achieve precise on/off drug release using thermoresponsive poly(N-isopropylacrylamide) hydrogels, we prepared nano-structured semi-IPNs, which consisted of thermosensitive PNIPAAm networks penetrated by pH-responsive poly(acrylic acid) (PAAc) linear chains and perforated to create nano-tracts as a molecular pathway. The present nano-tracted semi-IPNs show a rapid deswelling response to both temperature and pH. Model drug releases were investigated when simultaneous changes in temperature and pH were applied. We observed that the cationic drug was rapidly released and then abruptly discontinued from the nano-tracted semi-IPNs in response to the dual stimuli, and clear release and stopping cycles were repeatedly observed on successive steps. Moreover, the release rates and amount of drug released were controllable by the deswelling speed of the gels and the PAAc content inside the gels. This novel release system using the nano-tracted semi-IPNs may be useful for the high performance, pulsed release of molecules.Release profiles of MB from semi-IPNs at pH = 5.5, 20 degrees C (white region) and pH = 2, 40 degrees C (gray region).

Nanoparticles for Two-Photon Photodynamic Therapy in Living Cells.
Gao D, Agayan RR, Xu H, Philbert MA, Kopelman R.
Nano Lett. 2006 Nov 8;6(11):2383-2386.
[ expand abstract ]

We describe here a nontoxic two-photon photodynamic nanoparticle platform and its cellular application. We demonstrate that the dye's potential toxicity can be circumvented by its permanent encapsulation into a biocompatible nanoparticle polymer matrix; this was examined by dye leaching experiments and confirmed by cell uptake experiments. Infrared two-photon nanoplatform phototoxicity was demonstrated for rat C6 glioma cells, while the controls showed no dark toxicity for these living cells.

A single dose of doxorubicin-functionalized bow-tie dendrimer cures mice bearing C-26 colon carcinomas.
Lee CC, Gillies ER, Fox ME, Guillaudeu SJ, Frechet JM, Dy EE, Szoka FC.
Proc Natl Acad Sci USA. 2006 Nov 7;103(45):16649-54.
[ expand abstract ]

The antitumor effect of doxorubicin (DOX) conjugated to a biodegradable dendrimer was evaluated in mice bearing C-26 colon carcinomas. An asymmetric biodegradable polyester dendrimer containing 8-10 wt % DOX was prepared. The design of the dendrimer carrier optimized blood circulation time through size and molecular architecture, drug loading through multiple attachment sites, solubility through PEGylation, and drug release through the use of pH-sensitive hydrazone linkages. In culture, dendrimer-DOX was >10 times less toxic than free DOX toward C-26 colon carcinoma cells after exposure for 72 h. Upon i.v. administration to BALB/c mice with s.c. C-26 tumors, dendrimer-DOX was eliminated from the serum with a half-life of 16 +/- 1 h, and its tumor uptake was ninefold higher than i.v. administered free DOX at 48 h. In efficacy studies performed with BALB/c mice bearing s.c. C-26 tumors, a single i.v. injection of dendrimer-DOX at 20 mg/kg DOX equivalents 8 days after tumor implantation caused complete tumor regression and 100% survival of the mice over the 60-day experiment. No cures were achieved in tumor-implanted mice treated with free DOX at its maximum tolerated dose (6 mg/kg), drug-free dendrimer, or dendrimer-DOX in which the DOX was attached by means of a stable carbamate bond. The antitumor effect of dendrimer-DOX was similar to that of an equimolar dose of liposomal DOX (Doxil). The remarkable antitumor activity of dendrimer-DOX results from the ability of the dendrimer to favorably modulate the pharmacokinetics of attached DOX.

Preparation of poly varepsilon-caprolactone nanoparticles containing magnetite for magnetic drug carrier.
Yang J, Park SB, Yoon HG, Huh YM, Haam S.
Int J Pharm. 2006 Nov 6;324(2):185-90.
[ expand abstract ]

Magnetic poly varepsilon-caprolactone (PCL) nanoparticles were prepared in a well shaped spherical form by the o/w emulsion method. The influence of some preparative variables on the size and surface property was investigated. Nanoparticles were smooth, well individualized and homogeneous in size. The presence of magnetite and its superparamagnetic characteristic were confirmed by transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR) and vibrating sample magnetometer (VSM), respectively. The anti-cancer drug was encapsulated in the magnetic nanoparticle during preparation. A typical release behavior was observed for 30 days. In vitro experiment of magnetic susceptibility under external magnetic field demonstrated that the magnetic PCL nanoparticles have sufficient magnetic susceptibility for a potential magnetic drug carrier for targeted delivery.

Thermally associating polypeptides designed for drug delivery produced by genetically engineered cells.
Hart DS, Gehrke SH.
J Pharm Sci. 2006 Nov 1; [Epub ahead of print].
[ expand abstract ]

Thermally associating polymers, including gelatin, cellulose ethers (e.g., Methocels(R) and poloxamers (e.g., Pluronics(R)) have a long history of use in pharmacy. Over the past 20 years, significant advances in genetic engineering and the understanding of protein secondary and tertiary structures have been made. This has led to the development of a variety of polypeptides that do not occur naturally but can be expressed in recombinant cells and have useful properties that lend themselves to novel applications where current materials cannot perform. The most intensively studied motifs are derived from the consensus repeats of elastin and silk, as well as coiled-coil helices. Many of these designed polypeptides or 'artificial proteins' are thermally associating materials. This property can be exploited to develop solid dosage forms, injectable drug delivery systems, micro- or nanoparticle drug carriers, triggered or targeted release systems, or as a means of simplifying the purification process and thus reducing costs of production of these materials. This review focuses on the development and characterization of this novel class of biomaterials and examines their potential for pharmaceutical applications.

A Parenteral Econazole Formulation Using a Novel Micelle-to-Liposome Transfer Method: In Vitro Characterization and Tumor Growth Delay in a Breast Cancer Xenograft Model.
Cogswell S, Berger S, Waterhouse D, Bally MB, Wasan EK.
Pharm Res. 2006 Nov;23(11):2575-85.
[ expand abstract ]

PURPOSE: The purpose of this study was to develop a parenteral liposomal formulation of econazole, a poorly water-soluble compound not previously available in an intravenous form. We are investigating econazole as an anticancer agent based on its unique mechanism of action to which cancer cells are preferentially sensitive. An intravenous formulation of econazole was desired for preclinical toxicity and efficacy studies of econazole. METHODS: Liposomal econazole was prepared using a novel micelle exchange technique to incorporate the drug into the lipid bilayer of pre-formed liposomes using a poly(ethylene) glycol-linked phospholipid, distearoyl phosphatidylethanolamine (DSPE-PEG). This method allowed for stable and efficient drug incorporation into DPPC and DMPC liposomes at a final drug:lipid ratio of 0.05 (w/w) and increased solubility in saline from <0.1 to 5 mg/ml. RESULTS: Stability over 14 days at 4 degrees C in buffer was demonstrated as well as in vitro plasma stability at 37 degrees C. Plasma elimination studies of micelle-loaded liposomal econazole showed a half-life of approximately 35 min and plasma AUC of 281 mug/ml min. In MCF-7 human breast cancer xenografts in Rag2M mice. Liposomal econazole did not induce significant hepatoxicity, renal toxity or weight loss compared to empty liposomes. Tumor growth was slightly delayed in liposomal econazole-treated mice, with approximately 10-day lag time to reach 300 mm(3) compared to vehicle controls. CONCLUSIONS: The micelle transfer method provided an efficient means of preparing liposomal econazole suitable for intravenous administration. Liposomal econazole was successfully administered to tumor bearing mice at 50 mg/kg, and no significant toxicities attributable to econazole were observed.

Pathotropic nanoparticles for cancer gene therapy Rexin-G IV: three-year clinical experience.
Gordon EM, Lopez FF, Cornelio GH, Lorenzo CC 3rd, Levy JP, Reed RA, Liu L, Bruckner HW, Hall FL.
Int J Oncol. 2006 Nov;29(5):1053-64.
[ expand abstract ]

Metastatic cancer is a life-threatening illness with a predictably fatal outcome, thereby representing a major unmet medical need. In 2003, Rexin-G became the world's first targeted injectable vector approved for clinical trials in the treatment of intractable metastatic disease. Uniquely suited, by design, to function within the context of the human circulatory system, Rexin-G is a pathotropic (disease-seeking) gene delivery system bearing a designer killer gene; in essence, a targeted nanoparticle that seeks out and selectively accumulates in metastatic sites upon intravenous infusion. The targeted delivery of the cytocidal gene to primary tumors and metastatic foci, in effective local concentrations, compels both cancer cells and tumor-associated neovasculature to self-destruct, without causing untoward collateral damage to non-target organs. In this study: i) we report the results of three distinctive clinical studies which demonstrate the initial proofs of concept, safety, and efficacy of Rexin-G when used as a single agent for advanced or metastatic cancer, ii) we introduce the quantitative foundations of an innovative personalized treatment regimen, designated the 'Calculus of Parity', based on a patient's calculated tumor burden, iii) we propose a refinement of surrogate end-points commonly used for defining success in cancer therapy, and iv) we map out a strategic plan for the accelerated approval of Rexin-G based on the oncologic Threshold of Credibility paradigm being developed by the Food and Drug Administration.

Busulfan loading into poly(alkyl cyanoacrylate) nanoparticles: physico-chemistry and molecular modeling.
Layre AM, Couvreur P, Chacun H, Aymes-Chodur C, Ghermani NE, Poupaert J, Richard J, Requier D, Gref R.
J Biomed Mater Res B Appl Biomater. 2006 Nov;79(2):254-62.
[ expand abstract ]

The busulfan is an alkylating agent widely used for the treatment of haematological malignancies and nonmalignant disorders. For a long time, it has been available only in an oral form. This treatment leads to a wide variability in bioavailability and side effects such as the veino-occlusive disease. Thus, an intravenous formulation of busulfan-loaded nanoparticles may be considered as a major progress. This study deals with busulfan entrapment by nanoprecipitation into five different types of poly(alkyl cyanoacrylate) polymers. The polymers leading to the highest busulfan loading efficiencies were poly(isobutyl cyanoacrylate) (PIBCA) and poly(ethyl cyanoacrylate). Molecular modeling along with energy minimization process was employed to identify the nature of the interactions occurring between busulfan and PIBCA. Further, optimization studies enabled to obtain PIBCA nanoparticles displaying busulfan loading ratios equal to 5.9% (w/w) together with nanoparticle yields of 71% (w/w). Since busulfan is a highly reactive molecule, we performed (1)H-NMR spectroscopy experiments showing that chemical integrity of the drug was preserved after loading into nanoparticles. The in vitro release studies under sink conditions, in water, or in rat plasma showed a fast release in the first 10 min followed by a slower one over 6 h. This phenomenon could be explained by the semi-polar characteristics of busulfan.

Vaccination with liposome--DNA complexes elicits enhanced antitumor immunity.
U'Ren L, Kedl R, Dow S.
Cancer Gene Ther. 2006 Nov;13(11):1033-44.
[ expand abstract ]

Cationic liposomes have been shown to potentiate markedly the ability of plasmid DNA to activate innate immune responses. We reasoned therefore that liposome-DNA complexes (LDC) could be used to produce more effective plasmid DNA vaccines for cancer. To test this hypothesis, tumor-bearing mice were vaccinated with conventional plasmid DNA vaccines or with LDC vaccines encoding model tumor antigens and CD8(+) T-cell responses and antitumor activity were assessed. We found that although plasmid DNA vaccines generated large increases in antigen-specific CD8(+) T cells, they failed to elicit significant antitumor immunity. In contrast, LDC vaccines elicited large numbers of antigen-specific CD8(+) T cells and also generated significant antitumor activity against established tumors. The antitumor activity elicited by immunization with LDC vaccines was mediated primarily by CD8(+) T cells. Studies of the interaction of LDC with antigen-presenting cells found that LDC triggered dendritic cell production of interleukin-12 and interferon (IFN)-gamma production by natural killer cells in vivo. Activation by LDC was also accompanied by upregulation of costimulatory molecule expression. These findings suggest that by concurrently activating strong systemic innate immune responses and generating cytotoxic T-lymphocyte responses, LDC may be used to increase the effectiveness of therapeutic plasmid DNA vaccination for cancer.

Cytotoxicity of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles in rat glioma cell lines using different assays.
Sanchez De Juan B, Von Briesen H, Gelperina SE, Kreuter J.
J Drug Target. 2006 Nov;14(9):614-22.
[ expand abstract ]

The cytotoxicity of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles (Dox-PBCA-NP) was investigated in the rat glioma cell lines GS-9L, F-98 and RG-2. MTT and LDH assays were used as cytotoxic assays. In general, the cytotoxicity of nanoparticle-bound doxorubicin (Dox) was enhanced compared to the free drug in solution. However, responses of the cell lines towards the drug effects were different. In the case of free Dox in solution, this difference correlated with different intracellular concentrations of Dox, which in turn, depended on the level of P-glycoprotein (P-gp) expression in these cell lines. Accordingly, the 9L gliosarcoma (GS-9L) cells, which appeared to be most resistant towards Dox, were characterized by the highest P-gp expression.Additionally, the influence of surfactants on the cytotoxic effect was investigated at different Dox concentrations. It was shown that the presence of polysorbate 80 (Tween(R) 80) in the nanoparticle formulation significantly enhanced the cytotoxicity, whereas poloxamer 188 (Pluronic(R) F68) and poloxamine 908 (Tetronic(R) 908) had a negligible influence.

Biodistribution and pharmacokinetic analysis of long-circulating thiolated gelatin nanoparticles following systemic administration in breast cancer-bearing mice.
Kommareddy S, Amiji M.
J Pharm Sci. 2006 Oct 30; [Epub ahead of print].
[ expand abstract ]

The objective of the present study was to modify thiolated gelatin nanoparticles with poly(ethylene glycol) (PEG) chains and examine their long circulating and tumor-targeting properties in vivo in an orthotopic a human breast adenocarcinoma xenograft model. The crosslinked nanoparticle systems were characterized to have a size of 150-250 nm with rapid payload release properties in a highly reducing environment. Upon PEG modification, the nanoparticle size increased to 300-350 nm in diameter. The presence of PEG chains on the surface was confirmed by characterization with electron spectroscopy for chemical analysis. The in vivo long-circulating potential, biodistribution and passive tumor targeting of the controls, and PEG-modified thiolated gelatin nanoparticles were evaluated by injecting indium-111 ((111)In)-labeled nanoparticles into breast tumor (MDA-MB-435)-bearing nude mice. Upon modification with PEG, the nanoparticles were found to have longer circulation times, with the plasma and tumor half-lives of 15.3 and 37.8 h, respectively. The results also showed preferential localization of thiolated nanoparticles in the tumor mass. The resulting nanoparticulate systems with long circulation properties could be used to target encapsulated drugs and genes to tumors passively by utilizing the enhanced permeability and retention effect of the tumor vasculature.

Nanomedicine: Developing smarter therapeutic and diagnostic modalities.
Farokhzad OC, Langer R.
Adv Drug Deliv Rev. 2006 Oct 27; [Epub ahead of print].
[ expand abstract ]

The early impact of nanotechnology on medicine is beginning to get realized, with novel nanoscale therapeutic and diagnostic modalities under development or in clinical practice today. In this commentary the field of "nanomedicine" is briefly reviewed form the perspective of where we were; where we are today; and where we are likely to go tomorrow.

Aclarubicin-loaded cationic albumin-conjugated pegylated nanoparticle for glioma chemotherapy in rats.
Lu W, Wan J, Zhang Q, She Z, Jiang X.
Int J Cancer. 2006 Oct 25; [Epub ahead of print].
[ expand abstract ]

Traditional glioma chemotherapy with those second-line drugs such as anthracyclines usually failed because they are inaccessible to blood-brain barrier (BBB) in tumor. In our study, we incorporated aclarubicin (ACL) into cationic albumin-conjugated pegylated nanoparticle (CBSA-NP-ACL) to determine its therapeutic potential of rats with intracranially implanted C6 glioma cells. When labeled with fluorescent probe, 6-coumarin, CBSA-NP was shown to accumulate much more in tumor mass than nanoparticle without conjugated CBSA (NP) 1 hr post intravenous injection, as well as better retention after 24 hr. Tumor drug concentration of CBSA-NP-ACL displayed 2.6- and 3.3-fold higher than that of NP-ACL and ACL solution 1 hr post injection, while 2.7 and 6.6-fold higher after 24 hr, respectively. Moreover, using tumor microdialysis sampling, AUC(0-24 hr) of free drug amount in tumor interstitium delivered by CBSA-NP-ACL was about 2.0- and 2.7-fold higher than that of NP-ACL and ACL solutions, respectively. When the tumor rat model was subjected to 4 cycles of 2 mg/kg of ACL in different formulations, a significant increase of median survival time was found in the group of CBSA-NP-ACL compared with that of saline control animals, animals treated with NP-ACL and ACL solution. By terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, CBSA-NP-ACL can extensively make the tumor cell apoptosis. Histochemical evaluation by periodic acid Shiff staining and biochemical analysis depicted that the incorporation of ACL into CBSA-NP reduced its toxicity to liver, kidney and heart. Besides, CBSA-NP-ACL was not shown to open tight junction evaluated by BBB coculture. It was concluded that CBSA-NP-ACL could have a therapeutic potential for treatment of glioma.

Transition Metal-Mediated Liposomal Encapsulation of Irinotecan (CPT-11) Stabilizes the Drug in the Therapeutically Active Lactone Conformation.
Ramsay E, Alnajim J, Anantha M, Taggar A, Thomas A, Edwards K, Karlsson G, Webb M, Bally M.
Pharm Res. 2006 Oct 25; [Epub ahead of print].
[ expand abstract ]

PURPOSE: To determine whether entrapped transition metals could mediate the active encapsulation of the anticancer drug irinotecan into preformed liposomes. Further, to establish that metal complexation could stabilize liposomal irinotecan in the therapeutically active lactone conformation. MATERIALS AND METHODS: Irinotecan was added to preformed 1,2-distearoyl-sn-glycero-phosphocholine/cholesterol (DSPC/chol) liposomes prepared in CuSO(4), ZnSO(4), MnSO(4), or CoSO(4) solutions, and drug encapsulation was determined over time. The roles of the transmembrane pH gradient and internal pH were evaluated. TLC and HPLC were used to monitor drug stability and liposome morphology was assessed by cryo-TEM. RESULTS: Irinotecan was rapidly and efficiently loaded into preformed liposomes prepared in unbuffered ( approximately pH 3.5) 300 mM CuSO(4) or ZnSO(4). For Cu-containing liposomes, results suggested that irinotecan loading occurred when the interior pH and the exterior pH were matched; however, addition of nigericin to collapse any residual transmembrane pH gradient inhibited irinotecan loading. Greater than 90% of the encapsulated drug was in its active lactone form and cryo-TEM analysis indicated dark intravesicular electron-dense spots. CONCLUSION: Irinotecan is stably entrapped in the active lactone conformation within preformed copper-containing liposomes as a result of metal-drug complexation.

In vitro and in vivo suppression of hepatocellular carcinoma growth by chitosan nanoparticles.
Qi L, Xu Z, Chen M.
Eur J Cancer. 2006 Oct 16; [Epub ahead of print].
[ expand abstract ]

Chitosan nanoparticles (CNP), a kind of widely used drug carrier, have shown potent cytotoxic effects on various tumour cell lines in vitro and in vivo. This study sought to evaluate the antitumour effect of CNP on growth of human hepatocellular carcinoma (BEL7402) and the possible mechanisms involved. Cells were grown in the absence and presence of various concentrations of CNP with mean particle size of about 40nm. Cell viability, ultrastructural changes, surface charge, mitochondrial membrane potential, reactive oxygen species (ROS) generation, lipid peroxidation, DNA fragmentation and fatty acid composition were analysed by MTT assay, electron microscopy, zetasizer analysis, flow cytometry, spectrophotometric thiobarbituric (TBA) assays, DNA agarose gel electrophoresis and GC/MS respectively. For in vivo experiments, male BABL/c nude mice were implanted with BEL7402 cells subcutaneously to establish human hepatoma model. Chitosan, saline, and CNP with different mean particle size (40, 70 and 100nm) were administrated by oral administration (1mg/kg body weight). Tumour and body weight were measured, morphologic changes of tumour and liver tissues were studied under electron microscope. In vitro, CNP exhibited high antitumour activities with an IC(50) value of 15.01mug/ml, 6.19mug/ml and 0.94mug/ml after treatment for 24h, 48h and 72h respectively. CNP could induce cell necrosis observed by electron microscope and DNA fragmentation. The antitumour mechanism was mediated by neutralisation of cell surface charge, decrease of mitochondrial membrane potential and induction of lipid peroxidation. The tumour growth inhibitory rates on BEL7402 cells in nude mice treated with chitosan and CNP with different mean particle size (40, 70 and 100nm) were 24.07%, 61.69%, 58.98% and 34.91% respectively. Typical necrotic morphological changes of tumour tissues and no liver abnormalities were found under electron microscope. In this paper, results show a strong antitumour effect of CNP on human hepatoma cell line BEL7402 in vitro and in vivo. These findings suggest that CNP could be a kind of promising agent for further evaluations in the treatment of hepatocellular carcinoma.

Multivalent effects of RGD peptides obtained by nanoparticle display.
Montet X, Funovics M, Montet-Abou K, Weissleder R, Josephson L.
J Med Chem. 2006 Oct 5;49(20):6087-93.
[ expand abstract ]

The binding of RGD peptides to integrins offers an excellent system to study the multivalent mediated changes in affinity that arise when peptides, displayed on the surface of a nanoparticle carrier, bind to integrins displayed on the cell membrane. The IC50 of an RGD nanoparticle for endothelial adhesion was 1.0 nM nanoparticle or 20 nM peptide (20 peptide/nanoparticle) and was associated with strong multivalent effects, defined as a multivalent enhancement factor (MVE) of 38 (MVE=IC50 (peptide)/IC50 (peptide when displayed by nanoparticle)). The attachment of RGD peptides to nanoparticles resulted in an extension of the peptide blood half-life from 13 to 180 min. Based on the multivalent enhancement of affinity and extension of blood half-life, multivalent RGD nanoparticle-sized materials should be potent inhibitors of the alpha(V)beta(3) function on endothelial cells in vivo.

Amine-containing core-shell nanoparticles as potential drug carriers for intracellular delivery.
Feng M, Li P.
J Biomed Mater Res A. 2006 Oct 3; [Epub ahead of print] .
[ expand abstract ]

The present study aimed at exploring the use of amine-containing core-shell nanoparticles as potential drug carriers for intracellular delivery. Stable nanoparticles (100-200 nm in diameter) that consisted of poly(methyl methacrylate) (PMMA) cores with hydrophilic poly(ethyleneimine) (PEI) shells were synthesized and used to study their complexation with model drug, ibuprofen (IB), and release it under various electrolyte concentrations. The complexed IB/PEI-PMMA nanoparticles were characterized with FTIR, photon correlation spectroscopy, zeta-potential, and transmission electron microscopy (TEM). Results suggested that the PEI-PMMA nanoparticles could effectively complex with the IB via electrostatic interaction. The thick PEI shells ( approximately 30 nm) significantly enhanced the drug loading capacity up to 23% (w/w) of the complexed nanopartricle. In vitro release of the drug from the complexed nanoparticles was sensitive to the ionic strength of the media. Study of cellular entry of fluorescently labeled IB/nanoparticle complexes using a confocal laser scanning microscopy demonstrated that the entry of the complexed nanoparticles strongly depended on the complexing ratio between IB and PEI-PMMA nanoparticles.

Liposome-mediated RNA transfection should be used with caution.
Barreau C, Dutertre S, Paillard L, Osborne HB.
RNA. 2006 Oct;12(10):1790-3.
[ expand abstract ]

Liposome-mediated RNA transfection appears to present a number of advantages for studying the metabolism of reporter mRNAs in mammalian cells. This method is also widely used to transfect siRNAs. Here we describe results indicating that reporter mRNAs introduced into HeLa cells by liposomes do not present the expected behaviors. Namely, the stability of reporter mRNAs was independent of the presence or absence of an AUUUA instability element, a poly(A) tail, or even a 5' methylated cap. Confocal microscopy showed that fluorescent RNAs introduced by liposome-mediated transfection were present in discrete particles. These observations imply that a number of control experiments are required when using liposome to mediated RNA transfection, and the possible consequences are discussed.

Liposomal plasmid DNA encoding human thymosin alpha and interferon omega potently inhibits liver tumor growth in ICR mice.
Chen PF, Fu GF, Zhang HY, Xu GX, Hou YY.
J Gastroenterol Hepatol. 2006 Oct;21(10):1538-43.
[ expand abstract ]

Aim: To evaluate the potential therapeutic effect of liposomal gene delivery, genes encoding for human thymosin alpha1 (Talpha(1)) and interferon omega(1) were injected via the tail vein into mice bearing a Hep-A-22 liver tumor. The cDNA of human Talpha(1) and interferon omega(1) were obtained by synthesis or reverse transcription-polymerase chain reaction (RT-PCR), respectively. Eukaryotic expressing vectors pIRES2, encoding Talpha(1) and/or interferon omega(1), were constructed and injected with liposome via the tail vein into ICR mice bearing a Hep-A-22 tumor. The potency of tumor inhibition was evaluated when three treated groups were compared with the group receiving the empty vector. Apoptosis of tumor cells was investigated by analyzing DNA fragmentation. Only the group treated with dual-gene plasmid reached an eligible level of tumor inhibition (43%). The difference in tumor weight was statistically significant between the Talpha(1) gene or the interferon omega(1) gene treated groups and the control (P < 0.05), and highly significant between the dual-gene treated group and the control (P < 0.01). DNA ladder was observed in the tumor cells from the purpose gene treated groups but not from the control. The dual-gene plasmid-liposome complex showed more potent inhibition than the single gene constructs on the growth of Hep-A-22 tumor cells in mice, which may be attributed to indirect and additive induction of apoptosis in tumor cells by increased expression of Talpha(1) and interferon omega(1).

Optimized ultrasound-mediated gene transfection in cancer cells.
Feril LB Jr, Ogawa R, Tachibana K, Kondo T.
Cancer Sci. 2006 Oct;97(10):1111-4.
[ expand abstract ]

Ultrasound-mediated gene transfection (sonotransfection) is a promising physical method for gene therapy, especially for cancer gene therapy. To investigate the optimal sonotransfection conditions and to determine whether the optimal transfection rate using sonotransfection is comparable to that of electrotransfection or liposome-mediated transfection, we sonicated different cancer cell lines (U937, HeLa, PC-3, Meth A and T-24) using a 1-MHz unfocused ultrasound at different intensities, pulse repetition frequencies and exposure times. The ideal ultrasound conditions were noted to be at 1.5 Watt/cm(2) pulsed at 0.5 Hz with a duty factor of 50%. The results showed that transfection rate increased with the number of pulses, and peaked between 10 and 15 pulses before it started to decline. Using such optimal conditions, we have shown that sonotransfection is superior to electrotransfection and liposome-mediated transfection at the fixed conditions used in the present study. These findings suggest that sonotransfection could be a better alternative to other non-viral methods (e.g. electroporation and liposome-mediated transfection) of gene transfection, particularly in cancer gene therapy.

Combining cell therapy and nanotechnology.
Halberstadt C, Emerich DF, Gonsalves K.
Expert Opin Biol Ther. 2006 Oct;6(10):971-81.
[ expand abstract ]

Cell transplantation to treat diseases characterised by tissue and cell dysfunction, ranging from diabetes to spinal cord injury, has made great strides preclinically and towards clinical efficacy. In order to enhance clinical outcomes, research needs to continue in areas including the development of a universal cell source that can be differentiated into specific cellular phenotypes, methods to protect the transplanted allogeneic or xenogeneic cells from rejection by the host immune system, techniques to enhance cellular integration of the transplant within the host tissue, strategies for in vivo detection and monitoring of the cellular implants, and new techniques to deliver genes to cells without eliciting a host immune response. Overcoming these obstacles will be of considerable benefit, as it allows understanding, visualising and controlling cellular interactions at a submicron level. Nanotechnology is a multidisciplinary field that allows us to manipulate materials, tissues, cells and DNA at the level of and within the individual cell. As such, nanotechnology may be well suited to optimise the generally encouraging results already achieved in cell transplantation. This review presents some of the ways that nanotechnology can directly contribute to cell transplantation.

RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system.
Howard KA, Rahbek UL, Liu X, Damgaard CK, Glud SZ, Andersen MO, Hovgaard MB, Schmitz A, Nyengaard JR, Besenbacher F, Kjems J.
Mol Ther. 2006 Oct;14(4):476-84.
[ expand abstract ]

This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interference in vitro and in vivo. The formation of interpolyelectrolyte complexes between siRNA duplexes (21-mers) and chitosan polymer into nanoparticles, ranging from 40 to 600 nm, was shown using atomic force microscopy and photon correlation spectroscopy. Rapid uptake (1 h) of Cy5-labeled nanoparticles into NIH 3T3 cells, followed by accumulation over a 24 h period, was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (EGFP) was demonstrated in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively). In addition, Western analysis showed approximately 90% reduced expression of BCR/ABL-1 leukemia fusion protein while BCR expression was unaffected in K562 (Ph(+)) cells after transfection using nanoparticles containing siRNA specific to the BCR/ABL-1 junction sequence. Effective in vivo RNA interference was achieved in bronchiole epithelial cells of transgenic EGFP mice after nasal administration of chitosan/siRNA formulations (37% and 43% reduction compared to mismatch and untreated control, respectively). These findings highlight the potential application of this novel chitosan-based system in RNA-mediated therapy of systemic and mucosal disease.

Targeting gene therapy for prostate cancer cells by liposomes complexed with anti-prostate-specific membrane antigen monoclonal antibody.
Ikegami S, Yamakami K, Ono T, Sato M, Suzuki S, Yoshimura I, Asano T, Hayakawa M, Tadakuma T.
Hum Gene Ther. 2006 Oct;17(10):997-1005.
[ expand abstract ]

Prostate-specific membrane antigen (PSMA) is a membrane-bound antigen expressed on the surface of prostate cancer cells, and this paper describes the use of an antibody against PSMA for targeting gene therapy. We coupled anti-PSMA monoclonal antibody with poly-L-lysine and then incubated it with plasmids. These complexes were then transfected with cationic liposomes into cells. The transfection efficiency of anti-PSMA- liposome complex was higher than that of normal IgG-liposome complex in PSMA-positive LNCaP cells. Furthermore, anti-PSMA-liposome complex containing a suicide gene, thymidine kinase, demonstrated a selective growth-inhibitory effect on LNCaP cells in vitro, but did not exert a significant effect on PSMA-negative cells. In an in vivo xenograft model of LNCaP cells in nu/nu mice, we administered the complexes via the tail vein. Judging on the basis of both 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining and luciferase assay findings, a significant enrichment of plasmid DNA was observed in LNCaP xenografts with anti-PSMA-liposome complex in comparison with normal IgG-liposome complex. However, the distribution of plasmid DNA did not change substantially in any other organs including the liver, kidney, lung, and spleen. Moreover, in suicide gene therapy, anti-PSMA-liposome complex exerted a significant inhibitory effect on the growth of LNCaP xenograft, in contrast to normal IgG-liposome complex.

Crossing cellular barriers using dendrimer nanotechnologies.
Najlah M, D'Emanuele A.
Curr Opin Pharmacol. 2006 Oct;6(5):522-7.
[ expand abstract ]

Dendrimers represent a class of polymers characterised by their well-defined structure, with a high degree of molecular uniformity and low polydispersity. They have found a wide-range of pharmaceutical applications; however, more recently, they have been shown to function as effective intracellular carriers for drugs. In addition, dendrimers have been shown to be capable of bypassing efflux transporters. A new generation of dendrimer-based delivery systems will enable the efficient transport of drugs across cellular barriers.

Co-delivery of drugs and DNA from cationic core-shell nanoparticles self-assembled from a biodegradable copolymer.
Wang Y, Gao S, Ye WH, Yoon HS, Yang YY.
Nat Mater. 2006 Oct;5(10):791-6.
[ expand abstract ]

Non-viral gene-delivery systems are safer to use and easier to produce than viral vectors, but their comparatively low transfection efficiency has limited their applications. Co-delivery of drugs and DNA has been proposed to enhance gene expression or to achieve the synergistic/combined effect of drug and gene therapies. Attempts have been made to deliver drugs and DNA simultaneously using liposomes. Here we report cationic core-shell nanoparticles that were self-assembled from a biodegradable amphiphilic copolymer. These nanoparticles offer advantages over liposomes, as they are easier to fabricate, and are more readily subject to modulation of their size and degree of positive charge. More importantly, they achieve high gene-transfection efficiency and the possibility of co-delivering drugs and genes to the same cells. Enhanced gene transfection with the co-delivery of paclitaxel has been demonstrated by in vitro and in vivo studies. In particular, the co-delivery of paclitaxel with an interleukin-12-encoded plasmid using these nanoparticles suppressed cancer growth more efficiently than the delivery of either paclitaxel or the plasmid in a 4T1 mouse breast cancer model. Moreover, the co-delivery of paclitaxel with Bcl-2-targeted small interfering RNA (siRNA) increased cytotoxicity in MDA-MB-231 human breast cancer cells.

Blood progenitor cell separation from clinical leukapheresis product by magnetic nanoparticle binding and magnetophoresis.
Jing Y, Moore LR, Williams PS, Chalmers JJ, Farag SS, Bolwell B, Zborowski M.
Biotechnol Bioeng. 2006 Sep 28; [Epub ahead of print] .
[ expand abstract ]

Positive selection of CD34+ blood progenitor cells from circulation has been reported to improve patient recovery in applications of autologous transplantation. Current magnetic separation methods rely on cell capture and release on solid supports rather than sorting from flowing suspensions, which limits the range of therapeutic applications and the process scale up. We tested CD34+ cell immunomagnetic labeling and isolation from fresh leukocyte fraction of peripheral blood (leukapheresis) using the continuous quadrupole magnetic flow sorter (QMS), consisting of a flow channel (SHOT, Greenville, IN) and a quadrupole magnet with a maximum field intensity (B(o)) of 1.42 T and a mean force field strength (S(m)) of 1.45 x 10(8)TA/m(2). Both the sample magnetophoretic mobility (m) and the inlet and outlet flow patterns highly affect the QMS performance. Seven commercial progenitor cell labeling reagent combinations were quantitatively evaluated by measuring magnetophoretic mobility of a high CD34 expression cell line, KG-1a, using the cell tracking velocimeter (CTV). The CD34 Progenitor Cell Isolation Kittrade mark (Miltenyi Biotec, Bergisch Gladbach, Germany) showed the strongest labeling of KG-1a cells and was selected for progenitor cell enrichment from 11 fresh and 11 cryopreserved clinical leukapheresis samples derived from different donors. The CD34+ cells were isolated with a purity of 60%-96%, a recovery of 18%- 60%, an enrichment rate of 12-169 and a throughput of (1.7-9.3) x 10(4) cells/s. The results also showed a highly regular dependence of the QMS performance on the flow conditions that agreed with the theoretical predictions based on the CD34+ cell magnetophoretic mobility.

N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs: Endocytosis, exocytosis and drug release.
Park JS, Han TH, Lee KY, Han SS, Hwang JJ, Moon DH, Kim SY, Cho YW.
J Control Release. 2006 Sep 28;115(1):37-45.
[ expand abstract ]

Nano-sized vesicular systems (nanoparticles), ranging from 10 nm to 1000 nm in size, have potential applications as drug delivery systems. Successful clinical applications require the efficient intracellular delivery of drug-loaded nanoparticles. Here we describe N-acetyl histidine-conjugated glycol chitosan (NAcHis-GC) self-assembled nanoparticles as a promising system for intracytoplasmic delivery of drugs. Because N-acetyl histidine (NAcHis) is hydrophobic at neutral pH, the conjugates formed self-assembled nanoparticles with mean diameters of 150-250 nm. In slightly acidic environments, such as those in endosomes, the nanoparticles were disassembled due to breakdown of the hydrophilic/hydrophobic balance by the protonation of the imidazole group of NAcHis. Cellular internalization and drug release of the pH-sensitive self-assembled nanoparticles were investigated by flow cytometry and confocal microscopy. NAcHis-GC nanoparticles internalized by adsorptive endocytosis were exocytosed or localized in endosomes. The amount of exocytosed nanoparticles was dependent on the pre-incubation time prior to removal of free nanoparticles from the culture media. Flow cytometry and confocal microscopy showed that NAcHis-GC nanoparticles released drugs into the cytosol and cell cycle analysis demonstrated that paclitaxel-incorporated NAcHis-GC nanoparticles were effective in inducing arrest of cell growth.

Supramolecular assembly of cyclodextrin-based nanoparticles on solid surfaces for gene delivery.
Park IK, von Recum HA, Jiang S, Pun SH.
Langmuir. 2006 Sep 26;22(20):8478-84.
[ expand abstract ]

In this work, a new approach for surface-mediated gene delivery based on inclusion complex formation between the solid surface and delivery vehicles is presented. beta-Cyclodextrin (CD) molecules form high-affinity inclusion complexes with adamantane. This complexation ability was used to specifically immobilize beta-CD-modified poly(ethylenimine) (CD-PEI) nanoparticles on adamantane- (AD-) modified self-assembled monolayers. To investigate the nanoparticle/surface interaction, CD-PEI-based and PEI-based nanoparticles were passed through a surface plasmon resonance flow cell containing the monolayers. CD-PEI nanoparticles are specifically immobilized on the chip surface by cyclodextrin-adamantane inclusion complex formation. Minimal nanoparticle adsorption was detected with PEI-based nanoparticles or on control surfaces. Competition studies with free cyclodextrins reveal that the multivalent interactions between CD-PEI nanoparticles and the adamantane-modified surface results in significantly higher binding affinity than single cyclodextrin-adamantane complexes. Immobilized nanoparticles were characterized by atomic force microscopy and quantified by fluorescence assay. Thus, the ability of CD-PEI nanoparticles to form inclusion complexes can be exploited to attain specific, high-affinity loading of delivery vehicles onto solid surfaces.

Surface functionalization of inorganic nano-crystals with fibronectin and E-cadherin chimera synergistically accelerates trans-gene delivery into embryonic stem cells.
Kutsuzawa K, Chowdhury EH, Nagaoka M, Maruyama K, Akiyama Y, Akaike T.
Biochem Biophys Res Commun. 2006 Sep 25; [Epub ahead of print] .
[ expand abstract ]

Stem cells holding great promises in regenerative medicine have the potential to be differentiated to a specific cell type through genetic manipulation. However, conventional ways of gene transfer to such progenitor cells suffer from a number of disadvantages particularly involving safety and efficacy issues. Here, we report on the development of a bio-functionalized inorganic nano-carrier of DNA by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high affinity interactions with embryonic stem cell surface and accelerated trans-gene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced trans-gene delivery with a value notably higher than that of commercially available lipofection system. The involvement of both cell surface integrin and E-cadherin in mediating intracellular localization of the hybrid carrier was verified by blocking integrin binding site with excess free fibronectin and up-regulating both integrin and E-cadherin through PKC activation. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.

Synthesis and characterization of chitosan-g-poly(ethylene glycol)-folate as a non-viral carrier for tumor-targeted gene delivery.
Chan P, Kurisawa M, Chung JE, Yang YY.
Biomaterials. 2006 Sep 22; [Epub ahead of print] .
[ expand abstract ]

Poor water solubility and low transfection efficiency of chitosan are major drawbacks for its use as a gene delivery carrier. PEGylation can increase its solubility, and folate conjugation may improve gene transfection efficiency due to promoted uptake of folate receptor-bearing tumor cells. The aim of this study was to synthesize and characterize folate-poly(ethylene glycol)-grafted chitosan (FA-PEG-Chi) for targeted plasmid DNA delivery to tumor cells. Gel electrophoresis study showed strong DNA binding ability of modified chitosan. The pH(50) values, defined as the pH when the transmittance of a polymer solution at 600nm has reached 50% of the original value, suggested that the water solubility of PEGylated chitosan had improved significantly. Regression analysis of pH(50) value as a function of substitution degree of PEG yielded an almost linear correlation for PEG-Chi and FA-PEG-Chi. The solubility of PEGylated chitosan decreased slightly by further conjugation of folic acid due to the relatively more hydrophobic nature of folic acid when compared to PEG. In addition, the chitosan-based DNA complexes did not induce remarkable cytotoxicity against HEK 293 cells. FA-PEG-Chi can be a promising gene carrier due to its solubility in physiological pH, efficiency in condensing DNA, low cytotoxicity and targeting ability.

Cell Biological and Biophysical Aspects of Lipid-mediated Gene Delivery.
Rao NM, Gopal V.
Biosci Rep. 2006 Sep 22; [Epub ahead of print].
[ expand abstract ]

Cationic lipids are conceptually and methodologically simple tools to deliver nucleic acids into the cells. Strategies based on cationic lipids are viable alternatives to viral vectors and are becoming increasingly popular owing to their minimal toxicity. The first-generation cationic lipids were built around the quaternary nitrogen primarily for binding and condensing DNA. A large number of lipids with variations in the hydrophobic and hydrophilic region were generated with excellent transfection efficiencies in vitro. These cationic lipids had reduced efficiencies when tested for gene delivery in vivo. Efforts in the last decade delineated the cell biological basis of the cationic lipid gene delivery to a significant detail. The application of techniques such as small angle X-ray spectroscopy (SAXS) and fluorescence microscopy, helped in linking the physical properties of lipid:DNA complex (lipoplex) with its intracellular fate. This biological knowledge has been incorporated in the design of the second-generation cationic lipids. Lipid-peptide conjugates (peptoids) are effective strategies to overcome the various cellular barriers along with the lipoplex formulations methodologies. In this context, cationic lipid-mediated gene delivery is considerably benefited by the methodologies of liposome-mediated drug delivery. Lipid mediated gene delivery has an intrinsic advantage of being a biomimetic platform on which considerable variations could be built to develop efficient in vivo gene delivery protocols.

Controlled release from a nanocarrier entrapped within a microcarrier.
Rojas EC, Sahiner N, Lawson LB, John VT, Papadopoulos KD.
J Colloid Interface Sci. 2006 Sep 15;301(2):617-23.
[ expand abstract ]

This study illustrates the entrapment of the dye molecule fluorescein sodium salt (FSS) by hydrogel nanoparticles, which are in turn confined inside a water-in-oil-in-water double-emulsion globule, and its subsequent release by the action of the competing agent hydrochloric acid (HCl). Thus, a "double carrier" concept is being introduced in which a nanoscale delivery vehicle is being transported by a microscale delivery vehicle in order to simultaneously take advantage of both systems. This may facilitate storage and handling while protecting the active substance and improving its action upon application.

PEG-modified gold nanorods with a stealth character for in vivo applications.
Niidome T, Yamagata M, Okamoto Y, Akiyama Y, Takahashi H, Kawano T, Katayama Y, Niidome Y.
J Control Release. 2006 Sep 12;114(3):343-7.
[ expand abstract ]

Gold nanorods prepared in hexadecyltrimethylammonium bromide (CTAB) solution are expected to provide novel materials for photothermal therapy and photo-controlled drug delivery systems. Since gold nanorods stabilized with CTAB show strong cytotoxicity, we developed a technique to modify these with polyethyleneglycol (PEG) for medical applications. PEG-modification was achieved by adding mPEG-SH in the CTAB solution, then, excess CTAB was removed by dialysis. PEG-modified gold nanoparticles showed a nearly neutral surface, and had little cytotoxicity in vitro. Following intravenous injection into mice, 54% of injected PEG-modified gold nanoparticles were found in blood at 0.5 h after intravenous injection, whereas most of gold was detected in the liver in the case of original gold nanorods stabilized with CTAB.

Heparin-deoxycholic acid chemical conjugate as an anticancer drug carrier and its antitumor activity.
Park K, Lee GY, Kim YS, Yu M, Park RW, Kim IS, Kim SY, Byun Y.
J Control Release. 2006 Sep 12;114(3):300-6.
[ expand abstract ]

A chemically modified heparin-DOCA (HD) conjugate was developed as a drug carrier for cancer therapy. HD conjugate was found to have markedly low anticoagulant activity and to form self-assembled nanoparticles in aqueous condition. We observed that HD conjugate prevented squamous cell carcinoma (SCC) and human umbilical vascular endothelial cell (HUVEC) proliferation during BrdU incorporation assays. Here, we prepared doxorubicin-loaded heparin nanoparticles by entrapping doxorubicin into the amphiphilic HD conjugate by physical interaction and characterized the properties of these nanoparticles using Dynamic Light Scattering (DLS) and Atomic Force Microscope (AFM). In this study, doxorubicin-loaded heparin nanoparticles were designed to improve the antitumor effects of nano-sized particles (range of 180 to 210 nm) at high drug-loading efficiencies in the range 64% to 96%. These doxorubicin-loaded heparin nanoparticles displayed sustained drug release patterns. It was confirmed in vivo toxicity studies that HD conjugate did not induce unexpected side effects and that DHN 20 was safer than free DOX. An in vivo study showed that HD conjugate, doxorubicin and DHN 20 (one of doxorubicin-loaded heparin nanoparticles) induced tumor volume reductions of 43%, 56% and 74%, respectively, relative to the saline treated control. These results suggest that the drug-entrapped with heparin nanoparticles might provide a novel therapy for SCC.

Cisplatin Nanoliposomes for Cancer Therapy: AFM and Fluorescence Imaging of Cisplatin Encapsulation, Stability, Cellular Uptake, and Toxicity.
Ramachandran S, Quist AP, Kumar S, Lal R.
Langmuir. 2006 Sep 12;22(19):8156-62.
[ expand abstract ]

Cisplatin is the most effective cytotoxic agent against many cancers. Its usage, however, is limited due to inefficient uptake by the target cells. A liposomal formulation of cisplatin is reported to partly overcome this limitation. Physicochemical characteristics of the liposome-cisplatin preparation, including its size, stability, encapsulation efficiency, and cytoplasmic internalization efficiency, play a significant role in an effective usage of liposomal formulations. We have used atomic force microscopy (AFM) to determine physicochemical characteristics of cisplatin-encapsulated liposomes, AFM and fluorescence microscopy to examine their cytoplasmic internalization, and Live/Dead assay to examine their cell toxicity. Nonencapsulated cisplatin is globular and 10-50 nm in size. AFM force-dissection and stiffness measurements show that cisplatin-encapsulated liposomes are significantly stiffer ( approximately 100%) and more stable than liposomes without encapsulated cisplatin. Cisplatin-encapsulated liposomes of approximately 250 nm diameter (nanoliposomes) are most efficiently internalized and induce cell toxicity in a time-dependent manner. Liposomes without cisplatin of similar dimensions, although internalized in the cell cytoplasm, do not induce cell toxicity.

A dual-ligand approach for enhancing targeting selectivity of therapeutic nanocarriers.
Saul JM, Annapragada AV, Bellamkonda RV.
J Control Release. 2006 Sep 12;114(3):277-287.
[ expand abstract ]

Conjugation of ligands to nano-scale drug carriers targeting over-expressed cell surface receptors is a promising approach for delivery of therapeutic agents to tumor cells. However, most commonly utilized ligands are directed at receptors expressed not only on target cells but also on other cells in the body, leading to unintended uptake in these off-target cells. In this study, a novel, dual-ligand approach is reported, which targets tumor cells while sparing off-target cells by exploiting the fact that tumor cells typically over-express multiple types of surface receptors. This approach was tested in the human KB cell line, which over-expresses both folate receptor (FR) and the epidermal growth factor receptor (EGFR). Liposomal nanocarriers loaded with doxorubicin and bearing controlled numbers of both folic acid and a monoclonal antibody against the EGFR were designed. Cytotoxicity was used to determine targeting selectivity of the designed carriers in vitro by utilizing KB cells expressing both FR and EGFR and off-target control cells in which one or both receptors were blocked. The data demonstrates that nanocarriers can be designed to achieve toxicity only when all targeted receptors are available, providing an approach to improve selectivity over current single-ligand approaches.

Macrophage Uptake of Core-Shell Nanoparticles Surface Modified with
Poly(ethylene glycol).
Zahr AS, Davis CA, Pishko MV.
Langmuir. 2006 Sep 12;22(19):8178-8185.
[ expand abstract ]

The in vitro uptake of core-shell nanoparticles encapsulated in a bio-macromolecular nanoshell assembled from multilayered polyelectrolytes was studied. Sulfate modified fluorescent polystyrene nanobeads (diameter 200 nm) were used as a solid core upon which charged multilayers of poly-l-lysine, chitosan, and heparin sulfate are electrostatically deposited utilizing a layer-by-layer (LbL) self-assembly process. The nanoshell composed of the multilayered polyelectrolytes was modified with poly(ethylene glycol) (PEG) of varying molecular weights (either MW 2000, 5000, or 20 000 Da) to form a hydrophilic and long-circulating nanoparticle. The assembly of the nanoshell was confirmed by zeta potential, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The reversal in charge upon the deposition of alternating polyelectrolytes was observed by zeta potential measurements. The nanometer thickness of the nanoshell was confirmed by TEM. The presence of the (C-C-O)(n)() backbone in PEG at the surface of the nanoshell was confirmed by the increase in (C-O,N) peak area concentrations compared to (C-C) peak area, and these results were gathered from XPS. In vitro studies between suspension macrophages and core-shell nanoparticles were performed to determine how the hydrophilicity and the charge on the nanoshell can promote or reduce uptake. Results showed that after 24 h uptake was decreased 3-fold when PEGs of 2000 and 20 000 Da were chemisorbed to the nanoshell, as opposed to a nanoshell with either a positive or highly negative charge. Confocal microscopy aided in verifying that core-shell nanoparticles were internalized within the cell cytoplasm and were not attached to the cell surface. Protein adhesion studies with bovine serum albumin were performed to determine the relationship between surface charge and opsonization of core-shell nanoparticles. It was found that a hydrophilic surface with a low negative charge reduced protein adsorption and uptake. The in vitro uptake of macrophages and protein adsorption onto core-shell nanoparticles formed using layer-by-layer assembly has not been previously studied.

Conjugates of poly(DL-lactide-co-glycolide) on amino cyclodextrins and their nanoparticles as protein delivery system.
Gao H, Wang YN, Fan YG, Ma JB.
J Biomed Mater Res A. 2006 Sep 7; [Epub ahead of print] .
[ expand abstract ]

Poly(DL-lactide-co-glycolide) (PLG) was chemically conjugated on two amino cyclodextrins, mono(6-(2-aminoethyl)amino-6-deoxy)-beta-cyclodextrin and ethylenediamino bridged bis(beta-cyclodextrin), to afford novel amphiphilic conjugates. Those conjugates were then characterized with infrared spectrometry (IR), proton nuclear magnetic resonance ((1)H NMR) and gel permeation chromatography (GPC). A repeat-nanoprecipitation (RP-NP) method was also developed to fabricate the nanoparticles of the conjugates with a water-soluble model protein, bovine serum albumin (BSA). At the end of RP-NP process, the availability of BSA was over 80% while the entrapment efficiency was 40-50% for each nanoprecipitation. The nanoparticles were rigid and spherical with diameters of 110-180 nm determined by transmission electron microscope (TEM), atomic force microscopy (AFM) and particle size analyzer. Nanoparticles possessed good steric stability during freeze-drying and resuspensions due to the existence of cyclodextrins corona. Interactions between BSA and the conjugates in the nanoparticles were then elucidated with IR experiments. About 25% BSA adsorbed on the surface of nanoparticles due to the interaction and was easy to release in the first day. The release of BSA from the nanoparticles was in three phases: a burst effect in the first day, a followed plateau in about a week, and a sustained release of the protein over 14 days. By changing the lactide/glycolide ratio, the degradation time of the conjugates and the release rate of BSA could be controlled. The loss of CDs content was faster than that of overall Mw during degradation since CDs formed outer corona of the nanoparticles. Both the novel biomaterials and the nanosphere fabrication technique contributed to the maintenance of protein structure.

NK105, a paclitaxel-incorporating micellar nanoparticle, is a more potent radiosensitising agent compared to free paclitaxel.
Negishi T, Koizumi F, Uchino H, Kuroda J, Kawaguchi T, Naito S, Matsumura Y.
Br J Cancer. 2006 Sep 4;95(5):601-6.
[ expand abstract ]

NK105 is a micellar nanoparticle formulation designed to enhance the delivery of paclitaxel (PTX) to solid tumours. It has been reported to exert antitumour activity in vivo and to have reduced neurotoxicity as compared to that of free PTX. The purpose of this study was to investigate the radiosensitising effect of NK105 in comparison with that of PTX. Lewis lung carcinoma (LLC)-bearing mice were administered a single intravenous (i.v.) injection of PTX or NK105; 24 h after the drug administration, a proportion of the mice received radiation to the tumour site or lung fields. Then, the antitumour activity and lung toxicity were evaluated. In one subset of mice, the tumours were excised and specimens were prepared for analysis of the cell cycle distribution by flow cytometry. Combined NK105 treatment with radiation yielded significant superior antitumour activity as compared to combined PTX treatment with radiation (P=0.0277). On the other hand, a histopathological study of lung sections revealed no significant difference in histopathological changes between mice treated with PTX and radiation and those treated with NK105 and radiation. Flow-cytometric analysis showed that NK105-treated LLC tumour cells showed more severe arrest at the G2/M phase as compared to PTX-treated tumour cells. The superior radiosensitising activity of NK105 was thus considered to be attributable to the more severe cell cycle arrest at the G2/M phase induced by NK105 as compared to that induced by free PTX. The present study results suggest that further clinical trials are warranted to determine the efficacy and feasibility of combined NK105 therapy with radiation.

Biodegradable nanoparticles of amphiphilic triblock copolymers based on
poly(3-hydroxybutyrate) and poly(ethylene glycol) as drug carriers.
Chen C, Yu CH, Cheng YC, Yu PH, Cheung MK.
Biomaterials. 2006 Sep;27(27):4804-14.
[ expand abstract ]

New amorphous amphiphilic triblock copolymers of poly(3-hydroxybutyrate)-poly(ethylene glycol)-poly(3-hydroxybutyrate) (PHB-PEG-PHB) were synthesized using the ring-opening copolymerization of beta-butyrolactone monomer. They were characterized by fluorescence, SEM and (1)H NMR. These triblock copolymers can form biodegradable nanoparticles with core-shell structure in aqueous solution. Comparing to the poly(ethylene oxide)-PHB-poly(ethylene oxide) (PEO-PHB-PEO) copolymers, these nanoparticles exhibited much smaller critical micelle concentrations and better drug loading properties, which indicated that the nanoparticles were very suitable for delivery carriers of hydrophobic drugs. The drug release profile monitored by fluorescence showed that the release of pyrene from the PHB-PEG-PHB nanoparticles exhibited the second-order exponential decay behavior. The initial biodegradation rate of the PHB-PEG-PHB nanoparticles was related to the enzyme amount, the initial concentrations of nanoparticle dispersions and the PHB block length. The biodegraded products detected by (1)H NMR contained 3HB monomer, dimer and minor trimer, which were safe to the body.

Nanoparticulate drug carriers based on hybrid poly(d,l-lactide-co-glycolide)-dendron structures.
Costantino L, Gandolfi F, Bossy-Nobs L, Tosi G, Gurny R, Rivasi F, Angela Vandelli M, Forni F.
Biomaterials. 2006 Sep;27(26):4635-45; Epub 2006 May 22.
[ expand abstract ]

We describe a general method for incorporating target moieties in a well-defined arrangement into the surface of biocompatible polyester poly(d,l-lactic-co-glycolic acid) (PLGA) materials using dendrons. In this way it is possible to obtain nanoparticles (NPs) with a high degree of surface coverage. This new strategy was successfully applied to the preparation of peptide- and beta-d-glucose-covered NPs. The first application is based on the discovery of NPs made of conjugates between PLGA and short peptidic sequences able to cross the blood-brain barrier (BBB) after systemic administration. In this paper, we used a branched structure (dendron) in order to prepare a derivative of PLGA able to form, by simple nanoprecipitation, NPs with a higher degree of surface coverage than previously reported by us, characteristic that could influence the uptake by the liver and spleen. The NPs thus obtained retain the ability to cross the BBB and possess a core-shell structure, as evidenced from zeta-potential, X-ray photoelectron (ESCA) spectroscopy and elemental analyses. These results are comparable with the NPs obtained by the derivatization of preformed NPs. The same strategy, namely the use of a branched spacer (a dendron or a G1 dendrimer) inserted between one end of the PLGA chain and a derivatizing molecule, was also successfully applied to obtain beta-d-glucose-covered NPs; in this case, the surface analysis of the NPs was performed by using high resolution magic angle spinning (HRMAS) NMR spectroscopy and zeta-potential measurements.

The pinpoint promise of nanoparticle-based drug delivery and molecular diagnosis.
Emerich DF, Thanos CG.
Biomol Eng. 2006 Sep;23(4):171-84.
[ expand abstract ]

Nanotechnology, or systems/device manufacture at the molecular level, is a multidisciplinary scientific field undergoing explosive development. The genesis of nanotechnology can be traced to the promise of revolutionary advances across medicine, communications, genomics and robotics. Without doubt one of the greatest values of nanotechnology will be in the development of new and effective medical treatments (i.e., nanomedicine). This review focuses on the potential of nanomedicine as it specifically relates to (1) the development of nanoparticles for enabling and improving the targeted delivery of therapeutic agents; (2) developing novel and more effective diagnostic and screening techniques to extend the limits of molecular diagnostics providing point-of-care diagnosis and more personalized medicine.

Liposome formulation of a novel hydrophobic aryl-imidazole compound for anti-cancer therapy.
Liu J, Lee H, Huesca M, Young A, Allen C.
Cancer Chemother Pharmacol. 2006 Sep;58(3):306-18.
[ expand abstract ]

PURPOSE: A cholesterol-free liposome formulation formed from mixtures of egg phosphatidylcholine (ePC) and poly (ethylene glycol) conjugated distearoylphosphatidylethanolamine (DSPE-PEG 2000) was optimized and evaluated for delivery of a novel anti-cancer agent ML220 (2-(5-bromo-1H-indol-3-yl)-1H-phenanthro [9,10-d] imidazole). RESULTS AND DISCUSSION: ML220 is highly lipophilic with a water solubility of 0.14 mug/ml and calculated log P of 5.69. The ML220-loaded liposomes had a unimodal size-distribution and a mean diameter of 89 nm. The drug to lipid ratio in the formulation was 1:3.5 (mol:mol) and the drug loading efficiency was 83% providing a more than 50,000-fold increase in the water solubility of ML220. The formulation was demonstrated to be stable in vitro at 37 degrees C for over 2 weeks with a delayed drug release profile. Evaluation of the subacute toxicity of the liposome formulated drug in C3H mice revealed no overt signs of toxicity. Also, a biexponential drug plasma concentration pattern was found upon evaluation of the pharmacokinetics in Balb/C mice. The in vivo evaluation of the anti-cancer activity in a human colon HT29 carcinoma model revealed a significant delay in tumor growth. CONCLUSION: Overall, the ePC/DSPE-PEG liposomes were demonstrated to be a suitable delivery system for ML220. These studies also highlight the potential of cholesterol-free liposomes as a formulation strategy for highly lipophilic drugs.

Enhancement of anticancer activity in antineovascular therapy is based on the intratumoral distribution of the active targeting carrier for anticancer drugs.
Maeda N, Miyazawa S, Shimizu K, Asai T, Yonezawa S, Kitazawa S, Namba Y, Tsukada H, Oku N.
Biol Pharm Bull. 2006 Sep;29(9):1936-40.
[ expand abstract ]

We previously observed the enhanced anticancer efficacy of anticancer drugs encapsulated in Ala-Pro-Arg-Pro-Gly-polyethyleneglycol-modified liposome (APRPG-PEG-Lip) in tumor-bearing mice, since APRPG peptide was used as an active targeting tool to angiogenic endothelium. This modality, antineovascular therapy (ANET), aims to eradicate tumor cells indirectly through damaging angiogenic vessels. In the present study, we examined the in vivo trafficking of APRPG-PEG-Lip labeled with [2-(18)F]2-fluoro-2-deoxy-D-glucose ([2-(18)F]FDG) by use of positron emission tomography (PET), and observed that the trafficking of this liposome was quite similar to that of non-targeted long-circulating liposome (PEG-Lip). Then, histochemical analysis of intratumoral distribution of both liposomes was performed by use of fluorescence-labeled liposomes. In contrast to in vivo trafficking, intratumoral distribution of both types of liposomes was quite different: APRPG-PEG-Lip was colocalized with angiogenic endothelial cells that were immunohistochemically stained for CD31, although PEG-Lip was localized around the angiogenic vessels. These results strongly suggest that intratumoral distribution of drug carrier is much more important for therapeutic efficacy than the total accumulation of the anticancer drug in the tumor, and that active delivery of anticancer drugs to angiogenic vessels is useful for cancer treatment.

Nanocarriers: promising vehicle for bioactive drugs.
Rawat M, Singh D, Saraf S, Saraf S.
Biol Pharm Bull. 2006 Sep;29(9):1790-8.
[ expand abstract ]

Development of new delivery systems that deliver the potential drug specifically to the target site in order to meet the therapeutic needs of the patients at the required time and level remains the key challenge in the field of pharmaceutical biotechnology. Developments in this context to achieve desired goal has led to the evolution of the multidisciplinary field nanobiotechnology which involves the combination of two most promising technologies of 21st century--biotechnology and nanotechnology. Nanobiotechnology encompasses a wide array of different techniques to improve the delivery of biotech drugs, and nanoparticles offer the most suitable form whose properties can be tailored by chemical methods. This review highlights the different types of nanoparticulate delivery systems employed for biotech drugs in the field of molecular medicine with a short overlook at its applications and the probable associated drawbacks.

Nanoparticles of 5-fluorouracil (5-FU) loaded N-succinyl-chitosan (Suc-Chi) for cancer chemotherapy: preparation, characterization--in-vitro drug release and anti-tumour activity.
Yan C, Chen D, Gu J, Qin J.
J Pharm Pharmacol. 2006 Sep;58(9):1177-81.
[ expand abstract ]

N-Succinyl-chitosan has favourable properties as a drug carrier, such as biocompatibility, low toxicity and long-term retention in the body. It is a good candidate for cancer chemotherapy as a polymeric drug carrier. This study describes the preparation and characterization of 5-fluorouracil-loaded N-succinyl-chitosan nanoparticles (5-FU-Suc-Chi/NP) by an emulsification solvent diffusion method. The influence of the initial 5-FU concentration on the nanoparticle characteristics and release behaviour in phosphate-buffered saline solution (PBS) was evaluated. The Suc-Chi nanoparticles had a particle diameter (Z-average) in the range 202 approximately 273 nm and a negative zeta-potential (approx. -27 to -18 mV). The formulation with an initial 5-FU concentration of 1000 microg mL-1 provided the highest loading capacity (19%) and the highest extent of release (61% at 24 h). The 5-FU-Suc-Chi/NP showed good anti-tumour activity against Sarcoma 180 solid tumour and mild toxicity. According to the data obtained, this Suc-Chi-based nanotechnology opens new and interesting perspectives for cancer chemotherapy.

Susceptibility of nanoparticle-encapsulated paclitaxel to P-glycoprotein-mediated drug efflux.
Chavanpatil MD, Patil Y, Panyam J.
Int J Pharm. 2006 Aug 31;320(1-2):150-6.
[ expand abstract ]

Overexpression of P-glycoprotein (P-gp) is a key factor contributing to the development of multidrug resistance (MDR) in cancer cells. The objective of the study is to investigate whether a P-gp substrate, paclitaxel, delivered to MDR tumor cells in poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles is susceptible to P-gp - mediated drug efflux. Paclitaxel-loaded nanoparticles were formulated by emulsion-solvent evaporation technique. Nanoparticles had a mean hydrodynamic diameter of about 195nm, and demonstrated sustained release of paclitaxel. In vitro cell culture studies indicated that paclitaxel nanoparticles result in sustained, dose-dependent and significant cytotoxicity in drug-sensitive MCF-7 tumor cells but not in drug-resistant NCI-ADR/RES cells. Resistance to nanoparticle-encapsulated paclitaxel was reversed by verapamil, a P-gp inhibitor. Further, sustained inhibition of P-gp was necessary for sustaining the cytotoxicity of nanoparticle-encapsulated paclitaxel in drug-resistant cells. Inhibition of P-gp by verapamil did not significantly affect the uptake or retention of nanoparticles in drug-resistant cells. In conclusion, our studies suggest that P-gp substrates, such as paclitaxel, delivered to MDR cells by PLGA nanoparticles, are susceptible to efflux by P-gp. Inhibition of P-gp restores sensitivity to paclitaxel; however, sustained inhibition of P-gp is required for sustained therapeutic efficacy of nanoparticle-encapsulated drug.

Template synthesis of multifunctional nanotubes for controlled release.
Son SJ, Bai X, Nan A, Ghandehari H, Lee SB.
J Control Release. 2006 Aug 28;114(2):143-52.
[ expand abstract ]

In the past few decades, nanoscale materials have been widely used for controlled release applications. Importantly, many researches have focused on multifunctional nanoparticles for targeted delivery of bioactive and imaging agents as therapeutics and diagnostics. Recent advances in nanotechnology have made possible the design and development of tubular nanoscale particles called nanotubes. The tubular shape of such particles is highly attractive since it is possible to differentially functionalize the inner and outer surfaces to facilitate drug loading, biocompatibility and biorecognition. Novel synthetic strategies allow the fabrication of tubular structures with well-defined diameters and lengths. This can have important implications in biodistribution, subcellular trafficking and drug release. In this article the biomedical applications of nanotubes will be discussed with emphasis on the template synthesis of composite nanotubes containing silica and iron oxide that have potential use in drug delivery, magnetic resonance imaging (MRI), and chemical and biochemical separations.

Multicentre phase II pharmacokinetic and pharmacodynamic study of OSI-7904L in previously untreated patients with advanced gastric or gastroesophageal junction adenocarcinoma.
Falk S, Anthoney A, Eatock M, Van Cutsem E, Chick J, Glen H, Valle JW, Drolet DW, Albert D, Ferry D, Ajani J.
Br J Cancer. 2006 Aug 21;95(4):450-6.
[ expand abstract ]

A two-stage Simon design was used to evaluate the response rate of OSI-7904L, a liposome encapsulated thymidylate synthase inhibitor, in advanced gastric and/or gastroesophageal adenocarcinoma (A-G/GEJA), administered intravenously at 12 mg m(-2) over 30 min every 21 days. Fifty patients were treated. Median age was 64 years (range 35-82), 62% were male and 89% had ECOG PS of 0/1. A total of 252 cycles were administered; median of 4 per patient (range 1-21). Twelve patients required dose reductions, mainly for skin toxicity. Investigator assessed response rate was 17.4% (95% CI 7.8-31.4) with one complete and seven partial responses in 46 evaluable patients. Twenty-one patients (42%) had stable disease. Median time to progression and survival were 12.4 and 36.9 weeks, respectively. NCI CTCAE Grade 3/4 neutropenia (14%) and thrombocytopenia (4%) were uncommon. The main G3/4 nonhaematological toxicities were skin-related 22%, stomatitis 14%, fatigue/lethargy 10%, and diarrhea 8%. Pharmacokinetic data showed high interpatient variability. Patients with higher AUC were more likely to experience G3/4 toxicity during cycle 1 while baseline homocysteine did not predict toxicity. Response did not correlate with AUC. Elevations in 2'-dU were observed indicating target inhibition. Analysis of TS genotype, TS protein and expression did not reveal any correlation with outcome. OSI-7904L has activity in A-G/GEJA similar to other active agents and an acceptable safety profile.

Antigen Chemically Coupled to the Surface of Liposomes Are Cross-Presented to CD8+ T Cells and Induce Potent Antitumor Immunity.
Taneichi M, Ishida H, Kajino K, Ogasawara K, Tanaka Y, Kasai M, Mori M, Nishida M, Yamamura H, Mizuguchi J, Uchida T.
J Immunol. 2006 Aug 15;177(4):2324-30.
[ expand abstract ]

We have previously demonstrated that liposomes with differential lipid components display differential adjuvant effects when Ags are chemically coupled to their surfaces. In the present study, Ag presentation of liposome-coupled OVA was investigated in vitro, and it was found that OVA coupled to liposomes made using unsaturated fatty acid was presented to both CD4(+) and CD8(+) T cells, whereas OVA coupled to liposomes made using saturated fatty acid was presented only to CD4(+) T cells. Confocal laser scanning microscopic analysis demonstrated that a portion of the OVA coupled to liposomes made using unsaturated, but not saturated fatty acid, received processing beyond the MHC class II compartment, suggesting that the degradation of OVA might occur in the cytosol, and that the peptides generated in this manner would be presented to CD8(+) T cells via MHC class I. The ability to induce cross-presentation of an Ag coupled to liposomes consisting of unsaturated fatty acid was further confirmed by in vivo induction of CTL and by the induction of tumor eradication in mice; E.G7 tumors in mice that received combined inoculation with OVA(257-264)-liposome conjugates, CpG, and anti-IL-10 mAbs were completely eradicated. In those mice, the frequency of CD8(+) T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased. These results suggested that, by choosing lipid components for liposomes, surface-coupled liposomal Ags might be applicable for the development of tumor vaccines to present tumor Ags to APCs and induce antitumor responses.

In vivo antitumor activity of chitosan nanoparticles.
Qi L, Xu Z.
Bioorg Med Chem Lett. 2006 Aug 15;16(16):4243-5.
[ expand abstract ]

Chitosan nanoparticles have been synthesized as potential anticancer agents, and evaluated, in vitro, against various cancer cell lines. In this study, in vivo antitumor activity of chitosan nanoparticles against Sarcoma-180 and mouse hepatoma H22 was investigated. Chitosan nanoparticles showed significant antitumor activity in vivo. The doses and particle size made a great effect on their efficacy.

Studies on the oridonin-loaded poly(d,l-lactic acid) nanoparticles in vitro and in vivo.
Xing J, Zhang D, Tan T.
Int J Biol Macromol. 2006 Aug 7; [Epub ahead of print] .
[ expand abstract ]

The purpose of this paper was to investigate the possibility of developing a polymeric nanoparticle delivery system for ORI to increase its solubility, blood circulation time and tissue targeting. Oridonin-loaded poly(d,l-lactic acid) nanoparticles (ORI-PLA-NP) were prepared by the further modified spontaneous emulsion solvent diffusion (MSESD) method. Studies were carried out to characterize and evaluate the produced ORI-PLA-NP both in vitro and in vivo. The experimental results showed that the mean size of the nanoparticles were 137.3nm, with 87.2% of the nanoparticles distributed between the range of 107 and 195nm. The entrapment efficiency and actual drug loading of the nanoparticles were 91.88+/-1.83 and 2.32+/-0.05%, respectively. It was demonstrated by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD) that ORI existed in the form of amorphous in the nanoparticles. The in vitro release profile of ORI-PLA-NP could be expressed well by the Higuchi equation: Q=8.944t(1/2)+11.246. The results of pharmacokinetics demonstrated that being encapsulated in PLA nanoparticles was remarkably effective for ORI to prolong its blood circulation time. After the i.v. administration of ORI-PLA-NP, we could observe a stable and high concentration of ORI in liver, lung and spleen, while its distribution in heart and kidney decreased.

Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach.
Zeisberger SM, Odermatt B, Marty C, Zehnder-Fjallman AH, Ballmer-Hofer K, Schwendener RA.
Br J Cancer. 2006 Aug 7;95(3):272-81.
[ expand abstract ]

Tumour-associated macrophages, TAMs, play a pivotal role in tumour growth and metastasis by promoting tumour angiogenesis. Treatment with clodronate encapsulated in liposomes (clodrolip) efficiently depleted these phagocytic cells in the murine F9 teratocarcinoma and human A673 rhabdomyosarcoma mouse tumour models resulting in significant inhibition of tumour growth ranging from 75 to >92%, depending on therapy and schedule. Tumour inhibition was accompanied by a drastic reduction in blood vessel density in the tumour tissue. Vascular endothelial growth factor (VEGF) is one of the major inducers of tumour angiogenesis and is also required for macrophage recruitment. The strongest effects were observed with the combination therapy of clodrolip and a VEGF-neutralising antibody, whereas free clodronate was not significantly active. Immunohistologic evaluation of the tumours showed significant depletion of F4/80(+) and MOMA-1(+) and a less pronounced depletion of CD11b(+) TAMs. Blood vessel staining (CD31) and quantification of the vessels as well as TAMs and tumour-associated dendritic cells (TADCs) in the A673 model showed reduction rates of 85 to >94%, even 9 days after the end of therapy. In addition, CD11c(+) TADCs, which have been shown to potentially differentiate into endothelial-like cells upon stimulation by tumour released growth and differentiation factors, were similarly reduced by clodrolip or antibody treatment. These results validate clodrolip therapy in combination with angiogenesis inhibitors as a promising novel strategy for an indirect cancer therapy aimed at the haematopoietic precursor cells that stimulate tumour growth and dissemination and as a tool to study the role of macrophages and dendritic cells in tumorigenesis.

Crossing cellular barriers using dendrimer nanotechnologies.
Najlah M, D'Emanuele A.
Curr Opin Pharmacol. 2006 Aug 2; [Epub ahead of print] .
[ expand abstract ]

Dendrimers represent a class of polymers characterised by their well-defined structure, with a high degree of molecular uniformity and low polydispersity. They have found a wide-range of pharmaceutical applications; however, more recently, they have been shown to function as effective intracellular carriers for drugs. In addition, dendrimers have been shown to be capable of bypassing efflux transporters. A new generation of dendrimer-based delivery systems will enable the efficient transport of drugs across cellular barriers.

Parameters influencing the stealthiness of colloidal drug delivery systems.
Vonarbourg A, Passirani C, Saulnier P, Benoit JP.
Biomaterials. 2006 Aug;27(24):4356-73.
[ expand abstract ]

Over the last few decades, colloidal drug delivery systems (CDDS) such as nano-structures have been developed in order to improve the efficiency and the specificity of drug action. Their small size permits them to be injected intravenously in order to reach target tissues. However, it is known that they can be rapidly removed from blood circulation by the immune system. CDDS are removed via the complement system and via the cells of the mononuclear phagocyte system (MPS), after their recognition by opsonins and/or receptors present at the cell surface. This recognition is dependent on the physicochemical characteristics of the CDDS. In this study, we will focus on parameters influencing the interactions of opsonins and the macrophage plasma membrane with the surface of CDDS, whereby parameters of the polymer coating become necessary to provide good protection.

Intracellular photodynamic therapy with photosensitizer-nanoparticle conjugates: cancer therapy using a 'Trojan horse'.
Wieder ME, Hone DC, Cook MJ, Handsley MM, Gavrilovic J, Russell DA.
Photochem Photobiol Sci. 2006 Aug;5(8):727-34.
[ expand abstract ]

Phthalocyanine-nanoparticle conjugates have been designed and synthesised for the delivery of hydrophobic photosensitizers for photodynamic therapy (PDT) of cancer. The phthalocyanine photosensitizer stabilized gold nanoparticles have an average diameter of 2-4 nm. The synthetic strategy interdigitates a phase transfer reagent between phthalocyanine molecules on the particle surface that solubilises the hydrophobic photosensitizer in polar solvents enabling delivery of the nanoparticle conjugates to cells. The phthalocyanine is present in the monomeric form on the nanoparticle surface, absorbs radiation maximally at 695 nm and catalytically produces the cytotoxic species singlet oxygen with high efficiency. These properties suggest that the phthalocyanine-nanoparticle conjugates are ideally suited for PDT. In a process that can be considered as cancer therapy using a 'Trojan horse', when the nanoparticle conjugates are incubated with HeLa cells (a cervical cancer cell line), they are taken up thus delivering the phthalocyanine photosensitizer directly into the cell interior. Irradiation of the nanoparticle conjugates within the HeLa cells induced substantial cell mortality through the photodynamic production of singlet oxygen. The PDT efficiency of the nanoparticle conjugates, determined using colorimetric assay, was twice that obtained using the free phthalocyanine derivative. Following PDT with the nanoparticle conjugates, morphological changes to the HeLa cellular structure were indicative of cell mortality via apoptosis. Further evidence of apoptosis was provided through the bioluminescent assay detection of caspase 3/7. Our results suggest that gold nanoparticle conjugates are an excellent vehicle for the delivery of surface bound hydrophobic photosensitizers for efficacious photodynamic therapy of cultured tumour cells.

Emerging use of nanoparticles in diagnosis and treatment of breast cancer.
Yezhelyev MV, Gao X, Xing Y, Al-Hajj A, Nie S, O'Regan RM.
Lancet Oncol. 2006 Aug;7(8):657-67.
[ expand abstract ]

The biological application of nanoparticles is a rapidly developing area of nanotechnology that raises new possibilities in the diagnosis and treatment of human cancers. In cancer diagnostics, fluorescent nanoparticles can be used for multiplex simultaneous profiling of tumour biomarkers and for detection of multiple genes and matrix RNA with fluorescent in-situ hybridisation. In breast cancer, three crucial biomarkers can be detected and accurately quantified in single tumour sections by use of nanoparticles conjugated to antibodies. In the near future, the use of conjugated nanoparticles will allow at least ten cancer-related proteins to be detected on tiny tumour sections, providing a new method of analysing the proteome of an individual tumour. Supermagnetic nanoparticles have exciting possibilities as contrast agents for cancer detection in vivo, and for monitoring the response to treatment. Several chemotherapy agents are available as nanoparticle formulations, and have at least equivalent efficacy and fewer toxic effects compared with conventional formulations. Ultimately, the use of nanoparticles will allow simultaneous tumour targeting and drug delivery in a unique manner. In this review, we give an overview of the use of clinically applicable nanoparticles in oncology, with particular focus on the diagnosis and treatment of breast cancer.

Fullerene (C60) immunoconjugates: interaction of water-soluble C60 derivatives with the murine anti-gp240 melanoma antibody.
Ashcroft JM, Tsyboulski DA, Hartman KB, Zakharian TY, Marks JW, Weisman RB, Rosenblum MG, Wilson LJ.
Chem Commun (Camb). 2006 Jul 28;(28):3004-6.
[ expand abstract ]

The first fullerene (C60) immunoconjugates have been prepared and characterized as an initial step toward the development of fullerene immunotherapy (FIT).

Method of laser activated nano-thermolysis for elimination of tumor cells.
Lapotko D, Lukianova E, Potapnev M, Aleinikova O, Oraevsky A.
Cancer Lett. 2006 Jul 28;239(1):36-45.
[ expand abstract ]

We describe novel ex vivo method for elimination of tumor cells from cell suspension, Laser Activated Nanothermolysis and propose this method for purging of bone marrow and blood transplants. K562 and human lympholeukemia cells were eliminated in experiments by laser-induced micro-bubbles that emerge inside individual target cells around selectively formed clusters of light-absorbing gold nanoparticles. Pretreatment of tumor cells with specific monoclonal antibodies and Ig-conjugated 30-nm gold particles allowed the formation of clusters of 10-20 on the surface of cell membrane. Electron microscopy found the nanoparticulate clusters inside the cells. Total (100%) elimination of K562 cells targeted with specific antibodies was achieved with single laser pulses with optical fluence of 5J/cm(2) at the wavelength of 532 nm without damage to the same cells targeted without specific antibodies. Total elimination of human lymphoblasts from suspension of normal stem cells was achieved by a single laser pulse with the optical fluence of 1.7J/cm(2), while the damage level of normal cells was 16%.

Synthesis of branched antisense oligonucleotides having multiple specificities Treatment of hormone insensitive prostate cancer.
Rubenstein M, Anderson KM, Tsui P, Guinan P.
Med Hypotheses. 2006 Jul 24; [Epub ahead of print] .
[ expand abstract ]

Antisense oligonucleotides (oligos) directed against transforming growth factor-alpha (TGF-alpha) and its binding site, the epidermal growth factor receptor (EGFR), have demonstrated in vitro and in vivo efficacy against both the PC-3 and LNCaP prostate tumor models. In an attempt to increase the efficiency of these oligos a new type of antisense compound called a bispecific oligo has been evaluated in vitro both alone and in combination with traditional chemotherapeutic agents. These bispecifics, which were first proposed in this journal in 2004, include binding sites for both TGF-alpha and EGFR along the same stretch of complementary DNA. Such bispecifics are able to deliver essentially two antisense activities in an equal molar ratio and can be directed against mRNA encoding proteins of different biochemical pathways. The first bispecifics were developed against two proteins regulating a single autocrine loop. Subsequent bispecifics have been developed which target both EGFR and the apoptosis regulating protein bcl-2. Bispecific activity of a single linear sequence oligo has already been shown to have efficacy. To further develop this multispecific approach, we now propose a branched antisense compound, again, having multiple binding site activities (to complementary sequenced mRNA). Active oligos would be attached to a fat soluble backbone which might enhance targeting and also intracellular entry, release and activity. Such a structure would also permit the customization of these branched forms to include oligos targeting specific proteins related to the growth of various tumor types. Problems associated with the development of antisense oligos have included both membrane solubility and specific targeting. By designing this branched form of antisense structure, multiple activities can be retained (added), solubility improved and delivery enhanced. Such a new formulation would include several antisense oligos covalently bound to and branching off from a lipid-like backbone. An elongated hydrocarbon chain would increase fat solubility and would permit oligo incorporation into nanoparticles or liposome derived delivery vehicles. Specific delivery of oligos could also be enhanced by the tendency of these nanoparticle or liposomal microbubbles to be disrupted under the influence of ultrasonic waves beamed at the targeted tissue.

Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs: part 3. Therapeutic efficacy and safety studies in ovarian cancer xenograft model.
Devalapally H, Shenoy D, Little S, Langer R, Amiji M.
Cancer Chemother Pharmacol. 2006 Jul 22; [Epub ahead of print].
[ expand abstract ]

PURPOSE: The objective of this study was to evaluate the anti-tumor efficacy and lack of systemic toxicity of paclitaxel when administered in pH-sensitive poly(ethylene oxide) (PEO)-modified poly(beta-amino ester) (PbAE) nanoparticles in mice bearing human ovarian adenocarcinoma (SKOV-3) xenograft. METHODS: Paclitaxel-encapsulated PEO-modified PbAE (PEO-PbAE) nanoparticles were prepared by the solvent displacement method. PEO-modified poly(epsilon-caprolactone) (PCL) (PEO-PCL) nanoparticles were used as a non pH-responsive control formulation. Efficacy studies were conducted in SKOV-3 tumor-bearing athymic (Nu/Nu) mice at an equivalent paclitaxel dose of 20 mg/kg with the control and nanoparticle formulations. Safety of the drug when administered in the control and nanoparticle formulation was determined from blood cell counts and changes in body weight of the animals. RESULTS: The formulated paclitaxel-containing PEO-PbAE and PEO-PCL nanoparticles had a particle size in the range of 100-200 nm and a surface charge of + 39.0 and - 30.8 mV, respectively. After intravenous administration of paclitaxel in these formulations, the tumor growth was inhibited significantly. Both of the formulated nanoparticles tested have shown improved therapeutic efficacy as compared to the paclitaxel aqueous solution. Additionally, significantly lower toxicity profile of paclitaxel was observed with PEO-modified nanoparticles as compared to the aqueous solution formulation CONCLUSION: PEO-modified PbAE nanoparticles are a unique pH-sensitive drug delivery system that elicits enhanced efficacy and safety profile in solid tumor therapy.

Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach.
Zeisberger SM, Odermatt B, Marty C, Zehnder-Fjallman AH, Ballmer-Hofer K, Schwendener RA.
Br J Cancer. 2006 Jul 11; [Epub ahead of print] .
[ expand abstract ]

Tumour-associated macrophages, TAMs, play a pivotal role in tumour growth and metastasis by promoting tumour angiogenesis. Treatment with clodronate encapsulated in liposomes (clodrolip) efficiently depleted these phagocytic cells in the murine F9 teratocarcinoma and human A673 rhabdomyosarcoma mouse tumour models resulting in significant inhibition of tumour growth ranging from 75 to >92%, depending on therapy and schedule. Tumour inhibition was accompanied by a drastic reduction in blood vessel density in the tumour tissue. Vascular endothelial growth factor (VEGF) is one of the major inducers of tumour angiogenesis and is also required for macrophage recruitment. The strongest effects were observed with the combination therapy of clodrolip and a VEGF-neutralising antibody, whereas free clodronate was not significantly active. Immunohistologic evaluation of the tumours showed significant depletion of F4/80(+) and MOMA-1(+) and a less pronounced depletion of CD11b(+) TAMs. Blood vessel staining (CD31) and quantification of the vessels as well as TAMs and tumour-associated dendritic cells (TADCs) in the A673 model showed reduction rates of 85 to >94%, even 9 days after the end of therapy. In addition, CD11c(+) TADCs, which have been shown to potentially differentiate into endothelial-like cells upon stimulation by tumour released growth and differentiation factors, were similarly reduced by clodrolip or antibody treatment. These results validate clodrolip therapy in combination with angiogenesis inhibitors as a promising novel strategy for an indirect cancer therapy aimed at the haematopoietic precursor cells that stimulate tumour growth and dissemination and as a tool to study the role of macrophages and dendritic cells in tumorigenesis.

RNA Interference in Vitro and in Vivo Using a Novel Chitosan/siRNA Nanoparticle System.
Howard KA, Rahbek UL, Liu X, Damgaard CK, Glud SZ, Andersen MO, Hovgaard MB, Schmitz A, Nyengaard JR, Besenbacher F, Kjems J.
Mol Ther. 2006 Jul 7; [Epub ahead of print] .
[ expand abstract ]

This work introduces a novel chitosan-based siRNA nanoparticle delivery system for RNA interference in vitro and in vivo. The formation of interpolyelectrolyte complexes between siRNA duplexes (21-mers) and chitosan polymer into nanoparticles, ranging from 40 to 600 nm, was shown using atomic force microscopy and photon correlation spectroscopy. Rapid uptake (1 h) of Cy5-labeled nanoparticles into NIH 3T3 cells, followed by accumulation over a 24-h period, was visualized using fluorescence microscopy. Nanoparticle-mediated knockdown of endogenous enhanced green fluorescent protein (EGFP) was demonstrated in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively). In addition, Western analysis showed approximately 90% reduced expression of BCR/ABL-1 leukemia fusion protein while BCR expression was unaffected in K562 (Ph(+)) cells after transfection using nanoparticles containing siRNA specific to the BCR/ABL-1 junction sequence. Effective in vivo RNA interference was achieved in bronchiole epithelial cells of transgenic EGFP mice after nasal administration of chitosan/siRNA formulations (37% and 43% reduction compared to mismatch and untreated control, respectively). These findings highlight the potential application of this novel chitosan-based system in RNA-mediated therapy of systemic and mucosal disease.

pH-sensing nano-crystals of carbonate apatite: Effects on intracellular delivery and release of DNA for efficient expression into mammalian cells.
Chowdhury EH, Maruyama A, Kano A, Nagaoka M, Kotaka M, Hirose S, Kunou M, Akaike T.
Gene. 2006 Jul 5;376(1):87-94; Epub 2006 Apr 5.
[ expand abstract ]

Two unique and fascinating properties of carbonate apatite which are well-known in hard tissue engineering, have been unveiled, for the first time, for the development of the simplest, but most efficient non-viral gene delivery device - ability of preventing the growth of crystals needed for high frequency DNA transfer across a plasma membrane and a fast dissolution rate for effective release of DNA during endosomal acidification, leading to a remarkably high transgene expression (5 to 100-fold) in mammalian cells compared to the widely used transfecting agents. Moreover, by modulating the crystal dissolution rate of carbonate apatite through incorporation of fluoride or strontium into it, transfection activity could be dramatically controlled, thus shedding light on a new barrier in the non-viral route, which was overlooked so far. Thus we have developed an innovative technology with significant insights, that would come as a promising tool for both basic research laboratories and clinical settings.

Phase I trial of Doxorubicin-containing low temperature sensitive liposomes in spontaneous canine tumors.
Hauck ML, Larue SM, Petros WP, Poulson JM, Yu D, Spasojevic I, Pruitt AF, Klein A, Case B, Thrall DE, Needham D, Dewhirst MW.
Clin Cancer Res. 2006 Jul 1;12(13):4004-10.
[ expand abstract ]

PURPOSE: To determine the maximum tolerated dose, dose-limiting toxicities, and pharmacokinetic characteristics of doxorubicin encapsulated in a low temperature sensitive liposome (LTSL) when given concurrently with local hyperthermia to canine solid tumors. EXPERIMENTAL DESIGN: Privately owned dogs with solid tumors (carcinomas or sarcomas) were treated. The tumors did not involve bone and were located at sites amenable to local hyperthermia. LTSL-doxorubicin was given (0.7-1.0 mg/kg i.v.) over 30 minutes during local tumor hyperthermia in a standard phase I dose escalation study. Three treatments, given 3 weeks apart, were scheduled. Toxicity was monitored for an additional month. Pharmacokinetics were evaluated during the first treatment cycle. RESULTS: Twenty-one patients were enrolled: 18 with sarcomas and 3 with carcinomas. Grade 4 neutropenia and acute death secondary to liver failure, possibly drug related, were the dose-limiting toxicities. The maximum tolerated dose was 0.93 mg/kg. Other toxicities, with the possible exception of renal damage, were consistent with those observed following free doxorubicin administration. Of the 20 dogs that received >/=2 doses of LTSL-doxorubicin, 12 had stable disease, and 6 had a partial response to treatment. Pharmacokinetic variables were more similar to those of free doxorubicin than the marketed liposomal product. Tumor drug concentrations at a dose of 1.0 mg/kg averaged 9.12 +/- 6.17 ng/mg tissue. CONCLUSION: LTSL-doxorubicin offers a novel approach to improving drug delivery to solid tumors. It was well tolerated and resulted in favorable response profiles in these patients. Additional evaluation in human patients is warranted.

Shell cross-linked stearic acid grafted chitosan oligosaccharide self-aggregated micelles for controlled release of paclitaxel.
Hu FQ, Ren GF, Yuan H, Du YZ, Zeng S.
Colloids Surf B Biointerfaces. 2006 Jul 1;50(2):97-103; Epub 2006 May 5.
[ expand abstract ]

Stearic acid grafted chitosan oligosaccharide (CSO-SA) with different degree of amino substitution (SD) was synthesized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction. The critical micelle concentration (CMC) of CSO-SA with different SD was about 0.06, 0.04, 0.01mg/ml, respectively. With the increase of micelle concentration, the micelle size decreased, and the zeta potential increased. On the other hand, with the increase of SD of CSO-SA, the micelle size and zeta potential decreased due to the increased hydrophobic interaction of SA and the reduced free amino groups. To increase the stability of the micelle in vivo and controll drug release, the shells of micelles were cross-linked by glutaraldehyde. By controlling the molar ratio of CSO-SA to glutaraldehyde, the cross-linking of intra-micelle could be reached, and the nanoparticle with smaller size than that of its initial micelle was obtained. Paclitaxel was then used as model drug to incorporate into the micelles, and the surfaces of the micelles were further cross-linked by glutaraldehyde to form drug loaded and shell cross-linked nanoparticles. The effects of drug loading, SD of CSO-SA and cross-link degree on the size, zeta potential, drug entrapment efficiency and in vitro drug release behavior of micelles and its cross-linked nanoparticles were investigated. The higher drug entrapment efficiencies (above 94%) were observed in all case. The charged amounts of drug did not affect the drug release behavior. The drug release rate decreased with the increase of SD of CSO-SA and cross-link degree.

Antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models.
Kirpotin DB, Drummond DC, Shao Y, Shalaby MR, Hong K, Nielsen UB, Marks JD, Benz CC, Park JW.
Cancer Res. 2006 Jul 1;66(13):6732-40.
[ expand abstract ]

We describe evidence for a novel mechanism of monoclonal antibody (MAb)-directed nanoparticle (immunoliposome) targeting to solid tumors in vivo. Long-circulating immunoliposomes targeted to HER2 (ErbB2, Neu) were prepared by the conjugation of anti-HER2 MAb fragments (Fab' or single chain Fv) to liposome-grafted polyethylene glycol chains. MAb fragment conjugation did not affect the biodistribution or long-circulating properties of i.v.-administered liposomes. However, antibody-directed targeting also did not increase the tumor localization of immunoliposomes, as both targeted and nontargeted liposomes achieved similarly high levels (7-8% injected dose/g tumor tissue) of tumor tissue accumulation in HER2-overexpressing breast cancer xenografts (BT-474). Studies using colloidal gold-labeled liposomes showed the accumulation of anti-HER2 immunoliposomes within cancer cells, whereas matched nontargeted liposomes were located predominantly in extracellular stroma or within macrophages. A similar pattern of stromal accumulation without cancer cell internalization was observed for anti-HER2 immunoliposomes in non-HER2-overexpressing breast cancer xenografts (MCF-7). Flow cytometry of disaggregated tumors posttreatment with either liposomes or immunoliposomes showed up to 6-fold greater intracellular uptake in cancer cells due to targeting. Thus, in contrast to nontargeted liposomes, anti-HER2 immunoliposomes achieved intracellular drug delivery via MAb-mediated endocytosis, and this, rather than increased uptake in tumor tissue, was correlated with superior antitumor activity. Immunoliposomes capable of selective internalization in cancer cells in vivo may provide new opportunities for drug delivery.

Drug delivery system based on covalently bonded poly[N-isopropylacrylamide-co-2-hydroxyethylacrylate]-based nanoparticle networks.
Mathews AS, Ha CS, Cho WJ, Kim I.
Drug Deliv. 2006 Jul-Aug;13(4):245-51.
[ expand abstract ]

The present work is focused on investigating the controlled drug release behavior of Poly [N-isopropylacrylamide-co-2-Hydroxyethylacrylate] (P [NIPAm-HEAc])-based hydrogel. The synthesis process includes the preparation of monodispersed hydrogel nanoparticles containing specific functional groups, followed by cross-linking to neighboring spheres to stabilize the entire network. The color and volume of these crystalline hydrogel networks can reversibly change in response to external stimuli such as temperature, pH, and other environmental conditions. The feasibility of the hydrogel as a controlled release vehicle for 5-fluorouracil is evaluated.

Evaluation of firefly luciferase bioluminescence mediated photodynamic toxicity in cancer cells.
Schipper ML, Patel MR, Gambhir SS.
Mol Imaging Biol. 2006 Jul-Aug;8(4):218-25.
[ expand abstract ]

PURPOSE: This work investigated whether fLuc-catalyzed oxidation of D: -luciferin generates sufficient light to induce photodynamic toxicity in cancer cells. PROCEDURES: Light emission was assessed via cooled CCD (charge-coupled device) camera. Parental and fLuc expressing cancer cells were exposed to subtoxic concentrations of photosensitizers (Rose Bengal or hypericin) and D: -luciferin, sunlight, or lamplight. Toxicity was assessed by MTT assay. RESULTS: fLuc expressing cells emitted up to 500-fold higher levels of photons than parental cell lines. Although exposure to photosensitizer and sunlight reduced survival of various cell lines, survival of fLuc expressing cells incubated with photosensitizer and D: -luciferin, or photosensitizer and lamplight, did not differ significantly from parental or untreated cells. CONCLUSIONS: Contesting recent reports, fLuc bioluminescence does not generate sufficient photons to induce Rose Bengal or hypericin photodynamic toxicity in a range of malignant and nonmalignant cell lines, and is not suitable as a generalizable approach to antineoplastic therapy.

Effects of siRNAs in combination with Gleevec on K-562 cell proliferation and Bcr-Abl expression.
Baker BE, Kestler DP, Ichiki AT.
J Biomed Sci. 2006 Jul;13(4):499-507; Epub 2006 Mar 18.
[ expand abstract ]

RNA interference (RNAi) is the repression of gene expression through a cellular mechanism of transcript-specific mRNA degradation. RNAi has been observed in human cells and applied to the modulation of a variety of human transcripts. Our goals were to deliver small interfering RNA (siRNA) using a liposome-based method, and to show Bcr-Abl siRNA specificity against K-562 cells, alone or in combination with Gleevec. Both synthetic (syn) siRNA, consisting of homogeneous 21-nucleotide-long RNA duplexes specific for the Bcr-Abl fusion site, and recombinant (r)-generated Bcr-Abl siRNA were employed. siRNA was transfected into K-562 cells with greater than 90% efficiency using RNAiFecttrade mark, as judged by fluorescence analysis. The Bcr-Abl transcript was inhibited using either siRNA preparation as measured by RT-PCR or real-time PCR. The IC(50) of Gleevec in the K-562 subline F(1) was lowered over 3-fold from 0.2 to 0.06 muM in cells transfected with either syn or rBcr-Abl siRNA. No effect was observed in cells after transfection with an irrelevant control siRNA. Therefore, K-562 cells transfected with RNAifect deliver Bcr-Abl siRNA efficiently and the Bcr-Abl siRNA decreased the IC(50) of Gleevec required to inhibit the high levels of Bcr-Abl protein found in K-562 cells.

Nanotechnology: intelligent design to treat complex disease.
Couvreur P, Vauthier C.
Pharm Res. 2006 Jul;23(7):1417-50 ; Epub 2006 Jun 21.
[ expand abstract ]

The purpose of this expert review is to discuss the impact of nanotechnology in the treatment of the major health threats including cancer, infections, metabolic diseases, autoimmune diseases, and inflammations. Indeed, during the past 30 years, the explosive growth of nanotechnology has burst into challenging innovations in pharmacology, the main input being the ability to perform temporal and spatial site-specific delivery. This has led to some marketed compounds through the last decade. Although the introduction of nanotechnology obviously permitted to step over numerous milestones toward the development of the "magic bullet" proposed a century ago by the immunologist Paul Ehrlich, there are, however, unresolved delivery problems to be still addressed. These scientific and technological locks are discussed along this review together with an analysis of the current situation concerning the industrial development.

Plasmid size up to 20 kbp does not limit effective in vivo lung gene transfer using compacted DNA nanoparticles.
Fink TL, Klepcyk PJ, Oette SM, Gedeon CR, Hyatt SL, Kowalczyk TH, Moen RC, Cooper MJ.
Gene Ther. 2006 Jul;13(13):1048-51; Epub 2006 Mar 9.
[ expand abstract ]

Nanoparticles consisting of single molecules of DNA condensed with polyethylene glycol-substituted lysine 30-mers efficiently transfect lung epithelium following intrapulmonary administration. Nanoparticles formulated with lysine polymers having different counterions at the time of DNA mixing have distinct geometric shapes: trifluoroacetate or acetate counterions produce ellipsoids or rods, respectively. Based on intracytoplasmic microinjection studies, nanoparticle ellipsoids having a minimum diameter less than the 25 nm nuclear membrane pore efficiently transfect non-dividing cells. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids, whereas the 8-11 nm diameter of rod-like particles is smaller than the nuclear pore diameter. In mice, up to 50% of lung cells are transfected after dosing with a rod-like compacted 6.9 kbp lacZ expression plasmid, and correction of the CFTR chloride channel was observed in humans following intranasal administration of a rod-like compacted 8.3 kbp plasmid. To further investigate the potential size and shape limitations of DNA nanoparticles for in vivo lung delivery, reporter gene activity of ellipsoidal and rod-like compacted luciferase plasmids ranging in size between 5.3 and 20.2 kbp was investigated. Equivalent molar reporter gene activities were observed for each formulation, indicating that microinjection size limitations do not apply to the in vivo gene transfer setting.Gene Therapy (2006) 13, 1048-1051. doi:10.1038/sj.gt.3302761; published online 9 March 2006.

Microfabricated nanochannel implantable drug delivery devices: trends, limitations and possibilities.
Gardner P.
Expert Opin Drug Deliv. 2006 Jul;3(4):479-87.
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This is a review of the application of microfabrication technologies, borrowed from the semiconductor industry, to drug delivery implants incorporating structures in the nanometer dimension. In the futuristic ideal, these systems would involve the implantation of precisely microfabricated drug delivery systems with nanopores, nanochannels and/or nanoreservoirs fabricated from silicon, coupled with electronic sensing and actuator systems, for the precise, timed and/or targeted delivery of drugs. After more than a decade in conceptualisation and experimentation, four systems that have commercial potential are discussed: i) implantable microchips with on-demand microdosage for one or more therapeutic agents under internal control or external control using a wireless link; ii) nanopore pumps, implantable titanium pumps, consisting of a drug reservoir with a nanopore-release membrane, capable of delivering potent small or macromolecules at constant serum levels for sustained periods of time; iii) nanocages, microfabricated nanopore immunoisolation chambers for cellular implants, capable of natural feedback-controlled delivery of proteins and peptides; and iv) nanobuckets, micromachined silicon porous particles with drug-loading capacity and targeting ligands for localised delivery. Each of the systems, along with future trends in microfabrication manufacturing, limitations and possibilities, are discussed.

A pilot study of the liposomal MUC1 vaccine BLP25 in prostate specific antigen failures after radical prostatectomy.
North SA, Graham K, Bodnar D, Venner P.
J Urol. 2006 Jul;176(1):91-5.
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PURPOSE: Men with biochemical failure after radical prostatectomy have few therapeutic options other than androgen deprivation therapy. Targeted therapies in this group are appropriate because the optimal timing of the initiation of hormonal therapy in this patient population is unknown. A single institution pilot trial was performed using BLP25 liposome vaccine in hormone naive patients with prostate specific antigen failure after radical prostatectomy to determine if prostate specific antigen progression could be halted. MATERIALS AND METHODS: Men with biochemical failure after radical prostatectomy were enrolled. Primary end points were efficacy and safety of the MUC1 BLP25 liposomal vaccine. Changes in prostate specific antigen doubling time were also evaluated. Patients received a single intravenous dose of cyclophosphamide, followed by vaccinations with BLP25 liposome vaccine for up to 1 year. Prostate specific antigen was measured at baseline and during treatment, and prostate specific antigen doubling time was calculated for these intervals. RESULTS: A total of 16 patients with a median age of 60 years were enrolled. All patients received cyclophosphamide and 15 of 16 completed the primary treatment period. Ten patients completed the maintenance period. After the 8-week primary treatment period 8 of 16 patients had stable or decreased prostate specific antigen. At the last on-study prostate specific antigen measurement 1 patient maintained stable prostate specific antigen but all others had progression. However, 6 of the 16 patients had greater than 50% prolongation of prostate specific antigen doubling time compared to pre-study prostate specific antigen doubling time. CONCLUSIONS: BLP25 liposome vaccine shows promise for prolonging prostate specific antigen doubling time in hormone naive men with biochemical failure after prostatectomy and little morbidity. This could potentially translate into the deferral of hormonal therapy. Further testing in this population of patients is warranted.

Convection-enhanced delivery of Ls-TPT enables an effective, continuous, low-dose chemotherapy against malignant glioma xenograft model.
Saito R, Krauze MT, Noble CO, Drummond DC, Kirpotin DB, Berger MS, Park JW, Bankiewicz KS.
Neuro-oncol. 2006 Jul;8(3):205-14; Epub 2006 May 24.
[ expand abstract ]

Treatment of malignant gliomas represents one of the most formidable challenges in oncology. The combination of surgery, radiation, and chemotherapy yields median survivals of less than one year. Here we demonstrate the use of a minimally invasive surgical technique, convection-enhanced delivery (CED), for local administration of a novel nanoparticle liposome containing topotecan. CED of this liposomal topotecan (Ls-TPT) resulted in extended brain tissue retention (t((1/2)) = 1.5 days), whereas free topotecan was rapidly cleared (t((1/2)) = 0.1 days) after CED. The favorable pharmacokinetic profile of extended topotecan release for about seven days, along with biodistribution featuring perivascular accumulation of the nanoparticles, provided, in addition to the known topoisomerase I inhibition, an effective antiangiogenic therapy. In the rat intracranial U87MG tumor model, vascular targeting of Ls-TPT with CED was associated with reductions in laminin expression and vascular density compared to free topotecan or control treatments. A single CED treatment on day 7 showed that free topotecan conferred no survival benefit versus control. However, Ls-TPT produced a significant (P = 0.0002) survival benefit, with six of seven complete cures. Larger U87MG tumors, where CED of Ls-TPT on day 12 resulted in one of six cures, indicated the necessity to cover the entire tumor with the infused therapeutic agent. CED of Ls-TPT was also efficacious in the intracranial U251MG tumor model (P = 0.0005 versus control). We conclude that the combination of a novel nanoparticle Ls-TPT and CED administration was very effective in treating experimental brain tumors.

A phase I study with oral SU5416 in patients with advanced solid tumors: a drug inducing its clearance.
Salzberg M, Pless M, Rochlitz C, Ambrus K, Scigalla P, Herrmann R.
Invest New Drugs. 2006 Jul;24(4):299-304.
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Vascular endothelial growth factor (VEGF) is a potent stimulant of angiogenesis. SU5416, is a small molecule tyrosine kinase inhibitor, and a potent inhibitor of VEGF-mediated Flk-1 receptor signaling. Intravenous agent SU5416 has shown evidence of biological activity against a variety of tumor types. The current intravenous dosing regimen is not optimal for long-term administration, which is needed for optimal efficacy. The aim of this study was to evaluate the safety profile and pharmacokinetics of a Nanocrystal Colloidal Dispersion (NCD) SU5416 formulation in humans. Patients with advanced and/or metastatic solid organ tumors were included in the trial; various SU5416 regimens were tested for tolerability, safety and were evaluated concerning pharmacokinetics. The results of this study indicate that induction of clearance after oral dosing of NCD SU5416 in humans occurs and is greater than following i.v. administration. It has been confirmed that SU5416 is a high clearance compound, also as an oral NCD formulation. The NCD formulation was well tolerated, but no effective drug serum levels could be achieved. These data help to understand the ADME (Absorption, Distribution, Metabolism, Excretion) properties of indoline chemical class compounds. The lessons learned should be applied in the development of next generation indoline anti-angiogenic and anti-tumor compounds.

A new polymer-lipid hybrid nanoparticle system increases cytotoxicity of Doxorubicin against multidrug-resistant human breast cancer cells.
Wong HL, Rauth AM, Bendayan R, Manias JL, Ramaswamy M, Liu Z, Erhan SZ, Wu XY. Pharm Res. 2006 Jul;23(7):1574-85; Epub 2006 Jun 24.
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PURPOSE: This work is intended to develop and evaluate a new polymer-lipid hybrid nanoparticle system that can efficiently load and release water-soluble anticancer drug doxorubicin hydrochloride (Dox) and enhance Dox toxicity against multidrug-resistant (MDR) cancer cells. METHODS: Cationic Dox was complexed with a new soybean-oil-based anionic polymer and dispersed together with a lipid in water to form Dox-loaded solid lipid nanoparticles (Dox-SLNs). Drug loading and release properties were measured spectrophotometrically. The in vitro cytotoxicity of Dox-SLN and the excipients in an MDR human breast cancer cell line (MDA435/LCC6/MDR1) and its wild-type line were evaluated by trypan blue exclusion and clonogenic assays. Cellular uptake and retention of Dox were determined with a microplate fluorometer. RESULTS: Dox-SLNs were prepared with a drug encapsulation efficiency of 60-80% and a particle size range of 80-350 nm. About 50% of the loaded drug was released in the first few hours and an additional 10-20% in 2 weeks. Treatment of the MDR cells with Dox-SLN resulted in over 8-fold increase in cell kill when compared to Dox solution treatment at equivalent doses. The blank SLN and the excipients exhibited little cytotoxicity. The biological activity of the released Dox remained unchanged from fresh, free Dox. Cellular Dox uptake and retention by the MDR cells were both significantly enhanced (p < 0.05) when Dox was delivered in Dox-SLN form. CONCLUSIONS: The new polymer-lipid hybrid nanoparticle system is effective for delivery of Dox and enhances its efficacy against MDR breast cancer cells.

Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs: part 3. Therapeutic efficacy and safety studies in ovarian cancer xenograft model.
Devalapally H, Shenoy D, Little S, Langer R, Amiji M.
Cancer Chemother Pharmacol. 2006 Jul 22; [Epub ahead of print].
[ expand abstract ]

PURPOSE: The objective of this study was to evaluate the anti-tumor efficacy and lack of systemic toxicity of paclitaxel when administered in pH-sensitive poly(ethylene oxide) (PEO)-modified poly(beta-amino ester) (PbAE) nanoparticles in mice bearing human ovarian adenocarcinoma (SKOV-3) xenograft. METHODS: Paclitaxel-encapsulated PEO-modified PbAE (PEO-PbAE) nanoparticles were prepared by the solvent displacement method. PEO-modified poly(epsilon-caprolactone) (PCL) (PEO-PCL) nanoparticles were used as a non pH-responsive control formulation. Efficacy studies were conducted in SKOV-3 tumor-bearing athymic (Nu/Nu) mice at an equivalent paclitaxel dose of 20 mg/kg with the control and nanoparticle formulations. Safety of the drug when administered in the control and nanoparticle formulation was determined from blood cell counts and changes in body weight of the animals. RESULTS: The formulated paclitaxel-containing PEO-PbAE and PEO-PCL nanoparticles had a particle size in the range of 100-200 nm and a surface charge of + 39.0 and - 30.8 mV, respectively. After intravenous administration of paclitaxel in these formulations, the tumor growth was inhibited significantly. Both of the formulated nanoparticles tested have shown improved therapeutic efficacy as compared to the paclitaxel aqueous solution. Additionally, significantly lower toxicity profile of paclitaxel was observed with PEO-modified nanoparticles as compared to the aqueous solution formulation CONCLUSION: PEO-modified PbAE nanoparticles are a unique pH-sensitive drug delivery system that elicits enhanced efficacy and safety profile in solid tumor therapy.

The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene glycol succinate nanoparticles.
Zhang Z, Feng SS.
Biomaterials. 2006 Jul;27(21):4025-33; Epub 2006 Mar 27.
[ expand abstract ]

Paclitaxel is one of the most effective antineoplastic drugs. Its current clinical administration is formulated in Cremophor EL, which causes serious side effects. Nanoparticle (NP) technology may provide a solution for such poisonous adjuvant problems and promote a sustained chemotherapy, in which biodegradable polymers play a key role. Our group has successfully synthesized novel poly(lactide)-tocopheryl polyethylene glycol succinate (TPGS) (PLA-TPGS) copolymers of desired hydrophobic-hydrophilic balance for NP formulation of anticancer drugs. The present work is focused on effects of the PLA:TPGS composition ratio on drug encapsulation efficiency, in vitro drug release, in vitro cellular uptake and viability of the PLA-TPGS NP formulation of paclitaxel. The PLA-TPGS copolymers of various PLA:TPGS ratios were synthesized by the ring-opening polymerization method and characterized by GPC and (1)H NMR for their molecular structure. Paclitaxel-loaded PLA-TPGS NPs were prepared by a modified solvent extraction/evaporation method and characterized by laser light scattering for size and size distribution, scanning electron microscopy for surface morphology and zeta potential for surface charge. High performance liquid chromatography was used to measure the drug encapsulation efficiency and in vitro drug release profile. Cancer cell lines HT-29 and Caco-2 were used to image and measure the cellular uptake of fluorescent PLA-TPGS NPs. Cancer cell viability of the drug-loaded PLA-TPGS was measured by MTT assay. It was found that the PLA:TPGS composition ratio has little effects on the particle size and size distribution. However, the PLA-TPGS NPs of 89:11 PLA:TPGS ratio achieved the best effects on the drug encapsulation efficiency, the cellular uptake and the cancer cell mortality of the drug-loaded PLA-TPGS NPs. This research was also carried out in close comparison with the drug-loaded PLGA NPs.

Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery.
Nishiyama N, Kataoka K.
Pharmacol Ther. 2006 Jun 29; [Epub ahead of print] .
[ expand abstract ]

Polymeric micelles, self-assemblies of block copolymers, are promising nanocarrier systems for drug and gene delivery. Until now, several micellar formulations of antitumor drugs have been intensively studied in preclinical and clinical trials, and their utility has been demonstrated. Even compared with long-circulating liposomes, polymeric micelles might have several advantages, such as controlled drug release, tissue-penetrating ability and reduced toxicity such as hand-foot syndrome and hypersensitivity reaction. Importantly, critical features of the polymeric micelles as drug carriers, including particle size, stability, and loading capacity and release kinetics of drugs, can be modulated by the structures and physicochemical properties of the constituent block copolymers. Also, nano-engineering of block copolymers might allow the preparation of polymeric micelles with integrated smart functions, such as specific-tissue targetability, as well as chemical or physical stimuli-sensitivity. Thus, polymeric micelles are nanotechnology-based carrier systems that might exert the activity of potent bioactive compounds in a site-directed manner, ensuring their effectiveness and safety in the clinical use.

Poly(ethylene glycol)/poly(epsilon-caprolactone) diblock copolymeric nanoparticles for non-viral gene delivery: The role of charge group and molecular weight in particle formation, cytotoxicity and transfection.
Jeong Soon Jang, So Yeon Kim, Sang Bong Lee, Kyung Ok Kim, Joong Soo Han, Young Moo Lee.
J Control Release. 2006 Jun 28;113(2):173-82; Epub 2006 Apr 26.
[ expand abstract ]

Two types of nanoparticles containing pGL3-Control (plasmid DNA) were prepared using nonionic amphiphlic block copolymers and ionic amphiphilic block copolymers containing a terminal cationic group to investigate the effect of charge on the vehicle properties for systemic gene delivery. Methoxy poly(ethylene glycol) (MPEG)/poly(epsilon-caprolactone) (PCL) diblock copolymers were synthesized by the ring-opening polymerizatrion of epsilon-caprolactone in the presence of a catalyst-free MPEG homopolymer. The hydroxy groups of MPEG/PCL block copolymer were then modified into an amine group to synthesize an amine-terminated MPEG/PCL diblock copolymer (AMPEG/PCL). DNA was incorporated into the polymeric nanoparticles by physical entrapment and electrostatic interaction. All nanoparticle samples exhibited spherical structures and although their sizes increased slightly after DNA-loading, they remained less than 160 nm. The AMPEG/PCL nanoparticles exhibited smaller particle sizes than the MPEG/PCL nanoparticles of the same molecular weight after DNA-loading. The optimum mixing ratio of MPEG/PCL and AMPEG/PCL copolymers to DNA ranged from 4:1 to 1:2 depending on the molecular weight of the block copolymer, the composition of MPEG and PCL and terminal amine group. Based on in vitro cytotoxicity tests, the DNA-loaded MPEG/PCL and AMPEG/PCL nanoparticles did not induce any remarkable cytotoxicity against normal human fibroblasts. Transfection efficiencies of DNA-loaded nanoparticles were improved about 3.4 approximately 12.9 times under serum conditions.

Trehalose click polymers inhibit nanoparticle aggregation and promote pDNA delivery in serum.
Srinivasachari S, Liu Y, Zhang G, Prevette L, Reineke TM.
J Am Chem Soc. 2006 Jun 28;128(25):8176-84.
[ expand abstract ]

Herein, three new glycopolymers have been synthesized via "click polymerization" to promote nucleic acid delivery in the presence of biological media containing serum. These structures were designed to contain a trehalose moiety to promote biocompatibility, water solubility, and stability against aggregation, amide-triazole groups to enhance DNA binding affinity, and an oligoamine unit to facilitate DNA encapsulation, phosphate neutralization, and interactions with cell surfaces. A 2,3,4,2',3',4'-hexa-O-acetyl-6,6'-diazido-6,6'-dideoxy-D-trehalose (4) monomer was polymerized via copper(I)-catalyzed azide-alkyne cycloaddition with a series of dialkyne-amide comonomers that contain either one, two, or three Boc-protected secondary amines (7a, 7b, or 7c, respectively). After deprotection, three water-soluble polycations (9a, 9b, or 9c) were obtained with similar degrees of polymerization (n = 56-61) to elucidate the role of amine number on nucleic acid binding, complex formation, stability, and cellular delivery. Gel electrophoresis and ethidium bromide experiments showed that 9a-9c associated with plasmid DNA (pDNA) and formed complexes (polyplexes) at N/P ratios dependent on the amine number. TEM experiments revealed that 9a-9c polyplexes were small (50-120 nm) and had morphologies (spherical and rodlike) associated with the polymer chain stiffness. Dynamic light scattering studies in the presence of media containing serum demonstrated that 9c polyplexes had a low degree of flocculation, whereas 9a and 9b polyplexesd aggregate rapidly. Further biological studies revealed that these structures were biocompatible and deliver pDNA into HeLa cells. Particularly, 9c polyplexes promoted high delivery efficacy and gene expression profiles in the presence of serum.

Adenoviral Gene Vector Tethering to Nanoparticle Surfaces Results in Receptor-Independent Cell Entry and Increased Transgene Expression.
Chorny M, Fishbein I, Alferiev IS, Nyanguile O, Gaster R, Levy RJ.
Mol Ther. 2006 Jun 23; [Epub ahead of print] .
[ expand abstract ]

The present studies investigated the hypothesis that affinity immobilization of replication-defective adenoviruses (Ad) on the surfaces of biodegradable nanoparticles (NP) can improve transduction through uncoupling cellular uptake from the coxsackie-adenovirus receptor (CAR). Ad was tethered to the surfaces of polylactide-based NP that were surface-activated using a photoreactive polyallylamine-benzophenone-pyridyldithiocarboxylate polymer, which enabled (via thiol chemistry) the covalent attachment of Ad-binding proteins, either the recombinant D1 domain of CAR or an adenoviral knob-specific monoclonal antibody. Gene transfer by NP-Ad complexes was studied in relation to cellular uptake as a function of cell type and the character of NP-Ad binding. NP-Ad complexes, but not Ad applied with or without control nonimmune IgG-modified NP, significantly increased green fluorescent protein reporter expression in endothelioma and endothelial and arterial smooth muscle cells (SMC) in direct correlation to the extent of NP-Ad internalization. CAR-independent uptake of NP-Ad was confirmed by demonstrating inhibition of free Ad- but not NP-Ad complex-mediated transduction by knob protein. Complexes formulated with an Ad encoding inducible nitric oxide synthase inhibited growth of cultured SMC to a significantly greater extent than those with (GFP)Ad or (NULL)Ad or free vector. It is concluded that Ad-specific affinity tethering to biodegradable NP can significantly increase the level of gene expression via a CAR-independent uptake mechanism.

Potential use of drug carried-liposomes for cancer therapy via direct intratumoral injection.
Bao A, Phillips WT, Goins B, Zheng X, Sabour S, Natarajan M, Ross Woolley F, Zavaleta C, Otto RA.
Int J Pharm. 2006 Jun 19;316(1-2):162-9; Epub 2006 Mar 31.
[ expand abstract ]

Liposomes have recognized advantages as nano-particle drug carriers for tumor therapy. In this study, the pharmacokinetics and distribution of intratumorally administered liposomes were investigated as drug carriers for treating solid tumors via direct intratumoral administration. 99mTc-liposomes were administered intratumorally to nude rats bearing human head and neck squamous cell carcinoma xenografts. Planar gamma camera images were analyzed to evaluate the local retention of the intratumorally administered liposomes. Co-registered pinhole micro-single photon emission computed tomography (SPECT)/computed tomography (CT) images were acquired of the whole animal as well as the dissected tumors to determine intratumoral distribution of the 99mTc-liposomes. For 99mTc-liposomes, there was an initial retention of 47.4 +/- 11.0% (n = 4) in tumors and surrounding tissues. At 20 h, 39.2 +/- 10.6% (n = 4) of 99mTc-activity still remained in the tumor. In contrast, only 18.7 +/- 3.3% (n = 3) of the intratumoral 99mTc-activity remained for unencapsulated 99mTc-complex at 20 h. Pinhole micro-SPECT images demonstrated that 99mTc-liposomes also have a superior intratumoral 99mTc-activity diffusion compared with unencapsulated 99mTc-complex. Higher intratumoral retention of 99mTc-liposomes accompanied by an improved intratumoral diffusion suggests that intratumorally administered liposomal drugs are potentially promising agents for solid tumor local therapy.

A folate receptor-targeted liposomal formulation for paclitaxel.
Wu J, Liu Q, Lee RJ.
Int J Pharm. 2006 Jun 19;316(1-2):148-53; Epub 2006 Mar 6.
[ expand abstract ]

A novel liposomal formulation of paclitaxel targeting the folate receptor (FR) was synthesized and characterized. This formulation was designed to overcome vehicle toxicity associated with the traditional Cremophor EL-based formulation and to provide the added advantages of prolonged systemic circulation time and selective targeting of the FR, which is frequently overexpressed on epithelial cancer cells. The formulation had the composition of dipalmitoyl phosphatidylcholine/dimyristoyl phosphatidylglycerol/monomethoxy-polyethylene glycol (PEG)2000-distearoyl phosphatidylethanolamine/folate-PEG3350-distearoyl phosphatidylethanolamine (DPPC/DMPG/mPEG-DSPE/folate-PEG-DSPE) at molar ratios of (85.5:9.5:4.5:0.5) and a drug-to-lipid molar ratio of 1:33. The liposomes were prepared by polycarbonate membrane extrusion. The mean particle size of the liposomes was 97.1 nm and remained stable for at least 72 h at 4 degrees C. FR-targeted liposomes of the same lipid composition entrapping calcein were shown to be efficiently taken up by KB oral carcinoma cells, which are highly FR+. FR-targeted liposomes containing paclitaxel showed 3.8-fold greater cytotoxicity compared to non-targeted control liposomes in KB cells. Plasma clearance profiles of paclitaxel in the liposomal formulations were then compared to paclitaxel in Cremophor EL formulation. The liposomal formulations showed much longer terminal half-lives (12.33 and 14.23 h for FR-targeted and non-targeted liposomes, respectively) than paclitaxel in Cremophor EL (1.78 h). In conclusion, the paclitaxel formulation described in this study has substantial stability and favorable pharmacokinetic properties. The FR-targeted paclitaxel formulation is potentially useful for treatment of FR+ tumors and warrants further investigation.

PAMAM dendrimers for efficient siRNA delivery and potent gene silencing.
Zhou J, Wu J, Hafdi N, Behr JP, Erbacher P, Peng L.
Chem Commun (Camb). 2006 Jun 14;(22):2362-4; Epub 2006 May 10.
[ expand abstract ]

Genuine, nondegraded PAMAM dendrimers self-assemble with siRNA into nanoscale particles that are efficient for siRNA delivery and induce potent endogenous gene silencing.

Anti-angiogenic effects of liposomal prednisolone phosphate on B16 melanoma in mice.
Banciu M, Schiffelers RM, Fens MH, Metselaar JM, Storm G.
J Control Release. 2006 Jun 12;113(1):1-8; Epub 2006 Apr 7.
[ expand abstract ]

Prednisolone phosphate (PLP) encapsulated in long-circulating liposomes can inhibit tumor growth after intravenous administration (i.v.). These antitumor effects of liposomal PLP are the result of the tumor-targeting property of the liposome formulation. The mechanism by which liposomal PLP inhibits tumor growth is unclear. We investigated the effects of liposome-encapsulated PLP versus free PLP on angiogenic protein production in tumor tissue in vivo and on viability and proliferation of tumor and endothelial cells in vitro. In vivo, liposomal PLP had a stronger reducing effect on pro-angiogenic protein levels than free PLP, whereas levels of anti-angiogenic proteins were hardly affected. Cell viability was only slightly affected with either treatment. Liposomal PLP had strong anti-proliferative effects on human umbilical vein endothelial cells, whereas free PLP had hardly any effect. Taken together, the present study points to a strong inhibitory effect of liposomal PLP on tumor angiogenesis by reduction of the intratumoral production of the majority of pro-angiogenic factors studied and direct inhibition of endothelial cell proliferation, which is the result of high prolonged levels of prednisolone in the tumor by liposomal delivery.

In vivo antitumor activity of chitosan nanoparticles.
Qi L, Xu Z.
Bioorg Med Chem Lett. 2006 Jun 5; [Epub ahead of print] .
[ expand abstract ]

Chitosan nanoparticles have been synthesized as potential anticancer agents, and evaluated, in vitro, against various cancer cell lines. In this study, in vivo antitumor activity of chitosan nanoparticles against Sarcoma-180 and mouse hepatoma H22 was investigated. Chitosan nanoparticles showed significant antitumor activity in vivo. The doses and particle size made a great effect on their efficacy.

Lipoplexes prepared from cationic liposomes and mammalian DNA induce CpG-independent, direct cytotoxic effects in cell cultures and in mice.
Khazanov E, Simberg D, Barenholz Y.
J Gene Med. 2006 Jun 2; [Epub ahead of print] .
[ expand abstract ]

BACKGROUND: Recent studies demonstrated the cytotoxic activity of bacterial DNA (pDNA) complexed with cationic lipids. This cytotoxicity is related to the ability of pDNA to induce potently the immune system, which is associated with release of inflamatory cytokines. Both activities seem to be related to the nonmethylated CpG sequences present in the pDNA. Here we study the cytotoxic activity of nonbacterial DNA complexed with cationic lipids against various tumor cell lines. METHODS: Various nucleic acids complexed with cationic liposomes were prepared and their cytotoxic activity was studied in cell cultures and in tumor-bearing mice. Cell uptake of lipoplexes was evaluated, and mechanism of DNA cytotoxic activity was studied. RESULTS: We found that nonbacterial (vertebrate) genomic DNA when complexed with cationic lipids is highly cytotoxic against C-26 and M-109 tumor cells. Cationic lipids alone were not toxic to these cells. The cytotoxic activity does not result from nonspecific acidification of the intracellular milieu, as substitution of DNA by poly-L-glutamate did not result in cytotoxicity, although the level of uptake of anionic charges per cell was similar to that of the nucleic acids, suggesting that this cytotoxic effect is specific to nucleic acids. By studying the nucleic acid fate using confocal microscopy, we found that cytotoxicity correlated with the release of DNA into the cytoplasm following uptake of lipoplexes. Injection of calf thymus DNA-based lipoplexes to mice with peritoneal C-26 metastases resulted in doubling of median survival time and long-term survival in 20% of the tumor-bearing mice. Judging by low levels of IFN-gamma, TNF-alpha and IL-6 in the treated mice, this effect cannot be ascribed to Th-1 inflammation, but rather to a direct cytotoxic effect on the tumor cells. CONCLUSIONS: The above data provide a new insight into the mechanisms of lipoplex-mediated antitumor effects in vitro and in vivo and new perspectives in cancer therapy.

Protein transduction by lipidic peptide dendrimers.
Bayele HK, Ramaswamy C, Wilderspin AF, Srai KS, Toth I, Florence AT.
J Pharm Sci. 2006 Jun;95(6):1227-37.
[ expand abstract ]

We investigated the potential of a new family of lipidic peptide dendrimers in protein transduction into cultured cells. Dendrimer-protein interaction was determined by gel retardation assays using purified recombinant protein. To assess intracellular protein delivery, two marker proteins were used: recombinant firefly luciferase and a Cy3-labeled monoclonal antibody to the c-myc proto-oncogene. Protein delivery was determined by luciferase assays and fluorescence microscopy, respectively. While there was minimal delivery of luciferase or antibody in the absence of the dendrimers, the latter increased protein delivery substantially. Luciferase delivery was concentration and cell type-dependent; the efficiency of delivery also varied with the number of terminal amino groups on the dendrimers. In previous reports, we showed that these dendrimers could be used for gene and drug delivery; the data we report herein suggest that they may also be capable of intracellular protein delivery. This finding has important implications for the use of these dendrimers in protein therapeutics and vaccinology. (c) 2006 Wiley-Liss, Inc. and the American Pharmacists Association.

Pegylated liposomal doxorubicin (Lipo-Dox(R)) for platinum-resistant or refractory epithelial ovarian carcinoma: A Taiwanese gynecologic oncology group study with long-term follow-up.
Chou HH, Wang KL, Chen CA, Wei LH, Lai CH, Hsieh CY, Yang YC, Twu NF, Chang TC, Yen MS.
Gynecol Oncol. 2006 Jun;101(3):423-8; Epub 2005 Dec 1.
[ expand abstract ]

OBJECTIVES.: To evaluate the efficacy and safety of a distearoylphosphatidylcholine pegylated liposomal doxorubicin, Lipo-Dox(R), in platinum-resistant or refractory epithelial ovarian cancer. METHODS.: A multicenter phase II trial enrolled women with platinum-resistant or refractory epithelial ovarian carcinoma and naive to anthracycline. Eligible patients had either measurable tumor(s) or elevated serum CA 125 titer. Lipodox was initiated with a dose of 45 mg/m(2) at a 4-week interval with subsequent escalation or reduction. A total of six cycles were scheduled. RESULTS.: 29 patients, 20 with platinum-resistant and 9 with platinum refractory tumors, were enrolled. Lipo-Dox was given for an average of 4.6 cycles per patient with a total of 134 cycles. Among the 26 evaluable patients, one achieved CR, 5 PR and 9 SD. The overall response rate was 23.1% (95% CI, 6.8%-39.3%) with a median response duration of 11.6 weeks. 5 of the 6 responses were in patients with resistant disease. The median progression-free duration in the SD patients was 25.7 weeks. With a median follow-up of 13.8 months, the median progression-free and median overall survivals in the 26 patients were 5.4 months and 13.8 months, respectively. Hand-foot skin reaction occurred in 4.5% and skin pigmentation in 11.2% of all treatment cycles, all were Grade 1/2. Nausea and vomiting occurred in 14.2%, while anemia, leukopenia and thrombocytopenia occurred in 20.9%, 32.8% and 9% of cycle, respectively, and were mostly Grade 1 or 2. CONCLUSION.: Lipo-Dox, the third liposome encapsulated doxorubicin, at 45 mg/m(2) every 4 weeks, is effective against recurrent, platinum-resistant epithelial ovarian cancers.

Nano- and micro-particulate formulations of poorly water-soluble drugs by using a novel optimized technique.
Douroumis D, Fahr A.
Eur J Pharm Biopharm. 2006 Jun;63(2):173-5; Epub 2006 Mar 6.
[ expand abstract ]

A novel technique for the production of nano- and micro-particulate formulations of poorly water-soluble drugs has been developed. This technique involves the use of static mixer elements to provide fast precipitation by continuous turbulent mixing of two liquid flows, an aqueous phase and an organic phase, respectively. The objective of this study was to develop the mixer technique by investigating the influence of the element number on the particle size of the resulting dispersions. Four model active pharmaceutical ingredients (APIs) with a variety of polymers, lipids or surfactants underwent intensive mixing and the final suspensions showed a narrow size distribution. Parameters such as the flow rate and the temperature of the precipitated organic-aqueous phases were also significant in the reduction of particle size. Further development of the mixing technique led to reproducible and stable formulations with minimal excipient amounts. These formulations were spray- or freeze-dried to improve stability.

Optimization of preparation of DHAQ-loaded PEG-PLGA-PEG nonaparticles using central composite design.
Duan Y, Xu S, Wang Q, Liu J, Zhang Z.
J Mater Sci Mater Med. 2006 Jun;17(6):559-63.
[ expand abstract ]

Mitoxantrone (DHAQ)-loaded poly (ethylene glycol)-poly (lactic acid-co-glycolic acid) -poly (ethyleneglycol) (PELGE) nanoparticles (NP) were fabricated using an emulsification/solvent evaporation technique. A central composite design (CCD) was applied to evaluate the joint influence of three formulation variables: the amounts of polymer, concentration of the DHAQ, and the ratio of the organic phase (inner-phase) and the aqueous phase (outer-phase). In this study, we optimize the preparation technology on the basis of the single factor evaluation. The optimal conditions for the preparation of DHAQ-loaded nanoparticle were found to be: the concentration of PELGE was 9 mg/mL, the concentration of inner-phase of DHAQ was 27.5 mg/L, and the ratio of inner-phase/outer-phase was 8.5/1. The results showed that CCD is an ideal technique for formulation studies. The entrapment efficiency ratio (ER) was 90% and particle sizes are less than 500 nm. The nanoparticles, as examined by transmission electron microscopy (TEM), have a smooth and spherical surface. The DHAQ could be loaded into PELGE copolymers. In this study, the DHAQ nanoparticle-polymer delivery system was established by using PELGE polymers as carrier material.

Toward the emergence of nanoneurosurgery: part III--nanomedicine: targeted nanotherapy, nanosurgery, and progress toward the realization of nanoneurosurgery.
Leary SP, Liu CY, Apuzzo ML.
Neurosurgery. 2006 Jun;58(6):1009-26; discussion 1009-26.
[ expand abstract ]

The notion of nanotechnology has evolved since its inception as a fantastic conceptual idea to its current position as a mainstream research initiative with broad applications among all divisions of science. In the first part of this series, we reviewed the structures and principles that comprise the main body of knowledge of nanoscience and nanotechnology. In the second part, we discussed applications of nanotechnology to the emerging field of nanomedicine, with specific attention on medical diagnostics and imaging. This article further explores the applications of nanotechnology to nanomedicine. Specific attention is given to developments in therapeutic modalities, including advanced drug delivery systems and targeted nanotherapy, which will form the basis for the treatment arm of mature nanomedicine. A variety of modalities are discussed, including polymeric nanoparticles, micelles, liposomes, dendrimers, fullerenes, hydrogels, nanoshells, and smart surfaces. Applications of nanotechnology to nanosurgery and nanoneurosurgery are presented. Femtosecond laser systems, nanoneedles, and nanotweezers are presented as technologies that are operational at the nanoscale level and have the potential to revolutionize the practice of neurosurgery in a profound and momentous way.

Mannosylated liposomes as antigen delivery vehicles for targeting to dendritic cells.
White KL, Rades T, Furneaux RH, Tyler PC, Hook S.
J Pharm Pharmacol. 2006 Jun;58(6):729-37.
[ expand abstract ]

The immune stimulating ability of mannosylated liposomes containing FITC-ovalbumin as a model antigen and displaying either a branched tri-mannose or a mono-mannose ligand on the liposome surface was investigated in human monocyte-derived dendritic cells (MoDCs) and murine bone-marrow-derived dendritic cells (BMDCs). Uptake of liposomes, dendritic cell activation and proliferation of CD8(+) T cells from OT-I transgenic mice were determined by flow cytometry. Uptake of liposomes displaying the tri-mannose ligand was enhanced in human MoDCs compared with both non-mannosylated liposomes and liposomes displaying mono-mannose ligands. However, this increased uptake did not result in an increase in expression of CD80 or CD86 on the surface of the MoDCs. In contrast, neither tri-mannose- nor mono-mannose-containing liposomes were taken up by murine BMDCs to a greater extent than non-mannose-containing liposomes. The expression of CD86 and CD40 on the surface of BMDCs was not increased after exposure to mannosylated liposomes and BMDCs incubated with mannosylated liposomes were not able to stimulate proliferation of CD8(+) T cells to any greater extent than BMDCs incubated with non-mannosylated liposomes. These findings suggest that while mannose-containing ligands can enhance the uptake of antigen-containing liposomes by some dendritic cells, important differences in the affinity of carbohydrate-binding receptors for mannose-containing ligands do exist between species. In addition, the increase in uptake of antigen by dendritic cells using mannosylated liposomes does not necessarily result in enhanced dendritic cell activation.

A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system.
Wong HL, Bendayan R, Rauth AM, Xue HY, Babakhanian K, Wu XY.
J Pharmacol Exp Ther. 2006 Jun;317(3):1372-81; Epub 2006 Mar 17.
[ expand abstract ]

The objectives of this study were to evaluate the potential of a polymer-lipid hybrid nanoparticle (PLN) system to enhance cellular accumulation and retention of doxorubicin (Dox), a widely used anticancer drug and an established P-glycoprotein (Pgp) substrate, in Pgp-overexpressing cancer cell lines and to explore the underlying mechanisms. Nanoparticles containing Dox complexed with a novel anionic polymer (Dox-PLN) were prepared using an ultrasound method. Two Pgp-overexpressing breast cancer cell lines (a human cell line, MDA435/LCC6/MDR1, and a mouse cell line, EMT6/AR1) were used to investigate the effect of nanoparticles on cellular uptake and retention of Dox. Endocytosis inhibition studies and fluorescence microscopic imaging were performed to elucidate the mechanisms of cellular drug uptake. Treatment of Pgp-overexpressing cell lines with Dox-PLNs resulted in significantly enhanced Dox uptake and more substantial increases in drug retention after the end of treatment compared with free Dox solutions (p < 0.05). Fluorescence microscopic images showed improved nuclear localization of Dox and uptake of lipid when the drug was delivered in the Dox-PLN form to MDA435/LCC6/MDR1 cells. Endocytosis inhibition studies revealed that phagocytosis is an important pathway in the membrane permeability of the nanoparticles. These findings suggest that some of the Dox physically associated with the nanoparticles bypass the membrane-associated Pgp when delivered as Dox-PLNs, and in this form, the drug is better retained within the Pgp-overexpressing cells than the free drug. The present study suggests a new mechanism for overcoming drug resistance in Pgp-overexpressing tumor cells using lipid-based nanoparticle formulations.

Selective laser nano-thermolysis of human leukemia cells with microbubbles generated around clusters of gold nanoparticles.
Lapotko DO, Lukianova E, Oraevsky AA.
Lasers Surg Med. 2006 May 30; [Epub ahead of print] .
[ expand abstract ]

BACKGROUND AND OBJECTIVE: Previously reported studies on laser nano-thermolysis of cancerous cells demonstrated insufficient efficacy and specificity of malignant cell damage. Safety, that is, absence of damage to normal cells in the course of the laser thermolysis was also low due to less than optimal protocol of cancer cell targeting with nanoparticles (NP). The objective of this study was two-fold: to optimize NP targeting to real tumor (human) cells and to better understand physical mechanisms of cell damage for improved control of the laser ablation. STUDY DESIGN/MATERIALS AND METHODS: We have suggested (1) two-stage targeting method to form clusters of light-absorbing gold NPs selectively in target cells, and (2) the cell damage mechanism through laser-induced generation of vapor bubbles around NP clusters. Experimental investigation of laser nano-thermolysis of leukemia cells was performed using 30 nm spherical gold nanoparticles as a light absorbing agent, and photothermal and fluorescent microscopies as well as flow cytometry as methods to monitor microbubble formation and resulting damage of leukemia cells in human bone marrow specimens. RESULTS: NP clusters were formed and visualized using fluorescence microscopy at cell membranes and in cytoplasm of B-lymphoblasts. Laser irradiation of cells (532 nm, 10 nanoseconds, 0.6 J/cm(2)) induced microbubbles selectively in leukemia cells with large clusters, but not in cells with single NPs or small clusters. Quantitative analysis demonstrated that only 0.1%-1.5% of tumor cells and 77%-84% of normal bone marrow cells survived laser pulse. CONCLUSIONS: Two-stage cell targeting method permits formation of NP clusters selectively in diagnosis-specific tumor cells. The clusters serve as effective sources of photothermally-induced microbubbles, which kill individual target cells after a single laser pulse. The laser fluence threshold for generation of microbubbles is inversely proportional to the volume of NP clusters.

Construction, gene delivery, and expression of DNA tethered nanoparticles.
Prow T, Smith JN, Grebe R, Salazar JH, Wang N, Kotov N, Lutty G, Leary J.
Mol Vis. 2006 May 26;12:606-15.
[ expand abstract ]

PURPOSE: Layered nanoparticles have the potential to deliver any number of substances to cells both in vitro and in vivo. The purpose of this study was to develop and test a relatively simple alternative to custom synthesized nanoparticles for use in multiple biological systems, with special focus on the eye. METHODS: The biotin-labeled transcriptionally active PCR products (TAP) were conjugated to gold, semiconductor nanocrystals, and magnetic nanoparticles (MNP) coated with streptavidin. The process of nanoparticle construction was monitored with gel electrophoresis. Fluorescence microscopy followed by image analysis was used to examine gene expression levels from DNA alone and tethered MNP in human hepatoma derived Huh-7 cells. Adult retinal endothelial cells from both dog (ADREC) and human (HREC) sources were transfected with nanoparticles and reporter gene expression evaluated with confocal and fluorescent microscopy. Transmission electron microscopy was used to quantify the concentration of nanoparticles in a stock solution. Nanoparticles were evaluated for transfection efficiency, determined by fluorescence microscopy cell counts. Cells treated with MNP were evaluated for increased reactive oxygen species (ROS) and necrosis with flow cytometry. RESULTS: Both 5' and 3' biotin-labeled TAP bound equally to MNP and there were no differences in functionality between the two tethering orientations. Free DNA was easily removed by the use of magnetic columns. These particles were also able to deliver genes to a human hepatoma cell line, Huh-7, but transfection efficiency was greater than TAP. The semiconductor nanocrystals and MNP had the highest transfection efficiencies. The MNP did not induce ROS formation or necrosis after 48 h of incubation. CONCLUSIONS: Once transfected, the MNP had reporter gene expression levels equivalent to TAP. The nanoparticles, however, had better transfection efficiencies than TAP. The magnetic nanoparticles were the most easily purified of all the nanoparticles tested. This strategy for bioconjugating TAP to nanoparticles is valuable because nanoparticle composition can be changed and the system optimized quickly. Since endothelial cells take up MNP, this strategy could be used to target neovascularization as occurs in proliferative retinopathies. Multiple cell types were used to test this technology and in each the nanoparticles were capable of transfection. In adult endothelial cells the MNP appeared innocuous, even at the highest doses tested with respect to ROS and necrosis. This technology has the potential to be used as more than just a vector for gene transfer, because each layer has the potential to perform its own unique function and then degrade to expose the next functional layer.

Convection-enhanced delivery of Ls-TPT enables an effective, continuous, low-dose chemotherapy against malignant glioma xenograft model.
Saito R, Krauze MT, Noble CO, Drummond DC, Kirpotin DB, Berger MS, Park JW, Bankiewicz KS.
Neuro-oncol. 2006 May 24; [Epub ahead of print] .
[ expand abstract ]

Treatment of malignant gliomas represents one of the most formidable challenges in oncology. The combination of surgery, radiation, and chemotherapy yields median survivals of less than one year. Here we demonstrate the use of a minimally invasive surgical technique, convection-enhanced delivery (CED), for local administration of a novel nanoparticle liposome containing topotecan. CED of this liposomal topotecan (Ls-TPT) resulted in extended brain tissue retention (t1/2 = 1.5 days), whereas free topotecan was rapidly cleared (t1/2 = 0.1 days) after CED. The favorable pharmacokinetic profile of extended topotecan release for about seven days, along with biodistribution featuring perivascular accumulation of the nanoparticles, provided, in addition to the known topoisomerase I inhibition, an effective antiangiogenic therapy. In the rat intracranial U87MG tumor model, vascular targeting of Ls-TPT with CED was associated with reductions in laminin expression and vascular density compared to free topotecan or control treatments. A single CED treatment on day 7 showed that free topotecan conferred no survival benefit versus control. However, Ls-TPT produced a significant (P = 0.0002) survival benefit, with six of seven complete cures. Larger U87MG tumors, where CED of Ls-TPT on day 12 resulted in one of six cures, indicated the necessity to cover the entire tumor with the infused therapeutic agent. CED of Ls-TPT was also efficacious in the intracranial U251MG tumor model (P = 0.0005 versus control). We conclude that the combination of a novel nanoparticle Ls-TPT and CED administration was very effective in treating experimental brain tumors.

Paclitaxel nanoparticle inhibits growth of ovarian cancer xenografts and enhances lymphatic targeting.
Lu H, Li B, Kang Y, Jiang W, Huang Q, Chen Q, Li L, Xu C.
Cancer Chemother Pharmacol. 2006 May 23; [Epub ahead of print] .
[ expand abstract ]

Objectives: Ovarian cancer has the highest mortality of all the gynecologic cancers. The antitumor agent paclitaxel has been proved to be efficient in the treatment of ovarian cancer. Our study is to develop a polymeric drug delivery system for paclitaxel and determine whether paclitaxel nanoparticle can inhibit growth of ovarian carcinoma xenografts in Fisher344 (F344) rats by intraperitoneal administration. The mechanism of paclitaxel nanoparticles in rats bearing ovarian cancer has been investigated in this study. Methods: Synthesize paclitaxel loading nanoparticle (PLA) by ultrasonic emulsification; MTT analysis identified cytotoxic activity of paclitaxel nanoparticle in vitro; rat ovarian carcinoma cells were injected into the peritoneal cavity of F344 rats. The antitumor effect of paclitaxel nanoparticle in vivo has been evaluated by measuring tumor weight and ascite volume. At the end of the procedure rats were sacrificed; tumors were excised and processed for PCNA staining, tissue terminal deoxynucleotide transferase-mediated dUTP nick and labeling assay and RT-PCR to evaluate the proliferative and apoptotic changes and cancer transfer-related gene expression induced by PLA. Paclitaxel concentration in plasma, pelvic lymph nodes, liver, and heart were determined by high-performance liquid chromatography. Results: Paclitaxel nanoparticle and PTX (Cremophor) showed equivalent cytotoxic activity in vitro. In rats implanted carcinoma cells, paclitaxel nanoparticles significantly reduced tumor weight and ascites volume, and induced apoptosis of tumor cells. PLA also inhibited cell proliferation and matrix metalloproteinase 9 mRNA expression. The paclitaxel concentration of pelvic lymph nodes in PLA treated animals was 20-fold higher than that of free PTX treated animals at 48 h after intraperitoneal administration. Conclusion: The intraperitoneal administration of paclitaxel nanoparticle can significantly inhibit the progression of ovarian carcinoma in peritoneal cavity of female F344 rat. The paclitaxel nanoparticle is safe and lymphatic targeting.

Oligonucleotide-modified gold nanoparticles for intracellular gene regulation.
Rosi NL, Giljohann DA, Thaxton CS, Lytton-Jean AK, Han MS, Mirkin CA.
Science. 2006 May 19;312(5776):1027-30.
[ expand abstract ]

We describe the use of gold nanoparticle-oligonucleotide complexes as intracellular gene regulation agents for the control of protein expression in cells. These oligonucleotide-modified nanoparticles have affinity constants for complementary nucleic acids that are higher than their unmodified oligonucleotide counterparts, are less susceptible to degradation by nuclease activity, exhibit greater than 99% cellular uptake, can introduce oligonucleotides at a higher effective concentration than conventional transfection agents, and are nontoxic to the cells under the conditions studied. By chemically tailoring the density of DNA bound to the surface of gold nanoparticles, we demonstrated a tunable gene knockdown.

Biodistribution and tumor-accumulation of gadolinium (Gd) encapsulated in long-circulating liposomes in tumor-bearing mice for potential neutron capture therapy.
Le UM, Cui Z.
Int J Pharm. 2006 May 17; [Epub ahead of print] .
[ expand abstract ]

To deliver and maintain a sufficient amount of Gd into tumors is required for a successful Gd neutron capture therapy (Gd-NCT), but it has been proven to be rather challenging to achieve. Previously, we have reported a Gd-encapsulated liposome formulation that has the potential to overcome this challenge. In the present study, we sought to systemically evaluate the biodistribution and the tumor-accumulation of the Gd in model tumor-bearing mice. The Gd-encapsulated liposomes were injected into mice pre-grafted with two different model tumors. The Gd content in the tumors and other organs were determined at various time after the injection. A sufficient amount of Gd was readily delivered into those two different model tumors. Increasing the dose of Gd by injecting the Gd-encapsulated liposomes multiple times tended to increase the uptake of the Gd by the tumors. Finally, the uptake of Gd by tumors was inversely correlated with the size of the tumors. The Gd-encapsulated liposomes hold great potentials as a Gd delivery system for NCT of small- and medium-size tumors. Alternative strategies may have to be adopted in order to use NCT to treat large, advanced solid tumors, although for which, Gd-NCT might be advantageous over boron-NCT.

Efficient delivery of a Bcl-2-specific antisense oligodeoxyribonucleotide (G3139) via transferrin receptor-targeted liposomes.
Chiu SJ, Liu S, Perrotti D, Marcucci G, Lee RJ.
J Control Release. 2006 May 15;112(2):199-207; Epub 2006 Mar 6.
[ expand abstract ]

A novel transferrin receptor (TfR)-targeted liposomal formulation was synthesized and evaluated for the delivery of a phosphorothioate antisense oligodeoxyribonucleotide (ODN) (G3139, oblimerson sodium, or Genasensetrade mark) to Bcl-2 in K562 leukemia cells. Liposomes composed of DC-Chol/egg PC/PEG-DSPE (25:73.5:1.5, mol/mol/mol) were loaded with G3139 with high efficiency (70-80%). To prepare targeted liposomes, transferrin was first coupled to PEG-DSPE and then incorporated into the bilayer by post-insertion. The liposomes had a mean diameter of 100 to 150 nm and exhibited colloidal stability for up to 8 weeks. Uptake of Tf-conjugated G3139-containing liposomes in TfR positive K562 cells was found to be more efficient than that of the non-targeted control formulation and could be blocked by excess free Tf. Treatment with Tf-conjugated liposomes resulted in Bcl-2 protein downregulation in K562 cells that was approximately 2-fold greater than with non-targeted liposomes (p<0.05) and 10-fold greater than with free G3139. Treatment with 2 muM G3139 in Tf-conjugated liposomes resulted in >80% reduction in Bcl-2 transcript. In addition, Tf-conjugated liposomal G3139-sensitized K562 cells to daunorubicin, lowering IC(50) from 1.8 muM to 0.18 muM. In conclusion, Tf-conjugated liposomes are effective delivery vehicles for G3139 antisense oligos in TfR positive K562 cells and warrant further investigation as an in vivo oligo delivery vehicle.

Development and in vitro validation of a targeted delivery vehicle for DNA vaccines.
Talsma SS, Babensee JE, Murthy N, Williams IR.
J Control Release. 2006 May 15;112(2):271-9; Epub 2006 Mar 6.
[ expand abstract ]

Usage of DNA vaccination has been limited by inefficient cellular expression of plasmid constructs used in DNA vaccines. We describe a novel system for enhancing delivery of DNA vaccine plasmids into cells and their nuclei. This delivery system uses recombinant reovirus type 3 sigma1 attachment protein genetically modified with a nuclear localization sequence (sigma1-NLS) as a targeting ligand. Purified sigma1-NLS was covalently conjugated to the polycation polyethyleneimine (PEI) using a carboxyl-reactive cross-linking agent and complexed with plasmid DNA. The benefit of the NLS in enhancement of protein delivery into the nucleus was demonstrated by liposome-mediated loading of cells with sigma1 or sigma1-NLS. In L929 fibroblasts loaded with sigma1-NLS, 69% of the internalized protein was recovered in the nuclear fraction after 6 h compared to just 10% when using unmodified sigma1. Transfection of L929 cells with sigma1-NLS-conjugated PEI complexed with a luciferase expression plasmid resulted in a mean 16-fold increase in luciferase activity over complexes made with unmodified PEI, compared to a mean 3-fold boost obtained using sigma1-conjugated PEI. These results suggest that sigma1-NLS is a useful bifunctional targeting ligand suitable for enhancing DNA delivery and subsequent gene expression for both DNA vaccine applications and nonviral gene therapy.

siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene.
Zhang C, Tang N, Liu X, Liang W, Xu W, Torchilin VP.
J Control Release. 2006 May 15;112(2):229-39; Epub 2006 Mar 20.
[ expand abstract ]

Development of RNA interference (RNAi) technology utilizing the short interfering RNA sequences (siRNA) based 'targeted' therapeutics has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA encapsulated into liposomes additionally bearing arginine octamer (R8) molecules attached to their outer surface (R8-liposomes). The R8-liposomal human double minute gene 2 (HDM2)-siRNA demonstrated a significant stability against degradation in the blood serum (siRNA-loaded R8-liposomes remained intact even after 24-h incubation), and higher transfection efficiency into all three tested lung tumor cell lines. siRNA delivery successfully proceeds in the presence of plasma proteins, and R8-liposomes demonstrate low non-specific toxicity. The mechanism of action of R8-liposome-encapsulated siRNA is associated with the RNAi-mediated degradation of the target mRNA. siRNA in R8-liposomes effectively inhibited the targeted gene and significantly reduced the proliferation of cancer cells. The approach offers the potential for siRNA delivery for various in vitro and in vivo applications.

Polyelectrolyte Nanoparticles with High Drug Loading Enhance the Oral Uptake of Hydrophobic Compounds.
Cheng WP, Gray AI, Tetley L, Hang TL, Schatzlein AG, Uchegbu IF.
Biomacromolecules. 2006 May 8;7(5):1509-1520.
[ expand abstract ]

In the pharmaceutical industry, orally active compounds are required to have sufficient water solubility to enable dissolution within the gastrointestinal tract prior to absorption. Limited dissolution within the gastrointestinal tract often reduces the bioavailability of hydrophobic drugs. To improve gastrointestinal tract dissolution, nonaqueous solvents are often used in the form of emulsions and microemulsions. Here, we show that oil-free polyelectrolyte nanosystems (micellar dispersions and 100-300 nm particles) prepared from poly(ethylenimines) derivatized with cetyl chains and quaternary ammonium groups are able to encapsulate high levels of hydrophobic drug (0.20 g of drug per g of polymer) for over 9 months, as demonstrated using cyclosporine A (log P = 4.3). The polyelectrolytes facilitate the absorption of hydrophobic drugs within the gastrointestinal tract by promoting drug dissolution and by a hypothesized mechanism involving paracellular drug transport. Polyelectrolyte nanoparticle drug blood levels are similar to those obtained with commercial microemulsion formulations. The polyelectrolytes do not promote absorption by inhibition of the P-glycoprotein efflux pump.

Water Soluble Nanoparticles from PEG-Based Cationic Hyperbranched Polymer and RNA That Protect RNA from Enzymatic Degradation.
Khan JA, Kainthan RK, Ganguli M, Kizhakkedathu JN, Singh Y, Maiti S.
Biomacromolecules. 2006 May 8;7(5):1386-1388.
[ expand abstract ]

Recent advances in understanding biological systems have proven that RNA is not merely the carrier of genetic information, but also a key molecule in regulation of gene expression and other crucial metabolic processes. Therefore, it is being considered as an ideal therapeutic candidate both for metabolic and genetic disorders. However, research involving RNA molecules faces a practical limitation since RNA is highly labile. We have developed a novel method to protect RNA from cleavage by complexing it with a hyperbranched cationic polymer. It was found that total cellular RNA isolated from yeast spontaneously interacts with the positively charged polymer to form a spherical nanoparticle morphology. This interaction protects the RNA against enzymatic degradation. This methodology can be easily adapted for long-term storage of RNA, long distance transfer of RNA, and genetic engineering using RNA as a building block.

Application of Micro- and Nano-Electromechanical Devices to Drug Delivery.
Staples M, Daniel K, Cima MJ, Langer R.
Pharm Res. 2006 May 5; [Epub ahead of print] .
[ expand abstract ]

Micro- and nano-electromechanical systems (MEMS and NEMS)-based drug delivery devices have become commercially-feasible due to converging technologies and regulatory accommodation. The FDA Office of Combination Products coordinates review of innovative medical therapies that join elements from multiple established categories: drugs, devices, and biologics. Combination products constructed using MEMS or NEMS technology offer revolutionary opportunities to address unmet medical needs related to dosing. These products have the potential to completely control drug release, meeting requirements for on-demand pulsatile or adjustable continuous administration for extended periods. MEMS or NEMS technologies, materials science, data management, and biological science have all significantly developed in recent years, providing a multidisciplinary foundation for developing integrated therapeutic systems. If small-scale biosensor and drug reservoir units are combined and implanted, a wireless integrated system can regulate drug release, receive sensor feedback, and transmit updates. For example, an "artificial pancreas" implementation of an integrated therapeutic system would improve diabetes management. The tools of microfabrication technology, information science, and systems biology are being combined to design increasingly sophisticated drug delivery systems that promise to significantly improve medical care.

In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles.
Reddy ST, Rehor A, Schmoekel HG, Hubbell JA, Swartz MA.
J Control Release. 2006 May 1;112(1):26-34; Epub 2006 Mar 10.
[ expand abstract ]

Delivery of biodegradable nanoparticles to antigen-presenting cells (APCs), specifically dendritic cells (DCs), has potential for immunotherapy. This study investigates the delivery of 20, 45, and 100nm diameter poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. These nanoparticles consist of a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades within oxidative environments. 20 nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45nm nanoparticles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of a targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. Finally, an increase in nanoparticle-containing DCs (and other APCs) was seen at 96h vs. 24h, suggesting an infiltration of these cells to lymph nodes. Thus, PPS nanoparticles of 20-45nm have the potential for immunotherapeutic applications that specifically target DCs in lymph nodes.

Quantum Dot-Based Energy Transfer: Perspectives and Potential for Applications in Photodynamic Therapy.
Samia AC, Dayal S, Burda C.
Photochem Photobiol. 2006 May 1; [Epub ahead of print] .
[ expand abstract ]

Quantum dots have emerged as an important class of material that offers great promise to a diverse range of applications ranging from energy conversion to biomedicine. Here, we review the potential of using quantum dots and quantum dot conjugates as sensitizers for photodynamic therapy (PDT). The photophysics of singlet oxygen generation in relation to quantum dot-based energy transfer is discussed and the possibility of using quantum dots as photosensitizer in PDT is assessed, including their current limitations to applications in biological systems. The biggest advantage of quantum dots over molecular photosensitizers that comes into perspective is their tunable optical properties and surface chemistries. Recent developments in the preparation and photophysical characterization of quantum dot energy transfer processes are also presented in this review, to provide insights on the future direction of quantum dot-based photosensitization studies from the viewpoint of our ongoing research.

Nano-fibrous scaffold for controlled delivery of recombinant human PDGF-BB.
Wei G, Jin Q, Giannobile WV, Ma PX.
J Control Release. 2006 May 1;112(1):103-10; Epub 2006 Mar 3.
[ expand abstract ]

The localized and temporally controlled delivery of growth factors is key to achieving optimal clinical efficacy. In sophisticated tissue engineering strategies, the biodegradable scaffold is preferred to serve as both a three-dimensional (3-D) substrate and a growth factor delivery vehicle to promote cellular activity and enhance tissue neogenesis. This study presents a novel approach to fabricate tissue engineering scaffolds capable of controlled growth factor delivery whereby growth factor containing microspheres were incorporated into 3-D scaffolds with good mechanical properties, well-interconnected macroporous and nano-fibrous structures. The microspheres were uniformly distributed throughout the nano-fibrous scaffold and their incorporation did not interfere the macro-, micro-, and nanostructures of the scaffold. The release kinetics of platelet-derived growth factor-BB (PDGF-BB) from microspheres and scaffolds was investigated using poly(lactic-co-glycolic acid) (PLGA50) microspheres with different molecular weights (6.5 and 64kDa, respectively) and microsphere-incorporated poly(l-lactic acid) (PLLA) nano-fibrous scaffolds. Incorporation of microspheres into scaffolds significantly reduced the initial burst release. Sustained release from several days to months was achieved through different microspheres in scaffolds. Released PDGF-BB was demonstrated to possess biological activity as evidenced by stimulation of human gingival fibroblast DNA synthesis in vitro. The successful generation of 3-D nano-fibrous scaffold incorporating controlled-release factors indicates significant potential for more complex tissue regeneration.

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1.
Yu Z, Xia W, Wang HY, Wang SC, Pan Y, Kwong KY, Hortobagyi GN, Hung MC.
Mol Carcinog. 2006 May 1; [Epub ahead of print] .
[ expand abstract ]

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, by downregulating the HER-2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and development into tumors of HER-2/neu-overexpressing ovarian cancer cells. Here, we establish stable clones of the human breast cancer cell line MDA-MB-361DYT2 that express PEA3 under the control of a tetracycline-inducible promoter. Ectopic expression of PEA3 in this cell line inhibited cell growth and resulted in cell cycle accumulation in the G1 phase. We demonstrate that expression of PEA3 in an orthotopic breast cancer model inhibited tumor growth and prolonged the survival of tumor-bearing mice. In a parallel experiment with another breast cancer cell line, BT474M1, we were unable to obtain stable PEA3-inducible transfectants, suggesting that PEA3 may exert a strong growth inhibition effect in this cell line. Indeed, PEA3 coupled with the liposome SN2 demonstrated therapeutic effects in mice bearing tumors induced by BT474M1. These results provide evidence for the antitumor activity of PEA3 in human breast cancers.

Nanoparticle-aptamer bioconjugates for cancer targeting.
Farokhzad OC, Karp JM, Langer R.
Expert Opin Drug Deliv. 2006 May;3(3):311-324.
[ expand abstract ]

The combination of targeted drug delivery and controlled-release technology may pave the road for more effective yet safer chemotherapeutic options for cancer therapy. Drug-encapsulated polymeric nanoparticle-aptamer bioconjugates represent an emerging technology that can facilitate the delivery of chemotherapeutics to primary and metastatic tumours. Aptamers are short nucleic acid molecules with binding properties and biochemical characteristics that may make them suitable for use as targeting molecules. The goal of this review is to summarise the key components that are required for creating effective cancer targeting nanoparticle-aptamer bioconjugates. The field of controlled release and the structure and properties of aptamers, as well as the criteria for constructing effective conjugates, will be discussed.

ICS-283: a system for targeted intravenous delivery of siRNA.
Schiffelers RM, Storm G.
Expert Opin Drug Deliv. 2006 May;3(3):445-454.
[ expand abstract ]

ICS-283 was developed within Intradigm Corporation as a system that is designed for the systemic delivery of therapeutic small interfering (siRNA) to sites of pathological angiogenesis. The non-viral siRNA delivery system is based on synthetic nanoparticles, known as TargeTrantrade mark (Intradigm Corporation), which functions as a broad-platform technology to deliver siRNA to specific target cells in diseased tissues. The system is constructed to incorporate different functionalities that address critical needs for successful nucleic acid delivery. The TargeTran synthetic vector is a self-assembling, layered nanoparticle that protects and targets siRNA to specific cell types in pathological tissues. At present, ICS-283 is the only antiangiogenic siRNA delivery system that is designed for intravenous administration to treat angiogenesis-driven diseases.

Pharmacokinetic and cytotoxic studies of pegylated liposomal daunorubicin.
Song H, Zhang J, Han Z, Zhang X, Li Z, Zhang L, Fu M, Lin C, Ma J.
Cancer Chemother Pharmacol. 2006 May;57(5):591-8; Epub 2005 Aug 30.
[ expand abstract ]

Pegylated liposomes have been studied for nearly two decades. However, fewer pharmacological studies about its application in daunorubicin (DNR) than those in doxorubicin have been reported. In order to conduct a complete pharmacokinetic study, radiolabeled DNR was encapsulated in pegylated liposomes. Its in vitro drug release kinetics was determined to be in a slow manner, which was reflected in its cytotoxic effect on four cell lines. The lethal dose, plasma pharmacokinetics as well as tissue distribution of the formulation were evaluated in comparison with free DNR. The results revealed that liposomal daunorubicin significantly reduced the toxicity of the drug, with a half lethal dose of 29.35 mg/kg, compared with 5.45 mg/kg for free drug. Pharmacokinetic study of liposomal DNR demonstrated a slower clearance rate, an elevated area under the concentration-time curve, as well as increased half-lives compared to free drug. In addition, an altered tissue distribution of liposomal DNR was observed, with lower cardiac accumulation. Taken together, pegylated liposome-loaded DNR may be a promising anticancer drug and worth further therapeutic study.

Pharmacokinetic and pharmacodynamic evaluation of a novel in situ forming poly(ethylene glycol)-based hydrogel for the controlled delivery of the camptothecins.
Lalloo A, Chao P, Hu P, Stein S, Sinko PJ.
J Control Release. 2006 Apr 29; [Epub ahead of print] .
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Inadequate drug delivery, due to problems associated with achieving constant therapeutic blood levels, has hampered the use of anticancer agents of the camptothecin (CPT) class. The objective of the current studies was to develop a depot delivery system for the water-soluble analog of CPT, topotecan (TPT). In this study, a 2-phase drug depot consisting of TPT-loaded liposomes entrapped in a poly(ethylene glycol) hydrogel was designed. Physically entrapped unaltered TPT displayed a rapid release rate from the hydrogel. Controlled release was demonstrated in vitro and in vivo from the 2-phase system with constant blood levels being achieved for several days in rats. Cytotoxicity and antitumor activity were also evaluated in rats inoculated with syngeneic MAT B III breast cancer cells. Rats treated with the liposome-loaded hydrogel displayed significantly longer tumor growth suppression and did not exhibit body weight loss compared to those treated with other delivery modes. These experiments constitute a proof-of-principle of the 2-phase depot concept and its potential value for enhancing safety and efficacy in chemotherapy.

In vitro enhanced cytotoxicity of tumor-infiltrating lymphocytes transfected with tumor necrosis factor-related apoptosis-inducing ligand and/or interleukin-2 gene in human renal cell carcinoma.
Tian JQ, Wang ZP, Rodriguez R, Fu JS, Lu JZ, Ma BL.
Urology. 2006 Apr 22; [Epub ahead of print] .
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OBJECTIVES: To investigate whether tumor-infiltrating lymphocytes (TILs) transfected with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and interleukin-2 (IL-2) genes are capable of improving the potency and efficacy of propagation and cytotoxicity against renal cell carcinoma (RCC) cells in vitro. METHODS: A mammal expression vector system was constructed. TILs were transfected by liposome-mediated gene transfection. The degree of cytokine mRNA expression was evaluated with Northern blot. Protein expression was determined with Western blot and enzyme-linked immunosorbent assay. Cytotoxicity of TILs against autologous RCC cells and the human RCC cell line (786-0) were examined by chromium release assay. Flow cytometric analyses were performed to determine the apoptosis of tumor cells. RESULTS: A high level of expression of the human TRAIL and IL-2 stable transfected TILs was observed. The mean IL-2 production was 22.6 +/- 5.2, 507.7 +/- 52.4, and 549.0 +/- 74.0 ng/10(6) cells/24 hours in the TIL/parental, TIL/IL-2, and TIL/TRAIL+IL-2 genes, respectively. The mean cytotoxicity (effector/target ratio 20:1) of TIL/parental, TIL/IL-2, TIL/TRAIL, and TIL/TRAIL+IL-2 against autologous RCC cells in the percentage of cytolysis was 21.2% +/- 4.8%, 32.1% +/- 5.5%, 63.5% +/- 6.6%, and 78.1% +/- 9.63%, respectively. These four groups showed cytotoxic activity against allogeneic 786-0 RCC cells; the corresponding values were 9.8% +/- 3.5%, 12.3% +/- 3.4%, 24.1% +/- 4.9%, and 30.4% +/- 6.2%. The number of apoptotic cells was significantly greater for autologous RCC cells than for 786-0 cells after TIL/TRAIL and TIL/TRAIL+IL-2 treatment. CONCLUSIONS: TIL/TRAIL+IL-2 and TIL/IL-2 were expanded by autocrine IL-2. TIL/TRAIL+IL-2 and TIL/TRAIL showed significant cytotoxicity that was induced by TRAIL. TILs, including parental TILs and transfected TILs, demonstrated a potent cytotoxicity against RCC cells with remarkable selectivity. Autologous RCC cells seemed more sensitive than allogeneic RCC cells.

Antiangiogenesis and damaging blood flow by antisense vascular endothelial growth factor oligodeoxynucleotides to suppress lung cancers.
Li C, Cheng X, Jiang H, Sun X.
Tumour Biol. 2006;27(3):158-65; Epub 2006 Apr 20.
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Angiogenesis plays a key role in the growth and metastasis of lung cancers, and vascular endothelial growth factor (VEGF) is one of the major angiogenic factors. The study aims to investigate whether phosphoro thioate-modified antisense VEGF oligodeoxynucleo tides (ASODN) formulated in cationic liposome could inhibit the growth of Lewis lung carcinoma (LLC) tumors by antiangiogenesis. The study demonstrated that ASODN downregulated the expression of VEGF in LLC cells at levels of protein and mRNA in vitro and in vivo. The conditioned media obtained from LLC cells treated with ASODN significantly inhibited the proliferation of bovine aortic endothelial cells. The ASODN therapy significantly suppressed the growth of established subcutaneous LLC tumors in mice by inhibiting angiogenesis and damaging the blood flow of tumors. In conclusion, our results suggest that ASODN targeting VEGF presents a potent therapeutic strategy to combat lung cancers.

Eradication of established HPV 16-expressing tumors by a single administration of a vaccine composed of a liposome-encapsulated CTL-T helper fusion peptide in a water-in-oil emulsion.
Daftarian P, Mansour M, Benoit AC, Pohajdak B, Hoskin DW, Brown RG, Kast WM.
Vaccine. 2006 Apr 18; [Epub ahead of print] .
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Human papillomavirus (HPV)-induced cervical cancer is the second most common cancer among women worldwide with half a million new cases per year. Despite the encouraging development of a preventive vaccine for HPV, a therapeutic vaccine for cervical cancer or pre-cancerous lesions remains a high priority. The preclinical study reported here used VacciMax((R)) (VM) to deliver a peptide-based vaccine composed of an HPV 16 E7-derived cytotoxic T lymphocyte (CTL) epitope fused to the T helper epitope PADRE (FP) and combined with CpG or lipopeptide adjuvant. In the study, C57BL/6 mice received 0.5million HPV 16-expressing C3 tumor cells. Mice were inoculated post-tumor challenge with a single s.c. injection of FP-CpG-VM on either day 4, 5, 6, 9, or 14. All mice that received the FP-CpG-VM vaccine were tumor-free to day 130 when the experiment was terminated. In contrast, only a minority of mice that received a control vaccine were tumor-free on day 60. Cytotoxicity assays, ELISPOT and intracellular staining for interferon (IFN)-gamma showed the immune response was specific for the selected CTL epitope. All mice that received the FP-CpG-VM vaccine remained tumor-free when re-challenged with 6million C3 cells. Cytotoxicity assays 4 months post-challenge showed that only splenocytes from mice inoculated with the FP-CpG-VM vaccine had high lysis activity. These results indicate that VacciMax((R)) causes a rapid, robust, durable and therapeutic CTL response to HPV 16 E7 protein expressing tumors.

Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo.
Farokhzad OC, Cheng J, Teply BA, Sherifi I, Jon S, Kantoff PW, Richie JP, Langer R.
Proc Natl Acad Sci U S A. 2006 Apr 18;103(16):6315-20; Epub 2006 Apr 10.
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Targeted uptake of therapeutic nanoparticles in a cell-, tissue-, or disease-specific manner represents a potentially powerful technology. Using prostate cancer as a model, we report docetaxel (Dtxl)-encapsulated nanoparticles formulated with biocompatible and biodegradable poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG) copolymer and surface functionalized with the A10 2'-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the prostate-specific membrane antigen (PSMA), a well characterized antigen expressed on the surface of prostate cancer ce