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Tumor-Targeted Gene Delivery Using Poly(Ethylene Glycol)-Modified Gelatin Nanoparticles: In Vitro and in Vivo Studies

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Purpose

To develop safe and effective systemically administered nonviral gene therapy vectors for solid tumors, DNA-containing poly(ethylene glycol)-modified (PEGylated) gelatin nanoparticles were fabricated and evaluated in vitro and in vivo.

Methods

Reporter plasmid DNA encoding for β-galactosidase (pCMV-β) was encapsulated in gelatin and PEGylated gelatin nanoparticles using a water-ethanol solvent displacement method under controlled pH and temperature. Lewis lung carcinoma (LLC) cells in culture were transfected with the pCMV-β in the control and nanoparticle formulations. Periodically, the expression of β-galactosidase in the cells was measured quantitatively using an enzymatic assay for the conversion of o-nitrophenyl-β-d-galactopyranoside (ONPG) to o-nitrophenol (ONP). Qualitative expression of β-galactosidase in LLC cells was observed by staining with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). Additionally, the plasmid DNA-encapsulated gelatin and PEGylated gelatin nanoparticles were administered intravenously (i.v.) and intratumorally (i.t.) to LLC-bearing female C57BL/6J mice. At various time points postadministration, the animals were sacrificed and transgene expression in the tumor and liver was determined quantitatively by the ONPG to ONP enzymatic conversion assay and qualitatively by X-gal staining.

Results

Almost 100% of the pCMV-β was encapsulated in gelatin and PEGylated gelatin nanoparticles (mean diameter 200 nm) at 0.5% (w/w) concentration. PEGylated gelatin nanoparticles efficiently transfected the LLC cells and the β-galactosidase expression, as measured by the ONPG to ONP enzymatic conversion assay at 420 nm absorbance, increased starting from 12 h until 96 h post-transfection. The efficient expression of LLC cells was also evident by the X-gal staining method that shows blue color formation. The in vivo studies showed significant expression of β-galactosidase in the tumor following administration of DNA-containing PEGylated gelatin nanoparticles to LLC-bearing mice by both i.v. and i.t. routes. Following i.v. administration of pCMV-β in PEGylated gelatin nanoparticles, for instance, the absorbance at 420 nm per gram of tumor increased from 0.60 after 12 h to 0.85 after 96 h of transfection. After i.t. administration, the absorbance values increased from 0.90 after 12 h to almost 1.4 after 96 h.

Conclusions

The in vitro and in vivo results of this study clearly show that a long-circulating, biocompatible and biodegradable, DNA-encapsulating nanoparticulate system would be highly desirable for systemic delivery of genetic constructs to solid tumors.

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Acknowledgments

This study was supported by a grant (RO1-CA095522) from the National Cancer Institute of the National Institutes of Health. Cell and tissue microscopy studies were performed with the assistance of Mr. William Fowle at the Electron Microscopy Center of Northeastern University. We deeply appreciate all the technical assistance provided by graduate students and postdoctoral associates in Professor Vladimir Torchilin’s laboratory at Northeastern University.

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  1. Goldie Kaul

    Present address: Boston Scientific Corporation, Oncology Group, Watertown, Massachussetts, USA

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, Massachussetts, USA

    Goldie Kaul & Mansoor Amiji

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  1. Goldie Kaul
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Correspondence to Mansoor Amiji.

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Kaul, G., Amiji, M. Tumor-Targeted Gene Delivery Using Poly(Ethylene Glycol)-Modified Gelatin Nanoparticles: In Vitro and in Vivo Studies. Pharm Res 22, 951–961 (2005). https://doi.org/10.1007/s11095-005-4590-3

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  • DOI: https://doi.org/10.1007/s11095-005-4590-3

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