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

Abstract

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. Reporter plasmid DNA encoding for beta-galactosidase (pCMV-beta) 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-beta in the control and nanoparticle formulations. Periodically, the expression of beta-galactosidase in the cells was measured quantitatively using an enzymatic assay for the conversion of o-nitrophenyl-beta-D: -galactopyranoside (ONPG) to o-nitrophenol (ONP). Qualitative expression of beta-galactosidase in LLC cells was observed by staining with 5-bromo-4-chloro-3-indolyl-beta-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. Almost 100% of the pCMV-beta 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 beta-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 beta-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-beta 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. 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.