Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Jun;22(6):951-61.
doi: 10.1007/s11095-005-4590-3. Epub 2005 Jun 8.

Tumor-targeted gene delivery using poly(ethylene glycol)-modified gelatin nanoparticles: in vitro and in vivo studies

Goldie Kaul  1 , Mansoor Amiji
Affiliations

Tumor-targeted gene delivery using poly(ethylene glycol)-modified gelatin nanoparticles: in vitro and in vivo studies

Goldie Kaul et al. Pharm Res. 2005 Jun.

Abstract

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 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.

Results: 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.

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
In vitro quantitative transgene expression measured in Lewis lung carcinoma cells after incubation with plasmid DNA encoding for β-galactosidase (pCMV-β) that was encapsulated in the gelatin and poly(ethylene glycol)-modified (PEGylated) gelatin nanoparticle formulations. DNA-complexed Lipofectin®, a cationic lipid transfection reagent, was used as a control. The transfection efficiency in the cell lysates as a function of time was quantitated by an enzymatic assay using visible absorbance at 420 nm upon conversion of the colorless o-nitrophenyl-β-D-galactopyranoside solution into yellow-colored o-nitrophenol. Results are expressed as absorbance or optical density (OD) at 420 nm normalized per 10,000 cells (mean ± S.D, n = 4).
Figure 2
Figure 2
Brightfield microscopy images of Lewis lung carcinoma cells in culture transfected with plasmid DNA encoding for β-galactosidase that was encapsulated in the gelatin and poly(ethylene glycol)-modified (PEGylated) gelatin nanoparticle formulations. DNA-complexed Lipofectin®, a cationic lipid transfection reagent, was used as a control. Transgene expression after 12 and 96 hours was qualitatively evaluated by staining the cells with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal®) that results in the blue color formation.
Figure 3
Figure 3
In vivo quantitative transgene expression measured in the tumor mass of Lewis lung carcinoma-bearing female C57BL/6J mice administered with plasmid DNA encoding for β-galactosidase (pCMV-β) that was encapsulated in the gelatin and poly(ethylene glycol)-modified (PEGylated) gelatin nanoparticle formulations. The control (naked plasmid DNA) and nanoparticle formulations were administered by intravenous (A) and intratumoral (B) routes. The transfection efficiency in the tumor homogenates as a function of time was quantitated using visible absorbance at 420 nm by an enzymatic assay upon conversion of the colorless o-nitrophenyl-β-D-galactopyranoside solution into yellow-colored o-nitrophenol. Results are expressed as absorbance or optical density (OD) at 420 nm normalized per gram of tumor mass (mean ± S.D, n = 4).
Figure 4
Figure 4
Brightfield microscopy images of tumor cryogenic sections from Lewis lung carcinoma-bearing female C57BL/6J mice administered with naked plasmid DNA encoding for β-galactosidase (pCMV-β) intravenously (A) and intratumorally (B) after 12 and 96 hours post-transfection. Transgene expression was qualitatively evaluated by staining the tumor sections with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal®) that results in the blue color formation.
Figure 5
Figure 5
Brightfield microscopy images of tumor cryogenic sections from Lewis lung carcinoma-bearing female C57BL/6J mice administered with plasmid DNA encoding for β-galactosidase (pCMV-β) in the gelatin and poly(ethylene glycol)-modified (PEGylated) gelatin nanoparticle formulations. The DNA-containing nanoparticle suspension was injected intravenously (A) and intratumorally (B) and the tumor sections were obtained after 12 and 96 hours post-transfection. Transgene expression was qualitatively evaluated by staining the tumor sections with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal®) that results in the blue color formation.
Figure 6
Figure 6
In vivo quantitative transgene expression measured in the liver of Lewis lung carcinoma-bearing female C57BL/6J mice administered with plasmid DNA encoding for β-galactosidase (pCMV-β) that was encapsulated in the gelatin and poly(ethylene glycol)-modified (PEGylated) gelatin nanoparticle formulations. The control (naked plasmid DNA) and nanoparticle formulations were administered by intravenous (A) and intratumoral (B) routes. The transfection efficiency in the liver homogenates as a function of time was quantitated using visible absorbance at 420 nm by an enzymatic assay upon conversion of the colorless o-nitrophenyl-β-D-galactopyranoside solution into yellow-colored o-nitrophenol. Results are expressed as absorbance or optical density (OD) at 420 nm normalized per gram of liver tissue (mean ± S.D, n = 4).
Figure 7
Figure 7
Brightfield microscopy images of liver cryogenic sections from Lewis lung carcinoma-bearing female C57BL/6J mice administered with naked plasmid DNA encoding for β-galactosidase (pCMV-β) intravenously (A) and intratumorally (B) after 12 and 96 hours post-transfection. Transgene expression was qualitatively evaluated by staining the liver sections with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal®) that results in the blue color formation.
Figure 8
Figure 8
Brightfield microscopy images of liver cryogenic sections from Lewis lung carcinoma-bearing female C57BL/6J mice administered with plasmid DNA encoding for β-galactosidase (pCMV-β) in the gelatin and poly(ethylene glycol)-modified (PEGylated) gelatin nanoparticle formulations. The DNA-containing nanoparticle suspension was injected intravenously (A) and intratumorally (B) and the liver sections were obtained after 12 and 96 hours post-transfection. Transgene expression was qualitatively evaluated by staining the liver sections with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal®) that results in the blue color formation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. von Eschenbach AC. A vision for the national cancer program in the United States. Nat Rev Cancer. 2004;4:820–828. - PubMed
    1. Baselga J. New horizons: gene therapy for cancer. Anticancer Drugs. 1999;10:S39–S42. - PubMed
    1. Huber BE. Gene therapy strategies for treating neoplastic diseases. Ann NY Acad Sci. 1994;716:6–11. - PubMed
    1. Roth JA, Christiano RJ. Gene therapy for cancer: what have we done and where are we going? J Natl Cancer Inst. 1997;89:21–39. - PubMed
    1. R.J. Mrsny. Tissue- and cell-specific targeting for the delivery of genetic information. In M.M. Amiji (ed.) Polymeric Gene Delivery: Principles and Applications. Chapter 2, CRC Press, LLC. Boca Raton, FL. 2004, pp. 5–27.

Publication types

MeSH terms