April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Nitrogen To Phosphate Ratio In Nanoparticle-plasmid Transfection Reagent Modulates Gene Delivery And Toxicity In The Cornea
Author Affiliations & Notes
  • Eric T. Hansen
    Mason Eye Institute, University of Missouri-Columbia, Columbia, Missouri
    Harry S. Truman Veterans Administration Hospital, Columbia, Missouri
  • Ajay Sharma
    Mason Eye Institute, University of Missouri-Columbia, Columbia, Missouri
    Harry S. Truman Veterans Administration Hospital, Columbia, Missouri
  • Ashish Tandon
    Mason Eye Institute, University of Missouri-Columbia, Columbia, Missouri
    Harry S. Truman Veterans Administration Hospital, Columbia, Missouri
  • Rangan Gupta
    Mason Eye Institute, University of Missouri-Columbia, Columbia, Missouri
    Harry S. Truman Veterans Administration Hospital, Columbia, Missouri
  • Jennifer A. Fortune
    Departments of Chemistry and Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts
  • Alexander M. Klibanov
    Departments of Chemistry and Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts
  • Jonathan C. Tovey
    Mason Eye Institute, University of Missouri-Columbia, Columbia, Missouri
    Harry S. Truman Veterans Administration Hospital, Columbia, Missouri
  • Yasaman J. Hemmat
    Mason Eye Institute, University of Missouri-Columbia, Columbia, Missouri
    Harry S. Truman Veterans Administration Hospital, Columbia, Missouri
  • Rajiv R. Mohan
    Mason Eye Institute, University of Missouri-Columbia, Columbia, Missouri
    Harry S. Truman Veterans Administration Hospital, Columbia, Missouri
  • Footnotes
    Commercial Relationships  Eric T. Hansen, None; Ajay Sharma, None; Ashish Tandon, None; Rangan Gupta, None; Jennifer A. Fortune, None; Alexander M. Klibanov, None; Jonathan C. Tovey, None; Yasaman J. Hemmat, None; Rajiv R. Mohan, None
  • Footnotes
    Support  VA Merit 1I01BX000357-01 (RRM), NEI RO1EY17294 (RRM), NIH RO1EB000244 (AMK) and Unrestricted Research to Prevent Blindness
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 483. doi:
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      Eric T. Hansen, Ajay Sharma, Ashish Tandon, Rangan Gupta, Jennifer A. Fortune, Alexander M. Klibanov, Jonathan C. Tovey, Yasaman J. Hemmat, Rajiv R. Mohan; Nitrogen To Phosphate Ratio In Nanoparticle-plasmid Transfection Reagent Modulates Gene Delivery And Toxicity In The Cornea. Invest. Ophthalmol. Vis. Sci. 2011;52(14):483.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Recently we demonstrated that PEI2-GNP nanoparticles (2-12nm) generated from polyethylenimine (PEI) conjugation to gold nanoparticles are capable of delivering genes in rabbit cornea in vivo with mild-to-moderate side effects. We sought to test whether PEI nitrogen (N) and plasmid DNA phosphate (P) molar ratio in PEI2-GNP transfection reagent modulates gene delivery and affects cytotoxicity in the cornea using in vitro model.

Methods: : Human corneal fibroblasts (HSF) cultures were established from donor human corneas. Transfection reagents having N-P molar ratio 60, 120, 150, 180, 210 and 240 were prepared incubating appropriate volume of 150 mM PEI2-GNP nanoparticles and 1-5µg plasmid DNA (pTRUF11 expressing green fluorescent protein under control of CMV+chicken-β-actin promoter) using 10% glucose and balanced salt solution at room temperature for 30 minutes. Cultures (60-70% confluent) were exposed to transfection reagent for various time points (30 minutes to 4hrs). Phase-contrast microscopy, trypan blue and TUNEL assays evaluated phenotype, cellular viability and toxicity. Delivered transgene expression in HSF was analyzed with immunocytochemistry, real-time PCR and immunoblotting. DNA complexation to nanoparticles was confirmed by gel-retardation assay.

Results: : Tested transfection reagents showed effective complexation between PEI2-GNP nanoparticles and plasmid DNA. Transfection reagent of N-P ratio ≤180 did not alter HSF viability or phenotype whereas transfection reagents of N-P ratio ≥210 significantly compromised phenotype and cellular viability (13-20%, P < 0.01). Highest transgene delivery was observed with transfection reagent with N-P ratio 180 (53-58%, p <0.001) followed by 150, 120 and 60 (23-41%, p <0.01). A maximum 3-fold increase in the expression of delivered-gene mRNA and protein was detected with transfection reagent N-P 180 vs. N-P ≤ 150. More comparative studies are underway.

Conclusions: : Nanoparticle-based nonviral gene transfer approaches for the cornea requires testing of N-P molar ratio in transfection reagent.

Keywords: cornea: stroma and keratocytes • gene transfer/gene therapy • cornea: basic science 
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