Abstract 5404: DNA-chitosan nanoparticles in gene delivery: Endocytotic pathways and intracellular trafficking
Authors: Catharina de Lange Davies, Zuzana Garaiova, Nina K. Reitan, Astrid Bjørkøy, Oladayo Folasire, Sigmund Størseth, Kristian Berg, Sabina P. Strand
Journal: Cancer Research (2011)

Abstract Background: The success of gene therapy depends on efficient delivery of DNA and efficient transcription of the transgene. This involves several steps, including penetration through the extracellular matrix to target cells, intracellular uptake, and trafficking to the nucleus. A DNA vector is necessary, packing DNA into smaller polyplexes and protecting the DNA from degradation. A promising non-viral vector is chitosan, a cationic polysaccharide derived from chitin. The interactions with DNA and ability to transfect cells depend strongly on intrinsic chitosan properties. We are tailoring the chitosan to optimize transfection and have synthesized self-branched and trisaccharide-substituted chitosan oligomers (SBTCO), which seem to compact DNA less tightly and have shown superior transfection efficiency compared to linear chitosan (LCO) of equivalent molecular weight (1-3). The aim of the present work was to compare the endocytotic pathways and intracellular trafficking of polyplexes formed by SBTCO and LCO, to see if such differences could explain the difference in transfection efficacy. Material and Methods: Uptake of the two polyplexes in HeLa cells was studied using flow cytometry. Clathrin- and caveolin-dependent endocytosis was compared by inhibiting the two pathways using chlorpromazine, genistein and dynasore. Intracellular trafficking was studied using confocal laser scanning microscopy. Quantitative colocalization of polyplexes with early endosomes and intracellular DNA-chitosan colocalization were performed. The impact of photochemical internalization on transfection was studied by flow cytometric analysis of GFP expression (4). Results: All three inhibitors reduced the uptake of LCO polyplexes and genistein and dynasore were somewhat more efficient than chlorpromazine. For SBTCO polyplexes chlorpromazine showed little inhibition, whereas genistein and dynasore efficiently inhibited endocytosis. Consistent with this, only a fraction of the polyplexes was found in early endosomes. 60-80% of the intracellular DNA was binding to chitosan the first 4 hrs. Polyplexes of LCO showing low transfection was increased significantly by photochemical internalization. Conclusion: LCO polyplexes are taken up by cells both by clathrin and caveolin mediated pathways, whereas SBTCO polyplexes are primarily taken up by caveolin mediated endocytosis. The difference in endocytotic pathway results in different intracellular routes and this may explain the difference in transfection efficacy. 1. Reitan et al. Biomacromolecules 10, 2009 2. Strand et al. Biomaterials 31, 2010 3. Strand et al. Biomacromolecules 9, 2008 4. Berg et al Curr. Pharma Biotech 8, 2007 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5404. doi:10.1158/1538-7445.AM2011-5404