Abstract
Chitosan has been extensively studied as a genetic drug delivery platform. However, its efficiency is limited by the strength of DNA and RNA binding. Expecting a reduced binding strength of cargo with chitosan, we proposed including heparin as a competing polyanion in the polyplexes. We developed chitosan–heparin nanoparticles by a one-step process for the local delivery of oligonucleotides. The size of the polyplexes was dependent on the mass ratio of polycation to polyanion. The mechanism of oligonucleotide release was pH-dependent and associated with polyplex swelling and collapse of the polysaccharide network. Inclusion of heparin enhanced the oligonucleotide release from the chitosan-based polyplexes. Furthermore, heparin reduced the toxicity of polyplexes in the cultured cells. The cell uptake of chitosan–heparin polyplexes was equal to that of chitosan polyplexes, but heparin increased the transfection efficiency of the polyplexes two-fold. The application of chitosan–heparin small interfering RNA (siRNA) targeted to vascular endothelial growth factor (VEGF) silencing of ARPE-19 cells was 25% higher. Overall, chitosan–heparin polyplexes showed a significant improvement of gene release inside the cells, transfection, and gene silencing efficiency in vitro, suggesting that this fundamental strategy can further improve the transfection efficiency with application of non-viral vectors.
Highlights
Progress in the field of gene medicine has been stimulated by basic research of novel vectors for DNA, RNA, and oligonucleotides
To encapsulate oligo- and polynucleotides inside chitosan–heparin nanoparticles, the anionic oligonucleotide was added to chitosan solution immediately after sonication under stirring, thereafter heparin was added to these complexes
The RNA interference potency of 27 bp ds small interfering RNA (siRNA) complexed with chitosan–heparin and directed against vascular endothelial growth factor (VEGF) was evaluated in ARPE-19 cells
Summary
Progress in the field of gene medicine has been stimulated by basic research of novel vectors for DNA, RNA, and oligonucleotides. Chitosan is water-soluble and positively charged polymer with pKa of 6.0 It forms pH-sensitive nano-sized hydrogels that may enable pH-triggered release siRNA or pDNA in the acidic medium of endosomes [7,8]. High N/P ratios in the complexes lead to high stability of the polyplexes that prevents release of the cargo within the cells and precludes translation of DNA or RNA action [15,16] To address this issue, we incorporated heparin as a stronger polyanionic competitor for polynucleotides. In this work we developed pH-sensitive chitosan–heparin nanoparticles and investigated the effect of different chitosan/heparin ratios on pH, particle size and structure, zeta potential, and encapsulation efficiency of model oligonucleotide. We unraveled specific role of competitive polyanion heparin in the complex and showed the advances of chitosan–heparin nanocarrier to deliver plasmid DNA and VEGF silencing RNA to epithelial cells
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