Exogenous chondroitin sulfate glycosaminoglycan associate with arginine-rich peptide–DNA complexes to alter their intracellular processing and gene delivery efficiency

AbstractArginine-rich peptides have been used extensively as efficient cellular transporters. However, gene delivery with such peptides requires development of strategies to improve their efficiency. We had earlier demonstrated that addition of small amounts of exogenous glycosaminoglycans (GAGs) like heparan sulfate or chondroitin sulfate to different arginine-rich peptide–DNA complexes (polyplexes) led to an increase in their gene delivery efficiency. This was possibly due to the formation of a ‘GAG coat’ on the polyplex surface through electrostatic interactions which improved their extracellular stability and subsequent cellular entry. In this report, we have attempted to elucidate the differences in intracellular processing of the chondroitin sulfate (CS)-coated polyplexes in comparison to the native polyplexes by using a combination of endocytic inhibitors and co-localization with endosomal markers in various cell lines. We observed that both the native and CS-coated polyplexes are internalized by multiple endocytic pathways although in some cell lines, the coated polyplexes are taken up primarily by caveolae mediated endocytosis. In addition, the CS-coat improves the endosomal escape of the polyplexes as compared to the native polyplexes. Interestingly, during these intracellular events, exogenous CS is retained with the polyplexes until their accumulation near the nucleus. Thus we show for the first time that exogenous GAGs in small amounts improve intracellular routing and nuclear accumulation of arginine-based polyplexes. Therefore, addition of exogenous GAGs is a promising strategy to enhance the transfection efficiency of cationic arginine-rich peptides in multiple cell types

Differences in DNA condensation and release by lysine and arginine homopeptides govern their DNA delivery efficiencies

Designing of nanocarriers that can efficiently deliver therapeutic DNA payload and allow its smooth intracellular release for transgene expression is still a major constraint. The optimization of DNA nanocarriers requires thorough understanding of the chemical and structural characteristics of the vector-nucleic acid complexes and its correlation with the cellular entry, intracellular state and transfection efficiency. l-Lysine and l-arginine based cationic peptides alone or in conjugation with other vectors are known to be putative DNA delivery agents. Here we have used l-lysine and l-arginine homopeptides of three different lengths and probed their DNA condensation and release properties by using a multitude of biophysical techniques including fluorescence spectroscopy, gel electrophoresis and atomic force microscopy. Our results clearly showed that although both lysine and arginine based homopeptides condense DNA via electrostatic interactions, they follow different pattern of DNA condensation and release in vitro. While lysine homopeptides condense DNA to form both monomolecular and multimolecular complexes and show differential release of DNA in vitro depending on the peptide length, arginine homopeptides predominantly form multimolecular complexes and show complete DNA release for all peptide lengths. The cellular uptake of the complexes and their intracellular state (as observed through flow cytometry and fluorescence microscopy) seem to be controlled by the peptide chemistry. The difference in the transfection efficiency of lysine and arginine homopeptides has been rationalized in light of these observations