494. Glycosaminoglycans in Gene Delivery: An Effective Strategy for Enhancement of Transfection Efficiency of Amphipathic Peptides for Localized and Systemic Applications?

Abstract

Glycosaminoglycans (GAGs) are negatively charged, linear, sulphated polysaccharides. Cell surface glycosaminoglycans have often been described as portals of cellular entry of cationic peptides and polymers used for gene delivery. On the other hand, exogenous glycosaminoglycans have generally been considered as impediments towards DNA delivery mediated through cationic vectors since they are likely to destabilize these electrostatically assembled complexes. However, recent reports have shown that exogenous glycosaminoglycans can also aid DNA delivery by reducing cytotoxicity and enhancing cellular uptake in some cationic polymers. Our laboratory has shown that addition of small amounts of exogenous GAGs to different cationic arginine-rich peptide-DNA nanocomplexes leads to an increase in their gene delivery efficiency. We observed formation of a ‘GAG coat’ on the nanocomplex surface which improved extracellular stability and subsequent cellular entry along with improved endosomal release and enhanced accumulation near the nucleus. However very little work has been done to understand the role of exogenous glycosaminoglycans on gene delivery by amphipathic peptides. We have developed a set of amphipathic peptides which exhibit high transfection efficiency in multiple cell lines. We explored the effect of addition of chondroitin sulphate to the peptide-DNA nanocomplexes. We observed an increase in nanocomplex size, decrease in surface charge and stability towards nuclease degradation at wt/wt ratios of 0.25 of chondroitin sulfate/peptide indicating its shielding effect. Enhanced transfection efficiency was also observed in multiple cell lines. Interestingly, maximum increase was observed in human keratinocyte cell line. Preliminary transfection results indicate that presence of CD44 could act as primary binding site for coated nanocomplexes leading to increased internalization. In addition, transfection in presence of inhibitors like Bafilomycin A1 indicates that the endosomal escape property of chondroitin sulphate might also play a part. This is being further verified through colocalization studies in presence of endosomal and lysosomal markers. Moreover, on topical application to human skin tissue, we observed increase in DNA delivery with the coated nanocomplexes. Since the skin tissue is also rich in glycosaminoglycans, we are exploring how the skin architecture might be helpful in aiding DNA delivery by GAG-coated nanocomplexes. Interestingly, while the transfection efficiency of the native nanocomplexes was compromised in presence of serum, addition of chondroitin sulfate was able to restore the transfection efficiency. Although the cellular uptake of the coated nanocomplexes was almost equal to that of the uncoated ones in presence of serum, the endosomal escape of the nanocomplexes was higher in coated nanocomplexes which could contribute towards the overall enhancement in transfection. All these results reveal the potential use of glycosaminoglycans to improve the transfection efficiency of amphipathic peptides for localized applications in skin as well as in vivo applications.