Deciphering the Role of Chondroitin Sulfate in Increasing the Transfection Efficiency of Amphipathic Peptide-Based Nanocomplexes.

Glycosaminoglycans, both cell-surface and exogenous, can interfere with DNA delivery efficiency of nonviral carrier systems. In this work, we report an extensive comparative study to explore the effect of exogenously added chondroitin sulfate on biophysical characteristics, cellular uptake, transfection efficiency, and intracellular trafficking of nanocomplexes formed using primary and secondary amphipathic peptides developed in our laboratory. Our results indicate that the presence of exogenous chondroitin sulfate exhibits differential enhancement in transfection efficiency of the amphipathic peptides depending upon their chemical nature. The enhancement was more pronounced in primary amphipathic peptide-based nanocomplexes as compared to the secondary counterpart. This difference can be attributed to possible alteration of the intracellular entry pathway in addition to increased extracellular stability, less cellular toxicity, and assistance in nuclear accumulation. These results imply potential use of glycosaminoglycans such as chondroitin sulfate to improve the transfection efficiency of primary amphipathic peptides for possible in vivo applications.

Efficient Cellular Entry of (r-x-r)-Type Carbamate-Plasmid DNA Complexes and Its Implication for Noninvasive Topical DNA Delivery to Skin.

Arginine-rich cell penetrating peptides are powerful tools for in vitro as well as in vivo delivery of a wide plethora of biomolecules. However, presence of consecutive arginine residues leads to enhanced amenability for proteolytic degradation as well as steric hindrances for membrane interactions which compromise its bioavailability. In order to overcome these limitations we previously reported a safe and stable octaarginine based oligomer, i.e., (r-x-r)4-carbamate, where the backbone amide linkages were replaced by carbamate linkages and 6-aminohexanoic acid based spacer moieties were incorporated for better flexibility, hydrophobicity, optimal spacing of guanidinium groups, and protection against proteolytic cleavage; resulting in improved transfection efficiency over its amide counterpart. In the present work we have investigated the mechanism behind this enhanced transfection efficiency and, based on our observations, demonstrate how the synergistic effect of rationalized oligomer designing, complex characteristics, and cell type contributes to overall effective intracellular delivery. Our results indicate that the (r-x-r)4-carbamate-plasmid DNA complexes primarily utilize lipid raft dependent pathway of cellular entry more than other pathways, and this possibly facilitates their increased entry in the lipid raft rich milieu of skin cells. We also emphasize the utility of oligomer (r-x-r)4-carbamate as an efficient carrier for topical delivery of nucleic acids in skin tissue. This carrier can be utilized for safe, efficient, and noninvasive delivery of therapeutically relevant macromolecular hydrophilic cargo like nucleic acids to skin.