Contrasting effects of cysteine modification on the transfection efficiency of amphipathic peptides.

Delivery of DNA to cells remains a key challenge towards development of gene therapy. A better understanding of the properties involved in stability and transfection efficiency of the vector could critically contribute to the improvement of delivery vehicles. In the present work we have chosen two peptides differing only in amphipathicity and explored how presence of cysteine affects DNA uptake and transfection efficiency. We report an unusual observation that addition of cysteine selectively increases transfection efficiency of secondary amphipathic peptide (Mgpe-9) and causes a drop in the primary amphipathic peptide (Mgpe-10). Our results point the effect of cysteine is dictated by the importance of physicochemical properties of the carrier peptide. We also report a DNA delivery agent Mgpe-9 exhibiting high transfection efficiency in multiple cell lines (including hard-to-transfect cell lines) with minimal cytotoxicity which can be further explored for in vivo applications.

Insight into the role of physicochemical parameters in a novel series of amphipathic peptides for efficient DNA delivery.

Amphipathic peptides constitute a class of molecules with the potential to develop as efficient and safer alternatives to viral and other nonviral vectors for intracellular delivery of therapeutics. These peptides can be useful for nucleic acid delivery and hence promise to have pharmaceutical application, particularly in gene therapy. In order to design novel amphipathic peptides and improve their efficiency of therapeutic cargo delivery, one needs to understand the role of the physicochemical properties of the peptide. There are very few reports in the literature where the physicochemical properties of the peptide have been correlated with efficiency of plasmid DNA delivery. In the present work we hunted out a naturally occurring amphipathic peptide termed Mgpe-1 (derived from HUMAN Protein phosphatase 1E) as a possible novel DNA delivery agent. We systematically altered the physicochemical parameters of this peptide to further enhance its DNA delivery efficiency. We changed its amphipathicity (from secondary to primary), the total charge (from +6 to +9), hydrophobicity, and the amino acid composition (lysine and serines to arginine; substitution of tryptophan) and studied which of these alterations affect DNA delivery efficiency. Our results showed that although Mgpe-1 exhibited very strong cellular uptake, its plasmid DNA delivery efficiency was poor. The presence of nine arginines improved the DNA delivery efficiency, and the effect was observed in both the primary and the secondary amphipathic variants. We further observed that the presence of tryptophan was important but not essential and the effect of its removal was stronger in the case of the secondary amphipathic peptide. However, increase in total hydrophobicity of the peptide led to a fall in transfection efficiency in the primary amphipathic peptide whereas the secondary amphipathic peptide having the same chemical composition was almost unaffected by this change. The primary amphipathic peptides with high positive charge and low hydrophobicity formed colloidally stable polyplexes with DNA and avoided a major impediment in DNA delivery, namely, the aggregation of polyplexes and cytotoxicity. The secondary amphipathic variants by virtue of the positional arrangement of the amino acids led to formation of polyplexes with partly hydrophilic surfaces which prevented aggregation and controlled particle size irrespective of the hydrophobicity. Two variants in the series Mgpe-3 and Mgpe-4 having nine positive charges with less hydrophobicity showed high transfection efficiency in multiple cell lines along with serum stability and much less cytotoxicity and promise to be novel and efficient DNA delivery vectors.