Reactive and bioactive cationic α-helical polypeptide template for nonviral gene delivery.

Polypeptides were the first set of materials considered for use as nonviral gene delivery vectors. With its ability to bind and condense anionic plasmid DNA, cationic poly-l-lysine (PLL) was one of the most well studied of the early gene delivery polypeptides. Unfortunately, as a DNA delivery vector, unmodified PLL suffered from low transfection efficiency. Although there have been tremendous efforts to increase the efficiency of PLL-mediated gene delivery by incorporating various motifs such as saccharide, imidazole, and guanidinium groups, the improvement has been limited. As such, enthusiasm for PLL and its modified variants as transfection agents has dwindled. As an alternative, many basic gene delivery studies now utilize a more efficient material such as polyethylenimine (PEI). As the use of PLL in gene delivery studies declined, the function of peptide-based materials gradually shifted to other roles relevant to transfection. For example, through covalent conjugation to existing vectors, peptides found use as bioactive agents capable of adding functionality such as cell targeting, nuclear localization, or membrane destabilization to existing gene delivery materials. Membrane destabilization, in particular, has been a large area for peptide use in nonviral gene delivery systems. The cell-penetrating peptides (CPPs) penetratin, transportan, melittin, GALA, TAT, and oligoarginine are some of the commonly used peptide-based materials for membrane destabilization. When incorporated into delivery vectors, these CPPs have been shown to lead to increased internalization, improved endocytic escape, and overall better transfection efficiency. While effective in promoting membrane destabilization as part of a larger vector, CPPs are often too small or lack an adequate cationic charge density to function as stand-alone gene delivery agents. All cationic polypeptides (PLL, modified PLL, or other polypeptide electrolytes) adopt random coil structures because strong intramolecular charge repulsion between side chains prohibits helix formation. However, a shared feature among many CPPs is a helical secondary structure that allows them to interact with and destabilize lipid bilayers such as the cell and endosomal membranes. Because of this discrepancy in secondary structure, there has been no report of cationic polypeptides that can function as both a gene delivery vector with comparable or better transfection efficiency than some of the leading nonviral delivery vectors, and a CPP that destabilizes cellular membranes. We recently reported a strategy for the facile generation of cationic and helical polypeptides. Typically, cationic polypeptides such as PLL are unable to adopt helical conformations at physiological pH because of charge disruption with the side chains. However, our findings revealed that the helical structure of cationic polypeptides can be stabilized by increasing the distance between the charged groups of the side chains and the backbone of the polypeptide, thus minimizing the effect of charge repulsion by reducing the charge density on the helix surface (Scheme 1A). Stable helical structures with very high helical content (> 90%) can be achieved by maintaining a minimum separation distance of 11 s bonds between the peptide backbone and the charged side-chain for a polypeptide having completely charged side chains and a reasonable length (degree of polymerization of 60). By following this general strategy, it is possible to generate polypeptide materials that are sufficiently large and positively charged to bind and condense DNA, but also retain the helical structure seen in many CPPs. The unique combination of material properties allows us to examine helicity as a functional motif in the backbone of gene delivery vectors and evaluate its impact on transfection efficiency. Herein we report our efforts to develop a library of cationic a-helical polypeptides with CPP-like properties for gene delivery through the well-known ring-opening polymerization (ROP) of amino acid N-carboxyanhydrides (NCAs). The ROP of g-(4-vinylbenzyl)-l-glutamate Ncarboxyanhydride (VB-Glu-NCA) was used to form poly(g(4-vinylbenzyl)-l-glutamate) (PVBLG; Scheme 1B). PVBLG served as a reactive template that, through subsequent ozonolysis and reductive amination, allowed us to create a library of cationic polypeptides (PVBLGn-X, where n [*] Dr. N. P. Gabrielson, Dr. H. Lu, Dr. L. Yin, Prof. Dr. J. Cheng Department of Materials Science and Engineering University of Illinois, Urbana-Champaign 1304 W. Green Street, Urbana, IL 61801 (USA) E-mail: [email protected] Homepage: http://cheng.matse.illinois.edu/