Cationic amphiphilic polyproline helices (CAPHs) for cell delivery

Iris M Geisler, Purdue University

Abstract

The transport of therapeutic agents across cell membranes remains a major challenge in the field of drug delivery. Currently, the use of cell penetrating peptides (CPPs), which are rich in basic amino acids, have been shown to be beneficial in the delivery of various cargoes across the cell membrane. We have developed novel CPPs with a rigid secondary structure, referred to as cationic amphiphilic polyproline helices (CAPHs). These newly developed CPPs have been designed and synthesized to introduce cationic and hydrophobic moieties along the rigid backbone of a polyproline type II helix (PPII) in an amphiphilic manner. The CAPHs are comprised of modified polyproline residues, which have been specifically designed to mimick the hydrophilic and/or hydrophobic side chains of natural amino acids and were synthesized via O-alkylation of hydroxyproline. Previous investigations, using MCF-7 cells indicated a dramatic increase in the cellular uptake of CAPHs containing up to six guanidinium groups in comparison to amine-only polyproline compounds. Amphiphilicity has also been identified to play a key role in the enhanced cell translocation. As a continuation of this study, we further investigated length effects and the alteration of the hydrophobic side chains on cellular internalization. Additionally, in an effort to elucidate the pathway of peptide internalization, a number of mechanistic investigations were performed as well. Increased CPP length and cationic/hydrophobic character was found to facilitate cellular uptake efficiency. Mechanistic investigations of the elongated CAPH suggested internalization to occur primarily via an energy independent mechanism. Uptake studies of CAPHs featuring two distinctive hydrophobic functionalities, namely isobutyl or benzyl groups and tested in seven different cell lines, including six cancerous cell lines (MCF-7, HOS, HT1080, HeLa, KB-FD, KB3-1) and one non-cancerous cell line (WI 38) revealed high specificity of P11LRR toward MCF-7 breast cancer cells. Co-culture experiments with P11LRR demonstrated almost exclusive internalization of the CAPH by MCF-7 cells with little internalization by the noncancerous WI38. The replacement of the isobutyl hydrophobic group with a benzyl moiety resulted in a shift in uptake efficiency and specificity across all cell lines. In addition, greater uptake efficiency was observed at low concentrations with CAPHs containing benzyl groups. These results demonstrate that the type of hydrophobic residues utilized in the creation of cell penetrating peptides can strongly influence the extent and specificity of cellular internalization. The CAPH investigation has been further expanded to include the effect of CAPH oligomerization on cellular uptake. This study included the synthesis of dimeric and trimeric CAPHs both of which resulted in a significant increase in uptake efficiency at low concentrations as compared to their monomeric counterparts. Interestingly, subcellular trimer localization was found to vary between trimers containing isobutyl groups versus those containing benzyl groups as the hydrophobic moieties. The trimer containing side chain isobutyl groups localized within the nucleus and endosomes/lysosomes of MCF-7 and HeLa cells. In contrast, the trimer containing benzyl groups was found to localize within the mitochondria of MCF-7 and HeLa cells. However, oligomerization also led to a substantial increase in cytotoxicity at peptide concentrations above 5 μM. Oligomerized CAPHs were also tested for their ability to non-covalently deliver a small fluorescent dye across the cellular membrane. While some dye internalization was facilitated in the presence of dimeric CAPHs, no noticeable increase in dye uptake was observed in the presence of trimeric CAPHs. An all-cationic CAPH has also been created to facilitate the intracellular delivery of DNA through electrostatic interactions between DNA and peptide. Two CAPHs of differing length were prepared in this regard, both of which noticeably facilitate the internalization of a fluorescently labeled 21mer DNA sequence in MCF-7 and HeLa cells.

Degree

Ph.D.

Advisors

Chmielewski, Purdue University.

Subject Area

Organic chemistry

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