(305f) The Identity of Cargo Charge and Length Affect Cell Penetrating Peptide Mediated Cellular Internalization

Cell penetrating peptides (CPPs) have been the subject to a wide range of research in recent years as they have emerged as powerful tools for delivering bioactive cargoes into the cytosol of intact cells. Conjugation of positively charged short peptide sequences to small molecules, biosensors, antimicrobial drugs, cancer therapeutics, and nanoparticles allows them to permeate the lipid membrane of intact cells without compromising the cellular integrity. Prior work by Safa et. al. identified a β-hairpin CPP (RWRWR: RWVRVpGOWIRQ) that was both long-lived and behaved as a CPP¹. Safa et. al. then incorporated this CPP into a novel fluorescent biosensor to measure deubiquitinating enzyme activity in cancer cells². The second study found that incorporation of a biosensor cargo diminished the uptake kinetics of the peptide biosensor. The majority of CPP literature focuses on two areas: (1) the identification of novel CPPs of various structures and sequences or (2) the utility of a CPP to enhance cellular uptake of bioactive compounds; however, there are limited studies on the role of the cargo on CPP uptake. This work aims to address this gap in knowledge by examining how peptide cargoes of varying length and net charge impact the overall uptake of the CPP-conjugated peptides. A small library of peptides was synthesized conjugating different cargoes to established CPPs: RWRWR and nona-arginine (RRRRRRRRR) X different cargoes we added to the CPPs with different combinations of glycine (G), glutamic acid (E) and arginine (R) focusing on three characteristics: net charge, length, and sequence (position of positively charged arginine residues). Time- and concentration-dependent fluorometry studies showed that peptide sequences with the highest net positive charge exhibited the most prominent uptake kinetics while cargoes with a higher number of negatively charged amino acids almost completely inhibited peptide uptake. Interestingly, cargo length did not appear to have a significant effect with 8 amino acid cargoes entering as approximately the same rate as 4 amino acid cargoes with a net +2 charge on the cargo. A similar trend was observed with respect to the two CPPs as reported by Safa et al with nona-arginine-conjugated peptides achieving greater uptake than RWRWR-conjugated peptides¹. However, the RWRWR-conjugated peptides exhibited enhanced intracellular stability due to the secondary structure of the β-hairpin. Viability studies confirmed that neither CPP, no matter the cargo identity, were cytotoxic validating their utility as delivery mechanism. Fluorescence microscopy studies showed a mostly homogeneous distribution of the RWRWR-conjugated peptides for multiple cargoes confirming endosomal escape and successful delivery of the cargo the cytosol. The findings here highlight additional design criteria when conjugating different cargoes to different CPPs and that the net charge of the cargo can dramatically affect the performance of the CPP.

References:

[1]: N. Safa, J.C. Anderson, M. Vaithiyanathan, J.H. Pettigrew, G.A. Pappas, D. Liu, T.J. Gauthier, A.T. Melvin, CPProtectides: Rapid uptake of well-folded β- hairpin peptides with enhanced resistance to intracellular degradation, Pept. Sci. (2018).

[2]: N. Safa, J.H. Pettigrew, T.J. Gauthier, A.T. Melvin, Direct measurement of deubiquitinating enzyme activity in intact cells using T a protease-resistant, cell-permeable, peptide-based reporter, BEJ (2019).