Cell penetrating peptides (CPPs) 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, and nanoparticles allows them to permeate the lipid membrane of intact cells without compromising the cellular integrity. 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 (RWVRVpGOWIRQ) and nona-arginine (RRRRRRRRR). Eight different cargoes were 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). 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 at approximately the same rate as 4 amino acid cargoes with like charges. A similar trend was observed with respect to the two CPPs as previously reported with nona-arginine-conjugated peptides achieving greater uptake then RWRWR-conjugated peptides. However, the RWRWR-conjugated peptides exhibited enhanced intracellular stability due to the β-hairpin secondary structure of the CPP. Viability studies confirmed that neither CPP, no matter the cargo identity, were cytotoxic validating their utility as a 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.

Identity of cargo charge and length affect cell penetrating peptide mediated cellular internalization

ACS Fall 2021

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