Cell-Penetrating and Enzyme-Responsive Peptides for Targeted Cancer Therapy: Role of Arginine Residue Length on Cell Penetration and In Vivo Systemic Toxicity

Abstract

For the improved delivery of cancer therapeutics and imaging agents, the conjugation of cell-penetrating peptides (CPPs) increases the cellular uptake and water solubility of agents. Among the various CPPs, arginine-rich peptides have been the most widely used. Combining CPPs with enzyme-responsive peptides presents an innovative strategy to target specific intracellular enzymes in cancer cells and when combined with the appropriate click chemistry can enhance theranostic drug delivery through the formation of intracellular self-assembled nanostructures. However, one drawback of CPPs is their high positive charge which can cause nonspecific binding, leading to off-target accumulation and potential toxicity. Hence, balancing cell-specific penetration, toxicity, and biocompatibility is essential for future clinical efficacy. We synthesized six cancer-specific, legumain-responsive R n AANCK peptides containing one to six arginine residues, with legumain being an asparaginyl endopeptidase that is overexpressed in aggressive prostate tumors. When conjugated to Alexa Fluor 488, R 1 −R 6 AANCK peptides exhibited a concentration-and timedependent cell penetration in prostate cancer cells, which was higher for peptides with higher R values, reaching a plateau after approximately 120 min. Highly aggressive DU145 prostate tumor cells, but not less aggressive LNCaP cells, self-assembled nanoparticles in the cytosol after the cleavage of the legumain-specific peptide. The in vivo biocompatibility was assessed in mice after the intravenous injection of R 1 −R 6 AANCK peptides, with concentrations ranging from 0.0125 to 0.4 mmol/kg. The higher arginine content in R 4−6 peptides showed blood and urine indicators for the impairment of bone marrow, liver, and kidney function in a dose-dependent manner, with instant hemolysis and morbidity in extreme cases. These findings underscore the importance of designing peptides with the optimal arginine residue length for a proper balance of cell-specific penetration, toxicity, and in vivo biocompatibility.

Conditional Cell-Penetrating Peptide Exposure as Selective Nanoparticle Uptake Signal

“…A higher theoretical net charge of the attached ligand was accompanied by a stronger influence on particle uptake, which was shown as an example of a constant DOM of 50% (Figure , bottom right). This is consistent with the literature, which describes an increase in cell penetration properties for longer arginine chains . However, this has not yet been quantitatively investigated for the modification of nanoparticle surfaces and the resulting changes in particle uptake.…”

“…Since high amounts of positive charges are also associated with cytotoxicity, , a compromise must be found between uptake enhancement and cytotoxic effects. Our ζ 1/2 value could serve as an orientation benchmark for this.…”

“…This is consistent with the literature, which describes an increase in cell penetration properties for longer arginine chains. 53 However, this has not yet been quantitatively investigated for the modification of nanoparticle surfaces and the resulting changes in particle uptake. Additionally, this aspect suggests that there is not only a correlation between the theoretical net charge of the ligand but also between the total nanoparticle ζ-potential influenced by the CPP modification and its uptake.…”