Cell-Penetrating
and Enzyme-Responsive Peptides for
Targeted Cancer Therapy: Role of Arginine Residue Length on Cell Penetration
and In Vivo Systemic Toxicity
posted on 2024-02-22, 13:03authored byBehnaz Ghaemi, Swati Tanwar, Aruna Singh, Dian R. Arifin, Michael T. McMahon, Ishan Barman, Jeff W. M. Bulte
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
RnAANCK 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, R1–R6AANCK peptides
exhibited a concentration- and time-dependent 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 R1–R6AANCK peptides, with concentrations ranging from 0.0125 to
0.4 mmol/kg. The higher arginine content in R4–6 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.