Endocytosis has been implicated in the cellular uptake of arginine-rich, cell-penetrating peptides (CPPs). However, accumulating evidence suggests that certain conditions allow the direct, non-endocytic penetration of arginine-rich peptides through the plasma membrane. We previously showed that Alexa Fluor 488-labeled dodeca-arginine (R12-Alexa488) directly enters cells at specific sites on the plasma membrane and subsequently diffuses throughout cells. In this study, we found that the peptide influx was accompanied by the formation of unique, “particle-like” multivesicular structures on the plasma membrane, together with topical inversion of the plasma membrane. Importantly, the conjugation of dodeca-arginine (R12) to Alexa Fluor 488 or a peptide tag derived from hemagglutinin (HAtag) significantly accelerated particle formation, suggesting that the chemical properties of the attached molecules (cargo molecules) may contribute to translocation of the R12 peptide. Coincubation with R12-HAtag allowed the membrane-impermeable R4-Alexa488 to permeate cells. These results suggest that R12 peptides attached to hydrophobic cargo molecules stimulate dynamic morphological alterations in the plasma membrane, and that these structural changes allow the peptides to permeate the plasma membrane. These findings may provide a novel mode of cell permeabilization by arginine-rich peptides as a means of drug delivery.

Dynamic single-chain polymeric nanoparticles (SCPNs) are intriguing, bioinspired architectures that result from the collapse or folding of an individual polymer chain into a nanometer-sized particle. Here we present a detailed biophysical study on the behavior of dynamic SCPNs in living cells and an evaluation of their catalytic functionality in such a complex medium. We first developed a number of delivery strategies that allowed the selective localization of SCPNs in different cellular compartments. Live/dead tests showed that the SCPNs were not toxic to cells while spectral imaging revealed that SCPNs provide a structural shielding and reduced the influence from the outer biological media. The ability of SCPNs to act as catalysts in biological media was first assessed by investigating their potential for reactive oxygen species generation. With porphyrins covalently attached to the SCPNs, singlet oxygen was generated upon irradiation with light, inducing spatially controlled cell death. In addition, Cu(I)- and Pd(II)-based SCPNs were prepared and these catalysts were screened in vitro and studied in cellular environments for the carbamate cleavage reaction of rhodamine-based substrates. This is a model reaction for the uncaging of bioactive compounds such as cytotoxic drugs for catalysis-based cancer therapy. We observed that the rate of the deprotection depends on both the organometallic catalysts and the nature of the protective group. The rate reduces from in vitro to the biological environment, indicating a strong influence of biomolecules on catalyst performance. The Cu(I)-based SCPNs in combination with the dimethylpropargyloxycarbonyl protective group showed the best performances both in vitro and in biological environment, making this group promising in biomedical applications.