Enzyme/pH-sensitive nanoparticles based on poly(β-l-malic acid) for drug delivery with enhanced endocytosis

Abstract

Nanoparticles (NPs) derived from branched copolymers of poly (β-L-malic acid) (PMLA) have been extensively investigated for drug delivery due to their high density of pendant carboxyl groups. This abundant functional group availability enhances their potential as effective drug delivery systems; however, the strong negative charge of PMLA poses a challenge in its uptake by cancer cells due to electrostatic repulsion. In this study, we developed novel enzyme- and pH-sensitive nanoparticles (EP-NPs) based on PMLA, demonstrating tumor-specific behavior and selective activation within tumor tissues. To enhance the cellular internalization of the nanoparticles, we incorporated transactivator of transcription (TAT). In summary, long-chain polyethylene glycol (PEG) was conjugated to PMLA to confer specificity to the TAT peptide. This was achieved using a tetrapeptide linker: alanine–alanine–asparagine–leucine (AANL), which serves as a substrate for legumain. Legumain is a highly conserved cysteine protease primarily found in lysosomes and blood vessels, initially discovered in legumes. It is markedly overexpressed in numerous solid tumors, as well as in endothelial cells and tumor-associated macrophages. The release of doxorubicin in tumor cells was sustained due to the low pH (5.0–5.5) and degradation of PMLA. The PEG modification optimized the particle size and shielded the nanoparticles from plasma proteins and detection by the reticuloendothelial system, thereby prolonging their long circulation time. Once the nanoparticles reached the tumor microenvironment, the AANL was cleaved by legumain, exposing the TAT peptide on the surface, which enhances cellular internalization. Both in vitro and in vivo efficacy studies demonstrated that these EP-NPs significantly inhibited tumor growth while exhibiting negligible systemic toxicity, thereby suggesting that the developed enzyme/pH-sensitive PMLA-based nanoparticle holds great promise as an anti-tumor drug delivery system.