Modular Design and Facile Synthesis of Enzyme-Responsive Peptide-Linked Block Copolymers for Efficient Delivery of Doxorubicin

Construction of efficient doxorubicin (DOX) delivery systems addressing a cascade of physiological barriers remains a great challenge for better therapeutic efficacy of tumors. Herein, we design well-defined enzyme-responsive peptide-linked block copolymer, PEG-GPLGVRGDG-P(BLA-co-Asp) [PEG and P(BLA-co-Asp) are poly(ethylene glycol) and partially hydrolyzed poly(β-benzyl l-aspartate) (PBLA), respectively] (P3), with modular functionality for efficient delivery of DOX. The block copolymers were successfully obtained via click reaction to introduce peptide (alkynyl-GPLGVRGDG) into the end of PEG for initiating ring-opening polymerization of β-benzyl l-aspartate N-carboxyanhydride (BLA-NCA) by terminal amino groups followed by partial hydrolysis of PBLA segments. P3 micelle was demonstrated to encapsulate DOX efficiently through synergistic effect of benzyl group-based hydrophobic and carboxyl moiety-based electrostatic interactions. Effective matrix metalloproteinase-2 (MMP-2)-triggered cleavage of peptide for dePEGylation of P3 micelles was confirmed and residual RGD ligands were retained on the surfaces. Against HT1080 cells overexpressing MMP-2, DOX-loaded P3 micelles showed approximately 4-fold increase of the cellular internalization amount as compared with free DOX and half maximal inhibitory concentration (IC₅₀) value of DOX-loaded P3 micelles was determined to be 0.38 μg/mL compared with 0.66 μg/mL of free DOX due to MMP-triggered dePEGylation, RGD-mediated cellular uptake, and rapid drug release inside cells. Binding and penetration evaluation toward HT1080 multicellular tumor spheroids (MCTs) confirmed high affinity and deep penetration of P3 micelles in tumor tissues. This modular design of enzyme-responsive block copolymers represents an effective strategy to construct intelligent drug delivery vehicles for addressing a cascade of delivery barriers.