Synthesis of glutathione-responsive, amphiphilic methotrexate-based prodrug nanoparticles: In vitro and in vivo assessment against metastatic breast cancer.

Regarding the limitations of free methotrexate (MTX), such as non-specific effects and systemic toxicity, this study introduces a novel folate-targeted and glutathione-responsive polyprodrug nanoparticle of methotrexate, created using reversible addition-fragmentation chain transfer (RAFT) polymerization. First, poly(N-[3-aminopropyl] methacrylamide hydrochloride) (PAPMA.HCl) was synthesized and chain-extended with a disulfide-containing methacrylate monomer, producing PAPMA-b-PHESEM diblock copolymer. Folic acid (FA) and PEG5000 were then conjugated to this copolymer to enhance tumor targeting and improve hydrophilicity, respectively. MTX was covalently attached to the copolymer via a disulfide bond, resulting in PPFAPM, which enables glutathione (GSH)-dependent release in the tumor microenvironment. This polymer system self-assembled into spherical nanoparticles with an approximate Z-average of 216 nm, a negative zeta potential, a low critical micelle concentration (CMC) of 25 μg/mL, and redox-sensitive MTX release. PPFAPM nanoparticles demonstrated dose-dependent cytotoxicity against 4T1 breast cancer cells. Receptor-mediated uptake was confirmed through folate receptor blocking with excess FA, while in vivo tests on 4T1 tumor-bearing BALB/c mice showed significant tumor growth inhibition by PPFAPM nanoparticles. Overall, this research highlights the potential of a GSH-responsive copolymer decorated with FA to target overexpressed folate receptors on 4T1 cells, serving as a promising platform for treating metastatic breast cancer.