Self-Assembled Amphiphilic Fluorinated Random Copolymers for the Encapsulation and Release of the Hydrophobic Combretastatin A-4 Drug

Water-soluble amphiphilic random copolymers composed of tri(ethylene glycol) methacrylate (TEGMA) or poly(ethylene glycol) methyl ether methacrylate (PEGMA) and perfluorohexylethyl acrylate (FA) were synthesized by ARGET-ATRP, and their self-assembling and thermoresponsive behavior in water was studied by dynamic light scattering (DLS) and UV-vis spectroscopy. The copolymer ability to self-fold in single-chain nano-sized structures (unimer micelles) in aqueous solutions was exploited to encapsulate Combretastatin A-4 (CA-4), which is a very hydrophobic anticancer drug. The cloud point temperature (T-cp) was found to linearly decrease with increasing drug concentration in the drug/copolymer system. Moreover, while CA-4 was preferentially incorporated into the unimer micelles of TEGMA-ran-FA, the drug was found to induce multi-chain, submicro-sized aggregation of PEGMA-ran-FA. Anyway, the encapsulation efficiency was very high (>= 81%) for both copolymers. The drug release was evaluated in PBS aqueous solutions both below and above T-cp for TEGMA-ran-FA copolymer and below T-cp, but at two different drug loadings, for PEGMA-ran-FA copolymer. In any case, the release kinetics presented similar profiles, characterized by linear trends up to approximate to 10-13 h and approximate to 7 h for TEGMA-ran-FA and PEGMA-ran-FA, respectively. Then, the release rate decreased, reaching a plateau. The release from TEGMA-ran-FA was moderately faster above T-cp than below T-cp, suggesting that copolymer thermoresponsiveness increased the release rate, which occurred anyway by diffusion below T-cp. Cytotoxicity tests were carried out on copolymer solutions in a wide concentration range (5-60 mg/mL) at 37 degrees C by using Balb/3T3 clone A31 cells. Interestingly, it was found that the concentration-dependent micro-sized aggregation of the amphiphilic random copolymers above T-cp caused a sort of "cellular asphyxiation" with a loss of cell viability clearly visible for TEGMA-ran-FA solutions (T-cp below 37 degrees C) with higher copolymer concentrations. On the other hand, cells in contact with the analogous PEGMA-ran-FA (T-cp above 37 degrees C) presented a very good viability (>= 75%) with respect to the control at any given concentration.