Well-Defined Cholesterol Polymers with pH-Controlled Membrane Switching Activity

Cholesterol has been used as an effective component of therapeutic delivery systems because of its ability to cross cellular membranes. Considering this, well-defined copolymers of methacrylic acid and cholesteryl methacrylate, poly(methacrylic acid-co-cholesteryl methacrylate) P(MAA-co-CMA), were generated as potential delivery system components for pH-controlled intracellular delivery of therapeutics. Statistical copolymers with varying cholesterol contents (2, 4, and 8 mol %) were synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization. Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameters of the copolymers in aqueous solutions ranged from 5 ± 0.3 to 7 ± 0.4 nm for the copolymers having 2 and 4 mol % CMA and 8 ± 1.1 to 13 ± 1.9 nm for the copolymer having 8 mol % CMA with increasing pH (pH 4.5–7.4). Atomic force microscopy (AFM) analysis revealed that the copolymer having 8 mol % CMA formed supramolecular assemblies while the copolymers having 2 and 4 mol % CMA existed as unimers in aqueous solution. The pH-responsive behavior of the copolymers was investigated via UV–visible spectroscopy revealing phase transitions at pH 3.9 for 2 mol % CMA, pH 4.7 for 4 mol % CMA, and pH 5.4 for 8 mol % CMA. Lipid bilayers and liposomes as models for cellular membranes were generated to probe their interactions with the synthesized copolymers. The interactions were determined in a pH-dependent manner (at pH 5.0 and 7.4) using surface plasmon resonance (SPR) spectroscopy and liposome leakage assay. Both the SPR analyses and liposome leakage assays indicated that the copolymer containing 2 mol % CMA displayed the greatest polymer–lipid interactions at pH 5.0, presenting the highest binding ability to the lipid bilayer surfaces, and also demonstrating the highest membrane destabilization activity. CellTiter–Blue assay showed that the copolymers did not affect the cell viability up to 30 μM over a period of 72 h.