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

Abstract

Cholesterol has been used as an effective component of therapeutic delivery systems because of its ability to cross cellular membranes. Considering this, welldefined 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.

Assessment of Cholesterol-Derived Ionic Copolymers as Potential Vectors for Gene Delivery

“…Copolymer compositions with increasing CMA units tend to form supramolecular structures, where lipid moieties are brought together exposing the charged molecules on the outer layer. 31 Additionally, with decreasing MAA moieties in the copolymer composition, fewer acidic groups occupy the oligolysine amine units triggering the complexes to exhibit overall higher surface charges. Table 2 represents the diameter and ζ-potential of Q-P(DMAEMA-co-CMA)-siRNA complexes prepared at N/P 20:1 ratio in aqueous solution.…”

“…The procedure for the RAFT-mediated polymerizations of anionic P(MAA-co-CMA) (A-2% CMA, cholesterol content 2 mol %, with number average molecular weight (M n ) GPC of 16500 g/mol and polydispersity index (PDI) of 1.19; A-4% CMA, cholesterol content 4 mol %, with (M n ) GPC 15800 g/mol and PDI 1.10; A-8% CMA, cholesterol content 8 mol %, with (M n ) GPC 18000 g/mol and PDI 1.11) copolymer series has been previously described. 31 The preparation of quarternized-P(DMAEMA-co-CMA) copolymer series with varying CMA units (Q-2% CMA, cholesterol content 2 mol %, with (M n ) GPC 23400 g/mol and PDI 1.13; Q-4% CMA, cholesterol content 4 mol %, with (M n ) GPC 24800 g/mol and PDI 1.15; Q-8% CMA, cholesterol content 8 mol %, with (M n ) GPC 19200 g/mol and PDI 1.14; Q-15% CMA, cholesterol content 15 mol %, with (M n ) GPC 21400 g/mol and PDI 1.16; Q-20% CMA, cholesterol content 20 mol %, with (M n ) GPC 16000 g/mol and PDI 1.17) is presented in Supporting Information ( Figure S1 and Table S1). Dulbecco's Modified Eagle Medium (DMEM) with 4.5 g/L glucose and L-glutamine, Roswell Park Memorial Institute (RPMI) medium with HEPES and L-glutamine, heat inactivated Fetal Calf Serum (FCS), trypsin solution (0.25% (w/v) trypsin in Hank's solution), Trypan Blue, Opti-MEM I Reduced Serum Medium, Lipofectamine 2000 and Geneticin were purchased from Invitrogen Life Technologies.…”

pH-labile sheddable block copolymers by RAFT polymerization: Synthesis and potential use as siRNA conjugates

“…Cholesterol was purchased from Sigma-Aldrich. Cholesterol methacrylate monomer (CMA) was synthesized according to the procedure described elsewhere [48,49] (2) All the glasswares and chemicals were dried prior to use. A mixture of 1 (0.522 g, 0.1 mmol), 3 Å A 0 molecular sieve (1 g), 1,1,1-tris(hydroxymethyl)ethane (tHME) (120 mg, 1 mmol), anhydrous PTSA (6.9 mg, 0.038 mmol), and anhydrous THF (10 mL) were heated to reflux for 8 h, and cooled to ambient temperature.…”

“…6B) confirmed the presence of the pyridyl disulfide group on the polymer chains, as evidenced by the existence of new signals at 8.4, 7.65 and 7.02 ppm which were ascribed to PCMA-b-siRNA segment under acidic conditions and siRNA under reductive conditions. Here it should be noted that under acidic conditions the release of siRNA together with a cellular membrane-destabilizing block such as PCMA [48] would be desirable for trafficking siRNA to cytosol from endocytic compartments such as endosomes or lysosomes. Subsequent to the release of the membrane-destabilizing block-siRNA conjugate, siRNA is expected to be released from the membrane destabilizing block in the reducing environment of cytosol.…”

Multitarget, quantitative nanoplasmonic electrical field-enhanced resonating device (NE 2 RD) for diagnostics

“…Likewise, electrical detection systems such as electrochemical sensors and interdigitated electrodes sense changes in molecular charges (14)(15)(16)(17) and can provide affordable, simple, and highthroughput measurements. Further, plasmonic-based platforms that use an electrical field around metal surfaces/nanoparticles coupled with light (5,(18)(19)(20)(21)(22) have minimized readout complexity and demonstrated quantitative and sensitive measurements (23)(24)(25)(26). In particular, surface plasmons on 3D-oriented surfaces generate highly sensitive spots that provide a sevenfold stronger electrical field than 2D-oriented plasmonic sensors (27)(28)(29).…”