Amphiphilic Copolymer Inhibition of PNIPAM-PS Aggregation is HLB Dependent

Abstract

Injury due to physical trauma is mediated by alteration in biomolecular structure and intra- and inter-molecular bonding. When primary molecular structure of proteins survives trauma, we postulate that disaggregation may be catalyzed using certain amphiphilic block copolymers (ABCs). The ABC's ability to disaggregate is highly affected by structure, molecular weight, and hydrophobicity (HLB). A range of engineered polystyrene- poly(N-isopropylacrylamide)-polystyrene (PS-PNIPAm-PS) protein mimics (PMs) with different hydrophobic domains were engineered to study effectiveness of PEO-PPO ABCs to disaggregate thermally denatured PMs. PMs were synthesized by reverse-addition-fragmentation-transfer (RAFT) polymerization. The chain transfer agent, 3,5-Bis(2-dodecylthiocarbonothioylthio-1-oxopropoxy)benzoic acid, NIPAm monomer, and azobisisobutylnitrile (AIBN) initiator were mixed in dichloromethane under nitrogen (70 °C, 24 h), purified by precipitation in hexanes, and filtered. The resulting macroCTA was mixed with AIBN and styrene under nitrogen (70 °C, 6 h). Conversion to final PM structure was confirmed by size exclusion chromatography (SEC) and 1HNMR analysis in CDCl3. Cloud point at 400 nm was measured by thermal ramping (Shimadzu UV-3600 Plus Spectrophotometer). Differential scanning calorimetry (DSC) (TA Instruments Discovery 2500) was used to measure heat flow. Dynamic Light Scattering (DLS) (637 nm, 35mW laser, Brookhaven BI-APD autocorrelator) was used to measure hydrodynamic radii (Rh). SEC analysis revealed complete conversion of monomers to PMs. NMR confirmed PM structure: 7.2-5.8ppm (aromatic PS), 4.0ppm (-CONHCH(CH3)), 2.7-0.9 ppm (PS/PNIPAM backbone, -CONHCH(CH3)2). DSC and UV-VIS indicated that PMs have transitions in the expected temperature range (30-33 °C) and are fully reversible. DLS showed the heated PMs (50 °C) had higher Rh (∼500-800nm) compared to 20 °C PMs (10 ± 0.5 nm). The addition of ABCs reduced the Rh 59-95%. With all PMs, the larger the Mw of the chaperone, the greater the effect in reducing aggregate size.