Aqueous Solution Behavior of Poly(styrene-<i>alt</i>-maleic acid)-<i>b</i>-poly(<i>N</i>-acryloylmorpholine) Double Hydrophilic Block Copolymers in the Absence and Presence of Divalent Cations and Phospholipids

The extraction of membrane proteins (MPs) from their native lipid environment into nanosized poly-(styrene-<i>co</i>-maleic acid) (PSMA)-lipid particles (SMALPs), allows for retention of MP structure and functionality. Furthermore, numerous proteins require metal ions (such as Mg²⁺ and Ca²⁺) to maintain their activity. Generally, SMALPs are destabilized at relatively low concentrations of divalent cation as chelation to PSMA carboxylate functional groups decreases electrostatic repulsion between SMALPs and ultimately facilitates precipitation of the copolymer and the contents of the SMALP. Double hydrophilic block copolymers (DHBCs), in which one block comprises PSMA or one of its analogues, form colloidally stable hybrid polyionic complexes (HPICs) upon exposure to M²⁺, thereby overcoming the divalent cation sensitivity of traditional PSMA macromolecular detergents. Therefore, poly-(styrene-<i>alt</i>-maleic acid)-<i>block</i>-poly-(<i>N</i>-acryloylmorpholine) (PSMA-<i>b</i>-PNAM) and its analogue poly-(4-<i>tert</i>-butylstyrene-<i>alt</i>-maleic acid)-<i>block</i>-poly-(<i>N</i>-acryloylmorpholine) (P<i>t</i>BuSMA-<i>b</i>-PNAM) were synthesized via reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization and the aggregation behavior of PSMA-<i>b</i>-PNAM/M²⁺ and P<i>t</i>BuSMA-<i>b</i>-PNAM/M²⁺ complexes investigated. Under appropriate conditions, these complexes were amenable to self-assembly, while PSMA and P<i>t</i>BuSMA precipitated at significantly lower Mg²⁺/Ca²⁺ concentrations. Additionally, PSMA-<i>b</i>-PNAM/M²⁺ and P<i>t</i>BuSMA-<i>b</i>-PNAM/M²⁺ were efficient solubilizers of synthetic lipid vesicles, facilitating the formation of electrostatically and sterically stabilized SMALPs at unprecedented M²⁺ concentrations. This study effectively highlights the untapped potential of DHBCs as a new class of polymers for membrane protein related research endeavors.