Kapelner Rachel A, Fisher Rachel S, Elbaum-Garfinkle Shana, Obermeyer Allie C
Department of Chemical Engineering, Columbia University New York NY 10027 USA
Structural Biology Initiative, CUNY Advanced Science Research Center New York NY USA.
Chem Sci. 2022 Nov 29;13(48):14346-14356. doi: 10.1039/d2sc00192f. eCollection 2022 Dec 14.
Proteins are an important class of biologics, but there are several recurring challenges to address when designing protein-based therapeutics. These challenges include: the propensity of proteins to aggregate during formulation, relatively low loading in traditional hydrophobic delivery vehicles, and inefficient cellular uptake. This last criterion is particularly challenging for anionic proteins as they cannot cross the anionic plasma membrane. Here we investigated the complex coacervation of anionic proteins with a block copolymer of opposite charge to form polyelectrolyte complex (PEC) micelles for use as a protein delivery vehicle. Using genetically modified variants of the model protein green fluorescent protein (GFP), we evaluated the role of protein charge and charge localization in the formation and stability of PEC micelles. A neutral-cationic block copolymer, poly(oligoethylene glycol methacrylate--quaternized 4-vinylpyridine), POEGMA--qP4VP, was prepared RAFT polymerization for complexation and microphase separation with the panel of engineered anionic GFPs. We found that isotropically supercharged proteins formed micelles at higher ionic strength relative to protein variants with charge localized to a polypeptide tag. We then studied GFP delivery by PEC micelles and found that they effectively delivered the protein cargo to mammalian cells. However, cellular delivery varied as a function of protein charge and charge distribution and we found an inverse relationship between the PEC micelle critical salt concentration and delivery efficiency. This model system has highlighted the potential of polyelectrolyte complexes to deliver anionic proteins intracellularly. Using this model system, we have identified requirements for the formation of PEC micelles that are stable at physiological ionic strength and that smaller protein-polyelectrolyte complexes effectively deliver proteins to Jurkat cells.
蛋白质是一类重要的生物制品，但在设计基于蛋白质的治疗药物时，有几个反复出现的挑战需要解决。这些挑战包括：蛋白质在制剂过程中易于聚集、在传统疏水递送载体中的负载相对较低以及细胞摄取效率低下。对于阴离子蛋白来说，最后一个标准尤其具有挑战性，因为它们无法穿过阴离子质膜。在这里，我们研究了阴离子蛋白与带相反电荷的嵌段共聚物形成聚电解质复合物（PEC）胶束作为蛋白质递送载体的复凝聚过程。使用模型蛋白绿色荧光蛋白（GFP）的基因改造变体，我们评估了蛋白质电荷和电荷定位在PEC胶束形成和稳定性中的作用。通过可逆加成-断裂链转移（RAFT）聚合制备了一种中性-阳离子嵌段共聚物聚（聚乙二醇甲基丙烯酸酯-季铵化4-乙烯基吡啶），即POEGMA-qP4VP，用于与一系列工程化阴离子GFP进行络合和微相分离。我们发现，相对于电荷定位于多肽标签的蛋白质变体，各向同性超荷电蛋白质在更高的离子强度下形成胶束。然后，我们研究了PEC胶束对GFP的递送，发现它们能有效地将蛋白质货物递送至哺乳动物细胞。然而，细胞递送因蛋白质电荷和电荷分布而异，并且我们发现PEC胶束的临界盐浓度与递送效率之间存在反比关系。这个模型系统突出了聚电解质复合物在细胞内递送阴离子蛋白的潜力。使用这个模型系统，我们确定了在生理离子强度下稳定的PEC胶束形成的要求，以及较小的蛋白质-聚电解质复合物能有效地将蛋白质递送至Jurkat细胞的要求。
