Biomedical Polymers Laboratory, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, PR China.
Langmuir. 2012 Jan 31;28(4):2056-65. doi: 10.1021/la203843m. Epub 2011 Dec 28.
pH and reduction dual-bioresponsive nanosized polymersomes based on poly(ethylene glycol)-SS-poly(2-(diethyl amino)ethyl methacrylate) (PEG-SS-PDEA) diblock copolymers were developed for efficient encapsulation and triggered intracellular release of proteins. PEG-SS-PDEA copolymers with PDEA-block molecular weights ranging from 4.7, 6.8, to 9.2 kg/mol were synthesized in a controlled manner via reversible addition-fragmentation chain transfer (RAFT) polymerization of 2-(diethyl amino)ethyl methacrylate (DEAEMA) using PEG-SS-CPADN (CPADN = 4-cyanopentanoic acid dithionaphthalenoate; M(n) PEG = 1.9 kg/mol) as a macro-RAFT agent. These copolymers existed as unimers in water at mildly acidic pH (<7.2) conditions, but readily formed monodisperse nanosized polymersomes (54.5-66.8 nm) when adjusting solution pH to 7.4. These polymersomes were highly sensitive to intracellular pH and reductive environments, which resulted in fast dissociation and aggregation of polymersomes, respectively. Notably, both fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (FITC-BSA) and cytochrome C (FITC-CC) proteins could facilely be encapsulated into polymersomes with excellent protein-loading efficiencies, likely as a result of electrostatic interactions between proteins and PDEA. The in vitro release studies showed that protein release was minimal (<20% in 8 h) at pH 7.4 and 37 °C. The release of proteins was significantly enhanced at pH 6.0 due to collapse of polymersomes. Notably, the fastest protein release was observed under intracellular-mimicking reductive environments (10 mM dithiothreitol, pH 7.4). MTT assays in RAW 264.7 and MCF-7 cells indicated that PEG-SS-PDEA (9.2 k) polymersomes had low cytotoxicity up to a polymer concentration of 300 μg/mL. Confocal laser scanning microscope (CLSM) observations revealed that FITC-CC-loaded PEG-SS-PDEA (9.2 k) polymersomes efficiently delivered and released proteins into MCF-7 cells following 6 h of incubation. Importantly, flow cytometry assays showed that CC-loaded PEG-SS-PDEA (9.2 k) polymersomes induced markedly enhanced apoptosis of MCF-7 cells as compared to free CC and CC-loaded PEG-PDEA (8.9 k) polymersomes (reduction-insensitive control). These dual-bioresponsive polymersomes have appeared to be highly promising for intracellular delivery of protein drugs.
基于聚乙二醇-SS-聚（2-（二乙氨基）乙基甲基丙烯酸酯）（PEG-SS-PDEA）两亲性嵌段共聚物的 pH 和还原双重响应纳米级聚合物囊泡被开发用于高效封装和触发蛋白质的细胞内释放。通过使用聚乙二醇-SS-CPADN（CPADN=4-氰基戊酸二硫代萘酸酯；M（n）PEG=1.9kg/mol）作为大分子RAFT 试剂，通过可逆加成-断裂链转移（RAFT）聚合 2-（二乙氨基）乙基甲基丙烯酸酯（DEAEMA），以可控方式合成了 PDEA 嵌段分子量分别为 4.7、6.8 和 9.2kg/mol 的 PEG-SS-PDEA 嵌段共聚物。这些共聚物在轻度酸性 pH（<7.2）条件下以单体形式存在于水中，但当调节溶液 pH 值至 7.4 时，很容易形成单分散纳米级聚合物囊泡（54.5-66.8nm）。这些聚合物囊泡对细胞内 pH 和还原环境高度敏感，分别导致聚合物囊泡快速解离和聚集。值得注意的是，荧光素异硫氰酸酯（FITC）标记的牛血清白蛋白（FITC-BSA）和细胞色素 C（FITC-CC）蛋白都可以很容易地封装到聚合物囊泡中，具有优异的蛋白载药效率，这可能是由于蛋白与 PDEA 之间的静电相互作用。体外释放研究表明，在 pH7.4 和 37°C 时，蛋白释放最小（<20%，8h）。由于聚合物囊泡的崩溃，在 pH6.0 时蛋白质的释放显著增强。值得注意的是，在模拟细胞内还原环境（10mM 二硫苏糖醇，pH7.4）下观察到最快的蛋白质释放。RAW264.7 和 MCF-7 细胞中的 MTT 测定表明，PEG-SS-PDEA（9.2k）聚合物囊泡的细胞毒性低，聚合物浓度高达 300μg/mL。共聚焦激光扫描显微镜（CLSM）观察表明，负载 FITC-CC 的 PEG-SS-PDEA（9.2k）聚合物囊泡在孵育 6 小时后能够有效地将蛋白递送到 MCF-7 细胞中并释放。重要的是，流式细胞术分析表明，与游离 CC 和负载 CC 的 PEG-PDEA（8.9k）聚合物囊泡（无还原敏感性对照）相比，负载 CC 的 PEG-SS-PDEA（9.2k）聚合物囊泡诱导 MCF-7 细胞明显增强的细胞凋亡。这些双重生物响应聚合物囊泡似乎非常有希望用于蛋白质药物的细胞内递送。
