Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China; Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Jiangsu Key Laboratory of Biomedical Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210046, China.
Biomaterials. 2014 Sep;35(27):7909-18. doi: 10.1016/j.biomaterials.2014.05.066. Epub 2014 Jun 20.
Safety and high efficacy of vectors are essential requirements for gene therapy. To address these challenges, poly(carboxy betaine methacrylate ethyl ester)-poly(carboxy betaine methacrylate) (PCBMAEE-PCBMA) diblock copolymers were synthesized to form core-shell vector for gene delivery. The hydrophobic PCBMAEE segment, a polyzwitterionic precursor, can condense plasmid DNA (pDNA) into a hydrophobic core, which improves pDNA protection from nuclease attack and maintains the condensed structure against dilution. Moreover, the hydrolysis of PCBMAEE in uptaken gene vectors can enhance the pDNA release and reduce the cytotoxicity caused by the cationic polymer accumulation in the host cells. The PCBMA segment, zwitterionic fouling resistant material, is utilized to stabilize the gene vector in the complex medium and reduce the interference from serum proteins without impeding the endocytosis of DNA vector like PEG protection layer. Results showed that the complex formed by PCBMAEE50-PCBMA14 with luciferase or pEGFP gene exhibit higher transfection efficacy of pDNA than that formed by PEI 25 kDa or Lipofectamine(®) 2000 in tested cell lines (COS-7, HepG-2, HeLa, and HUVEC), especially, in difficult-to-transfect ones, such as HeLa and HUVEC. The luciferase expression level infected by the vectors of PCBMAEE50-PCBMA14/pGL-4 at N/P = 20/1 is 27 times of the branched PEI 25 kDa in COS-7 cells and 16 times of Lipofectamine(®) 2000 in HUVEC. Furthermore, the complex formed by PCBMAEE50-PCBMA14 also show advantages in transfection rate, dosage effectiveness and preservation of transfecting activity in serum contained growth medium. The luciferase expression of the vectors of PCBMAEE50-PCBMA14/pGL-4 at N/P = 20/1 is 230 times higher than that of PEI complex at low vector dosage (the 5% standard dosage). And the transfection rate is 25 times higher than that of PEI complex in 10% serum contained growth medium. In short, all these results indicated that the polymeric gene vector, consisted of convertible hydrophobic polyzwitterionic precursor and fixed polyzwitterionic fouling resistant segment, is a promising candidate for high and stable gene transfection in complex growth medium.
安全高效的载体是基因治疗的基本要求。为了解决这些挑战，合成了聚（羧基甜菜碱甲基丙烯酸乙酯）-聚（羧基甜菜碱甲基丙烯酸）（PCBMAEE-PCBMA）两亲嵌段共聚物，以形成用于基因传递的核壳载体。疏水 PCBMAEE 段作为聚两性离子前体，可以将质粒 DNA（pDNA）浓缩到疏水区，从而提高 pDNA 对核酸酶攻击的保护作用，并保持浓缩结构不受稀释的影响。此外，在摄取的基因载体中 PCBMAEE 的水解可以增强 pDNA 的释放，并减少阳离子聚合物在宿主细胞中积累引起的细胞毒性。PCBMA 段，抗污染的两性离子材料，用于稳定复杂介质中的基因载体，并减少血清蛋白的干扰，而不会像 PEG 保护层一样阻碍 DNA 载体的内吞作用。结果表明，与 25 kDa 的 PEI 或 Lipofectamine（®）2000 相比，用 Luciferase 或 pEGFP 基因形成的 PCBMAEE50-PCBMA14 复合物在测试的细胞系（COS-7、HepG-2、HeLa 和 HUVEC）中表现出更高的 pDNA 转染效率，尤其是在难以转染的细胞系，如 HeLa 和 HUVEC。在 COS-7 细胞中，用 PCBMAEE50-PCBMA14/pGL-4 载体感染的 Luciferase 表达水平是 25 kDa 的 branched PEI 的 27 倍，在 HUVEC 中是 Lipofectamine（®）2000 的 16 倍。此外，PCBMAEE50-PCBMA14 形成的复合物在含血清的生长培养基中也具有转染率、剂量效率和转染活性保持方面的优势。在低载体剂量（标准剂量的 5%）下，PCBMAEE50-PCBMA14/pGL-4 载体的 Luciferase 表达是 PEI 复合物的 230 倍。在含 10%血清的生长培养基中，转染率是 PEI 复合物的 25 倍。总之，这些结果表明，由可转化的疏水聚两性离子前体和亲水聚两性离子抗污染段组成的聚合物基因载体是在复杂生长培养基中进行高效、稳定基因转染的有前途的候选物。
