Department of Orthopedic Surgery, West China Hospital, Sichuan University, Chengdu 610041, China.
Technology Center for Public Research, West China Hospital, Sichuan University, Chengdu 610041, China.
Biomaterials. 2015 Jun;52:1-13. doi: 10.1016/j.biomaterials.2015.02.024. Epub 2015 Feb 18.
Safe and high-efficiency gene therapy for nucleus pulposus (NP) regeneration was urgently desired to treat disc degeneration-associated diseases. In this work, an efficient nonviral cationic block copolymer gene delivery system was used to deliver therapeutic plasmid DNA (pDNA), which was prepared via complexation between the mixed cationic block copolymers, poly(ethylene glycol)-block-poly{N-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide} [PEG-b-PAsp(DET)] and poly(N-isopropylacrylamide)-block-PAsp(DET) [PNIPAM-b-PAsp(DET)], and pDNA at 25 °C. The mixed polyplex micelles (MPMs) containing heterogeneous coronas with hydrophobic and hydrophilic microdomains coexisting could be obtained upon heating from 25 to 37 °C, which showed high tolerability against nuclease and strong resistance towards protein adsorption. The gene transfection efficiency of MPMs in NP cells was significantly higher than that of regular polyplex micelles prepared from sole block copolymer of PEG-b-PAsp(DET) (SPMs) in in vitro and in vivo evaluation due to the synergistic effect of improved colloidal stability and low cytotoxicity. High expression of heme oxygenase-1 (HO-1) in NP cells transfected by MPMs loading HO-1 pDNA significantly decreased the expression activity of matrix metalloproteinases 3 (MMP-3) and cyclo-oxygenase-2 (COX-2) induced by interleukin-1β (IL-1β), and simultaneously increased the NP phenotype-associated genes such as aggrecan, type II collagen, and SOX-9. Moreover, the therapeutic effects of MPMs loading pDNA were tested to treat disc degeneration induced by stab injury. The results demonstrated that administration of HO-1 pDNA carried by MPMs in rat tail discs apparently reduced inflammatory responses induced by need stab and increased glycosaminoglycan (GAG) content, finally achieving better therapeutic efficacy as compared with SPMs. Consequently, MPMs loading HO-1 pDNA were demonstrated to be potential as a safe and high-efficiency nonviral gene delivery system for retarding or regenerating the degenerative discs.
安全高效的基因治疗方法是治疗椎间盘退变相关疾病的迫切需求。在这项工作中，采用了一种高效的非病毒阳离子嵌段共聚物基因传递系统来传递治疗性质粒 DNA(pDNA)，该系统是通过混合阳离子嵌段共聚物聚乙二醇-嵌段-聚[N-[N-(2-氨基乙基)-2-氨基乙基]天冬酰胺][PEG-b-PAsp(DET)]和聚(N-异丙基丙烯酰胺)-嵌段-PAsp(DET)[PNIPAM-b-PAsp(DET)]与 pDNA 在 25°C 下复合制备的。当温度从 25°C 升高到 37°C 时，混合的聚合物胶束(MPMs)可以得到含有疏水性和亲水性微区共存的异质冠，表现出对核酸酶的高耐受性和对蛋白质吸附的强抵抗力。在体外和体内评价中，MPMs 对 NP 细胞的基因转染效率明显高于由 PEG-b-PAsp(DET)的单一嵌段共聚物(SPMs)制备的常规聚合物胶束，这是由于胶体稳定性提高和低细胞毒性的协同作用。转染 HO-1 pDNA 的 MPMs 显著降低了白细胞介素 1β(IL-1β)诱导的 NP 细胞中基质金属蛋白酶 3(MMP-3)和环氧化酶 2(COX-2)的表达活性，同时增加了 NP 表型相关基因如聚集蛋白聚糖、Ⅱ型胶原和 SOX-9 的表达。此外，还测试了 MPMs 负载 pDNA 治疗刺伤引起的椎间盘退变的治疗效果。结果表明，MPMs 负载 HO-1 pDNA 处理大鼠尾椎间盘明显降低了针刺引起的炎症反应，增加了糖胺聚糖(GAG)含量，最终达到了比 SPMs 更好的治疗效果。因此，MPMs 负载 HO-1 pDNA 被证明是一种安全有效的非病毒基因传递系统，可用于延缓或再生退变的椎间盘。
