鱼精蛋白改性柞蚕丝素靶向基因载体用于血管再生治疗

We aim to build a novel and biodegradable DNA delivery system, with functions of cell targeting, endosomal escaping and nuclear localization, which will be used in the gene therapy of limb ischemic diseases. This novel delivery vector was prepared by conjugating protamine sulfate (PS) to Antheraea pernyi silk fibroin (ASF) through oxidization of thiols introduced for the formation of disulfide linkage and then complexed with VEGF165-Ang-1 double genes co-expression plasmid by electrostatic absorption to form PS-s-s-ASF/DNA nanoparticles. This smart delivery vector has the following characteristics: 1) good biocompatibility and biodegradability; 2) the controllable degree of surface charge density and modification in their formation; 3) the ability of effectively condensing and protecting DNA as well as high transfection efficiency; 4) the unique multiple targeting function: RGD and Histidine in ASF surface accelerates the cells targeting and endosomal escape, nuclear localization signals in PS endows the carrier nuclear localization function; 5) the broken of –s-s- in cytoplasm facilitates DNA releasing in the cytosolic reductive environment; 6) strong stimulating ability that makes cells secrete VEGF165 and Ang-1 factors to promote angiogenesis and stable neovascular structures, enhanceing the vascularization efficiently.

本项目设计构建了具有细胞靶向性、内涵体逃逸及细胞核定位多功能的可生物降解的新型柞蚕丝素基因传递载体用于下肢缺血性疾病的基因治疗。该载体由巯基化的柞蚕丝素（ASF-SH）偶联巯基化的鱼精蛋白（PS-SH）生成PS-s-s-ASF，并通过静电作用与VEGF-Ang-1双基因共表达质粒形成PS-s-s-ASF/DNA复合物。该载体具有以下特点：1）良好生物相容性和降解性；2）表面电荷密度及改性程度等都方便可控；3）有效压缩并保护DNA，具有较高的转染效率；4）独特的多重靶向功能：ASF上的RGD和His帮助提高细胞膜靶向性及内涵体逃逸性，PS上的核定位信号促进DNA快速定位到细胞核；5）胞浆还原环境下，-s-s-键断裂，DNA从复合物内部游离并释放；6）刺激细胞分泌VEGF165及Ang-1因子，促进血管再生的同时稳定新生血管结构，实现高效促血管再生作用。

本项目将含有核定位短肽的鱼精蛋白(PS)偶联到富含精氨酸-甘氨酸-天门冬氨酸(RGD)三肽序列和组氨酸(His)的柞蚕丝素蛋白(ASF)上，构建同时具有细胞靶向性、内涵体逃逸及细胞核定位多功能的可生物降解新型阳离子化丝素(AP)。利用(AP+PEI)共包被基因载体传递促血管再生的VEGF165-Ang-1双基因共表达质粒原位转染细胞，促进被转染细胞持续、局部地分泌VEGF、Ang-1等血管再生所需的生长因子，促进微血管再生，加快血管化速度，以解决缺血性疾病的血管化问题。研究利用柞蚕丝素蛋白的羧基与鱼精蛋白的氨基发生偶联反应生成酰胺键，建立对柞蚕丝素进行阳离子化改性的技术。当柞蚕丝素与鱼精蛋白质量比为100/10时，柞蚕丝素的Zeta电位从-5.2 mV上升到+15.5 mv，等电点pI从4.2变为8.7。采用(AP+PEI)共包被法能够高效得包裹pDNA，并保护pDNA免受核酸酶的降解。复合物表面Zeta电位为+20~+30 mV，平均直径分布为200~400 nm。(AP+PEI)共包被法能介导质粒pDNA成功转染EA.hy926细胞，并表现出较高的转染效率和较低的细胞毒性。将(AP+PEI)/pDNA复合物装载到丝素支架中体外鸡胚尿囊膜实验显示，该复合物能够刺激其生成丰富的血管，且血管形态呈多分支的弥漫放射状，细胞在该支架内部能正常生长并分泌表达VEGF，且随时间的延长VEGF分泌量显著增多。体内实验结果显示，该材料有助于引导新生组织的长入和毛细血管形成，且随着修复时间的延长，支架内新生毛细血管和胶原纤维的生成量明显多于空白材料组。在创面修复早期，材料内VEGF、Ang-1的阳性表达率也比空白材料组高，进一步说明装载鱼精蛋白阳离子化柞蚕丝素/DNA的材料能够原位转染周围细胞并表达和分泌促血管化生长因子，为组织修复细胞的迁移、粘附、生长及功能发挥创造良好的微环境，进而促进血管再生。