柞蚕丝素纳米纤维温敏复合膜制备及其生物相容性

Preparation and biocompatibility of temperature-sensitive composite membrane of tussah silk fibroin nanofiber

为探究柞蚕丝素蛋白温敏纳米纤维的水溶液静电纺丝工艺及其生物相容性,以烯丙基缩水甘油醚改性柞蚕丝素蛋白(ASF-AGE)为基材、N-异丙基丙烯酰胺(NIPAAm)为单体,通过原位溶液聚合和静电纺丝技术制备p(ASF-AGE-PNIPAAm)温敏纳米纤维膜,探究单体质量配比对纳米纤维膜形成的影响,分析纳米纤维膜的温度响应性、亲疏水性、体外降解及细胞相容情况。结果表明:ASF-AGE与NIPAAm以1∶1.5质量比进行聚合并经静电纺丝成功制备均匀连续的纳米纤维膜,平均直径为(452±120)nm;纳米纤维膜在32.7~33.4℃具有明显的温度响应性,在45℃具有显著的疏水性;纳米纤维膜在蛋白酶XIV中更易发生降解,降解28 d质量损失率可达39.6%;纳米纤维膜与小鼠脑微血管内皮细胞b End.3共培养未表现出细胞毒性,经5 d和7 d培养展现出良好的细胞相容性,有利于b End.3细胞增殖黏附。

Objective Antheraea pernyi silk fibroin(ASF)-based nanofibers have wide potential for biomaterial applications because it contains tripeptide sequences(Arg-Gly-Asp)known as RGD,whereby the integrin binding motif promotes the cell attachment.At present,electrospinning of regenerated ASF nanofibers is usually performed with volatile organic solvents,which may cause potential toxicity to the encapsulated cells,and residual organic solvents will cause safety hazards to cells and human bodies.Therefore,it is necessary to find a method for electrospinning of ASF in aqueous solutions to prepare nanofibers.Method Novel ASF-based thermo-responsive hydrogel nanofibers were developed using aqueous electrospinning without any harsh organic solvent,and the employability of such nanofibers as an in-vitro platform for cell culture was explored.In order to study the spinning process and biocompatibility of temperature-sensitive nanofiber membrane of tussah silk fibroin,p(ASF-AGE-PNIPAAm)nanofiber membrane was prepared by in-situ solution polymerization and electrospinning,allyl glycidyl ether(AGE)modified tussah silk fibroin was used as base material(ASF-AGE)and N-isopropylacrylamide was used as monomer.The influence of monomer ratio on the formation of nanofiber membrane was investigated,and the temperature response,hydrophilicity,in-vitro degradation and cell compatibility of nanofiber membrane were analyzed.Results ASF-based thermoresponsive nanofibers(p(ASF-AGE-NIPAAm))were successfully manufactured by aqueous electrospinning with the polymerization of ASF and N-isopropylacrylamide(NIPAAm).The results showed that when ASF-AGE and NIPAAm were polymerized at a mass ratio of 1∶1.5,uniform and continuous nanofiber membrane with an average diameter of(452±120)nm were successfully prepared by electrospinning.The nanofibers exhibited good thermoresponsive characteristics that the lower critical solution temperature(LCST)was similar with PNIPAAm at about 32℃.The nanofiber membrane showed obvious temperature responsiveness at 32.7-33.4℃,and significant hydrophobicity at 45℃.The nanofiber membrane was prone to degrade in protease XIV,and the weight loss rate reached 39.6%after 28 d of degradation.Excellent cell proliferation,viability and morphology were demonstrated for b End.3 cells on the nanofibers by the characteristic methylthiazolyldiphenyl-tetrazolium bromide assay and confocal laser scanning microscope.It was not cytotoxic in co-culture of nanofiber membrane with mouse brain microvascular endothelial cells b End.3,and good cytocompatibility was found in co-culture of 5 d and 7 d.It was demonstrated that b End.3 cells grown on nanofibers showed improved cell adhesion,proliferation,and viability.The result indicated that the nanofiber membrane was beneficial to b End.3 cells adhesion and proliferation.Conclusion Novel ASF-based thermoresponsive nanofibers were successfully fabricated by aqueous electrospinning for b End.3 cells culture.These nanofiber membranes have obvious thermoresponsive and the LCST is close to human body temperature.The utilization of a thermoresponsive polymer in the development of cell culture platforms allowed the dynamic control of cell adhesion and detachment in a desired manner by changing the temperature for targeted purposes.Furthermore,the nanofibers can be degraded in protease XIV solution,the degradation products of silk based materials are soluble peptides and free amino acids,which are easily metabolized and absorbed by the human body.By culturing brain microvascular endothelial cells in vitro,the nanofibers support cell adhesion and growth well.These degradable and thermoresponsive hydrogels will have potential applications for cells delivery device and tissue scaffold.This is a convenient and feasible approach to fabricate ASF-based functional nanofibers in the application of cell culture,presenting a valuable route for developing an ASF-based cell culture platform.