关键词: 柞蚕丝素蛋白, 纳米纤维膜, N-异丙基丙烯酰胺, 温敏性, 生物相容性

Abstract:

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(L_CST) 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 L_CST 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.

Key words: tussah silk fibroin, nanofiber membrane, N-isopropylacrylamide, thermosensitivity, biocompatibility