Preparation and drug release evaluation of spider fibroin membrane
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摘要: 天然蜘蛛丝具有优越的力学性能，其多种剂型具有优异的抗菌活性、生物相容性及良好的导热性。开发具有良好生物相容性的纯天然生物载药材料，对于降低患者的强烈免疫原性反应具有重要意义。通过对蛛丝在不同溶剂中溶解及成膜方式的探索，优化反应条件：以六氟异丙醇做溶解溶剂，以1∶1 (mg∶mL)的料液质量体积比进行投料，在60℃条件下溶解8 h，通过溶剂浇铸法成膜，并结合相关表征探索其以罗丹明B为模型药物的体外释药规律。通过浸提法测试材料细胞毒性，将蛛丝丝素蛋白膜浸提液与细胞共培养显示其对小鼠胚胎成骨细胞无潜在细胞毒性。制得的载药蛛丝丝素蛋白膜产率高、厚度均一、操作简单，为天然蜘蛛丝丝素蛋白的溶解及其膜剂的制备提供了一项简单易行的技术。Abstract: Natural spider silk has superior mechanical properties, and its various forms have excellent antibacterial activity, biocompatibility, and good thermal conductivity. It is of great significance to develop natural biological drug-loaded materials with good biocompatibility for reducing the strong immunogenicity reaction of patients. By exploring the dissolution and membrane-forming methods of spider silk in different solvents, the reaction conditions were optimized, hexafluoroisopropanol was used as the dissolution solvent, the material ratio was 1∶1 (mg∶mL), the spider silk was dissolved at 60℃ for 8 h, and the membrane was formed by solvent casting, and the drug release in vitro of rhodamine B (RhB) as the model drug was explored with the correlation characterization. The cytotoxicity of the material was tested by extraction method. The spider fibroin membrane extract co-culture with cells showed that the material had no potential cytotoxicity to mouse embryonic osteoblasts cells. The drug-loaded spider silk fibroin membrane prepared by this method has high yield, uniform thickness and simple operation, which provides a simple and feasible technology for the dissolution of natural spider silk protein and the preparation of membrane agent.
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Key words:
- spider silk /
- hexafluoroisopropanol /
- silk fibroin /
- solvent casting method /
- sustained release /
- RhB
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图 2 蛛丝丝素蛋白膜宏观图：(a) 2 mg/mL；(b) 6 mg/mL；(c) 10 mg/mL；(d) 15 mg/mL；(e) 20 mg/mL
Figure 2. Macroscopic plots of drug-loaded silk protein membrane: (a) 2 mg/mL; (b) 6 mg/mL; (c) 10 mg/mL; (d) 15 mg/mL; (e) 20 mg/mL
图 3 载罗丹明B (RhB)蛛丝丝素蛋白膜表面((a)~(e))及截面((a1)~(e1))的SEM图像：((a), (a1)) 2 mg/mL；((b), (b1)) 6 mg/mL；((c), (c1)) 10 mg/mL；((d), (d1)) 15 mg/mL；((e), (e1)) 20 mg/mL
Figure 3. SEM images of surface ((a)-(e)) and cross-section ((a1)-(e1)) for rhodamine B (RhB) loaded silk protein membrane: ((a), (a1)) 2 mg/mL; ((b), (b1)) 6 mg/mL; ((c), (c1)) 10 mg/mL; ((d), (d1)) 15 mg/mL; ((e), (e1)) 20 mg/mL
图 4 成膜蛛丝丝素蛋白液浓度与膜厚关系
Figure 4. Relation between filamentin protein solution concentration and membrane thickness
图 5 RhB及其载药蛛丝丝素蛋白膜的XRD图谱
Figure 5. XRD patterns of RhB and its loaded spider filament protein membrane
图 6 蛛丝丝素蛋白膜拉伸强度及断裂伸长率
Figure 6. Tensile strength and elongation at break of silk protein membrane
图 7 药物在HFIP中不同时间段的稳定性薄层色谱图
Figure 7. Thin-layer chromatogram of drug stability in HFIP for different time periods
图 8 不同浓度蛛丝丝素蛋白液成膜释药曲线
Figure 8. Membrane release curves of different concentrations of spider filamin protein solution
图 9 空白蛛丝丝素蛋白膜浸提液细胞共培养24 h结果
HDPE—High density polyethylene; DMSO—Dimethylsulfoxide
Figure 9. Results of cell co-culture of extracts for 24 h
表 1 蛛丝丝素蛋白膜正交试验因素水平表
Table 1. Level of factors in orthogonal test of silk fibroin protein membrane
Level A B C Time/h Solid-liquid
mass-to-volume
ratio/(mg∶mL)Temperature/℃ 1 1 1∶2 20 2 4 1∶1 40 3 8 2∶1 60 
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表 2 天然蛛丝在不同溶剂中的溶解率
Table 2. Solubility of natural spider silk in different solvents
Solvent CH2O2 TFA HFIP Membrane 
Dissolution rate 7.90% 82.50% 71.50% Notes: TFA—Trifluoroacetic acid; HFIP—Hexafluoroisopropanol.
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表 3 天然蛛丝溶解条件正交设计试验结果
Table 3. Results of orthogonal design test on dissolution conditions of natural spider silk
Group Factor Dissolution rate/% A B C 1 1 1 1 54.26 2 1 2 2 62.89 3 1 3 3 59.89 4 2 2 3 74.85 5 2 3 1 68.63 6 2 1 2 69.44 7 3 3 2 71.74 8 3 1 3 72.70 9 3 2 1 69.74 K1 177.04 196.40 192.63 K2 212.92 207.48 204.07 K3 214.18 200.26 207.44 R 37.14 11.08 14.81 Notes: K—The sum of experimental results corresponding to any level sign i (i =1, 2, 3); R—Range.
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表 4 正交实验方差分析
Table 4. Orthogonal experimental analysis of variance
Factor Sum of squares Degree of freedom Mean square F value P value A 296.48 2 148.24 65.01 0.02 B 21.09 2 10.54 4.62 0.18 C 40.17 2 20.09 8.81 0.10 Error 4.56 2 2.28 Notes: F—Variance ratio; P—Probability.
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表 5 不同成膜浓度载药蛛丝丝素蛋白膜厚度
Table 5. Membrane thickness of silk protein protein loaded at different membrane-forming concentrations
Group/(mg·mL−1) Average thickness
/μm2 312.3 6 212.7 10 143.6 15 83.3 20 65.4
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[1] GOSLINE J M, DEMONT M E, DENNY M W. The structure and properties of spider silk[J]. Endeavour,1986,10:37-43. doi: 10.1016/0160-9327(86)90049-9 [2] SALEHI S, KOECK K, SCHEIBEL T. Spider silk for tissue engineering applications[J]. Molecules,2020,25(3):737-756. doi: 10.3390/molecules25030737 [3] 温睿. 三种蜘蛛丝蛋白基因结构及仿生蛛丝的性能研究[D]. 上海: 东华大学, 2020.WEN Rui. The study of three spidroin gene structures and biomimetic spider silk properties[D]. Shanghai: Donghua University, 2020(in Chinese). [4] PEZHMAN M, FABIAN Z, WOLFGONG W, et al. Interfacial crystallization and supramolecular self-assembly of spider silk inspired protein at the water-air interface[J]. Materials (Basel),2021,14(15):4239-4252. doi: 10.3390/ma14154239 [5] ZEPLIN P H, BERNINGER A K, MAKSIMOVIKJ N C, et al. Improving the biocompatibility of silicone implants using spider silk coatings: Immunohistochemical analysis of capsule formation[J]. Handchirurgie, Mikrochirurgie, Plastische Chirurgie,2014,46(6):336-341. doi: 10.1055/s-0034-1395558 [6] HTUT K Z, ALICEA-SERRANO A M, SINGLA S, et al. Correlation between protein secondary structure and mechanical performance for the ultratough dragline silk of Darwin's bark spider[J]. Journal of the Royal Society Interface,2021,18(179):320-330. [7] 周逸中. 新型嵌合体蛛丝蛋白的外源表达、成丝机理及人工蛛丝纤维的纺制与应 [D]. 上海: 东华大学, 2020.ZHOU Yizhong. The heterogenous production, fiber-formation mechanism of a new type chimeric spidroins and the spinning, applications of artificial spider silks[D]. Shanghai: Donghua University, 2020(in Chinese). [8] YU Q Z, SUN S Z. A three-dimensional multiporous fibrous scaffold fabricated with regenerated spider silk protein/poly(l-lactic acid) for tissue engineering[J]. Journal of Biomedical Materials Research Part A,2015,103(2):721-730. doi: 10.1002/jbm.a.35224 [9] QING C, LI Q Y, XUE N N, et al. The outlook of the development of innovative products from biocompatible natural spider silk in the beauty thread-lifting industry[J]. Natural Products and Bioprospecting,2021,11(1):21-30. doi: 10.1007/s13659-020-00291-9 [10] 马跃, 胡艳磊, 刘亮, 等. 蚕丝蛋白基高强度水凝胶的制备及其生物应用[J]. 复合材料学报, 2023, 40(9): 4893-4916.MA Yue, HU Yanlei, LIU Liang, et al. Preparation and biological application of high strength hydrogels based on silk fibroin[J]. Acta Materiae Compositae Sinica, 2023, 40(9): 4893-4916(in Chinese). [11] HU Y, YU J, LIU L, et al. Preparation of natural amphoteric silk nanofibers by acid hydrolysis[J]. Journal of Materials Chemistry B,2019,7(9):1450-1459. doi: 10.1039/C8TB03005G [12] DING Z, CHENG W, MIA M S, et al. Silk biomaterials for bone tissue engineering[J]. Macromolecular Bioscience,2021,21(8):2100153-2100165. doi: 10.1002/mabi.202100153 [13] 李昶, 倪中石. 仿生蜘蛛丝微纳米复合材料的集水性能[J]. 复合材料学报, 2022, 39(6):2515-2526. doi: 10.13801/j.cnki.fhclxb.20220107.001LI Chang, NI Zhongshi. Water harvesting of bio-inspired micro/nano-structured spider silk[J]. Acta Materiae Compositae Sinica,2022,39(6):2515-2526(in Chinese). doi: 10.13801/j.cnki.fhclxb.20220107.001 [14] HARRIS T, PATERSON C, FARJOOD F, et al. Utilizing recombinant spider silk proteins to develop a synthetic bruch's membrane for modeling the retinal pigment epithelium[J]. ACS Biomaterials Science & Engineering, 2019, 5(8): 4023-4036. [15] MAZUREK Ł, SZUDZIK M, RYBKA M, et al. Silk fibroin biomaterials and their beneficial role in skin wound healing[J]. Biomolecules,2022,12(12):1852. doi: 10.3390/biom12121852 [16] 中国机械工业联合会. 游标、带表和数显卡尺: GB/T 21389—2008[S]. 北京: 中国标准出版社, 2008.China Machinery Industry Federation. Verniers, strap-on tables, and digital graphics calipers: GB/T 21389—2008[S]. Beijing: China Standards Press, 2008(in Chinese). [17] ERKAN Y, MUSTAFA S. A novel and simple deep eutectic solvent based liquid phase microextraction method for rhodamine B in cosmetic products and water samples prior to its spectrophotometric determination[J]. Spectrochim Ica Acta Part A—Molecular and Biomolecular Spectroscopy,2018,202:81-86. doi: 10.1016/j.saa.2018.04.073 [18] 国家药典委员会. 中华人民共和国药典: 2020年版[M]. (四部). 北京: 中国医药科技出版社, 2020: 121-124.National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China: 2020 edition[M]. (Part 4). Beijing: China Medical Science and Technology Press, 2020: 121-124(in Chinese). [19] International Organization for Standardization. Biological evaluation of medical devices—Part 12: Sample preparation and reference materials: ISO-10993-12-2021[S]. Switzerland: Geneva Publishers, 2007. [20] 国家药品监督管理局. 医疗器械生物学评价: GB/T 16886.5—2017[S]. 北京: 中国标准出版社, 2017.State Medical Products Administration. Biological evaluation of medical devices: GB/T 16886.12—2017[S]. Beijing: China Standards Press, 2017(in Chinese). [21] LI X G, WU L Y, HUANG M R, et al. Conformational transition and liquid crystalline state of regenerated silk fibroin in water[J]. Biopolymers,2008,89(6):497-505. doi: 10.1002/bip.20905 [22] ALEKSANDRA P K, PAVEL V K, ELENA F K. Recent advances in development of functional spider silk-based hybrid materials[J]. Frontiers In Chemistry,2020,8:554-574. doi: 10.3389/fchem.2020.00554 [23] 卿晨. 基于天然蛛丝蛋白制备载药口腔膜剂及其释药性能初探[D]. 大理: 大理大学, 2022.QING Chen. Preliminary investigation on preparation of drugloaded oral membrane based on natural spider silk protein and its drug release properties[D]. Dali: Dali University, 2022(in Chinese). [24] 何京亚, 桑广泽, 吴红. 芬太尼双层口颊片含量及释放度测定方法的研究[J]. 中国药师, 2016, 19(4):755-757.HE Jingya, SANG Guangze, WU Hong. Study on content and release determination of fentanyl bilayer buccal tables[J]. China Pharmacist,2016,19(4):755-757(in Chinese). -
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