Analysis of protective mechanism of silk protein based cryoprotectants_Journal of Biomedical Engineering

Authors：

 ZHOU Xinli , DU Yukun , TENG Yun , ZHANG Xiaomin

Institute of Biothermal Technology, University of Shanghai for Science and Technology, Shanghai 200093, P.R.China;

Corresponding author：

ZHOU Xinli, Email: zjulily@163.com

Keywords：

silk fibroin; cryoprotectant; mechanism analysis

DOI：

10.7507/1001-5515.201807061

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Dimethyl sulfoxide (Me₂SO) supplemented with fetal bovine serum (FBS) is a widely used cryoprotectant combination. However, high concentration of Me₂SO is toxic to cells, and FBS presents problems related to diseases such as bovine spongiform encephalopathy and viral infections. Silk protein is a kind of natural macromolecule fiber protein with good biocompatibility and hydrophilicity. The aim of this paper is to analyze the cryoprotective mechanism of silk protein as cryoprotectant. Firstly, differential scanning calorimetry (DSC) was used to measure the thermal hysteresis activity (THA) of silk protein. The THA of 10 mg/mL sericin protein was 0.96°C, and the THA of 10% (V/V) fibroin protein was 1.15°C. Then the ice recrystallization inhibition (IRI) of silk protein-PBS solution was observed with cryomicroscope. The cold stage was set at − 7°C, after 40 minutes’ incubation, the mean grain size rate (MGSR) of sericin protein and fibroin protein were 28.99% and 3.18%, respectively, which were calculated relative to phosphate buffer saline (PBS) control. It is indicated that sericin and silk fibroin have certain effects of inhibiting recrystallization of ice crystals. Finally, the structure and physicochemical properties of silk protein were analyzed by Fourier transform infrared spectroscopy (FTIR). The results showed that the content of the random coil was 75.62% and the β-sheet structure was 24.38% in the secondary of sericin protein. The content of the β-sheet structure was 56.68%, followed by random coil structure 22.38%, and α-helix 16.84% in the secondary of fibroin protein. The above analysis demonstrates the feasibility of silk fibroin as a cryoprotectant, and provides a new idea for the selection of cryoprotectants in the future.

Citation： ZHOU Xinli, DU Yukun, TENG Yun, ZHANG Xiaomin. Analysis of protective mechanism of silk protein based cryoprotectants. Journal of Biomedical Engineering, 2019, 36(6): 986-993. doi: 10.7507/1001-5515.201807061 Copy

1.	Baust J G, Gao Dayong, Baust J M. Cryopreservation: An emerging paradigm change. Organogenesis, 2009, 5(3): 90-96.
2.	Lovelock J E, Bishop M W. Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature, 1959, 183(4672): 1394-1395.
3.	Guillouzo A, Rialland L, Fautrel A, et al. Survival and function of isolated hepatocytes after cryopreservation. Chem Biol Interact, 1999, 121(1): 7-16.
4.	Nagahara Y, Sekine H, Otaki M, et al. Use of high concentrations of dimethyl sulfoxide for cryopreservation of HepG2 cells adhered to glass and polydimethylsiloxane matrices. Cryobiology, 2016, 72(1): 53-59.
5.	李佳佳, 程腾, 贺小英, 等. 二甲基亚砜和胎牛血清对293T细胞冷冻效果的影响. 信阳师范学院学报:自然科学版, 2013, 26(2): 217-220.
6.	Hunter N, Foster J, Chong A, et al. Transmission of prion diseases by blood transfusion. J Gener Virol, 2002, 83(Pt 11): 2897-2905.
7.	睢晓洁, 潘超, 杨静, 等. 以氧化三甲胺作为细胞冻存保护剂的研究. 中国科技论文, 2017, 12(12): 1341-1345.
8.	滕芸. 基于蚕丝蛋白的低温保护剂的研究. 上海: 上海理工大学, 2018.
9.	Halwani D O, Brockbank K G, Duman J G, et al. Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions. Cryobiology, 2014, 68(3): 411-418.
10.	Sun K H, Jung K E, Won Y H, et al. Improvement in ovarian tissue quality with supplementation of antifreeze protein during warming of vitrified mouse ovarian tissue. Yonsei Med J, 2018, 59(2): 331-336.
11.	Rubinsky B, Arav A, Mattioli M, et al. The effect of antifreeze glycopeptides on membrane potential changes at hypothermic temperatures. Biochem Biophys Res Commun, 1990, 173(3): 1369-1374.
12.	Arav A, Rubinsky B, Fletcher G, et al. Cryogenic protection of oocytes with antifreeze proteins. Mol Reproduct Dev, 1993, 36(4): 488-493.
13.	Carpenter J F, Hansent T N. Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth. Proc Natl Acad Sci U S A, 1992, 89(19): 8953-8957.
14.	Amir G, Horowitz L, Rubinsky B, et al. Subzero nonfreezing cryopresevation of rat hearts using antifreeze protein I and antifreeze protein III. Cryobiology, 2004, 48(3): 273-282.
15.	马庆保, 刘志东. 南极磷虾抗冻蛋白热滞活性的差示扫描量热法评价. 食品科学, 2018, 39(11): 1-7.
16.	纪瑞庆, 刘爱国, 陈龙, 等. 鱼类抗冻蛋白结构与抗冻活性的关系. 食品科学, 2015, 36(5): 274-282.
17.	Mao Xinfang, Liu Zhongyuan, Li Honglei, et al. Calorimetric studies on an insect antifreeze protein ApAFP752 from Anatolica polita. J Therm Anal Calorim, 2011, 104(1): 343-349.
18.	张换成, 胥义. 深低温保存生物材料快速复温方法的研究进展. 中国医学物理学杂志, 2015, 32(1): 144-148.
19.	Tomczak M M, Marshall C B, Gilbert J A, et al. A facile method for determining ice recrystallization inhibition by antifreeze proteins. Biochem Biophys Res Commun, 2003, 311(4): 1041-1046.
20.	Cao Tingting, Zhang Yuqing. Processing and characterization of silk sericin from Bombyx mori and its application in biomaterials and biomedicines. Mater Sci Eng C Mater Biol Appl, 2016, 61: 940-952.
21.	Rockwood D N, Preda R C, Yücel T, et al. Materials fabrication from Bombyx mori silk fibroin. Nat Protoc, 2011, 6(10): 1612-1631.
22.	尉姗姗, 尹林克, 牟书勇, 等. 新疆沙冬青抗冻蛋白的提取分离及其热滞活性测定. 云南植物研究, 2007, 29(2): 251-255.
23.	Stubbs C, Lipecki J, Gibson M I. Regioregular alternating polyampholytes have enhanced biomimetic ice recrystallization activity compared to random copolymers and the role of side chain versus main chain hydrophobicity. Biomacromolecules, 2017, 18(1): 295-302.
24.	Jia Chunli, Huang Weining, Wu Chao, et al. Characterization and yeast cryoprotective performance for thermostable icestructuring proteins from Chinese Privet (Ligustrum Vulgare) leaves. Food Research International, 2012, 49(1): 280-284.
25.	Wu Jinhong, Zhou Yanfu, Wang Shaoyun, et al. Laboratory-scale extraction and characterization of ice-binding sericin peptides. Eur Food Res Technol, 2013, 236(4): 637-646.
26.	任禾盛, 许娜飞, 华泽钊. 抗冻蛋白活性的差示扫描量热测定及其吸附-抑制机制. 细胞生物学杂志, 2004, 26(4): 413-416.
27.	Du N, Liu X Y, Hew C L. Ice nucleation inhibition: mechanism of antifreeze by antifreeze protein. J Biol Chem, 2003, 278(38): 36000-36004.
28.	Raymond J A, DeVries A L. Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proc Natl Acad Sci U S A, 1977, 74(6): 2589-2593.
29.	Li Ling, Wu Jinhong, Zhang Li, et al. Investigation of the physiochemical properties, cryoprotective activity and possible action mechanisms of sericin peptides derived frommembrane separation. LWT, 2017, 77: 532-541.
30.	Gupta D, Agrawal A, Chaudhary H, et al. Cleaner process for extraction of sericin using infrared. J Clean Prod, 2013, 52(4): 488-494.
31.	周小进, 董雪. 不同脱胶方法对蚕丝性能的影响分析. 针织工业, 2013(4): 44-48.
32.	Zhang D Q, Liu B, Feng D R, et al. Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein. Biochem J, 2004, 377(3): 589-595.
33.	Wierzbicki A, Madura J D, Salmon C, et al. Modeling studies of binding of sea raven type II antifreeze protein to ice. J Chem Inf Comput Sci, 1997, 37(6): 1006-1010.

1. Baust J G, Gao Dayong, Baust J M. Cryopreservation: An emerging paradigm change. Organogenesis, 2009, 5(3): 90-96.
2. Lovelock J E, Bishop M W. Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature, 1959, 183(4672): 1394-1395.
3. Guillouzo A, Rialland L, Fautrel A, et al. Survival and function of isolated hepatocytes after cryopreservation. Chem Biol Interact, 1999, 121(1): 7-16.
4. Nagahara Y, Sekine H, Otaki M, et al. Use of high concentrations of dimethyl sulfoxide for cryopreservation of HepG2 cells adhered to glass and polydimethylsiloxane matrices. Cryobiology, 2016, 72(1): 53-59.
5. 李佳佳, 程腾, 贺小英, 等. 二甲基亚砜和胎牛血清对293T细胞冷冻效果的影响. 信阳师范学院学报:自然科学版, 2013, 26(2): 217-220.
6. Hunter N, Foster J, Chong A, et al. Transmission of prion diseases by blood transfusion. J Gener Virol, 2002, 83(Pt 11): 2897-2905.
7. 睢晓洁, 潘超, 杨静, 等. 以氧化三甲胺作为细胞冻存保护剂的研究. 中国科技论文, 2017, 12(12): 1341-1345.
8. 滕芸. 基于蚕丝蛋白的低温保护剂的研究. 上海: 上海理工大学, 2018.
9. Halwani D O, Brockbank K G, Duman J G, et al. Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions. Cryobiology, 2014, 68(3): 411-418.
10. Sun K H, Jung K E, Won Y H, et al. Improvement in ovarian tissue quality with supplementation of antifreeze protein during warming of vitrified mouse ovarian tissue. Yonsei Med J, 2018, 59(2): 331-336.
11. Rubinsky B, Arav A, Mattioli M, et al. The effect of antifreeze glycopeptides on membrane potential changes at hypothermic temperatures. Biochem Biophys Res Commun, 1990, 173(3): 1369-1374.
12. Arav A, Rubinsky B, Fletcher G, et al. Cryogenic protection of oocytes with antifreeze proteins. Mol Reproduct Dev, 1993, 36(4): 488-493.
13. Carpenter J F, Hansent T N. Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth. Proc Natl Acad Sci U S A, 1992, 89(19): 8953-8957.
14. Amir G, Horowitz L, Rubinsky B, et al. Subzero nonfreezing cryopresevation of rat hearts using antifreeze protein I and antifreeze protein III. Cryobiology, 2004, 48(3): 273-282.
15. 马庆保, 刘志东. 南极磷虾抗冻蛋白热滞活性的差示扫描量热法评价. 食品科学, 2018, 39(11): 1-7.
16. 纪瑞庆, 刘爱国, 陈龙, 等. 鱼类抗冻蛋白结构与抗冻活性的关系. 食品科学, 2015, 36(5): 274-282.
17. Mao Xinfang, Liu Zhongyuan, Li Honglei, et al. Calorimetric studies on an insect antifreeze protein ApAFP752 from Anatolica polita. J Therm Anal Calorim, 2011, 104(1): 343-349.
18. 张换成, 胥义. 深低温保存生物材料快速复温方法的研究进展. 中国医学物理学杂志, 2015, 32(1): 144-148.
19. Tomczak M M, Marshall C B, Gilbert J A, et al. A facile method for determining ice recrystallization inhibition by antifreeze proteins. Biochem Biophys Res Commun, 2003, 311(4): 1041-1046.
20. Cao Tingting, Zhang Yuqing. Processing and characterization of silk sericin from Bombyx mori and its application in biomaterials and biomedicines. Mater Sci Eng C Mater Biol Appl, 2016, 61: 940-952.
21. Rockwood D N, Preda R C, Yücel T, et al. Materials fabrication from Bombyx mori silk fibroin. Nat Protoc, 2011, 6(10): 1612-1631.
22. 尉姗姗, 尹林克, 牟书勇, 等. 新疆沙冬青抗冻蛋白的提取分离及其热滞活性测定. 云南植物研究, 2007, 29(2): 251-255.
23. Stubbs C, Lipecki J, Gibson M I. Regioregular alternating polyampholytes have enhanced biomimetic ice recrystallization activity compared to random copolymers and the role of side chain versus main chain hydrophobicity. Biomacromolecules, 2017, 18(1): 295-302.
24. Jia Chunli, Huang Weining, Wu Chao, et al. Characterization and yeast cryoprotective performance for thermostable icestructuring proteins from Chinese Privet (Ligustrum Vulgare) leaves. Food Research International, 2012, 49(1): 280-284.
25. Wu Jinhong, Zhou Yanfu, Wang Shaoyun, et al. Laboratory-scale extraction and characterization of ice-binding sericin peptides. Eur Food Res Technol, 2013, 236(4): 637-646.
26. 任禾盛, 许娜飞, 华泽钊. 抗冻蛋白活性的差示扫描量热测定及其吸附-抑制机制. 细胞生物学杂志, 2004, 26(4): 413-416.
27. Du N, Liu X Y, Hew C L. Ice nucleation inhibition: mechanism of antifreeze by antifreeze protein. J Biol Chem, 2003, 278(38): 36000-36004.
28. Raymond J A, DeVries A L. Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proc Natl Acad Sci U S A, 1977, 74(6): 2589-2593.
29. Li Ling, Wu Jinhong, Zhang Li, et al. Investigation of the physiochemical properties, cryoprotective activity and possible action mechanisms of sericin peptides derived frommembrane separation. LWT, 2017, 77: 532-541.
30. Gupta D, Agrawal A, Chaudhary H, et al. Cleaner process for extraction of sericin using infrared. J Clean Prod, 2013, 52(4): 488-494.
31. 周小进, 董雪. 不同脱胶方法对蚕丝性能的影响分析. 针织工业, 2013(4): 44-48.
32. Zhang D Q, Liu B, Feng D R, et al. Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein. Biochem J, 2004, 377(3): 589-595.
33. Wierzbicki A, Madura J D, Salmon C, et al. Modeling studies of binding of sea raven type II antifreeze protein to ice. J Chem Inf Comput Sci, 1997, 37(6): 1006-1010.

Journal of Biomedical Engineering

Analysis of protective mechanism of silk protein based cryoprotectants

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content