Enzymatic Hydrolysis of Silk Fibroin Peptide for Hemorrhage Control

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1 nd International Conference on Material Engineering and Application (ICMEA 2015) ISBN: Enzymatic Hydrolysis of Silk Fibroin Peptide for Hemorrhage Control Jingjing Li 1,2, Hailin Zhu 1,2, *, Caihong Lei 1,2 & Jianyong Chen 1,2, * 1 Engineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education, Zhejiang Sci-Tech University, Hangzhou, , China 2 The Key Laboratory of Advanced Textile Materials and Manufacturing Technology, Zhejiang Sci-Tech University, Hangzhou, , China ABSTRACT: Effective hemorrhage control becomes increasingly significant in today s military and civilian trauma, while the topical hemostats currently available in market still have various disadvantages. In this study, the silk fibroin peptide (SFP) was prepared through alcalase hydrolysis of silk fibroin. The result of thromboelastographic (TEG) method showed that the SFP can significantly strengthen the coagulation in blood. The results of APTT measurements reflected the acceleration of activation of intrinsic pathway. In the murine hepatic injury model, the blood loss and the bleeding time significantly reduced with the use of SFP as compared with the equal masses of gauze. Based on these results, it can be suggested that the developed SFP has the excellent hemostatic effect, caused no observable tissue damage at the site of application and would be a promising material in clinical hemostatic application. 1. INTRODUCTION Homeostasis is an important step in emergency medical treatment, whether it is sudden trauma in daily life or surgery. While uncontrolled blood loss, or hemorrhaging, accounts for 30 40% of these deaths and is the leading cause of preventable deaths after hospital admission. Nowadays a variety of topical hemostatic agents are available ranging from Chitosan-based dressings such as HemCon, fibrin glues agents, non-protein materials based on polyethylene glycol as well as mineral zeolite. They all have certain effects; however, none are without drawbacks. Therefore, the development of an alternative effective homeostatic dressing which is cheap, effective and easy to get is still a great challenge. SF derived from silkworm Bombyx mori is a typical natural protein polymer. Thanks to its outstanding properties including good biocompatibility, biodegradability, and minimal inflammatory reactions, SF utilized in biomedical applications has been widely reported recently. 1 zhhailin@163.com (Hailin Zhu) 2 cjy@zstu.edu.cn (Jianyong Chen) 12

2 In addition, the silk fibroin peptide (SFP) has been obtained through the hydrolysis of SF by enzymes and considered for applications as a functional material for foods, cosmetics and pharmaceutical preparations. Given the previous study showed that silk-peg-based materials can be used as sealant, and it also has certain blood clotting effect [6]. So there is a great prospect to develop it into hemostatic material. In this study, a novel SFP biomaterial powder produced by enzymatic Hydrolysis of silk fibroin demonstrated notable hemostatic characteristics in early-stage tests. Our research focused on the effect of amount of enzyme on hemostatic efficacy of SFP. 2. RESULTS AND DISCUSSION 2.1 Structure and morphology of SFP The morphology of SFP was revealed by SEM in Figure. 1. It can be seen that the SFP-1 enzymatic hydrolyzed at 800 U/g exhibited a sheet structure with few pores and smooth surface. In the case of SFP-2, some of the powders were the spherical structures, the other have been hydrolyzed into the fragments. The micro-morphology of SFP-3 was observed as the spherical structure with many internal pores, suggesting that the SFP-3 has high porosity. The diameter of sphere was varying between 15 and 80 micrometer. Figure 1. The morphology of SFP enzymatic hydrolyzed at different enzyme concentration. 2.2 Thromboelastograph analysis of SFP Thrombelastography (TEG) is conducted as a point-of-care technique in clinical diagnostics as well as for in vivo and in vitro experimental research. The relative clotting parameters R, α and MA are listed in Table 1. According to the previous study, when the blood transfer to a hypercoagulable state, the R value will be significantly decreased, α and MA value increased. It can be seen that the rabbit blood without agent (control group) began to clot with an R of 10.7min, α of 52.9 degree and MA of 49.5 mm. When the SPF hemostatic agents were added, the R value decreased, and the α and MA values increased significantly, especially for the SFP-3. 13

3 The result indicated that the SFP hydrolyzed from silk at high enzyme concentration could significantly shorten the time of initial fibrin formation and increases the maximum fibrin clot strength. 2.3 APTT and PT measurement APTT is a sensitive and commonly used screening test for endogenous coagulation system, the content of which reflects the level of coagulation factor V, VIII, IX, XI, and XII in plasma, and PT for the extrinsic coagulation system, the content of which reflects the overall activity of coagulation factor II, VII, IX, and X in plasma. The APTT and PT measurement of the SFPs were investigated and the results are shown in Figure. 2. It can be seen that APTT was obviously shortened by the SFP-2 and SFP-3 compared with the control. As for PT, there were no significant differences among the SFPs and the control, suggesting that the SFP could accelerate the activation of the intrinsic pathway of coagulation cascade but had no significant effect on the extrinsic coagulation system. Table 1. In vitro thromboelastograph results. Sample R(min) α( ) MA(mm) SFP-1 8.9± ± ±2.3 SFP-2 6.1±1.9* 57.9±2.4* 53.5±2.8 SFP-3 6.0±1.2* 57.6±2.1* 59.0±2.7* control 10.7± ± ±3.3 All values are expressed as the mean±sd; n=6. *p<0.05 compared to control. Figure 2. Changes of APTT and PT for rat plasma with the SFPs. 14

4 2.4 Hemostasis in a severe liver injury The severe liver injury of rats was surgically exposed and transected, and treated with the SPFs or standard gauze and manual compression. Mass of blood loss and bleeding time in rat liver assessing the hemostatic ability of the SFPs are shown in Figure. 3. After 261.8±24.5s (SPF-1 group), 188.3±18.9s (SPF-2 group) and 117.5±25.8s (SPF-3 group), hemostasis occurred in three agent treated groups, whereas the negative control prolonged the time to 282.1±28.5s. When compared with the negative control group, the SFP-2 and SFP-3 agents offered a statistically significant (p<0.05) advantage by decreasing the blood loss from 0.69±0.06g (control group) to 0.48±0.06g (SFP-2) and 0.26±0.07g (SFP-3), respectively. Although the exact hemostatic mechanism of the action is not known, the results presented here can explain some of the mechanistic basis for the observations. While during the process of hemostasis, it can be found that when applied onto the wound site, the SFP sponge material absorbed a large amount of water. So it was speculated that the SFP therefore has the propensity to concentrate the cellular and plasma components in the hemorrhaging blood at the site of injury, moreover, the behavior allows the SFP to act as a physical barrier to blood loss, and this will assist in primary homeostasis by physically forcing the platelets toward damaged vessels and accelerate the turnover of coagulation cascade for thrombin generation, and thus help forming blood clots in the wound. Figure 3. Mass of blood loss and bleeding time in rat liver assessing the hemostatic ability of the SFPs. 3. CONCLUSION In the present study, the SFP enzymatic hydrolyzed from silk has been developed for hemorrhage control and its hemostatic together with in vitro cytotoxicity was evaluated was evaluated. The results demonstrated that the SFP can significantly activate the intrinsic pathway of coagulation cascade, promote the blood clotting and decrease the blood loss and bleeding 15

5 time. It is indicated that the SFP enzymatic hydrolyzed from silk may be a potential environment friendly biomaterial for hemostatic. ACKNOWLEDGMENTS We gratefully thank the National Natural Science Foundation of China (grant nos , ), the Natural Science Foundation of Zhejiang Province (grant nos. LY14E030012), the Graduate Innovation Research Project of Zhejiang Sci-Tech University (grant No. YCX 13008), for support of this program. REFERENCES D.B. Hoyt. A clinical review of bleeding dilemmas in trauma [J]. Semin Hematol.2004, 41: E. Buskens, M.J. Meijboom, H. Kooijman, B.A. Van Hout, C.P.S. Grp. The use of a surgical sealant (CoSeal (R)) in cardiac and vascular reconstructive surgery: an economic analysis [J]. Jouranl of Cardiovascular Surgery. 2006, 47: H.J. Jin, J. Park, D.L. Kaplan. Biomaterial Films of Bombyx Mori Silk Fibroin with Poly (ethylene oxide) [J]. Biomacromolecules. 2004, 5: S.R Jung, N.J. Song, D.K. Yang. Silk proteins stimulate osteoblast differentiation by suppressing the Notch signaling pathway in mesenchymal stem cells [J]. Nutrition Research. 2013, 33: A.S. Monica, P. Bruce, D.L. Kaplan. Silk fibroin and polyethylene glycol-based biocompatible tissue adhesives [J]. Biomaterials. 2011, 98: N.R. Danielle, C.P. Rucsanda, Y. Tuna, X.Q. Wang, L.L. Michael, D.L. Kaplan. Materials Fabrication from Bombyx mori Silk Fibroin [J]. NIH Public Access. 2013, 10: