1. The efficient hemostatic effect of Antarctic krill chitosan is related to its hydration property Shuai Wu a,d, ZhuoyaoHuang a, JianhuiYue a,d, DiLiu a,c, TingWang a, PierreEzanno a, Changshun Ruan a,d, XiaoliZhao a,d, ∗, WilliamW.Lu b, HaoboPan a,d Student : Kun-Yi Kao Advisor : Cheng-Ho Chen Date : 2015/12/22

2. # Outline Introduction Experimental Results and Discussion Conclusions

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4. Chitin is the most important natural polysaccharide usually distributed in marine invertebrates, insects, fungi and yeast. The hemostatic effect of chitosan has been demonstrated in many studies, hemostatic responses of chitosan are highly dependent on its physicochemical properties such as molecular weight, intrinsic viscosity, hydration and crystallinity. Introduction

5. Antarctic krill chitosan was first evaluated in the hemostatic effect experiments by using mice tail amputation model and blood coagulation timing experiment. The structure properties of strong water–polymer interaction found in Antarctic krill chitin as mentioned above may benefit the hemostatic effect of its derived chitosan. Introduction

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7. The shell with 1M NaOH at 100 ℃ for an hour at a solid/solvent ratio of 1:20 (w/v) and washed to neutrality in deionized water Experimental Dried at 60 ◦ C in a vacuum drying oven Deacetylation was performed at 110 ℃ in 50% NaOH solution with refluxing for an hour at a solid/solvent ratio of 1:50 (w/v) Demineralization was carried out in 2MHCl solution at room temperature for an hour at a solid/solvent ratio of 1:20 (w/v) and washed to neutrality in deionized water

8. The crude chitosan was purified by dissolving in 1% acetic acid solution, filtering, and then precipitating from the solution by adjusting the solution’s pH to 8.5 with 1M NaOH Experimental The obtained product was washed and dried by lyophilization to get the chitosan powder Chitosan thin film was prepared on the round cover slice by the spin-coating (Chemat Technology spin-coater),and dried at60 ◦ C in a vacuum oven for 12 h. Films were then sterilized by ultraviolet light

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10. # Fig. 1. The chemical structure of the different kinds of chitosans was investigated by FTIR (A) and 1H NMR (B).For 1 H NMR characterization,chitosan was dissolved in 2 wt% DCl/D 2 O solution.

11. # Fig. 1. The chemical structure of the different kinds of chitosans was investigated by FTIR (A) and 1H NMR (B).For 1 H NMR characterization,chitosan was dissolved in 2 wt% DCl/D 2 O solution.

12. # Fig. 2. The crystalline structure and the hydration property were investigated. The crystalline structure was investigated by X-ray diffractometry (XRD) (A). The equilibrium water binding capacity (WBC) (B) and the WBC at different incubation time (C)of chitosan were examined.

13. # Fig. 2. The crystalline structure and the hydration property were investigated. The crystalline structure was investigated by X-ray diffractometry (XRD) (A). The equilibrium water binding capacity (WBC) (B) and the WBC at different incubation time (C)of chitosan were examined.

14. # Fig. 3. The hemostatic effect of chitosan evaluated by mice tail amputation model. The segment of the mice tail was amputated transversely (A) and the cut ends were immediately immersed in the samples (B). The bleeding time (C) and blood loss amount (D) were measured.

15. # Fig. 3. The hemostatic effectof chitosan evaluated by mice tail amputation model. The segment of the mice tail was amputated transversely (A) and the cut ends were immediately immersed in the samples (B). The bleeding time (C) and blood loss amount (D) were measured.

16. # Fig. 4. The effect of chitosan on the blood coagulation. The timing was started after recalcifying the anticoagulated blood until the blood turned to self-supporting gel in the inverted tube (A), and blood coagulation time (B) was compared between different groups.

17. # Fig. 5. Chitosans’ effect on platelets adhesion. The adherent platelets on different films were observed by calcein-AM staining (A), and the number of adherent platelets (B) was analyzed by Image J software.

18. # Fig. 6. Effect of chitosans on the red blood cells (RBCs) aggregation. The adherent RBCs were observed under microscopy for their aggregation (A) and deformation (B). The RBCs adherent area (C) and aggregation area. Scalebar,20 μm.

19. # Fig. 6. Effect of chitosans on the red blood cells (RBCs) aggregation. The adherent RBCs were observed under microscopy for their aggregation (A) and deformation (B). The RBCs adherent area (C) and aggregation area. Scalebar,20 μm.

20. # Fig. 6. Effect of chitosans on the red blood cells (RBCs) aggregation. The adherent RBCs were observed under microscopy for their aggregation (A) and deformation (B). The RBCs adherent area (C) and aggregation area. Scalebar,20 μm.

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22. # A-Chitosan with showed low level of crystallinity and significant high water binding capacity as 1293%. A-Chitosan could accelerate the tail hemostasis by 55% and shortened the blood coagulation time by 38%. The physicochemical properties resulted in better hydration property of chitosan would improve its hemostatic effect. These results showed that A-Chitosan is a good candidate for hemostatic application.

23. # Thanks for your attention