Preparation of HAD

Snake-extract laden hemostatic bioadhesive gel crosslinked by visible light 

Yicheng Guo^1,5#, Ying Wang^1#, Xiaohong Zhao¹, Xue Li¹, Quan Wang², Wen Zhong³, Kibret Mequanint⁴, Rixing Zhan¹*, Malcolm Xing⁵*, Gaoxing Luo¹*

¹ Institute of Burn Research, State Key Laboratory of Trauma, Burn and Combined Injury, Southwest Hospital, the Third Military Medical University (Army Medical University), Chongqing 400038, China

² Department of Civil Engineering, Shantou University, Shantou 515063, China 

³ Department of Chemical and Biochemical Engineering, and School of Biomedical Engineering, the University of Western Ontario, London, N6A5B9, Canada

⁴ Department of Biosystems Engineering, University of Manitoba, Winnipeg, R3T 2N2, Winnipeg, Canada

⁵ Department of Mechanical Engineering, University of Manitoba, Winnipeg, R3T 2N2, Winnipeg, Canada

Materials

Type A gelatin from porcine skin, TEA, VC, Eosin Y, and methacrylic anhydride were obtained from Sigma-Aldrich. HC was purchased from Penglai Nuokang Pharmaceutical Co. Ltd. (Shandong, China). Dulbecco’s phosphate-buffered saline (DPBS) was obtained from Macklin Co. (Beijing, China). Fibrin glue was obtained from Shanghai RAAS Blood Products Co. Ltd. (Shanghai, China)

Preparation of GelMA

GelMA was synthesized by following the previously published protocol (1). Briefly, 1 g of gelatin was dissolved in 10 ml of DPBS and heated to 60°C（Magnetic stirrer） for 1 hour. Methacrylic anhydride (0.8 ml) was then added to the solution dropwise and stirred （1ml pipette tip, one drop in 2 seconds） for another 3 hours at 50°C. The reaction was stopped by adding 40 ml of DPBS, and the product was dialyzed (molecular weight cutoff, 12 to 14 kDa) in a deionized water bath at 55°C for 5 days (Change deionized water every 8 hours） to remove the unreacted methacrylic anhydride. Last, the solution was filtered and lyophilized for 12 hours.

Preparation of HAD adhesive

HAD precursor solutions were prepared by dissolving TEA [1.88% (w/v)], VC [1.25% (w/v)], Eosin Y (0.5 mM), and HC [1% (w/v)] in GelMA solution [20% (w/v)] (2). Firstly, dissolving TEA [1.88% (w/v)], VC [1.25% (w/v)], Eosin Y (0.5 mM), and HC [1% (w/v)] in 10 ml of DPBS to prepare the initiator solution. Then, 1 mL initiator solutions were added in a EP tube, which contained 200 mg GelMA. Finally, keep in dark place and incubation at 37 oC (Constant temperature incubator) for 2h to obtain the HAD adhesive recursor solutions. The HAD adhesive recursor solutions were then photocrosslinked with a dental curing lamp (430 to 530 nm, ~1000 mW/cm2) for 1 to 4 min.

REFERENCES

(1) D. Loessner, C. Meinert, E. Kaemmerer, L. C. Martine, K. Yue, P. A. Levett, T. J. Klein, F. P. Melchels, A. Khademhosseini, D. W. Hutmacher, Functionalization, preparation and use of cell-laden gelatin methacryloyl–based hydrogels as modular tissue culture platforms. Nat. Protoc. 11, 727–746 (2016).

(2) E. S. Sani, A. Kheirkhah, D. Rana, Z. Sun, W. Foulsham, A. Sheikhi, A. Khademhosseini, R. Dana, N. Annabi, Sutureless repair of corneal injuries using naturally derived bioadhesive hydrogels. Sci. Adv. 5, eaav1281 (2019).