Our protocol describes the synthesis of a new adhesive hydrogel, gelatin o-nitrosobenzaldehyde, in detail. It mainly solve the problem of how to maximize the adhesion of hydrogel. In our technology, chemical groups in the hydrogen are modified, thus having the activity of chemical reaction with chemical groups on animal tissue surface to achieve close bending and attention.
The product synthesized by our technique can maintain a strong long-term attention on the surface of the animal tissues, thus effectively promoting damage repair and tissue regeneration. This method could provide insight into a regenerative medicine, including damage in biosystems. We have previously reported the application of products synthesized by this method in corneal injury and IBD.
To begin, dissolve the prepared compounds as described in the manuscript in 40 milliliters of N, N-dimethylformamide, and stir at ambient temperature for 16 hours. Then add 200 milliliters of zero degree Celsius water to the mixture and precipitate the mixture to obtain the crude product. Repeatedly dissolve the crude product in N, N-dimethylformamide and then precipitate for five cycles.
After precipitating the crude product, dry it at 80 degrees Celsius for two hours to obtain the early product. Perform the ipso substitution of methyl 4-butanoic acid methyl ester by adding 9.4 grams of methyl 4-butanoate slowly to a pre-cool solution of 70%nitric acid and stir at minus two degrees Celsius for three hours. Then precipitate the mixture.
Filter with 200 milliliters of zero degree Celsius water and purify it in N, N-dimethylformamide to precipitate a solid product. Hydrolyzed the solid product in trifluoroacetic acid at 90 degrees Celsius and place it in a rotary evaporator before drying it in the oven. After dissolving the intermediate product in tetrahydrofuran, add 1.43 grams of sodium borohydride slowly at zero degrees Celsius.
After three hours, remove all solvents under a vacuum and suspend the residue in a one-to-one water/dichloromethane solution. Prepare dichloromethane to extract the product from the aqueous layer while removing the organic layer and dry over magnesium sulfate. Then purify the crude product by silica gel column chromatography using dichloromethane methanol at a 10:1 ratio.
Finally, obtain 5.31 grams of relatively pure yellowish powder NB carboxy. Prepare a homogenous gelatin solution by dissolving five grams of gelatin in 100 milliliters of deionized water and stored at 37 degrees Celsius for one batch of modification. After defining the feed ratio as described in the manuscript, dissolve 1, 060 milligrams of NB carboxy in five milliliters of dimethyl sulfoxide.
To activate the carboxyl groups of NB carboxy, add a 746 milligrams of 1-3-ethylcarbodimide hydrochloride into the NB carboxy methylsulfoxide solution and stir for five minutes. After 1-3-ethylcarbodimide hydrochloride has dissolved, add 448 milligrams of N-hydroxysuccinimide and stir for five minutes. Use a dropping funnel to slowly drop the mixture at a rate of 0.5 milliliters per minute into the dissolved gelatin solution with vigorous stirring to react at 45 degrees Celsius for four hours.
After dialyzing the gelatin NB solution against excess deionized water for at least three days, collect, freeze, and lyophilize it to obtain the gelatin-NB foams. Keep the foams in a desiccate in the dark for further use. Dissolve the freeze dry gelatin-NB foams in deionized water at 37 degrees Celsius immediately before use.
The SEM images of the injured corneal surface gelatin and gelatin-NB-4 protein coating treated corneal surface were obtained, showing that the morphology of gelatin-NB-4 protein stable adheres to the corneal surface. However, an injured corneal surface treated with nothing or gelatin appears smooth. Fluorescence images of the mice colonic surface labeled by gelatin and gelatin-NB molecular coating show that the fluorescently labeled gelatin-NB has the capability to adhere to the intestinal tissue and form a dense coating.
However, the fluorescence intensity of the gelatin group is very weak, indicating that it fails to adhere firmly to the intestinal wall. The fluorescence images of the label gelatin and the gelatin-NB molecular coating-treated aminated plates at zero and 24 hours show that both can initially adhere to the plate. After pouring PBS and changing the aminated plate every four hours for 24 hours, only gelatin-NB maintains a strong fluorescence, indicating that it adheres strongly.
X-ray photon spectroscopy of gelatin-NB-4 bonding demonstrated that the spectra of a corneal surface and a surface treated with gelatin are the same. However, an extra peak appears at 400 electron volt, indicating the formation of many CN bonds in the tissue after treatment with UV activated gelatin-NB. The most important thing to remember is that when synthesizing gelatin-NB in different application conditions, it is necessary to set up pre-experiment FR gradient to explore the best attention effect.
This technique may pave the way for developing biosynthetic and regenerative medicine. Researchers can refer to the video to modify the group to meet biomedical needs.
