Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Summary

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

Abstract

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH₂ on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

Introduction

Hydrogel is a type of three-dimensional polymer formed by water swelling. In particular, hydrogel derived from an extracellular matrix is widely used in the field of biosynthesis and regenerative medicine because of its excellent biocompatibility and therapeutic effectiveness¹. Hydrogels have been reported for the treatment of gastric ulcers, neuritis, myocardial infarction^2,3,4, and other diseases. Further, it has been proved that gelatin-NB can promote the outcome of inflammation ininflammatory bowel disease (IBD)⁵. Traditional hydrogels include gellan gum, gelatin, hyaluronic acid, polyethylene glycol (PEG), layered, hydrophobic/hydrophilic, alginate/polyacrylamide, double network, and polyamphoteric hydrogels⁶, all of which have good histocompatibility and mechanical properties. However, these traditional hydrogels are vulnerable to moisture and air in the environment. If they are exposed to air for a long time, they will lose water and dry; if they are immersed in the water for a long time, they will absorb water and expand⁷, thus reducing their flexibility and mechanical function. In addition, maintaining the tissue adhesion of conventional hydrogels is a major challenge⁸.

Based on this, we designed and synthesized a nanoscale hydrogel gelatin-NB, which is a novel hydrogel formed by modifying biological gelatin with NB (Figure 1). NB has a strong adhesion ability to -NH₂ on the tissue, which can form a large number of C = N bonds, thus increasing the adhesiveness of the hydrogel-tissue interface. This strong adhesion can make the hydrogel firmly adhere to the tissue surface, thus forming a nano-level molecular coating. In the team's previous studies, it has been confirmed that this kind of modified hydrogel coating has improved tissue adhesion⁹; it can stably adhere to corneal and intestinal organs and tissues and play anti-inflammation, barrier isolation, and regeneration promotion roles. The goal is to introduce the specific synthesis process of gelatin-NB in detail here, so that gelatin-NB can be applied in more scenarios of damage repair. Moreover, we encourage other researchers to further strengthen and expand the nature of this material to suit more application scenarios.

Protocol

The C57BL/6 mice were purchased from Zhejiang University School of Medicine Sir Run Run Shaw Hospital. The New Zealand rabbits were purchased from Zhejiang University. The animals were maintained in natural light-dark cycle conditions and given food and drinking water freely. All experimental procedures were approved ethically by the institutional guidelines of the Zhejiang University Ethics Committee standard guidelines (ZJU20200156) and Zhejiang University School of Medicine Sir Run Run Shaw Hospital Animal Care and Use Committee, which conformed to the NIH Guide for the Care and Use of Laboratory Animals (SRRSH202107106).

1. Synthesis of NB-COOH

1. Prepare 4-hydroxy-3-(methoxy-D3) benzaldehyde (8.90 g, 58.5 mM, 1.06 equivalents [eq.]), potassium carbonate (10.2 g, 73.8 mM, 1.34 eq.), and methyl 4-bromobutyrate (9.89 g, 55.0 mM, 1.0 eq.) based on the protocol proposed in the previous study¹⁰. Dissolve the compounds in 40 mL of N, N-dimethylformamide (DMF) and stir at ambient temperature for 16 h.
2. Add 200 mL of 0 °C water to the mixture and precipitate the mixture to obtain a crude product.
3. Repeatedly dissolve the crude product in DMF and then precipitate for five cycles. Precipitate the crude product and dry it at 80 °C for 2 h to obtain the early product.

2. Chemical modification and processing

1. Perform the ipso substitution of methyl 4-(4-formyl-2-methoxyphenoxy methoxyphenyl) butanoic acid methyl ester as described below.
2. Add 9.4 g of methyl 4-(4-formyl-2-methoxyphenoxy) butanoate (37.3 mM, 1 eq.) slowly to a precooled (-2 °C) solution of 70% nitric acid (140 mL) and stir at -2 °C for 3 h.
   NOTE: Depending on the temperature of the nitration reaction, the ipso substitution of the formyl moiety will occur.
3. Filter the mixture (~9.0 g) with 200 mL of 0 °C water, then purify it in DMF to precipitate a solid product.
4. Hydrolyze the solid product in trifluoroacetic acid (TFA)/H₂O, 1:10 v/v (100 mL) at 90 °C and dry. Remove the solvent under 80 kPa to obtain the final intermediate product, a dry pale-yellow powder.
5. Dissolve the intermediate product (7.4 g, 23.8 mM, 1.0 eq.) in tetrahydrofuran (THF)/ethanol, 1:1 v/v (100 mL). Then add 1.43 g of NaBH₄ (35.7 mM, 1.5 eq.) slowly at 0 °C. After 3 h, remove all solvents under a vacuum and suspend the residue in a 1:1 water and dichloromethane solution (50 mL each).
6. Prepare dichloromethane to extract the product from the aqueous layer. Remove the organic layer and dry over magnesium sulfate.
7. Purify the crude product by silica gel column chromatography using DCM/MeOH at a 10:1 ratio (1% TEA). Finally, obtain 5.31 g (18.6 mM, 78.3%) of relatively pure yellowish powder NB-COOH.

3. Synthesis of gelatin-NB

1. Prepare 5 g of gelatin for one batch of modification. Prepare a homogeneous gelatin solution by dissolving 5 g of gelatin in 100 mL of deionized water and store at 37 °C.
   NOTE: Here, the original 33 x 10^-5 moles ε-amino groups/g gelatin¹¹ is defined.
2. Define the feed ratio (FR) as the molar ratio between NB groups and primary amino groups in gelatin. In this study, 53 mg of NB with 1 g of gelatin was defined as FR_NB = 1.
3. Dissolve 1,060 mg of NB-COOH in 5 mL of dimethyl sulfoxide (DMSO) to activate the carboxyl groups of NB-COOH. Since the NB-group is sensitive to ultraviolet (UV) light when in solution, always keep it away from light.
4. Add 746 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDC) into the NB-COOH DMSO solution and stir for 5 min. After EDC has dissolved, add 448 mg of N-hydroxysuccinimide (NHS) and stir for 5 min.
5. Use a dropping funnel to slowly drop the mixture at a rate of 0.5 mL/min into the dissolved gelatin solution with vigorous stirring to react at 45 °C for 4 h.

4. Purification and storage of the product

1. Dialyze the gelatin-NB solution against excess deionized water for at least 3 days, then collect, freeze, and lyophilize it to obtain the gelatin-NB foams. Keep the foams in a desiccator in the dark for further use.
2. Dissolve the freeze-dried gelatin-NB foams in deionized water at 37 °C, immediately before use.

Results

Figure 2A shows a schematic of the main chemical reactions involved in the synthesis of gelatin-NB, which promotes tissue integration by grafting NB groups onto gelatin. Figure 2B shows that the O-nitrobenzene of the gelatin-NB hydrogel converts to an NB group immediately after UV irradiation, and then the active aldehyde group can be crosslinked with an amino group to form a Schiff base. Figure 2C indicates that different ratios of...

Discussion

Adhesive materials are a new class of material. More and more researchers are committed to the synthesis of various types of adhesive materials, and are trying to find their applications in biotechnology, tissue engineering, regenerative medicine, and other fields, which has led to vigorous development in recent years. In addition to focusing on the strong adhesion of adhesive materials, researchers are also paying more attention to other properties, such as injectability, self-healing, hemostatic, antibacterial, control...

Materials

Name	Company	Catalog Number	Comments
1-(3Dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDC)	Aladdin	L287553
4-Hydroxy-3-(methoxy-D3) benzaldehyde	Shanghai Acmec Biochemical Co., Ltd	H946072
DCM	Aladdin	D154840
Dichloromethane	Sigma-Aldrich	270997
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	20-139
dimethylformamide (DMF)	Sigma-Aldrich	PHR1553
gelatin	Sigma-Aldrich	1288485
magnesium sulfate	Sigma-Aldrich	M7506
MeOH	Sigma-Aldrich	1424109
methyl 4-(4-formyl-2-methoxyphenoxy methoxyphenyl) butanoic acid methyl ester	chemsrc	141333-27-9
methyl 4-bromobutyrate	Aladdin	M158832
NaBH4	Sigma-Aldrich	215511
N-hydroxysuccinimide (NHS)	Aladdin	D342712
nitric acid	Sigma-Aldrich	225711
potassium carbonate	Sigma-Aldrich	209619
SEM (Nova Nano 450)	Thermo FEI	17024560
THF/EtOH	Aladdin	D380010
trifluoroacetic acid (TFA)	Sigma-Aldrich	8.0826