Name: synthesis of strong adhesive hydrogel, gelatin o-nitrosobenzaldehyde
Uploaded: 2022-11-11T14:50:00
Description: Watch this Scientific Journal Video about Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde at JoVE.com

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 Us...

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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...

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 infarction2,3,4, 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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-(3Dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDC)</td>
			<td>Aladdin</td>
			<td>L287553</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Hydroxy-3-(methoxy-D3) benzaldehyde</td>
			<td>Shanghai Acmec Biochemical Co., Ltd</td>
			<td>H946072</td>
			<td></td>
		</tr>
		<tr>
			<td>DCM</td>
			<td>Aladdin</td>
			<td>D154840</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>20-139</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethylformamide (DMF)</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1553</td>
			<td></td>
		</tr>
		<tr>
			<td>gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>1288485</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>MeOH</td>
			<td>Sigma-Aldrich</td>
			<td>1424109</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl 4-(4-formyl-2-methoxyphenoxy methoxyphenyl) butanoic acid methyl ester</td>
			<td>chemsrc</td>
			<td>141333-27-9</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl 4-bromobutyrate</td>
			<td>Aladdin</td>
			<td>M158832</td>
			<td></td>
		</tr>
		<tr>
			<td>NaBH4</td>
			<td>Sigma-Aldrich</td>
			<td>215511</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (NHS)</td>
			<td>Aladdin</td>
			<td>D342712</td>
			<td></td>
		</tr>
		<tr>
			<td>nitric acid</td>
			<td>Sigma-Aldrich</td>
			<td>225711</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>209619</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM (Nova Nano 450)</td>
			<td>Thermo FEI</td>
			<td>17024560</td>
			<td></td>
		</tr>
		<tr>
			<td>THF/EtOH</td>
			<td>Aladdin</td>
			<td>D380010</td>
			<td></td>
		</tr>
		<tr>
			<td>trifluoroacetic acid (TFA)</td>
			<td>Sigma-Aldrich</td>
			<td>8.0826</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of strong adhesive hydrogel, gelatin o-nitrosobenzaldehyde

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.

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...

Watch this Scientific Journal Video about Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde at JoVE.com

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

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.

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...

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.

Research

JoVE Journal

Bioengineering

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Rat Model of Adhesive Capsulitis of the Shoulder

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other ...

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...