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 – 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.
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'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.
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
2. Chemical modification and processing
3. Synthesis of gelatin-NB
4. Purification and storage of the product
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 NB groups can lead to different cross-linked structures of gelatin-NB.
At the same time, a preliminary characterization of the physical properties of gelatin-NB was also conducted. As shown in Figure 3, gelatin-NB has a strong gelation when the feed ratio (FR) of NB is low. This means that gelatin-NB with low FR forms a soft hydrogel due to the presence of a large number of amino groups that can react with photogenerated aldehyde groups, while gelatin-NB with high FR can maintain a flexible droplet. We also observed that the overall morphology of gelatin-NB stably adheres to the corneal surface by scanning electron microscopy (SEM), as shown in Figure 3C. However, an injured corneal surface treated with nothing or gelatin appears to be smooth. Figure 3D shows 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. Figure 3E shows that both gelatin and gelatin-NB are initially able to adhere to the aminated plate. However, after pouring phosphate buffered saline (PBS) into the aminated plate and changing it every 4 h for 24 h, only gelatin-NB maintains a strong fluorescence, indicating that it adheres strongly. These results indicate that gelatin-NB can adhere to the tissue surface to form a uniform and stable dense layer. As shown in Figure 3F, the spectra of a corneal surface and a surface treated with gelatin are almost the same. However, in the gelatin-NB treated group, there is an extra peak appearing at 400 eV, indicating the formation of many C = N bonds in the tissue after treatment with UV-activated gelatin-NB12.
Figure 1: Steps of the NB-COOH synthesis reaction. The figure provides a schematic representation of synthesis reaction Please click here to view a larger version of this figure.
Figure 2: Design and synthesis of gelatin-NB. (A) Schematic of the chemical reaction for gelatin-NB formation. (B) Schematic diagram illustrating the photo-triggered chemical structure transformation of the gelatin-NB hydrogel. O-nitrobenzene is converted to NB groups under UV exposure. Then the active aldehyde group could subsequently crosslink with amino groups to form Schiff bases. (C) Schematic of gelatin-NB forming hydrogel and coating under different feed ratios. This figure has been modified from12. Please click here to view a larger version of this figure.
Figure 3: Characterization of Gelatin-NB. (A) The varying gelling performance of gelatin-NB with various feed ratios of NB. (1-4) represent 0.5, 1, 2, and 4 NB feed ratios of gelatin-NB, respectively. (B) Gross view of the inactivated modified gelatin-NB solution, and the gelatin-NB solution after UV illumination. (C) SEM images of the injured corneal surface, gelatin, and gelatin-NB-4 protein coating-treated corneal surface. Scale bars: 30 µm (top panels); 40 µm (bottom panels, enlarged). (D) Fluorescence images of the mice colonic surface labeled by gelatin and gelatin-NB molecular coating. Scale bars: 200 µm. (E) Fluorescence images of the labeled gelatin and gelatin-NB molecular coating-treated aminated plates at 0 h and 24 h. Scale bars: 20 µm. (F) X-ray photon spectroscopy (XPS) of gelatin-NB-4 bonding to tissue. The bond energies of the peptide -C-NH- and amino amine group C-NH2 shifting due to the appearance of a C = N bond peak reveals the UV-induced formation of Schiff bases. This figure has been modified from5 and12. Please click here to view a larger version of this figure.
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, controlled removal, and so on13. These new applications greatly expand the application scope of adhesive materials with promising application prospects.
In this paper, the synthesis method of a novel hydrogel gelatin-NB was introduced. Gelatin-NB has strong adhesion, and it is reported that it can be applied to the repair of corneal injury and intestinal injury in clinical practice5,12. Therefore, it is of great academic and application value to popularize the preparation method of gelatin-NB.
The key step for preparing gelatin-NB is the synthesis process. We propose the concept of feed ratio (FR), which is the molar ratio between the NB group and the primary amino group in gelatin. The FR for synthesizing gelatin-NB is not a constant and can be adjusted according to the nature of the adherent tissue interface. For rabbit corneas, the FR of gelatin-NB eye drops is FRNB = 2, while the number of amino groups on the surface of mouse colons is relatively high12; FRNB = 2 proves not to be the optimal FR for this, and usually needs to be increased to about 4 to achieve the optimal adhesion effect. When synthesizing gelatin-NB in different application scenarios, it is necessary to set a pre-experiment FR gradient to explore the best adhesion effect. In addition, we mentioned in the article that NB needs to be kept away from UV light at all times, because NB groups are very sensitive to UV; we suggest avoiding all direct light sources as much as possible during the synthesis process, to minimize the impact of UV light on NB groups.
At the same time, this synthesis technology has some limitations. For example, the low yield of crude products leads to the need for a greater consumption of raw materials. We are trying to change various reaction conditions to improve the yield, such as adjusting the reaction temperature and further extending the reaction time. We will update the research progress in time. Researchers can refer to the video to improve the preparation strategy of gelatin o-nitrosobenzaldehyde or further modify the group on this basis to meet further biomedical needs. We believe that the synthesis method of gelatin o-nitrosobenzaldehyde described in this paper will accelerate the development of biosynthetic and regenerative medicine. In addition, gelatin-NB is expected to be further applied in critical clinical cases, such as bleeding caused by acute vascular injury, rupture of liver and spleen, and gastric perforation in the future.
The authors have nothing to disclose.
None.
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 |