An Adhesive Double-network Hydrogel Loaded With MCC950 Used For Hemostasis And Repair

Traumatic hemorrhage is the leading cause of death after severe trauma.Systemic blood volume deficiency will trigger a series of stress responses,seriously affect the recovery of patients,and even endanger life.So stopping hemorrhage in time is the first priority of first aid.Bioadhesive is a kind of biomedical material,which can be widely used in hemostasis,tissue suture,preventing the leakage of air and liquid during the operation,etc.Because of its good adhesion property and biocompatibility,it shows a broad application prospect in the field of tissue engineering organ repair.Hydrogels are hydrophilic cross-linked polymeric networks,which are similar to natural soft tissues and extracellular matrix.Hydrogels have many advantages such as high safety,good air permeability and degradability,making it an ideal material choice for biological adhesive.However,in the case of continuous blood flow,general hydrogels have problems such as weak adhesion ability,insufficient mechanical strength on the surface of moist and movable tissue,and cannot effectively adhere to the wound surface to stop bleeding.Therefore,we use the synergistic effect of covalent network and hydrogen bond to construct a doublenetwork hydrogel with a double-layer structure.On the one hand,covalent bonds give high layer overall mechanical strength to the hydrogel matrix,and hydrogen bonds can improve its toughness through energy dissipation.On the other hand,at the interface adhesion,hydrogen bonds promote the rapid adhesion of hydrogels to biological tissues,and further covalency enhances the adhesion strength of hydrogels.Both of the two aspects jointly promote the strong adhesion of hydrogel to biological tissue and improve its own mechanical strength,so as to achieve the adhesion and hemostasis.Hydrogels also provide a scalable and controllable platform for drug delivery applications.Molecular self-assembly use the interaction of attraction and repulsion,so drugs and other reactive molecules can be combined by mediating or covalent integration and mixed materials.Especially the double network hydrogels can also have good characteristics of selfassembly and covalent network,it means the double network hydrogels have a broad prospect for development in the aspect of drug targeting delivery.The healing of the wound after hemostasis is as important as the recovery of the function for the damaged tissues and organs.Especially when blood vessels are injured,excessive inflammatory reaction in the early stage will seriously affect the repair of the damaged site,intimal hyperplasia will occur,which will lead to vascular stenosis at the rupture site and affect the long-term patency of blood vessels.The key to the inflammatory response is the activation of NLRP3 inflammasomes.Early inhibition of the activation process can control the development of inflammation and avoid the vascular blockage caused by excessive proliferation.MCC950 is a highly effective inhibitor of NLRP3 inflammasome,which is modified in the constructed double-network hydrogel matrix layer.The hydrogel is used as a carrier to release the small molecule MCC950 at the wound site,so as to control the occurrence and development of early inflammation and ensure the healing and long-term patency of blood vessels after hemostatic adhesion.Methods:1.Preparation and performance test of double network hydrogelFirst,we used the HEAA,PLL and CS as the main raw material to prepare a double network structure of the double-layer hydrogel patch.Then we used scanning electron microscopy,infrared spectroscopy and TGA to characterize it,and we used universal testing machine to test its mechanical strength and other various adhesion properties of biological tissue,CCK-8 cell activity experiment verified hydrogel for cell toxicity,and the patch subcutaneously implanted in rats.After a few weeks,we took the hydrogel patch out and observed it by freezing section and HE staining verify the biocompatibility.These results could provide the basis for the next step of animal experiments.2.Large animal experiments verified the adhesion and hemostasis effect of double network hydrogelWe applied the prepared double-network hydrogel patch to the organ injury model of large animals to verify the effect of its adhesion and hemostasis.We first cut the livers of beagles and small pigs with a scalpel,and then applied a hydrogel patch to the bleeding wound to observe whether it could adhere to the organ and stop bleeding.At the same time,we choosed the artery injury model which have stronger impact.The femoral artery of beagles and small pig was also cut with a scalpel,and then the hydrogel patch was adhered around the damaged area to observe its adhesion and hemostasis effect on the bleeding artery surface.3.Hemostasis and repair effect of the adhesive double-network hydrogel loaded with MCC950 on vascular injuryIn order to get a better repair after the hemostasis,we added a small molecule inhibitor MCC950 to the synthetic hydrogel matrix layer to achieve the targeted release of the drug,so as to inhibit the excessive inflammatory response of the wound in the early stage.First,the experiments in vitro were carried out.The toxicity of small molecule drug MCC950 was tested by CCK-8 cell activity experiment,and the drug release amount was determined by drug standard concentration curve and hydrogel drug release curve.Then,the femoral artery injury model of small pigs was also used.We adhered the double-network hydrogel loaded with MCC950 to the wound and stop bleeding.Two weeks later,the degree of vascular patency was detected by Doppler ultrasound,and samples were collected for HE and Masson staining.The repair effect of MCC950 on vascular injury was evaluated by comparing with the group without drug addition.Results:1.We have successfully prepared a double network hydrogel with a double layer structure.SEM results showed that there is a uniform covalent network structure,Infrared spectra and TGA results confirmed the existence of hydrogen bonds and the synthesis of covalent bonds,which provide the possibility for the covalent bond and hydrogen bond interaction during adhesion.Mechanical tensile experiment and adhesion experiment proved that the hydrogel network we build can provide adequate mechanical support,and can adhesive the biological tissue strongly,CCK-8 cell activity detection tested the toxicity of hydrogel,the rats subcutaneous implant test section showed the hydrogel has good compatibility with biological tissue,without obvious rejection reaction.2.A large number of large animal experiments were conducted to verify whether the constructed double-network hydrogel patch could be successfully used for hemostasis of tissues and organs.In the arterial injury model,arterial bleeding stopped and the patch adhered tightly without falling off after slightly pressing the coated hydrogel patch around the wound.In the liver injury model,hydrogel could also adhere to the bleeding wound surface and play a hemostasis effect.3.In vitro CCK-8 cell assay results showed that MCC950 at a certain concentration was not cytotoxic.So we added drugs into the synthetic double-network hydrogel matrix layer at an appropriate concentration and determined to be capable of drug release.In animal experiments,compared with the hydrogel group without adding MCC950,HE and Masson staining showed that the vascular repair was better in the drug group,and the infiltration of inflammatory cells were significantly reduced.Doppler ultrasound showed that the vascular patency in the drug group was better than that in the non-drug group.Conclusion:The double-network hydrogel patch loaded with MCC950 can closely adhere to the traumatic bleeding wound of tissues and organs,and at the same time,it can release the small molecule MCC950,which can inhibit the inflammatory response.So when the hydrogel achieves the purpose of emergency hemostasis,at the same time,it is also conducive to wound healing and functional recovery of tissues and organs.