CN115400262A

CN115400262A - Injectable rapid hemostatic hydrogel containing TCP-25 and preparation method thereof

Info

Publication number: CN115400262A
Application number: CN202211163298.9A
Authority: CN
Inventors: 温娜; 李双双; 蒋鸿志; 杨佳超; 龙金林
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2022-09-23
Filing date: 2022-09-23
Publication date: 2022-11-29

Abstract

The invention discloses an injectable rapid hemostatic hydrogel containing TCP-25 and a preparation method thereof, the hydrogel is prepared by natural polymers and has multiple functions of antibiosis, rapid hemostasis, gastric ulcer wound healing promotion and the like, and the effect is superior to that of commercial omeprazole; the preparation method comprises the following steps: dissolving xanthan gum and konjac gum in deionized water, and adding NaIO ₄ The C-C bond of the cis-diol group is cracked to generate an aldehyde group, then, on the basis of the action of a dynamic imine bond, a xanthan gum derivative and a konjac gum derivative with the aldehyde group at the end group are used as gel factors, thrombin derivative peptide is introduced, and carboxymethyl chitosan is crosslinked to obtain the hydrogel containing the TCP-25. The hydrogel raw material obtained by the invention is easy to obtainThe gel has the advantages of simple and quick preparation, quick gelling, injectability, self-healing property and antibacterial property, can be directly injected into wounds or stomach, and has multiple functions of promoting wound healing, quickly stopping bleeding, promoting gastric ulcer wound healing and the like.

Description

Injectable rapid hemostatic hydrogel containing TCP-25 and preparation method thereof

Technical Field

The invention relates to a biomedical material, belongs to the technical field of wound dressing, hemostasis or gastric ulcer healing and the like of organisms, and particularly relates to a rapid hemostasis hydrogel containing TCP-25 based on carboxymethyl chitosan and a preparation method thereof.

Background

Gastric ulcer seriously affects the health of people. In clinical research, gastric ulcer patients have complications such as ulcer perforation, pyloric obstruction, cancer and the like, and are accompanied by upper gastrointestinal hemorrhage, and serious patients even endanger the life of the patients. Even if the gastric ulcer heals under the endoscope, the histology and the ultrastructure of the gastric ulcer still have the possible abnormality, such as reduction of the thickness of mucous membrane, reduction of glands and capillaries and the like, which easily causes the recurrence of the gastric ulcer. Bleeding is a necessary consequence of gastric ulcers, can cause significant pain to patients and even life-threatening, and bleeding from peptic ulcers invariably leads to significant morbidity and mortality. In order to deal with the great challenge of rapid bleeding after gastric ulcer, a series of hemostatic drugs, such as intravenous proton pump inhibitors and hemostatic agents, oral gastric mucosa protective agents and the like, have been developed in recent years. However, the effects of drug treatment on hemostasis and healing are limited and the data show that delayed bleeding occurs in 20% of cases. On the one hand, these agents generally last for a relatively short time due to their poor acid resistance and their relatively rapid dissolution; on the other hand, some powder drugs must be delivered to the lesion through a catheter, which places additional burden on the surgical procedure; in addition, as too many treatment options are sought during the treatment of the patient, too much treatment cost is consumed under the advice of the doctor, and great economic stress is brought to the patient and the family members thereof. To date, hemostatic treatment after bleeding ulcers has presented significant challenges.

The purpose of hydrogel as a primary wound dressing is to promote wound healing and reduce postoperative infection. Meanwhile, after the hydrogel is formed, the hydrogel can be directly injected to a wound part, so that the operation process is reduced, and the wound healing is accelerated. Therefore, hydrogels have unparalleled advantages when the treatment of bleeding from gastric ulcers is inconvenient. Guo et al reported an injectable hydrogel based on acryloyl-6-aminocaproic acid and AA-g-N-hydroxysuccinimide formed by in situ free radical polymerization for gastric hemostasis and wound healing. (Guo Y, dundas CM, zhou X, et al, molecular Engineering for hydrogel Water discovery and Sustainable solvent Vapor Generation [ J ]. Advanced Materials, 2021, 33 (35): 2102994.) summer et al reported a hemostatic hybrid hydrogel that further demonstrated good clotting capabilities (-60 s). (Xia X, xu X, wang B, et al, adhesive prosthetic Hydrogel with ultra fast tissue accumulation Instrument in Pigs [ J ]. Advanced Functional Materials, 2021: 2109332.) however, these hydrogels use complex raw Materials and have a long Hemostatic time for patients with Acute gastric ulcer bleeding, which may endanger the life of the patient in the severe case, thus limiting their use in treating critical Upper Gastrointestinal Hemorrhage.

To overcome these limitations, we have designed an ideal self-healing injectable hemostatic hydrogel dressing for hemostasis and wound healing after bleeding from acute gastric ulcers. The chitosan and the derivatives thereof are approved by food and drug administration and can be used as local hemostatic drugs, and meanwhile, the thrombin C-terminal derived peptide-GKYGFYTHVFRLKKWIQKVIDQFGE is marked as TCP-25, has the capabilities of healing wounds and inhibiting bacterial growth, and is proved to be a culprit for causing inflammation formation by removing Lipopolysaccharide (LPS) in pathogens in an experimental animal model. In addition, thrombin, a key factor in the coagulation process, is widely used at bleeding sites where hemostasis is difficult. The main reason why the thrombin can stop bleeding is to promote the fibrinogen to be converted into fibrin, which directly acts on the last step of blood coagulation, thus being capable of stopping bleeding rapidly, and being doped into hydrogel as a functional factor to prepare a hemostatic hydrogel dressing.

Disclosure of Invention

Aiming at the technical problems at present, the invention aims to prepare the self-healing injectable hemostatic hydrogel dressing, which has simple steps, cheap and easily obtained raw materials, universality and application in biomedical materials, sterile treatment, in-vitro wound dressing, rapid hemostasis, gastric ulcer wound healing, postoperative wound treatment in memory and the like. The self-healing injectable hemostatic hydrogel has extremely short gel time, good biocompatibility and excellent hemostatic performance; in addition, the wound surface formed after the operation in the organism can be protected, the purpose of early healing is achieved, the postoperative pain and other relevant clinical symptoms of a patient are reduced, and the postoperative wound surface protecting device is more beneficial to preventing the occurrence of postoperative complications such as perforation, stenosis, bleeding and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of an injectable rapid hemostatic hydrogel containing TCP-25 comprises the following steps: dissolving xanthan gum or konjac gum in deionized water, and adding appropriate amount of NaIO ₄ Cracking C-C bond of the cis-diol group to generate aldehyde group, introducing TCP-25 by taking xanthan gum derivative or konjac gum derivative with aldehyde group at the end group as a gel factor based on dynamic imine bond action, and crosslinking carboxymethyl chitosan to obtain series of self-healing hydrogel TCP-CX and TCP-CO containing TCP-25; the method specifically comprises the following steps:

(1) Dissolving carboxymethyl chitosan in water to obtain a first component solution;

(2) Dissolving xanthan gum or konjac gum in water, adding NaIO4, fully stirring for reaction, dialyzing, centrifugally precipitating, freeze-drying, and dissolving in water to obtain a second component solution;

(3) Adding the thrombin derivative peptide into the first component solution, mixing with the second component solution, and forming the injectable rapid hemostatic hydrogel containing TCP-25 after crosslinking.

Further, the concentration of the first component solution is 50-80mg/mL; the concentration of the second component solution is 5-50mg/mL.

Further, the mass ratio of NaIO4 to xanthan gum or konjac glucomannan in the step (2) is 1-100.

Further, the reaction in the step (2) is an oxidation reaction, the reaction temperature is 20-80 ℃, the reaction time is 4-24h, ethylene glycol is added to terminate the reaction after the reaction time is reached, preferably, 1-3mL of ethylene glycol is added to terminate the reaction in the oxidation reaction, and the reaction time is 1-5h to terminate the oxidation reaction.

Further, the crosslinking time in the step (3) is 8-20s.

Further, the concentration of the thrombin-derived peptide in step (3) is 0.3-1mM.

Furthermore, the carboxymethyl chitosan selected by the invention has active amino groups on the molecular chain, can provide active sites for Schiff base reaction, and has good foundation for forming hydrogel by crosslinking; and the carboxymethyl chitosan has a series of excellent physicochemical properties and biological characteristics, such as good water solubility, antibacterial property, blood coagulation property, cell proliferation promotion and the like, and has wide application prospects.

Furthermore, the raw material selected by the invention is xanthan gum which is used as a water-soluble polysaccharide, has multiple functions due to the special structure and the alternating characteristic of macromolecules, and simultaneously contains a large amount of active groups such as hydroxyl, carboxyl and the like on main and side chains, so that new performance can be endowed by modification.

Furthermore, the invention selects another matrix konjac gum. Konjac gum is a water-soluble non-ionic natural polysaccharide. The molecular of the modified polyvinyl alcohol has rich hydroxyl groups, so the modified polyvinyl alcohol has good reaction activity, is convenient for various modifications to improve the application performance, and has good affinity to water and good sol property. After the hydrogel is subjected to hydroformylation modification, the hydrogel can be subjected to Schiff base reaction with the first component solution to form the hydrogel with a stable three-dimensional network structure.

Furthermore, the cis-diol groups in the molecular chains of the xanthan gum and the konjac gum selected by the invention can be oxidized by a strong oxidant NaIO ₄ The aldehyde group on the oxidized derivative and the amino group on the carboxymethyl chitosan generate dynamic imine bond, and the dynamic imine bond generated by the aldehyde group and the amino group can bring self-healing property and injectability to the hydrogel, thereby meeting the requirementsVarious medical requirements, mild reaction conditions and high reaction rate.

Has the advantages that:

(1) One purpose of the invention is to prepare an antibacterial hemostatic hydrogel material based on TCP-25 peptide through surface interface colloid interaction, and the selected matrix material is non-toxic, green and healthy and has no potential harm to organisms;

(2) The second purpose of the invention is to mix the oxidized natural polymer and carboxymethyl chitosan and prepare the hydrogel material by adopting a one-pot method, thereby avoiding adding other toxic substances, and improving the biocompatibility of the hydrogel material.

(3) The third purpose of the invention is that the TCP-25 hydrogel prepared by natural polymer has the functions of fast hemostasis (8-9 s) and promoting the healing of gastric ulcer, can promote the healing of wound surface and reduce infection rate and inflammation rate. Also, it is intended to develop a treatment for the upper digestive tract of a living body after surgery, and it can be applied to the stomach, duodenum, etc.

(4) The invention also aims at the injectability, and can be widely applied to surgical operation or Endoscopic Submucosal Dissection (ESD), endoscopic tunneling resection (STER) under mucosa, endoscopic full-thickness resection (EFTR) and the like.

(5) The fifth purpose of the invention is that the invention is expected to have potential application in various fields of antibacterial hemostatic materials, anti-inflammatory antibacterial materials, drug sustained-release materials, biological medicines, rapid hemostasis, upper gastrointestinal gastric ulcer healing and the like.

Drawings

FIG. 1 is a schematic diagram of the preparation of carboxymethyl chitosan-hydroformylated xanthan gum hydrogel;

FIG. 2 is a reaction mechanism diagram of the hydroformylation process of carboxymethyl chitosan, xanthan gum and hydrogel;

FIG. 3 is a digital photograph of a hydrogel;

FIG. 4 is a schematic diagram of the preparation process of carboxymethyl chitosan-hydroformylation konjac gum gel;

FIG. 5 is a reaction mechanism diagram of a hydroformylation process of carboxymethyl chitosan and konjac gum and a hydrogel;

FIG. 6 is an infrared spectrum of a hydrogel;

FIG. 7 is a graph of the self-healing and injectability properties of hydrogels;

FIG. 8 is a hepatic trauma hemostasis map of a hydrogel;

figure 9 is a diagram of in vivo gastric ulcer treatment of a hydrogel.

Detailed Description

The invention discloses a preparation method and application of an injectable rapid hemostatic hydrogel containing TCP-25. The preparation material comprises the following steps: carboxymethyl chitosan, xanthan gum, konjac gum and NaIO ₄ Ethylene glycol, TCP-25 and ultrapure water.

Examples 1 to 3:

stirring and dissolving carboxymethyl chitosan (1.5 to 2.4 g) in sterile water (30 mL) until the carboxymethyl chitosan is completely dissolved to obtain a carboxymethyl chitosan solution of 50-80mg/mL as a first component solution; in this embodiment, the concentration of the selected first component solution is 80mg/mL, and the reaction scheme is shown in FIG. 1.

Xanthan gum (1 g) was dissolved in sterile water (100 mL) with stirring, and then an ultrasonically dissolved sodium periodate solution (10% (w/v); 3 mL) was added to the aqueous xanthan gum solution, and sufficiently stirred to allow oxidation reaction to occur under dark conditions at T =45 ℃ for 8h, and then ethylene glycol (1 mL) was added to terminate the oxidation reaction for 1h. The reaction solution was then dialyzed for 72h, followed by centrifugation in absolute ethanol to give the reaction product, which was finally freeze-dried to give the final hydroformylation xanthan gum product.

Continuously dissolving the hydroformylation xanthan gum product in sterile water to obtain a hydroformylation xanthan gum solution of 5-30mg/mL as a second component solution; in this embodiment, the concentrations of the second component solutions are 5mg/mL, 15mg/mL, and 30mg/mL, respectively.

And mixing the first functional component solution and the second component solution in equal volume, and crosslinking to form the carboxymethyl chitosan-hydroformylation xanthan gum hydrogel.

The reaction process is shown in a mechanism diagram in figure 2, a plurality of amino active reaction sites are arranged on carboxymethyl chitosan, a plurality of aldehyde active reaction sites are arranged on hydroformylation xanthan gum, and amino and aldehyde groups undergo Schiff base reaction to generate imine bonds, so that the imine bonds are gradually crosslinked to form the hydrogel material with a stable three-dimensional network structure. The digital image of the hydrogel is shown in FIG. 3, and the hydrogel was placed in an inverted state, and the sample did not flow.

Examples 4-6:

stirring and dissolving carboxymethyl chitosan (1.5-2.4 g) in sterile water (30 mL) until the carboxymethyl chitosan is completely dissolved to obtain a carboxymethyl chitosan solution of 50-80mg/mL as a first component solution; in this embodiment, the concentration of the first component solution is selected to be 60mg/mL, and the reaction scheme is shown in FIG. 4.

Konjak gum (1 g) is stirred and dissolved in sterile water (100 mL), then an ultrasonic-dissolved sodium periodate solution (10% (w/v); 3 mL) is added to the konjak gum solution, and the mixture is stirred sufficiently to cause oxidation reaction under the dark condition and the condition of T =30 ℃ for 4h, and then ethylene glycol (1 mL) is added to terminate the oxidation reaction for 3h. Then dialyzing the reaction solution for 48h, then centrifuging and precipitating to obtain a reaction product, and finally freeze-drying to obtain a final hydroformylation konjac glucomannan product.

Dissolving the hydroformylation konjac glucomannan product in sterile water to obtain a hydroformylation konjac glucomannan solution of 30-50mg/mL as a second component solution; in this embodiment, the concentrations of the second component solutions are 30mg/mL, 40mg/mL, and 50mg/mL, respectively.

And (3) mixing the first functional component solution and the second component solution in equal volume, and forming carboxymethyl chitosan-hydroformylation konjac gum gel after crosslinking.

The reaction process is shown in a schematic diagram in fig. 5, a plurality of amino active reaction sites are arranged on carboxymethyl chitosan, a plurality of aldehyde active reaction sites are arranged on the hydroformylation konjac glucomannan, and the amino and the aldehyde are subjected to Schiff base (Schiff) reaction to generate imine bonds, so that the imine bonds are gradually crosslinked to form the hydrogel material with a stable three-dimensional network structure. The digital image of the hydrogel is shown in FIG. 3, and the hydrogel was placed in an inverted state, and the sample did not flow.

Examples 7 to 9:

the aqueous first component solution and the aqueous second component solution of the hydroformylated xanthan gum were prepared in accordance with the procedure of example 1.

Dissolving thrombin derivative peptide (TCP-25, c =0.3 mM) as a third functional component into the first component solution to obtain a first functional component solution; in the embodiment, the concentration of the first functional component solution is 80mg/mL; in this embodiment, the concentrations of the second component solutions are 5mg/mL, 15mg/mL, and 30mg/mL, respectively.

And mixing the first functional component solution and the second functional component solution in equal volume, and crosslinking to form the carboxymethyl chitosan-hydroformylation xanthan gum-thrombin derivative peptide hydrogel, wherein the gel forming schematic diagram is shown in figure 2, and the infrared spectrum of the hydrogel is shown in figure 6.

The carboxymethyl chitosan has a plurality of amino active reaction sites, the hydroformylation xanthan gum has a plurality of aldehyde active reaction sites, the amino and aldehyde groups generate Schiff base reaction to generate imine bonds, so that gradual crosslinking is carried out to form a hydrogel material with stable three-dimensional network structure, meanwhile, thrombin derived peptide (TCP-25) is used as a functional component to play a role in antibiosis and hemostasis, and the obtained hydrogel not only has excellent self-healing property and excellent injectability (figure 7), but also has a rapid hemostasis function (figure 8), and has the functions of promoting gastric ulcer wound hemostasis and wound healing (figure 9).

Examples 10 to 12:

the first-component aqueous solution and the second-component hydroformylation konjac gum aqueous solution were prepared in accordance with the method of example 1.

Dissolving thrombin derivative peptide (TCP-25, c =0.3 mM) as a third functional component into the first component solution to obtain a first functional component solution; in the embodiment, the concentration of the first functional component solution is 60mg/mL; in this embodiment, the concentrations of the second component solutions are 30mg/mL, 40mg/mL, and 50mg/mL, respectively.

Mixing the first functional component solution and the second functional component solution in equal volume, and crosslinking to obtain carboxymethyl chitosan-hydroformylation konjac glucomannan-thrombin derived peptide hydrogel, wherein the gel formation schematic diagram is shown in FIG. 5, and the infrared spectrum of the hydrogel is shown in FIG. 6.

The carboxymethyl chitosan has a plurality of amino active reaction sites, the hydroformylation xanthan gum has a plurality of aldehyde active reaction sites, the amino and aldehyde groups generate Schiff base reaction to generate imine bonds, so that gradual crosslinking is carried out to form a hydrogel material with stable three-dimensional network structure, and the thrombin derived peptide is used as a functional component to play a role in antibiosis and hemostasis, so that the obtained hydrogel not only has excellent self-healing property and excellent injectability (figure 7), but also has a rapid hemostasis function (figure 8), and a function of promoting gastric ulcer wound hemostasis and wound healing (figure 9).

The above description is only a preferred embodiment of the present invention, and does not limit the scope of the present invention, and all other embodiments obtained without inventive labor by those skilled in the art after reading the present invention belong to the scope of the present invention.

Claims

1. A preparation method of an injectable rapid hemostatic hydrogel containing TCP-25 is characterized by comprising the following steps:

(2) Dissolving xanthan gum or konjac gum in water, and adding NaIO ₄ After fully stirring and reacting, dialyzing, centrifugally precipitating, freeze-drying and dissolving in water to obtain a second component solution;

2. The method for preparing an injectable rapid hemostatic hydrogel according to claim 1 comprising TCP-25, wherein the concentration of the first component solution is 50 to 80mg/mL; the concentration of the second component solution is 5-50mg/mL.

3. The method for preparing injectable rapid hemostatic hydrogel comprising TCP-25 according to claim 1, wherein NaIO in step (2) ₄ Mixing with xanthan gum or konjac gumThe ratio is 1-100.

4. The method for preparing injectable rapid hemostatic hydrogel containing TCP-25 according to claim 1, wherein the reaction in step (2) is oxidation reaction at 20-80 ℃ for 4-24h, and after the reaction time is reached, ethylene glycol is added to terminate the reaction.

5. The method for preparing injectable rapid hemostatic hydrogel comprising TCP-25 according to claim 1, wherein the cross-linking time in step (3) is 8-20s.

6. The method for preparing injectable rapid hemostatic hydrogel according to claim 1, wherein the thrombin-derived peptide in step (3) is present in a concentration of 0.3-1mM.

7. An injectable rapid hemostatic hydrogel containing TCP-25 prepared by the method of any one of claims 1 to 6.