CN117720697A - Antibacterial anti-adhesion chondroitin sulfate gel and preparation method and application thereof - Google Patents
Antibacterial anti-adhesion chondroitin sulfate gel and preparation method and application thereof Download PDFInfo
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- CN117720697A CN117720697A CN202311671127.1A CN202311671127A CN117720697A CN 117720697 A CN117720697 A CN 117720697A CN 202311671127 A CN202311671127 A CN 202311671127A CN 117720697 A CN117720697 A CN 117720697A
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- chondroitin sulfate
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- 229920001287 Chondroitin sulfate Polymers 0.000 title claims abstract description 54
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Materials For Medical Uses (AREA)
Abstract
The invention discloses an antibacterial anti-adhesion chondroitin sulfate gel and a preparation method and application thereof, and belongs to the field of medical biological materials. The preparation method of the antibacterial anti-adhesion chondroitin sulfate gel adopts a free radical polymerization method, takes methacryloxyethyl phosphorylcholine, epigallocatechin gallate, glycidyl methacrylate and 3-aminophenylboric acid modified chondroitin sulfate as raw materials, and initiates double bond polymerization to form hydrogel under the action of heating and a thermal initiator, so that the cost is low and the preparation method is simple. The chondroitin sulfate hydrogel disclosed by the invention has good mechanical properties, shear thinning characteristics and excellent antibacterial capability, and can effectively prevent bacterial infectious abdominal cavity wound adhesion.
Description
Technical Field
The invention belongs to the field of medical biological materials, and in particular relates to a preparation method and application of antibacterial anti-adhesion chondroitin sulfate hydrogel.
Background
Unavoidable peritoneal injuries, bleeding and infection during surgery can lead to fibrous bands at the site of injury and adjacent tissues or organs and to adhesions. Among them, peritoneal injury causes inflammatory reactions, producing large amounts of inflammatory cells, inflammatory mediators and fibrin, disrupting the balance between fibrin production and degradation, leading to the occurrence and development of abdominal adhesions. Bleeding at the wound site of the peritoneum after operation, blood clots and insoluble fibrin can be formed after solidification, fibroblast proliferation is promoted, and the formation and development of adhesion are aggravated. Accidental abdominal infections during surgery often affect the abdominal microenvironment, leading to fibrin exudates and fibrin formation and exacerbating adhesion formation. Intra-abdominal surgery typically requires extensive manipulation of the microorganism-rich intestinal tract, resulting in leakage of intestinal microorganisms into the abdominal environment. Intestinal microorganisms can increase adhesion severity by driving the epidermal growth factor receptor signaling pathway of the peritoneal mesothelial cells. Postoperative adhesions tapes have a variety of phenotypes, with slight adhesions being simply the formation of a thin fibrous membrane between adjacent tissues, and severe even adhesions involving fibrous tissues, blood vessels and nerves. Post-operative adhesion formation can be accompanied by a range of complications such as chronic pain, infertility, ileus or dysfunction of adjacent organs, and the like. Because the existing in-vitro diagnostic equipment such as X-ray and magnetic resonance imaging, computer tomography or ultrasonic detection cannot diagnose the postoperative adhesion well, a patient or doctor can only perceive the occurrence of the adhesion through adhesion related complications, so that the optimal treatment time is often missed, and the life safety of the patient is greatly threatened. Thus, post-operative adhesions, particularly bacterial infectious adhesions, have long been a significant clinical challenge.
The existing anti-adhesion method is mainly divided into two major categories of drug treatment and physical barrier, and the two major categories have obvious advantages and disadvantages. Currently, drugs clinically used for preventing postoperative abdominal adhesions include anticoagulants, fibrinolytics, anti-inflammatory agents, antihistamines, collagen inhibitors, proteases, and the like. But these areThe medicine is not designed for abdominal adhesion, but only for a certain part in the adhesion formation process, and is insufficient for completely preventing adhesion. Furthermore, the mode of administration limits the effect of these drugs on abdominal adhesions. Compared with oral administration, the topical injection administration can reduce the administration amount and improve the bioavailability. However, too fast absorption of the peritoneum will distribute the drug to other parts of the body, leading to limited anti-adhesion effects and certain side effects. The biomaterial barrier may isolate the damaged surface from other tissues or organs until the mesothelial cells are repaired. Although some biomaterials have been developed as related products for preventing adhesions, various problems have been difficult to avoid. Such as solid barriersAnd->It is difficult to completely cover the wound due to its physical characteristics, and the effect for laparoscopic surgery is poor; liquid barriers such as->The adhesion failure is prevented due to the problems of over-fast in vivo degradation and the like. In addition, the existing anti-adhesion agents have no antibacterial capability, and can not treat clinically complex operation conditions such as intestinal microbial leakage and the like. Therefore, a treatment method capable of effectively preventing bacterial infectious abdominal adhesions is a currently urgent problem to be solved.
Disclosure of Invention
The invention aims to avoid the defects of poor anti-adhesion effect and no antibacterial effect of solid and liquid biological material barriers, and provides antibacterial anti-adhesion chondroitin sulfate hydrogel, a preparation method and application thereof.
In order to achieve the purpose, the invention is realized by adopting the following technical scheme:
the preparation method of the antibacterial anti-adhesion chondroitin sulfate hydrogel comprises the following steps:
1) 2g of chondroitin sulfate is dissolved in 100mL of ultrapure water to form a chondroitin sulfate solution, and 1.6g of tetrabutylammonium bromide and 700 mu L of triethylamine are added for catalytic reaction; stirring at room temperature for 1h, adding 10mL of glycidyl methacrylate, reacting at room temperature for 24h, dialyzing with ultrapure water for 4 days (molecular weight of dialysis bag is 3500), and freeze-drying to obtain glycidyl methacrylate grafted chondroitin sulfate (CS-GMA).
2) 1.1 gCS-GMA was dissolved in 100mL of ultrapure water, and after complete dissolution, 1.15g of N-hydroxysuccinimide and 1.9g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were added in this order, and reacted at room temperature for 1 hour, and 0.32g of 3-aminophenylboric acid was added for 48 hours. After the reaction, the reaction solution was transferred into a dialysis bag having a molecular weight of 3500, dialyzed against ultrapure water for 4 days, and freeze-dried to obtain glycidyl methacrylate and 3-aminophenylboronic acid grafted chondroitin sulfate (CGA).
3) Dissolving methacryloxyethyl phosphorylcholine, epigallocatechin gallate, glycidyl methacrylate and 3-aminophenylboric acid grafted chondroitin sulfate in ultrapure water, and uniformly vortex mixing to obtain a uniform solution.
4) The mixed solution comprises, by mass, 5% -10% of methacryloyloxyethyl phosphorylcholine, 0.5% -5% of glycidyl methacrylate and 3-aminophenylboric acid grafted chondroitin sulfate, 0.15% -1.5% of epigallocatechin gallate and the balance of water.
5) A thermal initiator corresponding to 1% of the mass of the solute was added to the mixed solution, and the mixture was heated at 60℃for 0.5 hours to obtain the hydrogel.
6) The hydrogel was immersed in physiological saline for 48 hours to remove unreacted monomers, and passed. 22G sterile needle extrusion.
Further, the molecular weight of the chondroitin sulfate is 5000-50000.
Further, the thermal initiator in the step 3) is azo diiso Ding Mi hydrochloride.
Further, 1mg to 2mg of azobisiso Ding Mi hydrochloride was added to each 1mL of the mixed solution.
The application of the antibacterial anti-adhesion chondroitin sulfate hydrogel disclosed by the invention is used as a biological material barrier for preventing postoperative abdominal adhesion.
Compared with the prior art, the invention has the following beneficial effects:
according to the preparation method of the antibacterial anti-adhesion chondroitin sulfate hydrogel, a free radical copolymerization method is adopted, double bond functionalized chondroitin sulfate and methacryloyloxyethyl phosphorylcholine are initiated to polymerize to form the hydrogel under the action of heating and a thermal initiator, the cost is low, the preparation method is simple, and the problems of biotoxicity and the like caused by the introduction of a chemical crosslinking agent are avoided.
The antibacterial anti-adhesion chondroitin sulfate hydrogel disclosed by the invention has good mechanical properties, shear thinning characteristics, excellent antibacterial capability and good cell compatibility.
The antibacterial anti-adhesion chondroitin sulfate hydrogel is used for preventing bacterial infectious abdominal adhesions after abdominal operation, and is injected to a wound site through a syringe to isolate peritoneum and surrounding tissues, so that the purpose of preventing adhesions is achieved.
Drawings
FIG. 1 is a morphology of the hydrogel of example 1 after injection after complete swelling;
FIG. 2 is a microstructure of the hydrogel of example 1;
FIG. 3 is a graph showing the shear viscosity profile of the hydrogel of example 1;
FIG. 4 is a stress-strain curve of the hydrogel of example 1;
FIG. 5 is a time-scanning test chart of the hydrogel of example 1;
FIG. 6 is a self-healing test chart of the hydrogel of example 1;
FIG. 7 is a graph showing the results of in vitro antibacterial experiments on E.coli cells of the hydrogel of example 1;
FIG. 8 is a graph of HMrSV5 cell compatibility results for the hydrogels of example 1;
FIG. 9 is a graph showing the effect of the hydrogel of example 1 on preventing bacterial infectious abdominal adhesions, wherein FIG. 9 (a) shows a model set of adhesions, FIG. 9 (b) shows a gel set of commercial hyaluronic acid, and FIG. 9 (c) shows a gel set of chondroitin sulfate;
FIG. 10 is a graph showing the effect of the hydrogel of example 1 on bacterial infectious abdominal adhesions infection;
Detailed Description
In order that those skilled in the art will better understand the present invention, a detailed description of embodiments of the present invention will be given below with reference to the accompanying drawings in which the described embodiments are not all embodiments of the present invention, but only some embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that the terms "comprises" and "comprising," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but includes other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the attached drawing figures:
example 1
Dissolving methacryloxyethyl phosphorylcholine, epigallocatechin gallate, glycidyl methacrylate and 3-aminophenylboric acid grafted chondroitin sulfate in ultrapure water, and uniformly vortex mixing to obtain a uniform solution. The concentration of methacryloyloxyethyl phosphorylcholine in the mixed solution was 5%, the concentration of glycidyl methacrylate and 3-aminophenylboronic acid grafted chondroitin sulfate was 1%, and the concentration of epigallocatechin gallate was 0.3%. 1mL of the mixed aqueous solution was taken, 1mg of azobisiso Ding Mi hydrochloride was added, and the mixture was completely dissolved by ultrasonic wave, and heated at 60℃for 0.5h to prepare a hydrogel. The resulting hydrogel was immersed in physiological saline for 48 hours, with water being exchanged every 12 hours to remove unreacted monomers and initiator. The hydrogel was then extruded through a 22G needle to give the final injectable hydrogel.
Referring to fig. 1, hydrogel fragments extruded from a 22G needle were able to self-fuse together rapidly, forming an "XJTU" word and still maintaining a transparent uniform morphology, demonstrating excellent injectability and self-fusion properties of the hydrogel.
1. Microcosmic morphology of the hydrogel of example 1
The antibacterial anti-adhesion chondroitin sulfate hydrogel prepared in example 1 was placed in a vacuum freeze dryer for freeze drying, the surface of the gel was sprayed with gold, and then the microscopic morphology of the surface of the gel was observed using a scanning electron microscope. Referring to fig. 2, the chondroitin sulfate hydrogel has an obvious three-dimensional pore structure and is honeycomb-shaped, so that the chondroitin sulfate hydrogel is beneficial to the diffusion and transportation of oxygen and nutrients. The formation mechanism of the three-dimensional pore structure of the hydrogel is probably that a large number of hydrophilic groups existing in the chondroitin sulfate molecule can form hydrogen bonds with other hydrophilic groups or water molecules, so that a compact three-dimensional pore structure is formed.
2. Rheological Property test of hydrogels of example 1
The hydrogels were subjected to shear viscosity testing using a An Dongpa rheometer (MCR 302 for samples). As shown in fig. 3, in the strain sweep test, when the strain<The storage modulus (G ') of the chondroitin sulfate hydrogel was greater than the loss modulus (G') at 200%, indicating that it exhibited a stable gel state. When strained>At 200%, G "was found">G', indicating that the gel network is broken and the sol state is presented. Referring to FIG. 4, the viscosity of the chondroitin sulfate hydrogel gradually decreases with increasing shear rate at a shear rate of 0.1 to 100S -1 Within this range, the viscosity was reduced by nearly two orders of magnitude, indicating that the chondroitin sulfate hydrogel has stable shear thinning behavior, indicating excellent injectability. Referring to fig. 5, in the time-sweep test, G' and G "of the chondroitin sulfate hydrogel were almost constant, indicating that the chondroitin sulfate hydrogel can maintain a stable gel state for a long period of time.
3. Self-healing Performance test of hydrogels of example 1
Referring to fig. 6, when 500% high strain and 0.5% low strain are alternately applied, both G' and G "of the chondroitin sulfate hydrogel can be restored to the original levels, and there is no obvious change after 3 cycles of cyclic test, and the result proves that the structure of the chondroitin sulfate hydrogel can be quickly reconstructed after being damaged by external force, and has high-efficiency self-fusion capability.
4. In vitro antibacterial experiments
The chondroitin sulfate hydrogel prepared in example 1 was irradiated with ultraviolet rays for 0.5h to remove the foreign bacteria. OD is set to 600 Bacterial liquid 10-fold diluted with =0.1 (1×10) 6 CFU/mL), 10 μl was added to the hydrogel, incubated at 37 ℃ for 1h, 1mL of sterile physiological saline was added, and 20 μl was plated on agar plates. As a result of the antibacterial effect, referring to fig. 7, epigallocatechin gallate in chondroitin sulfate gel can kill more than 99% of escherichia coli, showing excellent antibacterial ability of chondroitin sulfate hydrogel, compared with normal saline.
5. Hydrogel cell compatibility evaluation of example 1
The present experiment uses thiazole blue colorimetry (MTT) to evaluate the cytocompatibility of chondroitin sulfate hydrogels to peritoneal mesothelial cells (HMrSV 5). HMrSV5 cells were cultured in DMEM medium (10% foetal calf serum and 1% diabody) at 37℃with 5% CO 2 Culturing in an incubator. The cells were mixed at 1X 10 4 Is inoculated in 96-well plates and incubated for 24h. The medium was aspirated and DMEM medium containing different concentrations (0, 50, 100, 200, 400, 800 μg/mL) of gel powder was added. Incubation was performed for 24h, medium was aspirated off, 100. Mu.L of 0.5mg/mL MTT solution was added and incubated for 4h at 37 ℃. The medium was removed and 150 μl of dimethyl sulfoxide solution was added to each well, shaken for 10min in the dark, its absorbance was measured at 490nm and HMrSV5 cell viability was calculated.
Referring to FIG. 7, the results show that the chondroitin sulfate hydrogel has no obvious effect on the survival rate of HMrSV5 cells after being incubated with the cells for 24 hours in the concentration of 0-800 mug/mL, and the chondroitin sulfate hydrogel has good cell compatibility.
6. Evaluation of hydrogel-preventive Abdominal adhesion Effect of example 1
1) Male SD rats were used as experimental animals, after isoflurane anesthesia, the cecum was cut off along the center of the abdomen, rubbed with sterile gauze until punctate bleeding occurred, and the cecum was ligated, and the cecum was pierced with a 22G needle to simulate intestinal microbial leakage. The corresponding peritoneal position is cut off by a surgical knife of 1X 2cm 2 Placing the rubbed cecum at the injured peritoneal siteTo simulate the unavoidable peritoneal injury after operation, less than 1mL of solution is applied to the wound surface, the abdomen is closed layer by layer, and penicillin is injected subcutaneously.
The 18 rats were randomly divided into three groups of 6:
the first group is a model group, and 1mL of physiological saline is injected on the wound surface;
the second group is commercial hyaluronic acid gel group, and 1mL commercial hyaluronic acid gel is injected on the wound surface;
the third group is chondroitin sulfate hydrogel group, and 1mL of chondroitin sulfate hydrogel is injected on the wound surface.
Photographing at 7 days and evaluating the abdominal adhesion degree and the bacterial infection degree, referring to fig. 9 and 10, it can be observed that rats of the chondroitin sulfate hydrogel group are not subjected to abdominal adhesion, and no bacterial colony is formed after the flat plate is coated, so that the chondroitin sulfate hydrogel prepared by the invention has good postoperative adhesion prevention and antibacterial effects.
The embodiments of the present invention are disclosed as preferred embodiments, but not limited thereto, and those skilled in the art will readily appreciate from the foregoing description that various extensions and modifications can be made without departing from the spirit of the present invention.
Claims (6)
1. The preparation method of the antibacterial anti-adhesion chondroitin sulfate hydrogel comprises the following steps:
1) Dissolving methacryloxyethyl phosphorylcholine, epigallocatechin gallate, glycidyl methacrylate and 3-aminophenylboric acid grafted chondroitin sulfate in ultrapure water, and uniformly vortex mixing to obtain a uniform solution;
2) The mixed solution comprises, by mass, 5% -10% of methacryloyloxyethyl phosphorylcholine, 0.5% -5% of glycidyl methacrylate and 3-aminophenylboric acid grafted chondroitin sulfate, 0.15% -1.5% of epigallocatechin gallate and the balance of water;
3) Adding a thermal initiator accounting for 1% of the mass of the solute into the mixed solution, and heating at 60 ℃ for 0.5h to obtain the hydrogel;
4) The hydrogel was immersed in physiological saline for 48 hours to remove unreacted monomers, and squeezed through a 22G sterile needle.
2. The method for preparing antibacterial anti-adhesion chondroitin sulfate hydrogel according to claim 1, wherein the molecular weight of the chondroitin sulfate is 5000-50000.
3. The method for preparing the antibacterial anti-adhesion chondroitin sulfate hydrogel according to claim 1, wherein the thermal initiator in the step 3) is azo diiso Ding Mi hydrochloride.
4. The method for preparing an antibacterial anti-adhesion chondroitin sulfate hydrogel according to claim 3, wherein 1mg-2mg of azobisis Ding Mi hydrochloride is added to 1mL of the mixed solution.
5. An antibacterial anti-blocking chondroitin sulfate hydrogel according to any one of claims 1 to 4, which is a hydrogel formed by free radical copolymerization of double bond functionalized chondroitin sulfate and methacryloyloxyethyl phosphorylcholine.
6. Use of an antibacterial anti-adhesion chondroitin sulfate hydrogel according to claim 5 as a biomaterial barrier for the prevention of postoperative bacterial infectious abdominal adhesions.
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