Preparation method and application of blood cross-linked injection type hemostatic antibacterial hydrogel

Abstract

The invention relates to the technical field of hemostatic antibacterial hydrogel, and particularly discloses a preparation method and application of blood cross-linked injection type hemostatic antibacterial hydrogel. The hydrogel is composed of chitosan quaternary ammonium salt with the concentration of 3-5%, sodium alginate with the concentration of 3-5%, beta-sodium glycerophosphate with the concentration of 1-5%, calcium chloride with the concentration of 0.2-2% and a proper amount of deionized water. According to the method, rapid crosslinking can be achieved at the bleeding part of the wound surface through injection, the antibacterial effect is achieved, blood coagulation can be promoted, and the problems that an existing hydrogel method is insufficient in mechanical strength, poor in biocompatibility and inconvenient to use in emergency are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of hemostatic and antibacterial hydrogels, and specifically to a preparation method and application of an injectable hemostatic and antibacterial hydrogel crosslinked by blood. Background Art

[0002] As a material with good moisture retention performance, hydrogels are widely used in wound repair and treatment. Especially under the hydrated crosslinked network structure, they can effectively keep the wound moist and promote the healing process. One of the common raw materials of hydrogels is polysaccharide substances. These long-chain macromolecules have excellent biocompatibility and are easy to modify and graft branches, so they become ideal hydrogel matrices. Among many polysaccharides, chitosan, as a product after deacetylation of chitin, not only has good biocompatibility but also exhibits excellent antibacterial properties due to its rich positively charged amino groups. The amino groups of chitosan can bind to the negative charges in the bacterial cell wall to inhibit the growth of bacteria. Therefore, chitosan is often used as an antibacterial agent during the wound repair process.

[0003] In order to further improve the antibacterial effect of chitosan, researchers grafted quaternary ammonium salt molecules onto the branches of chitosan to enhance its positive charge content and construct chitosan quaternary ammonium salts. These modified chitosan quaternary ammonium salts have significantly improved antibacterial properties, and some products have successfully entered the market. However, there are still certain technical bottlenecks in the application of existing chitosan quaternary ammonium salt hydrogels in wound repair. First of all, the crosslinking between chitosan quaternary ammonium salts and other components (such as sodium alginate) usually relies on ionic crosslinking, which is relatively weak, resulting in insufficient mechanical strength of the hydrogel. To make up for this deficiency, researchers usually need to add crosslinking agents such as glutaraldehyde to enhance the crosslinking degree of the hydrogel. However, glutaraldehyde has poor biocompatibility and certain toxicity, especially when it comes into contact with living tissues, it may cause adverse reactions. Therefore, this method is limited in clinical applications.

[0004] In addition, sodium alginate, as a negatively charged polysaccharide, can form hydrogels through chelation with calcium ions. This kind of hydrogel has wide applications in the fields of food and biomedicine. However, when sodium alginate is ionically crosslinked with chitosan quaternary ammonium salts, the crosslinking strength is limited, resulting in the formed hydrogel being difficult to meet the clinical requirements for high mechanical properties. At the same time, the existing hydrogel preparation methods often require advance preparation and cannot respond in time to the wound treatment needs in emergency trauma, resulting in their limited application in emergency scenarios.

[0005] Aiming at the deficiencies of the existing technology, the present invention proposes a preparation method and application of an injectable hemostatic and antibacterial hydrogel crosslinked by blood. Summary of the Invention

[0006] The object of the present invention is to address the deficiencies of the prior art and provide a method for preparing and applying an injectable hemostatic and antibacterial hydrogel for blood crosslinking, so as to solve the problems raised in the above-mentioned background art.

[0007] To achieve the above object, the present invention provides the following technical solution: A method for preparing an injectable hemostatic and antibacterial hydrogel for blood crosslinking, which is composed of chitosan quaternary ammonium salt with a concentration of 3-5%, sodium alginate with a concentration of 3-5%, β-glycerophosphate with a concentration of 1-5%, calcium chloride with a concentration of 0.2-2% and an appropriate amount of deionized water.

[0008] A method for preparing an injectable hemostatic and antibacterial hydrogel for blood crosslinking as described above, characterized in that the specific steps are as follows: Preparation of HACC / CaCl2 / SA / β-GP hydrogel;

[0009] Step 1: Add chitosan quaternary ammonium salt to deionized water at a concentration of 3-5% (w / v), and stir until completely dissolved to obtain a chitosan quaternary ammonium salt solution;

[0010] Step 2: Further add calcium chloride with a concentration of 0.2-2% (w / t) to the chitosan quaternary ammonium salt solution;

[0011] Step 3: Add sodium alginate to deionized water at a concentration of 3-5% (w / v), and stir until completely dissolved to obtain a sodium alginate solution. Then, continue to add β-glycerophosphate (1-5%, w / t) to this solution and stir until fully dissolved;

[0012] Step 4: Transfer the solutions described in Step 1 and Step 3 into syringes respectively, connect them with a Y-shaped tube, inject the two solutions simultaneously and mix and extrude them in the Y-shaped tube to obtain the HACC / CaCl2 / SA / β-GP hydrogel.

[0013] An application of an injectable hemostatic and antibacterial hydrogel for blood crosslinking as described above in the preparation of hemostatic and antibacterial drugs.

[0014] As a preferred technical solution of the present invention, one end of the wire rope fixing rod is connected with two metal clips. A through hole is provided in the middle of the metal clip, and a bolt passes through the through hole. A nut is threadedly connected to the bolt.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] The method of the present invention can achieve rapid crosslinking by injection at the bleeding site of the wound surface, has both antibacterial effects and can promote blood coagulation, and solves the problems of insufficient mechanical strength, poor biocompatibility and inconvenient emergency use in the existing hydrogel methods.

[0017] The injectable quaternary ammonium salt chitosan-sodium alginate hydrogel involved in the present invention enhances the crosslinking strength of the hydrogel by introducing multiple crosslinking systems, specifically including:

[0018] (1) Crosslinking strength: The present invention enhances the crosslinking strength of the hydrogel through the chelation of calcium chloride and sodium alginate. In practical applications, it is not necessarily required to directly add calcium chloride to the solution because calcium ions in the blood can play a similar crosslinking role during wound repair. This feature makes the hydrogel more convenient in clinical applications, reduces the dependence on external additives, and improves the flexibility and universality of its application.

[0019] (2) Thermosensitivity: The combination of β-glycerophosphate sodium introduced in the present invention and quaternary ammonium salt chitosan has thermosensitivity. In the application in human tissues, the hydrogel can crosslink at body temperature (32 - 37 °C), further enhancing the mechanical strength of the hydrogel. This thermosensitive property makes the hydrogel more stable on the wound surface, can rapidly enhance the mechanical properties at body temperature, and meet the high-strength requirements in wound repair.

[0020] (3) Antibacterial property: Compared with the traditional chitosan-sodium alginate hydrogel, the quaternary ammonium salt chitosan used in the present invention has stronger antibacterial properties. The quaternary ammonium salt structure in quaternary ammonium salt chitosan endows the hydrogel with stronger positive charges, enabling it to effectively bind to the cell wall of bacteria, thereby inhibiting the growth of bacteria, reducing the risk of infection, and improving the wound repair effect.

[0021] (4) Simple preparation process: The present invention provides an injectable crosslinking method, which simplifies the preparation process of the hydrogel. Traditional hydrogel preparation methods usually require complex crosslinking agent addition and curing processes, while the present invention can directly form a hydrogel at the wound surface through an injectable crosslinking method, reducing the operation difficulty and improving the preparation efficiency. This method makes the hydrogel easier to use, especially in first aid or clinical applications, and can quickly and conveniently complete wound repair. Description of the Drawings

[0022] Figure 1 It is the rheological shear thinning characterization diagram of the present invention;

[0023] Figure 2 It is the storage modulus and loss modulus diagram of the hydrogel after injection of the present invention;

[0024] Figure 3 It is the hemostasis time diagram of the rat liver bleeding model of the present invention;

[0025] Figure 4 It is the antibacterial rate diagram of the hydrogel of the present invention. Detailed Embodiments

[0026] The following elaborates on the preferred embodiments of the present invention in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0027] The present invention provides a technical solution: a preparation method of an injectable hemostatic and antibacterial hydrogel for blood crosslinking, which is composed of chitosan quaternary ammonium salt with a concentration of 3-5%, sodium alginate with a concentration of 3-5%, β-glycerophosphate sodium with a concentration of 1-5%, calcium chloride with a concentration of 0.2-2%, and an appropriate amount of deionized water.

[0028] A preparation method of an injectable hemostatic and antibacterial hydrogel for blood crosslinking as described above, the specific steps are as follows: preparation of HACC / CaCl2 / SA / β-GP hydrogel;

[0029] Step 1: Add chitosan quaternary ammonium salt to deionized water at a concentration of 3-5% (w / v), and stir until completely dissolved to obtain a chitosan quaternary ammonium salt solution;

[0030] Step 2: Further add calcium chloride with a concentration of 0.2-2% (w / t) to the chitosan quaternary ammonium salt solution; the addition amount of calcium chloride (0.2-2% w / t) depends on the urgent need for wound repair. The role of calcium chloride in the hydrogel is to further supplement on the basis of the existing calcium ions in the blood, promote the ionic crosslinking between sodium alginate and chitosan quaternary ammonium salt, so as to enhance the stability and formation effect of the hydrogel. In the case of a large wound or severe bleeding, the proportion of calcium chloride can be appropriately increased to ensure that the hydrogel can quickly form on the wound surface and has good adhesiveness, quickly closing the wound;

[0031] Step 3: Add sodium alginate to deionized water at a concentration of 3-5% (w / v), and stir until completely dissolved to obtain a sodium alginate solution. Then, continue to add β-glycerophosphate sodium (1-5%, w / t) to this solution and stir until fully dissolved;

[0032] Step 4: Transfer the solutions described in Step 1 and Step 3 into syringes respectively, connect them with a Y-shaped tube, inject the two solutions simultaneously and mix and extrude them in the Y-shaped tube to obtain the HACC / CaCl2 / SA / β-GP hydrogel.

[0033] An application of an injectable hemostatic and antibacterial hydrogel for blood crosslinking as described above in the preparation of hemostatic and antibacterial drugs.

[0034] Control group: Preparation of HACC / SA hydrogel;

[0035] Step 1: Add chitosan quaternary ammonium salt to deionized water at a concentration of 3-5% (w / v), and stir until completely dissolved to obtain a chitosan quaternary ammonium salt solution;

[0036] Step 2: Add sodium alginate into deionized water at a concentration of 3 - 5% (w / v), and stir until completely dissolved to obtain a sodium alginate solution;

[0037] Step 3: Transfer the chitosan quaternary ammonium salt solution and the sodium alginate solution into syringes respectively, connect them with a Y-shaped tube, inject the two solutions simultaneously and mix and extrude them in the Y-shaped tube to obtain the HACC / SA hydrogel;

[0038] Preparation of HACC / SA / β-GP hydrogel: for wound hemostasis and antibacterial applications;

[0039] Step 1: Add chitosan quaternary ammonium salt into deionized water at a concentration of 3 - 5% (w / v), and stir until completely dissolved to obtain a chitosan quaternary ammonium salt solution;

[0040] Step 2: Add sodium alginate into deionized water at a concentration of 3 - 5% (w / v), and stir until completely dissolved to obtain a sodium alginate solution; then, continue to add β-glycerophosphate (1 - 5%, w / t) into this solution and stir until fully dissolved; the addition amount of β-glycerophosphate is used to adjust the coagulation performance of the hydrogel and the local physiological reaction during wound repair, and endow the hydrogel with thermosensitivity. In the case of acute trauma or more bleeding, a higher concentration of β-glycerophosphate helps to enhance the cross-linking strength of the hydrogel, accelerate the coagulation process, reduce the bleeding volume, and thus promote wound healing; for milder trauma or non-bleeding wounds, the concentration of β-glycerophosphate can be appropriately reduced to balance other properties of the hydrogel;

[0041] Step 3: Transfer the solutions described in Step 2.1 and Step 2.2 into syringes respectively, connect them with a Y-shaped tube, inject the two solutions simultaneously and mix and extrude them in the Y-shaped tube to obtain the HACC / SA / β-GP hydrogel.

[0042] Example 1: Preparation of injectable chitosan quaternary ammonium salt sodium alginate hydrogel and exploration of its gel-forming properties under different conditions:

[0043] 1. Weigh 300 mg of chitosan quaternary ammonium salt and add it into 10 ml of deionized water, stir and dissolve to prepare a chitosan quaternary ammonium salt solution (HACC);

[0044] 2. Weigh 300 mg of chitosan quaternary ammonium salt and 20 mg of calcium chloride, add them into 10 ml of deionized water, stir and dissolve to prepare a chitosan quaternary ammonium salt - calcium chloride solution (HACC + CaCl2);

[0045] 3. Weigh 300 mg of chitosan quaternary ammonium salt, add it into 9.5 ml of deionized water, stir and dissolve, then add 0.5 ml of New Zealand white rabbit peripheral blood plasma to prepare a chitosan quaternary ammonium salt - plasma solution (HACC + Plasma);

[0046] 4. Weigh 300 mg of sodium alginate and add it to 10 ml of deionized water, stir to dissolve to prepare a sodium alginate solution (SA);

[0047] 5. Weigh 300 mg of sodium alginate and 100 mg of β-glycerophosphate and add them to 10 ml of deionized water, stir to dissolve to prepare a sodium alginate-β-GP glycerophosphate solution (SA + β-GP);

[0048] Test the shear thinning properties of HACC, HACC + CaCl2, SA and SA + β-GP solutions respectively. As Figure 1 shown, the results show that the viscosities of the four groups of solutions decrease with the increase of shear force, indicating that the four groups of solutions all have good injectability. Among them, the viscosities of HACC + CaCl2 and SA + β-GP are significantly greater than those of HACC and SA. This phenomenon is caused by CaCl2 and β-GP. Since the molecular weight of β-GP is much higher than that of CaCl2, the viscosity of SA + β-GP is higher than that of other groups.

[0049] Further, the gelation properties of the injectable chitosan quaternary ammonium salt sodium alginate hydrogel are verified by testing the storage modulus (G') and loss modulus (G") of the hydrogels of each group after gelation with a rheometer. As Figure 2 shown, the rheological results of the four groups of hydrogels all show that G' > G", indicating that the four groups of hydrogels have all formed solid hydrogel structures. With the increase of components, G' and G" increase accordingly, indicating that the addition of CaCl2 and β-GP can effectively improve the crosslinking degree of the hydrogel, and this hydrogel can react with plasma to enhance the crosslinking strength. In addition, by comparing HACC / Plasma / SA / β-GP with HACC / CaCl2 / SA / β-GP, it is found that the addition of CaCl2 can improve the strength of the hydrogel more than plasma, indicating that the calcium ions contained in the blood cannot completely chelate the sodium alginate in the hydrogel. Therefore, further adding CaCl2 during the hemostasis process can further improve the crosslinking strength of the hydrogel.

[0050] Example 2: Application of injectable chitosan quaternary ammonium salt sodium alginate hydrogel in rat liver hemostasis:

[0051] (1). Weigh 300 mg of chitosan quaternary ammonium salt and add it to 10 ml of deionized water, stir to dissolve to prepare a chitosan quaternary ammonium salt solution (HACC);

[0052] (2). Weigh 300 mg of sodium alginate and 100 mg of β-glycerophosphate and add them to 10 ml of deionized water, stir to dissolve to prepare a sodium alginate-β-glycerophosphate solution (SA + β-GP);

[0053] (3). Add the solutions in steps (1) and (2) into syringes respectively and connect them with a Y-tube for later use;

[0054] (4). Select 200 - 250 g SD rats. After anesthesia, make a 1-cm longitudinal incision on the abdomen to expose the liver. Use a skin drill with a diameter of 6 mm to create a defect in the center of the liver, and perform coagulation experiments using gauze (Control), gelatin sponge, and injectable hydrogel (HACC + SA + β-GP) respectively.

[0055] As Figure 3 shown in the figure, the results show that the hemostasis times of the gauze, gelatin sponge, and the hydrogel of the present invention are 218.4 s, 163 s, and 63.4 s respectively. And there are significant statistical differences between the hemostasis time of the hydrogel and that of the gauze and gelatin sponge, indicating that the hydrogel can effectively accelerate the hemostasis process and the effect is significantly better than that of the commonly used gauze and gelatin sponge in clinic.

[0056] Example 3: Antibacterial application of injectable quaternary ammonium chitosan alginate hydrogel:

[0057] (1) Prepare the hydrogel according to Example 1, sterilize it with ethylene oxide and reserve it for use.

[0058] (2) Use Staphylococcus aureus (S.aureus, S.a), Escherichia coli (Escherichia coli, E.coli), methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa (P.a). Dilute the concentration to 10^6 cfu / ml. Take 10 μl and drop it on the surface of the hydrogel respectively, and co-culture at 37°C for 6 hours; directly drop 10 μl of the bacterial solution into an ep tube as a blank control.

[0059] (3) After the co-culture, add 990 μl of phosphate buffer PBS respectively, and use a 300W ultrasonic cleaner to ultrasonically wash the bacteria on the surface of the hydrogel for 10 minutes. Take 100 μl and evenly coat it on the surface of the nutrient agar plate, and observe the colony formation after culturing at 37°C for 18 hours to evaluate the antibacterial performance of the hydrogel.

[0060] As Figure 4As shown, each group of hydrogels has good antibacterial effects against Gram-positive bacteria S.a, MRSA and Gram-negative bacteria E.coli, P.a. After 6 hours of co-culture, the bacteria can be effectively killed, and the antibacterial rates of each group of hydrogels against Gram-positive bacteria S.a, Gram-negative bacteria E.coli, P.a and drug-resistant bacteria MRSA are all above 99%. At the same time, there are no statistical differences in the antibacterial rates of each group of hydrogels against each bacterium, indicating that the addition of sodium β-glycerophosphate and calcium chloride will not affect the good antibacterial properties of the hydrogels themselves.

[0061] The above embodiments only express the implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A blood-crosslinked injectable hemostatic antibacterial hydrogel, characterized by: The hydrogel is composed of chitosan quaternary ammonium salt with a concentration of 3-5%, sodium alginate with a concentration of 3-5%, beta-sodium glycerophosphate with a concentration of 1-5%, calcium chloride with a concentration of 0.2-2% and a proper amount of deionized water.

2. A method for preparing the blood-crosslinked injectable hemostatic antibacterial hydrogel as claimed in claim 1, characterized in that: The specific steps are as follows; Step 1: adding chitosan quaternary ammonium salt to deionized water at a concentration of 3-5% (w / v), stirring until completely dissolved, to obtain a chitosan quaternary ammonium salt solution; Step 2: further adding calcium chloride with a concentration of 0.2-2% (w / t) to the chitosan quaternary ammonium salt solution; Step 3: adding sodium alginate at a concentration of 3-5% (w / v) to deionized water, stirring until completely dissolved, to obtain a sodium alginate solution, and then, continuing to add sodium β-glycerophosphate (1-5%, w / v) to the solution, stirring until fully dissolved; Step 4: The solutions in step 1 and step 3 are transferred into syringes respectively and connected with a Y-shaped tube, and the two solutions are injected simultaneously and mixed and extruded in the Y-shaped tube to obtain HACC / CaCl2 / SA / β-GP hydrogel.

3. Use of the blood-crosslinked injectable hemostatic antibacterial hydrogel as claimed in claim 1 in the preparation of hemostatic antibacterial drugs.

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