CN107349464B

CN107349464B - Preparation method of novel medical hemostatic gel dressing

Info

Publication number: CN107349464B
Application number: CN201710503854.5A
Authority: CN
Inventors: 郑晓燕; 范代娣; 惠俊峰; 朱晨辉; 马晓轩; 姜西娟
Original assignee: Northwestern University
Current assignee: Northwestern University
Priority date: 2017-06-28
Filing date: 2017-06-28
Publication date: 2020-02-18
Anticipated expiration: 2037-06-28
Also published as: CN107349464A

Abstract

The invention relates to a preparation method of a medical hemostatic gel dressing, which is characterized in that human-like collagen (LHC) is dissolved in ultrapure water to prepare a protein solution, inorganic salt and a composite cross-linking agent β -diimine zinc complex and 1,2,7, 8-diepoxyoctane are added, the pH value of the solution is adjusted to 2-5.5, the mixed solution is uniformly stirred and then is placed in a water bath at the temperature of 40-80 ℃ for 0.5-5h, and then the mixed solution is sequentially subjected to high-temperature steam treatment and water washing twice, drying treatment and Co-60 irradiation sterilization to obtain a novel sterile medical hemostatic gel dressing.

Description

Preparation method of novel medical hemostatic gel dressing

Technical Field

The invention relates to a preparation method of a medical hemostatic gel dressing, and belongs to the field of medical materials.

Background

The body is suffered from various skin or tissue wounds caused by factors such as war, traffic accident, sports or diseases, and the like to bleed. In recent years, a lot of researches show that patients who die from excessive blood loss clinically account for a large proportion, so that the preparation of the hemostatic material capable of rapidly stopping bleeding without tissue adhesion is an urgent problem to win time for later rescue and treatment.

The main characteristics of the ideal dressing currently used for wounds are generally: no toxicity, insensitivity and no allergy; the dressing is tightly attached to the wound, has good moisture absorption characteristic and can maintain the moist environment of the joint surface of the wound and the dressing; frequent replacement is not required; impermeable to microorganisms; the cost performance is high; provide good mechanical protection and are not easy to adhere to wounds, etc. The hydrogel is a high molecular polymer rich in water and having a three-dimensional network structure, and is widely applied to the fields of tissue engineering, drug sustained release and the like. Compared with other materials, the hydrogel as the hemostatic material has the advantages of good fitting property, infection resistance and the like. However, the traditional hydrogel has the defects of poor hemostatic effect, poor air permeability and the like due to lack of pore structures.

Human-like collagen (LHC) is a human-derived collagen produced by fermentation. The biomaterial has the characteristics of good cell adhesion, formation of new cells, good processing performance, no virus hidden trouble, good water solubility (avoiding cytotoxicity caused by acid-base solvent residue), low rejection reaction and the like.

Disclosure of Invention

Aiming at the defects of poor air permeability, easy wound adhesion and the like of the hemostatic material in the market, the novel medical hemostatic gel dressing which can rapidly stop bleeding and can not adhere to tissues is prepared, the method is simple in process, and the prepared material is good in biocompatibility and is expected to be widely used in wartime first-aid kits, clinical operation hemostasis and the like.

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

a preparation method of a novel medical hemostatic gel dressing comprises the steps of dissolving water-soluble macromolecular protein human collagen (LHC) in water, adding a composite cross-linking agent β -diimine zinc complex, an aqueous solution of 1,2,7, 8-diepoxyoctane and an inorganic salt aqueous solution in a mass ratio of 1:3-3:1, uniformly mixing, adjusting the pH value of the solution to 2-5.5, placing the solution in a water bath environment for cross-linking reaction to obtain a saline hydrogel, carrying out high-pressure steam treatment and distilled water soaking washing on the cross-linked saline hydrogel, removing inorganic salt and residual monomer cross-linking agent, drying and carrying out Co-60 sterilization treatment to obtain the novel medical hemostatic gel dressing.

The concentration of the human-like collagen solution is 50-400 mg/mL.

The inorganic salt is selected from sodium chloride, sodium phosphate, sodium hydrogen phosphate, ammonium sulfate, ammonium nitrate, potassium sulfate and potassium chloride, the concentration of the inorganic salt aqueous solution is 10-350 mg/mL, and the ratio of the addition volume of the inorganic salt aqueous solution to the volume of the protein solution is 1:2-1: 20.

The mass percentage concentration of the cross-linking agent β -diimine zinc complex solution and the 1,2,7, 8-diepoxyoctane solution is 0.1-5.0%, preferably 0.5-2.0%, and the volume is 1-20%, preferably 5-10% of the volume of the protein solution.

Regulating the pH value of the reaction solution to 2-5.5 by using hydrochloric acid and sodium hydroxide solution; the temperature of the crosslinking reaction can be 40-80 ℃ and the holding time is 0.5-5h, preferably 1-3 h.

In the two times of high-pressure steam treatment and distilled water soaking and washing, the sample is kept for 5-30min at the temperature of 110-121 ℃ for the first time, preferably for 10-20min, and then is soaked and washed by ultrapure water for 2-5 days; keeping the sample at the temperature of 110-121 ℃ for 1-3 h for 1.5-2.5h for the second time, and then soaking and washing the sample for 1-3 days by using ultrapure water, wherein the residual total amount of the cross-linking agent is controlled to be lower than 2 mu g/g.

The drying method of the novel medical hemostatic gel dressing can be a vacuum freeze drying method or a supercritical carbon dioxide drying method.

The aforementioned β -diimine zinc complexes as crosslinkers are described in the literature (catalysis Reactions Involving C)₁New High-Activity Zn (II) -Based Catalysts for the copolymerization of Carbon Dioxide and Epoxides, J. Am. chem.Soc. 1998, 120, 11018-11019) and the formula is shown in FIG. 1.

The invention relates to a forming mechanism of a medical hemostatic porous gel dressing, which is characterized in that protein molecules contain rich carboxyl and amino and provide two good functional groups for intermolecular crosslinking, β -diimine zinc complex molecular empty orbits of chelated zinc can form coordinate bonds with amino in protein molecules to realize intermolecular crosslinking of protein molecules, β -diimine zinc complex has an asymmetric ring-opening catalysis function on epoxy alkyl, can accelerate the ring opening of epoxy alkyl at two ends of 1,2,7, 8-diepoxyoctane molecules and form covalent bonds with carboxyl in protein molecules to realize intermolecular crosslinking of protein molecules, so that the ring-opening catalysis of 1,2,7, 8-diepoxyoctane by β -diimine zinc complex and the double-crosslinking action of two crosslinking agents on different groups between the protein molecules effectively realize and strengthen the crosslinking between the protein molecules.

The invention utilizes human-like collagen as a main raw material to prepare the novel superporous hydrogel through the synergistic co-crosslinking effect of double crosslinking agents. The hydrogel material has a super-porous structure, high porosity, good connectivity and reasonable pore size, and can rapidly absorb water in blood without absorbing cells, thereby promoting hemagglutination and realizing rapid hemostasis. The hydrogel material has good biocompatibility and pore passage connectivity, ensures the air permeability of the wound and is beneficial to promoting the healing of the wound. Compared with the traditional gauze or hemostatic sponge, the surface of the hydrogel material has smaller roughness and is not easy to adhere; the good connectivity of the pore passage ensures that the drug delivery is very convenient; while the smaller pore size of the material also prevents wound infection. The novel medical hemostatic gel dressing prepared by the invention is expected to be used in the fields of wartime first-aid kits, clinical operation hemostasis and the like.

The invention also has the following advantages: (1) the hemostatic material prepared by the invention can obviously shorten the hemostatic time, which is important for wound or postoperative recovery; (2) the material prepared by the invention has good air permeability and antibacterial property, and does not cause wound infection; (3) in addition, the hemostatic material prepared by the invention can not adhere to wounds after hemostasis is finished, so that the pain of a patient is obviously reduced; (4) the material prepared by the invention has a porous structure, so that the drug administration and the drug supplementation are very convenient.

Drawings

FIG. 1 is a molecular structure diagram of a synthesized β -diimine zinc complex;

FIG. 2 is an external view of a sample of the medical hemostatic porous gel dressing prepared in example 1;

FIG. 3 is an SEM image of a sample of the medical hemostatic porous gel dressing prepared in example 1;

FIG. 4 is a chart of the air permeability of the medical hemostatic porous gel dressing prepared in example 1;

FIG. 5 is a graph showing the hemostatic effect of the medical hemostatic porous gel dressing prepared in example 1 on rabbit auricular artery and liver;

FIG. 6 is a graph of the hemostatic effect on rabbit liver of two commercial brands of hemostatic sponges used in example 1.

Detailed Description

The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

EXAMPLE 1 preparation of medical hemostatic porous gel dressing

Dissolving water-soluble human-like collagen HLC in 10 mL of distilled water to obtain a solution with the concentration of 100 mg/mL, adding 1% of cross-linking agent β -diimine zinc complex and 1,2,7, 8-diepoxyoctane respectively in 1 mL, adding 10mg/mL of KCl 2mL, uniformly mixing, and adjusting the pH of the solution to 4.5 by using dilute hydrochloric acid and sodium hydroxide;

step two: the mixed solution in the step one is subpackaged in a mould, kept for 2 hours at the temperature of 50 ℃, then transferred into a high-pressure steam sterilization pot and kept for 20 minutes at the temperature of 121 ℃ to obtain a gel primary product;

step three: soaking and washing the primary product gel in distilled water for 5 days, changing washing water every 6 hours, and removing salt and residual cross-linking agent; transferring the washed primary gel into an autoclave for continuously keeping at 121 ℃ for 2h, then soaking and washing with distilled water for 2 days again, and controlling the total residual amount of the cross-linking agent to be less than 2 mug/g to obtain a porous wet gel sample.

Step four: and pre-freezing the wet gel in the third step for 3 h at-80 ℃, then carrying out vacuum freeze-drying and Co-60 irradiation sterilization to obtain a finished product of the sterile medical hemostatic porous gel dressing.

The physicochemical property characterization of the medical hemostatic porous gel dressing prepared in the example is performed, and fig. 2 is an appearance photograph of the hemostatic gel dressing freeze-dried sample prepared in example 1, which shows that the hemostatic gel dressing freeze-dried sample is opaque and milky in appearance, has a smooth surface texture and is in a porous structure; fig. 3 is a Scanning Electron Microscope (SEM) image of the prepared porous hydrogel hemostatic dressing. The SEM image shows that the inside of the hemostatic dressing is of a porous structure, the hole walls are formed by mutually adhering spherical particles, the hole diameter is generally different from dozens of nanometers to several micrometers, and the hole structure is uniform and compact and communicated.

The prepared medical hemostatic porous gel dressing block is placed in a filtering device fixed on an injector, 5 mL of air is filled into the injector, then the injector is extruded for air filtration, and as a result, the process has almost no resistance, and a push rod of the injector can be easily pushed to the bottom. The syringe squeeze air filtration experiment demonstrated the air permeability of the hemostatic dressing, as shown in fig. 4, illustrating the interconnected pore structure within the hemostatic material. The material has good air permeability and anti-adhesion property, and if the antibacterial drug can be directly added through the material and slowly released to the wound, the wound infection caused by air permeability can be avoided in the process of hemostasis, and the wound can not be festered due to long-time soaking.

The medical hemostatic porous gel dressing prepared in the example has good elasticity, strong compression resistance (the maximum compressive strain and the compressive stress are respectively about 68.9 percent and 5.6 MPa), high swelling rate (8 s basically achieves the swelling balance), and porosity of about 85.4 percent; the result of cytotoxicity test shows that the medical hemostatic porous gel dressing prepared in the example has good biocompatibility and no cytotoxicity.

EXAMPLE 2 preparation of medical hemostatic porous gel dressing

Step one, dissolving water-soluble human-like collagen HLC in 10 mL of distilled water to obtain a solution with the concentration of 100 mg/mL, adding 1 mL of each of 2% cross-linking agent β -diimine zinc complex and 1,2,7, 8-diepoxyoctane, and adding 10mg/mL of Na₂SO₄2mL of solution is uniformly mixed, and the pH value of the solution is adjusted to 4 by using dilute hydrochloric acid and sodium hydroxide;

step two: the mixed solution in the step one is subpackaged in a mould, kept for 2 hours in a water bath environment at 60 ℃, then transferred into a high-pressure steam sterilization pot and kept for 20 minutes at 110 ℃ to obtain a gel primary product;

step three: soaking and washing the primary product gel in distilled water for 5 days, changing washing water every 6 hours, and removing salt and residual cross-linking agent; transferring the washed primary gel into an autoclave for keeping at 110 ℃ for 2h, then soaking and washing the primary gel with distilled water for 2 days again to remove the residual cross-linking agent, and controlling the total residual amount of the cross-linking agent to be lower than 2 mu g/g to obtain a porous wet gel sample.

The physical and chemical properties and biological properties of the sterile medical hemostatic porous gel dressing obtained in the example are similar to those of the medical hemostatic gel dressing obtained in the example 1.

EXAMPLE 3 preparation of medical hemostatic porous gel dressing

Step one, dissolving water-soluble human-like collagen HLC in 20 mL of distilled water to obtain a solution with the concentration of 200 mg/mL, adding 4 mL of cross-linking agent β -diimine zinc complex and 1,2,7, 8-diepoxyoctane with the concentrations of 1%, respectively, and then adding 10mg/mL of NaH₂PO₄2mL of solution is uniformly mixed, and the pH value of the solution is adjusted to 5.5 by using dilute hydrochloric acid and sodium hydroxide;

step two: the mixed solution in the step one is subpackaged in a mould, kept for 2 hours at the temperature of 70 ℃, then transferred into a high-pressure steam sterilization pot and kept for 20 minutes at the temperature of 121 ℃ to obtain a gel primary product;

step three: soaking and washing the primary product gel in distilled water for 4 days, changing washing water every 6 hours, and removing salt and residual cross-linking agent; transferring the washed primary product gel to a high-pressure steam sterilization pot, keeping the temperature at 121 ℃ for 3 h, then soaking and washing the primary product gel for 1 day again by using distilled water, removing the residual cross-linking agent, and controlling the total residual amount of the cross-linking agent to be lower than 2 mu g/g to obtain a porous wet gel sample;

step four: and drying the wet gel in the third step by a supercritical carbon dioxide drying method to prepare an ultra-porous xerogel sample, and performing Co-60 irradiation sterilization on the dry sample to obtain a finished product of the sterile medical hemostatic porous gel dressing.

Example 4 hemostasis test of medical hemostatic porous gel dressing

New Zealand white rabbits are selected as experimental animals, rabbit ear wound bleeding and liver wound bleeding models are established, and the hemostatic gel dressing obtained in the embodiment 1 of the invention and hemostatic sponges of A, B brands on the market are selected for hemostasis comparison tests.

Fig. 5 shows the effect of the sterile medical hemostatic porous gel dressing prepared in example 1 of the present invention on rabbit ear hemostasis (a-c) and liver hemostasis (d-f), and it can be clearly seen that the material does not penetrate and adhere to the tissue after hemostasis. Comparative liver hemostasis test results using main stream A, B two brands of hemostatic sponges are shown in fig. 6, where it can be seen that the two brands of hemostatic materials selected have been substantially completely penetrated by the hemorrhage and require longer hemostasis times. The experimental process shows that the hemostatic time of the sterile medical hemostatic porous gel dressing prepared in example 1 on rabbit ears and livers is 20-30 s and 30-40 s respectively, and the hemostatic time of selected A, B two brands of hemostatic sponges on rabbit livers is about 80-90 s.

The result shows that the hemostatic gel prepared by the invention has excellent hemostatic function, mainly due to the high porosity, uniform pore diameter and good connectivity of the material, the hemostatic gel can rapidly absorb water in blood, so that blood cells in the blood are coagulated on the surface of the material to block ruptured blood vessels, thereby realizing rapid hemostasis and promoting wound healing, and is particularly suitable for osmotic bleeding caused in the operation process and massive bleeding caused by artery rupture. In addition, the material has the characteristics of bacteriostasis, antibiosis and wound infection prevention due to the small pore diameter of the material.

The invention is not limited to the above embodiments, and any equivalent changes to the technical solution of the invention by a person skilled in the art after reading the description of the invention, and modifications without changing the basic principle are all covered by the claims of the present invention.

Claims

1. A preparation method of a medical hemostatic gel dressing is characterized by dissolving water-soluble macromolecular protein human collagen in water, adding a composite cross-linking agent β -diimine zinc complex, an aqueous solution of 1,2,7, 8-diepoxyoctane and an inorganic salt aqueous solution in a mass ratio of 3:1-1:3, uniformly mixing, adjusting the pH value of the solution to 2-5.5, placing the solution in a water bath environment for cross-linking reaction to obtain a saline hydrogel, performing high-pressure steam treatment and distilled water soaking washing on the cross-linked saline hydrogel, removing inorganic salt and residual monomer cross-linking agent, drying and performing Co-60 sterilization treatment to obtain the medical hemostatic gel dressing,

the structural formula of the crosslinking agent β -diimine zinc complex is shown as (I):

（I）。

2. the method for preparing the medical hemostatic gel dressing according to claim 1, wherein: the concentration of the human-like collagen solution is 50-400 mg/mL.

3. The method for preparing the medical hemostatic gel dressing according to claim 1, wherein: the inorganic salt is selected from sodium chloride, sodium phosphate, sodium hydrogen phosphate, ammonium sulfate, ammonium nitrate, potassium sulfate and potassium chloride, the concentration of the inorganic salt aqueous solution is 10-350 mg/mL, and the ratio of the addition volume of the inorganic salt aqueous solution to the volume of the protein solution is 1:2-1: 20.

4. The preparation method of the medical hemostatic gel dressing according to claim 1, wherein the mass percentage concentration of the cross-linking agent β -diimine zinc complex solution and the 1,2,7, 8-diepoxyoctane solution is 0.1-5.0%, and the volume of the cross-linking agent β -diimine zinc complex solution and the volume of the diepoxyoctane solution are 1-20% of the volume of the protein solution.

5. The method for preparing the medical hemostatic gel dressing according to claim 1, wherein: regulating the pH value of the reaction solution to 2-5.5 by using hydrochloric acid and sodium hydroxide solution; the temperature of the crosslinking reaction is 40-80 ℃, and the holding time is 0.5-5 h.

6. The method for preparing the medical hemostatic gel dressing according to claim 1, wherein: carrying out high-pressure steam treatment and distilled water soaking and washing on the crosslinked hydrogel containing water, keeping the sample at the temperature of 110-121 ℃ for 5-30min for the first time, and then soaking and washing with ultrapure water for 2-5 days; keeping the sample at the temperature of 110-121 ℃ for 1-3 h for the second time, and then soaking and washing the sample for 1-3 days by using ultrapure water, wherein the residual total amount of the cross-linking agent is controlled to be lower than 2 mu g/g.

7. The method for preparing the medical hemostatic gel dressing according to claim 1, wherein: the drying method of the medical hemostatic gel dressing is a vacuum freeze drying method or a supercritical carbon dioxide drying method.