CN112870429A

CN112870429A - Chitosan-based polyelectrolyte composite hemostatic sponge, preparation method and application

Info

Publication number: CN112870429A
Application number: CN202011579534.6A
Authority: CN
Inventors: 孟欣; 卢阳; 刘涛; 高颖
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2021-06-01

Abstract

The invention relates to a chitosan-based polyelectrolyte composite hemostatic sponge, which comprises chitosan, sodium alginate, hyaluronic acid and genipin. The invention utilizes the characteristics of polyelectrolyte shape, obtains a series of chitosan-based electrolyte composite sponges by a freeze-drying method, and determines and evaluates the apparent structure, water absorption, mechanical property, biocompatibility and wound healing effect of the sponges, and the result shows that the screened composite hemostatic sponges have good mechanical property, liquid absorption property and biocompatibility and can obviously accelerate the wound healing process. The development of hemostatic materials provides an effective approach.

Description

Chitosan-based polyelectrolyte composite hemostatic sponge, preparation method and application

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a chitosan-based polyelectrolyte composite hemostatic sponge, a preparation method and application.

Background

Wound healing is a dynamic process that mainly involves four stages such as blood stasis, inflammation, proliferation and extracellular matrix (ECM) remodeling. These four stages of healing involve interactions between different types of cells, bioactive factors, and a supporting platform (usually the native ECM secreted by the cells). The accumulation of wound secretions, such as blood and body fluids, can promote bacterial growth, which in turn can cause infections and lead to a delay in the wound healing process. Millions of people suffer skin damage each year from excessive physicochemical factors or diseases. With consequent wound infection and severe tissue necrosis, endangering human life.

At present, the clinical hemostatic dressings are various, and comprise zeolite powder, Arista hemostatic microspheres, fibrin dressings, oxidized cellulose and oxidized regenerated cellulose dressings, medical gelatin sponges, chitosan bandages, alginate calcium fiber mats and the like. The Chitosan (CS) hemostatic sponge has great application potential due to the advantages of good hemostatic effect, strong antibacterial effect, good tissue adhesion, good biocompatibility and the like. The polyelectrolyte composite material is a high-hydrophilicity synthetic material consisting of polycation and polyanion, and is a biomedical material with excellent performance. The chitosan polyelectrolyte composite material applied to the wound dressing is developed rapidly in recent years. Wherein chitosan and alginate can form polyelectrolyte complex with bioactivity due to the ionic property thereof, and the polyelectrolyte complex is widely applied to hemostatic materials.

The brittleness of chitosan is the most obvious disadvantage in the preparation of wound healing materials.

Through searching, no patent publication related to the present patent application has been found.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a chitosan-based polyelectrolyte composite hemostatic sponge, a preparation method and application thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a chitosan-based polyelectrolyte composite hemostatic sponge comprises chitosan, sodium alginate, hyaluronic acid and genipin.

Moreover, the molecular weight of the chitosan is 800kDa, the deacetylation degree is 75 percent, the molecular weight of the sodium alginate is 1200kDa, and the purity of genipin is more than or equal to 98 percent.

The preparation method of the chitosan-based polyelectrolyte composite hemostatic sponge comprises the following steps:

preparing a chitosan-sodium alginate polyelectrolyte compound;

preparing the chitosan-based polyelectrolyte composite hemostatic sponge.

The step of searching specifically includes the steps of:

adding chitosan into deionized water, adding 1% by volume of acetic acid solution, stirring until the chitosan is completely dissolved, and preparing into 2% (w/v) chitosan solution; dissolving sodium alginate in deionized water to obtain 2% (w/v) sodium alginate solution; mixing the prepared chitosan solution and the sodium alginate solution according to the proportion of 1: 10-10: 1, and uniformly mixing and stirring to obtain the polyelectrolyte compounds with different proportions.

The method comprises the following steps:

dissolving genipin in deionized water to prepare 0.05-1.0% (w/v) genipin aqueous solution; dissolving hyaluronic acid in deionized water to prepare 1% (w/v) hyaluronic acid solution; adding the prepared genipin solution and the hyaluronic acid solution into the polyelectrolyte compound obtained in the step, wherein the polyelectrolyte compound comprises the following components in parts by weight: the mass ratio of the genipin solution is 1: 5-1: 1, polyelectrolyte complex: the mass ratio of the hyaluronic acid solution is 2:1, the obtained gel is fully and uniformly mixed by using a machine, poured into a polystyrene mould, placed at room temperature for 2 hours to enable genipin and chitosan to be crosslinked for 2 hours, and then the obtained gel is placed at the temperature of minus 20 ℃ for pre-freezing, wherein the pre-freezing time is 12 hours; and (3) carrying out vacuum freeze drying on the pre-frozen sample to obtain the chitosan-based polyelectrolyte composite sponge.

The chitosan-based polyelectrolyte composite hemostatic sponge is applied to the aspect of hemostatic dressing.

The invention has the advantages and positive effects that:

1. the invention discloses a composite hemostatic sponge, belonging to a novel hemostatic material, which comprises chitosan, sodium alginate, hyaluronic acid and genipin. The chitosan has the advantages of good hemostatic effect, strong antibacterial effect, good tissue adhesion, good biocompatibility and the like. Sodium alginate is non-toxic, good in biocompatibility, degradable and good in biological stability, and is a hydrophilic biopolymer. By utilizing the characteristics of the polyelectrolyte complex, the chitosan solution with positive charges and the sodium alginate solution with negative charges are mixed to prepare the chitosan-based polyelectrolyte complex solution. In order to improve the mechanical property of the chitosan, a natural biological crosslinking agent genipin is added into the compound to perform chemical crosslinking reaction with the chitosan. Meanwhile, hyaluronic acid is added, and the good hydrophilicity and the unique viscoelasticity of the hyaluronic acid can enable the composite material to provide a moist environment for wounds, promote the proliferation of fibroblasts and promote the healing of wound surfaces without scars.

2. The sponge with a porous structure is prepared by the vacuum freeze drying technology, has good mechanical property and imbibition property, and can promote gas exchange, thereby being beneficial to preventing wound infection and also being beneficial to discharging wound secretion and promoting wound healing.

3. The method has simple operation and mild reaction conditions in the whole process, and can realize large-scale quantitative production.

4. The composite hemostatic sponge can be effectively applied to the field of biomedical hemostatic materials.

5. The invention utilizes the characteristics of polyelectrolyte shape, obtains a series of chitosan-based electrolyte composite sponges by a freeze-drying method, and determines and evaluates the apparent structure, water absorption, mechanical property, biocompatibility and wound healing effect of the sponges, and the result shows that the screened composite hemostatic sponges have good mechanical property, liquid absorption property and biocompatibility and can obviously accelerate the wound healing process. The development of hemostatic materials provides an effective approach.

6. Since the brittleness of chitosan is the most significant disadvantage in preparing wound healing materials, defects can be ameliorated by forming polyelectrolyte complexes with other materials by exploiting the polycationic nature of chitosan itself. The chitosan, sodium alginate and Hyaluronic Acid (HA) are blended, and a natural biological cross-linking agent genipin (Ge) is used for cross-linking. Genipin has higher biocompatibility, and hydrogen bonds formed between genipin and chitosan can enable the material to have better performance. The Hyaluronic Acid (HA) HAs good hydrophilicity. It can provide a moist environment for the wound and protect the surface of the injured tissue from drying. Meanwhile, hyaluronic acid can promote the proliferation of fibroblasts, increase the collagen secretion of the wound surface and promote the healing of the wound surface without scars. A series of hemostatic sponges are obtained by different proportions, and the performances of the hemostatic sponges are evaluated.

Drawings

FIG. 1 is an apparent morphology chart of different chitosan-based polyelectrolyte composite hemostatic sponges prepared in example 1 of the present invention; wherein A is CAHS1, B is CAHS2, C is CAHS3, wherein the left figure is a sponge side surface profile figure, and the right figure is a sponge front surface profile figure;

FIG. 2 is a scanning electron microscope image of different chitosan-based polyelectrolyte composite hemostatic sponges prepared in example 1 of the present invention;

FIG. 3 porosity of different chitosan-based polyelectrolyte composite hemostatic sponges tested in example 3 of the present invention;

FIG. 4 is a graph of the water distribution of different chitosan-based polyelectrolyte composite hemostatic sponges tested in example 4 of the present invention at swelling equilibrium;

FIG. 5 is a swelling curve of different chitosan-based polyelectrolyte composite hemostatic sponges tested in example 4 of the present invention;

FIG. 6 is a graph of the hemolysis rate of different chitosan-based polyelectrolyte complex hemostatic sponges tested in example 5 of the present invention at different concentrations;

FIG. 7 is a graph of the evaluation of the effects of different chitosan-based polyelectrolyte composite hemostatic sponges and gauze groups on wound healing in example 6 of the present invention; wherein, A is a diameter change diagram of wounds treated by three groups of sponges and gauzes, B is a direct view diagram of wound healing treated by three groups of sponges and gauzes, and C is a tissue morphology diagram of wound regeneration.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.

The raw materials used in the invention are all conventional commercial products if no special description is provided, the method used in the invention is all conventional methods in the field if no special description is provided, and the mass of all the materials used in the invention is the conventional use mass.

Preferably, the molecular weight of the chitosan is 800kDa, the deacetylation degree is 75 percent, the molecular weight of the sodium alginate is 1200kDa, and the purity of genipin is more than or equal to 98 percent.

preparing a chitosan-sodium alginate polyelectrolyte compound;

preparing the chitosan-based polyelectrolyte composite hemostatic sponge.

Preferably, the step of making includes the following steps:

Preferably, the steps specifically include the following steps:

Specifically, the preparation and detection are as follows:

example 1: preparation of chitosan-based polyelectrolyte composite hemostatic sponge

The specific method comprises the following steps:

(1) adding the chitosan into deionized water, adding an acetic acid solution with the volume percentage of 1%, stirring until the chitosan is completely dissolved, and preparing a chitosan solution with the concentration of 2% (w/v); dissolving the sodium alginate in deionized water to prepare a 2% (w/v) sodium alginate solution; and mixing and stirring the prepared chitosan solution and the sodium alginate solution uniformly according to the volume ratio of 1:3, 2:3 and 1:1 to obtain the polyelectrolyte compound with different proportions.

(2) Dissolving the genipin in deionized water to prepare 0.05% genipin aqueous solution; dissolving the hyaluronic acid in deionized water to prepare a 1% hyaluronic acid solution; mixing the prepared genipin solution (M)_CS:M_Ge1:5) and hyaluronic acid solution (M)_CS:M_HAAdding the gel obtained in the step (1) into the polyelectrolyte compound obtained in the step (2: 1), fully and uniformly mixing the obtained gel by using a mechanical stirrer, pouring the mixture into a polystyrene mold, and standing the polystyrene mold at room temperature for 2 hours to enable genipin and chitosan to be crosslinked. After 2h, putting the obtained gel into a refrigerator with the temperature of-20 ℃ for pre-freezing, wherein the pre-freezing time is about 12 h; vacuum freeze drying the pre-frozen sample to obtain different samplesThe chitosan-based polyelectrolyte composite sponge (named as CAHS1, CAHS2 and CAHS3 according to the content of CS) in proportion. The apparent morphology of the sponges of the three groups of samples is shown in FIG. 1.

Example 2: microscopic morphology observation of the composite hemostatic sponge prepared in example 1

Microscopic morphology observations were made using a Scanning Electron Microscope (SEM) on three different component ratios of the composite sponge. As shown in fig. 2, the bottom surface and cross-sectional micro-morphograms of CAHS1, CAHS2, and CAHS3, respectively, prepared from example 1 are at 400 times. As can be seen from the figure, the three materials can form a structure with certain pores, and the surface and the section are almost the same, which can prove that the surface and the inner part of the material are relatively uniform. In comparison, the surface and the cross section of the CAHS2 are more uniform than those of the CAHS1 and the CAHS3, and have finer and denser porous structures, which play a vital role in absorbing body fluid and blood at wounds and influence the mechanical properties of the material to a certain extent. The structures of CAHS1 and CAHS3 are more sheet-like, and the structures have poor influence on the porosity and the hardness of the material. Therefore, based on SEM analysis of the surface and cross-section of the material, CAHS2 is more suitable for making hemostatic sponges.

Example 3: the porosity of the composite hemostatic sponge prepared in example 1 was tested

The method comprises the following steps: the prepared sponge samples were immersed in absolute ethanol until saturated, and the sponges before and after immersion were weighed.

The porosity (P) is calculated as follows:

wherein P represents porosity, W₁And W₂The weight of the sponge sample before and after immersion in ethanol is indicated. V₁Is the volume of ethanol before immersion of the sample, V₂Is the volume of ethanol after immersion of the sample, and ρ is a constant (ethanol density at room temperature). All samples were run in triplicate.

As a result: the porosity of the samples, CAHS1, CAHS2 and CAHS3 composite sponges, was evaluated using the ethanol displacement method as shown in fig. 3. It can be seen that the porosity of the CAHS3 sponge is 76% minimum and the porosity of the CAHS1 sponge is 115% maximum. The porosity of the sponge decreases slightly with increasing chitosan concentration. From the microscopic morphological image of the sponge under the scanning electron microscope of example 2, it can also be seen that the pores of the CAHS1 sponge are larger but sparse, while the pores of the CAHS2 sponge are tiny and dense.

Example 4: characterization of the swelling Properties of the composite hemostatic sponge prepared in example 1

The method comprises the following steps: the swelling properties of the sponges were characterized using low field nuclear magnetic resonance imaging (LF-NMR). Wherein T is utilized₂The LF-NMR data was analyzed by curve inversion. To test the initial T₂Distribution and amplitude A₀Taking 20mg (W) of each sponge₀) Placed in a 15mm diameter NMR tube. Subsequently, 100 μ L of phosphate buffer pH 7.0 was added to the tube.

The Swelling Ratio (SR) is calculated as follows:

wherein A is₀Initial amplitude of the sample before swelling, A_totalIs the amplitude of all the water contained in the system, A_texwaterThe amplitude of the water outside the material at time t min of the swelling process is indicated. W_waterIs the mass of water added.

As a result: in the present invention, LF-NMR was used to measure the water distribution of CAHS1, CAHS2 and CAHS3 at swelling equilibrium, and the results are shown in FIG. 4. In the LF-NMR image analysis, the amplitude of each T2 component corresponds to the amount of the water component, and is influenced by the molecular properties and three-dimensional structure of the material. And the component within 100ms is defined as the internal water (at about 20 ms), which is absorbed during the swelling process. Components greater than 100ms are attributable to external water (at about 140ms and 1400 ms) and can be removed gravimetrically. The Swelling Ratio (SR) of the sponge with different component ratios at different times can be calculated by using the formula in the method, and the swelling ratio is drawn into a curve as shown in FIG. 5. It is clear from the graph that when the swelling equilibrium (120min) is reached, the swelling ratio of CAHS3 is highest, reaching 166.67%, followed by CAHS2, reaching 158.46% and the swelling ratio of CAHS1 is lowest, 77.37%. However, throughout the process from the beginning of swelling to the equilibrium of swelling, the curves of CAHS1 and CAHS3 are relatively flat, while the swelling ratio of CAHS2 increases significantly before 30 min. From this it can be concluded that although the swelling ratio of CAHS2 at swelling equilibrium is not as high as CAHS3, the overall swelling process of CAHS2 is more satisfactory for the expected requirements of the material and therefore the swelling effect of CAHS2 is also more excellent.

Example 5: in vitro hemolytic performance test of the composite hemostatic sponge prepared in example 1

The method comprises the following steps: the hemocompatibility of the composite sponge was evaluated by in vitro hemolysis. Citric acid rabbit whole blood (2mL) was centrifuged at 200 Xg for 15min and erythrocytes were collected. The sample (1mg) and PBS (0.8mL) were added to the red blood cell suspension (0.2mL), respectively, as treatment groups. Deionized water and PBS without specimen are added into the red blood cell suspension respectively to serve as a positive control group and a negative control group. After incubation at 37 ℃ for 1h, the treated and control groups were centrifuged at 2000 Xg for 10min and the absorbance of the supernatant at 540nm was measured using an ultraviolet-visible spectrophotometer.

The Hemolysis rate (Hemolysis ratio) is calculated as follows:

as a result: hemolysis is a very simple and reliable method for assessing the blood compatibility of a hemostatic sponge. Generally, the lower the rate of hemolysis of a hemostatic sponge, the better the blood compatibility. As the concentration of the sample increased, the hemolysis rate substantially increased, as shown in fig. 6. At low concentrations, the hemolysis rate of CAHS2 is higher than that of CAHS1 and CAHS 3. As the concentration increases, the hemolysis rate of CAHS1 and CAHS3 increases. It can be seen that the haemolysis rate was less than 7% for all the sample sponges, indicating that the haemocompatibility of the haemostatic sponges is still acceptable.

Example 6: evaluation of the Effect of the composite hemostatic sponge prepared in example 1 on wound healing

The method comprises the following steps: carrying out in-vivo wound healing research on Wistra rats weighing 180-220 g and being 3-8 weeks old. The 24 rats were divided into 4 groups and normal rats were given feed and water without limitation. After depilation and anesthesia, a circular full-thickness wound of 2 cm in diameter was established on the back of each rat. Wounds from three groups of rats were bandaged with CAHS1, CAHS2, CAHS3 and gauze, respectively. The control group was wrapped with pure gauze. Photographs were taken twice weekly and wound area was measured. Rat skin wound tissue was excised, fixed with 4% paraformaldehyde, and stained with hematoxylin-eosin (H & E) for histological observation on

days

7, 14, and 21.

As a result: wound healing studies were performed in Wistra rats to evaluate the effects of skin tissue reconstruction. As can be seen from fig. 7(a), the wound areas of the groups gradually decreased at 7 days, 14 days, and 21 days. After 7 days, the CAHS3 group sponge-wrapped wounds began to heal. After 14 days, the contraction of the wound surface of the CAHS2 group is obviously better than that of the gauze group, the CAHS1 group and the CAHS3 group. Fig. 7(B) visually shows the effect of each group on wound healing. At 21 days, the CAHS2 group treated wounds healed completely, while the CAHS1 and CAHS3 groups were less effective than the gauze group. To further investigate the effect of the sponge on wound healing, the histomorphological evaluation of wound regeneration was as shown in fig. 7 (C). From the figure, it can be analyzed that inflammatory cell infiltration and fibroblast migration occurred at the wound surface of each group at day 7, where the CAHS1 group and the gauze group accumulated a large amount of inflammatory cells. On day 14, the CAHS1 group and gauze group still accumulated a large amount of inflammatory cells, while the CAHS2 group and CAHS3 group reduced inflammation and developed epithelial cells and a part of connective tissue. At 21 days, the gauze group also formed regenerated epithelial tissue, while the dermis still contained a large amount of inflammatory cells and fibroblasts. The CAHS1 group also formed epithelial cells, but inflammatory cells were still active. The CAHS2 group created stable complex reticular fibers that regenerate hair follicles and blood vessels. After connective tissue formation, inflammatory cell infiltration again occurred in the CAHS3 group, but no fibroblasts were formed, probably due to the high chitosan concentration in the sponge.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.

Claims

1. A chitosan-based polyelectrolyte composite hemostatic sponge is characterized in that: the composite hemostatic sponge comprises chitosan, sodium alginate, hyaluronic acid and genipin.

2. The chitosan-based polyelectrolyte composite hemostatic sponge according to claim 1, characterized in that: the molecular weight of the chitosan is 800kDa, the deacetylation degree is 75 percent, the molecular weight of the sodium alginate is 1200kDa, and the purity of genipin is more than or equal to 98 percent.

3. The method for preparing chitosan-based polyelectrolyte composite hemostatic sponge according to claim 1 or 2, wherein: the method comprises the following steps:

preparing a chitosan-sodium alginate polyelectrolyte compound;

preparing the chitosan-based polyelectrolyte composite hemostatic sponge.

4. The method for preparing chitosan-based polyelectrolyte composite hemostatic sponge according to claim 3, characterized in that: the step includes the following steps:

5. The method for preparing chitosan-based polyelectrolyte composite hemostatic sponge according to claim 3, characterized in that: the method comprises the following steps:

6. The use of the chitosan-based polyelectrolyte complex hemostatic sponge as claimed in claim 1 or 2 in hemostatic dressing.