CN111973802A

CN111973802A - CHI-HA/SH with rapid hemostasis function4Preparation and application of-PEG hydrogel

Info

Publication number: CN111973802A
Application number: CN202010914794.8A
Authority: CN
Inventors: 汪丹丹; 钟源; 陈树帆
Original assignee: Gansu Agricultural University
Current assignee: Gansu Agricultural University
Priority date: 2020-09-03
Filing date: 2020-09-03
Publication date: 2020-11-24

Abstract

The invention provides a CHI-HA/SH₄PEG gel, which is made using the following five steps: (1) hyaluronic acid dissolution, (2) CHI-HA synthesis reaction, (3) dialysis drying, (4) CHI-HA dissolution, and (5) CHI-HA/SH₄PEG synthesis reaction. The CHI-HA/SH₄The PEG gel has the characteristics of high hemostasis speed, high hemostasis efficiency, high mechanical strength, strong wet adhesion capability and good biocompatibility, and is generally superior to other hydrogel hemostasis materials.

Description

CHI-HA/SH with rapid hemostasis function4Preparation and application of-PEG hydrogel

Technical Field

The invention relates to the field of biomedicine, in particular to CHI-HA/SH for quickly stopping bleeding₄-preparation and application of PEG hydrogel.

Background

Sudden life accidents and surgical operations can cause acute bleeding, and if effective hemostatic measures are not taken, the life can be threatened when the bleeding amount exceeds 40 percent of the blood of a human body in a short time. Therefore, the bleeding can be quickly and effectively controlled, and the life of the patient can be saved in time.

The existing local hemostatic agents, such as fibrin glue and collagen glue, hydrogel hemostatic materials and the like, have certain limitations on the application range and cannot completely satisfy users. Fibrin glue and collagen glue have good biocompatibility, are difficult to cause inflammation and foreign body reaction, but the hemostatic glue has poor wet adhesion capacity, low gel strength, insufficient mechanical property, long curing time, failure in rapid heavy bleeding and incapability of meeting the emergency hemostatic requirement in case of excessive blood loss. Hydrogel hemostatic materials usually incorporate metal ions as a cross-linking agent, which results in poor cell compatibility, long time (more than 10min) for hemostasis, poor wet adhesion, and no compliance with the requirement of rapid bleeding control. In a word, a biological hemostatic material which has the advantages of high hemostatic speed, high hemostatic efficiency, high mechanical strength, strong wet adhesion capability and good biocompatibility is lacked at present.

The problems existing in the prior art are as follows: the existing hemostatic material has slow hemostasis speed, poor hemostatic effect and poor biocompatibility.

Disclosure of Invention

The key technical problem to be solved by the invention is to provide a preparation method of hydrogel for rapid hemostasis. In order to solve the technical problems, the invention adopts the following technical scheme:

1. CHI-HA/SH₄-a method for the preparation of a PEG gel comprising:

(1) dissolving hyaluronic acid: 500mg of hyaluronic acid was weighed out and dissolved in 100mL of deionized water to a final concentration of 0.5% (w/v).

(2) CHI-HA Synthesis reaction: after stirring at room temperature until hyaluronic acid was completely dissolved, 287.5mg of NHS (N-hydroxysuccinimide) and 479.5mg of EDC (ethyl (3- (dimethylamino) propyl) carbodiimide hydrochloride) were added, the molar ratio of hyaluronic acid to EDC was 1.5:1 and NHS was 1:1, and after stirring at room temperature for 1h, 718mg of dopamine hydrochloride was added, and the molar ratio of hyaluronic acid to hyaluronic acid was 1.5: 1. The reaction was stirred at room temperature for 24h, and nitrogen was introduced during the reaction.

(3) And (3) dialysis and drying: dialyzing for 48h after the reaction is finished, wherein the dialysate is deionized water, and changing the dialysate every 2h for 1 time. After dialysis, the mixture is frozen and dried to obtain the product which is stored at-4 ℃ for later use.

(4) CHI-HA dissolution: the CHI-HA lyophilized sample was dissolved in PBS solution (pH 5.8) at a concentration of 3 wt%. After the mixture is fully dissolved, the mixture is centrifuged at 5000r/min for 20min to remove bubbles.

(5)CHI-HA/SH₄PEG synthesis reaction: placing 500 μ L of sample on a glass plate at room temperature, adding different concentrationsSH ₄40 μ L of PEG (125, 250, 500mg/ml) was stirred uniformly, NaOH (0.1M) was dropped to adjust the pH of the gel to 7.5, and the mixture was stirred uniformly and left to stand for about 60 seconds to form a hydrogel.

2. CHI-HA/SH₄PEG gel, obtained as described above, having the following characteristics:

(1) hydrogel ultrastructure observation

Transferring the hydrogel into a 24-pore plate, fixing the shape, standing at-80 deg.C for 10h, and vacuum freeze-drying. The hydrogel was fluffy after freeze-drying, cut into thin pieces (about 1mm in thickness) rapidly with a razor blade under a dry environment, adhered and fixed on an observation box, and observed under an S-3400 type electron microscope at 5kV, and the results are shown in FIG. 5. The pore size of the hydrogel is capable of reacting to the extent of crosslinking. The larger the pore size, the lower the degree of crosslinking, and the poorer the mechanical strength of the hydrogel; conversely, the smaller the pore size, the higher the degree of crosslinking and the greater the mechanical strength of the hydrogel. With SH₄Increase in PEG concentration, CHI-HA/SH₄PEG gel pore sizes are getting smaller and smaller, reflecting an increased degree of gel cross-linking. In which SH₄-PEG₅₀₀The mechanical properties of (2) are strongest.

(2) Rheological Properties of hydrogels

The rheological properties of the hydrogels were determined by rheometer (HR-1, TA, USA). The test fixture is a stainless steel round flat plate (phi 20mm), and the measurement temperature is 25 ℃. Cleaning the test table, placing the hydrogel prepared above, and testing the magnitude and variation of the elastic modulus (G ') and viscous modulus (G') in order to ensure that the measurement results are in the linear viscoelastic region of the fluid, and performing dynamic stress sweep at a frequency set to 1 rad/s. The results are shown in FIGS. 6 to 7. The elastic modulus (G ') of the 3 groups of gels is larger than the respective viscous modulus (G'), and the typical characteristics of the gels are shown; the elastic modulus (G ') and viscous modulus (G') curves of group 3 are all kept parallel in the strain range of 0.1% -10%, indicating that the colloid structure is kept stable in the range, the gel can keep the structure intact and the colloid is not destroyed. In the elastic range, with SH₄Increase in PEG concentration, CHI-HA/SH₄Increase in the elastic modulus (G') of the PEG gel, at most CHI-HA/SH₄-PEG₅₀₀Gelling to 2195.22 Pa; with SH₄Increase in PEG concentration, CHI-HA/SH₄Increase in viscous modulus (G') of PEG gels, maximum CHI-HA/SH₄-PEG₅₀₀Gelling to 745.41 Pa; this result reflects CHI-HA/SH₄-PEG₅₀₀The gel has higher gel strength and stronger viscosity, and has good performance of a hemostatic material.

3. CHI-HA/SH₄-a method for determining the wet adhesion of PEG gels to animal skin and uses thereof, comprising:

the ability of the hydrogels to adhere wet to the skin of animals was measured using a texture analyzer (TMS-Pro, FTC, usa). Fresh pig skin was cut into 15mm × 25mm rectangles after removing excess fat, and immersed in PBS buffer to keep the pig skin moist. In the test, the pig skin was removed, the hydrogel (200. mu.L) prepared above was spread on a moist pig skin having an area of 15mm × 15mm, and another pig skin was quickly covered, and the magnitude of the adhesive force was measured after standing at room temperature for 1 hour. The tensile rate during the measurement was set to 5mm/min, and the adhesive strength was calculated by dividing the maximum load by the covered area, and the results were shown in FIG. 8 for each set of 5 replicates. With SH₄Increase in PEG concentration, CHI-HA/SH₄Increase in Wet adhesion of PEG gel to Pigskin, maximum CHI-HA/SH₄-PEG₅₀₀Gelling to 11.28 kPa. This result reflects CHI-HA/SH₄-PEG₅₀₀The gel has strong wet adhesion capability and good performance of hemostatic materials.

5. CHI-HA/SH₄-PEG gel biocompatibility assay methods and uses comprising:

(1) 5mg of the hydrogel prepared above is weighed and placed in an ultra-clean bench for ultraviolet sterilization for 30min, 2.5mL of DMEM culture medium containing fetal calf serum (10%, v/v), penicillin (100 units/mL) and streptomycin (100 mu g/mL) is respectively added into each group, the mixture is leached for 24h at 37 ℃, and the leaching solution is filtered and sterilized through a 0.22 mu m filter membrane.

(2) Rat L929 cells with good logarithmic phase growth state are selected, digested by trypsin and diluted to 3X 10 by DMEM medium (10% fetal bovine serum)⁴one/mL, inoculated into 96-well plates, 100 μ L per well. At 37 ℃ and 5％CO₂Culturing in an incubator for 24 h. The culture medium in the 96-well plate was discarded and 100. mu.L of the extract was added to each well, with 4 replicates per set.

(3) The cell culture plate is placed in a cell culture box at 37 ℃ for culture, after 24, 48 and 72 hours of culture, 10 mu L of MTT solution (5mg/mL) is added into each hole, the cell culture plate is incubated at 37 ℃ for 4 hours, the culture solution in each hole is discarded, 10% SDS is added, and the absorbance at 570nm is measured by a microplate reader and recorded.

6. CHI-HA/SH₄-a method for evaluating the hemostatic properties of PEG gels and the use of hemostasis.

Compared with the prior art, the method has the following beneficial effects:

1. a high molecular compound (CHI-HA) with DOPA as a functional group is developed to become a component of the novel waterproof hydrogel.

2. By selecting SH₄PEG is a cross-linker, which reacts with DOPA on the basis of Michael addition to form a hydrogel. Because the molecular weight is large and the mercapto content is higher than that of the common mercapto high polymer, the gelling time is only about 60s, and the gel can be used as a rapid hemostatic material.

3. First synthesized CHI-HA/SH₄PEG hydrogel has the characteristics of high hemostasis speed, high hemostasis efficiency, high mechanical strength, strong wet adhesion capability and good biocompatibility.

4. The elastic modulus of the existing reported hydrogel can only reach about 1000Pa, and the newly synthesized CHI-HA/SH₄The PEG hydrogel elastic die can reach 2195.22Pa, has high gel strength and strong gel deformation resistance, and has good hemostatic effect when being applied to massive hemorrhage.

5. The wet adhesion of the hydrogel reported in the prior art can reach about 5kPa, while the CHI-HA/SH₄The PEG hydrogel can reach 11kPa, has strong wet adhesion capability, is easy to adhere to bleeding parts, is not easy to fall off, and has high hemostasis efficiency.

6. The cell compatibility of the hydrogel reported in the prior art is poor, the relative cell proliferation rate is between 80% and 90%, and the hydrogel has certain inhibition on cell growth. And CHI-HA/SH₄The PEG gel has good biocompatibility, the relative cell proliferation rate is between 100 and 120 percent, and the PEG gel has certain function of promoting cell growth。

7. The hydrogel reported in the prior art has slow gelling speed, which is mostly required to be more than 10min, and some hydrogel even needs about 10 h. And CHI-HA/SH₄The PEG gel only needs about 60s for gelling, and has high gelling speed and high hemostasis speed.

8. CHI-HA/SH in liver bleeding and leg arterial bleeding models₄PEG gel has good hemostatic effect, high hemostatic speed and high hemostatic efficiency. Is generally superior to other hydrogel hemostatic materials.

Drawings

FIG. 1 shows the synthesis of hyaluronic acid-dopamine (CHI-HA);

wherein EDC is ethyl (3- (dimethylamino) propyl) carbodiimide hydrochloride; NHS is N-hydroxysuccinimide, both of which are reaction activators, and dialyzed to remove the NHS after the reaction is finished.

FIG. 2 is a nuclear magnetic characterization of CHI-HA;

wherein, the black frame is the area (6.5-6.8 ppm) of the characteristic absorption peak.

FIG. 3 shows CHI-HA/SH₄The principle of synthesis of PEG gels.

FIG. 4 shows CHI-HA/SH₄Appearance morphology of PEG gels.

FIG. 5 shows CHI-HA/SH₄-microscopic morphology of PEG gels.

FIG. 6 shows CHI-HA/SH₄-rheological analysis of the elastic modulus of PEG gels.

FIG. 7 shows CHI-HA/SH₄-rheological analysis of the viscous modulus of PEG gels.

FIG. 8 shows CHI-HA/SH₄-the ability of PEG gel to adhere to the skin of wet animals.

FIG. 9 shows CHI-HA/SH₄-cell compatibility of PEG gel.

FIG. 10 shows CHI-HA/SH₄The hemostatic effect of the rat liver of the PEG gel.

FIG. 11 shows CHI-HA/SH₄PEG gel rat leg artery hemostatic effect.

Detailed description of the invention

The methods and devices used in the following examples of the present invention are conventional methods and devices unless otherwise specified; the equipment and the reagent are all conventional equipment and reagents purchased by a reagent company. In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the embodiments of the present invention is provided in connection with the specific embodiments. Examples of these preferred embodiments are illustrated in the specific examples.

It should be noted that, in order to avoid obscuring the technical solutions of the present invention with unnecessary details, only the technical solutions and/or processing steps closely related to the technical solutions of the present invention are shown in the embodiments, and other details that are not relevant are omitted.

Example 1

This example provides CHI-HA/SH₄-a method for preparing hyaluronic acid-dopamine (CHI-HA) as a cross-linking substrate for PEG gel, comprising:

(1) dissolving: 500mg of hyaluronic acid was weighed out and dissolved in 100mL of deionized water to a final concentration of 0.5% (w/v).

(2) And (3) synthesis reaction: after stirring at room temperature until hyaluronic acid was completely dissolved, 287.5mg of NHS (N-hydroxysuccinimide) and 479.5mg of EDC (ethyl (3- (dimethylamino) propyl) carbodiimide hydrochloride) were added, the molar ratio of hyaluronic acid to EDC was 1.5:1 and NHS was 1:1, and after stirring at room temperature for 1h, 718mg of dopamine hydrochloride was added, and the molar ratio of hyaluronic acid to hyaluronic acid was 1.5: 1. The reaction was stirred at room temperature for 24h, and nitrogen was introduced during the reaction.

(3) And (3) dialysis and drying: dialyzing for 48h after the reaction is finished, wherein the dialysate is deionized water, and changing the dialysate every 2h for 1 time. After dialysis, the mixture is frozen and dried to obtain the product which is stored at-4 ℃ for later use. The synthetic schematic diagram is shown in figure 1. The nuclear magnetic identification shows that a characteristic peak appears at 6.5-6.8 ppm, which proves that the dopa grafting is successful, and is shown in figure 2.

Example 2

This example provides CHI-HA/SH₄Preparation of PEG gel, CHI-HA/SH₄The principle of preparation of the PEG gel is shown in FIG. 3. The synthesis method comprises the following steps:

(1) dissolving: the CHI-HA lyophilized sample was dissolved in PBS solution (pH 5.8) at a concentration of 3 wt%. After the mixture is fully dissolved, the mixture is centrifuged at 5000r/min for 20min to remove bubbles.

(2) Reaction: 500 μ L of the sample was placed on a glass plate at room temperature, and SH at different concentrations were added to the sample ₄40 μ L of PEG (125, 250, 500mg/ml) was stirred uniformly, NaOH (0.1M) was dropped to adjust the pH of the gel to 7.5, and the mixture was stirred uniformly and left to stand for about 60 seconds to form a hydrogel. The results are shown in FIG. 4. CHI-HA/SH₄PEG gel has high gelling speed and has the potential of rapid hemostasis.

Example 3

This example provides CHI-HA/SH₄-characterization of the properties of PEG gels, including:

(1) hydrogel ultrastructure observation

Transferring the hydrogel into a 24-pore plate, fixing the shape, standing at-80 deg.C for 10h, and vacuum freeze-drying. The hydrogel was fluffy after freeze-drying, cut into thin pieces (about 1mm in thickness) rapidly with a razor blade under a dry environment, stuck and fixed on an observation box, and observed under an S-3400 type electron microscope at 5kv, and the results are shown in FIG. 5. The pore size of the hydrogel is capable of reacting to the extent of crosslinking. The larger the pore size, the lower the degree of crosslinking, and the poorer the mechanical strength of the hydrogel; conversely, the smaller the pore size, the higher the degree of crosslinking and the greater the mechanical strength of the hydrogel. With SH₄Increase in PEG concentration, CHI-HA/SH₄PEG gel pore sizes are getting smaller and smaller, reflecting an increased degree of gel cross-linking. In which SH₄-PEG₅₀₀The mechanical properties of (2) are strongest.

(2) Rheological Properties of hydrogels

The rheological properties of the hydrogels were determined by rheometer (HR-1, TA, USA). The test fixture is a stainless steel round flat plate (phi 20mm), and the measurement temperature is 25 ℃. Cleaning the test table, placing the hydrogel prepared above, and testing the magnitude and variation of the elastic modulus (G ') and viscous modulus (G') in order to ensure that the measurement results are in the linear viscoelastic region of the fluid, and performing dynamic stress sweep at a frequency set to 1 rad/s. The results are shown in FIGS. 6 to 7. The elastic modulus (G ') of the 3 groups of gels is larger than the respective viscous modulus (G'), and the typical characteristics of the gels are shown; the elastic modulus (G ') and viscous modulus (G') curves of the 3 groups are kept flat in the strain range of 0.1-10%The line shows that the colloidal structure remains stable in this range, the gel is able to maintain structural integrity and the colloid is not destroyed. In the elastic range, with SH₄Increase in PEG concentration, CHI-HA/SH₄Increase in the elastic modulus (G') of the PEG gel, at most CHI-HA/SH₄-PEG₅₀₀Gelling to 2195.22 Pa; with SH₄Increase in PEG concentration, CHI-HA/SH₄Increase in viscous modulus (G') of PEG gels, maximum CHI-HA/SH₄-PEG₅₀₀Gelling to 745.41 Pa; this result reflects CHI-HA/SH₄-PEG₅₀₀The gel has higher gel strength and stronger viscosity, and has good performance of a hemostatic material.

Example 4

This example provides CHI-HA/SH₄-wet adhesion performance test of PEG gels on animal skin, comprising:

Example 5

This example provides CHI-HA/SH₄-a PEG gel biocompatibility assay comprising:

(2) Rat L929 cells with good logarithmic growth phase state are selected, digested by trypsin and diluted to 3X 10 by DMEM medium (10% fetal bovine serum)⁴one/mL, inoculated into 96-well plates, 100 μ L per well. At 37 deg.C, 5% CO₂Culturing in an incubator for 24 h. The culture medium in the 96-well plate was discarded and 100. mu.L of the extract was added to each well, with 4 replicates per set.

(3) The cell culture plate is placed in a cell culture box at 37 ℃ for culture, after 24, 48 and 72 hours of culture, 10 mu L of MTT solution (5mg/mL) is added into each hole, the cell culture plate is incubated at 37 ℃ for 4 hours, the culture solution in each hole is discarded, 10% SDS is added, and the absorbance at 570nm is measured by a microplate reader and recorded. The relative cell proliferation rate (RGR) was calculated by the following formula:

in the formula: OD₀Blank group without inoculated cells; OD₁A blank group of cells was seeded as a control group; OD₂Are test groups.

The results of the experiments are shown in FIG. 9, and after 24, 48, and 72h of culture, CHI-HA/SH₄The relative cell proliferation rate of PEG gel is above 95%, and CHI-HA/SH₄-PEG₂₅₀And CHI-HA/SH₄-PEG₅₀₀The relative proliferation rate of the gel cells is more than 100%, which shows that the two gels can promote the cell growth to a certain extent. According to the standard of medical apparatus biological evaluation of national Standard of the people's republic of China, CHI-HA/SH₄PEG gel with a cytotoxicity rating of 0 is a biologically safe material and can be used as a medical hydrogel.

Example 6

This example provides CHI-HA/SH₄-PEG gel hemostatic performance evaluation comprising:

due to CHI-HA/SH₄-PEG₅₀₀The gel has excellent gel strength, adhesion ability and cell compatibility, and is selectedAnd (4) evaluating the hemostasis performance. We established two representative bleeding models (rat liver bleeding model and rat leg arterial bleeding model).

Rat liver bleeding model: chloral hydrate was used to anaesthetize healthy SD rats (around 150 g), the abdomen was opened to expose the liver, and the liver was separated from the abdominal cavity with a preservative film before the filter paper was carefully placed under the liver to prevent contamination of the filter paper by blood leakage. Immediately after bleeding by puncturing the liver with a sterile needle (18N), hydrogel (200. mu.L) was applied to the bleeding site. The recording was photographed after observation until the bleeding point no longer bleeds, and the amount of bleeding absorbed on the filter paper was weighed.

Rat leg arterial bleeding model: healthy SD rats (150g or so) were anesthetized with chloral hydrate, the leg portions were exposed by incising the skin of the leg portions, the saphenous artery of the rat leg portions was exposed, and the hydrogel (200. mu.L) was spread on the bleeding portions immediately after the bleeding was caused by puncturing the artery with a sterile needle (18N). And (5) observing, and taking a photograph and recording until the bleeding point does not bleed any more.

As shown in FIGS. 10 to 11, in the liver bleeding model, the blood loss of the untreated group was 60.95mg, while the blood loss of the CHI-HA/SH group was₄-PEG₅₀₀The gel rapidly controlled bleeding, the total blood loss was 8.55mg, and in the rat leg arterial bleeding model, the bleeding volume in the untreated group was large; and CHI-HA/SH₄-PEG₅₀₀Only a small bleeding point was observed in the gel. CHI-HA/SH₄-PEG₅₀₀The gel exhibits good hemostatic properties.

The foregoing is directed to embodiments of the present application and it is noted that numerous modifications and adaptations may be made by those skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.

Claims

1. CHI-HA/SH₄-a method for preparing a PEG gel, characterized in that it comprises:

(1) dissolving hyaluronic acid: weighing 500mg of hyaluronic acid, and dissolving in 100mL of deionized water to obtain a final concentration of 0.5%;

(2) CHI-HA Synthesis reaction: stirring at room temperature until hyaluronic acid is completely dissolved, adding 287.5mg of NHS (N-hydroxysuccinimide) and 479.5mg of EDC (ethyl (3- (dimethylamino) propyl) carbodiimide hydrochloride), wherein the molar ratio of hyaluronic acid to EDC is 1.5:1 and the molar ratio to NHS is 1:1, stirring at room temperature for 1h, and adding 718mg of dopamine hydrochloride, wherein the molar ratio to hyaluronic acid is 1.5: 1; the reaction was stirred at room temperature for 24h, and nitrogen was introduced during the reaction.

(3) And (3) dialysis and drying: dialyzing for 48h after the reaction is finished, wherein the dialysate is deionized water, and changing the dialysate every 2h for 1 time. After dialysis, freeze-drying to obtain the product, and storing at-4 ℃ for later use;

(4) CHI-HA dissolution: dissolving the CHI-HA freeze-dried sample in a PBS solution at the concentration of 3 wt%, and centrifuging at 5000r/min for 20min after full dissolution to remove bubbles;

(5)CHI-HA/SH₄PEG synthesis reaction: 500 μ L of the sample was placed on a glass plate at room temperature, and SH at different concentrations were added to the sample₄40 mu L of PEG is evenly stirred, NaOH is dropped into the mixture to adjust the pH value of the gel to be 7.5, the mixture is evenly stirred and placed for about 60s to form hydrogel.

2. CHI-HA/SH₄-PEG gel, characterized in that it is obtained by the process according to claim 1.

3. CHI-HA/SH₄-use of PEG gel for high wet adhesion to animal skin, characterized in that said CHI-HA/SH₄-PEG gel as claimed in claim 2.

4. CHI-HA/SH₄-use of PEG gel with high biocompatibility, characterized in that said CHI-HA/SH₄-PEG gel as claimed in claim 2.

5. CHI-HA/SH₄-use of PEG gel for haemostasis, characterized in that said CHI-HA/SH₄-PEG gel as claimed in claim 2.