CN118895013A - A porous microsphere of giant salamander glycosaminoglycan and its preparation method and application - Google Patents

Abstract

The invention relates to giant salamander glycosaminoglycan porous microspheres and a preparation method and application thereof. The preparation method of the giant salamander glycosaminoglycan porous microsphere comprises the following steps: adding an activating agent into the giant salamander glycosaminoglycan solution to obtain an activated giant salamander glycosaminoglycan solution; dissolving methacryloylated gelatin and an initiator in water to obtain a methacryloylated gelatin solution; adding a methacryloylated gelatin solution into the activated giant salamander glycosaminoglycan solution to obtain a methacryloylated gelatin-giant salamander glycosaminoglycan conjugated solution; and (3) curing and crosslinking the methacryloylated gelatin-giant salamander glycosaminoglycan conjugated solution by using ultraviolet light, and freeze-drying to obtain the giant salamander glycosaminoglycan porous microspheres. The invention also provides the giant salamander glycosaminoglycan porous microsphere prepared by the preparation method. The invention also provides application of the giant salamander glycosaminoglycan porous microspheres in medicines for stopping bleeding of wound surfaces, reducing wound surface liquid seepage and promoting wound surface healing. The invention solves the problem that the seepage of the traditional giant salamander glycosaminoglycan is difficult to control for wound repair.

Description

A porous microsphere of giant salamander glycosaminoglycan and its preparation method and application

Technical Field

The invention relates to the technical field of biomaterials, and in particular to a porous microsphere of giant salamander glycosaminoglycan and a preparation method and application thereof.

Background Art

As an amphibian unique to China, the giant salamander is the longest-lived and largest amphibian in the world. During the long period of evolution, the giant salamander's skin contains a large number of mucus glands, which can secrete mucus containing a large amount of protein and polysaccharides after external stimulation to help it escape from danger or promote wound healing. It is based on this self-protection mechanism of the giant salamander that according to the "Compendium of Materia Medica" compiled by Hong Jingtao during the Southern and Northern Dynasties, the skin secretions of the giant salamander were used to treat burns. The skin secretions of the giant salamander can not only accelerate wound healing, but also improve the quality of wound repair, showing great medicinal potential. In recent years, with the increasing maturity of artificial breeding technology of giant salamanders and the increase in giant salamander production, the second generation of artificially bred giant salamanders has been approved for sale on the market.

In recent years, as the artificial breeding technology of giant salamanders has become increasingly mature and the production of giant salamanders has increased, the second generation of artificially bred giant salamanders has been approved for sale on the market. The application of giant salamander skin mucus has also gradually increased.

In the early stage, it was proved that the giant salamander glycosaminoglycan extracted from the skin mucus of giant salamander can promote the formation of blood vessels in the wound and thus has an excellent ability to promote wound repair. However, in actual application, it was found that it was difficult to control the large amount of exudate formed in the wound, especially in chronic wounds, so it was difficult to achieve the effect of continuous drug delivery.

Summary of the invention

In view of this, the object of the present invention is to provide a giant salamander glycosaminoglycan porous microsphere and a preparation method and application thereof, so as to solve the problem that the existing giant salamander glycosaminoglycan is difficult to control the exudate in wound repair.

In order to achieve the above object, the technical solution adopted by the present invention is as follows:

A method for preparing porous microspheres of glycosaminoglycans from giant salamander comprises the following steps:

adding an activator to the giant salamander glycosaminoglycan solution to activate the carboxyl groups in the giant salamander glycosaminoglycan to obtain an activated giant salamander glycosaminoglycan solution;

dissolving methacrylated gelatin and a polymerization initiator in water to obtain a methacrylated gelatin solution;

adding a methacrylic acid gelatin solution to the activated giant salamander glycosaminoglycan solution to obtain a methacrylic acid gelatin-giant salamander glycosaminoglycan conjugate solution;

The methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution was solidified and cross-linked by ultraviolet light, and freeze-dried to obtain giant salamander glycosaminoglycan porous microspheres.

According to the above technical means, an activator is first used to activate the carboxyl groups in the giant salamander glycosaminoglycan, and methacrylated gelatin is added to form a conjugated system between the amino groups on the methacrylated gelatin and the carboxyl groups, thereby forming a more stable chemical structure between the glycosaminoglycan and the methacrylated gelatin; after curing and cross-linking with ultraviolet light, the obtained microspheres are quickly frozen in liquid nitrogen for 3 to 10 minutes and then freeze-dried to form giant salamander glycosaminoglycan porous microspheres with a porous structure. The porous structure of the giant salamander glycosaminoglycan porous microspheres can fully absorb exudate, effectively solving the problem that the existing giant salamander glycosaminoglycan is difficult to control exudate for wound repair.

Preferably, the activator is selected from N-hydroxysuccinimide (NHS) and/or 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

Preferably, the activator is selected from a mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

By using a mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) as an activating agent, EDC acts as an active reagent for carboxyl groups in amide formation, and the combination with NHS can improve the coupling efficiency compared to the use of EDC alone to activate the carboxyl groups on glycosaminoglycans of giant salamander.

Preferably, the polymerization initiator is selected from lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

Preferably, the methacryloyl substitution degree of the methacryloyl gelatin is 71% to 90%.

By controlling the reaction time of methacrylic anhydride in gelatin, the degree of methacrylic substitution of methacrylic anhydride gelatin can be controlled between 71% and 90%, so that the microspheres obtained later can obtain better mechanical properties and hardness.

Preferably, the mass ratio of the methacrylated gelatin to the polymerization initiator is 50:1.

Preferably, the mass percentage of methacrylylated gelatin in the methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution is between 7% and 10%.

Preferably, the mass percentage of giant salamander glycosaminoglycan in the methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution is 0.4%.

By rationally controlling the ratio of methacryloylated gelatin to giant salamander glycosaminoglycan in the methacryloylated gelatin-giant salamander glycosaminoglycan conjugate solution, the giant salamander glycosaminoglycan microspheres can achieve the best mechanical properties while playing the role of glycosaminoglycan in promoting wound repair.

Preferably, the mass percentage of N-hydroxysuccinimide (NHS) in the methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution is 0.01%.

Preferably, the mass percentage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in the methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution is 0.04%.

Preferably, the methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution is cured and cross-linked by light to obtain giant salamander glycosaminoglycan porous microspheres, specifically comprising: using methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution as a continuous phase, mineral oil and a dispersant as a dispersed phase, feeding them into a microfluidic chip channel, curing and cross-linking them by light irradiation at the tail end of the channel, and freeze-drying to obtain giant salamander glycosaminoglycan porous microspheres.

By using microfluidic chips to prepare giant salamander glycosaminoglycan porous microspheres, the consistency and accuracy of the giant salamander glycosaminoglycan porous microspheres are effectively guaranteed, and the preparation time is shortened, the production efficiency is improved, the complexity and cost of the preparation process are reduced, and it has the advantages of being more flexible, convenient, fast and low-cost.

Preferably, the activation temperature of the carboxylic acid groups in the activated giant salamander glycosaminoglycan is between 20° C. and 50° C., and the activation time is between 10 and 15 hours.

Preferably, the dissolution temperature of the methacrylated gelatin and the polymerization initiator in water is between 50°C and 60°C.

Preferably, the flow rate ratio of the continuous phase to the dispersed phase is between 1:3 and 1:5.

Preferably, the mineral oil is selected from paraffin oil, and the dispersant is selected from Span 80.

Preferably, the method for preparing the giant salamander glycosaminoglycan comprises:

S1, enzymatically hydrolyzing the mucus secreted by the defatted giant salamander skin, and then heating it to obtain a giant salamander skin mucus enzymatic hydrolyzate;

S2. The benzethonium chloride method is used to precipitate a first precipitation from the enzymatic hydrolyzate of the giant salamander skin mucus, and then anhydrous ethanol is added to the supernatant after the first precipitation to obtain a second precipitation, and the second precipitation is dialyzed to obtain giant salamander glycosaminoglycan.

By adopting the benzethonium chloride method, the enzymatic hydrolyzate of the giant salamander skin mucus is precipitated, which not only effectively ensures that the main effective components in the giant salamander skin mucus enzymatic hydrolyzate are not destroyed, so as to fully retain its biological effects, but also expands the comprehensive utilization of the giant salamander, and has the advantages of high biosafety and mild conditions.

Compared with the Sevage method, the benzethonium chloride method further removes the neutral glycosaminoglycan components in the giant salamander glycosaminoglycan and retains more acidic glycosaminoglycan components. Its higher content of carboxyl groups makes it more suitable for preparing glycosaminoglycan porous microspheres by conjugation with methacryloyl gelatin, while retaining the biological activity of glycosaminoglycans to the maximum extent.

Preferably, the step S1 specifically includes: obtaining the mucus secreted by the skin of the giant salamander by a physical stimulation method, and freeze-drying the mucus secreted by the skin of the giant salamander to obtain freeze-dried powder of the mucus secreted by the skin of the giant salamander;

The lyophilized powder of the skin secretion mucus of the giant salamander is washed and degreased by using anhydrous ethanol, and then dried to obtain the defatted skin secretion mucus of the giant salamander;

The alkaline protease solution is added to the defatted giant salamander skin secretion mucus for enzymolysis, and then the enzyme is inactivated by heating to obtain the giant salamander skin mucus enzymolysis solution.

Preferably, S2 specifically comprises: concentrating the giant salamander skin mucus enzymatic hydrolysate obtained in S1, then adding the benzethonium chloride solution to the concentrated giant salamander glycosaminoglycan solution until the primary precipitate is completely precipitated, then dissolving the primary precipitate with a saturated sodium chloride solution, and centrifuging to obtain a supernatant;

Add anhydrous ethanol to the supernatant for alcohol precipitation, let it stand at 4°C for 6-12 hours, and obtain secondary precipitation after centrifugation;

The secondary precipitate is dialyzed using a dialysis bag, and the dialysis time is between 48 and 72 hours to obtain giant salamander glycosaminoglycan.

Preferably, in S1, the amount of anhydrous ethanol used is 2 to 5 times the amount of the lyophilized powder of giant salamander skin mucus secretion by mass ratio.

Preferably, in S1, the freeze-drying temperature is -75°C to -85°C.

Preferably, in S1, the mass percentage of alkaline protease in the alkaline protease solution is 1% to 5%.

Preferably, in S1, the alkaline protease in the alkaline protease solution is Bacillus licheniformis protease.

Preferably, in S1, the mass percentage of the mucus secreted by the skin of giant salamander and the alkaline protease solution is 90%-95%:5%-10%.

Preferably, in S1, the enzymatic hydrolysis is specifically as follows: adding the mucus secreted by the skin of giant salamander to an alkaline protease solution, incubating for 5 to 8 hours at a temperature of 40°C to 60°C and a pH value of 8 to 10, then adding water and continuing to incubate for 2 to 5 hours.

Preferably, in S1, the enzyme inactivation is specifically performed by heating the incubated solution to 80° C. to 100° C. and boiling for 10 to 20 minutes.

Preferably, in S2, the concentration is specifically: rotary evaporating the giant salamander skin mucus enzymatic hydrolysate at a temperature of 45°C to 65°C, and the concentrated giant salamander skin mucus enzymatic hydrolysate is 30% to 50% of the giant salamander skin mucus enzymatic hydrolysate before concentration.

Preferably, in S2, the concentration of benzethonium chloride in the benzethonium chloride solution is 3% to 6%.

Preferably, in S2, the dialysis bag is selected from a dialysis bag with a molecular weight of 3000D~8000D.

The present invention also provides a porous microsphere of glycosaminoglycan of giant salamander prepared by the preparation method of the present invention.

The present invention also provides an application of the giant salamander glycosaminoglycan porous microspheres prepared by the preparation method of the present invention in a medicine for hemostasis of wound surface.

The present invention also provides an application of the giant salamander glycosaminoglycan porous microspheres prepared by the preparation method of the present invention in a medicine for reducing wound exudate.

The present invention also provides an application of the giant salamander glycosaminoglycan porous microspheres prepared by the preparation method of the present invention in a medicine for promoting wound healing.

Beneficial effects of the present invention:

The preparation method of the giant salamander glycosaminoglycan porous microspheres of the present invention realizes effective separation and purification of giant salamander glycosaminoglycans by sequentially subjecting giant salamander skin mucus to freeze drying, enzymolysis, enzyme inactivation, protein removal, precipitation and dialysis treatment, wherein the enzymolysis method and benzethonium chloride method are used to treat the giant salamander skin mucus under mild conditions, which will not destroy the main effective components in the giant salamander mucus, and can maximize the biological effect of giant salamander glycosaminoglycans. At the same time, the microfluidic technology used makes the prepared giant salamander glycosaminoglycan microspheres The microspheres have better consistency and precision, thus having excellent hemostasis, exudate control and wound healing abilities, which makes the giant salamander glycosaminoglycan porous microspheres have great application value in drugs for wound hemostasis, wound exudate reduction and wound healing promotion. In addition, the dialysis treatment used effectively removes ineffective small molecule components, retains large molecule effect components, improves the purity of giant salamander glycosaminoglycan, and has the advantages of simple preparation process, strong operability and suitability for large-scale mass production.

The giant salamander glycosaminoglycan porous microspheres prepared by the present invention have been proved by experiments to have excellent hemostatic ability and can absorb wound exudate at least ten times its own weight. Relying on its own degradation, it provides a good drug sustained release ability, and its conjugated loaded giant salamander glycosaminoglycan improves the therapeutic effect, simplifies the medication method, and reduces toxic side effects, so that the material has excellent ability to promote wound healing while stopping bleeding and controlling exudate, effectively expanding the application of giant salamander derivative products, changing the development idea of only giant salamander mucus products, better exploring the medical and medicinal value of giant salamander, and having the advantage of high biosafety, it has potential application value in the field of medical application technology. .

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an example diagram of a method for preparing porous microspheres of glycosaminoglycans from giant salamander;

FIG2 is a diagram showing the hemostatic effect of glycosaminoglycan porous microspheres in a rat liver bleeding model;

FIG3 is a statistical diagram of hemostasis time of glycosaminoglycan porous microspheres in a rat liver bleeding model;

FIG4 is a statistical diagram of the bleeding volume of glycosaminoglycan porous microspheres in a rat liver bleeding model;

FIG5 is an exemplary diagram of the simulated exudate absorption capacity of glycosaminoglycan porous microspheres in vitro;

FIG6 is an exemplary diagram of the weight change of glycosaminoglycan porous microspheres simulating the absorption capacity of exudate in vitro;

FIG7 is an exemplary diagram showing the change in diameter of porous glycosaminoglycan microspheres in vitro simulating the absorption capacity of exudate;

FIG8 is an exemplary diagram showing the ability of porous glycosaminoglycan microspheres to promote full-thickness skin wound healing in a diabetic mouse model;

Figure 9 is a statistical graph showing the percentage of wound healing at different time points in the diabetic mouse model in which glycosaminoglycan porous microspheres promote the healing of full-thickness skin wounds.

DETAILED DESCRIPTION

The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and thus the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

The present invention intends to disclose a giant salamander glycosaminoglycan porous microsphere and a preparation method and application thereof, so as to solve the problem that the existing giant salamander glycosaminoglycan has difficulty in controlling exudate in wound repair.

Among them, a method for preparing porous microspheres of giant salamander glycosaminoglycan comprises the following steps:

adding an activator to the giant salamander glycosaminoglycan solution to activate the carboxyl groups in the giant salamander glycosaminoglycan to obtain an activated giant salamander glycosaminoglycan solution;

dissolving methacrylated gelatin and a polymerization initiator in water to obtain a methacrylated gelatin solution;

adding a methacrylic acid gelatin solution to the activated giant salamander glycosaminoglycan solution to obtain a methacrylic acid gelatin-giant salamander glycosaminoglycan conjugate solution;

The methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution was solidified and cross-linked by ultraviolet light, and freeze-dried to obtain giant salamander glycosaminoglycan porous microspheres.

The carboxyl groups in the giant salamander glycosaminoglycan are first activated by an activator, and methacryloyl gelatin is added to form a conjugated system with the amino groups and carboxyl groups on the methacryloyl gelatin. The system is then cured and cross-linked with ultraviolet light, and then quickly frozen for 3 to 10 minutes for freeze-drying to form porous giant salamander glycosaminoglycan microspheres. The porous structure of the giant salamander glycosaminoglycan porous microspheres can fully absorb exudate, effectively solving the problem that the exudate of the existing giant salamander glycosaminoglycan is difficult to control for wound repair.

In some embodiments, the activator is selected from N-hydroxysuccinimide (NHS) and/or 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

Exemplarily, the activator is selected from a mixture of N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

In some embodiments, the polymerization initiator is selected from lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

Exemplarily, the methacryloylated gelatin has a methacryloylated degree of substitution of 90%.

Exemplarily, the mass ratio of methacrylated gelatin to the polymerization initiator is 1:50.

In some embodiments, the mass percentage of methacrylylated gelatin in the methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution is between 7% and 10%.

Exemplarily, the mass percentage of giant salamander glycosaminoglycan in the methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution is 0.4%.

For example, the mass percentage of N-hydroxysuccinimide (NHS) in the methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution is 0.01%.

For example, the mass percentage of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in the methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution is 0.04%.

In some embodiments, in order to ensure the consistency and accuracy of the giant salamander glycosaminoglycan porous microspheres, the methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution is cured and cross-linked by light to obtain giant salamander glycosaminoglycan porous microspheres, specifically comprising: using methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution as a continuous phase, mineral oil and a dispersant as a dispersed phase, feeding them into a microfluidic chip channel, curing and cross-linking them by light irradiation at the tail end of the channel, and freeze-drying to obtain giant salamander glycosaminoglycan porous microspheres.

In some embodiments, the activation temperature for activating the carboxylic acid groups in the glycosaminoglycan of the giant salamander is between 20° C. and 50° C., and the activation time is between 10 and 15 hours.

In some embodiments, the dissolution temperature of the methacrylated gelatin and the polymerization initiator in water is between 50°C and 60°C.

In some embodiments, the ultraviolet light has a wavelength of 405 nm.

In some embodiments, the flow rate ratio of the continuous phase to the dispersed phase is between 1:3 and 1:5.

Exemplarily, the flow rate ratio of the continuous phase to the dispersed phase is 1:3.

Exemplarily, the mineral oil is selected from paraffin oil, and the dispersant is selected from Span 80.

Exemplary, the method for preparing the porous microspheres of giant salamander glycosaminoglycan comprises the following steps:

Step (1), dissolving a polymerization initiator, phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium, and methacrylated gelatin in half the total volume of deionized water to obtain a methacrylated gelatin solution;

Step (2), dissolving the giant salamander glycosaminoglycan in the remaining deionized water, then adding N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and fully mixing under heating conditions to activate the carboxyl groups in the giant salamander glycosaminoglycan to obtain an activated giant salamander glycosaminoglycan solution;

Step (3), adding the methacrylic acid-acylated gelatin solution to the activated giant salamander glycosaminoglycan solution, stirring at room temperature overnight, to obtain a methacrylic acid-acylated gelatin-giant salamander glycosaminoglycan conjugate solution;

Step (4), using methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution as the continuous phase and mineral oil and dispersant as the dispersed phase, the continuous phase and the dispersed phase are simultaneously introduced into the microfluidic chip channel, and irradiated with ultraviolet light (405 nm) at the end of the channel for curing and cross-linking. The obtained microspheres are filtered with a filter, and then washed three times with anhydrous ethanol to remove the oil and dispersant (Span 80), and then the microspheres are freeze-dried to obtain giant salamander glycosaminoglycan porous microspheres.

In some embodiments, in order to ensure that the main effective components in the giant salamander skin mucus enzymatic hydrolysate are not destroyed and to fully retain its biological effects, the preparation method of giant salamander glycosaminoglycans includes:

S1, enzymatically hydrolyzing the mucus secreted by the defatted giant salamander skin, and then heating it to obtain a giant salamander skin mucus enzymatic hydrolyzate;

S2. The benzethonium chloride method is used to precipitate a first precipitation from the enzymatic hydrolyzate of the giant salamander skin mucus, and then anhydrous ethanol is added to the supernatant after the first precipitation to obtain a second precipitation, and the second precipitation is dialyzed to obtain giant salamander glycosaminoglycan.

In some embodiments, S1 specifically includes: obtaining the mucus secreted by the skin of the giant salamander by a physical stimulation method, and freeze-drying the mucus secreted by the skin of the giant salamander to obtain freeze-dried powder of the mucus secreted by the skin of the giant salamander;

The lyophilized powder of the skin secretion mucus of the giant salamander is washed and degreased by using anhydrous ethanol, and then dried to obtain the defatted skin secretion mucus of the giant salamander;

The alkaline protease solution is added to the defatted giant salamander skin secretion mucus for enzymolysis, and then the enzyme is inactivated by heating to obtain the giant salamander skin mucus enzymolysis solution.

In some embodiments, S2 specifically includes: concentrating the giant salamander skin mucus enzymatic hydrolysate obtained in S1, then adding the benzethonium chloride solution to the concentrated giant salamander glycosaminoglycan solution until the primary precipitate is completely precipitated, then dissolving the primary precipitate with a saturated sodium chloride solution, centrifuging, and obtaining a supernatant;

Add anhydrous ethanol to the supernatant for alcohol precipitation, let it stand at 4°C for 6-12 hours, and obtain secondary precipitation after centrifugation;

The secondary precipitate is dialyzed using a dialysis bag, and the dialysis time is between 48 and 72 hours to obtain giant salamander glycosaminoglycan.

In some embodiments, in S1, the amount of anhydrous ethanol used is 2 to 5 times the mass ratio of the lyophilized powder of giant salamander skin secretion mucus.

In some embodiments, in S1, the freeze-drying temperature is -75°C to -85°C.

Exemplarily, in S1, the freeze-drying temperature is -80°C.

In some embodiments, in S1, the mass percentage of alkaline protease in the alkaline protease solution is 1%-5%.

In some embodiments, in S1, the mass percentage of the mucus secreted by the skin of giant salamander and the alkaline protease solution is 90%~95%:5%~10%.

In some embodiments, in S1, enzymatic hydrolysis is specifically as follows: adding the mucus secreted by the skin of giant salamander to an alkaline protease solution, incubating with rotation for 5 to 8 hours at a temperature of 45°C to 60°C and a pH value of 8 to 10, then adding water to 1.5 to 3 times the volume of the alkaline protease solution, and continuing to incubate for 2 to 5 hours.

In some embodiments, in S1, the enzyme inactivation is specifically performed by heating the incubated solution to 80°C-100°C and boiling for 10-20 minutes.

In some embodiments, in S2, the concentration is specifically: rotary evaporating the giant salamander skin mucus enzymatic hydrolysate at a temperature of 45°C to 65°C, and the concentrated giant salamander skin mucus enzymatic hydrolysate is 30% to 50% of the giant salamander skin mucus enzymatic hydrolysate before concentration.

In some embodiments, in S2, the concentration of benzethonium chloride in the benzethonium chloride solution is 3% to 6%.

Exemplarily, in S2, the concentration of benzethonium chloride in the benzethonium chloride solution is 5%.

In some embodiments, in S2, the dialysis bag is selected from a dialysis bag with a molecular weight of 3000D~8000D.

Exemplarily, S1 specifically includes: step 1), using artificially raised healthy giant salamanders as sample sources, stimulating the skin of the giant salamanders with boiling water to collect the mucus secreted by the skin of the giant salamanders, and freeze-drying the collected mucus secreted by the skin of the giant salamanders at -80°C to obtain freeze-dried powder of the mucus secreted by the skin of the giant salamanders for later use;

Step 2), mix the lyophilized powder of the skin secretion mucus of giant salamander with anhydrous ethanol, mix thoroughly and wash 2 to 5 times, each time for about 3 to 8 hours, to perform washing and degreasing treatment;

Step 3), drying the washed and defatted giant salamander skin secretion mucus at a temperature of 40°C to 60°C for 8 to 12 hours, grinding it into powder, and obtaining the defatted giant salamander skin secretion mucus;

Step 4), adding alkaline protease solution to the defatted giant salamander skin secretion mucus for enzymatic hydrolysis, then heating to inactivate the enzyme, and cooling to obtain giant salamander skin mucus enzymatic hydrolyzate.

Exemplarily, S2 specifically includes: step 5), using rotary evaporation to concentrate the giant salamander skin mucus enzymatic hydrolyzate obtained in step 4) at a temperature of 45° C. to 65° C. to obtain a concentrated giant salamander glycosaminoglycan solution;

Step 6), slowly adding the benzethonium chloride solution to the concentrated giant salamander glycosaminoglycan solution until no primary precipitate is precipitated, centrifuging, dissolving the primary precipitate with a saturated sodium chloride solution to further precipitate free protein, centrifuging, and obtaining a supernatant;

Step 7), adding four times of anhydrous ethanol to the supernatant for alcohol precipitation, standing at 4°C for 6-12 hours, and centrifuging at 4000 r/min to obtain a secondary precipitate;

Step 8), dialyze the secondary precipitate using a dialysis bag with a molecular weight of 3000D~8000D, change the water every 4h~6h, and the dialysis time is between 48~72h to obtain a large molecular weight giant salamander glycosaminoglycan powder without impurities.

In some embodiments, a porous microsphere of glycosaminoglycan of Giant Salamander prepared by the preparation method in any of the above embodiments is also provided.

In some embodiments, there is also provided a use of porous microspheres of glycosaminoglycans from Giant Salamander prepared by the preparation method of any of the above embodiments in a drug for hemostasis of wounds.

In some embodiments, there is also provided a use of porous microspheres of glycosaminoglycans from Giant Salamander prepared by the preparation method of any of the above embodiments in a drug for reducing wound exudation.

In some embodiments, there is also provided a use of porous microspheres of glycosaminoglycans from Giant Salamander prepared by the preparation method of any of the above embodiments in a drug for promoting wound healing.

In order to make the technical problems, technical solutions and beneficial effects solved by the present application clearer, the preparation method of the porous microspheres of giant salamander glycosaminoglycan of the present invention will be further described in detail below in conjunction with specific embodiments and drawings. Obviously, the specific embodiments described are only a part of the embodiments of the present application, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present application and its application. Based on the specific embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without paying creative work belong to the scope of protection of the present application.

If no specific techniques or conditions are specified in the specific embodiments, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

Example 1

As shown in FIG1 , a method for preparing porous microspheres of glycosaminoglycans from giant salamander comprises the following steps:

Step 1), using artificially raised healthy giant salamanders as sample sources, stimulating the skin of the giant salamanders with boiling water to collect the mucus secreted by the skin of the giant salamanders, and freeze-drying the collected mucus secreted by the skin of the giant salamanders at -80°C to obtain freeze-dried powder of the mucus secreted by the skin of the giant salamanders for later use;

Step 2), mix the lyophilized powder of the skin secretion mucus of giant salamander with 3 times the volume of anhydrous ethanol, mix thoroughly and wash 2 to 5 times, each time for about 8 hours, to perform washing and degreasing treatment;

Step 3), the washed and defatted giant salamander skin secreted mucus is naturally dried and ground into powder to obtain 75g of defatted giant salamander skin secreted mucus powder;

Step 4), add 1500 mL of deionized water and 30 g of alkaline protease to 75 g of defatted giant salamander skin mucus powder, incubate for enzymatic hydrolysis at 60°C and pH 9 for 36 hours, then heat to 70°C for enzyme inactivation for 10 minutes, and cool to obtain giant salamander skin mucus enzymatic hydrolyzate.

Step 5), using rotary evaporation, the giant salamander skin mucus enzymatic hydrolyzate obtained in step 4) is concentrated to 500 mL at a temperature of 45° C. to obtain a concentrated giant salamander glycosaminoglycan solution;

Step 6), slowly adding a 5% benzethonium chloride solution to the concentrated giant salamander glycosaminoglycan solution until no more primary precipitate is precipitated, centrifuging the primary precipitate at 3000 rpm for 30 min, then dissolving the primary precipitate with 500 mL of saturated sodium chloride solution to further precipitate free protein, and then centrifuging at 3000 rpm for 30 min to obtain a supernatant;

Step 7), adding four times of anhydrous ethanol to the supernatant for alcohol precipitation, standing at 4°C for 10 hours, and centrifuging at 4000 r/min to obtain a secondary precipitate;

Step 8), dialyze the secondary precipitate for 72 hours using a dialysis bag with a molecular weight of 8000D, changing the water every 4 to 6 hours to obtain a macromolecular glycosaminoglycan powder of giant salamander without impurities, weighing 3.15 g;

Step 9), dissolving 0.05g of polymerization initiator phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium and 0.8g of methacrylated gelatin in 5mL of deionized water at a temperature of 55°C to obtain a methacrylated gelatin solution; dissolving 40mg of giant salamander glycosaminoglycan in 5mL of deionized water, then adding 1mg of N-hydroxysuccinimide (NHS) and 4mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), stirring at a temperature of 40°C for 30min to fully mix and activate the carboxyl groups in the giant salamander glycosaminoglycan to obtain an activated giant salamander glycosaminoglycan solution;

Step 10), adding the methacrylic gelatin solution in step 9) to the activated giant salamander glycosaminoglycan solution, stirring at room temperature for 12 hours, to obtain a methacrylic gelatin-giant salamander glycosaminoglycan conjugate solution;

Step 11), using the methacrylylated gelatin-giant salamander glycosaminoglycan conjugate solution obtained in step 10) as the continuous phase, and mineral oil and dispersant as the dispersed phase, and injecting the continuous phase and the dispersed phase into two parallel syringes respectively, setting the continuous phase flow rate to 10ul/min, and the dispersed phase flow rate to 30ul/min, the droplets flow through the outlet and the subsequently connected polytetrafluoroethylene tube, and are simultaneously sent into the microfluidic chip channel, and are irradiated with ultraviolet light (405 nm) at the end of the channel for curing and cross-linking. The obtained microspheres are filtered with a filter, and then washed three times with anhydrous ethanol to remove the oil and dispersant, and then the microspheres are freeze-dried to obtain giant salamander glycosaminoglycan porous microspheres.

Example 2

Hemostasis test of the porous microspheres of giant salamander glycosaminoglycan prepared in Example 1

Specifically: Rat liver hemostasis experiment of giant salamander mucus glycosaminoglycan porous microspheres: rats were anesthetized with isoflurane (1-3% isoflurane with oxygen) to perform hemostasis and closure on liver damage. The abdominal hair was removed and the rats were placed on a heating pad during surgery. The liver was exposed by laparotomy. A biopsy punch was used to inflict a 5 mm diameter and 2 mm depth injury on the liver. After waiting for natural bleeding for 5 seconds, 20 mg of giant salamander glycosaminoglycan microspheres (Gel-GAGs MPs) prepared in Example 1 were immediately sprayed on the bleeding site, and the blood loss and hemostasis time before hemostasis were recorded in each group. After the hemostasis seal was confirmed, the incision was sutured with interrupted sutures, 3-6 ml of saline was injected subcutaneously, and the liver healing was recorded by taking pictures for two weeks. Each group of samples was evaluated by 3 rats, and the natural hemostasis method was used as the blank control group, and the commercially available chitosan hemostatic powder (CHP) and methacryloyl gelatin microspheres (GelMPs) were used as the positive control group. The results are shown in Figures 2 to 4.

From the analysis of Figures 2 to 4, it can be seen that the control group (Control) had the largest amount of bleeding, reaching 737.20±53.07 mg, and the bleeding time was 218.33±23.44 s. Compared with the control group, the methacrylated gelatin microsphere group (Gel MPs) and the giant salamander glycosaminoglycan porous microsphere group (Gel-GAGs MPs) can significantly reduce the amount of bleeding and shorten the hemostasis time. The amount of liver bleeding in the two groups was reduced to 236.40±22.58 mg and 205.93±22.24) mg, respectively, and the bleeding time was 74.33±11.59 s and 71.33±8.02 s, respectively. The hemostatic effect of giant salamander glycosaminoglycan porous microspheres was not inferior to that of the commercial chitosan hemostatic powder group (Chitosan). Therefore, giant salamander glycosaminoglycan porous microspheres retain excellent hemostatic effects.

Example 3

The exudate control ability of the porous microspheres of giant salamander glycosaminoglycan prepared in Example 1

10 mg of glycosaminoglycan porous microspheres of Giant Salamander were placed in the upper chamber of the Transwell nested 12-well plate. 2 ml of deionized water was added to each well, and the upper chamber of the Transwell plate was taken out at 2h, 4h, 6h, 8h, 24h, and 48h, respectively. After wiping off the excess water outside the chamber, the changes in diameter and weight were observed under a microscope (scale bar: 200um). Methacrylated gelatin microspheres (Gel MPs) were used as a control, and the results are shown in Figures 5 to 7.

From the analysis of Figures 5 to 7, it can be seen that the giant salamander glycosaminoglycan porous microspheres (Gel-GAGs MPs) can absorb more than 15 times the body weight of PBS solution within 24 hours, showing excellent ability to control chronic wound exudate. However, the PBS absorbed by methacrylated gelatin microspheres is only about 6 times the body weight.

Example 4

Wound healing ability of the porous microspheres of giant salamander glycosaminoglycan prepared in Example 1

The chronic wound healing ability of porous microspheres of glycosaminoglycans from Giant Salamander (Gel-GAGs MPs) was evaluated in a streptozotocin (STZ)-induced type 1 diabetes C57 mouse model. C57 mice were randomly divided into 5 groups (>5 mice/group). Full-thickness circular skin wounds with a diameter of 6 mm were made on the dorsal wound surface using a disposable biopsy punch, and a rubber splint made of silicone (9 mm inner diameter and 11 mm outer diameter) was used to prevent the wound from closing naturally. The splint was fixed with tissue glue and 8 interrupted sutures centered on the wound. The blank control group (Control) was treated with normal saline, and commercial chitosan hemostatic powder (CHP) and methacryloyl gelatin microspheres (Gel MPs) were used as positive controls. The wound was covered with 3M transparent film to avoid dehydration due to water evaporation and to prevent scratching or biting of the specimen. After recovery from anesthesia, the mice were returned to the cage, and the wounds were disinfected, the drugs were changed, and the wound healing was monitored and photographed on the third, seventh, tenth, and fourteenth days. The wound size was measured blindly using Image J software. The percentage of the wound area to the initial wound size (set as 100 on day 0) was recorded and reported, and the results are shown in Figures 8 and 9.

From the analysis of Figures 8 and 9, it can be seen that compared with the Control group, Gel-GAGs microspheres significantly promoted wound healing at each test time point, and the effect was better than Gel MPs and commercially available CHP. On the 14th day, the wound healing rate of the Gel-GAGs microsphere group was 86.41%, which was higher than 27.87% in the Control group, 49.96% in the Gel MPs group, and 69.17% in the CHP group. The early hemostatic ability and the ability to absorb exudate help CHP to obtain good healing ability in the early stage. Gel-GAGs microspheres showed excellent ability to promote the healing of chronic wounds.

In summary, the giant salamander glycosaminoglycan porous microspheres prepared by the present invention have been proven by experiments to have excellent hemostatic ability, and can absorb wound exudate at least ten times its own weight. Relying on its own degradation, it provides a good drug sustained-release ability, and its conjugated loaded giant salamander glycosaminoglycan improves the therapeutic effect, simplifies the medication method, and reduces toxic side effects, so that the material has excellent ability to promote wound healing while having hemostasis and exudate control, effectively expanding the application of giant salamander derivative products, changing the development idea of only giant salamander mucus products, better exploring the medical and medicinal value of giant salamander, and has the advantage of high biosafety. It has potential application value in the field of medical application technology.

The method for preparing the porous microspheres of giant salamander glycosaminoglycans of the present invention realizes effective separation and purification of giant salamander glycosaminoglycans by sequentially subjecting giant salamander skin mucus to freeze drying, enzymolysis, enzyme inactivation, protein removal, precipitation and dialysis treatment, wherein the enzymolysis method and benzethonium chloride method are used to treat the giant salamander skin mucus, and the conditions are mild, and the main effective components in the giant salamander mucus will not be destroyed, and the biological effects of the giant salamander glycosaminoglycans can be guaranteed to the maximum extent. At the same time, the microfluidic technology used makes the prepared giant salamander glycosaminoglycan microspheres have better consistency and accuracy, so as to have excellent hemostatic ability, exudate control and wound healing promotion ability. The dialysis treatment used effectively removes invalid small molecule components, retains large molecule effect components, and improves the purity of giant salamander glycosaminoglycans, and has the advantages of simple preparation process, strong operability and suitability for large-scale mass production.

The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or change made by a person skilled in the art based on the present invention is within the protection scope of the present invention.

Claims (10)

1. The preparation method of the giant salamander glycosaminoglycan porous microsphere is characterized by comprising the following steps of:

adding an activating agent into the giant salamander glycosaminoglycan solution to activate carboxyl groups in the giant salamander glycosaminoglycan to obtain an activated giant salamander glycosaminoglycan solution;

Dissolving methacryloylated gelatin and a polymerization initiator in water to obtain a methacryloylated gelatin solution;

Adding a methacryloylated gelatin solution into the activated giant salamander glycosaminoglycan solution to obtain a methacryloylated gelatin-giant salamander glycosaminoglycan conjugated solution;

And (3) curing and crosslinking the methacryloylated gelatin-giant salamander glycosaminoglycan conjugated solution by using ultraviolet light, and freeze-drying to obtain the giant salamander glycosaminoglycan porous microspheres.

2. The method for preparing giant salamander glycosaminoglycan porous microspheres according to claim 1, characterized in that the activator is selected from N-hydroxysuccinimide and/or 1-ethyl- (3-dimethylaminopropyl) carbodiimide;

And/or the polymerization initiator is selected from phenyl-2, 4, 6-trimethyl benzoyl lithium phosphonate.

3. The method for preparing giant salamander glycosaminoglycan porous microspheres according to claim 1, characterized in that the degree of substitution of methacryloylation of the methacryloylated gelatin is 71% -90%;

and/or the mass ratio of the methacryloylated gelatin to the polymerization initiator is 50:1;

and/or the mass percentage of the methacryloylated gelatin in the methacryloylated gelatin-giant salamander glycosaminoglycan conjugated solution is 7% -10%;

And/or the mass percentage of the giant salamander glycosaminoglycan in the methacryloylated gelatin-giant salamander glycosaminoglycan conjugated solution is 0.4%;

And/or the mass percentage of the N-hydroxysuccinimide in the methacryloylated gelatin-giant salamander glycosaminoglycan conjugated solution is 0.01%;

and/or the mass percentage of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide in the methacryloylated gelatin-giant salamander glycosaminoglycan conjugated solution is 0.04%.

4. The method for preparing the giant salamander glycosaminoglycan porous microspheres according to claim 1, characterized by curing and crosslinking a methacryloylated gelatin-giant salamander glycosaminoglycan conjugated solution with light to obtain the giant salamander glycosaminoglycan porous microspheres, and specifically comprising: the method comprises the steps of taking a methacryloylated gelatin-giant salamander glycosaminoglycan conjugated solution as a continuous phase, taking mineral oil and a dispersing agent as disperse phases, sending the continuous phase into a microfluidic chip channel, and carrying out light irradiation curing crosslinking and freeze drying on the tail end of the channel to obtain the giant salamander glycosaminoglycan porous microsphere.

5. The method for preparing the giant salamander glycosaminoglycan porous microspheres according to claim 4, characterized in that the activation temperature of carboxylic acid groups in the activated giant salamander glycosaminoglycan is 20-50 ℃ and the activation time is 10-15 h;

and/or the dissolution temperature of the methacryloylated gelatin and the polymerization initiator in water is 50-60 ℃;

and/or the flow rate ratio of the continuous phase to the disperse phase is 1:3-1:5;

And/or the mineral oil is selected from paraffin oil, and the dispersing agent is selected from span 80.

6. The method for preparing the giant salamander glycosaminoglycan porous microspheres according to claim 1, characterized in that the method for preparing the giant salamander glycosaminoglycan comprises the following steps:

S1, performing enzymolysis on mucus secreted by defatted giant salamander skin, and then performing heat treatment to obtain giant salamander skin mucus enzymatic hydrolysate;

S2, separating out primary precipitate by adopting a benzethonium chloride method from skin mucus enzymolysis liquid of giant salamanders, adding absolute ethyl alcohol into supernatant after separating out the primary precipitate to obtain secondary precipitate, and dialyzing the secondary precipitate to obtain the giant salamander glycosaminoglycan.

7. The method for preparing giant salamander glycosaminoglycan porous microspheres according to claim 6, wherein the step S1 specifically comprises: the method comprises the steps of adopting a physical stimulation method to obtain skin mucus secretion of giant salamanders, and freeze-drying the skin mucus secretion of the giant salamanders to obtain freeze-dried powder of the skin mucus secretion of the giant salamanders;

Washing and degreasing freeze-dried powder of skin mucus secretion of giant salamander by absolute ethyl alcohol, and drying to obtain defatted skin mucus secretion of giant salamander;

Adding alkaline protease solution into defatted skin mucus of giant salamander for enzymolysis, and then heating for enzyme deactivation to obtain giant salamander skin mucus enzymolysis liquid;

and/or, in S2, specifically including: concentrating the giant salamander skin mucus enzymatic hydrolysate obtained in the step S1, adding a benzethonium chloride solution into the concentrated giant salamander skin mucus enzymatic hydrolysate until primary precipitation is completely separated out, dissolving the primary precipitation by using a saturated sodium chloride solution, and centrifuging to obtain a supernatant;

adding absolute ethyl alcohol into the supernatant to carry out alcohol precipitation, standing for 6-12 h at the temperature of 4 ℃, and centrifuging to obtain secondary precipitation;

and dialyzing the secondary precipitate by using a dialysis bag, wherein the dialysis time is 48-72 h, and thus the giant salamander glycosaminoglycan is obtained.

8. The preparation method of the giant salamander glycosaminoglycan porous microspheres according to claim 7, characterized in that in the step S1, the dosage of absolute ethyl alcohol is 2-5 times of the dosage of the giant salamander skin secretion mucus freeze-dried powder in terms of mass ratio;

and/or, in the step S1, the freeze-drying temperature is-75 ℃ to-85 ℃;

And/or in the step S1, the mass percentage of alkaline protease in the alkaline protease solution is 1% -5%;

and/or, in the step S1, the alkaline protease in the alkaline protease solution is Bacillus licheniformis protease;

And/or in the S1, the skin secretion mucus of the giant salamander and the alkaline protease solution account for 90-95% in percentage by mass: 5% -10%;

and/or, in the step S1, the enzymolysis is specifically as follows: adding skin mucus of giant salamander into alkaline protease solution, incubating for 5-8 hours at the temperature of 40-60 ℃ and the pH value of 8-10, adding water, and continuing incubating for 2-5 hours;

And/or, in the step S1, the enzyme deactivation is specifically as follows: heating the incubated solution to 80-100 ℃ and boiling for 10-20 min;

and/or, in the step S2, the concentration is specifically as follows: under the condition that the temperature is 45-65 ℃, the giant salamander skin mucus enzymatic hydrolysate is rotationally evaporated, and the concentrated giant salamander skin mucus enzymatic hydrolysate is 30-50% of the giant salamander skin mucus enzymatic hydrolysate before concentration;

and/or in the step S2, the concentration of benzethonium chloride in the benzethonium chloride solution is 3-6%;

and/or in the step S2, the dialysis bag is selected from the dialysis bags with molecular weights of 3000D-8000D.

9. A giant salamander glycosaminoglycan porous microsphere prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the giant salamander glycosaminoglycan porous microspheres prepared by the preparation method according to any one of claims 1-8, characterized in that the giant salamander glycosaminoglycan porous microspheres are used in a wound hemostatic drug;

and/or the giant salamander glycosaminoglycan porous microspheres are applied to medicines for reducing wound surface seepage;

and/or the giant salamander glycosaminoglycan porous microspheres are applied to medicines for promoting wound healing.