CN113679880A

CN113679880A - Gymnodinium polysaccharide hydrogel dressing as well as preparation method and application thereof

Info

Publication number: CN113679880A
Application number: CN202110983433.3A
Authority: CN
Inventors: 范代娣; 雷桓; 马晓轩
Original assignee: Northwest University
Current assignee: Northwest University
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2021-11-23

Abstract

The invention discloses a gymnema costatum polysaccharide hydrogel dressing and a preparation method and application thereof, wherein the raw materials of the hydrogel dressing comprise a solvent, gymnema costatum polysaccharide, alkali and 1, 4-butanediol diglycidyl ether, and the mass ratio of the solvent to the gymnema costatum polysaccharide to the alkali to the 1, 4-butanediol diglycidyl ether is 100: (1-20): (0.3-8): (3-10). The hydrogel dressing takes the euglena polysaccharide as a structural and functional main body, has the cross-linking agent residue of less than 0.1 percent, is sterile and non-cytotoxic, and has good cell compatibility and safety. The preparation method of the dressing comprises the steps of crosslinking a solvent, the euglena polysaccharide, alkali and 1, 4-butanediol diglycidyl ether in a water bath, dialyzing and cleaning twice and sterilizing to creatively form the hydrogel dressing taking the euglena polysaccharide as a structural and functional main body.

Description

Gymnodinium polysaccharide hydrogel dressing as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a gymnema costatum polysaccharide hydrogel dressing as well as a preparation method and application thereof.

Background

The immune inflammation stage in the wound repair process is the rate-limiting step in the wound repair process, particularly in the chronic wound repair process, the wound has inflammation and a large amount of active oxygen for a long time, so that the wound is always in the immune inflammation stage and cannot enter the cell proliferation stage, and the development of effective and safe anti-inflammatory and antioxidant materials for promoting wound healing is always a hot topic in the field of biomedical research.

In the existing dressing, generally, due to poor antioxidant and antioxidant effects, the clinical popularization and application process is difficult to follow, a hydrogel material is widely concerned by research and development personnel as a biological material with broad-spectrum compatibility and compatibility, the combination of the hydrogel material and an anti-inflammatory preparation is a common dressing design method at present, however, the influence of the introduced drug on the hydrogel structure enables the ductility, the adhesiveness and the drug effect of the hydrogel material not to meet the clinical requirements, a safer and more efficient hydrogel material and a preparation method are searched, and the hydrogel material is an important way for improving the clinical application of the hydrogel material.

Disclosure of Invention

The invention aims to solve the technical problem of providing a gymnema costatum polysaccharide hydrogel dressing and a preparation method and application thereof aiming at the defects of the prior art. The invention provides a gymnema costatum polysaccharide hydrogel dressing, the raw materials of the hydrogel dressing comprise a solvent, gymnema costatum polysaccharide, alkali and 1, 4-butanediol diglycidyl ether, the hydrogel dressing takes the gymnema costatum polysaccharide as a structure and a functional main body, the residue of a cross-linking agent is less than 0.1%, and the dressing is sterile, free of cytotoxicity, and good in cell compatibility and safety.

In order to solve the technical problems, the invention adopts the technical scheme that: the euglena polysaccharide hydrogel dressing is characterized in that raw materials comprise a solvent, euglena polysaccharide, alkali and 1, 4-butanediol diglycidyl ether, and the mass ratio of the solvent to the euglena polysaccharide to the alkali to the 1, 4-butanediol diglycidyl ether is 100: (1-20): (0.3-8): (3-10).

The euglena polysaccharide hydrogel dressing is characterized in that the mass ratio of the solvent to the euglena polysaccharide to the alkali to the 1, 4-butanediol diglycidyl ether is 100: (6-10): 3: (4-8).

The euglena polysaccharide hydrogel dressing is characterized in that the euglena polysaccharide is powdery euglena polysaccharide, the particle size of the powdery euglena polysaccharide is 625-650 meshes, and the weight average molecular weight is 190000 Da.

The euglena polysaccharide hydrogel dressing is characterized in that the solvent comprises water or a buffer solution, and the buffer solution is a phosphate buffer solution, a boric acid-potassium chloride buffer solution, a boric acid-calcium chloride buffer solution, an ammonia-ammonium chloride buffer solution, a barbital buffer solution, a tris buffer solution or a disodium hydrogen phosphate-sodium hydroxide buffer solution; the base comprises sodium hydroxide, barium hydroxide, potassium hydroxide or calcium hydroxide.

The euglena polysaccharide hydrogel dressing is characterized in that the pH value of the euglena polysaccharide hydrogel dressing is 6.8-7.2.

In addition, the invention also provides a method for preparing the gymnocyanine polysaccharide hydrogel dressing, which is characterized by comprising the following steps:

step one, adding euglena polysaccharide and alkali into a solvent to obtain a solution A;

step two, adding 1, 4-butanediol diglycidyl ether into the solution A obtained in the step one to obtain a solution B;

step three, standing the solution B obtained in the step two for 4 to 10 hours under the water bath condition of 40 to 80 ℃ to obtain a gymnocyanine polysaccharide hydrogel I;

step four, dialyzing and cleaning the euglena polysaccharide hydrogel I obtained in the step three with weak acid water for 2-5 times, and dialyzing and cleaning with neutral water for 8-15 times to obtain a euglena polysaccharide hydrogel II;

and fifthly, sterilizing the gymnema sylvestre polysaccharide hydrogel II obtained in the fourth step for 5-10 min to obtain the gymnema sylvestre polysaccharide hydrogel dressing.

The method described above, wherein the weakly acidic water in step four is a weakly acidic water having a ph of 6.0 to 6.8, and the neutral water is a neutral water having a ph of 7.0.

The method is characterized in that when the step four is dialyzed and cleaned by weak acid water, the cleaning time is 0.5-1 h; and step four, when the water is dialyzed and cleaned by neutral water, the cleaning time is 3-5 h.

The method is characterized in that the pH value of the euglena polysaccharide hydrogel II in the step four is 6.8-7.2, and the pH value is the pH value of a section obtained by cutting the middle part of the euglena polysaccharide hydrogel II.

Furthermore, the invention also provides a method for preparing the wound dressing by using the gymnocyanine polysaccharide hydrogel dressing.

Compared with the prior art, the invention has the following advantages:

1. the invention provides a gymnema costatum hydrogel dressing which is prepared from raw materials including a solvent, gymnema costatum, alkali and 1, 4-butanediol diglycidyl ether, wherein the hydrogel dressing takes the gymnema costatum polysaccharide as a structural and functional main body, the residue of a cross-linking agent is less than 0.1 percent, and the dressing is sterile, free of cytotoxicity and good in cell compatibility and safety.

2. The invention provides a method for preparing the dressing, which comprises the steps of crosslinking a solvent, euglena polysaccharide, alkali and 1, 4-butanediol diglycidyl ether in a water bath, dialyzing and cleaning twice and sterilizing to creatively form the hydrogel dressing taking the euglena polysaccharide as a structural and functional main body.

3. The preparation method comprises the steps of standing the system for crosslinking reaction for 4-10 h under the water bath condition of 40-80 ℃, and then sequentially carrying out weak acid water dialysis cleaning and neutral water dialysis cleaning, so that the residue of the crosslinking agent of the dressing is effectively reduced, and the overall performance of the dressing is improved.

4. The gymnocyanine polysaccharide hydrogel dressing prepared by the method has good mechanical property and uniform and compact pore size.

5. The invention firstly demonstrates that the euglena polysaccharide can resist inflammation and oxidation, reduce the expression of inflammatory factors, promote the expression of hypoxia inducible factors and promote the vascularization of skin wounds in the process of promoting the wound repair.

6. The method of the invention has simple operation, reliable principle and wide popularization and application prospect.

The technical solution of the present invention is further described in detail with reference to the accompanying drawings and embodiments.

Drawings

FIG. 1 is a schematic diagram of a euglena polysaccharide structure and a 1, 4-butanediol diglycidyl ether structure, as well as a cross-linked network.

FIG. 2 is a diagram showing the appearance of the Euglena polysaccharide hydrogel I obtained in the third step and the Euglena polysaccharide hydrogel II obtained in the fourth step of example 1.

FIG. 3 is a topographical view of the Euglena polysaccharide hydrogel dressing obtained in step five of example 1.

Fig. 4 is a scanning electron micrograph of the dressing after lyophilization.

Fig. 5 is a state diagram of the paramylon hydrogel dressing of example 1.

Fig. 6 is a graph showing the result of the blood compatibility test of the paramylon hydrogel dressing of example 1.

FIG. 7 shows the compatibility of the Euglena polysaccharide hydrogel dressing of example 1 after cell seeding.

FIG. 8 degradation experiments of the Euglena polysaccharide hydrogel dressing of example 1 after implantation in rats.

FIG. 9 shows the in vitro anti-inflammatory test results of the Euglena polysaccharide hydrogel dressing of example 1.

FIG. 10 shows the results of a rat ROS scavenging experiment using the Euglena polysaccharide hydrogel dressing of example 1.

Fig. 11 is a graph showing the repairing effect of the gymnema sylvestre polysaccharide hydrogel dressing of example 1 on the deep third degree scald of the rat.

FIG. 12 is a graph showing TNF- α and HIF-1 α expression during wound repair using the Euglena polysaccharide hydrogel dressing of example 1.

FIG. 13 is a graph of the results of the rheological mechanical property tests of the Euglena polysaccharide hydrogel dressings of examples 1-7.

Detailed Description

The invention provides a gymnema costatum polysaccharide hydrogel dressing, which comprises a solvent, gymnema costatum polysaccharide, alkali and 1, 4-butanediol diglycidyl ether as raw materials, wherein the buffer solution is a phosphate buffer solution, a boric acid-potassium chloride buffer solution, a boric acid-calcium chloride buffer solution, an ammonia-ammonium chloride buffer solution, a barbiturate buffer solution, a tris buffer solution or a disodium hydrogen phosphate-sodium hydroxide buffer solution; the alkali is sodium hydroxide, barium hydroxide, potassium hydroxide or calcium hydroxide. The structure of Euglena polysaccharide and 1, 4-butanediol diglycidyl ether, and the cross-linking schematic diagram are shown in FIG. 1, under alkaline conditions, 1, 4-butanediol diglycidyl ether opens the ring and reacts with the hydroxyl of Euglena polysaccharide to form Euglena polysaccharide intermolecular linkage, and cross-linked Euglena polysaccharide sugar chains are obtained to form hydrogel.

The present invention will be described in detail with reference to the following examples, which are not intended to limit the present invention.

A series of euglena polysaccharide hydrogels were prepared according to the method of the present invention, as follows.

Example 1

This example provides a paramylon hydrogel dressing, which comprises water, paramylon, sodium hydroxide and 1, 4-butanediol diglycidyl ether.

The embodiment also provides a preparation method of the gymnocyanine polysaccharide hydrogel dressing, which comprises the following steps:

step one, adding 10g of powdery euglena polysaccharide and 3g of sodium hydroxide into 100g of water, and stirring until the solution is semitransparent to obtain a solution A; the weight average molecular weight of the euglena polysaccharide is 190000Da, and the purity of the euglena polysaccharide is more than or equal to 96%; the particle size of the powdery euglena polysaccharide is 625-650 meshes;

step two, adding 6g of 1, 4-butanediol diglycidyl ether into the solution A obtained in the step one, and stirring until the mixture is uniformly mixed to obtain a solution B;

step three, standing the solution B obtained in the step two for 6 hours under the water bath condition of 50-60 ℃ to obtain a gymnocyanine polysaccharide hydrogel I; the pH value of the euglena polysaccharide hydrogel I is more than 7.5;

placing a dialysis cylinder with the volume of 5L on a magnetic stirrer, wrapping the gymnema sylvestre polysaccharide hydrogel I obtained in the step three with gauze, fixing the gymnema sylvestre polysaccharide hydrogel I in the dialysis cylinder, adding water until the gauze bag filled with the gymnema sylvestre polysaccharide hydrogel is immersed, starting magnetic stirring, driving the water to rotate by rotating a magneton to dialyze and clean the gymnema sylvestre polysaccharide hydrogel, wherein the dialysis and cleaning times are 12 times, and the water comprises weakly acidic water and neutral water; the dialysis cleaning process specifically comprises: dialyzing and cleaning with weak acid water for 2 times, wherein the dialysis and cleaning time is 1 hour each time, dialyzing and cleaning with neutral water for 10 times, and the dialysis and cleaning time is 5 hours each time to obtain euglena polysaccharide hydrogel II, cutting the euglena polysaccharide hydrogel II from the middle part, and measuring the pH of a section to be 6.8-7.2; the weak acidic water is prepared by adding hydrochloric acid into deionized water until the pH value is 6.2-6.4, and the neutral water is prepared by adding sodium hydroxide into deionized water until the pH value is 7.0; changing water after each dialysis cleaning in the dialysis cleaning process;

and step five, placing the euglena polysaccharide hydrogel II in the step four into an autoclave, and sterilizing for 5min at the temperature of 121 ℃ to obtain the euglena polysaccharide hydrogel dressing.

Example 2

step one, adding 6g of powdery euglena polysaccharide and 3g of sodium hydroxide into 100g of water, and stirring until the solution is semitransparent to obtain a solution A; the weight average molecular weight of the euglena polysaccharide is 190000Da, and the purity of the euglena polysaccharide is more than or equal to 96%; the particle size of the powdery euglena polysaccharide is 625-650 meshes;

The structure of the gymnema sylvestre polysaccharide hydrogel dressing of the embodiment is basically consistent with that of the embodiment 1.

Example 3

step one, adding 8g of powdery euglena polysaccharide and 3g of sodium hydroxide into 100g of water, and stirring until the solution is semitransparent to obtain a solution A; the weight average molecular weight of the euglena polysaccharide is 190000Da, and the purity of the euglena polysaccharide is more than or equal to 96%; the particle size of the powdery euglena polysaccharide is 625-650 meshes;

Example 4

step three, standing the solution B obtained in the step two for 4 hours under the water bath condition of 50-60 ℃ to obtain a gymnocyanine polysaccharide hydrogel I; the pH value of the euglena polysaccharide hydrogel I is more than 7.5;

Example 5

step three, standing the solution B obtained in the step two for 8 hours under the water bath condition of 50-60 ℃ to obtain a gymnocyanine polysaccharide hydrogel I; the pH value of the euglena polysaccharide hydrogel I is more than 7.5;

Example 6

step two, adding 4g of 1, 4-butanediol diglycidyl ether into the solution A obtained in the step one, and stirring until the mixture is uniformly mixed to obtain a solution B;

Example 7

step two, adding 8g of 1, 4-butanediol diglycidyl ether into the solution A obtained in the step one, and stirring until the mixture is uniformly mixed to obtain a solution B;

Example 8

This example provides a gymnocyanine hydrogel dressing, which comprises the raw materials of normal saline, gymnocyanine, barium hydroxide and 1, 4-butanediol diglycidyl ether.

step one, adding 8g of powdery euglena polysaccharide and 3g of barium hydroxide into 100g of physiological saline, and stirring until the solution is semitransparent to obtain a solution A; the weight average molecular weight of the euglena polysaccharide is 190000Da, and the purity of the euglena polysaccharide is more than or equal to 96%; the particle size of the powdery euglena polysaccharide is 625-650 meshes;

Example 9

This example provides a gymnema costatum polysaccharide hydrogel dressing, the raw materials include buffer solution, gymnema costatum, potassium hydroxide and 1, 4-butanediol diglycidyl ether.

step one, adding 10g of powdery euglena polysaccharide and 3g of potassium hydroxide into 100g of buffer solution, and stirring until the solution is semitransparent to obtain a solution A; the weight average molecular weight of the euglena polysaccharide is 190000Da, and the purity of the euglena polysaccharide is more than or equal to 96%; the particle size of the powdery euglena polysaccharide is 625-650 meshes; as a feasible embodiment, the buffer solution is phosphate buffer solution with pH value of 7.0-8.0, and the preparation method of the phosphate buffer solution is a preparation method commonly used in the field, for example, the preparation method of the phosphate buffer solution with pH value of 7.0 is as follows: mixing 0.68g of monopotassium phosphate with 29.1mL of 0.1mol/L sodium hydroxide solution, and diluting the mixture to 100mL by using deionized water;

Example 10

The embodiment provides a gymnema algae polysaccharide hydrogel dressing, and the raw materials comprise buffer solution, gymnema algae polysaccharide, barium hydroxide and 1, 4-butanediol diglycidyl ether.

step one, adding 6g of powdery euglena polysaccharide and 3g of barium hydroxide into 100g of buffer solution, and stirring until the solution is semitransparent to obtain a solution A; the weight average molecular weight of the euglena polysaccharide is 190000Da, and the purity of the euglena polysaccharide is more than or equal to 96%; the particle size of the powdery euglena polysaccharide is 625-650 meshes; as a possible embodiment, the buffer solution is ammonia-ammonium chloride buffer solution with pH of 7.4-11.2, and the preparation method of the ammonia-ammonium chloride buffer solution is a preparation method commonly used in the art, for example, the preparation method of the ammonia-ammonium chloride buffer solution with pH of 8.0 can be: taking 1.07g of ammonium chloride, adding deionized water to make the solution into 100mL, and adding a dilute ammonia solution to adjust the pH to 8.0 to obtain the ammonium chloride-based water treatment agent;

Example 11

step one, adding 6g of powdery euglena polysaccharide and 3g of barium hydroxide into 100g of buffer solution, and stirring until the solution is semitransparent to obtain a solution A; the weight average molecular weight of the euglena polysaccharide is 190000Da, and the purity of the euglena polysaccharide is more than or equal to 96%; the particle size of the powdery euglena polysaccharide is 625-650 meshes; as a feasible embodiment, the buffer solution is a barbital buffer solution with pH of 7.4-7.8, the preparation method of the barbital buffer solution is a preparation method commonly used in the field, for example, the preparation method of the barbital buffer solution with pH of 7.4 can be as follows: taking 4.42g of barbital sodium, adding water to dissolve and dilute the barbital sodium to 400mL, and adjusting the pH value to 7.4 by using 2mol/L hydrochloric acid solution to obtain the compound;

Example 12

step one, adding 6g of powdery euglena polysaccharide and 3g of barium hydroxide into 100g of buffer solution, and stirring until the solution is semitransparent to obtain a solution A; the weight average molecular weight of the euglena polysaccharide is 190000Da, and the purity of the euglena polysaccharide is more than or equal to 96%; the particle size of the powdery euglena polysaccharide is 625-650 meshes; as a feasible embodiment, the buffer solution is a tris buffer solution with the pH value of 8-9, and the preparation method of the tris buffer solution is a preparation method commonly used in the field, for example, the tris buffer solution with the pH value of 8 can be prepared by the following steps: taking 12.14g of tris (hydroxymethyl) aminomethane, adding 800mL of deionized water, dissolving and diluting to 1000mL, and adjusting the pH value to 8.0 by using 6mol/L hydrochloric acid solution to obtain the tris (hydroxymethyl) aminomethane aqueous solution; in this embodiment, the buffer solution may also be a disodium hydrogen phosphate-sodium hydroxide buffer solution, a boric acid-potassium chloride buffer solution, or a boric acid-calcium chloride buffer solution;

Example 13

step one, adding 1g of powdery euglena polysaccharide and 0.3g of sodium hydroxide into 100g of water, and stirring until the solution is semitransparent to obtain a solution A; the weight average molecular weight of the euglena polysaccharide is 190000Da, and the purity of the euglena polysaccharide is more than or equal to 96%; the particle size of the powdery euglena polysaccharide is 625-650 meshes;

step two, adding 3g of 1, 4-butanediol diglycidyl ether into the solution A obtained in the step one, and stirring until the mixture is uniformly mixed to obtain a solution B;

step three, standing the solution B obtained in the step two for 6 hours under the water bath condition of 70-80 ℃ to obtain a gymnocyanine polysaccharide hydrogel I; the pH value of the euglena polysaccharide hydrogel I is more than 7.5;

placing a dialysis cylinder with the volume of 5L on a magnetic stirrer, wrapping the gymnema sylvestre polysaccharide hydrogel I obtained in the step three with gauze, fixing the gymnema sylvestre polysaccharide hydrogel I in the dialysis cylinder, adding water until the gauze bag filled with the gymnema sylvestre polysaccharide hydrogel is immersed, starting magnetic stirring, driving the water to rotate by rotating a magneton to dialyze and clean the gymnema sylvestre polysaccharide hydrogel, wherein the dialysis and cleaning times are 13 times, and the water comprises weakly acidic water and neutral water; the dialysis cleaning process specifically comprises: dialyzing and cleaning with weak acid water for 5 times, wherein the time for each dialysis and cleaning is 0.5h, dialyzing and cleaning with neutral water for 8 times, and the time for each dialysis and cleaning is 3h to obtain a euglena polysaccharide hydrogel II, cutting the euglena polysaccharide hydrogel II from the middle part, and measuring the pH of a section to be 6.8-7.2; the weak acidic water is prepared by adding hydrochloric acid into deionized water until the pH value is 6.0-6.2, and the neutral water is prepared by adding sodium hydroxide into deionized water until the pH value is 7.0; changing water after each dialysis cleaning in the dialysis cleaning process;

and step five, placing the euglena polysaccharide hydrogel II in the step four into an autoclave, and sterilizing for 8min at the temperature of 121 ℃ to obtain the euglena polysaccharide hydrogel dressing.

Example 14

step one, adding 20g of powdery euglena polysaccharide and 8g of sodium hydroxide into 100g of water, and stirring until the solution is semitransparent to obtain a solution A; the weight average molecular weight of the euglena polysaccharide is 190000Da, and the purity of the euglena polysaccharide is more than or equal to 96%; the particle size of the powdery euglena polysaccharide is 625-650 meshes;

step two, adding 10g of 1, 4-butanediol diglycidyl ether into the solution A obtained in the step one, and stirring until the mixture is uniformly mixed to obtain a solution B;

step three, standing the solution B obtained in the step two for 10 hours under the water bath condition of 40-50 ℃ to obtain a euglena polysaccharide hydrogel I; the pH value of the euglena polysaccharide hydrogel I is more than 7.5;

placing a dialysis cylinder with the volume of 5L on a magnetic stirrer, wrapping the gymnema sylvestre polysaccharide hydrogel I obtained in the step three with gauze, fixing the gymnema sylvestre polysaccharide hydrogel I in the dialysis cylinder, adding water until the gauze bag filled with the gymnema sylvestre polysaccharide hydrogel is immersed, starting magnetic stirring, driving the water to rotate by rotating a magneton to dialyze and clean the gymnema sylvestre polysaccharide hydrogel, wherein the dialysis and cleaning times are 18 times, and the water comprises weakly acidic water and neutral water; the dialysis cleaning process specifically comprises: dialyzing and cleaning with weak acid water for 3 times, wherein the dialysis and cleaning time is 0.5h each time, dialyzing and cleaning with neutral water for 15 times, and the dialysis and cleaning time is 4h each time to obtain a euglena polysaccharide hydrogel II, cutting the euglena polysaccharide hydrogel II from the middle part, and measuring the pH of a section to be 6.8-7.2; the weak acidic water is prepared by adding hydrochloric acid into deionized water until the pH value is 6.6-6.8, and the neutral water is prepared by adding sodium hydroxide into deionized water until the pH value is 7.0; changing water after each dialysis cleaning in the dialysis cleaning process;

and step five, placing the euglena polysaccharide hydrogel II in the step four into an autoclave, and sterilizing for 10min at the temperature of 121 ℃ to obtain the euglena polysaccharide hydrogel dressing.

Performance evaluation:

table 1 shows the performance data of the gymnocyanine hydrogel i obtained in step three, the gymnocyanine hydrogel ii obtained in step four, and the gymnocyanine hydrogel dressing obtained in step five in example 1, and it can be seen from table 1 that the gymnocyanine hydrogel i has a higher content of 1, 4-butanediol diglycidyl ether (BDDE), exhibits toxicity, has a cell survival rate of only 0.26 ± 0.032%, exhibits a higher cell killing rate, and is not suitable for wound repair, without being subjected to pH adjustment and twice cleaning; the euglena polysaccharide hydrogel II has relatively low content of 1, 4-butanediol diglycidyl ether, but has the cell survival rate of 33.21 +/-8.32 percent and shows cell contamination; the gymnocyanine polysaccharide hydrogel dressing prepared by the method has the advantages of obviously lower 1, 4-butanediol diglycidyl ether content and cell survival rate of 110.12 +/-10.32%, and shows that the gymnocyanine polysaccharide hydrogel dressing can obviously promote cell proliferation.

TABLE 1 Performance data of different hydrogel materials

	pH	BDDE content	Cell survival rate	Cytotoxicity
					Step three	Greater than 14	5.2-5.8％	0.26±0.032％	Grade	5
Step four	6.8-7.2	0.5-1％	33.21±8.32％	Grade 3, cell contamination
					Step five	6.8-7.2	Less than 0.1 percent	110.12±10.32％	Level	0

FIG. 2 is a schematic diagram of a paramylon hydrogel I obtained in the third step and a schematic diagram of a paramylon hydrogel II obtained in the fourth step in example 1, and FIG. 3 is a schematic diagram of a paramylon hydrogel dressing obtained in the fifth step in example 1. As can be seen from FIG. 2, after two times of washing, the hydrogel material absorbs water and swells, the volume of the hydrogel material is increased, and the color of the hydrogel material is changed from yellow transparent to white transparent. As can be seen from fig. 3, the dressing of the present invention is in a transparent state.

The euglena polysaccharide hydrogel dressing obtained in the embodiment 1 is subjected to freeze drying to obtain a freeze-dried dressing, the microstructure of the freeze-dried dressing is shown in fig. 4, and a scanning electron microscope result shows that the freeze-dried dressing is of a porous structure, the pore size is distributed between 200 and 50 micrometers, and the pore structure is uniform.

Fig. 5 is a state diagram of the paramylon hydrogel dressing of example 1, and it can be seen from fig. 5 that the paramylon hydrogel dressing of the present invention has a soft texture and can be attached to the skin surface.

Fig. 6 is a graph showing the result of the blood compatibility test of the paramylon hydrogel dressing of example 1. The blood compatibility test was performed according to the in vitro hemolysis assay test method, comprising: adding the hydrogel dressing into normal saline, leaching for 48 +/-2 hours in a biochemical incubator at 37 ℃ to obtain hydrogel leaching liquor, and mixing the normal saline and anticoagulation blood according to a mass ratio of 1: 1, mixing, placing in three 2mL centrifuge tubes, adding 0.5mL of the hydrogel leaching liquor, 0.5mL of a negative control group (the negative control group is normal saline equal to the hydrogel leaching liquor) and 0.5mL of a positive control group (the positive control group is deionized water equal to the hydrogel leaching liquor), adding 0.3 mL of fresh diluted blood into each centrifuge tube, heating in a water bath at 37 ℃ for 1 hour, centrifuging at 1500rpm for 5 minutes, absorbing the supernatant, and measuring absorbance at 540 nm. The absorbance of the sample was calculated As the hemolysis ratio (%). 100% × ((As-An))/((Ap-An)) As, where Ap is the absorbance of the positive control and An is the absorbance of the negative control. As can be seen from FIG. 6, the sample containing the hydrogel leaching liquor is still positioned at the bottom of the centrifuge tube after being centrifuged, which indicates that no hemoglobin is precipitated and the blood cells are not ruptured, and indicates that the gymnocyanine hydrogel dressing of the present invention does not cause hemolysis of blood cells and has good blood compatibility.

The compatibility of the gymnocyanine polysaccharide hydrogel dressing of embodiment 1 of the present invention after being planted on cells is shown in fig. 7, and the experimental process includes: placing the Gymnodinium polysaccharide hydrogel dressing in 48-hole pore plate for cell culture, soaking in culture solution for 6 hr, removing culture solution by suction, and mixing at a ratio of 1 × 10⁵And (2) inoculating the L929 cell suspension to the material of each hole, culturing for 1 day, sampling, fixing with 2.5% glutaraldehyde overnight, performing gradient dehydration on the hydrogel sample which is fixed overnight by using alcohol, wherein the gradient concentration is 30%, 50%, 70%, 90%, 95% and 100%, the time for each gradient dehydration is 15min, then placing the hydrogel sample in an oven for drying for 5min, spraying gold, observing under a scanning electron microscope, wherein the cell adhesion growth condition is shown in figure 7, and according to figure 7, cells are adhered to the surface of the hydrogel dressing, which shows that the hydrogel dressing has good cell compatibility.

FIG. 8 degradation experiments of the Euglena polysaccharide hydrogel dressing of example 1 after implantation in rats. The results show that the hydrogel dressing of the invention can be completely degraded after 9 weeks of subcutaneous implantation in rats.

FIG. 9 shows the in vitro anti-inflammatory test results of the Euglena polysaccharide hydrogel dressing of example 1. The test procedure for the anti-inflammatory assay included: RAW264.7 cells were seeded in a six-well plate, the supernatant was removed after 24 hours, and DMEM medium (negative control, indicated as control group), LPS (lipopolysaccharide solution, concentration 10ng/mL, positive control, indicated as LPS group) and paramylon (paramylon hydrogel dressing solution, concentration 50mg/mL, indicated as paramylon group) and LPS and paramylon hydrogel extracts (concentration the same as LPS group and paramylon group, respectively, indicated as LPS + paramylon group) were added to the six-well plate, followed by culturing for 24 hours, cell climbing was washed with PBS, immunofluorescent staining was performed after fixation with 4% paraformaldehyde, then the expression amount of TNF-. alpha.was observed under a laser confocal microscope, and the fluorescence intensity of TNF-. alpha.was analyzed with image J software. And analyzing data by taking the control group as a relative expression amount. The results show that the gymnocyanine hydrogel dressing has significantly reduced inflammatory factor TNF-alpha relative to lipopolysaccharide.

FIG. 10 is the results of ROS scavenging experiments in rats with the Euglena polysaccharide hydrogel dressing of example 1. The testing procedure for the rat in vivo ROS scavenging assay included: the method comprises the steps of unhairing the back of a rat (250g), drawing a triangle on the back, taking different rats, injecting 100 mu L of LPS (lipopolysaccharide solution, 10mg/mL), paramylon (euglena polysaccharide hydrogel dressing) and LPS + paramylon at the top of the triangle respectively, injecting 50 mu L of LPS after 2h, then injecting 50 mu L of paramylon, killing the rat after 24 h and 72 h respectively, taking the skin of an injection part for ROS staining (DHE fluorescent staining), observing the ROS expression amount under a laser confocal microscope, analyzing the ROS fluorescence intensity by using image J software, taking normal skin tissue as a control group, and analyzing data by taking the ROS amount in the control group as relative expression amount. The experiments were performed in compliance with the legal and institutional guidelines regarding animal ethics, with approval from the animal ethics committee of northwest university. The result shows that the ROS content in the sample group injected with the hydrogel dressing is obviously reduced, and the hydrogel dressing has good ROS removing and oxidation resisting effects.

Fig. 11 is a graph showing the repairing effect of the hydrogel dressing of example 1 on deep three-degree scald of a rat. On day 20, the wounds of rats using the hydrogel dressing group of the present invention were completely closed.

FIG. 12 is a graph showing TNF- α and HIF-1 α expression during wound repair in the hydrogel dressing of example 1, as measured by full thickness skin defect repair in rats. The determination method specifically comprises the following steps: a full-thickness skin wound (8 mm in diameter) was cut from the back of each rat by punch biopsy, and the test group was fixed by attaching a hydrogel dressing to the skin wound and then covering each wound with gauze and a polyurethane film, and the control group was not treated after the wound was made and covered with gauze and a polyurethane film only. The inner gauze was replaced daily for both groups to ensure the water retention of the hydrogel and the moist environment of the wound. Mice were sacrificed and tissues were removed. Determining the expression of tumor necrosis factor TNF-alpha and hypoxia inducible factor (HIF-alpha) by immunofluorescence staining, comprising fixing and freezing tissue, staining the tissue section, incubating the tissue section and TNF-alpha (TNF-alpha, Abcam, ab1793) for 2 hours at room temperature, staining cell nuclei by using a mounting solution containing DAPI, observing a slide under a confocal microscope, and analyzing fluorescence intensity by using image J software to determine the expression of TNF-alpha; anti-HIF- α was determined using anti-HIF- α antibodies (Bioss, bs-1407R). Wherein TNF-alpha represents the inflammation condition in the wound repair process, HIF-1 alpha is hypoxia inducible factor, HIF-1 target gene is closed under the normal oxygen condition, HIF accumulates and expresses under the hypoxia condition, can promote erythropoiesis, angiogenesis, bring more oxygen, change organism hypoxia condition. Experimental results show that the hydrogel dressing of example 1 has low TNF-alpha and high HIF-1 alpha in the process of promoting wound repair, and the hydrogel dressing has the functions of reducing inflammation, resisting oxidation and promoting wound repair.

FIG. 13 is a graph showing the results of the rheological mechanical property test of the hydrogel dressings of examples 1 to 7. As can be seen from comparative examples 1 to 3, as the addition amount of the euglena polysaccharide in the dressing raw material increases, the storage modulus of the hydrogel dressing also called elastic modulus, which means the amount of energy stored due to elastic (reversible) deformation when the material deforms, reflects the elasticity of the material, and indicates that as the addition amount of the euglena polysaccharide in the raw material increases, the elasticity of the hydrogel dressing product increases accordingly. The comparative examples 3 to 5 show that the crosslinking time is within the range of 4h to 8h, the dressing performance is not greatly influenced, and the comparative examples 3 and 6 to 7 show that the hydrogel dressing storage modulus is increased along with the increase of the content of the 1, 4-butanediol diglycidyl ether.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, changes and equivalent structural changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. The euglena polysaccharide hydrogel dressing is characterized in that raw materials comprise a solvent, euglena polysaccharide, alkali and 1, 4-butanediol diglycidyl ether, and the mass ratio of the solvent to the euglena polysaccharide to the alkali to the 1, 4-butanediol diglycidyl ether is 100: (1-20): (0.3-8): (3-10).

2. The gymnema sylvestre polysaccharide hydrogel dressing as claimed in claim 1, wherein the mass ratio of the solvent, the gymnema sylvestre polysaccharide, the alkali and the 1, 4-butanediol diglycidyl ether is 100: (6-10): 3: (4-8).

3. The gymnema sylvestre hydrogel dressing of claim 1, wherein the gymnema sylvestre polysaccharide is powdery gymnema sylvestre polysaccharide, the particle size of the powdery gymnema sylvestre polysaccharide is 625-650 meshes, and the weight average molecular weight is 190000 Da.

4. The paramylon hydrogel dressing according to claim 1, wherein the solvent comprises water or a buffer solution, and the buffer solution is a phosphate buffer solution, a boric acid-potassium chloride buffer solution, a boric acid-calcium chloride buffer solution, an ammonia-ammonium chloride buffer solution, a barbiturate buffer solution, a tris buffer solution, or a disodium hydrogen phosphate-sodium hydroxide buffer solution; the base comprises sodium hydroxide, barium hydroxide, potassium hydroxide or calcium hydroxide.

5. The paramylon hydrogel dressing according to claim 1, wherein the paramylon hydrogel dressing has a pH of 6.8 to 7.2.

6. A method of making the paramylon hydrogel dressing of claim 1, comprising:

7. The method as claimed in claim 6, wherein the weakly acidic water in the fourth step is a weakly acidic water having a pH of 6.0 to 6.8, and the neutral water is a neutral water having a pH of 7.0.

8. The method as claimed in claim 6, wherein, when the step four is performed by dialysis cleaning with weak acid water, the cleaning time is 0.5 h-1 h; and step four, when the water is dialyzed and cleaned by neutral water, the cleaning time is 3-5 h.

9. The method as claimed in claim 6, wherein the pH of the Gymnodinium cells hydrogel II in step four is 6.8-7.2, and the pH is the pH of a section obtained by cutting the middle of Gymnodinium cells hydrogel II.

10. A method of making a wound dressing using the paramylon hydrogel dressing of claim 1.