CN118063636B

CN118063636B - Extracellular polysaccharide Riclin-based antibacterial hydrogel as well as preparation method and application thereof

Info

Publication number: CN118063636B
Application number: CN202410154562.5A
Authority: CN
Inventors: 孙琪; 武新怡; 苏雅文; 夏瑜蔓; 刘旭勤; 张锦润
Original assignee: BENGBU MEDICAL COLLEGE
Current assignee: BENGBU MEDICAL COLLEGE
Priority date: 2024-02-04
Filing date: 2024-02-04
Publication date: 2024-08-27
Anticipated expiration: 2044-02-04
Also published as: CN118063636A

Abstract

The invention discloses an extracellular polysaccharide Riclin-based antibacterial hydrogel and a preparation method thereof, wherein the method comprises the following steps: reacting aldehyde modified extracellular polysaccharide Riclin with an amino compound in a solution to obtain the product; the amino compound is at least one selected from dioxyamine, aromatic amine and hydrazide. The extracellular polysaccharide Riclin-based antibacterial hydrogel prepared by the invention has good mechanical property, antibacterial property and hemostatic property, and can be used as dressing to remarkably promote wound healing of diabetics.

Description

Extracellular polysaccharide Riclin-based antibacterial hydrogel as well as preparation method and application thereof

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to extracellular polysaccharide Riclin-based antibacterial hydrogel as well as a preparation method and application thereof.

Background

Diabetic wounds are serious complications of diabetes mellitus, and this chronic inflammatory state is mainly due to the conditions of granulocytosis, impaired chemotaxis and macrophage function, and reduced growth factor secretion and neovascular disorders, resulting in inhibited wound healing. In extreme cases, diabetic wound ulcers can even lead to amputation. Bacterial infection is still the most common complication in wound healing, often causing exudate formation, impeding wound repair. An ideal wound dressing should not only act as a physical barrier, covering an open wound, preventing contamination, but also should accelerate the healing process without causing secondary injury. Therefore, the hydrogel dressing with antibacterial and hemostatic capabilities has great application prospect in clinical practice of diabetic wound healing.

Current clinical practice guidelines suggest extensive debridement, initially and periodically, to remove sloughed tissue and surrounding callus material, and use a wound dressing, such as wet gauze, to maintain a moist wound environment, absorb excess exudates, and protect the surrounding skin from maceration. Polysaccharide-based hydrogels can provide suitable moisture to wounds and act as a barrier against bacteria, with natural polysaccharide-based hydrogels having been widely used in wound dressings due to their good water retention, degradability, and controlled release of therapeutic drugs. The ideal natural polysaccharide hydrogel should have good antibacterial properties without entrapping other biological products. Hydrogels with antibacterial action at present always require complicated interventions and high cost treatments such as the addition of silver ions etc., whereas most of the hydrogels developed are used only as carriers for packaging various biological products, not only poorly resistant but also at risk of immunogenicity. Although the common polysaccharide such as chitosan in the market has strong biological activity, the polysaccharide has the problems of poor biocompatibility, slow degradation and the like; while hyaluronic acid has good biocompatibility, it is expensive and difficult to remove. Therefore, a natural polysaccharide hydrogel which has excellent biological activity to resist chronic inflammation and bacterial infection and excellent material characteristics to meet the special requirements of bleeding after debridement of diabetes wounds and the like and can be produced in batches with low cost is found to interfere with the chronic wound healing of diabetes.

Previous studies reported extracellular polysaccharide Riclin and its preparation method, and pointed out that its excellent anti-inflammatory activity (Y.Yang,X.Sun,Y.Zhao,et al.,Anti tumAR activity and immunogenicity of asuccinoglycanriclin,Carbohydr.Polym.2021Mar 1;255:117370.;R.Cheng,L.Wang,J.Li.,et al.,In vitro and in vivo antiinflammatARy activity of asuccinoglycan Riclin from Agrobacterium sp.ZCC3656,J.Appl.Microbiol.2019Dec;127(6):1716-1726.). patent CN111286466a also discloses preparation method and anti-inflammatory activity of extracellular polysaccharide Riclin, but no report has been made about extracellular polysaccharide Riclin used for wound dressing for diabetes, on the one hand, because extracellular polysaccharide Riclin is difficult to form stable hydrogel structure; on the other hand, extracellular polysaccharide Riclin does not have antibacterial ability against common bacteria for wound infection.

Disclosure of Invention

Based on the technical problems, the extracellular polysaccharide Riclin is modified, so that the extracellular polysaccharide Riclin-based antibacterial hydrogel has a cross-linked interpenetrating three-dimensional network structure, good mechanical property, antibacterial property and hemostatic property, and can be used as a dressing to remarkably promote wound healing of diabetics.

The specific scheme of the invention is as follows:

the invention aims at providing a preparation method of extracellular polysaccharide Riclin-based antibacterial hydrogel, which is obtained by reacting aldehyde modified extracellular polysaccharide Riclin with amino compounds in a solution; the amino compound is at least one selected from dioxyamine, aromatic amine and hydrazide.

The aldehyde modified extracellular polysaccharide Riclin reacts with amino compounds, and the aldehyde modified extracellular polysaccharide Riclin is oxidized into aldehyde due to partial hydroxyl groups, can be crosslinked with residual hydroxyl groups in extracellular polysaccharide Riclin to form acetal, and further reacts with amino compounds in a crosslinking way to obtain the crosslinked interpenetrating three-dimensional network structure hydrogel. The extracellular polysaccharide Riclin-based hydrogel has excellent antibacterial, hemostatic and mechanical properties, can be used as wound dressing for diabetics, and meets the performance requirements of the extracellular polysaccharide Riclin-based hydrogel on bacteriostasis, hemostasis and promotion of wound healing.

Preferably, the amino compound is added in an amount of 1:2 to 2:1 in terms of the molar ratio of amino groups contained therein to aldehyde groups of the hydroformylation-modified extracellular polysaccharide Riclin.

Preferably, the concentration of the aldehyde-modified extracellular polysaccharide Riclin in the reaction solution is 0.1 to 6.0%.

Preferably, the dioxyamine compound is selected from O, O '- (ethane-1, 2-diyl) bis (hydroxylamine) dihydrochloride or O, O' -1, 3-propanediyl bis hydroxylamine dihydrochloride.

In the invention, the CAS number of the O, O' - (ethane-1, 2-diyl) bis (hydroxylamine) dihydrochloride is 104845-83-2, the molecular formula is C ₂H₁₀Cl₂N₂O₂, and the molecular weight is 165.02, which is called EBH for short.

Preferably, the aromatic amine compound is a sulfonamide.

Preferably, the hydrazide compound is at least one of adipic acid dihydrazide and acethydrazide.

Preferably, the extracellular polysaccharide Riclin is dissolved in water, and an oxidant is added to perform oxidation reaction to obtain the aldehyde modified extracellular polysaccharide Riclin; more preferably, the molar ratio of the oxidizing agent to extracellular polysaccharide Riclin is 0.25-6:1; particularly preferably, the oxidizing agent is selected from at least one of periodate, hypochlorite, hydrogen peroxide.

Preferably, the oxidation reaction is carried out under stirring at a pH of 7-9, in the absence of light and at a temperature of 15-30 ℃ for 2-12 hours.

Preferably, the reaction is terminated by adding a reducing agent after the oxidation reaction; more preferably, the reducing agent is ethylene glycol or glycerol.

Preferably, after the oxidation reaction is terminated, dialysis, freeze-drying and then dissolution by heating are performed.

Preferably, the dialysis is carried out for 0.5-2 days by using a dialysis bag with a molecular weight cut-off of 500-14000Da, and water is changed every 3-8 hours during dialysis.

Preferably, the freeze-drying comprises cooling to- (60-80) deg.C, then reducing the pressure to 0-0.1kPa, and freeze-drying at the temperature and pressure for 24-48 hours; more preferably, the cooling rate is 10-15 ℃/min, and the depressurization rate is 150-200kPa/min.

Preferably, the temperature of the heated dissolution is 40-80 ℃; preferably, the heating dissolution temperature is 60 ℃.

The second object of the invention is to provide an extracellular polysaccharide Riclin-based antibacterial hydrogel, which is prepared by adopting any one of the above methods.

The invention also aims at providing the application of the polysaccharide-based hydrogel prepared by any one of the methods in preparing wound dressing for diabetics.

Preferably, the diabetes is type two diabetes.

The extracellular polysaccharide Riclin is a natural extracellular polysaccharide extracted from agrobacterium ZCC 3656. The molecular chain contains a large number of hydroxyl groups, and has good biocompatibility and biodegradability. Riclin has been found to have biological functional activities such as tumor growth inhibition, antioxidation, hypercholesterolemia improvement, etc.

The structure of the extracellular polysaccharide Riclin is shown as a formula (I), wherein R is-OCCH ₂CH₂ COOH or H, and n is an integer greater than 0.

The extracellular polysaccharide Riclin has a molecular weight range of 2.0X10 ⁶-3.0×10⁶ Da.

The extraction method of extracellular polysaccharide Riclin according to the present invention is not particularly limited, and includes, but is not limited to, the following methods: inoculating agrobacterium tumefaciens ZCC3656 into a seed culture medium for culture to obtain a seed culture solution, and inoculating the seed culture solution into a fermentation culture medium for culture to obtain a fermentation solution containing extracellular polysaccharide Riclin; adding a precipitant into the fermentation liquor containing the extracellular polysaccharide Riclin for precipitation, and filtering or centrifuging to obtain a crude product of the extracellular polysaccharide Riclin; purifying the crude extracellular polysaccharide Riclin by an alkali-alcohol method to obtain extracellular polysaccharide Riclin.

The invention has the beneficial effects that:

the invention provides a preparation method of extracellular polysaccharide Riclin-based antibacterial hydrogel, which is simple, is suitable for rapid industrialization, adopts microbial polysaccharide as a raw material, can be prepared by fermentation, has high yield and low cost, almost has no pollution in the generation process, and is suitable for future technological development.

The extracellular polysaccharide Riclin-based antibacterial hydrogel disclosed by the invention has the characteristics of good anti-inflammatory effect, strong antibacterial capability, remarkable wound healing promoting effect, good hemostatic effect, high biocompatibility, low cost and the like, and can be applied to preparing wound hemostatic dressings for diabetics. The method comprises the following steps:

(1) Effectively heal wounds: the traditional Chinese medicine composition has remarkable effect in the healing process of the wound of the type II diabetes rat, and is suitable for safe and effective treatment of clinical diabetes wounds;

(2) Excellent antibacterial performance: can be used as a physical barrier to cover open wounds and prevent bacterial infection;

(3) Good biocompatibility: has in vivo and in vitro degradation function, can be absorbed by organisms, and has the advantages of environmental friendliness and reduction of immune rejection;

(4) Excellent hemostatic performance: the three-dimensional porous reticular structure not only provides a hemostatic function for venule hemorrhage caused by debridement, but also provides good structural support for the absorption of a large amount of exudates from wounds of diabetics.

Drawings

FIG. 1 is an infrared spectrum (FT-IR) of ARHY-Gel described in example 1;

FIG. 2 is a scanning electron microscope image of the macro structure and surface pore structure of ARHY-Gel described in example 1;

FIG. 3 is a graph showing compressive strength test of ARHY-Gel, sulfa-Gel, ADH-Gel described in examples 1-3;

FIG. 4 is a graph of rheological measurements of ARHY-Gel described in example 1;

FIG. 5 is a graph of an in vitro antimicrobial assay of ARHY-Gel described in example 1;

FIG. 6 is a graph showing the use of ARHY-Gel and commercially available products described in example 1 in a mouse liver injury bleeding model and a mouse tail-break bleeding model;

FIG. 7 is a graph of the dynamic tracking of the wound healing model of type II diabetic rats with the product described in example 1 and commercially available;

FIG. 8 is a graph of ARHY-Gel-promoting HUVEC cell proliferation experiments and in vitro erythrocyte hemolysis experiments described in example 1;

Detailed Description

The extracellular polysaccharide Riclin described below was prepared by the following method: inoculating single colony of Agrobacterium tumefaciens sp.ZCC3656 (with preservation number of CCTCCNO: M2018797) into LB culture medium for seed culture, inoculating the obtained seed culture solution into M9 fermentation culture medium (with carbon source of 3% sucrose) at a ratio of 2% after 16h, and fermenting at 30deg.C for 2 days; adding 3 times of isopropanol into the obtained fermentation broth for alcohol precipitation, centrifuging (6000 g,5 min) to collect precipitate, and drying at 40 ℃ to obtain Riclin polysaccharide crude product; the Riclin polysaccharide crude product is redissolved in deionized water to prepare an aqueous solution with the concentration of 10mg/mL, naOH aqueous solution is added to adjust the pH=9.0, the solution is sterilized at the temperature of 105 ℃ under high pressure (30 kPa) for 20min, the solution is centrifuged (10000 g,10 min) to obtain supernatant, 3 times of volume of precooled 95% (v/v) ethanol aqueous solution is added for alcohol precipitation, and the precipitate is collected by centrifugation (6000 g,10 min) to obtain extracellular polysaccharide Riclin (the average molecular weight is 2.5X10 ⁶ Da).

The technical scheme of the present invention will be described in detail by means of specific examples, which should be explicitly set forth for illustration, but should not be construed as limiting the scope of the present invention.

Example 1

An extracellular polysaccharide Riclin-based antibacterial hydrogel, the preparation method of which comprises the following steps:

(1) Adding extracellular polysaccharide Riclin into deionized water to dissolve to prepare a solution with the concentration of 2% (w/v), adding 5wt% of sodium periodate solution according to the mass ratio of Riclin polysaccharide to NaIO ₄, adjusting the pH to 8, stirring at room temperature away from light to react for 3 hours, adding excessive ethylene glycol to stop the reaction, dialyzing the reacted solution for 24 hours by adopting a dialysis bag (Mw 1000 Da) in the deionized water, changing water every 4 hours to obtain a homogeneous structure, putting the homogeneous structure into a freeze dryer to freeze dry, reducing the temperature to-70 ℃ and then reducing the pressure to 0kPa, freeze-drying at the temperature and the pressure for 48 hours, then adopting ethanol to wash for 10 hours, drying at the temperature of 10 ℃ for 6 hours (the product obtained in the step is named as AR), and heating and dissolving at the temperature of 60 ℃ to obtain an aldehyde modified extracellular polysaccharide Riclin aqueous solution;

(2) An aqueous solution of the aldehyde modified extracellular polysaccharide Riclin was mixed with O, O '- (ethane-1, 2-diyl) bis (hydroxylamine) dihydrochloride, wherein O, O' - (ethane-1, 2-diyl) bis (hydroxylamine) dihydrochloride was added in a molar ratio of amino groups contained therein to aldehyde groups of the aldehyde modified extracellular polysaccharide Riclin of 1:1, and the mixture was placed on a magnetic stirrer and stirred uniformly, and the solution was poured into an orifice plate mold and allowed to stand for 2 minutes, to obtain an extracellular polysaccharide Riclin-based antibacterial hydrogel, which was designated ARHY-Gel.

Example 2

An extracellular polysaccharide Riclin-based antibacterial hydrogel was prepared by the same method and parameters as in example 1 except that sulfanilamide was used instead of O, O' - (ethane-1, 2-diyl) bis (hydroxylamine) dihydrochloride.

The extracellular polysaccharide Riclin-based antibacterial hydrogel obtained in this example was designated as sulfofa-Gel.

Example 3

An extracellular polysaccharide Riclin-based antibacterial hydrogel was prepared by the same method and parameters as in example 1 except that adipic acid dihydrazide was used instead of O, O' - (ethane-1, 2-diyl) bis (hydroxylamine) dihydrochloride.

The extracellular polysaccharide Riclin-based antibacterial hydrogel obtained in this example was designated ADH-Gel.

The performance of the extracellular polysaccharide Riclin-based antibacterial hydrogel obtained in the above example was tested, and the specific method and test results are as follows:

1. FT-IR spectroscopy detection

FT-IR spectroscopy was performed on ARHY-Gel obtained in example 1, and the results are shown in FIG. 1. Wherein AR is hemostatic sponge obtained after freeze drying.

It can be seen that a specific peak of 1725cm ^-1 was observed in the FTIR infrared spectrum of the AR, confirming the presence of the-CHO group. Meanwhile, the characteristic peak of 1725cm ^-1 in ARHY-Gel group is observed to be smaller, and the characteristic peak appears at about 1634cm ^-1, which proves that the hydrogel taking the cross-linked structure as the framework is successfully synthesized in the embodiment.

2. Macrostructure testing

The macrostructure of the hydrogels obtained in examples 1-3 was tested and the results are shown in fig. 2, wherein fig. 2a shows that the gel was not formed by performing an inversion bottle test for unoxidized extracellular polysaccharide Riclin; FIG. 2b shows the addition of ethane-1, 2-dioxyamine (EBH) to oxidized extracellular polysaccharide Riclin; FIG. 2c shows the addition of sulfonamide (sulfa) to the oxidized extracellular polysaccharide Riclin of FIG. 2 b; FIG. 2d shows an inverted bottle test of oxidized extracellular polysaccharide Riclin crosslinked to gum with adipic Acid Dihydrazide (ADH).

Experiments show that when the bottle is inclined and inverted, substances in the bottle adhere to the bottom of the bottle in a colloid form and do not slide down due to liquid fluidity along with the position change of the penicillin bottle. FIG. 2e is a compression recoverability ARHY; fig. 2f is ARHY for fold recoverability.

3. Microscopic topography testing

The ARHY-Gel obtained in example 1 was subjected to a metal spraying treatment and then scanned under a scanning electron microscope, and the result is shown in FIG. 2 g.

It can be seen that ARHY-Gel obtained in example 1 has an interpenetrating pore structure, and the three-dimensional network structure provides pores that facilitate absorption of liquid and adhesion and binding of cells, and provides good water retention.

4. Mechanical property test

Hydrogels were prepared as cylindrical samples (diameter 8mm, height 10 mm). The different hydrogels were placed on the sample stage of a universal tester with 100N load cell (Instron 4202, instron, usa) and compression tested under extreme strain, fixed at 80%. The results of the universal tester under compressive load are shown in FIG. 3, where 3a is the corresponding ARHY-Gel hydrogel, and 3b is the corresponding sulfofa-Gel hydrogel and ADH-Gel hydrogel.

It can be seen that the maximum bearing pressure of ARHY-Gel hydrogel obtained in example 1 can also reach 549 kilopascals, the sulfa-Gel hydrogel obtained in example 2, the ADH-Gel hydrogel obtained in example 3 is respectively 4.1 and 0.9 Pa, and compared with other various hydrogels already reported, ARHY-Gel hydrogel has good large deformation resistance toughness and higher compressive strength, and meets the toughness requirement as hydrogel dressing.

5. Rheology test

The ARHY-Gel obtained in example 1 was placed on a rheometer and subjected to rheology testing; in the strain amplitude sweep test, set at a temperature of 37 ℃ and a frequency of 1hz, the strain was varied from 1 to 1000%, and a curve was recorded. In a continuous step-strain test, the storage modulus (G ') and the loss modulus (G') were tested in time series with a frequency of 1Hz and a strain of 1%. On this basis, the angular frequency sweep test (1-100 rad/S) at constant strain is measured and three cycles are repeated.

The test results are shown in FIG. 4. FIG. 4a shows that ARHY-Ge obtained in example 1 showed a flow point of ARHY-Gel at a position where the shear strain was 316%, i.e., the hydrogel network collapsed when the strain was higher than 316%. FIG. 4b shows the ARHY-Ge of the example in a continuous step-strain test, and from FIG. 3b, it can be seen that after the first high strain (1000%), the G ' of the hydrogel drops significantly from 63.1Pa to 12.6Pa, G ' > G ', indicating that the hydrogel network is disrupted. At low strain (1%), the G' of the hydrogel recovered to 39.8Pa, indicating recovery of the dynamic schiff base cross-linked network. After two additional high-low strain cycles, the recovered hydrogels showed nearly identical G' and G "values. In summary, the above experiments demonstrate that ARHY-Gel has good self-healing properties.

6. Antibacterial property test

The ARHY-Gel hydrogel (cylindrical, 20mg, diameter 1 cm) prepared in example 1 and commercially available chitosan antibacterial hydrogel (CS) were incubated with bacterial suspensions (5 mg/mL) of gram-negative E.coli and gram-positive Staphylococcus aureus having a concentration of 1X 106 CFU.mL ^-1 at 37℃for 12 hours, 100uL of the blended bacterial suspension was extracted, the blended bacterial suspension was diluted 1000-fold, 100uL of the diluted bacterial solution was applied to Luria-Bertani (LB) agar medium in a petri dish, and after incubation at 37℃for 24 hours, colony growth was observed.

The test results are shown in FIG. 5, and FIG. 5 is a dilution-spread culture chart after ARHY-Ge and commercially available chitosan antibacterial hydrogel (CS) bacteria are applied as described in example 1. Referring to FIG. 5, in the experiment using the dilution-coated plate, it was observed that the number of colonies generated after ARHY-Gel co-culture was significantly lower than that of the blanc group and CS group.

7. Hemostatic Capacity test

(1) Animal liver perforation hemostasis capability test: mice (body weight 20-25g,8 week old female mice) were fully anesthetized with isoflurane and the upper abdomen surgery exposed to the liver maximum lobe; a cross incision (length-0.5 cm, depth-0.3 cm) was made with a surgical blade No. 23, and immediately after bleeding, the incision was pressed with ARHY-Gel as described in example 1 for 10 seconds until it was adhesively secured; recording hemostasis time, removing ARHY-Gel from wound after 5-10min of bleeding, recording blood loss, and recording hemostasis process by a camera; the hemostatic test was also performed using a commercially available CS hydrogel (chitosan medical biogel, human blessing medicine) as a comparative experimental group, and the test results are shown in fig. 6;

(2) Mice tail-breaking hemostatic ability test: completely anesthetizing mice (weight 20-25g,8 week old female mice) with isoflurane, separating tail from root 0.5cm, pressing material at tail for 10 seconds, and pressing for 10 seconds to fix adhesion; the hemostatic time was recorded, the hydrogel was removed from the wound after 5-10min of bleeding, the blood loss was recorded, and the hemostatic process was recorded by a camera.

The test results are shown in fig. 6, wherein: FIG. 6a is a graph showing the use of ARHY-Gel and CS described in example 1 in a mouse liver cross-incision hemorrhage model. Referring to fig. 6c-d, in the mouse liver cross incision bleeding model, compared with Blnak (454.83 mg average blood loss) and CS (359.5 mg average blood loss), the liver cross incision blood loss (71.17 mg average blood loss) of the ARHY-Gel in the case of the relatively low dosage is only 20% of the commercial medical CS hydrogel, the compression-free hemostatic time is reduced to less than 60s, and the hemostatic effect of the polysaccharide-based hemostatic sponge in the example is far higher than that of the medical CS hydrogel in the animal venous bleeding model; FIG. 6b is a graph showing the use of ARHY-Gel and CS in a mouse tail-bleeding model as described in example 1.

Referring to fig. 6e-f, it can be seen that in the mouse tail-breaking bleeding model, compared with the comparison Blank (average blood loss amount 948.33 mg) and CS (average blood loss amount 539 g), the blood loss amount of ARHY-Gel (average blood loss amount 14.17 mg) in the case of relatively low dosage is only 3% of that of the commercial medical CS hydrogel, the compression hemostasis time is reduced to less than 175s, and the hemostatic effect of ARHY-Gel in the example on the venous bleeding model of animals in different shapes is higher than that of the medical CS hydrogel.

8. Test for ability to promote healing

And selecting a type II diabetes rat to establish a diabetic foot wound healing model. Rats were anesthetized 24 hours after fasting with 10% chloral hydrate (300 mg/kg). Rats were fixed on surgical plates and the backs of each rat were dehaired and sterilized with 75% alcohol. Rats were randomly divided into: the control group, ARHY-Gel group and chitosan hydrogel (CS) group are used as the comparison experiment group to observe the healing condition of the wound surface.

Each photograph was taken at the same location, the ruler was placed on the bottom and calibrated, and the wound area was measured using Image J software. Shooting the contraction condition of the wound on days 0, 3, 7, 14 and 21, and calculating the wound healing rate; wherein A ₀ and A _t are the initial area of the wound surface and the reserved area of the wound surface after t days of wound healing respectively.

The test results are shown in FIG. 7, and FIG. 7a is a graph showing the use of ARHY-Gel and CS described in example 1 in full thickness wounds of type II diabetic rats. Referring to fig. 7b, it can be seen that, on the seventh day of intervention under the same condition in the rat diabetic wound model, compared with Blnak (wound healing rate 33.3%) and CS (wound healing rate 36%), the ARHY-Gel described in example 1 (wound healing rate 72.3%), the healing rate is 2 times that of the commercial medical CS hydrogel; on the fourteenth day of intervention, the ARHY-Gel described in example 1 (wound healing rate 97%) had a 1.07-fold faster healing rate than Blnak (wound healing rate 78%) and CS (wound healing rate 90.33%). It can be seen that the ARHY-Gel effect described in the example on the wound model of type II diabetic rats is higher than that of the medical CS hydrogel.

9. Cell proliferation assay

1X 10 ⁶ Human Umbilical Vein Endothelial Cells (HUVECs) were seeded in 24-well plates containing 300. Mu.l of DMEM medium without fetal bovine serum with cell climbing plates placed and hydrogels placed in trans-well inserts. 1. After 2 and 3 days, staining is carried out by using a Calcein/PI cell activity and cytotoxicity detection reagent, then the climbing plate is taken out, and the climbing plate is observed under a fluorescence microscope and photographed. The test results are shown in FIG. 8, and FIG. 8a is a fluorescent staining chart of HUVEC cells after ARHY-Gel and commercially available chitosan hydrogel (CS) as described in example 1.

As can be seen, after staining with Calcein/PI, the densities of viable cells after ARHY-Gel co-culture were observed to be significantly higher than in the Blank group, CS group, for 1,2,3 days, demonstrating that ARHY-Gel has a promoting effect on HUVEC proliferation and is higher than in the CS hydrogels on the market.

10. Blood compatibility test

Fresh volunteer blood (ACD) was taken, red Blood Cells (RBC) were extracted, diluted to 2% with PBS, and then 20mg of ARHY-Gel prepared in the example and medical CS hydrogel were blended with 4mL of red blood cell suspension and incubated for 1h at 37 ℃; subsequently 1mL of the blend was centrifuged in an EP tube (1200 g,10 min), each group was hemolyzed, 100uL of supernatant was measured for OD value of each material (ODexperiment) at 540nm with a micro plate reader (Thermo Fisher) to add PBS as negative control (ODnegative) and 2% triton X-100 (triton X-100) as positive control; the hemolysis rate was calculated according to the following formula:

The rate of hemolysis/% = ("ODexperiment-ODnegative")/"ODpositive-ODnegative" 100%

The hemolysis was plotted according to the hemolysis rates of the different concentrations, and the results are shown in FIG. 8. FIG. 8b is a chart of the blood compatibility test of ARHY-Gel described in example 1. Referring to FIG. 8b, ARHY-Gel described in example 1 has good blood compatibility, and the hemolysis rate is less than 5%, and the blood compatibility is not changed basically, so that the blood compatibility is good.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

Claims

1. A preparation method of extracellular polysaccharide Riclin-based antibacterial hydrogel is characterized in that extracellular polysaccharide Riclin is dissolved in water, and an oxidant is added for oxidation reaction to obtain aldehyde modified extracellular polysaccharide Riclin; reacting the aldehyde modified extracellular polysaccharide Riclin with an amino compound in a solution to obtain extracellular polysaccharide Riclin-based antibacterial hydrogel;

The amino compound is at least one selected from dioxyamine, aromatic amine and hydrazide; the addition amount of the amino compound is 1:2-2:1 according to the molar ratio of the amino contained in the amino compound to aldehyde groups of the aldehyde modified extracellular polysaccharide Riclin;

The extracellular polysaccharide Riclin has a structure shown in a formula (I), wherein R is-OCCH ₂CH₂ COOH or H, and n is an integer greater than 0;

Formula (I)

2. The method for producing an antibacterial hydrogel based on extracellular polysaccharide Riclin according to claim 1, wherein the concentration of the hydroformylation-modified extracellular polysaccharide Riclin in the reaction solution is 0.1 to 6.0%.

3. The method for preparing an extracellular polysaccharide Riclin-based antibacterial hydrogel according to claim 1 or 2, wherein the dioxyamine is selected from O, O '- (ethane-1, 2-diyl) bis (hydroxylamine) dihydrochloride or O, O' -1, 3-propanediylbis hydroxylamine dihydrochloride.

4. The method for preparing an extracellular polysaccharide Riclin-based antibacterial hydrogel according to claim 1 or 2, wherein the aromatic amine is a sulfonamide.

5. The method for preparing an extracellular polysaccharide Riclin-based antibacterial hydrogel according to claim 1 or 2, wherein the hydrazide is at least one of adipic acid dihydrazide and acethydrazide.

6. The method for preparing an extracellular polysaccharide Riclin-based antibacterial hydrogel according to claim 1 or 2, wherein the molar ratio of the oxidizing agent to extracellular polysaccharide Riclin is 0.25-6:1.

7. The method for preparing an extracellular polysaccharide Riclin-based antibacterial hydrogel according to claim 1 or 2, wherein the oxidation reaction is carried out for 2-12 hours under stirring at a pH of 7-9, in the absence of light, at 15-30 ℃; adding a reducing agent to terminate the reaction after the oxidation reaction; after the oxidation reaction is terminated, dialyzing, freeze-drying, and then heating and dissolving to obtain an aldehyde modified extracellular polysaccharide Riclin solution.

8. An extracellular polysaccharide Riclin-based antibacterial hydrogel, which is prepared by the method of any one of claims 1 to 7.

9. Use of an extracellular polysaccharide Riclin-based antibacterial hydrogel prepared by the method according to any one of claims 1-7 in the preparation of a wound dressing for a diabetic patient.