CN112587717A

CN112587717A - Metal cation crosslinked alginate/bacterial cellulose composite hydrogel antibacterial dressing

Info

Publication number: CN112587717A
Application number: CN202011239486.6A
Authority: CN
Inventors: 洪枫; 史婷兰; 刘露; 陈琳
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2020-11-09
Filing date: 2020-11-09
Publication date: 2021-04-02

Abstract

The invention relates to a metal cation crosslinked alginate/bacterial cellulose composite hydrogel antibacterial dressing which is formed by immersing a bacterial cellulose membrane loaded with alginate into a metal cation aqueous solution. The preparation method of the composite hydrogel material is novel and simple, the raw material source is wide, and the obtained hydrogel material has the pH response antibacterial characteristic and can serve as an intelligent response dressing in an infected wound area.

Description

Metal cation crosslinked alginate/bacterial cellulose composite hydrogel antibacterial dressing

Technical Field

The invention belongs to the field of antibacterial auxiliary materials, and particularly relates to a metal cation crosslinked alginate/bacterial cellulose composite hydrogel antibacterial dressing.

Background

The skin is the largest organ of the body and plays a vital role in maintaining homeostasis and physiological balance. The structure and function of the skin are easily damaged by cuts, burns, surgery and diseases. After skin damage, its structure and function must be reestablished as soon as possible to ensure homeostasis of the body. The dressing is the most important medical product for curing skin wounds. For a long time, the main functions of traditional dressings (gauze, bandages, etc.), including keeping the wound dry, absorbing exudates, preventing bacterial invasion, etc., have been considered as the key to the process of healing wounds. However, this view has changed silently over the past decades. The dressing no longer only plays a role in passively healing wounds, but can actively control infection in the process of healing the wounds, play an antibacterial role and provide a proper healing environment.

It is well known that various metal cations have inhibitory effects on bacteria. The antibacterial effect of metal ions has been explored for thousands of years, and some metal ions (e.g., silver and copper) are more toxic to bacterial cells than humans, and cerium cannot penetrate the cell membrane of mammals. Therefore, the metal ions have less adverse effects on human cells. Zinc, copper and magnesium (detectable amounts 10)^-2To 10^-4mol) bind to DNA and RNA in cells due to their biological function. Different metal cations are used in the synthesis of textiles, food, agriculture, cosmetics, pharmaceuticals, etc.

Alginate (Alg) is a natural polymer extracted from brown algae. It is non-toxic, is a linear anionic polysaccharide, and has ion exchange properties. The alginate hydrogel is low in price, wide in source, strong in hydrophilicity, immunogenicity and certain biological inertia, is applied to various medical applications such as cell encapsulation, drug slow release and the like, and has a recognized clinical application value in the field of alginate wound healing and a great commercial prospect. However, the mechanical strength of single alginate is low, and dressing change in a gel state is difficult.

Bacterial cellulose (BNC) is a natural nano-cellulose material mainly produced by acetic acid bacteria, has a unique nano-scale fiber network and pore structure, has excellent water retention capacity (98-99%) compared with plant cellulose with the same molecular formula, and has excellent mechanical properties in a wet state, extremely large specific surface area, high crystallinity and purity and good biocompatibility. Has wide application prospect in the medical dressing industry. At present, some bacterial cellulose dressing products are available in the market, and the dressing products can reduce stimulation to wounds, effectively relieve pain, effectively cure burn and chronic ulcer wounds and promote the healing of the wounds more effectively than other dressing products. With the increasing demand of human beings, the corresponding functions need to be endowed through related technologies or methods, so that the medical dressing has better and wider application prospects.

CN110124098A discloses a bacterial cellulose/sodium alginate/polyvinyl alcohol composite antibacterial dressing and a preparation method thereof, wherein although healthy and environment-friendly high polymer materials such as sodium alginate and polyvinyl alcohol are compounded with bacterial cellulose, the dressing has no toxic or side effect on human bodies and does not contain chemical substances harmful to the human bodies, a plurality of components are introduced in the method, the experimental operation is complex, and the uniformity of the product is not good.

CN111012797A discloses a hydrogel dressing for treating psoriasis and a preparation method thereof, wherein rubidium, magnesium and zinc ions are chemically cross-linked in hydrogel with an alginate/polyacrylamide interpenetrating network structure, but compared with the hydrogel dressing disclosed by the invention, the preparation process of the hydrogel dressing is relatively complex, the preparation steps are more, the use of chemical reagents is more, and the hydrogel dressing comprises a cross-linking agent, an accelerant, an initiator and the like; although the metal ions are crosslinked, the release effect of the metal ions is not obvious after multiple compounding. In addition, polyacrylamide acts as a first network, and wet strength is much weaker than bacterial cellulose, and can still tear when the dressing is changed. It is worth noting that the monomer compound of polyacrylamide, acrylamide, has strong carcinogenicity and is basically prohibited in the cosmetic and medical industries, but the bacterial cellulose is nontoxic and has good biocompatibility.

Disclosure of Invention

The invention aims to solve the technical problem of providing the metal cation crosslinked alginate/bacterial cellulose composite hydrogel antibacterial dressing, and avoiding the problems of poor moisture retention, poor bacteria isolation, poor healing promoting performance and the like of the traditional dressing; the difficult problems of poor strength of a single alginate dressing and the need of a second dressing are solved.

The composite hydrogel is metal ion crosslinked alginate/bacterial cellulose interpenetrating network hydrogel, wherein the bacterial cellulose is a first network matrix, and the metal ion crosslinked alginate is a second network and is fixed in the bacterial cellulose network in an alternating and interpenetrating manner, so that a compound with a double-network structure is obtained.

The metal ion Mn²⁺、Co²⁺、Cu²⁺、Zn²⁺、Ag⁺、Ce³⁺One or more of the above; the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate.

The preparation method of the composite hydrogel comprises the following steps:

(1) placing the bacterial cellulose membrane in a soluble alginate aqueous solution, and carrying out oscillation and immersion overnight to obtain the bacterial cellulose membrane loaded with the alginate aqueous solution;

or extruding the sodium alginate aqueous solution into a network of the bacterial cellulose membrane by using a vacuum suction or pressure permeation mode to obtain the bacterial cellulose membrane loaded with the alginate aqueous solution;

(2) and (3) soaking the bacterial cellulose membrane loaded with the alginate aqueous solution into the aqueous solution of metal cations to obtain the composite hydrogel.

The preferred mode of the above preparation method is as follows:

the alginate in the step (1) is one or more of sodium alginate, potassium alginate and ammonium alginate.

The concentration of the alginate solution in the step (1) is 1 w/v%.

The step (1) of shaking and dipping overnight is to soak for 12 to 24 hours at the rotating speed of 160 rpm; the vacuum degree of vacuum suction or the pressure of pressure infiltration is 11-65 KPa.

The aqueous solution of metal cations in the step (2) is Mn²⁺、Co²⁺、Cu²⁺、Zn²⁺、Ag⁺、Ce³⁺One or more salt water solutions with the concentration of 0.05-0.5mol/L respectively.

The invention provides a composite hydrogel prepared by the method.

The invention provides a pH response antibacterial auxiliary material based on the composite hydrogel.

The invention provides application of the pH response antibacterial dressing in preparing an external preparation for treating an infected wound.

According to the invention, a soluble alginate aqueous solution is extruded into a bacterial cellulose membrane network by using a vacuum suction or pressure permeation mode to obtain the alginate/bacterial cellulose composite hydrogel; and then soaking the bacterial cellulose membrane loaded with the alginate aqueous solution into aqueous solutions containing different metal cations to obtain the metal cation crosslinked alginate/bacterial cellulose composite hydrogel dressing. The composite hydrogel dressing has the characteristics of broad-spectrum antibiosis, pH-sensitive metal ion release, high hydrogel mechanical strength and the like. The in vitro and in vivo tests and evaluations prove that the composite hydrogel dressing can accelerate the healing of skin wounds.

Advantageous effects

(1) The main raw materials used in the invention are bacterial cellulose and soluble alginate which are all healthy and environment-friendly high molecular materials and have no toxic or side effect on human bodies, the soluble alginate is uniformly dispersed in a bacterial cellulose network by using an impregnation or pressure diffusion method, then the bacterial cellulose membrane loaded with the alginate is immersed in a metal cation aqueous solution for crosslinking to form a second network hydrogel, and finally the composite hydrogel which is interpenetrating with the first network, has enhanced wet strength and has antibacterial activity is obtained. The structural principle is shown in fig. 13.

(2) The existing calcium alginate dressing has certain hemostatic performance, but the single alginate has low mechanical strength, and the dressing is difficult to replace in a gel state. After the gel-state wet strength is combined with the bacterial cellulose, the gel-state wet strength is increased, the dressing can be cut at will, the shape of the dressing is controlled, and the gel-state wet strength has a good application prospect; when the hemostatic powder is applied as a wound dressing, hemostasis is performed in time at the initial stage of a wound; the wound exudate is absorbed in a large amount in the middle and later periods, and the dressing is easy to replace.

(3) The cross-linked metal cations can improve the mechanical property, obtain broad-spectrum antibacterial activity, ensure the safe and rapid healing of the wound, do not adhere to the skin and can be replaced for many times as required. Based on the characteristics, the metal cation crosslinked alginate/bacterial cellulose composite hydrogel dressing has good commercial advantages.

(4) The metal ion crosslinked alginate/BNC composite hydrogel dressing has pH-responsive antibacterial activity, the release rate of metal ions is increased in a low-pH environment, the dressing can be used for infected wounds, and the obtained gel material has pH-responsive antibacterial property and can serve as an intelligent-responsive dressing in infected wound areas.

(5) According to the invention, the sodium alginate and the bacterial cellulose are combined by a vacuum suction or pressure permeation method, the method is simple, the requirement on experimental conditions is not high, compared with a conventional impregnation method, the compounding is efficient and uniform, the time consumption is short, the cost is low, and the mass production can be realized.

Drawings

FIG. 1 is a macroscopic picture (a) and compressive strength graphs (b), (c) of different kinds and concentrations of Alg crosslinked metal cations prepared in example 1.

FIG. 2 is an SEM topography observation of the surfaces of BNC, Alg hydrogel and different metal cation crosslinked Alg/BNC composite hydrogel dressings prepared in examples 2-7. A is a schematic diagram of a composite gel with a double-network structure of a second network formed by BNC as a first network and Alg; B1-B7 are the macro-morphology of alginate gel crosslinked by different metal ions; c is BNC, D1-D7 is Alg/BNC composite hydrogel dressing crosslinked by different metal ions and a corresponding SEM structure thereof.

FIG. 3 is a graph showing the antibacterial activity of the composite hydrogel dressing prepared in examples 2 to 7 against Staphylococcus aureus and Escherichia coli with a control group.

In FIG. 4, a and b are the characterization diagrams of the mechanical properties of the BNC and different metal cation crosslinked Alg/BNC composite hydrogel dressings prepared in examples 2 to 7.

FIG. 5 is a graph showing the cumulative release profiles of metal ions at different pH values for different metal cation crosslinked Alg/BNC composite hydrogel dressings prepared in examples 2-7 (wherein pH 5.5: black rectangle; pH 7.4: red circle; pH 8.4: blue triangle).

FIG. 6 is a whole blood coagulation experimental diagram of the BNC and different metal cation crosslinked Alg/BNC composite hydrogel dressing prepared in examples 2-7.

FIG. 7 is a graph of in vitro hemolysis experiments on the composite hydrogel dressings of BNC and different metal cations crosslinked Alg/BNC prepared in examples 2-7.

FIG. 8 is a cytotoxicity evaluation chart of different metal cation crosslinked Alg/BNC composite hydrogel dressings prepared in examples 2 to 7.

FIG. 9 is a fluorescence observation image of different metal cation crosslinked Alg/BNC composite hydrogel dressings prepared in examples 2 to 7.

FIG. 10 is a graph illustrating the evaluation of the healing performance of the BNC and different metal cation crosslinked Alg/BNC composite hydrogel dressing prepared in examples 2-7 in vitro.

FIG. 11 is a test evaluation chart of the in vitro wound healing rate of the BNC and different metal cation crosslinked Alg/BNC composite hydrogel dressing prepared in examples 2-7.

FIG. 12 is a flow chart of the present invention.

FIG. 13 is a schematic diagram of the gel structure principle.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

(1) Sodium alginate solution with mass concentration of 1 w/v% is prepared, and 1g of sodium alginate (national drug group chemical reagent Co., Ltd.) is dissolved in 100ml of deionized water.

(2) Respectively preparing MnSO with the concentration of 0.5mol/L, 0.3mol/L, 0.1mol/L and 0.05mol/L₄·4H₂O，CuSO₄·5H₂O，AgNO₃，CoSO₄·7H₂O，Ce(NO₃)₃·6H₂O，ZnCl₂(national chemical group chemical Co., Ltd.) of a metal cation solution.

(3) Transferring 5mL of the sodium alginate solution in step (1) into a six-well plate at 25 ℃, then slowly adding the cationic solution in step (2) to cover the sodium alginate solution, and shaking at 30rpm for 24h at room temperature.

(4) The hydrogel in step (3) was removed and washed 3 times with deionized water to remove excess cations.

(5) The hydrogel in step (4) was subjected to compression measurement by a universal material testing machine. Compression measurements were performed using a general purpose material testing machine (H5K-S, Huns field Test Equipment Ltd., England). The measurement of the compression is carried out at 25 ℃ at a speed of 0.5mm/min and a force of 10N until the deformation exceeds 50%. Each test was repeated eight times and the mean ± standard deviation was given.

FIG. 1 shows the macroscopic picture (a) and the compressive strength (b) and (c) of different types and concentrations of cations crosslinked by Alg. It can be seen from the figure that the properties of alginate hydrogels are highly influenced by the concentration of cations, i.e. at high concentrations cations form rigid hydrogels (a) with sodium alginate. All cationic crosslinked Alg showed uniform three-dimensional hydrogel formation. Compression measurements show that the deformation of the Alg-Cu hydrogel withstands compression pressures greater than other cationically crosslinked Alg, followed by Alg-Co, Alg-Ce, Alg-Zn, Alg-Mn and Alg-Ag. AgNO with concentration of 0.05mol/L₃The silver ion crosslinked Alg hydrogel was soft and could not withstand any pressure (VI, a). Depending on the highly desirable properties of the functional wound dressing, the concentration of cationic ionically crosslinked Alg is preferred, which, while regenerating a suitable hydrogel, can both promote fibroblast proliferation and have an antimicrobial effect. In FIG. 2, B1 is an Alg-Na aqueous solution, and B2-B7 are respectively Alg-Mn, Alg-Co, Alg-Cu, Alg-Zn, Alg-Ag and Alg-Ce hydrogels.

Example 2

(1) The sodium alginate solution was prepared in the same manner as in example 1.

(2) Preparing MnSO with the concentration of 0.05mol/L₄·4H₂And O metal cation solution.

(3) Acetobacter xylinum strain (the strain is Gluconobacter xylinus ATCC 23770 which is purchased from American strain collection center) is subjected to static culture at constant temperature of 30 ℃ for 10 days in a liquid culture medium, the bacterial cellulose membrane is taken out and placed in NaOH (national drug group chemical reagent Co., Ltd.) solution with the concentration of 10g/L, the bacterial cellulose membrane is taken out after being treated for 2 hours at 80 ℃, and the bacterial cellulose membrane is rinsed to be neutral by deionized water to obtain the bacterial cellulose membrane.

(4) The bacterial cellulose membrane obtained in step (1) was placed at 25 ℃ on the bottom of a funnel, and then 50mL of sodium alginate solution was added to the funnel, and a BNC/Alg hydrogel membrane was obtained by vacuum suction under a vacuum of 15 kPa. To prevent final overpressure, the BNC membrane was sucked flat and the vacuum stopped when 10mL of solution was left in the funnel.

(5) Soaking the bacterial cellulose/sodium alginate hydrogel film obtained in the step (4) in 0.05mol/L MnSO obtained in the step (2)₄·4H₂O (colorless solution), magnetic stirring at 25 ℃ for 24 h.

(6) And (3) taking out the hydrogel film in the step (5), and washing the hydrogel film for 3 times by using deionized water to remove excessive cations.

(7) Taking out the composite hydrogel dressing obtained in the step (6), and carrying out macroscopic observation: and after freeze drying for 48-72 h at minus 40-50 ℃, observing the microscopic morphology of the freeze-dried sample, including the synthetic cation crosslinked alginate/BNC dressing, BNC/Alg and original BNC.

(8) And (4) performing microscopic morphology observation on the composite hydrogel dressing freeze-dried sample in the step (7): the sample was fixed on a double conductive tape and sprayed with gold particles. Surface characterization was observed using a field emission scanning electron microscope.

(9) And (3) measuring the alginate content of the composite hydrogel dressing obtained in the step (6): a circular BNC/Alg film with a radius of 3cm was used. The water and alginate content of BNC/Alg dressings was obtained using the following formula:

W_B＝W_E-W_D

W_C＝W_A-W_D

wherein W_BIs the water content, W, of the original BNC film_EIs the weight of water-saturated BNC, W_DIs the weight of the same BNC film after drying at 105 ℃ for 8h, W_AIs the weight in the BNC film after the planned entry of Alg. W_CIs the weight of the actual Alg in the composite hydrogel dressing.

(10) Measurement of water holding capacity and water absorption capacity of the composite hydrogel dressing obtained in step (6): a circular composite hydrogel dressing with a radius of 3cm was used at 25 ℃. The dressing was placed on cotton gauze at the bottom of the centrifuge tube to absorb moisture. The Water Holding Capacity (WHC) and water uptake (WAC), water retention in the fibers (WBF) and water distribution in the material (WDWM) of the dressing were calculated using the following equations:

WHC＝W₀-W₃/W₃

WAC＝W₁-W₃/W₃

WBF＝W₁-W₂/W₃

WDWM＝(W₁-W₂)/(W2-W₃)

wherein W₀Is the weight of the composite hydrogel dressing in the wet state, W₁Is prepared by freeze-drying, and soaking in CaCl with concentration of 2.5mmol/L₂And the weight of the composite hydrogel dressing after 30 minutes in an aqueous solution of 142mmol/L NaCl. W₂Is the weight of the sample after centrifugation at 1200rpm for 15min, W₃Is the final weight of the composite hydrogel dressing completely dried at 105 ℃.

(11) And (4) testing the antibacterial activity of the composite hydrogel dressing obtained in the step (6): approximately 0.25mL (1X 105-3X 105CFU/mL) of the adjusted inoculum was placed in direct contact with a sterile control and composite dressing. After 24h of contact with the sample at 37 ℃, the sample was transferred into 20mL of autoclaved SCDLP medium and the viable bacterial cells were washed from the sample using a vortex mixer. After serial dilution, the number of viable bacterial cells was quantified by plating the dilutions on agar plates.

(12) And (4) carrying out mechanical property test on the composite hydrogel dressing obtained in the step (6): all samples were cut into 1.5 x 5cm rectangles and the mechanical strength of the dressing was evaluated at 25 ℃ using a universal materials testing machine. The stretching rate was 50 mm/min.

(13) pH response release cation test: the release of cations was measured using an inductively coupled plasma mass spectrometer and recorded for 48 h. The composite dressing was cut into a shape of a circular disc having a diameter of 3 cm. The discs were immersed in 10mL of phosphate buffer at pH 5.5, 7.4 and 8.4, respectively, at 37 ℃ and incubated for 12, 24, 48h, respectively, with stirring at 40 rpm. And at each time point, the supernatant was collected to measure the release of cations, and 10mL of fresh phosphate buffer was added to compensate for the decreased solution while collecting the supernatant.

(14) Drug release kinetics testing: the release profile of the cation was fitted using a kinetic model commonly used in drug release studies. The model used is as follows:

Q_t/Q_f＝K×t^1/2

Q_t/Q_f＝K×t

Ln(1-Q_t/Q_f)＝-K×t

wherein Q_tIs the amount of cation released (mg/mL) at time t, Q_fIs the final release amount of cation (mg/mL), t is the release time (min), and K is the kinetic constant. Using a correlation coefficient r²And (6) evaluating the adaptability.

(15) In vitro whole blood coagulation test: a sterile synthetic dressing (in the form of a 1cm diameter disc) was placed in the tube and preheated at 37 deg.C, after which 100. mu.L of recalcified whole blood solution (20. mu.L of 0.25M CaCl in 2mL of blood) was added₂) In direct contact with the dressing. The tubes were incubated at 37 ℃ for 5, 15, 25, 35 and 45 min. After a preset time, 10mL of deionized water was gently added to release unbound blood without disturbing the clot formed on the dressing surface. The absorbance of the collected supernatant was measured at 550nm using a microplate reader. At the same time, the absorbance of 100 μ L of whole blood recalcified on the original BNC surface was measured as a negative control.

(16) In vitro hemolysis rate measurement: the red blood cells were obtained by centrifuging whole blood at 116 Xg for 10min at 25 ℃. The erythrocytes were gently rinsed 3 times with Phosphate Buffered Saline (PBS). The washed erythrocytes were then diluted to a final concentration of 5% (v/v). Before this, the sterile dressing was placed in a 24-well plate, kept at 37 ℃ and immersed in a physiological saline solution for 2 h. After removal of the salt solution, 2mL of deionized water was added to the wells as a positive control and sodium chloride solution as a negative control. Then 500 μ L of red blood cell suspension was added to each well, followed by incubation at 37 ℃ for 2h, and finally 1mL of supernatant was gently removed with a pipette and transferred to a new tube. All samples were centrifuged at 115 Xg for 15 min. The obtained supernatant was gently transferred to a new 96-well plate. The absorbance of the solution at 550nm was read using a microplate reader. The hemolysis rate of the composite dressing was calculated using the following formula:

hemolysis rate (OD)_s-OD_n)/(OD_p-OD_n)

OD_sIs the absorbance value, OD, of the supernatant of the sample set_pIs the absorbance value, OD, of a positive control_nIs the absorbance value of the negative control.

(17) Cytotoxicity test: a sterile dressing (a 1cm diameter disc) was placed in a 24-well plate. The L929 fibroblast cells were cultured at 1X 10⁴The density of individual cells/well was seeded on the dressing surface and complete medium (DMEM) was added to the well. Then at 5% CO₂Incubate at 37 ℃ in a concentrated environment, and replace the medium every 24 h.

Cytotoxicity was measured by using a cell counting kit (CCK-8): after removing the medium and washing the cells 3 times with PBS, 400. mu.L of DMEM containing 10% CCK-8 reagent was added to each well of the 24-well plate. After 1h of incubation, the absorbance of 100 μ L of supernatant per well was measured at 450nm on

days

1, 3 and 5.

Cytotoxicity was measured by using live/dead staining method: after 72h of direct contact of the cells with the medium, the medium was removed and washed 3 times with PBS. Adding live/dead staining solution to the surface of the material, incubating at 37 deg.C for 30min, removing the staining solution, and observing the dressing material under confocal scanning microscope.

(18) In vivo repair test: rats were randomly selected, fed a standard diet, and acclimatized for one week prior to laboratory surgery (24 ℃ -27 ℃). For the surgical portion, all procedures were performed under sterile conditions. Rats were anesthetized by injection of chloral hydrate at a dose of 0.4mg/kg body weight. The dorsal area of the rats was shaved and sterilized with 75% ethanol as surgical preparation. A square full-thickness wound of 5X 5mm was created on the skin on both sides of the midline at 3cm intervals. The wound was wrapped with 2X 2cm sterile dressing. The same sterile dressing was changed daily. After surgery, the rats were observed until day 12, and photographs were taken periodically. Wound healing rate (%) was calculated using the following formula:

wound healing rate (%) [ (Area)₀-Area_n)/Area_n]×100％

Wherein Area₀Is the wound Area on day 0, and Area_nIs the wound area (in mm) on day n.

Example 3

(2) Except that CuSO with the preparation concentration of 0.05mol/L₄·5H₂The experimental procedure was the same as in example 2 except that the O metal cation solution and the degree of vacuum were 11 kPa.

(3) Macroscopic observations were made as in example 2.

(4) Microscopic morphology observation was performed as in example 2.

(5) Alginate content was tested according to the test method in example 2 (table 1).

(6) The water holding capacity and water absorption tests were carried out according to the test methods in example 2 (Table 2).

(7) The mechanical properties were measured according to the test method in example 2.

(8) The antibacterial activity test was carried out according to the test method in example 2.

(9) The release test of cations was performed according to the test method in example 2.

(10) The drug release kinetics test was performed according to the test method in example 2 (table 3).

(11) The in vitro whole blood coagulation test was performed according to the test method in example 2.

(12) The in vitro hemolysis rate test was performed according to the test method in example 2.

(13) Cytotoxicity experiments were performed according to the test method in example 2.

(14) The in vivo repair assay was performed according to the test method in example 2.

Example 4

(2) Except AgNO with the preparation concentration of 0.05mol/L₃The experimental procedure was the same as in example 2 except that the metal cation solution and the degree of vacuum were 65 kPa.

(3) Macroscopic observations were made as in example 2.

(4) Microscopic morphology observation was performed as in example 2.

Example 5

(1) The potassium alginate solution was prepared in the same manner as in example 1.

(2) Except that the CoSO with the preparation concentration of 0.05mol/L₄·7H₂The experimental procedure was the same as in example 2 except that the O metal cation solution and the degree of vacuum were 20 kPa.

(3) Macroscopic observations were made as in example 2.

(4) Microscopic morphology observation was performed as in example 2.

Example 6

(2) Except that Ce (NO) with the preparation concentration of 0.05mol/L₃)₃·6H₂The experimental procedure was the same as in example 2 except that the O metal cation solution and the degree of vacuum were 55 kPa.

(3) Macroscopic observations were made as in example 2.

(4) Microscopic morphology observation was performed as in example 2.

Example 7

(1) The ammonium alginate solution was prepared in the same manner as in example 1.

(2) Except that ZnCl with the preparation concentration of 0.05mol/L is prepared₂The experimental procedure was the same as in example 2 except for the metal cation solution.

(3) Macroscopic observations were made as in example 2.

(4) Microscopic morphology observation was performed as in example 2.

Table 1 alginate content test

Material	Mass of material (g)	Mass of liquid (g)	Mass of solid (g)
				BNC	3.35±0.15	3.28±0.08g	0.024±0.001g
BNC/Alg	2.85±0.01	2.85±0.13g	0.024±0.001g

As can be seen from the above table, the weight of BNC is 3.35. + -. 0.15g, which contains 3.28. + -. 0.08g of water and 0.024. + -. 0.001g of pure BNC fibers, and the weight of the same BNC membrane after imbibing the aqueous solution of Alg is 2.85. + -. 0.01g, which contains 0.024. + -. 0.001g of pure BNC fibers and 2.85. + -. 0.13g of 1% Alg. The results indicate that 85.66% of the saturated BNC internal moisture was replaced by Alg after vacuum suction, indicating that Alg filled the BNC interior. While 14.34g of water in the BNC that was not replaced by Alg was lost, probably because of the difference in viscosity between water and Alg.

Table 2 water holding capacity and water absorption testing of examples 2-7

Dressing material	WHC^*(g/g)	WAC^*(g/g)	WBF^*(g/g)	WDWM^*(g/g)
					Control(BNC)	20.54±0.15	15.24±0.04	13.24±0.03	5.14±0.04
BNC/Alg-Ag	49.55±0.06	46.52±0.14	37.13±0.1	5.13±0,02
					BNC/Alg-Co	23.34±0.02	19.26±0.03	17.22±0.08	8.32±0.04
BNC/Alg-Cu	20.16±0.09	17.51±0.04	14.63±0.05	6.24±0.03
					BNC/Alg-Zn	23.66±0.04	21.26±0.05	20.52±0.08	15.45±0.07
BNC/Alg-Mn	42.25±0.05	31.38±0.1	26.61±0.04	5.31±0.01
					BNC/Alg-Ce	37.38±0.07	33.89±0.04	31.94±0.08	15.89±0.065

WHC^*: water holding capacity; WAC^*: water absorption capacity; WBF^*: water holding capacity in the fiber; WDWM^*: the distribution amount of water in the material.

As can be seen from the above table, the composite dressings with BNC/Alg-Ag, BNC/Alg-Co, BNC/Alg-Cu, BNC/Alg-Zn, BNC/Alg-Mn and BNC/Alg-Ce have the water holding capacities of 49.55, 23.34, 20.16, 23.66, 42.25 and 37.38g/g, while the original BNC is 20.54g/g, which shows that the WHC of the obtained composite material is improved. WAC is an important factor for absorbing wound exudate and maintaining moist, clean and microenvironment of the wound. The WACs of the BNC/Alg-Ag, BNC/Alg-Co, BNC/Alg-Cu, BNC/Alg-Zn, BNC/Alg-Mn, BNC/Alg-Ce lyophilized hydrogels were 46.52, 19.26, 17.51, 21.26, 31.38 and 33.89g/g, respectively, compared to the WAC of the original BNC of 15.24g/g, indicating a stronger moisture absorption capacity compared to the BNC composite dressing. WBF embodies the moisture environment the material provides to the wound site. Measurements show that the WBF is greater than the original BNC in all composite dressings, and that BNC/Alg-Ag has the highest WBF of 37.13 g/g. WDWM reflects the distribution of liquid within the wound dressing, with BNC/Alg-Ce, BNC/Alg-Zn having almost three times the WDWM of the control BNC.

Table 3 pharmacokinetic testing of examples 2-7

As can be seen from the above table, in general, the release kinetics of copper is modeled on the zeroth order (r 2)>0.99), silver and cerium (r 2)>0.98), cobalt and manganese (r)²>0.97) fit well.

SEM topography observations of the surfaces of BNC, BNC/Alg and different cationic crosslinked Alg/BNC composite hydrogel dressings are shown in FIG. 2. Morphology observed by SEM indicated that the pore structure of the original BNCs after vacuum pumping of Alg, Alg molecules and impregnation into the BNC nanofiber network, the BNC nanofibers were squeezed due to the applied pressure, and SEM images all showed. All the cationic crosslinked Alg hydrogel adsorbed into the BNCs was uniformly distributed and filled the pores of the BNCs.

FIG. 3 shows the antibacterial activity of the composite hydrogel dressing and a control group against Staphylococcus aureus and Escherichia coli. After 24h exposure to bacterial cells, all dressings showed broad spectrum antibacterial effect compared to the original BNCs. BNC/Alg-Co can minimize the number of viable bacterial cells, followed by BNC/Alg-Ag, BNC/Alg-Zn, BNC/Alg-Cu, BNC/Alg-Mn and BNC/Alg-Ce.

Fig. 4 shows the mechanical property characterization chart of BNC and different cation crosslinked Alg/BNC composite hydrogel dressing. As can be seen, the ultimate tensile strength of the water-saturated pristine BNC hydrogel is only 0.05. + -. 0.06MPa, and the mechanical properties are improved by introducing cations into the BNC/Alg. The tensile strength of BNC/Alg-Cu, BNC/Alg-Co, BNC/Alg-Zn, BNC/Alg-Mn, BNC/Alg-Ag and BNC/Alg-Ce reaches 0.53 +/-0.02 MPa, 0.47 +/-0.06, MPa, 0.29 +/-0.06, 0.19 +/-0.04, 0.17 +/-0.07 and 0.157 +/-0.05 MPa respectively;

in fig. 4 a.

Fig. 5 shows the cumulative release profiles of metal ions at different pH for different cationic crosslinked Alg/BNC composite hydrogel dressings. More cations are released from the dressing at pH 5.5 than at pH 7.4 and 8.4. The release of cations from BNC/Alg-Co, BNC/Alg-Ag is faster than BNC/Alg-Zn, BNC/Alg-Mn, BNC/Alg-Cu and BNC/Alg-Ce.

As shown in figure 6, the whole blood coagulation experiment chart of BNC and different cation cross-linked Alg/BNC composite hydrogel dressings is shown. The coagulation rates of BNC/Alg-Cu and BNC/Alg-Zn were highest during the first 10min, followed by BNC/Al-Ce, BNC/Al-Co, BNC/Alg-Mn and BNC/Alg-Ag. The absorbance of BNC/Alg-Ag for the first 5min was about 0.53, higher than all other materials, but lower than the original BNC. The absorbance of BNC/Alg-Co, BNC/Alg-Ce, and BNC/Alg-Mn is higher than the original BNC, but the absorbance of the other materials is close to and less than the original BNC, during the second, third, fourth, and fifth 10 min.

FIG. 7 is a graph showing the in vitro hemolysis rate test of BNC and different cation crosslinked Alg/BNC composite hydrogel dressings. Although all dressings showed an absorbance below 3.1, which is well suited as wound dressings, the hemolysis rate of BNC/Alg-Cu was the lowest, 0.48, close to the original BNC, with the remaining groups in order: BNC/Alg-Ce < BNC/Alg-Mn < BNC/Alg-Zn < BNC/Alg-Co < BNC/Alg-Ag. The hemolysis rate results demonstrate excellent blood compatibility as a functional wound dressing material.

Fig. 8 shows a cytotoxicity evaluation chart of different cation-crosslinked Alg/BNC composite hydrogel dressings, fig. 9 shows a fluorescence observation chart of different cation-crosslinked Alg/BNC composite hydrogel dressings, and as shown in the figure, none of the dressings is toxic to L929 fibroblasts. The cell viability and proliferation of L929 fibroblasts on the surface of the dressing material indicates the safety of the material for use as a functional wound dressing. In contrast to other cations, fibroblasts contain Ce²⁺The proliferation on the dressing showed the best results live/dead staining results confirming that the dressing is non-toxic. (fluorescence Picture BNC/Alg-Cu repeat, BNC/Alg-Ce none)

Fig. 10 is a diagram showing the evaluation of the healing performance of BNC and different cation crosslinked Alg/BNC composite hydrogel dressing in vitro, and fig. 11 is a diagram showing the evaluation of the healing rate of BNC and different cation crosslinked Alg/BNC composite hydrogel dressing in vitro wound. Based on the results of the in vitro studies, healing evaluations were performed in a rat model. The composite dressing was applied as a wound dressing to a wound created in the dorsal region of the rat, and the original BNCs were used as control dressings for comparison. And regularly taking pictures, and monitoring the healing capacity of different materials for 12 days to show the wound healing capacity. The healing rate of BNC/Alg-Zn is more obvious after 8 days. All dressings can promote wound healing much faster than the original BNCs. The original BNCs showed the slowest healing rate in the test within 12 days.

Comparative example 1

A bacterial cellulose/sodium alginate/polyvinyl alcohol composite antibacterial dressing and a preparation method thereof are provided, the composite antibacterial dressing has a three-dimensional interpenetrating network structure formed by the sodium alginate, the polyvinyl alcohol and the bacterial cellulose, and boric acid is diffused in the network. The bacterial cellulose membrane is dipped in sodium alginate/polyvinyl alcohol mixed solution and then is fixed with boric acid/calcium chloride in a crosslinking way. Although the bacterial cellulose is compounded with healthy and environment-friendly high polymer materials such as sodium alginate, polyvinyl alcohol and the like, the bacterial cellulose has broad-spectrum antibacterial property, has no toxic or side effect on human bodies, and does not contain chemical substances harmful to the human bodies, the method introduces a plurality of components, and has more experimental processes and complicated operation steps.

Comparative example 2

Immersing collagen into bacterial cellulose by a negative pressure permeation method or a positive pressure method, crosslinking the collagen immersed into the bacterial cellulose composite material by a crosslinking agent to obtain a bacterial cellulose/collagen interpenetrating network composite material, and then immersing the material into a chitosan or derivative antibacterial agent solution thereof to prepare the bacterial cellulose/collagen-chitosan composite material. The method adopts a negative pressure osmosis method and a positive pressure method similar to the method of the invention to immerse collagen into a bacterial cellulose network, but chemical cross-linking agents such as glutaraldehyde are used in the later period, and if the cleaning is insufficient, cytotoxicity can be caused.

Comparative example 3

CN111012797A hydrogel dressing for treating psoriasis and a preparation method thereof, although rubidium, magnesium and zinc ions are chemically cross-linked in hydrogel with alginate/polyacrylamide interpenetrating network structure, the hydrogel dressing has the advantages of high viscosity and the like. But compared with the invention, the material preparation process is relatively complex and the preparation steps are more; with the use of cross-linking agents, there is low toxicity; although the metal ions are crosslinked, the release effect of the metal ions is not obvious after multiple compounding, and the pH responsiveness is not generated.

The BNC used in the invention is a natural material, is green and environment-friendly, has simple preparation process and lower operation requirement; the composite dressing prepared by combining BNC serving as a substrate and other materials is enough to provide a moist environment for a wound. Finally, the release rate of metal ions is increased in a low pH environment, and the metal ions can serve as an intelligent response antibacterial dressing in an infected wound area.

Claims

1. The composite hydrogel is characterized in that the composite hydrogel is metal ion crosslinked alginate/bacterial cellulose interpenetrating network hydrogel, wherein the bacterial cellulose is a first network matrix, and the metal ion crosslinked alginate is a second network and is fixed in the bacterial cellulose network in an alternating and alternating manner.

2. The composite hydrogel according to claim 1, wherein the metal ion Mn is²⁺、Co²⁺、Cu²⁺、Zn²⁺、Ag⁺、Ce³⁺One or more of the above; the alginate is one or more of sodium alginate, potassium alginate and ammonium alginate.

3. A method of making a composite hydrogel, comprising:

4. The preparation method according to claim 3, wherein alginate in step (1) is one or more of sodium alginate, potassium alginate and ammonium alginate.

5. The method according to claim 3, wherein the concentration of the alginate solution in step (1) is 1 w/v%.

6. The method according to claim 3, wherein the step (1) of shaking and immersing overnight is soaking at 160rpm for 12-24 h; the vacuum degree of vacuum suction or the pressure of pressure infiltration is 11-65 KPa.

7. The method according to claim 3, wherein the aqueous solution of metal cations in the step (2)Is Mn²⁺、Co²⁺、Cu²⁺、Zn²⁺、Ag⁺、Ce³⁺One or more salt water solutions with the concentration of 0.05-0.5mol/L respectively.

8. A composite hydrogel prepared by the method of claim 3.

9. A pH-responsive antimicrobial adjuvant based on the composite hydrogel of claim 1.

10. Use of the pH-responsive antimicrobial dressing according to claim 9 for the preparation of an external preparation for the treatment of infected wounds.