CN111303449B

CN111303449B - Degradable electroactive bacterial cellulose/MXene composite hydrogel and preparation and application thereof

Info

Publication number: CN111303449B
Application number: CN202010050848.0A
Authority: CN
Inventors: 杨光; 毛琳; 石志军; 龙笑; 叶伟亮; 杨跃梅; 周颖
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2021-10-08
Anticipated expiration: 2040-01-17
Also published as: CN111303449A

Abstract

The invention discloses degradable electroactive bacterial cellulose/MXene composite hydrogel and preparation and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the steps of dissolving bacterial cellulose into pre-cooled aqueous alkali containing urea to obtain a bacterial cellulose solution; adding the MXene nano material into a bacterial cellulose solution, and adding a cross-linking agent to obtain a mixed solution; and pouring the mixed solution into a mold, standing at 4-8 ℃ to crosslink the bacterial cellulose and the MXene nano material and crosslink the bacterial cellulose to obtain the degradable electroactive bacterial cellulose/MXene composite hydrogel. The composite hydrogel disclosed by the invention not only has good electrical activity and degradability, but also has good mechanical properties and biocompatibility; in addition, the cell-surface-modified chitosan hydrogel can promote cell adhesion, growth, spreading, proliferation and the like through the coupling and combined action with external electrical stimulation, can be applied to skin wound dressings, and has good curative effects on promoting skin wound healing and tissue regeneration.

Description

Degradable electroactive bacterial cellulose/MXene composite hydrogel and preparation and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to degradable electroactive bacterial cellulose/MXene composite hydrogel and preparation and application thereof.

Background

Skin wound healing is a precise and complex process involving four phases of inflammation, granulation tissue formation, matrix remodeling, and re-epithelialization. Wound dressings can accelerate wound healing, reduce scar formation and further enhance the barrier to skin tissue regeneration. The use of wound dressings has been made necessary to promote wound healing. Based on this, a variety of wound dressings such as cotton, wool, natural or synthetic bandages, gauze, etc. have been developed so far, and these conventional dry wound dressings, although having a very important role in the initial stage of wound healing, are relatively dry and do not provide a moist environment for the wound and easily adhere to the wound surface, thus causing secondary trauma to the wound upon removal.

To address these issues, hydrogel dressings have attracted considerable attention as a three-dimensional (3D) crosslinked hydrophilic polymer network with very high intrinsic water content. The hydrogel has high water content and three-dimensional space network structure, so that the moist environment of the wound surface can be maintained, sufficient gas exchange is provided, excessive tissue exudate is removed, and the wound surface is cooled, so that the problem of dehydration of the wound surface is solved, wound infection caused by excessive tissue exudate can be prevented, and pain of a patient is relieved. Further, hydrogel dressings do not adhere to the wound surface and thus can be easily removed without causing secondary trauma to the wound, and in addition, they have excellent biocompatibility, and thus, exhibit significant advantages over conventional dry dressings, and have great application prospects in promoting wound healing.

In addition, a number of studies have shown that during wound healing, a spontaneous endogenous direct current electric field (DC-EFs) is generated at the wound site, wherein physiological electric signals are generated to regulate cell behavior (such as cell adhesion, proliferation, migration and differentiation) and thus promote wound healing and tissue regeneration. Therefore, inspired by the spontaneous endogenous electric field, one achieves wound healing by simulating the exogenous electric field or electrical stimulation. The skin is one of electrically stimulated tissues, and has a conductivity value of 2.6mS/cm to 1 × 10^-4mS/cm. It has been reported that an external electric field or electric stimulation can induce re-epithelialization of skin wounds by promoting proliferation and migration of keratinocytes, fibroblasts, and epithelial cells of the skin, thereby improving the wound closure rate and the degree of tissue regeneration.

Based on the above two points, it is a trend to develop an electroactive hydrogel which can be stimulated by a coupled electric field to achieve the effect of treating skin injury more rapidly. Although many hydrogel dressings have been developed, the technology has certain defects, such as insufficient toughness and mechanical properties of the prepared hydrogel, and thus, the application of the hydrogel in wound dressing is limited. In addition, most dressings, including hydrogel wound dressings, are not electrically active, so that they cannot accelerate wound healing in response to physiological electrical signals or exogenous electrical field stimulation at the wound site during the wound healing process; furthermore, most hydrogel dressings are difficult to degrade and cause environmental pollution after use.

Disclosure of Invention

The invention solves the technical problems that the gel dressing in the prior art has no electric activity, insufficient toughness and mechanical strength and is difficult to degrade. In order to solve the technical problems, the invention aims to provide a degradable electroactive bacterial cellulose/MXene composite hydrogel and preparation and application thereof. The bacterial cellulose/MXene composite hydrogel prepared by the preparation method disclosed by the invention not only has biodegradability, good conductivity, mechanical property and water absorption property, but also has excellent biocompatibility. Furthermore, the composite hydrogel can promote the proliferation activity of skin cells under the combined action of electrical stimulation, so as to promote the healing of wounds, and therefore, the composite hydrogel has an important application value in the aspect of preparing skin wound dressings.

According to the first aspect of the invention, the preparation method of the degradable electroactive bacterial cellulose/MXene composite hydrogel comprises the following steps:

(1) crushing bacterial cellulose, and dissolving the crushed bacterial cellulose into a pre-cooled urea-containing alkali solution to obtain a transparent bacterial cellulose solution; the temperature in the dissolving process is-8 ℃ to-20 ℃;

(2) adding MXene nano materials into the transparent bacterial cellulose solution obtained in the step (1), and adding a cross-linking agent to obtain a mixed solution, wherein the mass fraction of the MXene nano materials in the mixed solution is 0.1-2%;

(3) and (3) pouring the mixed solution obtained in the step (2) into a mould, standing at the temperature of 4-8 ℃, and crosslinking the bacterial cellulose and the MXene nano material and crosslinking the bacterial cellulose to obtain the degradable electroactive bacterial cellulose/MXene composite hydrogel.

Preferably, the MXene nano material is Ti₃C₂And (3) nano materials.

Preferably, the standing time in the step (3) is 12-36 h.

Preferably, the mass percentage of the bacterial cellulose in the bacterial cellulose solution is 2-4%, and the mass volume ratio of the bacterial cellulose to the cross-linking agent is 4 g: 3-6 mL.

Preferably, the preparation method of the MXene nano material in the step (2) comprises the following steps: adding the titanium carbon aluminum ceramic material into a hydrofluoric acid solution, and etching the titanium carbon aluminum ceramic material under the action of magnetic stirring to obtain the MXene nano material.

Preferably, the mass fraction of the hydrofluoric acid solution is 30-49%, and the mass-volume ratio of the titanium-carbon-aluminum ceramic material to the hydrofluoric acid solution is 1g: 10-13 mL; the rotating speed of magnetic stirring is 400-600 rpm, the etching reaction temperature is 40-60 ℃, and the etching reaction time is 24-36 h.

According to another aspect of the invention, the degradable electroactive bacterial cellulose/MXene composite hydrogel prepared by any one of the preparation methods is provided.

Preferably, the composite hydrogel is internally provided with a three-dimensional network porous structure which is communicated with each other, and the pore diameter is 100-500 mu m.

According to another aspect of the invention, the application of the degradable electroactive bacterial cellulose/MXene composite hydrogel in preparing a skin wound dressing is provided.

Preferably, the composite hydrogel is coupled with an electrical stimulus to promote cell adhesion, growth, spreading and proliferation, wherein the voltage of the electrical stimulus is 50mV-400 mV.

Generally, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:

(1) the preparation method of the composite hydrogel has the advantages of simple process, easy control and low cost, is green and pollution-free from the use of raw materials to the preparation process, and is beneficial to industrial large-scale production. The degradable electroactive bacterial cellulose/MXene composite hydrogel prepared by the method has a double-layer structure, the compact outer layer of the hydrogel as a wound dressing is favorable for preventing wound dehydration and avoiding bacterial invasion, and the porous support layer provides mechanical strength through the pore walls formed by the bacterial cellulose and provides enough space for absorbing wound exudate and retaining a large amount of water.

(2) Compared with the traditional dry dressing, the composite hydrogel disclosed by the invention cannot be adhered to the surface of a wound, is easy to take out for changing the dressing, and avoids secondary wound caused by adhesion of the traditional dressing and the wound. Compared with the hydrogel in the prior art, the composite hydrogel has good biodegradability, can be degraded quickly under the action of cellulase, and can well avoid the pollution to the environment after use; the hydrogel has excellent mechanical properties, not only has good compressive and tensile strength, but also has very high compressive and tensile strain, and is hydrogel with excellent flexibility and elasticity; in addition, the electric conduction hydrogel also has good electric conductivity and water absorption performance and excellent biocompatibility, can promote the proliferation activity of skin cells under the combined action of electric stimulation, further promotes the healing of wounds, and has potential application prospect in the aspect of skin wound dressings.

(3) In the invention, the mixed solution of the MXene nano material and the bacterial cellulose solution contains 0.1-2% of the MXene nano material by mass. When the mass fraction of MXene is 1% or less, the compressive strength and the compressive modulus of the bacterial cellulose/MXene are improved along with the increase of the MXene content, and when the mass fraction of MXene is 2% or more, the compressive strength and the compressive modulus of the composite hydrogel still have strong mechanical properties, but the compressive strength and the compressive modulus are slightly reduced, so that the MXene is easy to agglomerate in a bacterial cellulose solution with viscosity to influence the mechanical properties, and the MXene content is not easy to be too high.

(4) In the invention, the standing is carried out at the temperature of 4-8 ℃, which is more beneficial to causing the cross-linking of the bacterial cellulose and the MXene nano material and causing the cross-linking of the bacterial cellulose, and the standing is not beneficial to causing the cross-linking of the bacterial cellulose and the MXene through the-OH interaction at the room temperature of 25 ℃ and the high temperature of 50 ℃.

(5) In the invention, the bacterial cellulose is dissolved in the pre-cooled aqueous alkali containing urea, the temperature in the dissolving process is-8 ℃ to-20 ℃, and under the low temperature condition, the urea alkali system can easily destroy the intra-chain hydrogen bonds of the bacterial cellulose and the bonding network of the inter-chain cellulose, thereby being more beneficial to the disentangling of cellulose polymer chains and finally being beneficial to the rapid dissolution of the cellulose chains in water.

Drawings

Fig. 1 is an exemplary flow chart of a method for preparing the degradable electroactive bacterial cellulose/MXene composite hydrogel of the present invention.

Fig. 2 is an optical representation of the regenerated BC hydrogel prepared in comparative example 1 and the degradable electroactive bacterial cellulose/MXene composite hydrogel with MXene contents of 0.1%, 0.2%, 0.5%, 1%, 2% prepared in examples 7-11 of the present invention, respectively.

FIG. 3 is a FSEM plot of the regenerated BC hydrogel prepared in comparative example 1 and the bacterial cellulose/MXene composite hydrogels prepared in inventive examples 7, 9 and 11 having MXene contents of 0.1%, 0.5% and 2%, respectively; wherein, FIGS. 3a, 3b and 3c are the surface structure diagrams of the regenerated BC hydrogel and the BC/MXene composite hydrogel with MXene contents of 0.1%, 0.5% and 2%, respectively; FIG. 3d, FIG. 3e, FIG. 3f are cross-sectional views of the regenerated BC hydrogel and the BC/MXene composite hydrogel having MXene contents of 0.1%, 0.5% and 2%, respectively, and FIG. 3g, FIG. 3h, FIG. 3i are enlarged cross-sectional views thereof, respectively.

Fig. 4 is a graph showing the results of the degradation performance test of the regenerated BC hydrogel prepared in comparative example 1 and the bacterial cellulose/MXene composite hydrogel having an MXene content of 2% prepared in example 11 of the present invention.

In fig. 5a, lines (a) - (f) are the compressive stress-strain curves of the regenerated BC hydrogel prepared in comparative example 1 and the degradable electroactive bacterial cellulose/MXene composite hydrogel with MXene contents of 0.1%, 0.2%, 0.5%, 1% and 2% prepared in examples 7-11 of the present invention, respectively, in sequence; FIG. 5b is a graph of the compression modulus results of bacterial cellulose/MXene composite hydrogels with different MXene contents.

Fig. 6 is a graph showing the results of conductivity tests on the regenerated BC hydrogel prepared in comparative example 1 and the degradable electroactive bacterial cellulose/MXene composite hydrogel having MXene contents of 0.1%, 0.2%, 0.5%, 1%, 2% prepared in examples 7 to 11 of the present invention, respectively. As shown in FIG. 6a, the BC/MXene composite hydrogel with MXene content of 1% is communicated with the circuit to light the LED lamp. FIG. 6b shows that when MXene content is increased from 0.1% to 2%, the bacterial fiber prepared by the present inventionThe conductivity of the vitamin/MXene composite hydrogel is from 2.83X 10^-5The S/cm is increased to 7.04X 10^-4S/cm。

FIG. 7 is a graph showing the results of the cytotoxicity test, i.e., Lactate Dehydrogenase (LDH) assay, of the regenerated BC hydrogel prepared in comparative example 1 and the degradable electroactive bacterial cellulose/MXene composite hydrogel having MXene contents of 0.1%, 0.2%, 0.5%, 1%, 2% prepared in examples 7-11 of the present invention, respectively, wherein TCP refers to a cell culture plate control group without hydrogel material.

FIG. 8 is a graph showing the results of measuring cell proliferation activity (CCK-8) of the regenerated BC hydrogel prepared in comparative example 1 and the degradable electroactive bacterial cellulose/MXene composite hydrogel having MXene contents of 0.1%, 0.2%, 0.5%, 1%, and 2% prepared in examples 7 to 11 of the present invention, respectively, wherein TCP refers to a cell culture plate control group without hydrogel material.

FIG. 9 is a graph showing the results of blood compatibility (hemolysis) tests of the regenerated BC hydrogel prepared in comparative example 1 and the degradable electroactive bacterial cellulose/MXene composite hydrogel having MXene contents of 0.1%, 0.2%, 0.5%, 1%, 2% prepared in examples 7-11 of the present invention, wherein distilled water and physiological saline were used for the positive and negative control groups, respectively.

FIG. 10 is a graph showing the results of cell adhesion and morphology detection on bacterial cellulose/MXene complex hydrogels with different MXene contents; wherein FIG. 10a is a cytoskeletal fluorescence picture of NIH3T3 cells on the regenerated BC hydrogel prepared in comparative example 1, and FIGS. 10b, 10c, 10d, 10e and 10f are cytoskeletal fluorescence pictures of NIH3T3 cells on the degradable electroactive bacterial cellulose/MXene composite hydrogel with MXene contents of 0.1%, 0.2%, 0.5%, 1% and 2% respectively prepared in examples 7-11 of the present invention from NIH3T3 cells.

Fig. 11 is a graph showing the effect of the regenerated BC hydrogel prepared in comparative example 1 and the bacterial cellulose/MXene composite hydrogel having an MXene content of 1% and 2% prepared in examples 10 and 11 of the present invention on cell proliferation activity under the coupling effect with electrical stimulation.

FIG. 12 is a photograph showing the gelling effect of the bacterial cellulose/MXene/ECH composite solutions having an MXene content of 0.5% prepared in example 12 and comparative examples 2 and 3 after standing at 4 deg.C, 25 deg.C and 50 deg.C, respectively, for 12 hours.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

MXene (transition metal carbide or carbonitride) is a new type of two-dimensional nanomaterial with a graphene-like two-dimensional structure obtained by selective etching of the original MAX phase having the general formula M_n+1AX_n(n-1, 2,3) wherein M is a transition metal, a is one of IIIA or IVA, and X is carbon or nitrogen. The MAX phase has a layered and P63/mmc symmetric hexagonal structure, M layers are almost hexagonally closed and aggregated, and X atoms fill in octahedral positions to form M layers which are alternately arranged_n+1X_nThe slice layer is connected with the tightly stacked A atomic layer to form a MAX phase structure. Wherein, M-X atom layers are mainly covalent bonds and ionic bonds, M-A atom layers are mainly connected by metal bonds, and compared with the M-X bonds, the M-A bonds have weaker binding force, so that the A atom layers have higher reactivity and are easy to strip. Namely, removing the A layer from the structure by a proper method (chemical liquid phase etching method) to obtain a novel two-dimensional stacked layered structure, wherein the MXene formed by the method has three different structures, namely M₂X、M₃X₂Or M₄X₃。Ti₃C₂T_xIs MXene synthesized for the first time in 2011, and two wet methods are generally adopted to prepare MXene: the layered precursor is etched by hydrofluoric acid (HF), or HF generated in situ by mixing strong acid and fluoride salt is etched. Such as etching Ti using 40 wt% HF₃AlC₂The weak bond in (A) can give Ti of a multilayer structure₃C₂T_x(T represents a terminal functional group-OH, -O or-F).

The invention provides a preparation method of degradable electroactive bacterial cellulose/MXene composite hydrogel, which comprises the following steps:

1) slowly adding the titanium-carbon-aluminum ceramic Material (MAX) into hydrofluoric acid (HF) solution, and etching to prepare Ti with a multilayer structure and good conductivity under the action of constant-temperature magnetic stirring₃C₂T_x-MXene nanomaterials; specifically, a water bath or an oil bath may be employed to maintain a constant temperature condition; titanium carbon aluminum ceramic Material (MAX) is available from scientific and technological Limited, Jilin province;

2) crushing dried bacterial cellulose, and adding the crushed bacterial cellulose into a low-temperature NaOH/urea alkali system for dissolving to form a transparent bacterial cellulose solution;

3) adding the MXene nano material prepared in the step 1) into the bacterial cellulose solution dissolved in the step 2), fully and uniformly mixing, and then adding a crosslinking agent Epichlorohydrin (ECH) to form a bacterial cellulose/MXene/ECH mixed solution;

4) pouring the bacterial cellulose/MXene/ECH mixed solution obtained in the step 3) into moulds (such as 24-hole plates or culture dishes) with different shapes and sizes, placing the moulds at the temperature of 4-8 ℃, and standing for a period of time to form the bacterial cellulose/MXene composite hydrogel with different shapes and sizes through crosslinking.

In some embodiments of the above preparation method of the present invention, in step 1), the mass fraction of the HF solution is 30 to 49%, preferably 49%; the mass-to-volume ratio (w/v) of MAX to HF solution is 1g: 10-13 mL, preferably 1g:12 mL.

In some embodiments of the above preparation method of the invention, in step 1), the reaction temperature of MAX and HF solution is 40-60 ℃, preferably 60 ℃; the rotating speed of the magnetic stirring is 400-600 rpm, preferably 500 rpm; the reaction time in the HF solution is 24-36 h, preferably 30 h.

In some embodiments of the above-mentioned preparation method of the present invention, in step 2), the bacterial cellulose may be secreted from Acetobacter gluconicum (Gluconacetobacter xylinum) strain ATCC 53582, which is an aerobic gram-negative bacterium purchased from American model culture Collection Bank(American Type Culture Collection, Manassas, Va., USA), in addition, other bacterial celluloses in the prior art can also be used in the present invention. Purifying the bacterial cellulose for later use; then, drying directly by using an oven, drying by vacuum heating and low-temperature freeze drying, preferably low-temperature freeze drying, so that a fluffy structure is formed; drying the bacterial cellulose, and crushing the bacterial cellulose by using a tissue crusher so as to facilitate subsequent dissolution; the NaOH/urea alkali system is W_NaOH：W_Urea：W_{Distilled water}＝7:12:81。

In some embodiments of the above preparation method of the present invention, in step 2), the NaOH/urea alkali solution needs to be pre-cooled in a refrigerator at 4 ℃ for 4 hours, and the low temperature dissolution temperature of the bacterial cellulose in the NaOH/urea alkali system is-8 ℃ to-20 ℃, preferably-12 ℃; the mass percentage of the bacterial cellulose prepared after dissolution is 2-4%, and the preferred mass percentage is 3%.

In some embodiments of the above preparation method of the present invention, in step 3), after MXene is added to the bacterial cellulose solution and mixed sufficiently, the mass fraction of MXene is 0.1 to 2%, preferably 2%; and the mass to volume ratio (w/v) of the bacterial cellulose to the ECH is 4 g: 3-6 mL, preferably 4 g: 4.2 mL.

In some embodiments of the above preparation method of the present invention, in step 4), the bacterial cellulose/MXene mixed solution is poured into molds (such as 24-well plates or petri dishes) of different shapes and sizes immediately after adding the ECH, and then is allowed to stand at 4-8 ℃ for 12-36 h, preferably at 4 ℃ for 24 h.

In some embodiments of the above preparation method of the present invention, in step 4), the bacterial cellulose/MXene mixed solution is poured into molds (such as a 24-well plate or a petri dish) with different shapes and sizes immediately after adding the ECH, and bacterial cellulose/MXene/ECH composite gels with different shapes and sizes are formed by controlling the mass of the bacterial cellulose/MXene/ECH mixed solution; for example, a bacterial cellulose/MXene composite hydrogel column with a cross section diameter of 15mm and a height of 8-14 mm (preferably a height of 14mm) or a composite hydrogel film with a diameter of 9cm and a thickness of 3-6 mm can be formed.

The invention also provides degradable electroactive bacterial cellulose/MXene composite hydrogel produced by the preparation method, the surface of the degradable electroactive bacterial cellulose/MXene composite hydrogel is a compact less-porous or non-porous structure, the interior of the degradable electroactive bacterial cellulose/MXene composite hydrogel is a three-dimensional network porous structure which is communicated with each other, and the pore diameter is 100-500 mu m.

The invention further provides application of the degradable electroactive bacterial cellulose/MXene composite hydrogel generated by the preparation method in preparation of skin wound dressings. Specifically, NIH3T3 fibroblasts can be well adhered, grown, spread and proliferated on the surface of the bacterial cellulose/MXene composite hydrogel; and under the action coupled with the electric stimulation, the compound has more positive promoting effect on the behaviors of NIH3T3 cells such as adhesion, growth, spreading, proliferation and the like.

Example 1: preparation and purification of Bacterial Cellulose (BC)

Bacterial cellulose is secreted by acetobacter gluconicum (g.xylinum) strain ATCC 53582. The culture medium for the growth of the strain is a liquid culture medium (Hestrin)&Schrammm, HS), the main components of which are: 5.0gL^-1Yeast extract, 20gL^-1Glucose, 1.5gL^-1Citric acid monohydrate, 20gL^-1Glucose and 6.8gL^-1Disodium phosphate dodecahydrate. The medium was prepared from Milli-Q ultrapure water and autoclaved at high temperature (121 ℃) for 20min before use. The gluconacetobacter is inoculated into the sterile Herstin-Schramm culture medium, and the volume of the inoculated strain liquid accounts for 10% of the total culture medium volume.

The synthesized bacterial cellulose was purified by the following method. Specifically, the obtained bacterial cellulose membrane was immersed in distilled water for 2 days to remove excess medium residue therein, treated with 0.1M NaOH solution at 100 ℃ for 30min to remove gluconacetobacter therein, and then immersed in distilled water for 3 days with water changed every day to further remove impurities and NaOH solution therein and to make the pH thereof neutral. Finally, the mixture is sterilized by high pressure at high temperature (121 ℃) for 20min and then stored.

Example 2

Fig. 1 is an exemplary flow chart of a method for preparing the degradable electroactive bacterial cellulose/MXene composite hydrogel of the present invention. The preparation method of the degradable electroactive bacterial cellulose/MXene composite hydrogel comprises the following steps:

1) slowly adding 10g of titanium-carbon-aluminum ceramic Material (MAX) into 100mL of 30% hydrofluoric acid solution, magnetically stirring and reacting for 24h at 400rpm under the condition of 40 ℃ oil bath, centrifugally cleaning, collecting precipitate, and freeze-drying at low temperature to obtain Ti with a multilayer structure and good conductivity₃C₂T_x-MXene nanomaterials;

2) the purified BC prepared in example 1 was freeze-dried at a low temperature, and then pulverized by a tissue crusher to obtain BC powder. 2g of the BC powder was added to a beaker containing 98g of a NaOH/urea base solution pre-cooled in a refrigerator at 4 deg.C (the NaOH/urea base system is W)_NaOH：W_Urea：W_{Distilled water}7:12: 81); then placing the beaker at-8 ℃ for dissolving to form a transparent bacterial cellulose solution with the mass fraction of 2%;

3) adding 0.1g of MXene material prepared in the step 1) into 99.9g of the bacterial cellulose solution obtained in the step 2), fully and uniformly mixing to obtain a bacterial cellulose/MXene mixed solution with the MXene content of 0.1%, and then mixing the bacterial cellulose and Epoxy Chloropropane (ECH) in a mass-to-volume ratio (w/v) of 4 g: adding ECH into 3mL of the mixed solution to form a BC/MXene/ECH mixed solution with MXene content of 0.1%;

4) pouring the BC/MXene/ECH mixed solution obtained in the step 3) into a 24-hole plate, and standing for 12 hours at 4 ℃ to obtain the BC/MXene composite hydrogel with the MXene content of 0.1%, the diameter of 15mm and the height of 9 mm.

Example 3

1) 10g of a titanium-carbon-aluminum ceramic Material (MAX) was slowly added to a solution containing 120mL of 40% hydrofluoric acid and the strip was oil-bathed at 50 deg.CMagnetically stirring at 500rpm for reaction etching for 24 hr, centrifugally cleaning, collecting precipitate, and freeze drying at low temperature to obtain Ti with multilayer structure and excellent conductivity₃C₂T_x-MXene nanomaterials.

2) The purified BC prepared in example 1 was freeze-dried at a low temperature, and then pulverized by a tissue crusher to obtain BC powder. 2.5g of the BC powder was added to a beaker containing 97.5g of a refrigerator-precooled NaOH/urea base solution at 4 ℃ (the NaOH/urea base system is W)_NaOH：W_Urea：W_{Distilled water}7:12: 81); and then placing the beaker at the temperature of-12 ℃ for dissolving to form a transparent bacterial cellulose solution with the mass fraction of 2.5%.

3) Adding 0.2g of MXene material obtained in the step 1) into 99.8g of the bacterial cellulose solution obtained in the step 2), fully and uniformly mixing to obtain a bacterial cellulose/MXene mixed solution with the MXene content of 0.2%, and then mixing the bacterial cellulose and Epoxy Chloropropane (ECH) in a mass-to-volume ratio (w/v) of 4 g: ECH was added in a proportion of 3.6mL to form a mixed BC/MXene/ECH solution having an MXene content of 0.2%.

4) Pouring the BC/MXene/ECH mixed solution obtained in the step 3) into a 24-hole plate, and standing for 24 hours at 6 ℃ to obtain the BC/MXene composite hydrogel with the MXene content of 0.2%, the diameter of 15mm and the height of 10 mm.

Example 4

1) slowly adding 10g of titanium-carbon-aluminum ceramic Material (MAX) into a hydrofluoric acid solution containing 130mL of 49%, magnetically stirring at 600rpm under the condition of 60 ℃ oil bath for reaction and etching for 36h, centrifugally cleaning, collecting precipitate, and freeze-drying at low temperature to obtain Ti with a multilayer structure and good conductivity₃C₂T_x-MXene nanomaterials;

2) purified BC prepared in example 1 was passed through at low temperatureAfter freeze-drying, the BC powder is obtained by crushing the mixture by a tissue crusher. 3g of the BC powder was added to a beaker containing 97g of a NaOH/urea base solution pre-cooled in a refrigerator at 4 deg.C (the NaOH/urea base system is W)_NaOH：W_Urea：W_{Distilled water}7:12: 81); and then placing the beaker in a refrigerator at the temperature of-20 ℃ for dissolving to form a transparent bacterial cellulose solution with the mass fraction of 3%.

3) Adding 0.5g of MXene material obtained in the step 1) into 99.5g of the bacterial cellulose solution obtained in the step 2), fully and uniformly mixing to obtain a bacterial cellulose/MXene mixed solution with the MXene content of 0.5%, and then mixing the bacterial cellulose and Epoxy Chloropropane (ECH) in a mass-to-volume ratio (w/v) of 4 g: ECH was added in a proportion of 5mL to form a mixed BC/MXene/ECH solution having an MXene content of 0.5%.

4) Pouring the BC/MXene/ECH mixed solution obtained in the step 3) into a 24-hole plate, and standing for 24 hours at the temperature of 8 ℃ to obtain the BC/MXene composite hydrogel with the MXene content of 0.5%, the diameter of 15mm and the height of 12 mm.

Example 5

1) slowly adding 10g of titanium-carbon-aluminum ceramic Material (MAX) into a hydrofluoric acid solution containing 110mL of 40%, magnetically stirring at 500rpm under the condition of 50 ℃ oil bath for reaction and etching for 26h, centrifugally cleaning, collecting precipitate, and freeze-drying at low temperature to obtain Ti with a multilayer structure and good conductivity₃C₂T_x-MXene nanomaterials;

2) the purified BC prepared in example 1 was freeze-dried at a low temperature, and then pulverized by a tissue crusher to obtain BC powder. 4g of the BC powder was added to a beaker containing 96g of a NaOH/urea base solution pre-cooled in a refrigerator at 4 ℃ (the NaOH/urea base system is W)_NaOH：W_Urea：W_{Distilled water}7:12: 81); then placing the beaker in a refrigerator at the temperature of-20 DEG CAnd (4) dissolving, namely forming a transparent bacterial cellulose solution with the mass fraction of 4%.

3) Adding 1g of MXene material obtained in the step 1) into 99g of the bacterial cellulose solution obtained in the step 2), fully and uniformly mixing to obtain a bacterial cellulose/MXene mixed solution with the MXene content of 1%, and then mixing the bacterial cellulose and Epoxy Chloropropane (ECH) in a mass-to-volume ratio (w/v) of 4 g: ECH was added at a ratio of 6mL to form a mixed BC/MXene/ECH solution having an MXene content of 1%.

4) Pouring the BC/MXene/ECH mixed solution obtained in the step 3) into a 24-hole plate, and standing the mixed solution at 8 ℃ for 30 hours to obtain the BC/MXene composite hydrogel with the MXene content of 1%, the diameter of 15mm and the height of 13 mm.

Example 6

1) slowly adding 10g of titanium-carbon-aluminum ceramic Material (MAX) into a hydrofluoric acid solution containing 120mL of 40%, magnetically stirring at 600rpm under the condition of 50 ℃ oil bath for reaction and etching for 30h, centrifugally cleaning, collecting precipitate, and freeze-drying at low temperature to obtain Ti with a multilayer structure and good conductivity₃C₂T_x-MXene nanomaterials;

2) the purified BC prepared in example 1 was freeze-dried at a low temperature, and then pulverized by a tissue crusher to obtain BC powder. 4g of the BC powder was added to a beaker containing 96g of a NaOH/urea base solution pre-cooled in a refrigerator at 4 ℃ (the NaOH/urea base system is W)_NaOH：W_Urea：W_{Distilled water}7:12: 81); and then placing the beaker in a refrigerator at the temperature of-12 ℃ for dissolving to form a transparent bacterial cellulose solution with the mass fraction of 4%.

3) Adding 2g of MXene material obtained in the step 1) into 98g of the bacterial cellulose solution obtained in the step 2), fully and uniformly mixing to obtain a bacterial cellulose/MXene mixed solution with the MXene content of 2%, and then mixing the bacterial cellulose and Epoxy Chloropropane (ECH) in a mass-to-volume ratio (w/v) of 4 g: ECH was added at a ratio of 6mL to form a BC/MXene/ECH mixed solution having an MXene content of 2%.

4) Pouring the BC/MXene/ECH mixed solution obtained in the step 3) into a 24-hole plate, and standing the mixed solution at 8 ℃ for 30 hours to obtain the BC/MXene composite hydrogel with the MXene content of 2%, the diameter of 15mm and the height of 14 mm.

Example 7

1) slowly adding 10g of titanium-carbon-aluminum ceramic Material (MAX) into hydrofluoric acid solution containing 120mL of 49%, magnetically stirring at 500rpm under the condition of 60 ℃ oil bath for reaction and etching for 30h, centrifugally cleaning, collecting precipitate, and freeze-drying at low temperature to obtain Ti with a multilayer structure and good conductivity₃C₂T_x-MXene nanomaterials;

2) the purified BC prepared in example 1 was freeze-dried at a low temperature, and then pulverized by a tissue crusher to obtain BC powder. 3g of the BC powder was added to a beaker containing 97g of a NaOH/urea base solution pre-cooled in a refrigerator at 4 deg.C (the NaOH/urea base system is W)_NaOH：W_Urea：W_{Distilled water}7:12: 81); and then placing the beaker at the temperature of-12 ℃ for dissolving to form a transparent bacterial cellulose solution with the mass fraction of 3%.

3) Adding 0.1g of MXene material obtained in the step 1) into 99.9g of bacterial cellulose solution obtained in the step 2), fully and uniformly mixing to obtain a bacterial cellulose/MXene mixed solution with the MXene content of 0.1%, and then mixing the bacterial cellulose and Epoxy Chloropropane (ECH) according to a mass-to-volume ratio (w/v) of 4 g: ECH was added at a rate of 4.2mL to form a mixed BC/MXene/ECH solution having an MXene content of 0.1%.

4) Pouring the BC/MXene/ECH mixed solution obtained in the step 3) into a 24-hole plate, and standing the mixed solution at 4 ℃ for 24 hours to obtain the BC/MXene composite hydrogel with the MXene content of 0.1%, the diameter of 15mm and the height of 14mm (shown in figure 2).

Example 8

Based on example 7, the difference is: changing the amount of MXene taken in the step 3) to 0.2g, changing the amount of the 3% BC solution to 99.8g, and the rest of the steps are the same as those in the example 7, so as to obtain the BC/MXene composite hydrogel with the MXene content of 0.2%, the diameter of 15mm and the height of 14mm (as shown in figure 2).

Example 9

Based on example 7, the difference is: changing the amount of MXene taken in the step 3) to 0.5g, changing the amount of the 3% BC solution to 99.5g, and the rest of the steps are the same as those in the example 7, so as to obtain the BC/MXene composite hydrogel with the MXene content of 0.5%, the diameter of 15mm and the height of 14mm (as shown in figure 2).

Example 10

Based on example 7, the difference is: changing the amount of MXene taken in the step 3) to 1g, changing the amount of the used 3% BC solution to 99g, and the rest of the steps are the same as those in the example 7, so as to obtain the BC/MXene composite hydrogel with the MXene content of 1%, the diameter of 15mm and the height of 14mm (as shown in figure 2).

Example 11

Based on example 7, the difference is: changing the amount of MXene taken in the step 3) to 2g, changing the amount of 3% BC solution to 98g, and the rest of the steps are the same as those in the example 7, so as to obtain the BC/MXene composite hydrogel with the MXene content of 2%, the diameter of 15mm and the height of 14mm (as shown in figure 2).

Example 12

3) adding 0.5g of MXene material prepared in the step 1) into 99.5g of the bacterial cellulose solution obtained in the step 2), fully and uniformly mixing to obtain a bacterial cellulose/MXene mixed solution with the MXene content of 0.5%, and then mixing the bacterial cellulose and Epoxy Chloropropane (ECH) according to a mass-to-volume ratio (w/v) of 4 g: adding ECH into 3mL of the mixed solution to form a BC/MXene/ECH mixed solution with MXene content of 0.5%;

4) pouring the BC/MXene/ECH mixed solution obtained in the step 3) into a 25mL glass bottle, and standing the glass bottle at 4 ℃ for 12 hours to obtain the BC/MXene composite hydrogel with the MXene content of 0.5% (as shown in figure 12).

Comparative example 1

The preparation method of the degradable bacterial cellulose regenerated hydrogel (without adding MXene conductive material) comprises the following steps:

1) the purified BC prepared in example 1 was freeze-dried at a low temperature, and then pulverized by a tissue crusher to obtain BC powder. 3g of BC powder was added to a beaker containing 97g of NaOH/urea base solution pre-cooled in a refrigerator at 4 ℃ (the NaOH/urea base system is W)_NaOH：W_Urea：W_{Distilled water}7:12: 81); and then, placing the beaker at a temperature of-12 ℃ for dissolving to obtain a transparent bacterial cellulose solution with the mass fraction of 3%.

2) In the bacterial cellulose solution obtained in the step 1), the mass-to-volume ratio (w/v) of the bacterial cellulose to the Epichlorohydrin (ECH) is 4 g: ECH was added at a ratio of 4.2mL to form a BC/ECH mixed solution.

3) Pouring the BC/ECH mixed solution obtained in the step 2) into a 24-well plate, and standing the mixed solution at 4 ℃ for 24 hours to form a regenerated BC hydrogel with the diameter of 15mm and the height of 14mm (shown in figure 2).

Comparative example 2

Based on example 12, the difference is: pouring the BC/MXene/ECH mixed solution in the step 4) into a 25mL glass bottle, and standing the mixed solution at 25 ℃ for 12 h. As a result, the BC/MXene composite solution with 0.5 percent of MXene content is not well gelatinized and still has certain fluidity. (see fig. 12)

Comparative example 3

Based on example 12, the difference is: pouring the BC/MXene/ECH mixed solution in the step 4) into a 25mL glass bottle, and standing the mixed solution at 50 ℃ for 12 h. As a result, the BC/MXene composite solution with 0.5 percent of MXene content is not well gelatinized and still has certain fluidity. (see fig. 12)

Results and analysis:

the regenerated BC hydrogel (without MXene) prepared in comparative example 1 and the BC/MXene composite hydrogel with MXene contents of 0.1%, 0.5% and 2% prepared in inventive examples 7, 9 and 11 were immersed in 75% alcohol solution for 30min at the same time, and then immersed in distilled water for 3 days to remove excess ECH in the hydrogel and to make the pH neutral. And (3) after the clean regenerated BC hydrogel and the BC/MXene composite hydrogel are treated, and the change of the structural appearance of the regenerated BC hydrogel and the BC/MXene composite hydrogel is observed after freeze drying. As can be seen from fig. 3, the prepared regenerated BC hydrogel and BC/MXene composite hydrogel both have a bilayer morphology structure, the surface thereof has a less-porous or non-porous relatively dense structure (fig. 3a, 3b, 3c), the interior thereof is a three-dimensional porous network structure (fig. 3d, 3e, 3f) which is interconnected, and fig. 3g, 3h, 3i are respectively enlarged corresponding cross-sectional views thereof. The aperture is 100-500 μm; and with the increase of the percentage content of MXene, the amount of MXene embedded on the surface and the pore wall of the BC/MXene composite hydrogel gradually increases, and the roughness of the BC/MXene composite hydrogel also gradually increases. White arrows indicate embedding of MXene in the BC gel matrix and black arrows indicate embedding of MXene on the surface of the gel as well as on the walls of the pores.

The results of the degradation performance test of the regenerated BC hydrogel prepared in comparative example 1 and the bacterial cellulose/MXene composite hydrogel having an MXene content of 2% prepared in example 11 of the present invention are shown in fig. 4. The result shows that the bacterial cellulose/MXene composite hydrogel with the MXene content of 2% prepared in the embodiment 11 can be completely degraded after reacting for 210min under the action of the cellulase; and the regenerated BC hydrogel is not completely degraded after reacting for 210min under the action of cellulase. The bacterial cellulose/MXene composite hydrogel prepared by the method has good biodegradability.

The mechanical property test results of the regenerated BC hydrogel prepared in comparative example 1 and the degradable electroactive bacterial cellulose/MXene composite hydrogel having MXene contents of 0.1%, 0.2%, 0.5%, 1%, 2% prepared in examples 7 to 11 of the present invention are shown in fig. 5. As a result, it is found that when the mass fraction of MXene is 1% or less, the compressive strength (FIG. 5a) and the compressive modulus (FIG. 5b) of the bacterial cellulose/MXene are increased with the increase of the MXene content, and when the mass fraction of MXene is 2%, the compressive strength and the compressive modulus of the composite hydrogel of the invention begin to slightly decrease but are still obviously higher than those of the raw BC hydrogel, which indicates that the bacterial cellulose/MXene composite hydrogel prepared by the invention has good mechanical properties, and also indicates that the MXene content in the composite hydrogel cannot be too high, and the MXene with high concentration is easy to agglomerate in a bacterial cellulose solution with viscosity, so the mechanical properties of the MXene can be influenced to a certain extent.

The conductivity test results of the regenerated BC hydrogel prepared in comparative example 1 and the degradable electroactive bacterial cellulose/MXene composite hydrogel having MXene contents of 0.1%, 0.2%, 0.5%, 1%, and 2% prepared in examples 7 to 11 of the present invention are shown in fig. 6. The results show that the combination of BC/MXene composite hydrogel with MXene content of 1% and the circuit can make the LED lamp light up (FIG. 6 a). In addition, the MXene is doped to ensure that the BC/MXene composite hydrogel has good electrical activity, for example, under the condition of keeping other preparation conditions unchanged, when the MXene content is increased from 0.1% to 2%, the electrical conductivity of the bacterial cellulose/MXene composite hydrogel prepared by the invention is increased from 0.1% to 2%2.83×10^-5The S/cm is increased to 7.04X 10^-4S/cm (FIG. 6 b).

The regenerated BC hydrogel prepared in comparative example 1 and the degradable electroactive bacterial cellulose/MXene composite hydrogel having MXene contents of 0.1%, 0.2%, 0.5%, 1% and 2% prepared in examples 7 to 11 of the present invention were subjected to biocompatibility test, and the results are shown in fig. 7, 8, 9 and 10. As can be seen from FIG. 7, after NIH3T3 cells were cultured on TCP, regenerated BC hydrogel and BC/MXene composite hydrogel with different MXene contents for 1 day and 3 days, the release amounts of Lactate Dehydrogenase (LDH) in each group of TCP, regenerated BC hydrogel and BC/MXene composite hydrogel were not significantly different, which indicates that the BC/MXene composite hydrogel with different MXene contents prepared by the present invention is non-toxic and biologically safe. As can be seen from FIG. 8, the viability of the cells on the BC/MXene composite hydrogel gradually increased with the increasing of MXene content, and the BC/MXene composite hydrogel with 2% of MXene content is most beneficial to promote the growth and proliferation of the cells. In addition, as can be seen from fig. 9, the hemolysis rate of all the BC/MXene composite hydrogels with different MXene contents is less than 2%, which is within the range of international safety standards, and thus, the hydrogel prepared by the present invention has good blood compatibility.

The fluorescence staining results of the morphology of NIH3T3 cells cultured on the degradable electroactive bacterial cellulose/MXene composite hydrogel with 0.1%, 0.2%, 0.5%, 1%, 2% of MXene content prepared in comparative example 1 and examples 7-11 of the present invention are shown in fig. 10. Fig. 10a is a cytoskeleton fluorescence picture of NIH3T3 cells on regenerated BC hydrogel, and fig. 10b, fig. 10c, fig. 10d, fig. 10e and fig. 10f are cytoskeleton fluorescence pictures of NIH3T3 cells on degradable electroactive bacterial cellulose/MXene composite hydrogel with MXene content of 0.1%, 0.2%, 0.5%, 1% and 2% prepared in examples 7-11 of the present invention by NIH3T3 cells, respectively. The result shows that after NIH3T3 cells are cultured on the gel for 7 days, the number of the cells on the BC/MXene composite hydrogel is gradually increased along with the gradual increase of the MXene content, and the spreading state is better and better; the BC/MXene composite hydrogel with the MXene content of 2% has the largest number of cells, the best cell growth and spreading and presents tight connection among cells. The results show that the bacterial cellulose/MXene composite hydrogel prepared by the method has good biocompatibility, and the BC/MXene composite hydrogel with the MXene content of 2% is most beneficial to the adhesion, growth, spreading and proliferation of cells, so that the BC/MXene composite hydrogel has potential application value in the aspect of wound dressing.

The regenerated BC hydrogel prepared in comparative example 1 and the bacterial cellulose/MXene composite hydrogel having an MXene content of 1% and 2% prepared in examples 10 and 11 of the present invention were coupled to an electrical stimulus, respectively, and after 3 days of culture of NIH3T3 cells, the result of staining of dead/live cells is shown in fig. 11, and it was found that the BC/MXene composite hydrogel significantly promoted the growth and proliferation of cells at a voltage of 100mV and an MXene content of 2%. Therefore, the electroactive composite hydrogel (BC/MXene-2%) can be used as a wound dressing and coupled with electrical stimulation to accelerate wound healing.

In addition, fig. 12 shows the gelling effect of the bacterial cellulose/MXene composite solution with 0.5% of MXene content prepared in example 12 of the present invention and comparative examples 2 and 3, and as a result, the BC/MXene/ECH mixed solution can well form BC/MXene composite hydrogel after the mixed solution is added and is kept standing for 12 hours at 4 ℃, and no flow of the composite solution is found when the glass bottle is inverted. And the BC/MXene/ECH mixed solution does not gelatinize well after standing for 12 hours at the temperature of 25 ℃ and 50 ℃ respectively, and shows certain liquid fluidity, which indicates that the low-temperature standing is more favorable for the gelatinization of the bacterial cellulose/MXene.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A preparation method of degradable electroactive bacterial cellulose/MXene composite hydrogel is characterized by comprising the following steps:

(1) pulverizing bacterial cellulose, and dissolving into precooled solutionObtaining a transparent bacterial cellulose solution in an aqueous alkali of urea; the temperature of the dissolution process is-8 deg.C^oC~-20 ^oC；

(2) Adding MXene nano material into the transparent bacterial cellulose solution obtained in the step (1), wherein the MXene nano material is Ti₃C₂Adding a crosslinking agent which is epoxy chloropropane into the nano material to obtain a mixed solution, wherein the mass fraction of the MXene nano material in the mixed solution is 0.1-2%;

the mass percentage of the bacterial cellulose in the bacterial cellulose solution is 2-4%, and the mass volume ratio of the bacterial cellulose to the cross-linking agent is 4 g: 3-6 mL;

the preparation method of the MXene nano material comprises the following steps: adding a titanium-carbon-aluminum ceramic material into a hydrofluoric acid solution, and etching the titanium-carbon-aluminum ceramic material under the action of magnetic stirring to obtain an MXene nano material with a multilayer structure;

the mass fraction of the hydrofluoric acid solution is 30-49%, and the mass volume ratio of the titanium-carbon-aluminum ceramic material to the hydrofluoric acid solution is 1g: 10-13 mL; the rotating speed of magnetic stirring is 400-600 rpm, and the etching reaction temperature is 40-60 DEG C^oC, etching reaction time is 24-36 h;

(3) pouring the mixed solution obtained in the step (2) into a mould and placing the mixed solution in a position 4^oC~8 ^oAnd C, standing for 12-36 hours under the condition of C, and crosslinking the bacterial cellulose and the MXene nano material with a multilayer structure and crosslinking the bacterial cellulose to obtain the degradable electroactive bacterial cellulose/MXene composite hydrogel.

2. The degradable electroactive bacterial cellulose/MXene composite hydrogel prepared by the preparation method of claim 1.

3. The degradable electroactive bacterial cellulose/MXene composite hydrogel according to claim 2, wherein the composite hydrogel has an interconnected three-dimensional network porous structure with a pore size of 100-500 μm.

4. Use of the degradable electroactive bacterial cellulose/MXene composite hydrogel according to claim 2 or 3 for preparing a skin wound dressing.

5. The use of claim 4, wherein the composite hydrogel is coupled to an electrical stimulus to promote cell adhesion, growth, spreading and proliferation, wherein the electrical stimulus has a voltage of from 50mV to 400 mV.