CN112979999B

CN112979999B - Biological macromolecule and modified halloysite composite hydrogel and preparation and application thereof

Info

Publication number: CN112979999B
Application number: CN202110211322.0A
Authority: CN
Inventors: 赵荟菁; 徐张鹏; 孟凯
Original assignee: Suzhou University; Nantong Textile and Silk Industrial Technology Research Institute
Current assignee: Suzhou University; Nantong Textile and Silk Industrial Technology Research Institute
Priority date: 2021-02-25
Filing date: 2021-02-25
Publication date: 2022-11-11
Anticipated expiration: 2041-02-25
Also published as: CN112979999A

Abstract

The invention relates to a biomacromolecule and modified halloysite composite hydrogel as well as preparation and application thereof, wherein the biomacromolecule and modified halloysite composite hydrogel comprises a chemical crosslinking network of silk fibroin and chitosan, and a plurality of acid-modified halloysite nanotubes are distributed in the chemical crosslinking network. The composite hydrogel comprises silk fibroin and chitosan with good biocompatibility and modified halloysite, and combines the physiological activity of biomacromolecules with the drug loading capacity of the halloysite to meet the requirements of antibiosis, hemostasis and healing promotion at each stage of wound healing. The thermal stability, swelling ratio, mechanical properties of hydrogels formed by chemical crosslinking were investigated. The in vitro biological performance of the hydrogel was evaluated by bacteriostatic, hemostatic and cell migration experiments. The effect of the hydrogel on wound healing was evaluated using the SD rat full-thickness skin wound model.

Description

Biological macromolecule and modified halloysite composite hydrogel and preparation and application thereof

Technical Field

The invention relates to the technical field of hydrogel dressings, in particular to a biomacromolecule and modified halloysite composite hydrogel and preparation and application thereof.

Background

The skin is the organ of the human body which is firstly affected by external stress factors, and once the skin is damaged, the barrier function of the skin is weakened, so that the tissue cells are damaged. Skin wound healing has a complex set of mechanisms that can accelerate the healing process by stimulating cell adhesion, migration and angiogenesis. The moist environment is more favorable for wound healing, and the novel wound dressing needs to absorb and maintain a proper amount of wound exudates, provides nutrition for a wound bed, provides an optimal environment for cell proliferation and migration, and promotes wound repair. Hydrogel is a translucent solid material that is widely used for wound management due to its ability to maintain a moist wound environment, replacing the skin as a physical barrier. The network structure of hydrogels provides water-swelling capacity, retaining moisture near or even above their weight, which enables them to be used for drug delivery, absorbing tissue exudates to treat skin wounds. Hydrogel dressings, which are novel wound dressings, are required to have more excellent properties to cope with various stages of wound healing, including hemostasis, inflammation, proliferation and remodeling. Whether the wound dressing can rapidly initiate coagulation cascade reaction is investigated in the hemostasis period; the inflammatory phase focuses on the destruction of bacteria in preparation for the growth of wound tissue; the proliferation stage needs to provide a scaffold for the growth of tissue cells and new blood vessels; the dressings should be easily degraded by the organism during the reconstitution phase and minimize scarring. Meeting the requirements of wounds at different stages of healing is a hot spot of current wound dressing research.

In order to solve the problems of different stages of wound healing, many materials having unique physiological functions have been applied to the preparation of wound dressings. Chitosan (CS) is a natural biological macromolecular substance, has excellent biocompatibility, antibacterial property, immunostimulation activity and blood coagulation function, can accelerate tissue repair and promote wound contraction, but pure CS gel has insufficient compression resistance and unstable property and needs to be compounded with other materials. The Silk Fibroin (SF) is a biological protein extracted from silkworm silk, has high mechanical strength, good biocompatibility and biodegradability, and is an excellent candidate material for wound dressings. The combination of SF and CS can enhance the mechanical property of the gel. The Halloysite (HNT) which is an inorganic mineral has a hollow tube cavity structure and has certain drug loading capacity, and the tube diameter can be further increased and hydroxyl groups can be exposed through heating and acid-base modification treatment, so that the combination of the halloysite and drugs is promoted, and the drug loading capacity is increased. An endogenous coagulation mechanism caused by HNT can effectively deal with wound bleeding, but the HNT is not degradable and is easy to remain in the wound position when being used alone, so that the healing effect is influenced.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a biomacromolecule and modified halloysite composite hydrogel as well as preparation and application thereof.

The invention provides a biomacromolecule and modified halloysite composite hydrogel, which comprises a chemical cross-linked network of silk fibroin and chitosan, wherein a plurality of acid-modified halloysite nanotubes are distributed in the chemical cross-linked network.

Further, the mass ratio of the silk fibroin to the chitosan is 9:1-3:2; the mass ratio of the chitosan to the halloysite nanotubes is 1:2-2:1. Preferably, the mass ratio of silk fibroin to chitosan is 4:1.

Further, the molecular weight of silk fibroin is 25-350kDa.

Furthermore, the viscosity average molecular weight of the chitosan is 550-1150kDa, and the deacetylation degree is 75-86%.

Furthermore, the halloysite nanotube has a length of 500 to 1500nm, an outer diameter of about 30 to 50nm and an inner diameter of about 15 to 20nm.

In the invention, the halloysite nanotube is connected with a chemical crosslinking network of silk fibroin and chitosan through non-covalent bonds.

The second purpose of the invention is to provide a preparation method of the biomacromolecule and modified halloysite composite hydrogel, which comprises the following steps:

uniformly mixing silk fibroin solution, chitosan solution and acid-modified halloysite nanotubes, and carrying out chemical crosslinking reaction on silk fibroin and chitosan under the action of a crosslinking agent and an alcohol solvent to obtain the biomacromolecule and modified halloysite composite hydrogel after complete reaction.

Further, the crosslinking agents are 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

Further, the alcohol solvent is ethanol.

Further, the silk fibroin solution is prepared by a lithium bromide (LiBr) dissolution method.

Further, the preparation method of the silk fibroin solution comprises the following steps:

boiling silk in alkaline aqueous solution for degumming to obtain degummed silk; and then drying the degummed silk, incubating the degummed silk in a lithium bromide solution for 4 hours at the temperature of 60 ℃, dialyzing, and centrifuging to remove supernatant to obtain the silk fibroin solution. Wherein the cut-off molecular weight of the dialysis bag is 3500Da.

Further, the chitosan solution is acetic acid solution of chitosan.

Further, the preparation method of the acid modified halloysite nanotube comprises the following steps:

and (3) carrying out heat treatment on the halloysite powder at 550 ℃ for 3h, then dispersing the heat-treated halloysite powder in an acid solution, acidifying at 60 ℃, and obtaining the acid-modified halloysite nanotube after complete reaction. Among them, the acid solution is preferably a sulfuric acid solution.

The forming principle of the biomacromolecule and modified halloysite composite hydrogel is as follows:

SF becomes a short peptide chain with a smaller molecular weight during extraction with a high temperature salt solution, a carboxyl group (-COOH) on the SF peptide chain is activated with EDC, a carbon-nitrogen double bond (-C = N-) on EDC reacts with the carboxyl group of SF to form an O-isoureide derivative, and then a hydroxyl group (-OH) on NHS undergoes a substitution reaction with the derivative to form a stable intermediate product. After mixing with CS, the activated carboxyl (-COOH) group is linked with the free amino (-NH) group on CS ₂ ) Form stable amido bond (-CO-NH-) connection. Meanwhile, SF has secondary structure transformation under the influence of polar alcohol solvents, hydrogen bonds are destroyed, and random coils thereof are transformed into silk I type andthe silk II type secondary structure and the unstable silk I type structure form a thermodynamically most stable silk II type structure under the dehydration action of ethanol to form a stable cross-linked network. Meanwhile, the surface of the modified halloysite nanotube (mHNT) contains a large number of hydrophilic groups, and the hydrophilic groups in SF and CS form physical crosslinking, so that the modified halloysite nanotube (mHNT) is dispersed in a chemical crosslinking network of silk fibroin and chitosan to form stable hydrogel.

The third purpose of the invention is to disclose the application of the biomacromolecule and modified halloysite composite hydrogel in preparing wound dressings.

By means of the scheme, the invention at least has the following advantages:

the invention prepares the hydrogel material with the functions of hemostasis, antibiosis and drug sustained release by chemically crosslinking biological macromolecular Silk Fibroin (SF) and Chitosan (CS) to form gel and uniformly dispersing inorganic mineral halloysite nanotubes (mHNT) modified by acid in the gel. The gel has a porous structure and can absorb 600-800% of the mass of water. SF is converted into a thermodynamically stable silk II structure under the action of a polar solvent, so that the thermal stability of the gel material is enhanced. The network structure of the gel and the ordered arrangement of HNT enhance the compressive mechanical property of the material, and the maximum compressive stress is 0.13-0.22 MPa. The combination of the mHNT and the gel enables the medicine to be released for a long time, the burst release rate is low in a neutral environment, and the sustained release period is as long as 14 days. The coagulation effect of the hydrogel was evaluated by in vitro coagulation experiments, and it was found that CS and mHNT promote the coagulation reaction through extrinsic and intrinsic pathways, respectively. The influence of the hydrogel on wound tissues is researched through full-layer skin wound experiments and histological analysis, and the biomacromolecule and modified halloysite composite hydrogel with excellent antibacterial and hemostatic performances and drug slow-release capacity is proved to be a potential skin wound dressing.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to make the technical solutions of the present invention practical in accordance with the contents of the specification, the following description is made with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a schematic diagram of the synthetic principle of SF/CS/mHNT hydrogel and a macro topography diagram of the hydrogel forming process;

FIG. 2 shows the cross-sectional microstructure of SF/CS/mHNT hydrogel under a scanning electron microscope and the scanning result of Si element;

FIG. 3 is an infrared spectrum plot of each hydrogel raw material and each hydrogel;

FIG. 4 is a thermogravimetric plot of the preparation of each hydrogel starting material and each hydrogel;

FIG. 5 is a compression curve of different hydrogels;

figure 6 is the dynamic swelling ratio test results for different hydrogels in PBS solution at pH = 7.4;

FIG. 7 is an in vitro drug release behavior curve for hydrogel and HNT;

FIG. 8 shows the results of bacteriostatic properties of filter paper sheets, SF/CS hydrogel, SF/CS/mHNT hydrogel, and S/C1/H1-CHD hydrogel against Staphylococcus aureus and Escherichia coli;

FIG. 9 is a result of migration behavior of human skin fibroblasts from the surface of different hydrogels to the interior;

FIG. 10 is the results of a coagulation index test of different hydrogels;

FIG. 11 is the results of a full-thickness skin wound healing experiment with different hydrogels;

figure 12 is the results of histological analysis of full-thickness skin wounds of different hydrogels.

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.

In the following examples of the invention, the reagents and sources used were as follows:

mulberry Silk (SF), lazhou, zhejiang; chitosan (CS), viscosity average molecular weight 550kDa, degree of deacetylation 75%, shanghai Sigma-Aldrich company; anhydrous sodium carbonate, analytically pure, national drug group chemical reagents ltd; lithium bromide, analytically pure, national chemical group chemical reagents ltd; halloysite Nanotubes (HNT) having a length of about 500-1500 nm, an outer diameter of about 30-50 nm, an inner diameter of about 15-20 nm, available from Shanghai Sigma-Aldrich; dialysis bag, molecular weight cut-off 3500Da, USA Spectrum company; absolute ethanol, analytically pure, shanghai Michelin Biochemical technology, inc.; 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), sigma-Aldrich, shanghai; n-hydroxysuccinimide (NHS), shanghai sahn chemical technology limited; sulfuric acid, bolinda technologies, inc., shenzhen; chlorhexidine gluconate (19% -21% aqueous solution), shanghai Michelin Biochemical technology, inc.

In the following examples of the present invention, the reaction solutions and the preparation methods thereof used were as follows:

1. SF solution

The SF solution is prepared by a LiBr dissolution method. Adding mulberry silk into 0.02M sodium carbonate aqueous solution at a mass volume ratio of 1. The degummed silk was removed, washed three times with deionized water to remove residual sericin, and dried overnight. Degummed silk was added to a 9.3M lithium bromide solution at a mass to volume ratio of 2.7. The dissolved SF was put into a dialysis bag and dialyzed in deionized water for 2 days. After dialysis, the SF solution was centrifuged twice, the supernatant was taken, diluted to 3% (w/v) with deionized water, and stored at 4 ℃ until use.

2. CS solution

2g of CS powder was put into 100mL of 1% acetic acid solution, and stirred on a magnetic stirrer until completely dissolved to obtain a CS solution with a concentration of 2% (w/v), which was stored at 4 ℃ for further use.

3. Alcohol solution of EDC/NHS cross-linking agent

0.3g of EDC powder and 0.1g of NHS powder were completely dissolved in 10mL of 95% ethanol solution and stored at 4 ℃ until use.

4. Hot acid modified HNT powders

Washing a certain amount of HNT powder with deionized water, centrifuging, purifying, and drying at 60 deg.C to constant weight. Grinding, sieving with 400 mesh sieve, refining, and heat treating in muffle furnace at 550 deg.C for 3 hr. The heat treated HNT was dispersed in a 0.01M sulfuric acid solution and magnetically stirred on a hot plate at 60 ℃ for 8h. After filtration, the filter cake was rinsed several times with deionized water until the supernatant pH was near neutral. Drying in an oven at 60 deg.C for 24h, grinding and sieving with 400 mesh sieve to obtain hot acid modified HNT powder (mHNT).

Unless otherwise specified, the solutions used in the following examples were prepared by the above methods.

Example 1: preparation of SF/CS/mHNT hydrogel

Mixing the SF solution and the CS solution according to a mass ratio of 80, taking a certain mass of mHNT powder, and adding the mHNT powder into the mixed solution, wherein the mass ratio of mHNT to CS is 1, 2, 1. Adding an alcoholic solution of EDC/NHS crosslinker (V) _{Crosslinking agent} :V _{SF solution} = 1:5) are stirred homogeneously. The mixed solution was poured into a centrifuge tube and centrifuged at 4 ℃ and 3000rpm for 15min to defoam. After incubation at 4 ℃ for a period of time different SF/CS/mHNT hydrogels were obtained.

In addition, for comparison, the present invention also prepared an SF/CS hydrogel by mixing an SF solution and a CS solution at a mass ratio of 80 _{Crosslinking agent} :V _{SF solution} = 1:5) are stirred homogeneously. The mixed solution was poured into a centrifuge tube and centrifuged at 4 ℃ and 3000rpm for 15min to defoam. Obtained after incubation at 4 ℃ for a certain period of time.

The nomenclature of the different SF/CS/mHNT hydrogels and the raw material ratios are shown in Table 1.

TABLE 1 different SF/CS/mHNT hydrogels

FIG. 1 (a) is a schematic diagram of the synthetic principle of SF/CS/mHNT hydrogel, which is prepared by physical and chemical crosslinking method; FIG. 1 (b) is a macro-topographic map of the hydrogel-forming process, demonstrating the successful gel production.

The internal structure of the hydrogel can be retained to the maximum extent by using a freeze drying method, fig. 2 shows the cross-sectional micro-morphology and Si element scanning results of various groups of hydrogels under a scanning electron microscope, the magnification of 200 μm of the ruler length in the figure is 400 times, the magnification of 50 μm of the ruler length in the figure is 1200 times, wherein fig. 2a1 and a2 are SEM images of S80/C20; FIGS. 2b1 and b2 are SEM images of S/C2/H1, and FIG. 2b3 is Si distribution diagram of S/C2/H1; FIGS. 2C1 and C2 are SEM images of S/C1/H1, and FIG. 2C3 is Si distribution diagram of S/C1/H1; FIGS. 2d1 and d2 are SEM images of S/C1/H2, and FIG. 2d3 is Si distribution diagram of S/C1/H2. From FIG. 2, it can be seen that each group of gels exhibited a reticulated porous structure. The S80/C20 porous structure is more obvious, the aperture is about 5-20 mu m, the hole wall is thinner, and through holes are mutually connected among the holes.

The surface of the mHNT contains a large number of hydrophilic groups, and a small amount of the hydrophilic groups can increase the hydrophilicity of the hydrogel, so that the surface structure of the hydrogel is loose and porous. The scanning result of Si element shows that the mHNT is uniformly distributed on the surface of the hydrogel and does not have serious agglomeration. When the mass ratio of mHNT to CS is 1:1, the pore structure is the most complete but the pore size is larger.

During the reaction, the centrifuge tube was tilted every 1min, and the time from the start of the addition of the crosslinking agent to the time when the mixed solution in the tube did not flow was recorded as the gelation time. The gelation time and porosity statistics for the 4 above-mentioned groups of reactions are shown in table 2.

TABLE 2 gelation time and porosity

The gelation time of the above 4 groups of reactions is 14-18min. From the porosity results, the porosity of the hydrogel is gradually reduced along with the increase of the content of the mHNT, the addition of a small amount of the mHNT can increase the specific surface area of the hydrogel and enhance the adsorption capacity, and the overhigh content of the mHNT can influence the morphology of the pore structure, cause the irregular pore shape and reduce the adsorption capacity.

FIG. 3 (a) is an infrared spectrum curve of a raw material for preparing a hydrogel, and FIG. 3 (b) is an infrared spectrum curve of each hydrogel. As can be seen from FIG. 3 (a), the infrared curve of CS is 3423cm ^-1 The left and right peaks are hydroxyl (-OH) and amino (-NH) ₂ ) Has a wide peak of extension vibration of 1650cm ^-1 And 1560cm ^-1 At the stretching vibration peak of carbonyl (C = O) and C-N bond, respectively, 894cm ^-1 The position is the characteristic absorption peak of beta glycosidic bond. SThe infrared curve of F is 1645cm ^-1 1530cm at (amide I, alpha helix) ^-1 (amide II) 1233cm ^-1 The (amide III) has a characteristic absorption peak for representing the secondary structure of the protein, which indicates that the secondary structure is mainly random coil. 3694cm on the infrared curve of mHNT ^-1 And 3619cm ^-1 The peak is the stretching vibration peak of hydroxyl (-OH) on the surface of HNT, 1064cm ^-1 Is characterized by a Si-O layer characteristic peak at 911cm ^-1 The peak is the characteristic peak of the Al-O layer.

In the infrared ray graphs of SF/CS and SF/CS/mHNT hydrogels (FIG. 3 (b)), 1645cm ^-1 The SF amide I peak is shifted to a low wave number, which shows that SF is subjected to conformational transition under the action of ethanol, and the secondary structure of the silk II is increased. At 3423cm ^-1 The absorption peak at (2) was narrowed to 1650cm ^-1 And 1560cm ^-1 Has an absorption peak of 894cm ^-1 The absorption peak at the beta-glycosidic bond disappears, indicating that the amino group (-NH) of CS ₂ ) Participate in acylation crosslinking reaction. The mHNT in the hydrogel is 1014cm ^-1 The peak intensity at (A) is remarkably enhanced along with the increase of the mHNT content, and the effective combination of the mHNT and the SF/CS gel network is proved.

FIG. 4 (a) is a thermogravimetric plot of the starting material for the preparation of the hydrogel, and FIG. 4 (b) is a thermogravimetric plot of each hydrogel. FIG. 4 (a) shows the thermogravimetric curves of SF, CS, mHNT. For SF and CS, the mass loss in the first stage (30-240 ℃) is mainly the loss of water molecules in the sample, and thermal decomposition does not occur; in the second stage, the mass loss speed is obviously accelerated, mainly the decomposition of biological macromolecules, the decomposition speed of CS begins to be slowed down at 370 ℃, the decomposition speed of SF is basically unchanged, and the CS is completely decomposed at 600 ℃. For the mHNT, the mass loss in the first stage (30-88 ℃) is mainly the rapid loss of the absorbed water on the surface of the mHNT, and accounts for 4.45 percent of the total mass; in the second stage (88-481 ℃) mainly the dissipation of interlayer combined water molecules, and the mass loss accounts for 6.71 percent of the total mass; the third stage (481-586 ℃) is the main mass loss, which accounts for 11.17% of the total mass, a large amount of hydroxyl (-OH) in the mHNT structure is dehydrated, which belongs to an irreversible process, and the mHNT is converted into an amorphous structure from a crystal structure.

According to the thermogravimetric curve (fig. 4 (b)) of the lyophilized sample of the S80/C20 hydrogel, as the SF content in the hydrogel decreases, the mass loss in the first stage (30 to 240 ℃) gradually decreases, because SF contains a large number of hydrophilic groups, the more the content is, the easier the water molecules are retained, and the more the water molecules are lost during heating; in the second stage (240-334 ℃), the rate of mass loss of the hydrogel is also significantly increased starting at 240 ℃ compared to the pyrolysis of pure SF, CS. The third stage (> 334 ℃) slows the mass loss rate of the hydrogel, which is mainly the thermal decomposition of CS. The thermogravimetric curve of the lyophilized SF/CS/mHNT hydrogel sample (FIG. 4 (b)) has a similar thermal decomposition law to that of the SF/CS hydrogel. Compared with the maximum weight loss temperature (292.3 ℃) of S80/C20, the maximum weight loss temperature of each gel is 301 ℃, 308 ℃ and 311 ℃ in sequence along with the increase of the HNT content, and the final mass loss is gradually reduced, which shows that the thermal stability of the gel is enhanced to a certain extent by adding the mHNT due to the electrostatic adsorption and interface interaction between the hydrogel network and the mHNT.

Hydrogel materials used as wound dressings need to have a certain compressive strength to cope with external compressive deformation. As can be seen in fig. 5, different hydrogels exhibited different compressive resistances as the strain increased. The S80/C20 hydrogel has the strongest compressive capacity, which may be due to the regular and dense pore structure of the S80/C20 hydrogel, and the network scaffold structure of the hydrogel has a certain relation with the compressive property. Compared with S/C2/H1 hydrogel, S/C1/H1 and S/C1/H2 hydrogel with high mHNT content has stronger compression resistance, which shows that the addition of mHNT has a reinforcing effect on the hydrogel, and is beneficial to improving the mechanical properties of the pore wall and even the whole gel scaffold.

Wounds produce a large amount of exudate in the early stages of healing, and rapid absorption and retention of exudate is a fundamental requirement of new wound dressings to provide a moist healing environment for wound repair. The dynamic swelling ratio of the hydrogel is measured by a gravimetric method. According to the swelling experiment result shown in FIG. 6, each hydrogel can absorb 600-700% of the liquid of the mass of the hydrogel within 10min after the beginning of swelling, and the swelling equilibrium is reached within 2 h. The swelling of SF/CS hydrogels may be associated with increased electrostatic repulsion between biopolymer chains due to deprotonation of CS amino groups in the hydrogel structure. The maximum swelling ratio increases with the increase of the mHNT content without changing the SF/CS mass ratio, because the addition of mHNT containing hydrophilic groups increases the specific surface area of the gel and water molecules more easily enter the interior of the gel, causing the gel to swell.

HNT has been widely used for drug loading and sustained release due to its hollow nanotube-like structure, large length-diameter ratio and abundant surface groups. After the thermal acid modification, the pore volume and the pore diameter of the hollow tubular structure are increased, more hydroxyl groups are exposed, and the drug is conveniently adsorbed. The invention takes biguanide chlorobenzene antibacterial agent chlorhexidine gluconate (CHD) as a model drug, and researches the in vitro drug release behavior of HNT and SF/CS/mHNT hydrogel.

The S/C1/H1-CHD hydrogel is prepared by soaking S/C1/H1 hydrogel in CHD solution (40 mg/mL), placing in a vacuum box, and vacuumizing for 30min to make hydrogel fully absorb the drug. After returning to atmospheric pressure, the hydrogel was removed from the solution. The other treatment methods of the drug-loaded gel are the same as the method.

In addition, a certain amount of mHNT powder is ultrasonically dispersed in CHD solution (40 mg/mL), and placed in a vacuum box to be vacuumized for 30min, so that the hollow lumen of the mHNT can fully absorb the medicine. After the normal pressure is recovered, centrifuging the liquid medicine dispersed with the mHNT, removing supernatant, drying and grinding to obtain CHD-loaded mHNT powder.

The drug loading rate of the mHNT can reach 13.14%. FIG. 7 (a) is the in vitro drug release behavior of S/C1/H1-CHD hydrogel at different pH values, and FIG. 7 (b) is the in vitro drug release behavior of mHNT at different pH values. As can be seen from fig. 7 (a), the drug-loaded gel can release most of the drug during the burst period (first 24 h) to cope with the early wound infection. Under the condition of pH =5.5, S80/C20 was still released continuously after 24h, unlike HNT drug release (fig. 7 (b)), SF/CS/mHNT hydrogel was drug released continuously for a longer time because the hydrogel has a uniform porous structure, which facilitates diffusion of CHD molecules from the inside of HNT to the gel structure and retention in the pore structure, thereby prolonging the drug release time.

The invention adopts the inhibition zone method to verify that S80/C20 hydrogel, SF/CS/mHNT hydrogel with different mHNT contents and drug-loaded hydrogel can treat golden yellow glucoseAntibacterial ability of staphylococcus and escherichia coli. FIG. 8 (a) ₁ ) The upper row of (A) is a graph showing the antibacterial effect of the filter paper sheet on Escherichia coli, the lower row is a graph showing the antibacterial effect of the S80/C20 hydrogel on Escherichia coli, and FIG. 8 (a) ₂ ) The upper row is a graph of the antibacterial effect of S/C2/H1 on Escherichia coli, the lower row is a graph of the antibacterial effect of S/C1/H2 hydrogel on Escherichia coli, and FIG. 8 (a) ₃ ) The upper row is a graph of the antibacterial effect of S/C1/H1 on Escherichia coli, the lower row is a graph of the antibacterial effect of S/C1/H1-CHD hydrogel on Escherichia coli, and FIG. 8 (b) ₁ ) The upper row of the (A) is a graph of the antibacterial effect of the filter paper sheet on staphylococcus aureus, the lower row is a graph of the antibacterial effect of the S80/C20 hydrogel on staphylococcus aureus, and a graph of fig. 8 (b) ₂ ) The upper row is a graph of the antibacterial effect of S/C2/H1 on Staphylococcus aureus, the lower row is a graph of the antibacterial effect of S/C1/H2 hydrogel on Staphylococcus aureus, and FIG. 8 (b) ₃ ) The upper row of the hydrogel is a graph of the antibacterial effect of S/C1/H1 on staphylococcus aureus, and the lower row is a graph of the antibacterial effect of the S/C1/H1-CHD hydrogel on staphylococcus aureus. Table 3 shows the statistical results of the zone widths of the antibacterial effect profiles. According to the table 3, the width of the inhibition zone of each hydrogel to escherichia coli and staphylococcus aureus is 0-1 mm, and no bacterial growth trace exists at the bottom of the gel, which indicates that the gel has good antibacterial effect on two kinds of bacteria. There was no significant difference between the inhibition rates of each group of gels against e.coli (fig. 8 (c)) at each level. With the increase of the mHNT content, the inhibition rate of the gel to staphylococcus aureus is slightly increased, and the significant difference (p < 0.05) exists among groups. The inhibition rate of the drug-loaded hydrogel to staphylococcus aureus is obviously higher than that of escherichia coli (p is less than 0.01), because the inhibition effect of the main antibacterial component CS of the drug-unloaded hydrogel to gram-positive bacteria is stronger than that of gram-negative bacteria, and the acidic extracellular environment and the hydrophilic surface of the positive bacteria are beneficial to the CS to enter the interior to interfere the growth and reproduction of bacteria.

TABLE 3 zone of inhibition width of hydrogel

The laser confocal 3D image can reflect the cell from the hydrogelMigration behavior of the surface towards the interior. FIG. 9 (a) ₁ ) FIG. 9 (a) is the result of cell migration on day 1 of S80/C20 ₂ ) FIG. 9 (b) is the result of cell migration of cells on day 3 in S80/C20 ₁ ) FIG. 9 (b) is the result of cell migration of cells on day 1 in S/C1/H1 ₂ ) As a result of cell migration of cells on day 3 in S/C1/H1, FIG. 9 (C) is a statistical result of migration distance of cells from the hydrogel surface toward the inside. From the cell migration data (FIG. 9 (C)), it was found that the migration distance of human skin fibroblasts into the hydrogel was increased and the cell migration distance of the S80/C20 hydrogel was greater as the culture time was prolonged. The displacement of the cells reflects the local degradation and recombination of the hydrogel surface, HNT is a non-degradable inorganic mineral, the porosity of the hydrogel after the HNT is added is reduced (table 2), the structure is tighter, and the speed of degrading SF and CS by the cells through secretion of protease and lysozyme is slow. During the wound healing process, fibroblasts are convenient to migrate from the wound edge to the interior of the hydrogel for proliferation, and a scaffold is provided for tissue regeneration.

The new wound dressings need to have certain hemostatic properties to cope with excessive bleeding in the early stage of the wound. The invention evaluates the hemostatic effect of the hydrogel material by the blood coagulation index, and the blood coagulation index is inversely proportional to the hemostatic effect. As can be seen from fig. 10, the hemostatic effect of the gel was significantly superior to that of the control group (gauze group ). The hemostatic capacity of the SF/CS/mHNT gel is enhanced along with the increase of HNT, which indicates that HNT has a promoting effect on the hemostatic performance. After HNT is added, the hemostatic effect of the SF/CS/mHNT gel is obviously superior to that of SF/CS hydrogel (p is less than 0.01), which is the result of the synergistic effect of the hemostatic substances of CS and HNT, the protonated amino group of CS is combined with the negatively charged amino acid on the surface of erythrocyte membrane to agglutinate erythrocytes, and HNT can initiate coagulation cascade reaction and activate intrinsic coagulation pathway.

A full-thickness skin wound model on the back of rats was constructed in vivo to study the effect of hydrogel dressings on wound healing. Fig. 11 (a) is a photograph of the wound at 1,3,5,7,9, 11, 13 days, fig. 11 (b) is a curve of the change in wound area with time, and fig. 11 (c) is a change in wound healing rate with time. The appearance of the treated wounds after different times is shown in fig. 11 (a), with all groups of wounds showing different degrees of contraction. The SF/CS hydrogel promotes wound healing more efficiently when administered for a long period of time. The formation of granulation tissue was observed for all groups. On day 13 of wound healing, compared with the control group (saline group ), the wound re-epithelialization of the hydrogel treatment group was significant, and the 13-day healing rate of the S80/C20 hydrogel was up to 99.34%. The wound healing rate of S/C1/H1 is lower than that of S80/C20 hydrogel during the healing period, which indicates that the addition of HNT has adverse effect on the wound repair, probably because the inorganic mineral HNT cannot be degraded by enzymes secreted by the wound, the HNT is easy to accumulate at the wound site after being replaced for many times, and the HNT concentration is too high to influence the growth of cells, thereby influencing the healing effect.

FIG. 12 (a) is a 13-day image of HE and Masson stain of wound tissue treated with different methods (saline treatment, S80/C20 hydrogel applied to the wound surface, S/C1/H1 hydrogel applied to the wound surface), (red arrow: blood vessel, blue arrow: hair follicle, green arrow: epidermal layer, scale 200 μm), FIG. 12 (b) is a statistical result of scar length of wound tissue treated with different methods. After histomorphological evaluation of wound tissue on day 13 of wound healing by hematoxylin-eosin (H & E) and Masson staining (FIG. 12 (a)), it was found that the S80/C20 hydrogel and the S/C1/H1 hydrogel accelerated tissue regeneration and reduced scar formation (FIG. 12 (b)). All groups had completed re-epithelialization, resulting in vascularization of the hair follicles and regenerative tissue areas. The scar length of the wound surface of the normal saline control group is the longest (2.21 +/-0.09 mm), and compared with the normal saline control group, the scar lengths of the S80/C20 hydrogel group (1.93 +/-0.18 mm) and the S/C1/H1 group (2.14 +/-0.15 mm) are reduced. Masson staining showed abundant collagen deposition in both S80/C20 and S/C1/H1 groups, with a higher degree of neovascularization and ordered arrangement in the S80/C20 group. The result shows that on the tissue level, the hydrogel has the promotion effect on the repair and regeneration of skin wounds and the structural reconstruction, and can reduce the generation of scars.

In conclusion, the biomacromolecule-inorganic mineral hydrogel is prepared by a physical and chemical crosslinking mode. The cross-linking of SF and CS forms a pore structure in the hydrogel, and the mechanical property of the CS gel is improved. The addition of the modified HNT provides larger drug loading capacity for the hydrogel, and the gel structure also enables the drug to be stably released for a long time. The addition of CS imparts antimicrobial activity to the hydrogel, and the synergistic clotting effects of CS and HNT help the hydrogel to address wound bleeding problems. Although the addition of HNT slows the rate of wound contraction, it does not adversely affect the revascularization and epithelialization of the wound, and HNT-containing hydrogels remain potential for short-term wound dressing applications. In summary, the S80/C20 hydrogel and the S/C1/H1 hydrogel can meet different requirements of medical dressings, and are good candidate materials for wound dressings.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims

1. A biomacromolecule and modified halloysite composite hydrogel is characterized in that: the silk fibroin/chitosan composite material comprises a chemical cross-linked network of silk fibroin and chitosan, wherein a plurality of acid-modified halloysite nanotubes are distributed in the chemical cross-linked network; the mass ratio of the silk fibroin to the chitosan is 9:1-3:2; the mass ratio of the chitosan to the halloysite nanotubes is 1:2-2:1;

the preparation method of the biomacromolecule and modified halloysite composite hydrogel comprises the following steps: uniformly mixing silk fibroin solution, chitosan solution and acid-modified halloysite nanotubes, and carrying out chemical crosslinking reaction on silk fibroin and chitosan under the action of a crosslinking agent and an alcohol solvent to obtain the biomacromolecule and modified halloysite composite hydrogel after complete reaction;

the cross-linking agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

2. The biomacromolecule and modified halloysite composite hydrogel according to claim 1, wherein: the molecular weight of the silk fibroin is 25-350kDa.

3. The biomacromolecule and modified halloysite composite hydrogel according to claim 1, wherein: the viscosity average molecular weight of the chitosan is 550-1150kDa, and the deacetylation degree is 75-86 percent.

4. The biomacromolecule and modified halloysite composite hydrogel according to claim 1, wherein: the silk fibroin solution is prepared by a LiBr dissolution method.

5. The biomacromolecule and modified halloysite composite hydrogel according to claim 1, wherein: the chitosan solution is acetic acid solution of chitosan.

6. The biomacromolecule and modified halloysite composite hydrogel according to claim 1, wherein: the preparation method of the acid modified halloysite nanotube comprises the following steps:

washing and drying the halloysite powder in deionized water, carrying out heat treatment at 350-550 ℃ for 2-3h, then dispersing the heat-treated halloysite powder in an acid solution, acidifying at 50-80 ℃, and obtaining the acid-modified halloysite nanotube after complete reaction.

7. Use of a biomacromolecule and modified halloysite composite hydrogel according to any one of claims 1-6 in the preparation of a wound dressing.