CN105778126B

CN105778126B - Genipin cross-linked biogel and preparation method and application thereof

Info

Publication number: CN105778126B
Application number: CN201610202049.4A
Authority: CN
Inventors: 窦桂芳; 高磊; 甘慧; 孟志云; 孙涛; 朱晓霞; 顾若兰; 吴卓娜; 孙文种; 李俭; 郑颖
Original assignee: Institute of Field Blood Transfusion Chinese Academy of Military Medical Sciences
Current assignee: Institute of Field Blood Transfusion Chinese Academy of Military Medical Sciences
Priority date: 2016-03-31
Filing date: 2016-03-31
Publication date: 2020-11-10
Anticipated expiration: 2036-03-31
Also published as: CN105778126A

Abstract

The invention discloses a genipin cross-linked biogel and a preparation method and application thereof, and the genipin cross-linked biogel comprises 0.05-0.2 wt% of genipin, 1-10 wt% of matrix and the balance of water. The gel is crosslinked by adopting the optimized genipin concentration, and the obtained gel has the properties of low toxicity, good water absorption, good mechanical property and the like. The gel can be directly used for protecting and isolating the surfaces of various wounds, bedsores and ulcers, can also be used as a dressing carrier for loading antibacterial drugs, analgesics, growth factors and the like, and is used for treating wound infection, wound analgesia, wound healing promotion and the like. The matrix and the cross-linking agent of the gel provided by the invention are all natural biological sources, are safe and nontoxic, and can be widely used for treating and repairing clinical acute and chronic wound surfaces.

Description

Genipin cross-linked biogel and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a biogel obtained by crosslinking by using genipin as a crosslinking agent, and a preparation method and application thereof.

Background

Trauma refers to any physical, chemical, or disease-related injury to the skin, including both anatomical and functional disruptions. Wounds can be classified into acute wounds and chronic wounds according to the natural healing process after the wounds. Acute wounds refer to wounds that generally heal within 8-12 weeks, most of which are caused by mechanical injury due to physical factors, such as abrasions, cuts, etc., and also include burns or erosive wounds caused by some radiation or chemicals, etc. Chronic wounds generally refer to wounds that heal more slowly, usually for more than 1 year, and may recur. The delay in healing may be due to various factors, such as the presence of complications like diabetes, blood circulation disorders, persistent infections, etc. Chronic wounds are often bedsores, traumatic ulcers and the like, and are often accompanied by excessive exudates, so that wound surface impregnation is caused, a series of physiological healing processes are delayed, and even the risk of causing systemic infection is caused. In addition, it can be divided into superficial wound (into epidermis), II degree wound (into dermis, and deep into blood vessel, sweat gland and hair follicle) and III degree wound (into subcutaneous tissue) according to the depth and area of the wound.

Wound healing is a very complex physiological process. Despite the inherent wound healing mechanisms of the skin, the wound healing process presents a number of obstacles, such as infection from exposure of the wound to microorganisms or other contaminants in the air or water, which in turn can penetrate into surrounding normal tissue causing tissue damage. The wound surface in a natural open state, particularly a deep wound surface, is slow to heal and difficult to achieve complete recovery structurally and functionally, and finally causes the burden of a patient in two aspects of physiology and psychology. Therefore, in order to improve the healing degree of the wound surface and reduce the risks of tissue necrosis and function loss, proper dressing is timely applied to the wound surface after the wound is wounded so as to timely protect the wound surface and accelerate the healing of the wound surface.

In recent years, many natural or synthetic polymer materials such as chitosan, gelatin, collagen, polyacrylamide, aminopolyethylene glycol and the like have been widely used in biomedical fields including wound dressings due to their good biocompatibility, such as chitosan gel, gelatin sponge, collagen sponge, polyacrylamide gel and the like made of the above materials.

However, the mechanical properties of these natural or synthetic polymer materials are often not satisfactory when they are used directly. Therefore, in order to increase the mechanical properties of the gel material, many scholars use crosslinking methods, mainly radiation crosslinking, ion crosslinking, chemical crosslinking, and the like. Radiation crosslinking requires special mechanical equipment, while ionic crosslinking results in gels that are brittle and brittle. The chemical crosslinking enables the crosslinking process to be controllable, and the obtained product is uniform and stable. Common chemical cross-linking agents include aldehyde substances such as glutaraldehyde, formaldehyde, glyoxal and the like, and although the cross-linking agents can obtain gel with good mechanical property, the cross-linking agents have high cytotoxicity, so that the obtained biological material can influence the growth of normal tissues after being applied to wound tissues. In recent years, various safer crosslinking agents have been tried to be used in the crosslinking reaction of biomaterials to improve the biocompatibility of the biomaterials.

Genipin is an iridoid compound obtained by separating and purifying natural plant gardenia fruits, has a multi-active functional group, and can perform cross-linking reaction with a macromolecular material containing amino in a single-molecule or multi-molecule form. Compared with the traditional cross-linking agent, the natural biological cross-linking agent genipin has good biocompatibility and certain anti-inflammatory activity, can inhibit the release of inflammatory factors and metal matrix protease, and has unique advantages when being used in wound treatment materials. However, the crosslinked biogel obtained by the reaction of genipin and a high molecular material has high brittleness and poor extensibility and elasticity, and still cannot be used as a wound dressing.

Disclosure of Invention

Aiming at the technical defects in the prior art, the invention provides the genipin cross-linked biogel which can effectively isolate and protect the wound surface, and the raw materials of the genipin cross-linked biogel comprise 0.05-0.2 wt% of genipin, 1-10 wt% of matrix and the balance of water; wherein the content of genipin is preferably 0.05wt% to 0.1wt%, most preferably 0.075 wt%.

The matrix can be one or more of macromolecular compounds containing amino groups, such as chitosan, carboxymethyl chitosan, carboxyethyl chitosan, collagen, gelatin, polyacrylamide, aminopolyethylene glycol and the like.

The matrix also comprises one or more of alginate, starch, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, carbomer, etc.

The drug carrier also comprises a load drug, wherein the load drug is preferably one or more of antibacterial drugs, analgesic drugs and growth factors.

The antibacterial drug accounts for 0.001-10% by mass and can be one or any combination of penicillin, cephalosporin, aminoglycoside, quinolone, macrolide, polypeptide, sulfonamide, tetracycline, chloramphenicol, silver salt (containing nano silver) and zinc salt (containing nano zinc).

The analgesic drug is 0.001-10 wt% and can be one or more of procaine, tetracaine, lidocaine, bupivacaine, etidocaine, ropivacaine, dyclonine, indomethacin, ibuprofen, diclofenac diethylamine, and diclofenac sodium.

The growth factor is 0.001-10 wt%, and may be one or several of recombinant human epidermal growth factor, mouse epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human acidic fibroblast growth factor, recombinant bovine basic fibroblast growth factor, etc.

In a second aspect, the present invention provides a method for preparing the genipin-crosslinked bio-gel, wherein the preparation process comprises: preparing 5wt% genipin solution; preparing 1wt% acetic acid aqueous solution of the matrix; uniformly mixing a 5wt% genipin solution and a 1wt% acetic acid aqueous solution of a matrix, and fixing the volume by using deionized water to obtain a gel solution; subpackaging in a mold, and reacting at a constant temperature of 37 ℃ for 24 hours to obtain the product.

The method comprises the following steps:

(1) and preparing a genipin solution:

dissolving 5g of genipin in 95g of deionized water, and uniformly mixing to obtain a genipin solution with the mass percentage of 5 wt%;

(2) and preparing a gel solution:

dispersing a matrix in a 1wt% acetic acid aqueous solution, fully swelling, adding a 5wt% genipin solution, uniformly stirring, and fixing the volume with deionized water to obtain a gel solution, wherein the final concentration of genipin in the gel solution is 0.05-0.2%, and the final concentration of the matrix in the gel solution is 1-10%; preferably, the drug loading (solid or solution) can be added while the 5wt% genipin solution is added;

(3) and forming the genipin crosslinked biological gel:

and (3) sub-packaging the gel solution obtained in the step (2) into a mold, and placing the mold at 37 ℃ for constant-temperature reaction for 24 hours to obtain the genipin cross-linked biological gel.

In a third aspect, the invention provides an application of the genipin crosslinked biological gel in preparing a medicament for treating wound infection, relieving pain of the wound or promoting wound healing.

The genipin cross-linked biological gel provided by the invention is cross-linked by adopting the optimized genipin concentration, is a biological gel applicable to acute and chronic wound treatment and nursing, and all the components of the matrix and the cross-linking agent which form the biological gel are natural biological sources, and are safe and non-toxic. The genipin cross-linked biological gel can be uniformly coated on a wound surface, has the properties of good water absorption, good mechanical property and the like, can effectively isolate and protect the surfaces of various wounds, bedsores and ulcers, and provides an absorbable exogenous matrix support for the repair of the wound surface; the dressing carrier can be used for loading antibacterial drugs, analgesics, growth factors and the like, and is used for treating wound infection, wound pain relief, wound healing promotion and the like.

Drawings

FIG. 1 is a microscopic view of a genipin-crosslinked biogel according to the present invention;

FIG. 2 is a microstructure diagram of the genipin-crosslinked biogel loaded with an antibacterial agent according to the present invention;

FIG. 3 is a drug release profile of genipin-crosslinked biogel loaded with antibacterial agents.

Detailed Description

The genipin in the genipin cross-linked biological gel provided by the invention is used as a cross-linking agent, and is a high molecular compound extracted from natural plant gardenia fruits, wherein amino groups can react with the genipin and are cross-linked with each other to form a three-dimensional network structure. The cytotoxicity of genipin is only one ten thousandth of that of glutaraldehyde, and the obtained cross-linked product has mechanical strength equivalent to that of aldehyde cross-linked products. In addition, genipin has been shown to have anti-inflammatory effects, inhibiting the release of inflammatory factors and metallomatrix proteases.

At present, genipin crosslinked polymer gel dressings are reported at home and abroad, wherein the genipin is used as a crosslinking agent, and the concentration of the crosslinking agent is more than 0.5-2 wt%, but the inventor finds through experiments that the crosslinked biological gel obtained by reacting genipin with the concentration and a polymer material has high brittleness and poor extensibility and elasticity, and cannot be used as a wound dressing. Thus, the inventors hoped to find a crosslinking technique that combines the mechanical and ductility properties of the material, so that the resulting biogel has better usability. Repeated tests show that: when the concentration of the genipin cross-linking agent is less than 0.05wt%, the gel becomes pasty and it is difficult to obtain sufficient mechanical strength, while when it is more than 0.2 wt%, the obtained cross-linked gel is too brittle and brittle to the touch. Therefore, the concentration of the genipin cross-linking agent is optimized between 0.05wt% and 0.2 wt% finally.

The genipin cross-linked biological gel comprises genipin and a matrix for forming the gel, wherein the genipin accounts for 0.05-0.2% by mass, and the matrix accounts for 1-10% by mass; the matrix can be one or more of macromolecular compounds containing amino groups, such as chitosan, carboxymethyl chitosan, carboxyethyl chitosan, collagen, gelatin, polyacrylamide, aminopolyethylene glycol and the like, or can be a mixture of the macromolecular compounds containing amino groups, which are randomly combined with one or more of alginate, starch, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, carbomer (Carbopol) and the like, and the combination proportion is not limited. The matrix is common auxiliary materials in the field of medicine, and is safe and nontoxic.

The biological gel related to the invention can be a gel material without any medicine, and can also be a gel material loaded with different medicines. The load medicine can be one or any combination of several types of antibacterial medicines, analgesic medicines and growth factors, and is used for controlling wound infection, relieving pain of wound, promoting wound healing, etc.

When there are some complications to the wound, such as infection, excessive inflammation, diabetic ulcer, etc., the conventional gel dressing may be difficult to fight against the more intractable complications. For example, in case of infection, the exudate existing in the wound surface is rich in nutrients such as protein and the like, and good culture conditions are provided for breeding of microorganisms, so that the wound surface is easy to cause concurrent infection. When infection occurs, microorganisms can decompose wound collagen and granular tissues, destroy new substrates, and cause continuous excessive aggregation of macrophages, inflammatory cells and the like, which can cause delay of wound healing, even deterioration of the wound and even cause general sepsis. The method has the advantages that the method is very important for controlling wound infection and timely and locally applying the antibacterial drugs, particularly when wound tissues are necrotic and lack of blood circulation, and systemic medication cannot reach the wound tissues, the antibacterial drugs can be applied to the genipin cross-linked biological gel for responding to local wound infection, and the method has very important significance for reversing local infection and accelerating the healing of infected wound. In addition, some analgesics, growth factors and the like can also be applied to the genipin cross-linked biological gel for wound surface pain relief, wound surface healing acceleration and the like.

The antibacterial drug accounts for 0.001-10% of the mass percent and can be one or any combination of penicillin, cephalosporin, aminoglycoside, quinolone, macrolide, polypeptide, sulfonamide, tetracycline, chloramphenicol, silver salt (containing nano silver) and zinc salt (containing nano zinc). The application of the antibacterial drug can control or prevent wound infection, reduce or even eliminate wound microorganism breeding, avoid the damage of microorganisms to wound granulation tissues and accelerate wound healing.

The analgesic drug is 0.001-10 wt% and can be one or more of procaine, tetracaine, lidocaine, bupivacaine, etidocaine, ropivacaine, dyclonine, indomethacin, ibuprofen, diclofenac diethylamine, and diclofenac sodium. The application of the analgesic can relieve the pain of deep wound surface to patients, and especially can reduce the pain of patients in dressing change.

The growth factor is 0.001-10 wt%, and may be one or several of recombinant human epidermal growth factor, mouse epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human acidic fibroblast growth factor, recombinant bovine basic fibroblast growth factor, etc. The application of the growth factor can accelerate the migration and proliferation of fibroblasts, accelerate the synthesis of collagen fibers and the formation of granulation tissues, and finally accelerate the healing of wound surfaces.

The method for preparing the genipin cross-linked biogel comprises the following steps:

(1) and preparing a genipin solution:

weighing 5g of genipin, dissolving the genipin in 95g of deionized water, and uniformly mixing to obtain a genipin solution with the mass percentage of 5 wt%.

(2) And preparing a gel solution:

dispersing a matrix in 1wt% acetic acid aqueous solution, fully swelling, adding a proper amount of 5wt% genipin solution, uniformly stirring, and fixing the volume with deionized water to obtain a gel solution, wherein the final concentration of genipin in the gel solution is 0.05wt% to 0.2 wt%, and the final concentration of the matrix in the gel solution is 1wt% to 10 wt%; when 5wt% genipin solution is added, a loaded drug can be added, and the loaded drug can be a solid drug or a solution dissolved with the drug.

(3) And forming the genipin crosslinked biological gel:

and (3) subpackaging the gel solution obtained in the step (2) into plastic molds of 50mm multiplied by 30mm multiplied by 2mm, and placing the molds into a thermostat at 37 ℃ for reaction for 24 hours to obtain the genipin cross-linked biological gel.

The present invention will be described more specifically and further illustrated with reference to specific examples, which are by no means intended to limit the scope of the present invention.

Examples 1 to 8:

a series of genipin cross-linked biogels without any drug were prepared according to the above method, and the raw material composition thereof is shown in Table 1.

TABLE 1 raw Material composition of genipin-crosslinked biogel

Meanwhile, two comparative examples of bio-gels without any drug were also prepared according to the above method, and the raw material composition thereof was identical to example 4 except that the final concentrations of genipin in the gel solutions were 0.01 wt% (comparative example 1) and 0.5 wt% (comparative example 2), respectively.

Determination of the Properties of the genipin-crosslinked biogel prepared in Table 1

1. Microstructure:

the genipin cross-linked biogel prepared from the raw materials in the table 1 is pre-frozen in a refrigerator at the temperature of-80 ℃ for 24 hours, freeze-dried for 48 hours, and the microstructure inside the section is inspected by adopting SEM. Taking examples 1-4 as examples, the microstructures of the genipin-crosslinked biogels of examples 1-4 are shown in fig. 1, F1-F4, respectively.

As can be seen from FIG. 1, the genipin crosslinked chitosan biogel microstructure is in a porous sponge shape, the crosslinking degree is increased along with the increase of the dosage of the crosslinking agent, the microstructure is more compact, the mechanical strength of the gel is increased, and more attachment points are provided for the growth and migration of cells.

Other embodiments have the same effects, which are not described in detail.

2. Physical and chemical properties:

the storage modulus, the contact angle and the water absorption swelling ratio of the genipin crosslinked biogel prepared from the raw materials in the table 1 are respectively considered, and the specific test method is as follows:

storage modulus: the storage (elastic) modulus G' of the genipin-crosslinked biogel of the invention was examined using a parallel plate rheometer. Before testing, the biological gel is transferred to the bottom of a sample plate of a parallel plate rheometer and is leveled, the temperature is set to be 37 ℃, the downward probing depth of the flat plate is set to be 1.5mm, and frequency scanning and deformation scanning are respectively carried out. The compression deformation of frequency scanning is set to be 1.0 percent, and the scanning frequency range is 0.1-10 Hz; the vibration frequency of deformation scanning is set to be 5.0Hz, and the scanning deformation range is 0.1-10%.

Contact angle: the hydrophilicity of the genipin crosslinked biogel surface of the invention was evaluated by static contact angle testing. 10 mul of purified water is dropped on the surface of the biological gel by a point contact method, and contact angles on two sides of the drop are tested after 5 s. Each bio-gel sample was tested for 6 zones and averaged.

Swelling ratio: the water absorption swelling ratio of the genipin crosslinked biogel in the PBS solution with the pH value of 7.4 is tested by adopting a weighing method. Mixing the living things with diameter of 20mm and thickness of 3mmAnd (3) after precisely weighing the gel, immersing the gel into a proper amount of PBS (phosphate buffer solution) with the pH value of 7.4, naturally swelling the gel for 24 hours, taking out the sample, absorbing excessive water by using filter paper, and precisely weighing the gel again. The water absorption swelling ratio is calculated by the following formula: (W) Water absorption swelling Rate%_{Weight after swelling}-W_{Initial weight})/W_{Initial weight}X 100%. Each bio-gel sample was tested in 3 replicates.

The storage modulus, contact angle and water absorption swelling ratio of the genipin-crosslinked biogels of examples 1-4 are shown in Table 2.

Table 2 storage modulus, contact angle and water absorption swell ratio of genipin-crosslinked biogels of examples 1 to 4

As can be seen from table 2, as the amount of genipin was increased (examples 1 to 4), the crosslink density was increased and the elastic modulus was gradually increased, indicating that the mechanical strength was gradually increased, and the obtained biogel could satisfy both the mechanical strength and the extensibility and elasticity of the wound dressing. As can also be seen from Table 2, the surface properties of the biogel of examples 1-4 changed gradually from slightly hydrophobic (contact angle 103.6) to moderately hydrophilic (contact angle 71.0), indicating that the increased structural integrity of the gel interior increases its hydrophilicity. The gel also shows moderate water absorption swelling property, the water absorption swelling rate is also related to the crosslinking degree, and the swelling rate is reduced along with the increase of the crosslinking degree, so that the gel can maintain certain viscoelasticity and is more favorable for the attachment of the gel on the wound surface. When genipin is used in an amount too low (comparative example 1), the gel becomes pasty and has an elastic modulus of only 560Pa, and it is difficult to obtain sufficient mechanical strength, while when it is used in an amount too high (comparative example 2), the obtained gel has an elastic modulus of > 3000Pa, is highly brittle, has poor extensibility and elasticity, is brittle to the touch, and cannot be used as a wound dressing.

Other embodiments have the same effects, which are not described in detail.

3. Evaluation of cytotoxicity

The genipin cross-linked biogels of examples 1-8 were sterilized by irradiation under an ultraviolet lamp for 30min,then respectively soaking in sterile DMEM culture solution at 37 deg.C for 24 hr with leaching ratio of 1.25cm²And/ml. L929 cells were seeded into 96-well plates at 5X 10 per well³Then, the 96-well plate was placed in a 37 ℃ cell incubator and incubated for 24 hours. Taking out 96-well plate, discarding old solution, adding collected leaching solution, adding 100 μ l per well, and culturing in incubator. After 48 hours, the reaction mixture was removed, 20. mu.l of MTT solution (5 mg/ml) was added to each well, and the reaction mixture was left in the incubator for 4 hours. And finally, taking out the 96-well plate, carefully sucking the old solution to remove, adding 150 mu l of DMSO into each well, shaking for 10min, and then placing the well in an enzyme-linked immunosorbent assay (ELISA) instrument to read the OD value. The test wavelength was 490nm and the reference wavelength was 630 nm. Each sample was prepared with 6 duplicate wells, and a blank medium (DMSO) and a 0.5 wt% aqueous solution of phenol were used as a negative control and a positive control, respectively. The Relative growth rate (RGR%) is calculated as: RGR% ═ OD_{Sample (I)}/OD_{Negative control}X 100%. The genipin-crosslinked biogels of examples 1-4 were used as examples, and the relative growth rates to cells are shown in Table 3.

Table 3 relative cell growth rates of genipin-crosslinked biogels of examples 1-4

	Example 1	Example 2	Example 3	Example 4	Positive control	Negative control
							OD	0.445	0.433	0.418	0.425	0.010	0.361
RGR/％	123.27	119.94	115.79	117.73	2.77	100.00
							SD	2.70	2.52	3.00	4.28	0.00	4.46
P value	0.0001	0.0008	0.0024	0.0075	0.0000	-

From the relative growth rate RGR% in Table 3, it can be seen that: compared with a negative control, the biological gel can obviously promote the proliferation of the L929 cells, wherein the gel of the examples 1-4 has an obvious proliferation promoting effect (P is less than 0.05) compared with a positive control, and the wound surface application can be favorable for promoting the proliferation of wound surface fibroblasts and endothelial cells, so that the wound surface healing is accelerated.

Example 9: antibacterial drug-loaded genipin cross-linked biogel

The method for preparing the genipin cross-linked biogel loaded with the antibacterial drugs comprises the following steps:

(1) and preparing a genipin solution:

(2) And preparing a gel solution:

dispersing a matrix in 1wt% acetic acid aqueous solution, fully swelling, adding a proper amount of 5wt% genipin solution and 20 wt% gentamicin sulfate solution, uniformly stirring, and fixing the volume with deionized water to obtain a gel solution, wherein the final concentration of genipin in the gel solution is 0.05wt% to 0.2 wt%, the final concentration of the matrix in the gel solution is 1wt% to 10wt%, and the final concentration of gentamicin sulfate in the gel solution is 0.001 wt% to 10 wt%.

(3) And forming the genipin crosslinked biological gel:

and (3) subpackaging the gel solution obtained in the step (2) into plastic molds of 50mm multiplied by 30mm multiplied by 2mm, and placing the molds into a thermostat of 37 ℃ for reaction for 24 hours to obtain the genipin cross-linked biogel loaded with the antibacterial drugs.

Examples 10 to 13: antibacterial drug-loaded genipin cross-linked biogel

The preparation method is the same as example 9, only the gentamicin sulfate solution in the step (2) is replaced by nano silver sulfadiazine suspension, and the final concentration of the nano silver sulfadiazine in the gel solution is 0.001-10 wt%.

Wherein the preparation of 20 wt% of nano sulfadiazine silver suspension comprises the following steps: dissolving and dispersing 2.5ml of tween 80 in 50g of deionized water to obtain tween 80 solution, dispersing 10g of silver sulfadiazine in the tween 80 solution, uniformly shearing, adding into a ball mill, adjusting the rotating speed to 1500rpm for 10min, and then treating at 2000rpm for 50min to obtain nano silver sulfadiazine suspension.

A series of antibacterial drug-loaded genipin cross-linked biogels of examples 10-13 were prepared according to the method of example 9, with the gel raw material composition of examples 1-4, and the final concentration of silver sulfadiazine in the genipin cross-linked biogel was 1 mg/g.

Microscopic features

1. Microstructure:

the prepared genipin crosslinked biological gel loaded with the sulfadiazine silver in the embodiment 10-13 is placed in a refrigerator at the temperature of-80 ℃ for pre-freezing for 24h, and is frozen and dried for 48h, and the morphology and the particle size of the sulfadiazine silver encapsulated in the gel are observed by a scanning electron microscope, and the morphology is shown in figure 2.

As can be seen from FIG. 2, the particle size of the silver sulfadiazine particles in the genipin crosslinked biogel loaded with silver sulfadiazine is about 100-400 nm. As can be seen from the Ostwald-Freundlich equation and the Noyes-Whitney equation, when the particle size of the drug particles is less than 1 μ M, the smaller the particle size, the higher the saturation solubility and the higher the dissolution rate. Therefore, the solubility of the sulfadiazine silver in the biological gel is obviously increased, which is beneficial to the release and the play of the medicine; in addition, the specific surface area of the medicine is obviously increased due to the fact that the particle size of the medicine is reduced to be nano, so that the contact area of the medicine and bacteria is greatly increased, and the antibacterial effect of the medicine is improved.

2. The drug release mechanism is as follows:

the prepared genipin crosslinked biogel loaded with sulfadiazine silver, example 10-13, is placed in a 250ml conical flask, fixed in a constant temperature oscillator at 37 ℃, added with 100ml of PBS solution, and the oscillation speed is adjusted to 100 rpm. 50 μ l of each flask was sampled at 5,20,40min and 1,2,3,6,12,24,36,48h and added to a blank EP tube and the same volume of PBS solution was immediately added to the flask. After each sample is diluted by adding a proper amount of mobile phase (0.1% phosphoric acid solution: acetonitrile: 94ml:6ml), the mixture is centrifuged at 14000rpm for 10min at a high speed, 20 mul of supernatant is taken, the content of the silver sulfadiazine is measured by UHPLC, the cumulative release rate of the silver sulfadiazine at each time point is calculated, and a drug release curve is made as shown in figure 3, wherein D1-D4 in the figure respectively correspond to the biological gels of examples 10-13. As shown in figure 3, the genipin crosslinked biological gel loaded with the sulfadiazine silver has a slow release function, the drug release time is longer than 48h, and the long-acting control of wound infection is facilitated.

3. Investigation of bacteriostatic effect

Taking activation and diluting to 1.5X 10⁸100 mul of staphylococcus aureus (S. aureus) per ml is added to the surface of the solid LB agar culture medium and is gently spread evenly, and the basically residue-free bacterium liquid is taken as the standard. A cavity with a diameter of about 3mm was punched out of agar plates arranged at a distance, 50. mu.l of a double antibody solution (containing 5U/ml penicillin and 5. mu.g/ml streptomycin), a commercially available silver sulfadiazine cream (AgSD cream), a blank gel (the biological gel without the drug loaded in example 1) and the inventive silver sulfadiazine-loaded genipin cross-linked biological gel of examples 10-13(AgSD/NCT gel) were added, respectively, and all samples were diluted with LB agar medium to a silver sulfadiazine concentration of 10. mu.g/ml. The culture dish is covered and placed in an incubator at 37 ℃ in an inverted mode, and the culture is carried out for 12 hours. Escherichia coli (e.coli), pseudomonas aeruginosa (p.aeruginosa) were inoculated as described above, and the samples were added in the same manner. Each inoculum was plated in 3 parallel plates. Taking the genipin-crosslinked biogel loaded with sulfadiazine silver of example 10 as an example, the average results of the inhibition zone experiments of the groups of staphylococcus aureus, escherichia coli and pseudomonas aeruginosa are shown in table 4.

TABLE 4 antibacterial Ring diameters (mm) of different AgSD preparations against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa

	S.aureus	E.coli	P.aeruginosa
				Double-antibody solution	13.7±0.9	12.8±1.9	10.2±0.6
AgSD cream	14.6±1.5	11.5±0.6	13.4±0.9
				Blank gel	4.9±0.5	4.5±0.5	4.3±0.2
AgSD/NCT gel	17.2±0.8^*#	12.7±1.1^*	14.7±0.3^*#

Note:^*(iii) P < 0.05, vs AgSD cream;^#p is less than 0.05, vs. double-resistant solution;

as can be seen from the results in Table 4, the three products showed significant bacteriostatic effects against three bacteria except the blank gel. The genipin cross-linked biological gel loaded with the sulfadiazine silver has a larger inhibition zone diameter on three bacteria than that of a commercially available sulfadiazine silver cream, and has higher inhibition on staphylococcus aureus and pseudomonas aeruginosa than that of a double-resistant solution, which is probably related to improvement of the dissolution rate of a medicament and slow release effect in the gel.

Example 14: biological gel loaded with analgesic drug

The preparation method comprises the following steps:

the preparation of the genipin solution in the step (1) and the forming of the genipin crosslinked biogel in the step (3) are the same as those in the example 9, except that in the preparation of the gel solution in the step (2), the matrix is dispersed in 1wt% of acetic acid aqueous solution, after the matrix is fully swelled, 5wt% of genipin solution and 20 wt% of lidocaine hydrochloride solution are added, after the matrix is uniformly stirred, deionized water is used for fixing the volume to obtain the gel solution, and the final concentration of the lidocaine hydrochloride in the gel solution is 0.001 wt% -10 wt%.

Example 15: preparation of growth factor-loaded biogel

The preparation method comprises the following steps:

the preparation of the genipin solution in the step (1) and the forming of the genipin crosslinked biogel in the step (3) are the same as those in the example 9, except that the matrix is dispersed in 1wt% acetic acid aqueous solution in the preparation of the genipin solution in the step (2), after the matrix is fully swelled, 5wt% genipin solution and 20 wt% recombinant human epidermal growth factor solution are added, the mixture is uniformly stirred and then fixed to the constant volume by deionized water to obtain a gel solution, and the final concentration of the recombinant human epidermal growth factor in the gel solution is 0.001 wt% -10 wt%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the content of the present invention.

Claims

1. A genipin cross-linked biogel is characterized in that the raw materials of the genipin cross-linked biogel consist of 0.05wt% -0.1wt% of genipin, 1wt% -10wt% of matrix, 1wt% of acetic acid aqueous solution and the balance of water; the matrix is one of chitosan, carboxymethyl chitosan, carboxyethyl chitosan, gelatin, polyacrylamide and amino polyethylene glycol; or one of alginate, starch, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and carbomer.

2. The genipin-crosslinked biogel of claim 1, wherein the genipin is present in an amount of 0.075 wt%.

3. The genipin-crosslinked biogel according to claim 1 or 2, further comprising a drug load.

4. The genipin cross-linked biogel of claim 3, wherein the loaded drug is selected from one or more of an antibacterial drug, an analgesic drug and a growth factor.

5. The genipin cross-linked biogel according to claim 4, wherein the antibacterial drug is 0.001% -10% by mass.

6. The genipin cross-linked biogel of claim 5, wherein the antibacterial drugs are one or any combination of penicillin, cephalosporin, aminoglycoside, quinolone, macrolide, polypeptide, sulfonamide, tetracycline, chloramphenicol, silver salt, zinc salt drugs.

7. The genipin-crosslinked biogel of claim 6, wherein the silver salt is a silver salt containing nano-silver, and the zinc salt is a zinc salt containing nano-zinc.

8. The genipin cross-linked biogel of claim 4, wherein the analgesic drug is present in an amount of 0.001% -10% by weight.

9. The genipin-crosslinked biogel of claim 8, wherein the analgesic drug is one or any combination of procaine, tetracaine, lidocaine, bupivacaine, etidocaine, ropivacaine, dyclonine, indomethacin, ibuprofen, diclofenac diethylamine, and diclofenac sodium.

10. The genipin cross-linked biogel according to claim 4, wherein the growth factor is 0.001-10% by weight.

11. The genipin cross-linked biogel according to claim 10, wherein the growth factor is one or more of recombinant human epidermal growth factor, murine epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human acidic fibroblast growth factor and recombinant bovine basic fibroblast growth factor.

12. A method of preparing the genipin-crosslinked biogel of claim 1 or 2, which is prepared by a process comprising: preparing 5wt% genipin solution; preparing 1wt% acetic acid aqueous solution of the matrix; uniformly mixing a 5wt% genipin solution and a 1wt% acetic acid aqueous solution of a matrix, and fixing the volume by using deionized water to obtain a gel solution; subpackaging in a mold, and reacting at a constant temperature of 37 ℃ for 24 hours to obtain the product.

13. The method of claim 12, comprising the steps of:

(1) and preparing a genipin solution:

(2) and preparing a gel solution:

dispersing a matrix in a 1wt% acetic acid aqueous solution, fully swelling, adding a 5wt% genipin solution, uniformly stirring, and fixing the volume with deionized water to obtain a gel solution, wherein the final concentration of genipin in the gel solution is 0.05-0.2%, and the final concentration of the matrix in the gel solution is 1-10%;

(3) and forming the genipin crosslinked biological gel:

14. A method of preparing a genipin-crosslinked biogel according to any one of claims 3 to 11, comprising the steps of:

(1) and preparing a genipin solution:

(2) and preparing a gel solution:

dispersing the matrix in 1wt% acetic acid water solution, and preparing 1wt% acetic acid water solution of the matrix after full swelling; adding 5wt% of genipin solution, stirring uniformly, and fixing the volume with deionized water to obtain gel solution, wherein the final concentration of genipin in the gel solution is 0.05% -0.2%, and the final concentration of the matrix in the gel solution is 1% -10%; adding a solid or solution loaded medicine while adding a 5wt% genipin solution;

(3) and forming the genipin crosslinked biological gel:

15. Use of the genipin-crosslinked biogel of any one of claims 1 to 11 in the preparation of a medicament for treating a wound infection, wound analgesia or promoting wound healing.