CN114149598A

CN114149598A - Diabetes microenvironment responsive composite intelligent hydrogel and preparation method and application thereof

Info

Publication number: CN114149598A
Application number: CN202111532147.1A
Authority: CN
Inventors: 陈陶; 李帝泽; 王华楠; 季平; 陈楷文
Original assignee: Dalian University of Technology; Chongqing Medical University
Current assignee: Dalian University of Technology; Chongqing Medical University
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-03-08
Anticipated expiration: 2041-12-14
Also published as: CN114149598B

Abstract

The invention discloses a micro-environment responsive composite intelligent hydrogel for diabetes, a preparation method and application thereof, and belongs to the technical field of biomedical materials. The invention relates to a double-grid hydrogel which comprises a first network and a second network, wherein the second network is encapsulated in the first network, and reversible interaction force is exerted between the first network and the second network; the first network comprises a polyvinyl alcohol polymer and the second network comprises gelatin particles. The double-network hydrogel composed of gelatin particle groups is prepared, and when a polyvinyl alcohol network is not covalently crosslinked, the hydrogel has the characteristics of shear thinning and self-repairing due to reversible interaction among particles; after further achieving injection and printing properties, the gel can achieve an increase in the mechanical strength of the gel by non-covalent crosslinking of the polyvinyl alcohol as desired. The characteristic enables the material to have wide application prospect in the fields of minimally invasive implantation materials, artificial extracellular matrix and the like.

Description

Diabetes microenvironment responsive composite intelligent hydrogel and preparation method and application thereof

Technical Field

The invention relates to a micro-environment responsive composite intelligent hydrogel for diabetes, a preparation method and application thereof, belonging to the technical field of biomedical materials.

Background

Diabetes is a chronic systemic disease characterized by an imbalance of metabolism, hyperglycemia, and B cell destruction or insulin resistance of the islets of langerhans. Diabetic patients have long-term exposure of their own tissues to the chronic inflammatory microenvironment, which adversely affects their healing when the tissues are damaged. In addition, many diabetic patients are in an uncontrolled fluctuating state of blood glucose, because they are often not treated effectively in a timely manner due to effects such as clinical inertia and patient compliance. The glycosylated hemoglobin is an important index for measuring the blood sugar stability of the diabetic, however, according to statistics, the standard reaching rate of the glycosylated hemoglobin in the diabetic in China is only 19.5%. With the progress of related studies in recent years, the negative effects of diabetes on skin defects, angiogenesis, bone mineral density and fracture risk are revealed, and there are experiments that show that blood glucose fluctuations are more damaging to bone tissues than persistent hyperglycemia. The back mechanism involves the damage of blood sugar fluctuation to autologous cell mitochondria, thereby causing the increase of active oxygen and the reduction of the activity of antioxidant enzyme of the body, and the like. In general, healing of autologous tissue defects involves a number of complex biological processes: in the initial stage of inflammatory reaction, the wound signal activates immune cells, so that chemical gradient of inflammatory factors is caused, more immune cells are recruited to a wound, and immune response is initiated; after necrotic tissues and bacteria are removed through acute inflammation, the defect part is converted into chronic inflammation, stem cells are recruited through immune regulation and control, the stem cells are induced to be differentiated into various functional cells, and new tissues are reconstructed. Under a healthy state, immune cells interact with osteoblasts, fibroblasts and the like to form a complex and fine regulation network so as to promote tissue reconstruction. However, when the general state is abnormal, such as diabetes, the immune system and the reparative stem cells are interfered by a plurality of ways, calcium and phosphorus metabolism is disturbed, the mineralization of the extracellular matrix is reduced and regulated, and the efficiency and the quality of new bone generation are reduced. Because of the huge diabetic patient population in the world, the poor blood sugar control level, the medical cost of tissue damage and the influence of the delayed defect on the quality of life, the preparation of a more intelligent and effective tissue defect treatment strategy aiming at the diabetic state is very important.

At present, due to the requirement of intelligent drug release at the injured part, many researches are focused on the research and development of a responsive intelligent drug release carrier, and the response conditions comprise magnetic response, thermal response, near infrared response and the like, however, the "autonomy" of the carrier cannot be guaranteed. Responsive materials such as photothermal therapy, magnetic field stimulation for drug delivery, etc., require the decision to apply stimulation based on the clinician's diagnostic judgment, and diabetic patients often fail to receive timely clinical therapeutic intervention due to problems of "clinical inertia" and patient compliance. The glucose oxidase is a representative self-response type drug carrier, and the problems of high immunogenicity, sensitivity, hysteresis and the like also exist, so that the research and development of a more accurate intelligent response type self-discipline drug release carrier has potential clinical significance for treating tissue defects under the blood sugar fluctuation state of poor blood sugar control.

Compared with glucose oxidase and biogenic sugar response molecules such as concanavalin and the like, the biological material based on the phenylboronate chemical bond is easy to combine with glucose, has non-immunogenicity, is easy to manufacture, transport, store and maintain, and is an excellent choice of glucose response materials. In addition, phenylboronic acid is also capable of producing specific degradation behavior for reactive oxygen species, represented by hydrogen peroxide, which are also highly expressed in chronic wound environments. In addition to blood sugar and active oxygen, inflammatory factor matrix metalloproteinase (MMP-9) is highly expressed around the tissue defect, MMP-9 is mainly produced by macrophages and is an important molecule of autoimmune response, but under the diabetic microenvironment, the expression is obviously increased, abnormal hydrolysis of self tissues is caused, and the wound burden is increased.

It is of central importance to accomplish local self-organisation interventions by rational drug loading and delivery strategies. Research shows that the regeneration of soft tissue and the healing of bone defect are all regulated and controlled by the autoimmune system. Research indicates that good tissue engineering biological materials should modulate immune responses, promote the release of cytokines to drive downstream stem cell-dominated reparative differentiation, and thus regulate tissue defects without departing from the co-intervention of the immune-repair biological cascade. In the biological cascade, different biological events occur at different times, for example, after the occurrence of body injury, usually in an acute inflammatory phase within 1-3 days, immune cells reach the defect site, pro-inflammatory factors are produced, proteases are secreted, antigen presentation and phagocytosis are performed, more immune cells are recruited based on chemokines, and the series of biological reactions aim at removing invading pathogenic microorganisms and tissue fragments. After 4-5 days, the acute inflammation stage is gradually changed into the chronic inflammation stage, the reparative immune cells gradually start to differentiate, stem cells are recruited to the defect part by inhibiting inflammation and secreting chemotactic factors, the differentiation of the stem cells is promoted by the secretion of the reparative cytokines, and the tissue healing is accelerated. Under the abnormal metabolic state of diabetes, the acute inflammation stage often cannot be stopped in time by the autoimmune regulation function, so that the local long-term inflammation microenvironment is caused, and pathological changes such as autologous tissue degradation, local active oxygen increase and the like are initiated. Therefore, by combining the time for releasing the drug with the time point when the biological behavior of the host appears, better treatment logic can be realized, and the biological reaction of each step can be accurately controlled, thereby achieving better tissue engineering treatment effect. However, currently, in the existing biomaterials for drug sustained release, one drug is generally loaded, or two drugs are loaded and delivered with similar release curves, and these strategies cannot precisely control drug release for various biological events in biological cascade. Further, even with biomaterials capable of achieving loading and differential drug release of two (or more) drugs, achieving programmed drug delivery in response to a variety of pathological stimuli in dynamic pathological microenvironments presents a number of challenges.

Disclosure of Invention

In order to solve the key clinical problems, the invention provides an injectable composite double-network hydrogel which has the characteristic of responding to various pathological stimuli of diabetes and can adjust the time-sequence release of medicines, and a preparation method and application thereof.

In order to realize the purpose, the invention adopts the following technical scheme:

the invention provides a double-grid hydrogel, which comprises a first network and a second network, wherein the second network is encapsulated in the first network, and reversible interaction force is exerted between the first network and the second network; the first network comprises a polyvinyl alcohol polymer and the second network comprises gelatin particles.

Optionally, the polyvinyl alcohol polymer is selected from a four-armed phenylboronic acid crosslinked polyvinyl alcohol polymer;

optionally, the reversible interaction forces include electrostatic forces, hydrophobic forces, hydrogen bonding forces.

The second aspect of the present invention provides a method for preparing a double-lattice hydrogel, comprising the steps of: and mixing the solution containing the polyvinyl alcohol with the material containing the gelatin particles, adding the material containing the cross-linking agent, mixing to obtain a material containing the pre-mixed colloidal gel, and curing to obtain the product containing the double-network hydrogel.

The third aspect of the present invention provides a method for preparing a double-lattice hydrogel, comprising the steps of: adding a material containing polyvinyl alcohol into a material containing gelatin particle suspension, freeze-drying to obtain a material containing double-grid hydrogel prepolymerization powder, adding an aqueous solution containing a cross-linking agent, mixing, and solidifying to obtain a product containing double-grid hydrogel.

Alternatively, the polyvinyl alcohol has a molecular weight of 47-145kDa and a concentration of 0.01-0.2 g/mL.

Optionally, the gelatin particle size is 50nm to 500 μm; the surface charge of the gelatin particles is-40-20 mV; the volume fraction of the gelatin-coated particles is

Optionally, the mass ratio of the gelatin particles to the polyvinyl alcohol is 0.1-10. The mass ratio of the gelatin particles to the cross-linking agent is 0.5-100.

Optionally, the curing temperature is room temperature and the curing time is 80-120 seconds.

Optionally, the cross-linking agent is named TSPBA, and the cross-linking agent has a structural formula shown in formula 1:

the invention provides an application of a double-grid hydrogel as a drug carrier.

The fifth aspect of the invention provides an application of the double-grid hydrogel in diabetic bone defects, wherein the application is used for repairing and filling defects of bone tissues, cartilage tissues, muscles and vascular tissues.

Optionally, when the double-network structure composite hydrogel is used as a human tissue wound repair filling material, the double-network structure composite hydrogel can be mixed with granular bone repair materials such as hydroxyapatite, calcium phosphate, bioactive ceramics, bioactive glass, acellular bone matrix and the like.

A sixth aspect of the present invention provides a use of a dual-lattice hydrogel as a hemostatic article.

Optionally, the applying comprises: is used for stopping bleeding, preventing adhesion, preventing infection, promoting tissue healing and/or sealing wound of blood wound surface of body surface tissue and organ in body cavity.

Optionally, the double-network hydrogel may be mixed with one or a combination of several of bioactive substances, bioactive protein drugs, living cell particles or drug molecules.

The principle is as follows: according to the invention, gelatin particles or gelatin core-shell particles are used as basic units, in the core-shell particles, the rigid core can increase the mechanical strength of the colloid particles, and the flexible shell enables the particles to retain the high deformability and surface charge of a gelatin polymer phase, so that reversible interaction (including electrostatic, hydrophobic and hydrogen bond acting forces) can be still established among composite material particles through the gelatin polymer phase, the self-repairing capability of a colloid gel network is maintained, the close packing degree and the volume fraction of colloid are increased, and the enhancement of the colloid gel is realized. Further, the complex of the rigid core particles also endows the colloid gel with more functions, such as osteogenesis, photothermal, magnetic response and the like. Further, a new material design concept of constructing the colloid gel with high strength and self-repairing capability by further introducing groups capable of constructing covalent bond crosslinking into the gelatin phase is introduced, and a theoretical basis is laid for the wide application of the colloid gel materials in the biomedical field.

The invention combines a reversible crosslinked polyvinyl alcohol (PVA) network with a colloid network assembled by gelatin nano particles in an electrostatic manner, and develops the hydrogel based on dual logic and with strong adaptability. The four-arm phenylboronic acid crosslinked polyvinyl alcohol hydrogel network can be decomposed in a high-active-oxygen or high-glucose environment and accelerate the release of drug molecules, and the gelatin colloid network has high cell compatibility and enzyme responsiveness induced by high metal matrix protease MMP-9. Therefore, the composite double-network hydrogel can control the controllable drug release of the hydrogel network according to the biological signal molecules in the dynamic diabetes microenvironment.

Has the advantages that:

(1) the double-network hydrogel composed of gelatin particle groups is prepared, and when a polyvinyl alcohol network is not covalently crosslinked, the hydrogel has the characteristics of shear thinning and self-repairing due to reversible interaction among particles; after further achieving injection and printing properties, the gel can achieve an increase in the mechanical strength of the gel by non-covalent crosslinking of the polyvinyl alcohol as desired. The characteristic enables the material to have wide application prospect in the fields of minimally invasive implantation materials, artificial extracellular matrix and the like.

(2) The covalent crosslinking gelatin particle gel prepared by the invention has excellent properties of injectability, self-repairing and plasticity, and can be used for obtaining a double-network hydrogel with high mechanical strength and structural stability through covalent crosslinking of polyvinyl alcohol, the mass fraction of the gel is regulated, the storage modulus of the covalent crosslinking group grafting degree gel can be regulated and controlled between 1-500 kPa, the gel is far higher than that of the conventional particle gel formed by physical interaction, and the structural integrity and the high mechanical strength of the gel after being implanted are ensured.

(3) The present invention is in a complex diabetic microenvironment, where chronic inflammation and impaired metabolism induce overexpression of ROS and increase glucose and MMPs concentrations at tissue defects. The double-network hydrogel prepared by the invention has the property of accurately responding to the micro-environment of diabetes, can simultaneously respond to the triple stimulation related to the diabetes, and controls the release of the drug. In contrast, conventional stimuli responsive biomaterials respond to a single stimulus. The current double-network hydrogel can respond to three specific biological clues of the diabetes microenvironment with remarkable superiority.

(4) According to the invention, the PVA network and the gelatin nanoparticle network both belong to porous networks, drug molecules are easy to permeate into hydrogel, the networks have a physical barrier effect on the drugs, the release of the drugs follows a non-Fick release model, and the differential release rate regulation of the two loaded drugs can be realized according to the dosage of the loaded drugs, the mixing time of the loaded drugs with the PVA or the gelatin nanoparticles and the proportion regulation of the PVA and the gelatin nanoparticles, so that the biological process from inflammation of a human body injury part to inflammation resistance can be better matched, and the drug release peak value has pertinence.

Drawings

FIG. 1 is an electron micrograph of (a) the colloidal gel prepared in comparative example 2, (b) the polyvinyl alcohol hydrogel prepared in comparative example 1, and (c) the double-network hydrogel prepared in example 1.

FIG. 2 is a compressive stress strain curve of the hydrogel material of example 5.

FIG. 3 is a cyclic compressive stress strain curve for the hydrogel material of example 5.

FIG. 4 is a tensile stress strain curve for the hydrogel material of example 6.

FIG. 5 is a cyclic tensile stress strain curve for the hydrogel material of example 6.

Fig. 6 is a graph of the adhesion stress strain for the materials of example 7 and comparative example 3.

FIG. 7 is the responsive degradation behavior of the hydrogel material at different glucose concentrations in example 8;

FIG. 8 is the responsive degradation behavior of the hydrogel material in example 9 at different matrix metalloproteinase concentrations.

FIG. 9 is the responsive degradation behavior of the hydrogel material of example 10 at various hydrogen peroxide concentrations.

FIG. 10 is the drug release behavior of the hydrogel material of example 11 loaded with two model drugs in a diabetes simulation environment.

FIG. 11 is the drug release behavior of the hydrogel material of example 12 under the hyperglycemic/hypoglycemic alternation after loading with the two model drugs.

FIG. 12 is the drug release behavior of the hydrogel material of example 13 after loading with two model drugs under two alternating matrix metalloproteinase/matrix metalloproteinase-free conditions.

FIG. 13 is a MicroCT reconstruction of healthy and diabetic rat cranial apical bone defects at 2 weeks, 4 weeks, and 8 weeks after implantation of the material in example 14.

FIG. 14 is an optical microscope photograph of H & E staining of healthy and diabetic rat femoral metaphysis at 4 weeks, 8 weeks after cylindrical defect preparation and implantation of material in example 15.

Detailed Description

The present invention is further illustrated by the following specific examples. Unless otherwise specified, the raw materials used in the examples were all purchased commercially.

Example 1

0.1g N, N, N, N-tetramethyl-1, 3-propanediamine (purchased from Aladdin reagent, China) and 0.5g4- (bromomethyl) phenylboronic acid (purchased from Aladdin reagent, China) were dissolved in 10mL of dimethylformamide and mixed together, respectively. After stirring overnight at 60 ℃, the mixture was poured into 100mL of tetrahydrofuran to give a white precipitate and filtered, and the white precipitate was washed with tetrahydrofuran. After drying overnight under vacuum, four-armed benzeneboronic acid (TSPBA) was obtained.

0.05g of PVA with the molecular weight of 47 and 145kDa is dissolved in 1mL of deionized water at 80 ℃ to obtain a uniform solution, and the uniform solution is uniformly blended with 0.1g of gelatin nanoparticles through a luer adapter and then is blended with 0.025g of TSPBA to obtain the injectable self-repairing pre-polymerized double-network hydrogel. The hydrogel was allowed to stand at room temperature for 100 seconds to give a double-network hydrogel.

The storage and loss moduli of the above-described double-network hydrogels (table 1) were obtained using a time-sweep mode of a rotational rheometer, with a frequency of 1Hz and a strain of 0.5%. The microstructure of the hydrogel was observed by scanning electron microscopy (fig. 1).

TABLE 1

	47kDa PVA	145kDa
			Storage modulus	1.2kPa	7.1kPa
Loss modulus	0.1kPa	0.4kPa
			Self-repair efficiency	0.74	0.78

Comparative example 1

0.1g N, N, N, N-tetramethyl-1, 3-propanediamine (purchased from Aladdin reagent, China) and 0.5g4- (bromomethyl) phenylboronic acid (purchased from Aladdin reagent, China) were dissolved in 10mL of dimethylformamide and mixed together, respectively. After stirring overnight at 60 ℃, the mixture was poured into 100mL of tetrahydrofuran to give a white precipitate and filtered, and the white precipitate was washed with tetrahydrofuran. After drying overnight under vacuum, TSPBA was obtained.

0.05g of PVA with a molecular weight of 47, 145kDa was dissolved in 1mL of deionized water at 80 ℃ to give a homogeneous solution, and blended with 0.025g of TSPBA via a luer adapter to obtain a polyvinyl alcohol hydrogel pre-polymerization solution. The hydrogel was allowed to stand at room temperature for 100 seconds to give a polyvinyl alcohol hydrogel.

The storage and loss moduli of the above-described double-network hydrogels (table 2) were obtained using a time-sweep mode of a rotational rheometer, with a frequency of 1Hz and a strain of 0.5%. The microstructure of the hydrogel was observed by scanning electron microscopy (fig. 1).

TABLE 2

	47kDa PVA	145kDa
			Storage modulus	0.21kPa	0.69kPa
Loss modulus	0.02kPa	0.02kPa
			Self-repair efficiency	0.81	0.86

Comparative example 2

And uniformly blending 0.1g of gelatin nanoparticles with 1mL of deionized water to obtain the injectable self-repairing colloidal gel. The storage and loss moduli of the colloidal gel (table 3) were obtained using a time-sweep mode of a rotary rheometer, with a frequency of 1Hz and a strain of 0.5%. The microstructure of the hydrogel was observed by scanning electron microscopy (fig. 1).

TABLE 3

Storage modulus	2.1kPa
		Loss modulus	0.2kPa
Self-repair efficiency	0.58

Example 2

0.025g of TSPBA was dissolved in 1mL of deionized water containing 0.1g of gelatin nanoparticles to obtain a homogeneous solution and stirred for 12hrs, followed by freeze-drying to obtain a granular powder, and after 0.025g of TSPBA was dissolved in 1mL of deionized water to obtain a homogeneous solution, it was blended with the above granular powder to obtain a pre-polymerized double network hydrogel. The hydrogel was allowed to stand at room temperature for 100 seconds to give a double-network hydrogel.

The storage and loss moduli of the above-described double-network hydrogels (table 4) were obtained using a time-sweep mode of a rotational rheometer, with a frequency of 1Hz and a strain of 0.5%.

TABLE 4

	47kDa PVA	145kDa
			Storage modulus	65.2kPa	64.1kPa
Loss modulus	1.7kPa	1.8kPa
			Self-repair efficiency	0.39	0.38

Example 3

0.1g of PVA with the molecular weight of 47 and 145kDa is dissolved in 1mL of deionized water at 80 ℃ to obtain a uniform solution, and the uniform solution is uniformly blended with 0.1g of gelatin nanoparticles through a luer adapter and then is blended with 0.025g of TSPBA to obtain the injectable self-repairing pre-polymerized double-network hydrogel. The hydrogel was allowed to stand at room temperature for 100 seconds to give a double-network hydrogel.

The storage and loss moduli of the above-described double-network hydrogels (table 5) were obtained using a time-sweep mode of a rotational rheometer, with a frequency of 1Hz and a strain of 0.5%. The compressive mechanical strain and the breaking strength were obtained by a mechanical testing machine, in which the compression rate was 0.0211/s.

TABLE 5

Example 4

0.05g of PVA with the molecular weight of 47 and 145kDa is dissolved in 1mL of deionized water at 80 ℃ to obtain a uniform solution, and the uniform solution is uniformly blended with 0.1g of gelatin nanoparticles through a luer adapter and then is blended with 0.05g of TSPBA to obtain the injectable self-repairing pre-polymerized double-network hydrogel. The hydrogel was allowed to stand at room temperature for 100 seconds to give a double-network hydrogel.

The storage and loss moduli of the above-described double-network hydrogels (table 6) were obtained using a time-sweep mode of a rotational rheometer, with a frequency of 1Hz and a strain of 0.5%. The compressive mechanical strain and the breaking strength were obtained by a mechanical testing machine, in which the compression rate was 0.0211/s.

TABLE 6

	47kDa PVA	145kDa
			Storage modulus	3.2kPa	9.1kPa
Loss modulus	0.2kPa	1.1kPa
			Self-repair efficiency	0.42	0.78

Example 5

Compression performance, compression recovery performance

Using the double-network hydrogel having a molecular weight of 145kDa of polyvinyl alcohol prepared in examples 1 to 4 and the gels prepared in comparative examples 1,2, a cylindrical scaffold (diameter 12mm, height 8mm) was obtained by molding in a three-dimensional printing mold. The gels were tested for compressive stress strain curves using a universal mechanical tester, as shown in figure 2. The double-network hydrogels prepared in examples 1-4 were further subjected to a cyclic compression test. FIG. 3 is a cyclic stress-strain curve of a double-network hydrogel.

Example 6

Tensile Properties

Using the double-network hydrogel with a molecular weight of 145kDa polyvinyl alcohol prepared in examples 1 to 4 and the gels prepared in comparative examples 1,2, standard uniaxial tensile test bars (design type 5B according to ISO527-2 standard) were obtained by gelling in a mold. And the hydrogel was subjected to a tensile test at a deformation speed of 50mm/min using a universal tester equipped with a 50N load cell, and FIG. 4 is a tensile stress-strain curve of the hydrogel. The double-network hydrogels prepared in examples 1-4 were further subjected to cyclic tensile testing. Figure 5 is a cyclic stress-strain curve for a double-network hydrogel.

Example 7

Adhesion test Performance

The double-network hydrogel obtained in examples 1 to 4 was lapped and adhered to the surfaces of two pigskins (5.0cm × 2.0cm rectangles), wherein the lapping overlapping region was (1.5cm × 2.0cm rectangles), the hydrogel was applied to the surfaces of the pigskins, lapped and pressed, and then left to stand for 10min, and then the lapped sample was subjected to shear peeling (peeling rate: 10mm/min) using a tensile tester with a 50N force sensor, to obtain a peeling process stress-strain curve, and the adhesive strength was defined using the stress maximum point of the curve. The maximum adhesion is shown in figure 6.

Comparative example 3

After lapping and adhering a commercial tissue adhesive fibrin glue (purchased from Shanghai Leishi) and a cyanoacrylate glue (purchased from China Kong-Sharp biology) on the surfaces of two pigskins (a 5.0cm × 2.0cm rectangle), wherein the lapping overlapping area is (a 1.5cm × 2.0cm rectangle), the gel was applied to the pigskin surface and was left to stand for 10min after lapping and pressing, the lapped sample was subjected to shear peeling (peeling rate: 10mm/min) using a tensile tester with a 50N force transducer to obtain a peeling process stress-strain curve, and the adhesive strength was defined by the stress maximum point of the curve. The maximum adhesion is shown in figure 6.

Example 8

The composite hydrogels of examples 1-4 were used to determine their ability to responsively degrade at different sugar concentrations. Specifically, the composite hydrogel was weighed as M1 on a dry weight basis before preparation, and was grouped and soaked in a phosphate buffered saline solution (PBS, pH 7.4) containing 25mM glucose to simulate an in vivo hyperglycemic environment while being kept on a horizontal shaker (30 rpm) at room temperature. The hydrogel is taken out at 1d, 4d, 7d and 14d respectively, and is subjected to low-temperature vacuum drying at-50 ℃ for 24h by a freeze dryer, the dry weight is recorded as M2, and the degradation rate of the hydrogel is calculated according to the proportion of M1 and M2.

Composite hydrogels were used to determine their ability to degrade in a sugar-free PBS environment, as well as in an environment that mimics physiological sugar concentrations. Specifically, the composite hydrogel was weighed as M1 before preparation, and was soaked in PBS (pH 7.4) containing 0mM and 5mM glucose while maintaining room temperature on a horizontal shaker (30 rpm) after grouping. The hydrogel is taken out at 1d, 4d, 7d and 14d respectively, and is subjected to low-temperature vacuum drying at-50 ℃ for 24h by a freeze dryer, the dry weight is recorded as M2, and the degradation rate of the hydrogel is calculated according to the proportion of M1 and M2.

As shown in FIG. 7, in pure PBS, the degradation rate of the composite hydrogel in 14 days is not more than 5%, the degradation rate is close to 10% at 5mM physiological blood glucose concentration, and the degradation degree of the hydrogel exceeds 35% at 25mM simulated high sugar concentration, which shows that the composite hydrogel has obvious blood glucose response degradation characteristics.

Example 9

The composite hydrogels of examples 1-4 were used to determine their ability to degrade responsively at different matrix metalloproteinase concentrations. Specifically, the composite hydrogel was weighed as M1 before preparation and soaked in PBS (pH 7.4) containing 10nM matrix metalloproteinase 9 to simulate a protease-rich microenvironment in the inflammatory defect region in vivo while maintaining room temperature on a horizontal shaker (30 rpm). The hydrogel is taken out at 1d, 4d, 7d and 14d respectively, and is subjected to low-temperature vacuum drying at-50 ℃ for 24h by a freeze dryer, the dry weight is recorded as M2, and the degradation rate of the hydrogel is calculated according to the proportion of M1 and M2.

Composite hydrogels were used to determine their ability to degrade in the matrix metalloproteinase-free state. Specifically, the composite hydrogel was weighed as M1 before preparation and soaked in PBS (pH 7.4) containing 0nM matrix metalloproteinase 9 to simulate a protease-rich microenvironment in the inflammatory defect region in vivo while maintaining room temperature on a horizontal shaker (30 rpm). The hydrogel is taken out at 1d, 4d, 7d and 14d respectively, and is subjected to low-temperature vacuum drying at-50 ℃ for 24h by a freeze dryer, the dry weight is recorded as M2, and the degradation rate of the hydrogel is calculated according to the proportion of M1 and M2.

As shown in fig. 8, the degree of degradation within the composite hydrogel 14d was less than 5% in PBS alone solution without matrix metalloproteinase, whereas the hydrogel achieved almost 30% material degradation under 10nM matrix metalloproteinase-mimicking conditions, and the composite hydrogel exhibited degradation characteristics in response to matrix metalloproteinase compared to PBS alone.

Example 10

The composite hydrogels of examples 1-4 were used to determine their ability to responsively degrade in the presence of high reactive oxygen species. Specifically, the composite hydrogel was weighed as M1 before preparation and soaked in PBS containing 1mM hydrogen peroxide (pH 7.4) to simulate a protease-rich microenvironment in the inflammatory defect region in vivo while maintaining room temperature on a horizontal shaker (30 rpm). The hydrogel is taken out at 1d, 4d, 7d and 14d respectively, and is subjected to low-temperature vacuum drying at-50 ℃ for 24h by a freeze dryer, the dry weight is recorded as M2, and the degradation rate of the hydrogel is calculated according to the proportion of M1 and M2.

The composite hydrogel was used to determine its ability to degrade in the absence of reactive oxygen species. Specifically, the composite hydrogel was weighed as M1 before preparation and soaked in PBS containing 0mM hydrogen peroxide (pH 7.4) to simulate a protease-rich microenvironment in the inflammatory defect region in vivo while maintaining room temperature on a horizontal shaker (30 rpm). The hydrogel is taken out at 1d, 4d, 7d and 14d respectively, and is subjected to low-temperature vacuum drying at-50 ℃ for 24h by a freeze dryer, the dry weight is recorded as M2, and the degradation rate of the hydrogel is calculated according to the proportion of M1 and M2.

As shown in fig. 9, in the pure PBS solution without hydrogen peroxide, the degradation degree in the composite hydrogel 14d is less than 5%, whereas under the condition of 1mM hydrogen peroxide, the hydrogel almost achieves 30% of material degradation, and compared with the pure PBS, the composite hydrogel body shows the degradation characteristic responding to hydrogen peroxide, and the response sensitivity is similar to the matrix metalloproteinase response.

Example 11

The composite hydrogels of examples 1-4 were used to determine their ability to deliver therapeutic logic-based programmed release of a loaded bioactive macromolecule in a diabetes simulation environment. Specifically, Bovine Serum Albumin (BSA) grafted with green fluorescent protein FITC and red fluorescent protein RBITC is used as a model drug (marked as model drug 1 and model drug 2). In the process of preparing the composite hydrogel, mixing the model drug 1 with gelatin nanoparticles to obtain a drug release system loaded on a second network; mixing the model drug 2 with PVA to obtain a drug release system loaded on the first network; the double-network hydrogel was soaked in a liquid simulating the diabetes microenvironment, specifically PBS (pH 7.4) containing 25mM glucose, 1mM hydrogen peroxide, 10nM matrix metalloproteinase 9, and placed on a horizontal shaker at room temperature. Supernatants from the release systems were aspirated at fixed times each day and supplemented with an equal amount of fresh PBS. And detecting the fluorescence intensity in the collected samples according to the excitation and emission wavelengths of the two fluorescent proteins, converting the fluorescence intensity into the concentration of the model drug according to a standard curve, detecting 3 samples in each group, and repeatedly detecting each sample for 3 times.

Model drug 1 exhibited a delayed release profile, indicating that it took longer to release from gelatin nanoparticles and reached a peak release on day 5 in the body fluid environment of the diabetes simulation; model drug 2 exhibited an initial higher and sustained steadily decreasing release profile, indicating that it took less time to release from the PVA network, and that both model drugs could sustain steady release for at least 2 weeks.

Example 12

The double-loaded composite hydrogel in example 11 was used to test its drug release capacity in PBS alone. The double-supported composite hydrogel was soaked in PBS alone (pH 7.4) and placed on a horizontal shaker at room temperature. Supernatants from the release systems were aspirated at fixed times each day and supplemented with an equal amount of fresh PBS. And detecting the fluorescence intensity in the collected samples according to the excitation and emission wavelengths of the two fluorescent proteins, converting the fluorescence intensity into the concentration of the model drug according to a standard curve, detecting 3 samples in each group, and repeatedly detecting each sample for 3 times.

The dual-loading composite hydrogel of example 11 was used to determine its responsive smart controlled release capability at alternating sugar concentrations. Specifically, the composite hydrogel is soaked in a pure PBS (pH 7.4), liquid is absorbed after 12 hours, an equal volume of PBS containing 25mM glucose (pH 7.4) is added, high-sugar PBS is absorbed after 12 hours, an equal volume of pure PBS is added, the degradation environment is changed every 12 hours by analogy, so as to simulate the microenvironment of dynamically changing blood sugar, and meanwhile, the composite hydrogel is placed on a horizontal shaking bed (30 revolutions per minute) at room temperature. The supernatant in the release system was aspirated at 12h, 24h, 36h, 48h, 60h, 72h, 84h, and 96h, respectively, and supplemented with an equal amount of fresh PBS or high-sugar PBS. And detecting the fluorescence intensity in the collected samples according to the excitation and emission wavelengths of the two fluorescent proteins, converting the fluorescence intensity into the concentration of the model drug according to a standard curve, detecting 3 samples in each group, and repeatedly detecting each sample for 3 times.

In a 96-hour high-sugar-low-sugar alternating liquid environment, the material-loaded model drug 1 and model drug 2 both showed significant responsiveness, specifically, the released drug concentration increased in the high-sugar environment (indicated by a gray background) and decreased in the pure PBS environment (indicated by a white background).

Example 13

The dual-loading composite hydrogel of example 11 was used to determine its responsive smart controlled release capability at alternating matrix metalloproteinase concentrations. Specifically, the composite hydrogel is soaked in a pure PBS (pH 7.4), liquid is absorbed after 12 hours, an equal volume of PBS (pH 7.4) containing 1mM hydrogen peroxide is added, high-sugar PBS is absorbed after 12 hours, an equal volume of pure PBS is added, the degradation environment is changed every 12 hours by analogy, so as to simulate the microenvironment of dynamically changing blood sugar, and meanwhile, the composite hydrogel is placed on a horizontal shaking bed (30 revolutions per minute) at room temperature. The supernatant in the release system is sucked at 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h respectively, and is supplemented by adding an equal amount of fresh PBS or PBS containing hydrogen peroxide. And detecting the fluorescence intensity in the collected samples according to the excitation and emission wavelengths of the two fluorescent proteins, converting the fluorescence intensity into the concentration of the model drug according to a standard curve, detecting 3 samples in each group, and repeatedly detecting each sample for 3 times.

In a 96-hour high active oxygen-no active oxygen alternating liquid environment, both the material-loaded model drug 1 and the model drug 2 showed significant responsiveness, specifically, the released drug concentration increased in a high sugar environment (indicated by a gray background) and decreased in a pure PBS environment (indicated by a white background).

Example 14

The healing promoting effect on the bone defect in the diabetes animal model is determined by the composite hydrogel loaded with interleukin 10(IL-10) and bone morphogenetic protein 2 (BMP-2). Specifically, a diabetic rat model was manufactured by injecting streptozotocin (40ng/kg) into SD rats (purchased from the animal experiment center of the university of Chongqing medicine), and a blood glucose fluctuation state of poor blood glucose control was manufactured by injecting recombinant insulin every 1 day, and blood glucose monitoring was performed using a blood glucose meter. Diabetic rats were anesthetized with 3% w/v sodium pentobarbital (1ml/kg), after complete anesthesia, rat hairs at the cranial parietal sites were removed, and the operative area was cleaned with iodophors and alcohol alternately. It was then locally anesthetized using a 2% lidocaine injection at the surgical site. After analgesia in the operated area of diabetic rats, an incision was made along the sagittal midline of the skull, including the full thickness flap of skin and periosteum. After opening the periosteum, a circular window was prepared bilaterally using a 5mm trephine on the sagittal midline on the basal bone. The bony wall of the circular window was carefully removed to avoid damaging the underlying dura mater. Adding the drug-loaded composite hydrogel at the defect position, and suturing the periosteum layer and the skin layer by layer. Penicillin is injected regularly for 3 days after operation, and the suture is removed after 7 days. The cells were sacrificed at 2w, 4w and 8w for imaging analysis.

MicroCT shows that the diabetic environment has an obvious negative effect on new bone formation, and the bone effect of the double-load composite hydrogel loaded with IL-10 and BMP-2 is obviously better than that of a pure composite hydrogel group without drug loading and a diabetic blank group. The experimental results show that the dual-load composite hydrogel loaded with IL-10 and BMP-2 can promote the healing of bone defects under diabetes.

Example 15

Diabetic rats in example 14, as well as healthy rats not molded with diabetes, were used to test the self-healing capacity of unfilled bone defects wounds, and the bone-promoting effect of the non-drug-loaded composite hydrogels of examples 1-4. Specifically, healthy rats and diabetic rats were anesthetized with 3% w/v sodium pentobarbital (1ml/kg), after complete anesthesia, rat hair at the cranial vertex was removed, and the operative area was cleaned alternately with iodophor and alcohol. It was then locally anesthetized using a 2% lidocaine injection at the operative site. After analgesia in the operated area of diabetic rats, an incision was made along the sagittal midline of the skull, including the full thickness flap of skin and periosteum. After opening the periosteum, a circular window was prepared bilaterally using a 5mm trephine on the sagittal midline on the basal bone. The bony wall of the circular window was carefully removed to avoid damaging the underlying dura mater. Adding the drug-loaded composite hydrogel at the defect position, and suturing the periosteum layer and the skin layer by layer. Penicillin is injected regularly for 3 days after operation, and the suture is removed after 7 days. The cells were sacrificed at 2w, 4w and 8w for imaging analysis.

Example 16

The IL-10 and BMP-2-loaded composite hydrogel of example 14, and the diabetic rat of example 14 were used to examine the effect of repairing a femoral metaphyseal defect in a femoral defect model. Specifically, 3% w/v sodium pentobarbital (1ml/kg) was used to anaesthetize diabetic rats, after complete anaesthesia, leg rat hairs were removed, and the operative area was cleaned alternately with iodophor and alcohol. It was then locally anesthetized using a 2% lidocaine injection at the surgical site. After the diabetic rats had no pain sensation in the operative field, a vertical incision was made along the lateral side of the knee joint, including a full thickness flap of skin and periosteum. After the periosteum is opened by using a stripper, a cylindrical defect with the diameter of 1mm and the depth of about 2mm is made in the center of the metaphysis of the femur by using a split drill with the diameter of 1 mm. Injecting the IL-10 and BMP-2 loaded composite hydrogel into the defect, and suturing the muscle layer and the skin layer by layer. Penicillin is injected regularly for 3 days after operation, and the suture is removed after 7 days. The samples were sacrificed at 4w and 8w, fixed and 8 μm thick tissue sections were taken from the specimens, H & E stained sections were taken, and histological analysis was performed.

Example 17

Diabetic rats in example 14, as well as healthy rats not molded with diabetes, were used to test the self-healing capacity of unfilled bone defects wounds, and the bone-promoting effect of the non-drug-loaded composite hydrogels of examples 1-4. Specifically, healthy rats and diabetic rats were anesthetized with 3% w/v sodium pentobarbital (1ml/kg), leg rat hairs were removed after complete anesthesia, and the operated area was cleaned with iodophor and alcohol alternately. It was then locally anesthetized using a 2% lidocaine injection at the operative site. After the diabetic rats had no pain sensation in the operative field, a vertical incision was made along the lateral side of the knee joint, including a full thickness flap of skin and periosteum. After the periosteum is opened by using a stripper, a cylindrical defect with the diameter of 1mm and the depth of about 2mm is made in the center of the metaphysis of the femur by using a split drill with the diameter of 1 mm. According to the grouping, the defect is not treated, or the non-drug-loaded composite hydrogel is injected, and the muscle layer and the skin layer are sutured layer by layer. Penicillin is injected regularly for 3 days after operation, and the suture is removed after 7 days. The samples were sacrificed at 4w and 8w, fixed and 8 μm thick tissue sections were taken from the specimens, H & E stained sections were taken, and histological analysis was performed.

Histological staining shows that no obvious immune cell infiltration is seen around the injection material, which indicates that the biological safety of the injection material is ideal. And compared with healthy rats, the self-healing capacity of the defects of the diabetic rats is obviously weakened, and the drug-free composite hydrogel shows extremely limited healing promotion capacity. Compared with the prior art, the composite hydrogel loaded with IL-10 and BMP-2 obviously promotes more braided bone formation and increases the number of trabeculae.

Claims

1. A dual-lattice hydrogel, wherein said dual-lattice hydrogel comprises a first network and a second network, said second network being encapsulated within said first network, said first network and said second network having reversible interaction forces therebetween; the first network comprises a polyvinyl alcohol polymer and the second network comprises gelatin particles;

preferably, the polyvinyl alcohol polymer is selected from the group consisting of a tetraarm phenylboronic acid crosslinked polyvinyl alcohol polymer;

preferably, the reversible interaction forces include electrostatic forces, hydrophobic forces, hydrogen bonding forces.

2. A method of making the hydrogel of claim 1, comprising the steps of: and mixing the solution containing the polyvinyl alcohol with the material containing the gelatin particles, adding the material containing the cross-linking agent, mixing to obtain a material containing the pre-mixed colloidal gel, and curing to obtain the product containing the double-network hydrogel.

3. A method of making the hydrogel of claim 1, comprising the steps of: adding a material containing polyvinyl alcohol into a material containing gelatin particle suspension, freeze-drying to obtain a material containing double-grid hydrogel prepolymerization powder, adding an aqueous solution containing a cross-linking agent, mixing, and solidifying to obtain a product containing double-grid hydrogel.

4. The method according to claim 2 or 3, wherein the polyvinyl alcohol has a molecular weight of 47-145kDa and a concentration of 0.01-0.2 g/mL.

5. The production method according to claim 2 or 3, wherein the gelatin particle size is 50nm to 500 μm; the surface charge of the gelatin particles is-40-20 mV; the volume fraction of the gelatin granules is

6. The production method according to claim 2 or 3, wherein the mass ratio of the gelatin particles to the polyvinyl alcohol is 0.1 to 10; the mass ratio of the polyvinyl alcohol to the cross-linking agent is 0.5-100.

7. The method of claim 2 or 3, wherein the cross-linking agent is designated TSPBA, and the cross-linking agent has a structural formula shown in formula 1:

8. use of a hydrogel according to claim 1 or a hydrogel prepared by a method according to any one of claims 2 to 7 in a pharmaceutical carrier.

9. Use of a hydrogel according to claim 1 or a hydrogel prepared by a method according to any one of claims 2 to 7 in a diabetic bone defect;

preferably, the application is used for repairing and filling defects of bone tissues, cartilage tissues, muscles and vascular tissues;

preferably, the hydrogel is added with at least one of hydroxyapatite, calcium phosphate, bioactive ceramics, bioactive glass and acellular bone matrix when being applied to repair and filling of tissue defects.

10. Use of a hydrogel according to claim 1 or a hydrogel prepared by a method according to any one of claims 2 to 7 as a haemostatic product;

preferably, the application comprises: the hemostatic, anti-adhesion and anti-infection dressing is used for hemostasis, anti-adhesion and anti-infection of a bloody wound surface of a tissue and an organ in a body cavity, promoting tissue healing and/or closing a wound;

preferably, at least one of bioactive substances, bioactive protein drugs, living cell particles and drug molecules is added into the hydrogel.