Double-network hydrogel, double-network hydrogel for packaging nano-carrier and medicine, and preparation method and application of double-network hydrogel

Abstract

The invention provides a double-network hydrogel, a double-network hydrogel for packaging a nano-carrier and a medicine, and a preparation method and application of the double-network hydrogel, and belongs to the technical field of hydrogel preparation. The preparation method of the nano-composite dual-network hydrogel comprises the following steps: (1) reacting a polymer monomer, a cross-linking agent, an initiator and a natural high-molecular compound to obtain first network hydrogel; and (2) dipping the first network hydrogel in an acid system with the pH value of 0.9-1.5, and carrying out a reaction to obtain the dual-network hydrogel. The dual-network hydrogel and the dual-network hydrogel encapsulating the nano-carrier and the medicine, provided by the invention, have relatively high swelling ratio, stability, active oxygen scavenging effect, inflammation treatment effect, mucous membrane repairing capability and biodegradability, and are suitable for the biomedical field of digestive tract inflammatory diseases such as peptic ulcer and inflammatory bowel disease; and the method can also be expanded to inflammation repair of other systems.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, and in particular to a double network hydrogel, a double network hydrogel encapsulating nano-carriers and drugs, and a preparation method and application thereof. Background Art

[0002] With the changes in social environment and people's living habits, inflammatory diseases related to the digestive system have gradually become a global health problem, seriously affecting the quality of human life. Among the inflammatory diseases related to the digestive tract, the prevention and treatment of inflammation related to the digestive tract is crucial to improving the health of people around the world.

[0003] Due to the complexity of the digestive system, the etiology and pathogenesis of inflammation are also complex and multifactorial, such as external harmful stimuli, emotional or physical stress, immune deficiency, family heredity, nutritional status and excessive immune response.

[0004] The main goal of treating inflammatory diseases of the digestive system is to reduce inflammation, promote mucosal healing, and improve the patient's quality of life. To achieve these goals, commonly used drugs in clinical practice include anti-inflammatory drugs, immunosuppressants, and antibiotic supplements. However, most of these drugs can only temporarily relieve the condition or reduce symptoms, and cannot achieve a fundamental cure.

[0005] Research data show that 80% of Crohn's disease (CD) patients and 45% of UC patients need to undergo at least one surgical intervention to alleviate their condition during their lifetime. In addition, most of these drugs also cause adverse side effects, such as drug resistance, infection, drug-related toxicity, etc. For example, in the clinical treatment of peptic ulcers, since anti-inflammatory drugs such as amoxicillin and clarithromycin are degraded by gastric acid, in order to achieve the desired therapeutic effect, higher than standard drug doses are often used, which leads to increased gastrointestinal side effects in patients, and adverse reactions such as diarrhea, nausea, vomiting, abdominal distension and abdominal pain occur, reducing compliance with treatment. In addition, the various organs of the digestive system have been stimulated by long-term inflammation but have not been truly cured, and the risk of inflammatory diseases deteriorating into cancer has risen sharply. Therefore, there is an urgent need to develop new drug delivery systems with ideal therapeutic effects and limited side effects.

[0006] Inflammatory diseases of the digestive tract are a common problem encountered in clinical practice. This disease not only has a high incidence rate, but is also prone to a variety of complications, mainly upper gastrointestinal bleeding, perforation, pyloric obstruction, and cancer, which can be life-threatening in severe cases. The inability of the digestive tract mucosa to achieve high-quality healing, increased drug toxicity, and increased drug resistance make the treatment of inflammatory diseases of the digestive tract more difficult. Currently, triple therapy consisting of a proton pump inhibitor and two antibiotics is the clinical first choice for the treatment of peptic ulcers. However, this method is prone to drug resistance, and as the body's resistance to antibiotics increases, the cure rate shows a trend of gradual decline.

[0007] Recent studies have shown that reactive oxygen species, including superoxide anions (O2·-), hydrogen peroxide (H2O2), hydroxyl radicals (OH·) and other chemically active small molecules, are associated with gastrointestinal inflammatory diseases to varying degrees. Therefore, scavenging free radicals and reactive oxygen metabolites may help protect the gastrointestinal mucosa from oxidative stress damage, thereby accelerating the healing of gastrointestinal inflammatory diseases.

[0008] Hydrogel is a composite material with a three-dimensional cross-linked network. It has now developed into a multifunctional therapeutic platform. After reasonable structural and functional design, materials engineering is used to adjust its physicochemical properties (such as hardness, pore size, viscoelasticity, microstructure, degradability, ligand presentation, stimulus response characteristics, etc.) and affect cell signal cascades. Hydrogel can act as a barrier to prevent bacterial infection and create a suitable microenvironment for tissue regeneration. It can serve as an ideal therapeutic platform for the repair of gastrointestinal inflammatory diseases. However, traditional hydrogels have disadvantages such as low mechanical strength and low swelling when treating gastrointestinal inflammatory diseases, and it is difficult to meet the requirements of continuous treatment.

[0009] Existing nanotherapy methods are designed to overcome the effects of the acidic environment in the digestive tract on drugs, protect drugs from being destroyed by gastric acid, and increase the drug's residence time on the digestive tract mucosa and penetrate into the mucus layer to improve the efficacy of inflammatory disease treatment. However, this therapy has great limitations and cannot fully cover the treatment of digestive tract inflammatory diseases caused by various factors. Therefore, a new technical therapy based on the pathogenesis and cellular level of digestive tract inflammatory diseases is urgently needed to better treat digestive tract inflammatory diseases. Summary of the invention

[0010] In view of this, the present invention provides a double network hydrogel, a double network hydrogel encapsulating nanocarriers and drugs, and a preparation method and application thereof. The present invention places the hydrogel in an acidic environment so that the natural polymer compound can be protonated, increase its solubility, further react to form new cross-linking points, generate a second network, and enhance the mechanical strength, stability and swelling of the hydrogel, which is suitable for a variety of biomedical applications, such as drug controlled release, tissue engineering, etc.

[0011] In order to achieve the above effect, the technical solution adopted by the present invention is as follows:

[0012] The first object of the present invention is to provide a method for preparing a double network hydrogel, comprising the following steps:

[0013] (1) reacting a polymer monomer, a cross-linking agent, an initiator, and a natural polymer compound to obtain a first network hydrogel;

[0014] (2) The first network hydrogel is immersed in an acid system with a pH of 0.9 to 1.5 to react and obtain a double network hydrogel.

[0015] Preferably, the polymer monomer includes any one of acrylic acid and its derivatives, methacrylic acid and its derivatives, acrylamide and its derivatives, vinyl pyridine, and N-vinyl formamide;

[0016] Preferably, the cross-linking agent is selected from compounds containing multiple unsaturated double bonds in the molecule, and preferably the cross-linking agent includes any one of N,N'-methylenebisacrylamide, glutaraldehyde, and sulfated acrylic acid;

[0017] Preferably, the initiator comprises a free radical polymerization initiator, more preferably, the initiator is an azo initiator, and more preferably, the initiator is azobisisobutyronitrile;

[0018] Preferably, the natural polymer compound includes any one of natural polysaccharides and short peptides; further preferably, the natural polysaccharide includes any one of sodium alginate, sodium hyaluronate and chitosan;

[0019] Preferably, the step (1) further comprises a coagulant, and the coagulant comprises at least one of N,N′-bis(acryloxy)cystamine, ethylene glycol diacrylate, and methyl methacrylate;

[0020] Preferably, the step (1) further comprises a solvent. Further preferably, the solvent comprises a mixed solution of deionized water and ethanol, or any one of deionized water. More preferably, the mass ratio of the ethanol to the mixed solution is 1 to 100 wt %, but not 100 wt %.

[0021] Preferably, the mass ratio of the polymer monomer to the natural polymer compound is 1 to 10:1, more preferably 1 to 9.9:1, and even more preferably 1:1.

[0022] Preferably, the pH of the acid system in step (1) is 1.2.

[0023] The second object of the present invention is to provide a method for preparing a double network hydrogel encapsulating nanocarriers, comprising the following steps:

[0024] (1) reacting a polymer monomer, a cross-linking agent, an initiator, Prussian blue nanoparticles, and a natural polymer compound to obtain a first network hydrogel;

[0025] (2) The first network hydrogel is immersed in an acid system with a pH of 0.9 to 1.5 to react and obtain a double network hydrogel.

[0026] When the natural polymer compound in step (1) is sodium alginate, the reaction principle is as follows:

[0027] The present invention relates to a first network hydrogel formed by the reaction of a polymer monomer, a crosslinking agent, an initiator, Prussian blue nanoparticles, and sodium alginate, and then the first network hydrogel is dried and orally swallowed, and the gastric acid environment is used to change the potential of the Prussian blue nanoparticles. In the gastric acid environment with a low pH value (0.9 to 1.5), the manganese ions and ferrous ions in the Prussian blue nanoparticles react with the Na+ on the gulonic acid unit of the sodium alginate. + The ions undergo ion exchange reactions, and the gulonic acid units accumulate to form a cross-linked network structure, forming a second network composed of Prussian blue nanoparticles and sodium alginate hydrogel, thereby forming a double-network hydrogel with high swelling rate and stability that can scavenge reactive oxygen species, reduce inflammatory responses, and promote mucosal repair encapsulated nanocarriers.

[0028] In the gelation process, there is a coordination effect between the manganese and iron ions in the Prussian blue nanoparticles and the carboxyl groups of the sodium alginate, and ion substitution is performed at the carboxyl site, which has an enhancing effect on the mechanical properties of the second network of the double network hydrogel encapsulating the nanocarrier after the formation. The double network hydrogel encapsulating the nanocarrier provided by the present invention can be used for long-term treatment and repair of inflammatory diseases of the digestive system, and is biodegradable, and is a good method for treating inflammatory diseases of the digestive tract by using swelling to achieve a long retention effect.

[0029] Preferably, in step (1), the mass ratio of the polymer monomer, the natural polymer compound, and the Prussian blue nanoparticles is 1-10:1:0.001-1, and more preferably 1:10:0.01.

[0030] Preferably, the Prussian blue nanoparticles in step (1) have a particle size of 100 to 200 nm and are spherical particles.

[0031] Preferably, the step (1) further comprises a coagulant, and the mass ratio of the coagulant to the Prussian blue nanoparticles is 1-50:1-100.

[0032] The third object of the present invention is to provide a method for preparing a double network hydrogel encapsulating nanocarriers and drugs, comprising the following steps:

[0033] (1) reacting a polymer monomer, a cross-linking agent, an initiator, Prussian blue nanoparticles, a drug, and a natural polymer compound to obtain a first network hydrogel;

[0034] (2) The first network hydrogel is immersed in an acid system with a pH of 0.9 to 1.5 to react and obtain a double network hydrogel.

[0035] Preferably, the drug in step (1) comprises sucralfate.

[0036] Preferably, in step (1), the mass ratio of the polymer monomer, the natural polymer compound, the Prussian blue nanoparticles and the drug is 1-10:1:0.001-1:0.001-1, and more preferably 1:10:0.01:0.01.

[0037] The present invention combines nanomaterials for removing active oxygen from the inflammatory site with hydrogel dressings, designs and synthesizes a double-network hydrogel with a high swelling coefficient as a carrier, combines an active oxygen scavenger-bionic Prussian blue analogue and a digestive tract inflammatory disease treatment drug sucralfate, utilizes the adhesion and swelling properties of the hydrogel, increases the action time with the inflammatory site, and combines with the active oxygen scavenger to eliminate oxidative stress in ulcer tissue cells. The high swelling coefficient of the hydrogel combined with the double-network structure formed in the digestive tract enhances the action time of the drug and the nanomaterial, and the sustained-release effect also reduces the toxicity of the drug, so that the ulcer is protected in multiple ways and treated for a long time, achieving the effect of promoting the healing of peptic ulcers.

[0038] Preferably, the reaction temperature of step (1) is 0-100°C, and the reaction time is 1-120 min. More preferably, the reaction temperature is 30-100°C.

[0039] Preferably, the preparation method of the Prussian blue nanoparticles comprises: dispersing potassium ferrocyanide and manganese chloride with bovine serum albumin in ultrapure water to obtain potassium ferrocyanide solution A and manganese chloride solution B respectively; adding potassium ferrocyanide solution A to manganese chloride solution B to obtain Prussian blue nanoparticles.

[0040] Preferably, the molar ratio of the potassium ferrocyanide to manganese chloride is 0.1-1000:0.1-1000; the mass ratio of the bovine serum albumin to potassium ferrocyanide or manganese chloride is 0.1-1000:1.

[0041] Preferably, the ambient temperature for preparing Prussian blue nanoparticles is controlled within the range of 0 to 100°C.

[0042] Furthermore, the double network hydrogel provided by the present invention can be applied in the biomedical field, and further, in the field of inflammation repair.

[0043] Furthermore, the double network hydrogel provided by the present invention is an interpenetrating structure.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The double network hydrogel provided by the present invention has a high swelling rate and stability. The double network hydrogel encapsulating nanocarriers and drugs provided by the present invention has active oxygen scavenging effect, inflammation treatment effect, mucosal repair ability and biodegradability. It is suitable for the biomedical field of gastrointestinal inflammatory diseases including gastrointestinal ulcers, inflammatory bowel disease, etc., and can also be expanded to the inflammation repair of other systems.

[0046] (2) The Prussian blue nanoparticles with high active oxygen scavenging and anti-inflammatory activity prepared by the present invention adopt a biomimetic strategy to form a second network of hydrogel with natural polymer compounds. On the basis of the first network, the main function of the second network is to increase the hardness of the hydrogel and maintain its high swelling rate, so that it has a long-term gastric retention effect and plays a mucosal repair function. Among them, the first network hydrogel has good water absorption and water retention; the natural polymer compound is an edible biomaterial, which enhances the stability and mechanical strength of the hydrogel when forming the second network; the Prussian blue nanoparticles have biomimetic properties, and sucralfate is a clinical first-line gastric mucosal repair drug. Therefore, the double-network hydrogel encapsulating the nanocarrier and the drug has excellent biocompatibility and can enter the digestive system by oral administration. While repairing the gastrointestinal mucosa, it can treat inflammatory diseases, especially gastrointestinal inflammatory diseases, for a long time without toxic side effects.

[0047] (3) The double-network hydrogel provided by the present invention can achieve efficient repair of digestive tract inflammatory diseases with a long retention period of one week, with rapid and obvious therapeutic effects. It has good application prospects as a biomedical material.

[0048] (4) The preparation method of the double network hydrogel provided by the present invention has a simple process, is easy to operate, does not require complex and expensive equipment, and is easy to realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the SEM characterization image of the vinylpyridine-chitosan composite double network hydrogel sample after freeze-drying in Example 1.

[0050] Figure 2 It is a comparison of the swelling rates of chitosan hydrogel, vinyl pyridine hydrogel and vinyl pyridine-chitosan composite double network hydrogel in gastric acid.

[0051] Figure 3 This is the SEM characterization image of the N-vinyl formamide-sodium hyaluronate composite double network hydrogel sample after freeze-drying in Example 2.

[0052] Figure 4 The swelling rates of sodium hyaluronate hydrogel, N-vinyl formamide hydrogel and N-vinyl formamide-sodium hyaluronate composite double network hydrogel in gastric acid are compared.

[0053] Figure 5 This is a TEM characterization image of the Prussian blue nanoparticles prepared in Example 3.

[0054] Figure 6 This is the SEM characterization image of the PB / S@H sample after freeze-drying in Example 3.

[0055] Figure 7 It is a line graph of the iron and manganese contents in feces at different times after the PB / S@H hydrogel prepared in Example 3 of the present invention was treated in the gastrointestinal tract of mice.

[0056] Figure 8 This is a comparison chart of the rheological properties of hydrogel materials in different groups.

[0057] Fig. 9 These are the mechanical properties of hydrogel materials in different groups before and after being immersed in SGF solution.

[0058] Fig.10 This is the Fourier infrared spectroscopy analysis graph of PB / SAnetwork; PB / SAmixture; SA; SApowder; AM; PAM network in the present invention.

[0059] Fig.11 It is a comparison chart of the swelling rates of the sodium alginate-acrylamide hydrogel (H) of the present invention, the double network acrylamide-sodium alginate hydrogel doped with Prussian blue (PB@H), and the acrylamide-sodium alginate hydrogel doped with Prussian blue and sucralfate (PB / S@H) in water.

[0060] Fig.12 It is a comparison chart of the swelling rates of the sodium alginate-acrylamide hydrogel (H) of the present invention, the double-network acrylamide-sodium alginate hydrogel doped with Prussian blue (PB@H), and the acrylamide-sodium alginate hydrogel doped with Prussian blue and sucralfate (PB / S@H) in simulated gastric fluid.

[0061] Fig.13 These are DHE staining images of the intestinal tissues of mice in different groups.

[0062] Fig.14 Quantification of fluorescence intensity of DHE staining images of the intestines of mice in different groups.

[0063] Fig.15 This is a fluorescence imaging image of PB / S@H hydrogel in the gastrointestinal tract of mice. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0065] The experimental methods or test methods described in the following examples are conventional methods unless otherwise specified; the raw materials and auxiliary agents are obtained from conventional commercial channels or prepared by conventional methods unless otherwise specified.

[0066] In the following descriptions, sucralfate is abbreviated as S, acrylamide is abbreviated as AM, sodium alginate is abbreviated as SA, and Prussian blue nanoparticles are abbreviated as PB.

[0067] The test method adopted by the present invention is as follows:

[0068] (1) Swelling rate test method: The hydrogel is placed in a constant temperature drying oven at 60°C to remove moisture; then the thoroughly dried hydrogel is immersed in 37°C water and simulated gastric fluid, respectively, and shaken at 100 revolutions per minute. At different time points, the hydrogel is rinsed with deionized water to remove the solvent on the surface, and then the hydrogel is gently wiped with absorbent paper to remove the residual solvent on the surface. The mass of the hydrogel at different time points is weighed. The swelling rate of the hydrogel is described by the following formula:

[0069] Swelling ratio (%) = (W t -W0) / W0×100%

[0070] Where W0 and W t are the masses of the hydrogel at the initial time and at time t, respectively. Three experimental groups of the same size were set up for each type of hydrogel to repeat the experiment to ensure accuracy.

[0071] (2) Mechanical properties test method:

[0072] The rheological properties of different hydrogels were tested on a rotational rheometer (Anton Paar MCR92, Austria). Hydrogels with a diameter of 30 mm and a thickness of 5 mm were placed on a PP302 plate and measured in dynamic oscillation mode. During the dynamic oscillation scan, the frequency and temperature were maintained at 10 rad / s and 25°C, respectively, and the oscillation was gradually increased from 0.1 to 100%. During this period, the storage (G′) and loss (G″) moduli were recorded as a function of the oscillation stress. The rheological properties of the hydrogels incubated under different incubation conditions (before and after immersion in SGF) were recorded separately to reveal how the formation of a double cross-linked network in the hydrogel induced by gastric juice enhances the formation of composite hydrogels.

[0073] Example 1

[0074] A method for preparing a double network hydrogel, the specific steps are as follows:

[0075] (1) 10 mL of vinyl pyridine (4-VP), 0.5 g of cross-linking agent N,N'-methylenebisacrylamide (MBA), 0.1 g of initiator azobisisobutyronitrile (AIBN), 0.2 g of chitosan, and 0.1 mL of glutaraldehyde were dispersed in 100 mL of deionized water, and then the premixed solution was placed in a mold and heated to 60° C. until a vinyl pyridine hydrogel as a first network was formed;

[0076] (2) The vinyl pyridine hydrogel was rinsed three times with deionized water to remove unreacted components, and then immersed in simulated gastric fluid with a pH of 1.2 to form a chitosan second network in the first network of the vinyl pyridine hydrogel, i.e., a vinyl pyridine-chitosan composite double network hydrogel.

[0077] Figure 1 This is a SEM characterization image of the freeze-dried vinylpyridine-chitosan composite double network hydrogel sample in Example 1. Figure 1 It can be seen that the pore size formed by the vinylpyridine-chitosan composite double network hydrogel after freeze-drying is about 500nm.

[0078] Figure 2 This is a comparison of the swelling rates of chitosan hydrogel, vinyl pyridine hydrogel, and vinyl pyridine-chitosan composite double network hydrogel in gastric acid. Figure 2 It can be seen that the swelling rate of chitosan hydrogel is low, does not change much over time, and is stable at about 100%. However, due to the strong water absorption of the vinyl pyridine network, the swelling rate of the vinyl pyridine hydrogel is high, and the vinyl pyridine-chitosan composite double network hydrogel undergoes secondary cross-linking in gastric acid, and the formation of the double network increases the swelling rate. Finally, the swelling rate of the vinyl pyridine hydrogel is about 1500%, and the swelling rate of the vinyl pyridine-chitosan composite double network hydrogel is close to about 2000%.

[0079] Example 2

[0080] A method for preparing a double network hydrogel, the specific steps are as follows:

[0081] (1) Disperse 10 mL of N-vinyl formamide (NVF), 0.5 g of cross-linker N,N'-methylenebisacrylamide (MBA), 0.1 g of initiator azobisisobutyronitrile (AIBN), 2.0 g of sodium hyaluronate, and 0.1 g of cross-linker sulfated acrylic acid in 100 mL of deionized water, and stir at room temperature with a magnetic stirrer to ensure that all ingredients are completely dissolved. In order to remove oxygen from the solution, degassing is performed by nitrogen displacement for 5 to 10 minutes, and the degassed solution is sealed in a reaction container, and the mold is placed in a constant temperature water bath with the temperature controlled at 60°C to 70°C. The polymerization time is 12 hours. After the polymerization is completed, the hydrogel is removed from the mold and soaked in a large amount of deionized water, and the water is replaced until there is no significant residual monomer or by-product;

[0082] (2) The N-vinyl formamide hydrogel was rinsed three times with deionized water to remove unreacted components, and then immersed in simulated gastric fluid with a pH of 1.2 to form a sodium hyaluronate second network in the first network of the N-vinyl formamide hydrogel, i.e., an N-vinyl formamide-sodium hyaluronate composite double network hydrogel.

[0083] Figure 3 This is a SEM characterization image of the N-vinyl formamide-sodium hyaluronate composite double network hydrogel sample after freeze drying in Example 2. Figure 3 It can be seen that the pore size formed by the N-vinyl formamide-sodium hyaluronate composite double network hydrogel after freeze-drying is about 300 nm.

[0084] Figure 4 The swelling rates of sodium hyaluronate hydrogel, N-vinyl formamide hydrogel and N-vinyl formamide-sodium hyaluronate composite double network hydrogel in gastric acid are compared.

[0085] Depend on Figure 4 It can be seen that the swelling rate of sodium hyaluronate hydrogel is low, does not change much over time, and is stable at about 250%. However, due to the strong water absorption of N-vinyl formamide, the swelling rate of N-vinyl formamide hydrogel is high, and the N-vinyl formamide-sodium hyaluronate composite double network hydrogel undergoes secondary cross-linking in gastric acid, and the formation of a double network increases the swelling rate. Finally, the swelling rate of N-vinyl formamide hydrogel is about 1600%, and the swelling rate of N-vinyl formamide-sodium hyaluronate composite double network hydrogel is close to about 2000%.

[0086] Example 3

[0087] A method for preparing a nanocomposite double network hydrogel, the specific steps are as follows:

[0088] (1) 1.5 g acrylamide (AM), 10 μL N,N,N′,N′-tetramethylethylenediamine, 15 mg N,N′-bis(acryloyloxy)cystamine, 0.2 g sodium alginate (SA), 10 mg Prussian blue nanoparticles (PB) and 20 mg sucralfate were dispersed in 10 mL of an aqueous solution containing 30 wt % ethanol, and then the premixed solution was heated to 60° C. until a polyacrylamide hydrogel as a first network was formed;

[0089] (2) The polyacrylamide hydrogel was rinsed three times with deionized water to remove the unreacted components and then immersed in simulated gastric fluid with a pH of 1.2 to form a second network of Prussian blue nanoparticles and sodium alginate in the first network of polyacrylamide hydrogel, namely, a nanocomposite double network hydrogel named PB / S@H.

[0090] The preparation method of the Prussian blue nanoparticles is as follows:

[0091] At a temperature of 60°C, potassium ferrocyanide (0.04 mmol) and manganese chloride (0.05 mmol) were dispersed in 10 ml of ultrapure water with bovine serum albumin (BSA) (50 mg), respectively; potassium ferrocyanide solution A and manganese chloride solution B were obtained after magnetic stirring for 0.5 hour; then potassium ferrocyanide solution A was slowly added to manganese chloride solution B and stirred for 1 hour; after washing with distilled water several times and freeze-drying, Prussian blue nanoparticles were obtained.

[0092] Figure 5 TEM characterization image of Prussian blue nanoparticles prepared in Example 3, Figure 5 It can be seen that the particle size of Prussian blue nanoparticles is about 100 nm, presenting a relatively regular sphere.

[0093] Figure 6 This is the SEM characterization picture of the PB / S@H sample after freeze drying in Example 3. Figure 6 It can be seen that the pore size formed after freeze-drying of PB / S@H hydrogel is about 50nm.

[0094] Figure 7 : is a line graph of the iron and manganese contents in feces at different times after the PB / S@H hydrogel prepared in Example 3 of the present invention was treated in the gastrointestinal tract of mice. Figure 7 It can be seen that the hydrogel achieved long-term residence in the gastrointestinal tract, with a residence time of up to one week (168h). The high swelling ability of the hydrogel ensured its long residence time in the stomach.

[0095] Figure 8This is a comparison chart of the rheological properties of hydrogel materials in different groups. Rheology is an effective method to characterize the viscoelasticity of hydrogels and an indicator for judging the stability of hydrogel materials. When Prussian blue nanoparticles participate in the formation of hydrogels, the elastic modulus of double network hydrogels and drug-loaded double network hydrogels increases, which means that the hydrogel has stronger elastic properties under stress and is not prone to plastic deformation or flow. Therefore, we believe that after adding sucralfate, the mechanical properties of the hydrogel are improved to a certain extent, and when Prussian blue nanoparticles are introduced, the mechanical strength of the hydrogel is greatly improved.

[0096] At the same time, the rheological test results also show that when the drug-loaded double network hydrogel of Example 3 is immersed in SGF and forms a second cross-linked network, the drug-loaded double network hydrogel of Example 3 always remains in the linear viscoelastic region within a strain range of less than 30%, which means that it exhibits a greater degree of strain-independent viscoelastic response than a hydrogel with only a single cross-linked network. The highest gastric pressure in the human stomach is about 10-13 kPa, and the strain formed by sodium alginate-acrylamide hydrogel, double network hydrogel and drug-loaded double network hydrogel of Example 3 within this compressive stress range is less than 0.06%, which indicates that their rigidity in the stomach makes them less likely to be expelled by gastric pressure, providing favorable conditions for their long-term residence in the stomach.

[0097] Figure 8 and Fig. 9 The preparation method of sodium alginate hydrogel is as follows:

[0098] Disperse 60 mg of sodium alginate in 4 mL of water and slowly add it dropwise into a 2 g / L calcium lactate aqueous solution (calcium lactate acts as a coagulant) to form a sodium alginate hydrogel.

[0099] Fig. 9 The mechanical properties of hydrogel materials in different groups before and after being immersed in SGF solution. The hydrogels were loaded onto a pressure sensor (CMT-500, SANS, China) and a certain pressure was applied to obtain a force (F)-displacement (ΔL) curve. This was then converted into a stress (F / A)-strain (ΔL / L) curve. Each hydrogel was tested three times. Fig. 9 It can be seen that the hydrogel swelled to a certain extent after being immersed in simulated gastric fluid, resulting in a decrease in compressive stress. However, after the second cross-linked network was formed, the compressive stress of the hydrogel increased. Therefore, it can be inferred that the change in compressive stress exhibited by the drug-loaded double network hydrogel in Example 3 after being immersed in SGF is the result of a balance between these two factors.

[0100] Figure 8 and Fig. 9 The preparation method of acrylamide hydrogel is as follows:

[0101] 1.5 g acrylamide, 10 μL N,N,N′,N′-tetramethylethylenediamine, and 15 mg N,N′-bis(acryloyloxy)cystamine were dispersed in 10 mL of an aqueous solution containing 30 wt % ethanol, and the premixed solution was heated to 60° C. until a polyacrylamide network hydrogel was formed.

[0102] Figure 8 , Fig. 9 , Fig.11 and Fig.12 The preparation method of sodium alginate-acrylamide hydrogel is as follows:

[0103] 1.5 g acrylamide, 10 μL N,N,N′,N′-tetramethylethylenediamine, 15 mg N,N′-bis(acryloyloxy)cystamine, and 0.2 g sodium alginate were dispersed in 10 mL of an aqueous solution containing 30 wt % ethanol, and the premixed solution was heated to 60 °C until a polyacrylamide network hydrogel was formed.

[0104] Figure 8 , Fig. 9 , Fig.11 and Fig.12 The preparation method of the double network hydrogel is as follows:

[0105] (1) 1.5 g acrylamide, 10 μL N,N,N′,N′-tetramethylethylenediamine, 15 mg N,N′-bis(acryloyloxy)cystamine, 0.2 g sodium alginate and 10 mg Prussian blue nanoparticles were dispersed in 10 mL of an aqueous solution containing 30 wt % ethanol, and then the premixed solution was heated to 60° C. until a polyacrylamide hydrogel as a first network was formed;

[0106] (2) The polyacrylamide hydrogel was rinsed three times with deionized water to remove the unreacted components and then immersed in simulated gastric fluid with a pH of 1.2. Prussian blue nanoparticles and a sodium alginate second network were formed in the hydrogel with the polyacrylamide first network, namely, a nanocomposite double network hydrogel named PB@H.

[0107] Figure 8 , Fig. 9 , Fig.11 and Fig.12 The mesosucralfate-double network hydrogel was prepared by Example 3.

[0108] (3) Fourier transform infrared spectroscopy analysis:

[0109] 1) Immerse the PB and sodium alginate mixture in simulated gastric fluid, wait for it to gel, and place it in a constant temperature drying oven at 60°C to remove moisture. Then, the thoroughly dried hydrogel is made into powder, referred to as PB / SA network;

[0110] 2) directly removing water from the mixture of PB and sodium alginate to prepare powder, referred to as PB / SA mixture;

[0111] 3) Sodium alginate is added to pure water and dried thoroughly in an oven to form a powder, referred to as SA;

[0112] 4) Directly obtain the finished sodium alginate powder, referred to as SApowder;

[0113] 5) Directly obtain the finished acrylamide powder, referred to as AM;

[0114] 6) 1.5 g acrylamide, 10 μL N,N,N′,N′-tetramethylethylenediamine, and 15 mg N,N′-bis(acryloyloxy)cystamine were dispersed in 10 mL of an aqueous solution containing 30 wt% ethanol, and the premixed solution was heated to 60°C until a polyacrylamide network hydrogel was formed, and placed in a constant temperature drying oven at 60°C to remove moisture. The thoroughly dried hydrogel was then made into a powder, referred to as PAMnetwork.

[0115] The above samples were all subjected to Fourier transform infrared spectroscopy analysis to analyze the changes in chemical bonds during the gelation process.

[0116] Fig.10 This is the Fourier infrared spectroscopy analysis graph of PB / SAnetwork; PB / SAmixture; SA; SApowder; AM; PAM network in the present invention.

[0117] Depend on Fig.10 It is known that in Fig.10 In (b), the PAM network is formed by free radical polymerization of AM. When AM polymerizes, the double bond -C=C- (989 cm -1 ) almost disappears, indicating the formation of a PAM network. Fig.10 In (c), for the PB / SA network, PB and SA were physically mixed in the hydrogel before immersion in gastric fluid. In the FTIR spectrum of SA, 1591 and 1409 cm -1 The peaks at are attributed to the stretching vibration of -COO- in SA, and these two peaks disappear in the mixture of PB and SA.

[0118] As far as the spectrum of PB is concerned, Fig.10 (a) and Fig.10In (c), there are clearly identifiable -CN and -CO-NH peaks. These two peaks are not very obvious in the mixture of PB and SA before immersion in gastric juice. These peaks are likely to be interfered by the strong peak of -COO- in SA. Since no new peaks were observed, it is assumed that there is no crosslinking between PB and SA in the absence of acidic medium. After immersion in gastric juice, the hydrogel gradually swells as the gastric juice diffuses. At the same time, the PB nanoparticles surrounded by gastric juice are dissolved and form a second network with SA through ionic crosslinking. The two peaks of PB nanoparticles reappear and shift to a low wavenumber, indicating that there is ionic crosslinking between PB and SA.

[0119] Fig.11 The figure is a comparison of the swelling rates of the sodium alginate-acrylamide hydrogel (H) of the present invention, the double network acrylamide-sodium alginate hydrogel doped with Prussian blue (PB@H), and the acrylamide-sodium alginate hydrogel doped with Prussian blue and sucralfate (PB / S@H) in water. Fig.11 It can be seen that all three have high swelling rates in water, among which H has a swelling rate of about 1000% after absorbing water, increasing to ten times of its original volume. The swelling rate of PB@H is about 1500%. The swelling rate of PB / S@H is about 750%, which is because sucralfate does not absorb water in a neutral solution, so after adding sucralfate, the swelling rate of PB / S@H is slightly reduced.

[0120] Fig.12 The figure is a comparison of the swelling rates of the sodium alginate-acrylamide hydrogel (H) of the present invention, the double network acrylamide-sodium alginate hydrogel doped with Prussian blue (PB@H), and the acrylamide-sodium alginate hydrogel doped with Prussian blue and sucralfate (PB / S@H) in simulated gastric fluid. Fig.12 It can be seen that there is no significant difference in the swelling rates of the three in simulated gastric fluid. The swelling rate of H in gastric fluid is about 500%, the swelling rate of PB@H after forming a double network is about 500%, and the swelling rate of PB / S@H after forming a double network is about 700%. This is because in an acidic solution with a low pH, the colloid formed by sucralfate has a certain water absorption capacity, so the swelling rate of PB / S@H is slightly increased.

[0121] (4) Active oxygen scavenging function test:

[0122] Balb / c male mice (about 22 g) were fasted one day before formal modeling. On the second day, each mouse was gavaged with 200 μL of 75% ethanol to form acute peptic ulcer. At the same time, normal mice without gavage were set as blank control group. In the formal treatment stage, mice in different groups (such as

[0123] Control: blank control group,

[0124] Stimulus: Peptic ulcer model group was formed by ethanol gavage.

[0125] Self-healing: The peptic ulcer model was formed by ethanol gavage and then the normal diet group,

[0126] PB: Prussian blue nanoparticles gavage group,

[0127] H: Acrylamide-sodium alginate hydrogel gavage group,

[0128] S: Sucralfate suspension gavage group,

[0129] PB@H: sodium alginate-acrylamide hydrogel loaded with Prussian blue nanoparticles was intragastrically administered.

[0130] PB / S@H: sodium alginate-acrylamide hydrogel loaded with Prussian blue nanoparticles and sucralfate (gavage group)

[0131] After different treatments, the gastrointestinal tract of mice was embedded in paraffin and stained with dihydroethidium (DHE).

[0132] Dihydroethidium is a superoxide anion fluorescent probe with blue fluorescence in the cytoplasm. When it is taken up by living cells, it can be dehydrogenated under the action of superoxide anions in the cells to produce ethidium, which combines with RNA or DNA to produce red fluorescence. It can be used to detect oxidative activity in living cells. When the level of superoxide anions in the cells is high, more ethidium is produced, and the red fluorescence is stronger, otherwise it is weaker.

[0133] Fig.13 These are DHE staining images of the intestinal tissues of mice in different groups.

[0134] Fig.14 Quantification of fluorescence intensity of DHE staining images of the intestines of mice in different groups.

[0135] Combination Fig.13 and Fig.14 The Control group reflects the oxidative stress level of gastric tissue in normal mice. When inflammation occurs,

[0136] As shown in the Stimulus group, there was more red fluorescence, and the degree of inflammation was not reduced after seven days of self-eating.

[0137] The red fluorescence signal in the stomach tissue of mice in the Self-healing group increased slightly.

[0138] After H treatment, the oxidative stress level in gastric tissue returned to near normal levels due to the gastric mucosal protective effect of the hydrogel.

[0139] After PB treatment, the oxidative stress level was close to normal due to the ROS scavenging effect of PB.

[0140] After S treatment, due to its certain mucosal protective effect, the oxidative stress level of gastric tissue decreased after treatment, but it was still higher than the normal level.

[0141] After PB@H treatment, the oxidative stress level gradually returned to normal.

[0142] After PB / S@H treatment, the red signal almost disappeared, indicating that the level of oxidative stress in gastric tissue was extremely low.

[0143] Fig.15 This is a fluorescence imaging image of PB / S@H hydrogel in the gastrointestinal tract of mice.

[0144] As can be seen from the figure, the hydrogel achieved a long-term residence in the gastrointestinal tract, with a residence time of more than 96 hours. The high swelling capacity of the hydrogel ensures its long residence time in the stomach.

[0145] Test on the residence of the hydrogel nanoplatform in animals: 6-8 week old balb / c mice were fasted for 24 hours and then fed with PB / S@H containing the fluorescent dye Cy5.5. The fluorescence residence in the gastrointestinal tract was observed by in vivo imaging.

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a double network hydrogel, characterized in that: The following steps are involved: (1) reacting a polymer monomer, a cross-linking agent, an initiator, and a natural polymer compound to obtain a first network hydrogel; (2) Immersing the first network hydrogel in an acid system with a pH of 0.9 to 1.5 to react and obtain a double network hydrogel.

2. The method for preparing the double network hydrogel according to claim 1, characterized in that: The polymer monomer includes any one of acrylic acid and its derivatives, methacrylic acid and its derivatives, acrylamide and its derivatives, vinyl pyridine, and N-vinyl formamide; The cross-linking agent is selected from compounds containing multiple unsaturated double bonds in the molecule, and preferably the cross-linking agent includes any one of N,N'-methylenebisacrylamide, glutaraldehyde, and sulfated acrylic acid; The initiator includes a free radical polymerization initiator, preferably, the initiator is an azo initiator, and more preferably, the initiator is azobisisobutyronitrile; The natural polymer compound includes any one of natural polysaccharides and short peptides; preferably, the natural polysaccharide includes any one of sodium alginate, sodium hyaluronate and chitosan; The step (1) further comprises a coagulant, wherein the coagulant comprises at least one of N,N′-bis(acryloxy)cystamine, ethylene glycol diacrylate, and methyl methacrylate; The step (1) further comprises a solvent. Preferably, the solvent comprises a mixed solution of deionized water and ethanol, or deionized water. More preferably, the mass ratio of the ethanol to the mixed solution is 1 to 100 wt %, but not 100 wt %.

3. The method for preparing the double network hydrogel according to claim 1, characterized in that: The mass ratio of the polymer monomer to the natural high molecular compound is 1 to 10:

1.

4. A method for preparing a double network hydrogel encapsulating nanocarriers, characterized in that: The following steps are involved: (1) reacting a polymer monomer, a cross-linking agent, an initiator, Prussian blue nanoparticles, and a natural polymer compound to obtain a first network hydrogel; (2) Immersing the first network hydrogel in an acid system with a pH of 0.9 to 1.5 to react and obtain a double network hydrogel.

5. The method for preparing the double network hydrogel encapsulating nanocarriers according to claim 4, characterized in that: In step (1), the mass ratio of the polymer monomer, the natural polymer compound and the Prussian blue nanoparticles is 1-10:1:0.001-1.

6. The method for preparing the double network hydrogel encapsulating nanocarriers according to claim 4, characterized in that: The particle size of the Prussian blue nanoparticles in step (1) is 100 to 200 nm.

7. A method for preparing a double network hydrogel encapsulating nanocarriers and drugs, characterized in that: The following steps are involved: (1) reacting a polymer monomer, a cross-linking agent, an initiator, Prussian blue nanoparticles, a drug, and a natural polymer compound to obtain a first network hydrogel; (2) Immersing the first network hydrogel in an acid system with a pH of 0.9 to 1.5 to react and obtain a double network hydrogel.

8. The method for preparing the double network hydrogel encapsulating nanocarriers and drugs according to claim 7, characterized in that: The drug in step (1) includes sucralfate.

9. The method for preparing the double network hydrogel encapsulating nanocarriers and drugs according to claim 7, characterized in that: In step (1), the mass ratio of the polymer monomer, the natural polymer compound, the Prussian blue nanoparticles and the drug is 1-10:1:0.001-1:0.001-1.

10. A method for preparing the double network hydrogel according to claim 1, the double network hydrogel encapsulating nanocarriers according to claim 4, or the double network hydrogel encapsulating nanocarriers and drugs according to claim 7, characterized in that: The reaction temperature of step (1) is 0-100° C., and the reaction time is 1-120 minutes.

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