Preparation method and application of pH-responsive composite hydrogel
By constructing a dual-network hydrogel of sodium alginate and polyacrylamide-modified gelatin, the problems of uncontrollable mechanical properties and drug release of existing hydrogel materials were solved, enabling rapid drug release in the acidic tumor microenvironment and slow release in the normal environment, thus improving the accuracy and safety of drug delivery.
Patent Information
- Application Number
- CN202511937841.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-22
- Publication Date
- 2026-01-20
AI Technical Summary
Existing pH-responsive hydrogel materials suffer from poor mechanical properties, uncontrollable degradation behavior, and unsatisfactory drug release kinetics, making it difficult to achieve precise drug delivery.
By constructing a composite hydrogel with a dual-network interpenetrating structure of sodium oxidized alginate (OSA) and polyacrylamide-modified gelatin (GelMA), pH responsiveness is achieved through acylhydrazone bonds, and drug release is regulated by combining crosslinking agents and regulators.
It achieves rapid drug release in the acidic tumor microenvironment and slow release in the normal environment, exhibits excellent mechanical properties and biocompatibility, and reduces systemic toxicity.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of medical biomaterials, in particular to a preparation method and application of a pH-responsive composite hydrogel. BACKGROUND
[0002] At present, drug delivery systems play a crucial role in precision medicine, and hydrogel materials have become ideal carriers for local controlled release of drugs due to their three-dimensional network structure, good biocompatibility and controllable physicochemical properties. Especially, intelligent hydrogels with environmental response characteristics can achieve on-demand drug release according to external stimuli such as pH, temperature and enzymes, which can improve the therapeutic effect while reducing systemic toxicity, and show broad application prospects.
[0003] Among them, pH-responsive hydrogels have attracted widespread attention from researchers because they can achieve targeted drug release in the slightly acidic environment of tumor, infected site and diseased tissue. This kind of hydrogel usually introduces pH-sensitive chemical bonds or groups such as acylhydrazone bond and carboxyl group into the polymer network, so as to realize the swelling, degradation or bond rupture under acidic conditions, thereby triggering the release of the loaded drug.
[0004] However, the pH-responsive hydrogels in the prior art, especially the materials based on a single polymer system, still have several key defects, which seriously limit their practical application potential: (1) insufficient mechanical properties and structural stability. Most of the hydrogels prepared from single natural polymer such as hyaluronic acid, chitosan or oxidized sodium alginate have low mechanical strength, which is easy to collapse or degrade prematurely in the dynamic environment in vivo, and it is difficult to maintain the long-term stability of the drug carrier. (2) Poor controllability of drug release behavior. The degradation rate and drug release kinetics of single-component hydrogels are often difficult to accurately control, which easily leads to burst release or incomplete release of drugs, and cannot achieve smooth and continuous drug release matching the treatment cycle. (3) Limited response sensitivity and release efficiency. The pH response range of some hydrogels is wide and the sensitivity is insufficient, and the differential release effect between the physiological environment and the diseased microenvironment is not significant, which reduces the specificity and efficiency of targeted drug delivery. (4) Limited multifunctional integration and structural design. The existing systems often focus on single response characteristics, and there are still deficiencies in considering the multifunctional synergy of antibacterial property, self-repairing ability, pore uniformity and cell compatibility, which limits the application adaptability in complex biological environment.
[0005] Therefore, there is an urgent need in the art to develop a new type of composite hydrogel material, which can simultaneously improve its mechanical properties, degradation controllability, drug release kinetics and structural functional integration design on the basis of maintaining excellent biocompatibility and clear pH response, thereby providing a more reliable and efficient material platform for intelligent drug delivery systems. SUMMARY
[0006] The application aims to provide a preparation method and application of a pH-responsive composite hydrogel, so as to solve the technical problems of poor mechanical property, uncontrollable degradation behavior and unsatisfactory drug release kinetics of a single-network intelligent hydrogel material in the prior art.
[0007] To achieve the above-mentioned object, the application provides the following technical scheme. In one aspect, the application provides a preparation method of a pH-responsive composite hydrogel, comprising the following steps. (1) Preparation of oxidized sodium alginate (OSA): sodium alginate is dispersed in ultrapure water to form a suspension, and after oxidation reaction with sodium periodate under light shielding conditions, an oxidation reaction termination agent is used to terminate the oxidation reaction, and the oxidized sodium alginate with pH response of acylhydrazone bond is prepared through dialysis purification and freeze-drying. (2) Preparation of modified gelatin (GelMA): gelatin is dispersed in ultrapure water and stirred until completely dissolved, then an initiator and a modifier are sequentially added to obtain a mixed solution A, and then a crosslinking agent A is added and stirred until the crosslinking reaction is complete to obtain a modified gelatin precursor solution. (3) Preparation of OSA-GelMA drug-loaded composite hydrogel: the oxidized sodium alginate with pH response of acylhydrazone bond and a drug carrier are added to the modified gelatin precursor solution, and stirring is performed until complete dissolution to obtain a mixed solution B; a crosslinking agent B and an adjusting agent are added to the mixed solution B, and stirring and oscillation are performed until the solution is uniformly mixed to obtain an OSA-GelMA drug-loaded mixed solution. (4) The OSA-GelMA drug-loaded mixed solution is placed in an oven for reaction to obtain a pH-responsive OSA-GelMA drug-loaded hydrogel, and the gel is freeze-dried.
[0008] Preferably, in step (1), the concentration of sodium alginate is 0.01-1.00 mol / L, and the concentration of sodium periodate is 0.02-1.00 mol / L; the termination agent is a diethylene glycol solution with a concentration of 0.01-1.00 mol / L.
[0009] Preferably, in step (1), the oxidation reaction under light shielding conditions is 300-400 r / min magnetic stirring at 25-30℃ for 2-4 h; the termination of the oxidation reaction is 300-400 r / min magnetic stirring at 25-30℃ for 0.5-1.5 h; the dialysis purification is that the solution after termination of the oxidation reaction is loaded into a dialysis bag with a molecular weight of 3000-4000 Da, and dialysis is performed at 25℃ for 2-8 days; and the freeze-drying is performed at -40--20℃ for 48-72 h.
[0010] Preferably, the concentration of gelatin in the mixed solution A of step (2) is 0.01-1.5 g / mL, the initiator is potassium persulfate with a concentration of 0.001-0.1 mol / L, the modifier is acrylamide with a concentration of 0.1-2.0 mol / L, and the crosslinking agent A is N,N-methylene bisacrylamide with a concentration of 0.002-0.006 mol / L.
[0011] Preferably, the stirring and dissolving of step (2) is performed at 300-400 r / min under magnetic stirring for 1-2 h in a water bath at 30-40℃, and the crosslinking reaction is performed at 300-400 r / min under magnetic stirring for 0.5-1 h at a temperature of 50-70℃.
[0012] Preferably, the drug carrier in step (3) is bortezomib, the mass ratio of the pH-responsive acylhydrazone-oxidized sodium alginate to the modified gelatin in the mixed solution B is 1:1-1:5, the Z loading capacity of the drug carrier is 10-100 mg, the crosslinking agent B is adipic acid hexahydrazone with a concentration of 0.1-2.0 mol / L, and the adjusting agent is polyethylene glycol with a concentration of 5-50 mg / mL.
[0013] Preferably, the stirring and dissolving of step (3) is performed at 300-400 r / min under magnetic stirring for 1-3 h at a temperature of 50-70℃, and the stirring and oscillation is performed for 1-4 min under ultrasonic conditions at a power of 80-100 W, a frequency of 50-80 KHz, and a temperature of 24-27℃.
[0014] Preferably, the reaction in the oven of step (4) is performed at 50-70℃ for 1-4 h.
[0015] Preferably, the freeze-drying of step (4) is performed at -40--20℃ for 48-72 h.
[0016] In another aspect, the application provides a use of the pH-responsive OSA-GelMA drug-loaded hydrogel prepared by the method in the preparation of a pharmaceutical preparation for local drug delivery, wherein the pH-responsive OSA-GelMA drug-loaded hydrogel is loaded with a drug carrier and is used to trigger the release of an active substance of the drug carrier in an acidic microenvironment.
[0017] Specifically, the acidic microenvironment is the microenvironment of a solid tumor, and the active substance of the drug carrier is an active substance of an anti-tumor drug.
[0018] Specifically, a degradable antibacterial material is composed of the pH-responsive OSA-GelMA drug-loaded hydrogel prepared by the method.
[0019] The application has excellent intelligent responsiveness and precise drug delivery performance: the hydrogel has high sensitivity to pH changes based on the acylhydrazone bond in the OSA network of oxidized sodium alginate; under the simulated tumor acidic microenvironment pH 5.4, the hydrogel can quickly swell and realize efficient and rapid release of drugs (the cumulative release rate can reach 99 %); and under the normal physiological environment pH 7.4, the hydrogel remains stable in structure and slowly releases drugs, effectively realizing local targeted delivery of drugs and reduction of systemic toxic side effects.
[0020] The application has optimized mechanical properties and stable three-dimensional structure: by constructing the double-network interpenetrating structure of OSA and polyacrylamide modified gelatin GelMA, the defect of poor mechanical property of single-component hydrogel is overcome; the composite hydrogel has uniform and connected honeycomb porous structure, the pore distribution is concentrated, and the hydrogel has good mechanical strength, structural stability and ideal injectability and implantability, and can provide stable space for drug loading and cell growth.
[0021] The application has controllable degradation behavior and good biocompatibility: by adjusting the crosslinking density of the double network, the in vitro degradation rate of the hydrogel can be effectively controlled, which can match the tumor treatment cycle, ensure the sustained release of drugs, and avoid long-term residual of the carrier. The raw materials sodium alginate, gelatin and the final product all exhibit good biocompatibility.
[0022] The application has significant antibacterial performance and comprehensive treatment potential: the hydrogel itself shows obvious antibacterial effect on escherichia coli and staphylococcus aureus, which can effectively reduce the risk of local infection after implantation or injection; combined with its pH-triggered drug release characteristics, it provides a new integrated platform for realizing synergistic treatment of anti-tumor and anti-infection.
[0023] The preparation process of the application is simple, controllable and reproducible: the preparation method of the aqueous phase reaction and the medium-temperature crosslinking has mild conditions, the raw materials are cheap and easy to obtain, the steps are clear and controllable, and is suitable for large-scale production, and has good clinical transformation prospect.
[0024] In summary, the beneficial effects of the application are: 1. The pH-responsive OSA-GelMA drug-loaded hydrogel prepared by the application has clear pH response characteristics, and can achieve rapid release of drugs in the tumor acidic microenvironment and slow release in the neutral environment, realizing precise targeted delivery of drugs.
[0025] 2. The application adopts sodium alginate and polyacrylamide modified gelatin to construct a double-network structure, which has good mechanical properties, thermal stability and self-repairing performance, and the pore structure is beneficial to drug loading and release.
[0026] 3、The loaded bortezomib has significant toxicity to various tumor cells, and the hydrogel itself has obvious antibacterial effect on escherichia coli and staphylococcus aureus, and can reduce the risk of infection in the tumor treatment process.
[0027] 4、The preparation process is simple and controllable, the raw materials used have excellent biocompatibility, the degradation rate matches the tumor treatment cycle, and has good clinical transformation prospect. BRIEF DESCRIPTION OF DRAWINGS
[0028] Figure 1 The physical map of the BTZ@OSA-GelMA hydrogel prepared in embodiment 1 of the application; Figure 2 The infrared spectrum (FTIR) of the BTZ@OSA-GelMA hydrogel prepared in embodiment 1 of the application; Figure 3 The scanning electron microscope (SEM) of the BTZ@OSA-GelMA hydrogel prepared in embodiment 1 of the application; Figure 4 The scanning electron microscope (SEM) of the pH-responsive OSA hydrogel prepared in comparative example 1 of the application; Figure 5 The pore size distribution of the BTZ@OSA-GelMA and OSA hydrogels prepared in embodiment 1 and comparative example 1 of the application; Figure 6 The swelling property diagram of the BTZ@OSA-GelMA composite hydrogel prepared in embodiment 1 and comparative examples 2-4 of the application at pH=7.4; Figure 7 The in vitro degradation rate diagram of the BTZ@OSA-GelMA composite hydrogel prepared in embodiment 1 and comparative examples 2-4 of the application; Figure 8 The swelling property diagram of the BTZ@OSA-GelMA composite hydrogel prepared in embodiment 1 of the application under different pH conditions; Figure 9 The drug cumulative release rate diagram of the BTZ@OSA-GelMA composite hydrogel prepared in embodiment 1 of the application under different pH conditions. DETAILED DESCRIPTION
[0029] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application. Example 1
[0030] A pH-responsive BTZ@OSA-GelMA composite hydrogel was prepared, and the specific steps were as follows: (1) Preparation of oxidized sodium alginate: First, 5 g of sodium alginate was weighed and placed in 250 mL of ultrapure water, stirred at 300 rpm at 25°C until completely dissolved, and cut into a tin foil to completely package the beaker containing the sodium alginate suspension. Then, 2.5 g of sodium periodate was weighed and added to the sodium alginate suspension, which was stirred at 300 rpm at 25°C in the dark for 3 h. After 3 h, 2.5 mL of diethylene glycol was added to the beaker to quench the oxidation reaction for 1 h. The reacted oxidized sodium alginate solution was placed in a 3500 Da dialysis bag and dialyzed for 4 days. Finally, the dialyzed oxidized sodium alginate solution was pre-frozen at -40°C for 12 h, then the sample was placed in a freeze dryer at -40°C, and the oxidized sodium alginate was obtained after freeze-drying.
[0031] (2) Preparation of polyacrylamide-modified gelatin: 1.5 g of gelatin was weighed and placed in 30 mL of ultrapure water, and the water bath was placed in a 40°C, 400 rpm magnetic stirring environment until completely dissolved. 0.1 g of potassium persulfate was weighed and added to the gelatin solution, which was stirred at 400 rpm for 10 min. Acrylamide was added to the mixed solution, which was stirred at 400 rpm for 30 min. Then the temperature was adjusted to 60°C, and 20 mg of N,N-methylenebisacrylamide was added, and the reaction was carried out for 40 min to obtain a polyacrylamide-modified gelatin solution.
[0032] (3) Preparation of BZT@OSA-GelMA hydrogel: 5 mL of polyacrylamide-modified gelatin solution was placed in a beaker, and 0.45 g of oxidized sodium alginate, 50 mg of polyethylene glycol, and 20 mg of bortezomib were added to the beaker, which was stirred with a glass rod and ultrasonicated for 2 min to obtain a mixed solution B. 40 mg of adipic acid hexahydrate was weighed and added to 2 mL of polyethylene glycol solution, which was mixed and dissolved in another beaker to obtain solution C. Then 1 mL of mixed solution C was added to mixed solution B, which was stirred with a glass rod and ultrasonicated for 2 min until the solution was uniformly mixed to obtain BZT@OSA-GelMA mixed solution. The BZT@OSA-GelMA mixed solution was placed in a 60°C oven and reacted for 2 h, at which time the BTZ@OSA-GelMA composite hydrogel was obtained, and the product was cooled to room temperature. Finally, the composite hydrogel was pre-frozen for 24 h and freeze-dried at -40°C.
[0033] Figure 1The actual figure of the BZT@OSA-GelMA hydrogel prepared in this embodiment 1 is white and transparent jelly with good formability.
[0034] Figure 2 The infrared spectrum of the hydrogel in this example 1 is shown in the figure, which shows that the BZT@OSA-GelMA composite hydrogel has N-H / O-H stretching vibration peaks at 3438 cm Figure 2 , acylhydrazone bond characteristic absorption peaks at 1680 cm -1 , and C-H stretching vibration peaks of polyacrylamide at 2954 cm -1 . -1 The characteristic peaks (3436 cm -1 , 1055 cm -1 ) of BTZ are clearly present in the infrared spectrum of the composite hydrogel without obvious shift or disappearance, indicating that BTZ does not destroy the chemical structure of the hydrogel, but exists stably in the hydrogel network through physical loading or weak interaction, indicating that OSA, GelMA and BTZ are effectively compounded. Embodiment 2
[0035] A pH-responsive BTZ@OSA-GelMA composite hydrogel was prepared, and the specific steps were as follows: (1) Preparation of oxidized sodium alginate: first, weigh 5 g of sodium alginate and place it in 250 mL of ultrapure water, stir at 350 rpm at 27°C until completely dissolved, cut a suitable tin paper to completely package the beaker containing the sodium alginate suspension, then weigh 2.5 g of sodium periodate and pour it into the sodium alginate suspension, stir at 350 rpm at 27°C in the dark for 2 h. After 2 h, 2.5 mL of diethylene glycol is added to the dark reaction beaker, and the oxidation reaction is quenched for 0.5 h. The reacted oxidized sodium alginate solution is placed in a 3500 Da dialysis bag and dialyzed for 6 days. Finally, the dialyzed oxidized sodium alginate solution is pre-frozen at -40°C for 12 h, then the sample is placed in a freeze dryer at -40°C, and after freeze-drying, the oxidized sodium alginate is obtained.
[0036] (2) Preparation of polyacrylamide modified gelatin: weigh 1.5 g of gelatin and place it in 30 mL of ultrapure water, place it in a 30°C water bath environment with magnetic stirring at 400 rpm until completely dissolved. Weigh 0.1 g of potassium persulfate and add it to the gelatin solution, stir at 400 rpm for 10 min. Add acrylamide to the mixed solution and stir at 400 rpm for 30 min. Then adjust the temperature to 50°C, add 20 mg of N,N-methylenebisacrylamide, and react for 30 min to obtain a polyacrylamide modified gelatin solution.
[0037] (3) Preparation of BZT@OSA-GelMA hydrogel: 5 mL of polyacrylamide modified gelatin solution was taken into a beaker, 0.45 g of oxidized sodium alginate, 50 mg of polyethylene glycol and 20 mg of bortezomib were weighed and added to the beaker, a glass rod was used to stir and ultrasonic for 3 min to obtain a mixed solution B. 40 mg of adipic acid hexahydrate hydrazide was weighed and added to 2 mL of polyethylene glycol solution, and another beaker was used to mix and dissolve to obtain solution C. Then 1 mL of mixed solution C was taken into mixed solution B, stirred with a glass rod and ultrasonic for 2 min until the solution was uniformly mixed to obtain BZT@OSA-GelMA mixed solution. The BZT@OSA-GelMA mixed solution was placed in a 50°C oven for 4 h, at which time the BTZ@OSA-GelMA composite hydrogel was obtained, and the product was cooled to room temperature. Finally, the composite hydrogel was pre-frozen for 24 h and freeze-dried at -40°C.
[0038] The BTZ@OSA-GelMA hydrogel prepared in this example also has good drug loading effect and swelling rate. Example 3
[0039] A pH-responsive BTZ@OSA-GelMA composite hydrogel was prepared, and the specific steps were as follows: (1) Preparation of oxidized sodium alginate: first, 5 g of sodium alginate was weighed and placed in 250 mL of ultrapure water, stirred at 30°C and 400 rpm until completely dissolved, and then cut a suitable tin paper to completely package the beaker containing the sodium alginate suspension. Then 2.5 g of sodium periodate was weighed and added to the sodium alginate suspension, and stirred at 30°C and 3400 rpm in the dark for 4 h. After 4 h, 2.5 mL of diethylene glycol was added to the beaker for the reaction in the dark, and the oxidation reaction was quenched after 1.5 h. The reacted oxidized sodium alginate solution was placed in a 3500 Da dialysis bag and dialyzed for 8 days. Finally, the dialyzed oxidized sodium alginate solution was pre-frozen at -40°C for 12 h, then placed in a freeze dryer at -40°C, and freeze-dried to obtain oxidized sodium alginate.
[0040] (2) Preparation of polyacrylamide modified gelatin: 1.5 g of gelatin was weighed and placed in 30 mL of ultrapure water, and placed in a water bath environment with magnetic stirring at 35°C and 400 rpm until completely dissolved. 0.1 g of potassium persulfate was weighed and added to the gelatin solution, and stirred at 400 rpm for 10 min. Acrylamide was added to the mixed solution, and stirred at 400 rpm for 30 min. Then the temperature was adjusted to 70°C, 20 mg of N,N-methylene bisacrylamide was added, and the reaction was carried out for 1 h to obtain a polyacrylamide modified gelatin solution.
[0041] (3) Preparation of BZT@OSA-GelMA hydrogel: 5 mL of polyacrylamide modified gelatin solution was taken into a beaker, 0.45 g of oxidized sodium alginate, 50 mg of polyethylene glycol and 20 mg of bortezomib were weighed and added into the beaker, a glass rod was used for stirring and ultrasonic treatment for 4 min to obtain a mixed solution B. 40 mg of adipic acid hexahydropyridine was weighed and added into 2 mL of polyethylene glycol solution, and another beaker was used for mixing and dissolving to obtain solution C. Then 1 mL of mixed solution C was taken into mixed solution B, a glass rod was used for stirring and ultrasonic treatment for 2 min until the solution was uniformly mixed to obtain a BZT@OSA-GelMA mixed solution. The BZT@OSA-GelMA mixed solution was placed in a 70°C oven for 1 h, at which time the BTZ@OSA-GelMA composite hydrogel was obtained, and the product was cooled to room temperature. Finally, the composite hydrogel was pre-frozen for 24 h and freeze-dried at -40°C.
[0042] The BTZ@OSA-GelMA hydrogel prepared in this example also has good drug loading effect and swelling rate.
[0043] Comparison Table 1 Preparation of a pH-responsive oxidized sodium alginate hydrogel
[0044] (1) Preparation of oxidized sodium alginate: first, 5 g of sodium alginate was weighed into 250 mL of ultrapure water and stirred at 300 rpm at 25°C until completely dissolved. The beaker containing the sodium alginate suspension was completely wrapped with a suitable tin paper, and 2.5 g of sodium periodate was then weighed and added into the sodium alginate suspension. The mixture was stirred at 300 rpm at 25°C in the dark for 3 h. After 3 h, 2.5 mL of diethylene glycol was added to the beaker for the reaction in the dark, and the oxidation reaction was quenched after 1 h. The reacted oxidized sodium alginate solution was placed in a 3500 Da dialysis bag and dialyzed for 4 days. Finally, the dialyzed oxidized sodium alginate solution was pre-frozen at -40°C for 12 h, and then the sample was placed in a freeze dryer at -40°C for freeze-drying to obtain the oxidized sodium alginate.
[0045] (2) Preparation of pH-responsive oxidized sodium alginate hydrogel: 0.45 g of OSA raw material was weighed and added to 5 mL of polyethylene glycol solution. It was completely dissolved at 25°C under 300 rpm stirring to obtain mixed solution A. 40 mg of adipic acid hexahydropyridine was weighed and added to another beaker containing 2 mL of polyethylene glycol solution. It was ultrasonically treated for 2 min and stirred with a glass rod until completely dissolved to obtain mixed solution B. 1 mL of mixed solution B was further taken and added dropwise to mixed solution A. After ultrasonic treatment for 2 min, mixed solution C was obtained. Then mixed solution C was dropped into a 1 cm x 1 cm cylindrical hole mold to obtain OSA hydrogel. Finally, the OSA hydrogel was pre-frozen in a ultra-low temperature refrigerator at -40°C for 24 h and dried in a freeze dryer at -40°C for 60 h.
[0046] Figure 3 and Figure 4 The scanning electron microscope (SEM) images of the composite hydrogel of Example 1 and the single OSA hydrogel of Comparative Example 1 are shown respectively. As can be seen from the figure, the BTZ@OSA-GelMA composite hydrogel presents a uniform and interconnected honeycomb porous structure, while the single OSA hydrogel has a relatively sparse pore structure and poor connectivity, and part of the area has a pore collapse phenomenon.
[0047] The pore size distribution curve of Figure 5 It can be seen that the pore structure of the single OSA hydrogel is loose and the pore size distribution is chaotic, while the pore size distribution of the BTZ@OSA-GelMA composite hydrogel is uniform and concentrated. This uniform porous structure not only provides sufficient space for the efficient loading of BTZ, but also facilitates the uniform diffusion and release of drug molecules in an acidic environment, laying a structural foundation for the drug delivery performance of the material.
[0048] It can be seen that the pure pH-responsive material OSA itself cannot form an ideal structural carrier; and the present application introduces and cooperates another network GelMA, which not only retains the pH responsiveness, but also fundamentally overcomes the core structural defects of single material, thereby obtaining a new material with significantly improved comprehensive performance. Comparative Table 2 A pH-responsive anti-tumor BTZ@OSA-GelMA hydrogel was prepared, and the specific steps were as follows:
[0049] (1) Preparation of oxidized sodium alginate: First, weigh 5 g of sodium alginate into 250 mL of ultrapure water, stir at 300 rpm at 25°C until completely dissolved, cut a suitable tin paper to completely package the beaker containing the sodium alginate suspension, then weigh 2.5 g of sodium periodate into the sodium alginate suspension, stir at 300 rpm at 25°C in the dark for 3 h. After 3 h, 2.5 mL of diethylene glycol is added to the dark reaction beaker, and the oxidation reaction is quenched after 1 h. The reacted oxidized sodium alginate solution is placed in a 3500 Da dialysis bag and dialyzed for 4 days. Finally, the dialyzed oxidized sodium alginate solution is pre-frozen at -40°C for 12 h, then the sample is placed in a freeze dryer at -40°C, and the oxidized sodium alginate is obtained after freeze-drying.
[0050] (2) Preparation of polyacrylamide modified gelatin: Weigh 1.5 g of gelatin into 30 mL of ultrapure water, and place it in a 40°C water bath environment with magnetic stirring at 400 rpm until completely dissolved. Weigh 0.1 g of potassium persulfate into the gelatin solution and stir at 400 rpm for 10 min. Add acrylamide to the mixed solution and stir at 400 rpm for 30 min. Then adjust the temperature to 60°C, add 10 mg of N,N-methylene bisacrylamide, and react for 40 min to obtain a polyacrylamide modified gelatin solution.
[0051] (3) Preparation of BZT@OSA-GelMA hydrogel: Take 5 mL of polyacrylamide modified gelatin solution into a beaker, weigh 0.45 g of oxidized sodium alginate, 50 mg of diethylene glycol, and 20 mg of bortezomib into the beaker, stir with a glass rod and ultrasonic for 2 min to obtain a mixed solution B. Weigh 40 mg of adipic acid hexahydrate into 2 mL of polyethylene glycol solution, mix and dissolve in another beaker to obtain solution C. Then take 1 mL of mixed solution C into mixed solution B, stir with a glass rod and ultrasonic for 2 min until the solution is uniformly mixed to obtain BZT@OSA-GelMA mixed solution. Put the BZT@OSA-GelMA mixed solution into a 60°C oven and react for 2 h, at which time the BTZ@OSA-GelMA composite hydrogel is obtained, and the product is cooled to room temperature. Finally, the composite hydrogel is pre-frozen for 24 h and freeze-dried at -40°C. Comparative Table 3 A pH-responsive anti-tumor BTZ@OSA-GelMA hydrogel is prepared, and the specific steps are as follows:
[0052] (1) Preparation of oxidized sodium alginate: First, weigh 5 g of sodium alginate into 250 mL of ultrapure water, stir at 300 rpm at 25°C until completely dissolved, cut a suitable tin paper to completely package the beaker containing the sodium alginate suspension, then weigh 2.5 g of sodium periodate into the sodium alginate suspension, stir at 300 rpm at 25°C in the dark for 3 h. After 3 h, 2.5 mL of diethylene glycol is added to the dark reaction beaker, and the oxidation reaction is quenched after 1 h. The reacted oxidized sodium alginate solution is placed in a 3500 Da dialysis bag and dialyzed for 4 days. Finally, the dialyzed oxidized sodium alginate solution is pre-frozen at -40°C for 12 h, then the sample is placed in a freeze dryer at -40°C, and the oxidized sodium alginate is obtained after freeze-drying.
[0053] (2) Preparation of polyacrylamide modified gelatin: Weigh 1.5 g of gelatin into 30 mL of ultrapure water, and place it in a 40°C water bath environment with magnetic stirring at 400 rpm until completely dissolved. Weigh 0.1 g of potassium persulfate into the gelatin solution and stir at 400 rpm for 10 min. Add acrylamide to the mixed solution and stir at 400 rpm for 30 min. Then adjust the temperature to 60°C, add 15 mg of N,N-methylene bisacrylamide, and react for 40 min to obtain a polyacrylamide modified gelatin solution.
[0054] (3) Preparation of BZT@OSA-GelMA hydrogel: Take 5 mL of polyacrylamide modified gelatin solution into a beaker, weigh 0.45 g of oxidized sodium alginate, 50 mg of diethylene glycol, and 20 mg of bortezomib into the beaker, stir with a glass rod and ultrasonic for 2 min to obtain a mixed solution B. Weigh 40 mg of adipic acid hydrazide into 2 mL of polyethylene glycol solution, mix and dissolve in another beaker to obtain solution C. Then take 1 mL of mixed solution C into mixed solution B, stir with a glass rod and ultrasonic for 2 min until the solution is uniformly mixed to obtain BZT@OSA-GelMA mixed solution. Put the BZT@OSA-GelMA mixed solution into a 60°C oven and react for 2 h, at which time the BTZ@OSA-GelMA composite hydrogel is obtained, and the product is cooled to room temperature. Finally, the composite hydrogel is pre-frozen for 24 h and freeze-dried at -40°C. Table 4 A pH-responsive anti-tumor BTZ@OSA-GelMA hydrogel is prepared, and the specific steps are as follows:
[0055] (1) Preparation of oxidized sodium alginate: First, weigh 5 g of sodium alginate into 250 mL of ultrapure water, stir at 300 rpm at 25°C until completely dissolved, cut the appropriate tin paper to completely package the beaker containing the sodium alginate suspension, then weigh 2.5 g of sodium periodate into the sodium alginate suspension, stir at 300 rpm at 25°C in the dark for 3 h. After 3 h, 2.5 mL of diethylene glycol is added to the light-protected reaction beaker, and the oxidation reaction is quenched after 1 h. The oxidized sodium alginate solution is placed in a 3500 Da dialysis bag and dialyzed for 4 days. Finally, the dialyzed oxidized sodium alginate solution is pre-frozen at -40°C for 12 h, then the sample is placed in a freeze dryer at -40°C, and the oxidized sodium alginate is obtained after freeze-drying.
[0056] (2) Preparation of polyacrylamide modified gelatin: Weigh 1.5 g of gelatin into 30 mL of ultrapure water, and place it in a 40°C water bath environment with magnetic stirring at 400 rpm until completely dissolved. Weigh 0.1 g of potassium persulfate into the gelatin solution and stir at 400 rpm for 10 min. Add acrylamide to the mixed solution and stir at 400 rpm for 30 min. Then adjust the temperature to 60°C, add 25 mg of N,N-methylene bisacrylamide, and react for 40 min to obtain a polyacrylamide modified gelatin solution.
[0057] (3) Preparation of BZT@OSA-GelMA hydrogel: Take 5 mL of polyacrylamide modified gelatin solution into a beaker, weigh 0.45 g of oxidized sodium alginate, 50 mg of diethylene glycol, and 20 mg of bortezomib into the beaker, stir with a glass rod and ultrasonic for 2 min to obtain a mixed solution B. Weigh 40 mg of adipic acid hexahydrate into 2 mL of polyethylene glycol solution, mix and dissolve in another beaker to obtain solution C. Then take 1 mL of mixed solution C into mixed solution B, stir with a glass rod and ultrasonic for 2 min until the solution is uniformly mixed to obtain BZT@OSA-GelMA mixed solution. Put the BZT@OSA-GelMA mixed solution into a 60°C oven and react for 2 h, at which time the BTZ@OSA-GelMA composite hydrogel is obtained. Cool the product to room temperature. Finally, the composite hydrogel is pre-frozen for 24 h and freeze-dried at -40°C.
[0058] Figure 6The swelling rates of the composite hydrogels prepared in Example 1 and Comparative Examples 2-4 under normal physiological environment pH = 7.4 were compared. As can be observed from the figure, all the hydrogels showed a faster swelling rate in the first 8 hours, and then gradually reached a stable state. Among them, the equilibrium swelling rate of the hydrogel of Example 1 was about 500 %, which was in a reasonable range, avoiding the problem of excessive swelling and easy collapse of structure caused by insufficient crosslinking degree of Comparative Example 2, and overcoming the problem of insufficient swelling and blocked drug release caused by excessive crosslinking degree of Comparative Example 4. Example 1 can construct a hydrogel network with both structural stability and controllable swelling, providing protection for drug release under normal physiological environment.
[0059] Figure 7 The in vitro degradation rate of the composite hydrogels prepared in Example 1 and Comparative Examples 2-4 was compared. Comparative Example 2 had the lowest crosslinking degree and the fastest degradation rate, making it difficult to match the tumor treatment cycle; Comparative Example 4 had too high crosslinking degree and too slow degradation rate, which might lead to in vivo residue; while the degradation rate of the hydrogel of Example 1 was moderate, about 48 %, which could gradually degrade within the tumor treatment cycle, ensuring the sustainability of drug release and avoiding long-term carrier residue.
[0060] Figure 8 The swelling performance of the composite hydrogel prepared in Example 1 under different pH environments was demonstrated. Under the condition of pH = 5.4, the swelling rate was the highest and the rate of reaching equilibrium was the fastest, while under the condition of pH = 7.4, the swelling rate was the lowest. This phenomenon provides a structural basis for targeted drug release, achieving rapid swelling of the hydrogel at the tumor site to promote efficient release of BTZ, while in normal tissues, the low swelling rate of the hydrogel can reduce drug leakage and reduce systemic toxic side effects.
[0061] Figure 9 The drug cumulative release curve of BTZ@OSA-GelMA composite hydrogel under different pH environments directly reflects the pH-responsive drug delivery performance of the prepared composite hydrogel. In the simulated tumor acidic microenvironment pH = 5.4, the cumulative release rate of BTZ was the highest, reaching about 99 %, and the release rate was the fastest, which could achieve rapid response to pH and release an effective therapeutic concentration of drug. In the normal physiological environment pH = 7.4, the release rate of BTZ was the slowest and the cumulative release rate was the lowest. This phenomenon can effectively solve the key problem of non-specific release of traditional chemotherapy drugs.
[0062] It can be seen that the concentration of the crosslinking agent N,N-methylenebisacrylamide (MBA) is a key parameter for regulating the network structure of polyacrylamide modified gelatin (GelMA) and the performance of the final composite hydrogel; too high or too low concentration of the crosslinking agent N,N-methylenebisacrylamide will have a decisive influence on the comprehensive performance of the hydrogel; specifically, the concentration of the crosslinking agent N,N-methylenebisacrylamide is preferably 0.002-0.006 mol / L, and further preferably about 0.004 mol / L. When the concentration is lower than 0.002 mol / L, the hydrogel is insufficiently crosslinked, and the mechanical properties and degradation stability are poor; when the concentration is higher than 0.006 mol / L, the hydrogel is excessively crosslinked, which is not conducive to drug loading and release.
[0063] In summary, the application is a preparation method of a pH-responsive anti-tumor composite hydrogel, which has mild conditions, simple equipment, cheap and readily available raw materials, stable process, and the prepared hydrogel is white and transparent jelly-like, has a uniform and interconnected honeycomb porous structure in micro-morphology, and has excellent pH responsiveness to the acidic microenvironment of tumors, which can provide a safer and more effective local drug delivery platform for postoperative adjuvant therapy of tumors.
[0064] The specific embodiments of the application are described in detail above, but they are only examples, and the application is not limited to the above-described specific embodiments. Any equivalent modifications or alternatives to the application made by those skilled in the art are also within the scope of the application, and therefore, equivalent transformations, modifications, improvements, etc. made without departing from the spirit and principle range of the application should be covered within the scope of the application.
Claims
1. A method for preparing a pH-responsive composite hydrogel, characterized by, The method comprises the following steps: (1) dispersing sodium alginate in ultrapure water to form a suspension, and after oxidation reaction with sodium periodate under light-proof conditions, terminating the oxidation reaction with a terminating agent, and purifying through dialysis and freeze-drying to prepare pH-responsive acylhydrazone bond oxidized sodium alginate; (2) dispersing gelatin in ultrapure water and stirring until completely dissolved, then adding an initiator and a modifier in sequence to obtain a mixed solution A, and then adding a crosslinking agent A and stirring and dissolving until the crosslinking reaction is complete to obtain a modified gelatin precursor solution; (3) adding the pH-responsive acylhydrazone bond oxidized sodium alginate and a drug carrier into the modified gelatin precursor solution, and stirring until completely dissolved to obtain a mixed solution B; adding a crosslinking agent B and an adjusting agent into the mixed solution B, and stirring and oscillating until the solution is uniformly mixed to obtain an OSA-GelMA drug-loaded mixed solution; (4) placing the OSA-GelMA drug-loaded mixed solution into an oven for reaction to obtain a pH-responsive OSA-GelMA drug-loaded hydrogel; and freeze-drying the gel.
2. The method for preparing a pH-responsive composite hydrogel according to claim 1, wherein, In step (1), the concentration of sodium alginate is 0.01-1.00 mol / L, and the concentration of sodium periodate is 0.02-1.00 mol / L; the terminating agent is a diethylene glycol solution with a concentration of 0.01-1.00 mol / L.
3. The method for preparing a pH-responsive composite hydrogel according to claim 2, wherein, In step (1), the oxidation reaction under light-proof conditions is carried out at 25-30 DEG C under magnetic stirring at 300-400 r / min for 2-4 h; the termination of the oxidation reaction is carried out at 25-30 DEG C under magnetic stirring at 300-400 r / min for 0.5-1.5 h; the dialysis purification is carried out by placing the solution after the termination of the oxidation reaction into a dialysis bag with a molecular weight cut-off of 3000-4000 Da and dialyzing at 25 DEG C for 2-8 days; and the freeze-drying is carried out at -40--20 DEG C for 48-72 h.
4. The method for preparing a pH-responsive composite hydrogel according to claim 1, wherein In step (2), the concentration of gelatin in the mixed solution A is 0.01-1.5 g / mL, the initiator is potassium persulfate with a concentration of 0.001-0.1 mol / L, the modifier is acrylamide with a concentration of 0.1-2.0 mol / L, and the crosslinking agent A is N,N-methylenebisacrylamide with a concentration of 0.002-0.006 mol / L.
5. The method for preparing a pH-responsive composite hydrogel according to claim 4, wherein In step (2), the stirring and dissolving is carried out at 30-40 DEG C under water bath conditions under magnetic stirring at 300-400 r / min for 1-2 h; and the crosslinking reaction is carried out at a temperature of 50-70 DEG C under magnetic stirring at 300-400 r / min for 0.5-1 h.
6. The method for preparing a pH-responsive composite hydrogel according to claim 1, wherein, In step (3), the drug carrier is bortezomib; the mass ratio of the pH-responsive acylhydrazone bond oxidized sodium alginate to the modified gelatin in the mixed solution B is 1:1-1:5; the Z loading capacity of the drug carrier is 10-100 mg; the crosslinking agent B is adipic acid hexahydrazone with a concentration of 0.1-2.0 mol / L, and the adjusting agent is polyethylene glycol with a concentration of 5-50 mg / mL.
7. The method for preparing a pH-responsive composite hydrogel according to claim 6, wherein The stirring to complete dissolution in step (3) is 300-400 r / min magnetic stirring at 50-70 DEG C for 1-3 h; the stirring and oscillation is ultrasonic time of 1-4 min, power of 80-100 W, frequency of 50-80 KHz, and temperature of 24-27 DEG C.
8. The method for preparing the pH-responsive composite hydrogel as described in claim 1, characterized in that, The reaction in the oven of step (4) is 1-4 h at 50-70 DEG C.
9. The method for preparing a pH-responsive composite hydrogel according to claim 8, wherein, The freeze-drying of step (4) is 48-72 h at -40--20 DEG C.
10. Use of a pH-responsive OSA-GelMA drug-loaded hydrogel prepared according to the method of any one of claims 1-9 for the manufacture of a pharmaceutical preparation for topical drug delivery, characterized in that, The pH-responsive OSA-GelMA drug-loaded hydrogel carries a drug carrier and is used for triggering release of active substances of the drug carrier in an acidic microenvironment.