Chitosan beta-sodium glycerophosphate magnetic response temperature-sensitive hydrogel loaded with Fe3O4 particles and preparation method of chitosan beta-sodium glycerophosphate magnetic response temperature-sensitive hydrogel

Abstract

The invention discloses chitosan / beta-sodium glycerophosphate magnetic response temperature-sensitive hydrogel loaded with Fe3O4 particles and a preparation method of the chitosan / beta-sodium glycerophosphate magnetic response temperature-sensitive hydrogel. The hydrogel is obtained by taking a chitosan / beta-sodium glycerophosphate temperature-sensitive gel system as a carrier and loading Fe3O4 particles. Specifically, by controlling the concentration of Fe3O4, the ratio of sodium glycerophosphate powder to a Fe3O4 nano aqueous dispersion, the ratio of a chitosan solution to an iron-containing beta-glycerophosphoric acid aqueous solution and the acidity of a gelling microenvironment, the distribution of magnetic nanoparticles in a gel matrix is adjusted, and the magnetic nanoparticles which are in an injectable liquid state at room temperature and can be uniformly dispersed in a gel matrix are formed. The hydrogel can be rapidly cured into gel in a body temperature environment, and has a three-dimensional network structure for fixing high-load FeO nanoparticles. The hydrogel Fe8000 S < 1.5 > C28.3-A < 1.1 > and the hydrogel Fe8000 S < 1.5 > C46.7-A < 1.1 > which are prepared from the high-viscosity chitosan can be heated to 28 DEG C at the room temperature, and the magnetic heating performance is excellent.

Description

Technical Field

[0001] This invention relates to the field of magnetically responsive thermosensitive hydrogel preparation technology, specifically to a chitosan / β-glycerophosphate magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles. This invention also relates to a method for preparing the hydrogel. Background Technology

[0002] Currently, hydrogels capable of loading various materials have been approved for use in clinical settings, such as drug delivery, cardiac repair, wound covering, or implantation. They can form a stable three-dimensional network structure locally within the body and remain in situ for extended periods. Furthermore, due to their slow degradation rate, they can also carry nanomedicines, allowing nanomaterials to be stably "fixed" at specific sites within the body for longer periods, maintaining a high concentration of local drugs and thus improving the therapeutic effects of various treatments.

[0003] Regarding the selection of hydrogels, hydrogels prepared from biopolymers contain no chemical additives, possess biocompatibility and eco-friendliness, making them an ideal choice for biomedical applications. Among them, chitosan is the only naturally occurring cationic polysaccharide, obtained through the deacetylation of chitin, and exhibits superior biocompatibility and biodegradability. In recent years, both chitosan itself and gel materials composed of it have received widespread attention.

[0004] The chitosan / β-glycerophosphate sodium gel system is a thermosensitive hydrogel system. This hydrogel is liquid at room temperature and can be injected into the target treatment area, subsequently cross-linking rapidly at body temperature to form an in-situ gel. Its gelation mechanism is as follows: when chitosan dissolves in an acidic liquid, its amino groups accept protons under acidic conditions, forming positively charged ammonium ions. Subsequently, the phosphate ions in β-glycerophosphate sodium undergo a cross-linking reaction with these protonated ammonium ions. This process transforms the entire reaction system from an initial liquid state to a final solid gel. However, at lower temperatures, the interaction between the protonated ammonium ions and phosphate ions in the chitosan solution is weak, keeping the system liquid. As the temperature increases, molecular thermal motion intensifies, and the interaction between these ions gradually strengthens, eventually forming a stable hydrogel structure. Studies have reported that the storage modulus of this hydrogel after gelation is similar to that of human brain tissue, meaning that the gel can adapt well to the biomechanical environment of most tissues in the human body after gelation. In 2024 alone, this hydrogel system demonstrated significant potential in various applications. For example, Xu et al. used this gel system to deliver self-replicating RNA to alleviate osteoarthritis; Zhang et al. achieved effective treatment of periodontitis by encapsulating salicylic acid in the gel; and Yao et al. successfully inhibited the formation of glial scars in rats by loading all-trans retinoic acid into the gel system.

[0005] For magnetic nanoparticles loaded within gel systems, although Fe3O4 nanoparticles are often considered a traditional magnetic material with limited heating capacity under a magnetic field, their proven biocompatibility is undeniable, given that Fe3O4 nanoparticles are among the very few magnetic materials approved by the US Food and Drug Administration for human trials. While the heating effect of Fe3O4 nanoparticles in a dispersed state may not be as strong as that of novel magnetic materials, their heating performance could be improved by agglomerating them through modified methods, potentially allowing these traditional materials to regain their application value.

[0006] Patent CN 109364018 B discloses an injectable, temperature-curing, self-regulating thermotherapy magnetic hydrogel and its preparation method. The hydrogel is prepared by using an injectable, temperature-curing hydrogel as a matrix and loading it with magnetic nanoparticles. The hydrogel is injectable in a room-temperature liquid state and cures at body temperature, remaining stable within a specified thermotherapy temperature (42℃~45℃). After curing, it will not liquefy again upon further heating. The preparation method is as follows: chitosan powder is added to an acetic acid dispersion of self-regulating magnetic nanoparticles to obtain dispersion A; sodium glycerophosphate and sodium hyaluronate powders are added to deionized water to obtain a mixed solution B; mixed solution B is added dropwise to dispersion A, which is in an ice bath, to obtain the product. The applicant attempted to use this method to prepare a chitosan / β-glycerophosphate magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles, but in practical applications, it was found that the pre-gel solution prepared by this method would clump at room temperature, making subsequent injection impossible. Summary of the Invention

[0007] Based on the above, the purpose of this invention is to provide a chitosan / β-glycerophosphate sodium magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles. By using a chitosan / β-glycerophosphate sodium gel system, Fe3O4 nanoparticles loaded within it are aggregated, thereby significantly improving the heat generation effect of Fe3O4 nanoparticles in a magnetic field. The resulting hydrogel is an injectable liquid at room temperature and can be rapidly solidified into a gel at body temperature. It has a three-dimensional network structure that fixes a high load of Fe3O4 nanoparticles.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned magnetically responsive thermosensitive hydrogel.

[0009] To achieve its purpose, the present invention adopts the following technical solution: This invention provides a method for preparing a chitosan / β-glycerophosphate sodium magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles, comprising the following steps: (1) Preparation of Fe3O4 nano-aqueous dispersion: Weigh Fe3O4 nanoparticles, add them to physiological saline, and perform ultrasonic dispersion treatment to obtain Fe3O4 nano-aqueous dispersion with a concentration of 1000-8000mg / 100mL; (2) Preparation of iron-containing glycerol phosphate sodium solution: Weigh out sodium glycerophosphate powder and place it in a sample bottle. Add the Fe3O4 nano-aqueous dispersion from step (1) and stir thoroughly at room temperature until the sodium glycerophosphate is completely dissolved to form a uniform iron-containing sodium glycerophosphate solution. In the iron-containing sodium glycerophosphate solution, the mass-to-volume ratio of sodium glycerophosphate powder to Fe3O4 nano-aqueous dispersion is 3-4:7, where the mass unit is g and the volume unit is mL. (3) Preparation of chitosan solution Preparation of low-viscosity chitosan solution: Weigh low-viscosity chitosan powder, place it in a sample bottle, add physiological saline, then add acetic acid, mix well to obtain a chitosan solution with a concentration of 28.3 mg / ml (containing 1.1% acetic acid); Stir the chitosan solution magnetically at 35-45℃ for 1.5-2 h, then sonicate for 1-1.5 h to obtain a low-viscosity chitosan solution; Preparation of high-viscosity chitosan solution: Weigh two portions of high-viscosity chitosan powder and place them in two sample bottles; then, add physiological saline to each sample bottle; prepare two sets of the above chitosan powder samples, and add acetic acid to each set of samples respectively, and obtain the following four concentrations of chitosan solution by mixing: 28.3 mg / ml chitosan solution (containing 1% acetic acid); 28.3 mg / ml chitosan solution (containing 1.1% acetic acid); 46.7 mg / ml chitosan solution (containing 1% acetic acid); 46.7 mg / ml chitosan solution (containing 1.1% acetic acid); stir the four chitosan solutions slightly and let them stand at room temperature for 25-35 min to obtain high-viscosity chitosan solution; (4) Preparation of magnetically responsive thermosensitive hydrogel preform The liquids prepared in steps (2) and (3) are pre-cooled, and then, under ice bath conditions, the iron-containing sodium glycerophosphate solution in step (2) is slowly dripped into the low viscosity / high viscosity chitosan solution in step (3) while continuously stirring to mix thoroughly, to obtain the pre-formed gel solution; the volume ratio of the iron-containing sodium glycerophosphate solution to the low viscosity / high viscosity chitosan solution is 1:3; (5) Preparation of magnetically responsive thermosensitive hydrogel: The pre-gel solution in step (4) is put into a sample bottle and placed upside down in a constant temperature water bath at 36°C. Observe whether it can form a gel. If the liquid in the sample bottle stops flowing completely and can maintain a fixed shape, it is determined that the pre-gel solution has completely transformed into a gel state. Take it out from the sample bottle to obtain the magnetically responsive thermosensitive hydrogel.

[0010] As a further preferred embodiment of the technical solution of the present invention, in step (1), the ultrasonic frequency is 30kHz and the ultrasonic time is 15-25min.

[0011] Furthermore, in step (1), the concentration of the Fe3O4 nano-aqueous dispersion is preferably 8000 mg / 100 mL.

[0012] Further, in step (2), the preferred mass-to-volume ratio of sodium glycerophosphate powder to Fe3O4 nano-aqueous dispersion in the iron-containing sodium glycerophosphate solution is 3:7.

[0013] Furthermore, in step (3), when preparing the low-viscosity chitosan solution, the magnetic stirring speed is 200 r / min and the ultrasonic treatment frequency is 100 kHz.

[0014] Furthermore, in step (3), when preparing the high-viscosity chitosan solution, the magnetic stirring speed is 100 r / min and the stirring time is 20 min.

[0015] Further, in step (3), the concentration of the high-viscosity chitosan solution is preferably 28.3 mg / ml chitosan solution (containing 1.1% acetic acid) or 46.7 mg / ml chitosan solution (containing 1.1% acetic acid).

[0016] The above method can be used to prepare chitosan / β-glycerophosphate sodium magnetic responsive thermosensitive hydrogels that have a heat-generating effect in a magnetic field and are loaded with Fe3O4 particles.

[0017] Furthermore, the chitosan is preferably a high-viscosity chitosan with a higher Fe3O4 loading.

[0018] Further, the hydrogel is preferably Fe8000S1.5 C28.3-A1.1 or Fe8000 S1.5 C46.7-A1.1, which can be heated to close to 50°C at room temperature (about 22°C), where Fe represents the mass of Fe3O4 nanoparticles (mg), S represents the mass of sodium glycerophosphate (g), C represents the chitosan concentration (mg / ml), and A represents the volume fraction of acetic acid.

[0019] By adopting the above technical solution, the beneficial effects of the present invention are as follows: The hydrogel described in this invention uses a chitosan / sodium β-glycerophosphate thermosensitive gel system as a carrier to load Fe3O4 particles. Specifically, this invention adjusts the distribution of magnetic nanoparticles in the gel matrix by controlling the concentration of magnetic nanoparticles, the ratio of sodium glycerophosphate powder to Fe3O4 nanoparticle aqueous dispersion, the ratio of chitosan solution to iron-containing β-glycerophosphate aqueous solution, and the acidity of the gel-forming microenvironment. This results in a hydrogel that is injectable at room temperature, can rapidly solidify into a gel at body temperature, and has a three-dimensional network structure that fixes a high load of Fe3O4 nanoparticles. The hydrogels Fe8000 S1.5 C28.3-A1.1 and Fe8000 S1.5 C46.7-A1.1 prepared using high-viscosity chitosan can be heated to nearly 50°C at room temperature (approximately 22°C), exhibiting excellent magnetocaloric heating performance. Attached Figure Description

[0020] Figure 1 This is a method for evaluating the gelling performance of pre-gelled solutions. A: After inverting the sample bottle in a warm bath, the pre-gelled solution does not flow; B: After inverting the sample bottle in a warm bath, the pre-gelled solution remains fluid; C: Visual observation before and after gelation (left is the glossy liquid before gelation, right is the granular solid after gelation). Figure 2 For the injectionability assessment of the pre-formed adhesive; Figure 3 The image shows the infrared thermography results of the gel sample after gelation placed in a magnetic field within a centrifuge tube. The white part at the bottom of the tube represents the heated magnetic gel. Figure 4 Scanning electron microscopy characterization of the microstructure of the magnetically responsive thermosensitive hydrogel; Figure 5 The results are from an injection experiment of the hydrogel prepared using the disclosed method. Detailed Implementation

[0021] The preparation method and properties of the thermosensitive hydrogel of the present invention will be described in detail below.

[0022] 1. Experimental materials, reagents, and instrument consumables 1.1 Experimental Reagents The experimental reagents are shown in Table 1.

[0023] Table 1 List of Experimental Reagents 1.2 Experimental Instruments and Consumables The experimental instruments and consumables are shown in Table 2.

[0024] Table 2 List of Experimental Instruments and Consumables 2. Experimental Methods 2.1 Preparation of magnetically responsive thermosensitive hydrogels from chitosans of different molecular weights (1) The ultrasonic water bath and magnetic stirrer are preheated to above 15°C, and the glacial acetic acid is preheated to ensure that it exists in liquid form.

[0025] (2) Preparation of Fe3O4 nano-aqueous dispersion Weigh 1000 mg of Fe3O4 nanoparticles and add them to 100 mL of physiological saline. Then, use an ultrasonic disperser to sonicate the nanoparticles for 20 min.

[0026] (3) Preparation of iron-containing glycerol phosphate sodium solution Weigh out 1.5 g and 2.0 g of sodium glycerophosphate powder respectively, and place them in two sample vials. Then, add 3.5 mL of the Fe3O4 nanoparticle aqueous dispersion prepared in step (2) to each vial. Stir with a magnetic stirrer at room temperature until the sodium glycerophosphate is completely dissolved to form a homogeneous iron-containing sodium glycerophosphate solution.

[0027] (4) Preparation of chitosan solution 1) Preparation of low-viscosity chitosan solutions: Weigh 170 mg and 280 mg of low-viscosity chitosan powder respectively, place them in two sample vials, and add 6 mL of physiological saline to each. Prepare three groups of the above chitosan powder samples, and add different volumes of acetic acid (60 μl, 66 μl, and 72 μl, respectively) to each group of samples. By mixing, the following six chitosan solution groups with different conditions were obtained: 28.3 mg / ml chitosan solution (containing 1% acetic acid); 28.3 mg / ml chitosan solution (containing 1.1% acetic acid); 28.3 mg / ml chitosan solution (containing 1.2% acetic acid); 46.7 mg / ml chitosan solution (containing 1% acetic acid); 46.7 mg / ml chitosan solution (containing 1.1% acetic acid); 46.7 mg / ml chitosan solution (containing 1.2% acetic acid). The prepared chitosan solution was heated to 40°C and stirred at 200 rpm for 1.5 hours using a magnetic stirrer, followed by sonication for 1 hour to remove air bubbles. Finally, the chitosan mucus with a small number of residual air bubbles in the upper layer was carefully aspirated using a syringe to obtain the final chitosan solution.

[0028] 2) Preparation of high-viscosity chitosan solution: The high-viscosity chitosan solution is prepared using the same component ratios and grouping scheme as the low-viscosity chitosan solution. However, unlike the treatment method for the low-viscosity chitosan solution, the high-viscosity chitosan solution only requires slight stirring (100 r / min, stirring time for 20 min) after preparation and standing at room temperature for about 30 min to form a transparent and clear chitosan solution system.

[0029] (5) Preparation of magnetically responsive thermosensitive hydrogel preform The liquids prepared in steps (3) and (4) were pre-cooled, and then, under ice bath conditions, 1 mL of the iron-containing sodium glycerophosphate solution in step (3) was added dropwise to 3 mL of the chitosan solution in step (4), while continuously stirring to ensure that the two solutions were fully mixed.

[0030] (6) Modify the Fe3O4 nanoparticle content in step (2) to study the maximum Fe3O4 nanoparticle content that can be carried in a gel system composed of chitosan with different viscosities. The specific operation is as follows: For the low viscosity chitosan group, weigh 2000 mg of Fe3O4 nanoparticles, disperse them in 100 mL of physiological saline, and sonicate for 20 min to ensure uniform dispersion. Then repeat steps (3) to (5) to observe the maximum amount of Fe3O4 nanoparticles that the carrier can carry. For the high viscosity chitosan group, weigh 2000 mg and 8000 mg of Fe3O4 nanoparticles respectively, place them in 100 mL of physiological saline, and sonicate for 20 min. Then repeat steps (3) to (5) to compare the maximum carrying capacity of the gel carrier composed of chitosan with different viscosities for Fe3O4 nanoparticles.

[0031] 2.2 Gelation performance testing, injectability testing, and evaluation of the heat generation performance of the gel under magnetic field. (1) Detection of gelation properties of magnetically responsive thermosensitive hydrogels The various pregel solutions prepared in section 2.1 were placed in a constant temperature water bath to observe whether the samples could gel at 36℃. The gelation performance was observed and analyzed using the inversion method: the sample bottle containing the pregel solution was carefully inverted and the liquid flow was observed. If the liquid completely stopped flowing and maintained a fixed shape, the pregel solution was determined to have completely transformed into a gel state. At the same time, the time required for the solution to transform from a liquid state to a gel state was recorded, and photos of the state before and after gelation were taken for comparison.

[0032] (2) Injectability test of magnetically responsive thermosensitive hydrogel Two different sizes of medical syringes were used for testing. First, the pre-cooled pre-formed gel solution was loaded into a 1 mL ordinary syringe (with a 21G needle) and a 1 mL insulin-specific syringe (with a 31G needle), respectively. The solution was observed to see if it could be smoothly discharged through the needle at room temperature.

[0033] (3) Evaluation of the heat generation performance of magnetically responsive thermosensitive hydrogels under magnetic field To evaluate the heat generation capacity and performance differences of various gel-forming and injectable magnetically responsive thermosensitive hydrogels under alternating magnetic fields, 80 μl of the prepared pre-gel sample was injected into a small plastic centrifuge tube. After gelation at 36°C, the tube was placed in an alternating magnetic field generator for testing. The experiment was conducted under 200 A AC power conditions with magnetic field radiation. Simultaneously, an infrared thermal imager was used to monitor the temperature changes on the surface and inside of the centrifuge tube in real time, recording the temperature rise of each sample from the start of magnetic field application until the temperature reached a stable value. This comprehensively evaluated the heat generation capacity and performance differences of different types of gels.

[0034] 2.3 Scanning electron microscopy analysis of the microstructure of the magnetically responsive thermosensitive hydrogel First, the hydrogel samples were frozen in liquid nitrogen for 20 minutes to ensure the stability of their internal structure during subsequent processing. Then, they were transferred to a freeze-drying oven and freeze-dried at -60°C for 24 hours to remove moisture without damaging the structure. Next, the hydrogel samples were cut using a precision blade to expose fresh cross-sections. To enhance the conductivity of the sample surface and thus improve image quality, these cross-sections were sputter-coated with gold. Finally, their microstructure was observed using a high-resolution scanning electron microscope.

[0035] 3. Experimental Results 3.1 Evaluation of gelation behavior, injectability, and heat generation characteristics of the gel under alternating magnetic field Methods for evaluating gelling performance Figure 1 As shown, this is to determine whether the pre-gel solution can be transformed into a solid, magnetically responsive, temperature-sensitive hydrogel. Specifically, if the pre-gel solution can gel (e.g.... Figure 1 As shown in Figure A), after incubation in a 36℃ water bath for 5-10 minutes, inverting the sample vial will result in the pre-gel solution, which was originally free-flowing due to gravity, transforming into a non-flowing solid gel that remains at the bottom of the inverted vial. Conversely, if gelation fails (as shown in Figure A), the pre-gel solution will not flow due to gravity. Figure 1 (As shown in B), after incubation, the pre-formed adhesive liquid maintains its original fluidity and cannot form a solid structure.

[0036] Figure 1 C illustrates the process of material transformation from liquid to solid and the changes in its morphological characteristics. The sample on the left is the pre-gelled liquid before gelation, with a smooth surface and a liquid state. The sample on the right shows the state after gelation, which presents an irregular, nearly spherical mass with a noticeable granular texture on the surface.

[0037] Injectability assessment criteria Figure 2 As shown: If the pre-formed adhesive can be smoothly expelled from the needle, the sample is considered to have injectable properties.

[0038] Evaluation criteria for magnetocaloric heating performance Figure 3 As shown: Infrared thermal imaging results of gel samples after gelation placed in a magnetic field within a centrifuge tube are used to evaluate the differences in heat generation capacity of different types of gels. The white part at the bottom of the test tube is the heated magnetic gel.

[0039] The magnetocaloric heating properties, injectability, and heat generation properties of magnetically responsive thermosensitive hydrogels prepared with chitosan of different viscosities, sodium glycerophosphate of different concentrations, and under different acidic environments were investigated. Figure 1 , 2 After evaluation, the results are summarized in Tables 3 and 4 (in the sample group, Fe represents the mass of Fe3O4 nanoparticles in mg, S represents the mass of sodium glycerophosphate in g, C represents the concentration of chitosan in mg / ml, and A represents the volume fraction of acetic acid in %).

[0040] Table 3. Gel-forming properties, injectability, and magnetocaloric heating performance of gel samples prepared from low-viscosity chitosan. Table 3 shows that for the pregel solutions prepared for the low-viscosity chitosan group, the vast majority of samples failed to successfully form magnetically responsive thermosensitive hydrogels. Compared to the Fe1000 group, none of the pregel solutions in the Fe2000 group showed gel-forming ability, while in the Fe1000 group, only two samples, "Fe1000 S1.5 C46.7-A1" and "Fe1000 S1.5 C46.7-A1.1", were able to form gels. Among these gel-forming samples, only the "Fe1000 S1.5 C46.7-A1.1" sample possessed good injectability; the remaining samples failed to meet the injection requirements. Further evaluation of the sample's heating performance under a magnetic field (magnetic-thermal performance evaluation was not performed on pre-gel samples that did not meet the requirements for gelation or were not injectable) showed that although the magnetic nanoparticle content was low, the "Fe1000 S1.5 C46.7-A1.1" sample could still produce a slight temperature rise, but its heating capacity was far from sufficient to reach the temperature threshold required for tumor magnetic induction hyperthermia. Given that all pre-gel solutions in the Fe2000 group could not simultaneously meet the requirements for gelation and injection due to the influence of high-concentration nanoparticles, further tests were not conducted to increase the nanoparticle content in the pre-gel solutions prepared from the low-viscosity chitosan group.

[0041] Table 4. Gel-forming characteristics, injectability, and magnetocaloric heating properties of gel samples prepared from high-viscosity chitosan. As can be seen from the experimental results in Table 4, most samples in the high-viscosity chitosan group exhibited superior gelling ability compared to the low-viscosity chitosan group, successfully forming magnetically responsive thermosensitive hydrogels. However, when the microenvironment for gel formation exhibited excessive acidity, the gelling process of the samples was significantly inhibited, making effective gelation difficult, as shown in the "Fe1000S1.5 C28.3-A1.2" and "Fe1000 S1.5C46.7-A1.2" groups. This phenomenon indicates that pH value has a decisive influence on the gelling process, emphasizing the importance of controlling the acidity of the microenvironment during preparation.

[0042] Regarding injectability, most successfully gelled samples also exhibited good injectability. However, when the gelation microenvironment was weakly acidic, particulate matter appeared in the pregel solution, causing syringe blockage and making injection difficult, as shown in samples "Fe1000S1.5 C28.3-A1" and "Fe1000 S1.5 C46.7-A1". Furthermore, a high number of nanoparticles and a high amount of dissolved matter in the system also interfered with the injectability of the samples. These results highlight the importance of optimizing the formulation to ensure that the material possesses both excellent gelation properties and good injectability.

[0043] Regarding magnetocaloric properties, pregel samples that did not meet the requirements for gelation properties or / and injectability were not further evaluated. Because the gel systems prepared by the high-viscosity chitosan group could accommodate more magnetic nanoparticles, the two samples, "Fe8000 S1.5 C28.3-A1.1" and "Fe8000 S1.5C46.7-A1.1", not only exhibited excellent magnetocaloric properties by heating to nearly 50°C from room temperature (approximately 22°C), but also successfully gelled and possessed excellent injectability, standing out among all samples prepared using both low-viscosity and high-viscosity chitosan. Therefore, these two samples meet the requirements for tumor magnetic induction hyperthermia, providing ideal experimental conditions for subsequent research. Based on the above findings, all subsequent experiments used the experimental conditions of the "Fe8000 S1.5 C28.3-A1.1" group within the high-viscosity chitosan group to prepare magnetically responsive thermosensitive hydrogels.

[0044] The above results indicate that optimizing the formulation and precisely controlling the pH value of the gelation microenvironment are crucial for achieving ideal gelation performance, injection performance, and magnetothermal heating performance when preparing magnetically responsive thermosensitive hydrogels.

[0045] 3.2 Scanning electron microscopy characterization of the microstructure of the magnetically responsive thermosensitive hydrogel Figure 4Scanning electron microscopy (SEM) images revealed the microstructure of the magnetically responsive thermosensitive hydrogel, exhibiting a porous morphology with a complex three-dimensional network. The pore sizes ranged from tens to hundreds of micrometers and were unevenly distributed, with some pores interconnected to form channels. The pore wall thickness varied considerably, showing a degree of randomness, while the hydrogel surface appeared rough, indicating an uneven surface.

[0046] This invention develops a magnetically responsive thermosensitive hydrogel that combines thermosensitive gelation properties, intratumoral injectability, and magnetic field-responsive heating capability. The preparation method, key performance characterization, and biocompatibility of the gel are described in detail, and its biocompatibility is verified.

[0047] The preparation of magnetically responsive hydrogels is particularly challenging. This invention systematically validated over 60 different parameter selections and ratios, ultimately determining a preparation process that meets all requirements (thermosensitive gelation properties, intratumoral injectability, and magnetic field-responsive heating capability). This iterative experimentation and optimization process is crucial and necessary in the design of magnetic nanogels. For example, the thermosensitive gel used by Che et al. had a phase transition temperature of 26°C before the addition of magnetic nanoparticles; however, after incorporating magnetic nanoparticles, the phase transition temperature decreased to 21.9°C. Furthermore, environmental temperature and pH significantly affect various gel properties. Therefore, a key issue in the design of magnetic nanogels is that the overall gel significantly influences the properties of individual components, and vice versa. For instance, the gel matrix may restrict the rotational freedom of magnetic nanoparticles under a magnetic field, thus affecting their heat generation efficiency. On the other hand, since magnetic nanoparticles occupy a portion of the internal volume of the nanogel, their presence also significantly interferes with the phase transition process. Therefore, understanding the interaction between the gel matrix and the encapsulated magnetic nanoparticles, and continuously optimizing the preparation process of this composite system, is a key approach to achieving the desired performance of magnetic nanogels.

[0048] In the preparation process of this invention, the acidity of the reaction system simultaneously determines the solubility of chitosan and whether the pregel solution can form a gel. When the acidity is slightly high, although the chitosan is not completely dissolved, the gelling performance of the entire system is good. However, in this case, the pregel solution cannot be injected due to the insufficient dissolution of chitosan. Conversely, when the acidity is slightly low, although the chitosan is more completely dissolved, the gelling performance of the entire gel system will be significantly reduced due to the acidic environment. Similarly, the content of magnetic nanoparticles in the reaction system determines the final heating result of the gel under a magnetic field and its injection and gelling performance. When the content of magnetic nanoparticles in the system is low, the gel cannot achieve the heating effect required for magnetic induction thermotherapy. However, if the content of magnetic nanoparticles in the system is high, its injection and gelling performance will be significantly affected. Therefore, continuously matching and understanding the influence and interaction between various components on the magnetically responsive thermosensitive hydrogel is crucial.

[0049] Comparative experiment The chitosan / β-glycerophosphate magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles was prepared using the method described in patent CN 109364018 B. The specific process is as follows: (1) Fe3O4 magnetic nanoparticles were added to deionized water, and after ultrasonic treatment, they were mixed with pure acetic acid solution to prepare an acetic acid dispersion of magnetic nanoparticles, wherein the concentration of Fe3O4 was 160 mg / mL and the concentration of acetic acid was 0.1 mol / L.

[0050] (2) Add chitosan powder (the concentration of chitosan in the dispersion is 35 mg / mL) to the above dispersion, and use magnetic stirring to promote its complete dissolution to obtain self-temperature controlled magnetic nanoparticle dispersion A, and then place it in a constant temperature oven at 4℃ for 2 hours.

[0051] (3) Mix sodium glycerophosphate and sodium hyaluronate powder in a sample vial, add deionized water, and stir magnetically until completely dissolved to obtain mixed solution B. Let the solution stand in a 4℃ incubator for 1 hour before use. The concentration of sodium hyaluronate in solution B is 20 mg / mL, and the concentration of sodium glycerophosphate is 800 mg / mL.

[0052] (4) The mixed solution B is slowly added dropwise to the dispersion A under ice bath conditions, with a volume ratio of 15:1. During the dropwise addition, the magnetic stirring is continuously carried out to ensure uniform mixing, and finally a pre-formed gel is obtained.

[0053] The injection test results of the pre-formed adhesive are shown in Figure 5 . Figure 5 The results show that, even before injection, the liquid pre-formed gel can be drawn into the syringe and has a certain degree of fluidity. Figure 5 B shows a gap at the tip of the needle, indicating that no injection has been administered yet; Figure 5 C indicates that when force is injected, the pre-formulated adhesive clogs the pillow and is almost impossible to expel smoothly from the needle, resulting in injection failure. Figure 5 The D-display shows that there is no gap at the tip of the needle, but only a barely visible amount of liquid is being expelled from the needle tip, indicating injection failure. Therefore, the chitosan / β-glycerophosphate sodium magnetic responsive thermosensitive hydrogel loaded with 20nm Fe3O4 particles prepared using the method in patent CN 109364018B will exhibit clumping at room temperature, making subsequent injection impossible and rendering it unusable in practical applications.

Claims

1. A method for preparing a chitosan / β-glycerophosphate sodium magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles, characterized in that, Includes the following steps: (1) Preparation of Fe3O4 nano-aqueous dispersion: Weigh Fe3O4 nanoparticles, add them to physiological saline, and perform ultrasonic dispersion treatment to obtain Fe3O4 nano-aqueous dispersion with a concentration of 1000-8000mg / 100mL; (2) Preparation of iron-containing glycerol phosphate sodium solution: Weigh out sodium glycerophosphate powder and place it in a sample bottle. Add the Fe3O4 nano-aqueous dispersion from step (1) and stir thoroughly at room temperature until the sodium glycerophosphate is completely dissolved to form a uniform iron-containing sodium glycerophosphate solution. In the iron-containing sodium glycerophosphate solution, the mass-to-volume ratio of sodium glycerophosphate powder to Fe3O4 nano-aqueous dispersion is 3-4:7, where the mass unit is g and the volume unit is mL. (3) Preparation of chitosan solution Preparation of low-viscosity chitosan solution: Weigh low-viscosity chitosan powder, place it in a sample bottle, add physiological saline, then add acetic acid, mix well to obtain a chitosan solution with a concentration of 28.3 mg / ml (containing 1.1% acetic acid); Stir the chitosan solution magnetically at 35-45℃ for 1.5-2 h, then sonicate for 1-1.5 h to obtain a low-viscosity chitosan solution; Preparation of high-viscosity chitosan solution: Weigh two portions of high-viscosity chitosan powder and place them in two sample bottles; then, add physiological saline to each sample bottle; prepare two sets of the above chitosan powder samples, and add acetic acid to each set of samples respectively, and obtain the following four concentrations of chitosan solution by mixing: 28.3 mg / ml chitosan solution (containing 1% acetic acid); 28.3 mg / ml chitosan solution (containing 1.1% acetic acid); 46.7 mg / ml chitosan solution (containing 1% acetic acid); 46.7 mg / ml chitosan solution (containing 1.1% acetic acid); stir the four chitosan solutions slightly and let them stand at room temperature for 25-35 min to obtain high-viscosity chitosan solution; (4) Preparation of magnetically responsive thermosensitive hydrogel preform The liquids prepared in steps (2) and (3) are pre-cooled, and then, under ice bath conditions, the iron-containing sodium glycerophosphate solution in step (2) is slowly dripped into the low viscosity / high viscosity chitosan solution in step (3) while continuously stirring to mix thoroughly, to obtain the pre-formed gel solution; the volume ratio of the iron-containing sodium glycerophosphate solution to the low viscosity / high viscosity chitosan solution is 1:3; (5) Preparation of magnetically responsive thermosensitive hydrogel: The pre-gel solution in step (4) is put into a sample bottle and placed upside down in a constant temperature water bath at 36°C. Observe whether it can form a gel. If the liquid in the sample bottle stops flowing completely and can maintain a fixed shape, it is determined that the pre-gel solution has completely transformed into a gel state. Take it out from the sample bottle to obtain the magnetically responsive thermosensitive hydrogel.

2. The method for preparing the chitosan / β-glycerophosphate sodium magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles as described in claim 1, characterized in that, In step (1), the ultrasonic frequency is 30 kHz and the ultrasonic time is 15-25 min.

3. The method for preparing the chitosan / β-glycerophosphate sodium magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles as described in claim 1, characterized in that, In step (1), the concentration of Fe3O4 nano-aqueous dispersion is 8000 mg / 100 mL.

4. The method for preparing the chitosan / β-glycerophosphate sodium magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles as described in claim 3, characterized in that, In step (2), the mass-volume ratio of sodium glycerophosphate powder to Fe3O4 nano-aqueous dispersion in the iron-containing sodium glycerophosphate solution is 3:

7.

5. The method for preparing the chitosan / β-glycerophosphate sodium magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles as described in claim 1, characterized in that, In step (3), when preparing the low-viscosity chitosan solution, the magnetic stirring speed is 200 r / min and the ultrasonic treatment frequency is 100 kHz.

6. The method for preparing the chitosan / β-glycerophosphate sodium magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles as described in claim 4, characterized in that, In step (3), when preparing the high-viscosity chitosan solution, the magnetic stirring speed is 100 r / min and the stirring time is 20 min.

7. The method for preparing the chitosan / β-glycerophosphate magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles as described in claim 6, characterized in that, In step (3), the concentration of the high-viscosity chitosan solution is 28.3 mg / ml chitosan solution (containing 1.1% acetic acid) or 46.7 mg / ml chitosan solution (containing 1.1% acetic acid).

8. Chitosan / β-glycerophosphate sodium magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles prepared by the method according to any one of claims 1-7.

9. The chitosan / β-glycerophosphate sodium magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles as described in claim 8, characterized in that, The chitosan is a high-viscosity chitosan.

10. The chitosan / β-glycerophosphate sodium magnetically responsive thermosensitive hydrogel loaded with Fe3O4 particles as described in claim 9, characterized in that, The hydrogel is Fe8000 S1.5 C28.3-A1.1 or Fe8000 S1.5 C46.7-A1.1, where Fe represents the mass of Fe3O4 nanoparticles (mg), S represents the mass of sodium glycerophosphate (g), C represents the chitosan concentration (mg / ml), and A represents the volume fraction of acetic acid.

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