Magnetic polysaccharide gel and preparation method and application thereof

Abstract

The application discloses a magnetic polysaccharide gel and a preparation method and application thereof, which is prepared from natural polysaccharide, magnetic nanoparticles and boric acid at room temperature; wherein, borate ions provided by the boric acid and catechol groups on the natural polysaccharide chain are cross-linked to form a three-dimensional network structure through forming dynamic borate ester bonds, and the magnetic nanoparticles are uniformly dispersed and physically embedded in the three-dimensional network structure. The application uses the dynamic borate ester bond to replace the traditional toxic chemical cross-linking agent, and constructs an intelligent gel material with high magnetic responsiveness, self-healing property, injectability, tissue adhesion, excellent mechanical property and good biocompatibility. The gel can realize complex motion and targeted delivery under the precise driving of an external magnetic field, and exhibits a rapid hemostatic capacity in vivo and in vitro. The preparation method is simple, mild, green and environment-friendly, and has a wide application prospect in the biomedical field, such as targeted drug delivery, tissue engineering, minimally invasive surgery, foreign body removal and wound hemostasis.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to an injectable magnetic polysaccharide gel based on dynamic borate ester crosslinking, its preparation method, and its applications. Background Technology

[0002] Magnetic hydrogels, as a type of smart soft material, have shown great potential in targeted drug delivery, tissue engineering, minimally invasive surgery, and biosensors. Existing magnetic polysaccharide hydrogels are typically composed of polysaccharide matrices (such as chitosan, alginate, hyaluronic acid, and starch) and magnetic nanoparticles (mostly Fe3O4) through physical blending, in-situ precipitation, or chemical grafting. Their structural characteristic is that the magnetic nanoparticles are dispersed within a three-dimensional network structure of the polysaccharide, enabling the hydrogel to respond to external magnetic fields. Common cross-linking methods include chemical cross-linking (such as glutaraldehyde cross-linking) and physical cross-linking (such as ionic cross-linking and hydrogen bonding).

[0003] Despite some progress in existing magnetic polysaccharide hydrogel technology, the following prominent problems still exist: 1. Existing magnetic polysaccharide hydrogels mostly use chemical cross-linking agents such as glutaraldehyde and divinyl sulfone to construct three-dimensional network structures. Although these cross-linking agents can form stable cross-linking systems, their residues have significant cytotoxicity, easily causing denaturation and inactivation of embedded drugs, growth factors, or cells, which seriously restricts the clinical translation and application of the materials.

[0004] 2. Existing technologies for preparing polysaccharide-based magnetic materials (especially nano / microsphere materials) generally suffer from low magnetic flux and low magnetic particle content. This is because magnetic nanoparticles tend to aggregate in gel networks, making uniform dispersion difficult; to prevent particle leakage, the amount of magnetic particles added is often reduced. This results in insufficient driving force under an applied magnetic field, making it difficult to achieve precise targeting, complex trajectory movements, and foreign object grasping.

[0005] 3. Existing polysaccharide hydrogels generally suffer from poor mechanical strength and low thermal stability, making it difficult to meet the mechanical requirements of applications such as tissue engineering scaffolds and injectable materials. Furthermore, existing magnetic polysaccharide materials mostly focus on a single function (such as a simple drug carrier or a simple scaffold), lacking a design concept that integrates multiple functions.

[0006] 4. Existing research is mostly limited to in vitro experiments, with insufficient studies on the in vivo degradation behavior, long-term biocompatibility, and immune response of the materials. In addition, the controllability of movement and tissue self-filling ability of existing magnetic hydrogels in complex in vivo environments (such as gastrointestinal peristalsis, interstitial spaces, and blood flow) are still unclear.

[0007] 5. Existing preparation processes for magnetic polysaccharide hydrogels often involve multi-step reactions, the use of organic solvents, and stringent condition control, resulting in complex processes, high costs, and difficulties in large-scale production.

[0008] In summary, existing magnetic polysaccharide hydrogels still have significant shortcomings in terms of safety, magnetic response intensity, mechanical properties, multifunctional integration, and in vivo application effects. There is an urgent need to develop a new type of magnetic polysaccharide gel material that is safe and non-toxic, has strong magnetic responsiveness, excellent mechanical properties, and multiple integrated functions. Summary of the Invention

[0009] To address the aforementioned problems in existing technologies, this invention provides a magnetic polysaccharide gel, its preparation method, and its applications. By utilizing dynamic borate ester bonds to replace traditional toxic chemical crosslinking agents, a smart gel material is constructed that possesses high magnetic responsiveness, self-healing properties, injectability, tissue adhesion, excellent mechanical properties, and good biocompatibility. This gel can achieve complex movements (such as crawling, rolling, rotating, and grasping) and targeted delivery under precise external magnetic field driving, and exhibits rapid hemostasis capabilities both in vivo and in vitro. Its preparation method is simple, mild, and environmentally friendly, and it has broad application prospects in targeted drug delivery, tissue engineering, minimally invasive surgery, foreign body removal, and trauma hemostasis in the biomedical field.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for preparing magnetic polysaccharide gel, comprising the following steps: (1) Dissolve the natural polysaccharide in a buffer solution to obtain a natural polysaccharide solution; (2) Dissolve boric acid in a buffer solution to obtain a boric acid solution; (3) Add the magnetic nanoparticles to the natural polysaccharide solution in step (1) and mix them evenly to obtain a magnetic polysaccharide mixture; (4) Add the boric acid solution from step (2) to the magnetic polysaccharide mixture from step (3), stir and mix evenly, and let it stand to react to obtain the magnetic polysaccharide gel. The natural polysaccharides are selected from any one or more combinations of giant kelp polysaccharides, seaweed polysaccharides, nori polysaccharides, and spirulina polysaccharides. These marine-derived natural polysaccharides are rich in catechol groups, which are ideal backbones for forming borate ester bonds.

[0011] The core concept of this invention lies in utilizing the dynamic borate ester bonds formed between borate ions provided by boric acid and catechol groups on natural polysaccharide chains as the crosslinking core to construct a three-dimensional network structure. Simultaneously, magnetic nanoparticles are uniformly and physically embedded within this network through a key process. The key process feature is that the boric acid solution must be added to initiate crosslinking only after the magnetic nanoparticles and polysaccharide solution have been thoroughly mixed. If crosslinking is performed before adding the magnetic particles, it will lead to aggregation and uneven distribution of the magnetic particles. Similarly, if the magnetic nanoparticles are mixed with the boric acid solution first and then added to the polysaccharide solution, uneven distribution of the magnetic particles will also occur.

[0012] Furthermore, the mass-volume percentage concentration of the natural polysaccharide solution is 4-6%, more preferably 4.5-5.5%. This concentration range ensures the formation of a stable gel network with excellent mechanical properties, while avoiding a decrease in process stability due to excessively high concentrations.

[0013] Furthermore, the boric acid solution has a mass-volume percentage concentration of 0.5-2%, more preferably 1-2%. This range enables rapid gelation (from a few seconds to tens of seconds) and avoids incomplete dissolution of the boric acid.

[0014] Furthermore, the volume ratio of the natural polysaccharide solution to the boric acid solution is (8-12):1. Preferably, it is 10:1.

[0015] Furthermore, the magnetic nanoparticles are Fe3O4 nanoparticles with a particle size of 5 μm; the mass-volume percentage concentration of Fe3O4 nanoparticles in the gel is 40-50% based on the total weight of the gel. This loading ensures uniform dispersion while imparting excellent magnetic response properties to the gel.

[0016] Furthermore, the buffer solution is a phosphate buffer solution.

[0017] The present invention also provides a magnetic polysaccharide gel, which is prepared by the above-described preparation method.

[0018] Furthermore, the magnetic polysaccharide gel is prepared from natural polysaccharides, magnetic nanoparticles, and boric acid; wherein, the borate ions provided by boric acid and the catechol groups on the natural polysaccharide chains cross-link through dynamic borate ester bonds to form a three-dimensional network structure, and the magnetic nanoparticles are uniformly dispersed and physically embedded in the three-dimensional network structure.

[0019] The present invention also provides the application of the above-mentioned magnetic polysaccharide gel in the preparation of magnetically controlled targeted delivery systems, magnetically controlled micromanipulation instruments, tissue engineering scaffolds, wound hemostatic materials or in vivo foreign body removal agents.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention mixes natural polysaccharide extracts (Giant Kelp Polysaccharide MPP, Ulva Prolifera Polysaccharide EPP, Nori Polysaccharide PP, Spirulina Polysaccharide SP) with boric acid. The borate ions in the boric acid rapidly form dynamic and reversible borate ester bonds with the catechol groups on the polysaccharide chains, constructing a three-dimensional cross-linked network. A hydrogel is formed within seconds at room temperature. The formation conditions are mild (room temperature, aqueous phase) and do not require toxic catalysts. More importantly, the bond has dynamic and reversible properties: it remains solid under no stress; the network is destroyed under shear force (such as injection through a needle) or large strain (>100%), and the gel flows; after the external force is removed, the borate ester bonds can reform, achieving self-healing. This fundamentally solves the toxicity problem of traditional chemical cross-linking agents and endows the material with injectability and adaptive filling ability.

[0021] (2) This invention optimizes the composite process of magnetic particles and polysaccharide matrix by employing a mixing-then-crosslinking process. This ensures that Fe3O4 nanoparticles are uniformly dispersed in a high-viscosity polysaccharide solution before the formation of the crosslinking network. The rapidly formed crosslinking network then fixes the magnetic particles in situ, effectively inhibiting their aggregation. This allows for a significant increase in the magnetic particle loading (up to approximately 45%) without sacrificing mechanical properties, resulting in a magnetic response driving force far exceeding that of existing technologies. Experiments have shown that the gel of this invention can undergo network rearrangement under stress, ensuring structural integrity while allowing for adaptive shape changes. By adjusting the intensity, direction, and trajectory of the external magnetic field, complex motion behaviors such as rolling, crawling, rotating, and grasping of the gel can be achieved, significantly improving magnetic control precision and controllability.

[0022] (3) Experiments have shown that the gel prepared by this invention has synergistically enhanced mechanical properties and multifunctional integration: The dynamic borate ester bond network provides sufficient mechanical strength (storage modulus G' > loss modulus G''), while its dynamic properties prevent brittle fracture and endow the gel with high elasticity; After the gel is cut and the cut surfaces come into contact, it can heal itself within minutes, restoring structure and function. Polysaccharide backbones (such as MPP, EPP, etc.) have good biocompatibility and procoagulant activity. Combined with the self-adaptive filling ability of gel (which can closely fit irregular wound surfaces), they can quickly cover liver wounds, significantly reduce bleeding, and achieve magnetically guided targeted hemostasis. Hemolysis assay (hemolysis rate <5%), MTT assay and live-dead staining assay confirmed that the gel is non-cytotoxic and has good blood compatibility. Attached Figure Description

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0024] Figure 1The preparation and characterization of four natural polysaccharide gels are shown in the figures (A: gelation photograph; B: schematic diagram of cross-linking mechanism; C: FT-IR spectrum; D: SEM image). Figure 2 Adaptive deformation capability diagrams of four natural polysaccharide gels (A: in-vitro flow remodeling; B: filling molecular sieve pores; C: adhering to pigskin wound). Figure 3 The preparation and rheological properties of the magnetic polysaccharide gel are shown in the diagram (A: Schematic diagram of the preparation process; B: SEM image; CF: Strain scan curve). Figure 4 This is a diagram illustrating the self-healing properties of the magnetic polysaccharide gel (photograph of healing after cutting). Figure 5 The magnetic response motion properties of the magnetic polysaccharide gel are shown in the diagram (AB: complex trajectory motion in air; CE: movement in isolated rat intestine / stomach). Figure 6 Diagrams illustrating the precise manipulation and grasping capabilities of magnetic polysaccharide gels (A: grasping foreign objects inside a shaped tube; B: writing letters using magnetic control). Figure 7 A diagram illustrating the multi-environment grasping capabilities of the magnetic polysaccharide gel (A: grasping various objects in open environments; BC: grasping glass and skin surfaces, underwater). Figure 8 The in vivo hemostatic properties of the magnetic polysaccharide gel are shown in the diagram (A: hemostasis process in mouse liver; B: blood loss statistics). Figure 9 The diagram shows the biocompatibility evaluation of the magnetic polysaccharide gel (A, B: hemolysis rate, C: MTT, D: live / dead staining). Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1 Effects of different polysaccharide concentrations on gel formation and mechanical properties: PBS solutions containing 2%, 4%, 6%, and 8% by weight (w / v) of giant kelp polysaccharide (MPP), seaweed polysaccharide (EPP), nori polysaccharide (PP), and spirulina polysaccharide (SP) were prepared respectively.

[0027] A gel was prepared using a subsequently optimized method (see Example 4) with boric acid solution and Fe3O4 nanoparticles. The gelation was evaluated by observing the gelation process and the compressive strength of the gel (compression test using a universal testing machine).

[0028] In addition, polysaccharides at concentrations of 4.5%, 5%, and 5.5% were tested, and the results are shown in Table 1.

[0029] Table 1 As shown in Table 1, none of the four polysaccharides could form a three-dimensional network structure at a concentration of 2% (marked as N), indicating that the polysaccharide concentration was too low to construct a three-dimensional network structure.

[0030] All polysaccharides can form gels at a concentration of 4%, but EPP, PP, and SP gels have low compressive strength and insufficient mechanical support.

[0031] When the polysaccharide concentration is 6%, although the strength of some systems is improved, errors generally increase and repeatability is poor. Furthermore, the strength of EPP and PP no longer increases with increasing concentration after 6%.

[0032] When the polysaccharide concentration is 8%, the strength does not improve or even decreases, and the process stability is poor with large errors.

[0033] Subsequent compression tests were conducted using polysaccharide concentrations of 4.5%, 5%, and 5.5%. No significant differences in compressive strength were observed among the systems, and the repeatability was good. This indicates that once the polysaccharide concentration reaches 4.5%, the cross-linking network is essentially complete. Further increasing the concentration contributes very little to the mechanical strength and reduces process stability. Therefore, the optimal polysaccharide concentration is 4.55% (or any concentration within this range, such as 4.5-5.5%).

[0034] Example 2 Effect of different boric acid concentrations on gelation time: Boric acid PBS solutions with mass-volume percentage concentrations of 0.5%, 1.0%, 2.0%, and 3.0% were prepared. Using giant kelp polysaccharide (MPP), nori polysaccharide (PP), and spirulina polysaccharide (SP) as examples, gels were prepared according to the subsequent method (see Example 4). The time required from the addition of the boric acid solution to the system losing its fluidity (gel formation) was recorded. The test results are shown in Table 2.

[0035] Table 2 Boric acid (BA) was prepared using a concentration gradient from low to high at 0.5%, 1.0%, 2.0%, and 3.0%. After gelation, the optimal BA concentration for the hydrogel was determined by the gelation time. At a BA concentration of 0.5%, M-SP failed to gel. As the BA concentration increased from 0.5% to 1.0%, the gelation time decreased. When the BA concentration was further increased to 2.0%, the gelation time for M-MPP, M-PP, and M-SP no longer decreased. At a BA concentration of 3.0%, the BA could not be completely dissolved.

[0036] Therefore, rapid gelation can be achieved with boric acid concentrations ranging from 1.0% to 2.0%. Considering both solubility and raw material costs, the optimal boric acid concentration is 1.36%.

[0037] Example 3 The effect of preparation order on gel uniformity and magnetic responsiveness: Preparation sequence 1: Add boric acid solution to polysaccharide (MPP) solution, crosslink to form blank gel, and then stir and add Fe3O4 nanoparticles.

[0038] Preparation sequence 2: Fe3O4 nanoparticles are first mixed with boric acid solution, and then mixed with polysaccharide (MPP) solution to prepare gel.

[0039] Preparation sequence 3: Mix polysaccharide (MPP) solution with Fe3O4 nanoparticles, then add boric acid solution and mix to prepare gel.

[0040] Table 3-5 shows the three preparation sequences mentioned above, and records the observations and records of whether gelation occurred during the preparation process and the uniformity of the dispersion of Fe3O4 nanoparticles.

[0041] Table 3 1 polysaccharides 4.55 N 2 BA 1.36 Y 3 <![CDATA[Fe3O4]]> 45.5 <![CDATA[ Fe3O4 cannot be evenly dispersed ]]> Table 4 1 <![CDATA[Fe3O4]]> 45.5 N 2 BA 1.36 <![CDATA[Fe3O4 cannot be evenly dispersed]]> 3 polysaccharides 4.55 <![CDATA[Fe3O4 cannot be evenly dispersed]]> Table 5 1 polysaccharides 4.55 N 2 <![CDATA[Fe3O4]]> 45.5 N 3 BA 1.36 Y As shown in Table 3, using preparation sequence 1, Fe3O4 nanoparticles cannot be uniformly dispersed in the formed dense gel network. Instead, they tend to agglomerate on the gel surface or form uneven clumps, resulting in uneven magnetic response and decreased mechanical properties. Under an applied magnetic field, the gel movement is discontinuous, and some areas show no response.

[0042] As shown in Table 4, when using preparation sequence 2, Fe3O4 nanoparticles are first mixed with boric acid solution, which results in the inability of Fe3O4 nanoparticles to be effectively and uniformly dispersed. Subsequently, they are mixed with polysaccharide (MPP) solution, which causes some Fe3O4 nanoparticles in the prepared gel to agglomerate and become unevenly dispersed. Moreover, the reduction of free borate ions used to crosslink the polysaccharide leads to an incomplete gel network and poor mechanical properties.

[0043] As shown in Table 5, the gel prepared using the sequence of this invention (corresponding to preparation sequence 3) exhibits a uniform distribution of Fe3O4 particles with no visible agglomeration. Under an applied magnetic field, the entire gel responds rapidly and moves smoothly and controllably.

[0044] Example 4 A general method for preparing magnetic polysaccharide gels: 1. Polysaccharide extract solution: Accurately weigh 50 mg of polysaccharide extract (giant kelp polysaccharide (MPP)) in advance and dissolve it in 950 μL of PBS buffer solution.

[0045] 2. Boric acid solution: Accurately weigh 0.3g of boric acid in advance and dissolve it in 2mL of PBS buffer at 80℃.

[0046] 3. Add 500 mg of Fe3O4 nanoparticles to 1 mL of polysaccharide extract solution, add 100 μL of boric acid solution (the volume ratio of extract solution to boric acid solution is 10:1), stir, mix evenly, and let stand for 10 s to obtain magnetic hydrogel M-MPP.

[0047] Using the exact same method described above, M-EPP, M-PP, and M-SP gels can be prepared by replacing MPP with Ursula polysaccharide (EPP), Nori polysaccharide (PP), and Spirulina polysaccharide (SP), respectively.

[0048] Performance testing: The pre-crosslinking solution and the final gel prepared in Example 4 were respectively placed into vials and inverted. The results showed that the pre-crosslinking solution was a flowable liquid, while the M-MPP, M-EPP, M-PP, and M-SP formed after crosslinking were all stably adhered to the bottom of the vial and did not flow, proving that the gelation was successful (see [link to example]). Figure 1 A).

[0049] Figure 1 B elucidates the cross-linking mechanism: borate ions form dynamic borate ester bonds with the ortho-dihydroxy groups on the polysaccharide chains, constructing a three-dimensional network.

[0050] Fourier transform infrared spectroscopy (FT-IR) was used to analyze the lyophilized gels. The results showed that, compared with pure polysaccharides, all magnetic gels exhibited a new characteristic absorption peak in the 1340-1370 cm⁻¹ range. This peak was attributed to the stretching vibration of the BOC bond, clearly confirming the formation of borate ester bonds (see [link to article]). Figure 1 C).

[0051] Scanning electron microscopy (SEM) was used to observe the cross-sections of the lyophilized gels. The results showed that all four magnetic gels exhibited typical porous three-dimensional network structures. Specifically, M-SP gel had a dense structure, M-PP gel showed a honeycomb-like structure, M-EPP gel had regular pores, and M-MPP gel had relatively large pore sizes. Fe3O4 nanoparticles were uniformly attached to or embedded in the pore walls in the form of nano-sized particles, and no obvious large-sized aggregates were observed (see [link to SEM]). Figure 1 D).

[0052] Figure 2 This demonstrates the adaptability of natural hydrogels. Figure 2 A shows that the four gels (M-MPP, M-EPP, M-PP, and M-SP) can flow in a glass bottle and reshape into the container shape; Figure 2 As shown in B, the gel can fill the tiny pores between molecular sieves; Figure 2 C further confirmed that the gel could closely adhere to irregular wounds on pigskin, and dye labeling showed that it uniformly filled the wound. The results indicate that the gel has good adaptive deformation capabilities, can conform to wounds with complex contours, and provides important support for filling irregular wounds and tissue repair.

[0053] Figure 3 A illustrates the preparation process of the magnetically responsive hydrogel in this invention. Figure 3 SEM images of B show that the four magnetic gels maintained a porous three-dimensional network structure after freeze-drying, with magnetic particles uniformly dispersed on the backbone without obvious aggregation, proving that the magnetic particles were successfully loaded and did not damage the gel network.

[0054] Rheological testing: Strain scanning (strain range 1%-1000%) was performed on M-MPP, M-EPP, M-PP and M-SP gels using a rotational rheometer.

[0055] The results show (see) Figure 3CF): In the low strain region (1-10%), the storage modulus (G') of all gels is consistently greater than the loss modulus (G''), indicating that the gel maintains a solid elastic network. When the strain increases to approximately 100%, the G' and G'' curves intersect, and subsequently G''>G', indicating that the three-dimensional network structure is disrupted and the gel begins to flow. This strain-dependent behavior is consistent across the four magnetic gels, demonstrating that the material retains good injectability and self-healing capabilities, and that the introduction of magnetic particles does not impair the mechanical properties of the gel.

[0056] Figure 4 The self-healing properties of the gels were demonstrated by cutting each of the four gels into several pieces with a blade and gently touching the fresh cut surfaces at room temperature. The cut surfaces healed spontaneously into a whole within minutes at room temperature and could be picked up with tweezers without breaking at the healed site, proving that they have excellent self-healing properties.

[0057] Figure 5 The magnetic response motion properties of the magnetic hydrogel were demonstrated: Precise in vitro movement: When M-EPP gel is placed in a culture dish and driven by a neodymium magnet (surface magnetic field strength of approximately 300 mT), the gel can precisely move along the trajectories of letters such as "W, H, B, O, T" in an air environment and can pass through channels of different widths, demonstrating that its movement is precise and controllable (see [reference]). Figure 5 AB).

[0058] Motion in complex environments: When M-EPP gel was placed on the surface and inside isolated rat stomach and intestinal tissues, guided by an external magnet, the gel was able to move directionally along the natural contours and peristaltic direction of the tissues, and could move from the stomach to a designated location in the intestine, demonstrating good adaptability and magnetic navigation ability in complex physiological environments (see [link to relevant documentation]). Figure 5 CE).

[0059] Figure 6 A shows that four types of magnetic gels can move directionally and grasp foreign objects (water-absorbing silica gel) inside irregularly shaped glass tubes, demonstrating their potential for targeted delivery and foreign object removal in confined spaces. Figure 6 In B, four magnetic gels (M-MPP, M-EPP, M-PP, and M-SP) accurately wrote the letters "M", "E", "P", and "S" under the control of a magnetic field, which intuitively demonstrated their excellent motion precision and programmability, providing strong support for micromanipulation and targeted therapy.

[0060] Figure 7 This demonstrates the multi-environment gripping capabilities of magnetic hydrogels. Figure 7 A shows that M-EPP gel can adhere to and move objects of different materials, such as suture needles, sunflower seeds, cotton, toothpicks, and breadcrumbs, in an open environment, proving its universal foreign object grasping performance. Figure 7BC further confirmed that the gel can adhere to moving targets not only in air on glass and skin / mucous membrane surfaces, but also successfully grasp objects in underwater environments. These results fully demonstrate the strong adhesion and grasping ability of the magnetic gel in both dry and wet environments, providing important support for in vivo targeted delivery and foreign body removal applications.

[0061] Evaluation of the in vivo hemostatic properties of magnetic polysaccharide gel: Animal model: Establish a mouse model of liver trauma and hemorrhage.

[0062] Experimental groups: blank control group (no treatment), M-MPP group, M-EPP group, M-PP group, M-SP group.

[0063] Methods: A linear incision approximately 1 cm long and 2 mm deep was made on the surface of the mouse liver using a scalpel. After active bleeding occurred, approximately 0.2 g of the corresponding magnetic gel was immediately applied to the wound, or approximately 0.2 g of the corresponding magnetic gel was placed near the wound and a small magnet was applied to guide the gel to cover the wound. The bleeding process was recorded and blood was collected to calculate the amount of blood loss.

[0064] Test results show that: Figure 8 The hemostasis process photos of A showed that all four gels, M-MPP, M-EPP, M-PP and M-SP, could quickly cover the liver wound in mice and achieve effective hemostasis, while the blank control group continued to bleed. Furthermore, the magnetic polysaccharide gel could achieve magnetically guided targeted hemostasis. Figure 8 Quantitative analysis of blood loss in group B confirmed that the control group had the highest blood loss, while all four gel groups significantly reduced blood loss, with the M-SP group showing the best hemostatic effect. These results indicate that all four marine polysaccharide hydrogels possess good in vivo hemostatic capabilities, can rapidly seal wounds and reduce bleeding, and hold promise as novel injectable hemostatic materials for trauma treatment.

[0065] Biocompatibility evaluation of magnetic polysaccharide gels: Hemolysis Assay: Mouse blood was used for the hemolysis assay. A suitable amount of fresh blood was collected from healthy mice using the ocular blood collection method and placed in an anticoagulant tube. After proper mixing, the blood was transferred to a centrifuge tube and centrifuged at 3000 rpm for 5 min. The supernatant was removed, and the bottom red blood cells were washed with physiological saline. Centrifugation was repeated until the supernatant was clear and transparent. The centrifuged red blood cells were collected and prepared as a 5% (v / v) red blood cell solution using physiological saline. 100 μL of each natural hydrogel was immersed in 300 μL of blood cell solution, followed by the addition of 300 μL of physiological saline. The positive control group consisted of a mixture of 300 μL blood cell solution and 300 μL purified water, while the negative control group consisted of a mixture of 300 μL blood cell solution and 300 μL physiological saline. All mixtures were incubated at 37°C for 4 h, followed by centrifugation at 3000 rpm for 5 min. After centrifugation, photographs were taken and the supernatant was collected. The absorbance of each group of supernatant was measured at 545 nm using a full-band microplate reader, and the hemolysis rate was calculated. (Hemolysis rate = (A sample group - A negative control) / (A positive control - A negative control) * 100%).

[0066] MTT cytotoxicity assay: 8000 NIH 3T3 cells were seeded in 96-well plates and placed in a cell culture incubator (37℃, 5% CO2). After 24 h, the cell status and number in the wells were observed. After 24 h, the culture medium was aspirated, and the cells were washed 1-2 times with PBS buffer. 10 μL of MPP, EPP, PP, and SP hydrogel extract and 90 μL of 0.5% serum medium were added, and the cells were co-cultured in a cell culture incubator (37℃, 5% CO2) for 24 h. Finally, 20 μL of 5 mg / mL MTT solution (prepared with PBS) was added to each well, and the cells were incubated for another 4 h. The mixed culture medium containing MTT was then aspirated, and 150 μL of DMSO was added. The absorbance wavelength was measured at 490 nm using a multi-mode microplate reader (Spark10M, TECAN, Swit). Wells containing cells but without MTT were used as blank wells.

[0067] Cell viability (%) = (A S -A0) / (A c -A0)×100%; Where A S A c A0 and A0 represent the absorbance values ​​of the hydrogel sample, control sample, and blank well at 450 nm, respectively.

[0068] Live / dead cell staining assay: The cytotoxicity of the prepared MPP, EPP, PP, and SP hydrogel extracts was investigated using the Calcein-AM / PI cell viability kit. First, 5 × 10⁶ cells were... 43T3 cells were seeded into 24-well cell culture chambers and placed in a cell culture incubator (37℃, 5%). After 24 hours, the culture medium was aspirated, and 1 mL of drug-treated medium (complete medium: extract = 9:1) was added. The cells were then placed in a cell culture incubator (37℃, 5%) and cultured for another 24 hours. The culture medium was then aspirated, and the cells were gently washed twice with PBS. 500 μL of a mixed dye of Calcein-AM and PI was added, and the cells were placed in a cell culture incubator protected from light. After 30 minutes, the cells were removed, the plate solution was aspirated, and the cells were washed three times with PBS to remove the dye. 300 μL of PBS buffer was added, and cell growth was observed using a fluorescence inverted microscope (488 nm green fluorescence - live cells; 595 nm red fluorescence - dead cells).

[0069] Figure 9 The in vitro biocompatibility evaluation results of the polysaccharide gels were presented. Hemolysis experiments showed that the hemolysis rates of the polysaccharide gels (MPP, EPP, PP, SP) were all below the 5% requirement of medical material standards, indicating no risk of hemolysis and good blood compatibility. MTT cytotoxicity assays confirmed that cells co-cultured with the gels had high survival rates and their proliferation activity was not inhibited, indicating no cytotoxicity. Live / dead cell staining results further visually demonstrated that the vast majority of cells in the field of view were green (live cells), with only a very small number of red (dead cells), indicating good cell condition. The overall results indicate that this series of natural polysaccharide hydrogels has good in vitro biocompatibility, with no risk of hemolysis or cytotoxicity, providing crucial safety evidence for their biomedical applications such as in vivo injection and implantation.

[0070] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a magnetic polysaccharide gel, characterized in that, Includes the following steps: (1) Dissolve the natural polysaccharide in a buffer solution to obtain a natural polysaccharide solution; (2) Dissolve boric acid in a buffer solution to obtain a boric acid solution; (3) Add the magnetic nanoparticles to the natural polysaccharide solution in step (1) and mix them evenly to obtain a magnetic polysaccharide mixture; (4) Add the boric acid solution from step (2) to the magnetic polysaccharide mixture from step (3), stir and mix evenly, and let it stand to react to obtain the magnetic polysaccharide gel. Among them, the natural polysaccharides are selected from any one or more combinations of giant kelp polysaccharides, seaweed polysaccharides, and spirulina polysaccharides.

2. The preparation method according to claim 1, characterized in that, The mass-volume percentage concentration of the natural polysaccharide solution is 4-6%.

3. The preparation method according to claim 2, characterized in that, The boric acid solution has a mass-volume percentage concentration of 0.5-2%.

4. The preparation method according to claim 3, characterized in that, The volume ratio of the natural polysaccharide solution to the boric acid solution is (8-12):

1.

5. The preparation method according to claim 1, characterized in that, The magnetic nanoparticles are Fe3O4 nanoparticles; the mass-volume percentage concentration of Fe3O4 nanoparticles in the gel is 40-50% based on the total weight of the gel.

6. The preparation method according to claim 1, characterized in that, The buffer solution is a phosphate buffer solution.

7. A magnetic polysaccharide gel, characterized in that, It is prepared by any one of claims 1-6.

8. The magnetic polysaccharide gel according to claim 7, characterized in that, The magnetic polysaccharide gel is prepared from natural polysaccharides, magnetic nanoparticles and boric acid; wherein, the borate ions provided by boric acid and the catechol groups on the natural polysaccharide chains cross-link through dynamic borate ester bonds to form a three-dimensional network structure, and the magnetic nanoparticles are uniformly dispersed and physically embedded in the three-dimensional network structure.

9. The application of the magnetic polysaccharide gel as described in claim 7 in the preparation of magnetically controlled targeted delivery systems, magnetically controlled micromanipulation instruments, tissue engineering scaffolds, wound hemostatic materials, or in vivo foreign body removal agents.

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