Injectable polysaccharide hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable polysaccharide hydrogel as well as a preparation method and application thereof. The method comprises the following steps: dissolving hyaluronic acid to obtain a hyaluronic acid solution; dissolving gellan gum to obtain a gellan gum solution; the hyaluronic acid solution and the gellan gum solution are mixed, the hyaluronic acid and the gellan gum are subjected to an esterification reaction to prepare the hydrogel, and the mass ratio of the hyaluronic acid to the gellan gum is 1: 2-1: 6. The hydrogel system composed of hyaluronic acid and gellan gum disclosed by the invention not only has excellent flowing property, mechanical property and injection force, but also can be used as a high-performance carrier of a contrast agent, and can realize efficient loading, uniform dispersion and stable retention of the contrast agent; a solution is provided for the filling material with visual diagnosis and excellent filling treatment performance, and the filling material has wide application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical polymer materials, specifically to an injectable polysaccharide hydrogel, its preparation method, and its applications. Background Technology

[0002] Enophthalmos, or enophthalmos, is a pathological condition where the eyeball is abnormally displaced posteriorly relative to the bony orbital rim, resulting in a sunken appearance within the orbit. It is typically secondary to orbital trauma, orbital hypoplasia, orbital inflammation, and metastatic ocular tumors. This condition not only affects facial aesthetics but can also lead to functional impairments such as diplopia and eyelid misalignment. One of the main goals of enophthalmos correction is to restore facial appearance by filling the orbital cavity with implants.

[0003] Existing implants for treating enophthalmos present several unresolved challenges. First, clinical implantation of devices that completely fill orbital defects often requires large surgical incisions, potentially causing greater trauma to patients. Second, because contrast agents can affect implant performance—for example, they may be difficult to disperse evenly, impact implant stability, or become inactive—current implants typically do not carry contrast agents, making it difficult to visually assess their precise alignment with the defect morphology after implantation. Third, existing implants also suffer from drawbacks such as absorption, displacement, and inflammatory reactions, making it difficult for them to remain stably present at the defect site for extended periods. Furthermore, the high cost and complex manufacturing process of these implants further limit their application. Summary of the Invention

[0004] One objective of this invention is to provide a method for preparing injectable polysaccharide hydrogels, which has mild reaction conditions and a simple process, making it suitable for mass production and application. Furthermore, the hydrogel prepared by this method not only possesses excellent mechanical properties and fluidity, allowing it to be precisely injected into the defects within the orbit by being made into an injection solution, significantly reducing trauma, but also exhibits good stability and biocompatibility. In addition, this hydrogel can also serve as a high-performance carrier for contrast agents.

[0005] This invention is achieved through the following technical solution:

[0006] A method for preparing an injectable polysaccharide hydrogel includes the following steps:

[0007] Dissolve hyaluronic acid to obtain a hyaluronic acid solution;

[0008] Dissolve gellan gum to obtain a gellan gum solution;

[0009] A hydrogel is prepared by mixing the hyaluronic acid solution and the gellan gum solution and then esterifying the hyaluronic acid with the gellan gum.

[0010] The mass ratio of hyaluronic acid to gellan gum is 1:2 to 1:6.

[0011] In this technical solution, hyaluronic acid and gellan gum are first prepared into solutions. In some preferred embodiments, deionized water is used as the solvent for preparing the solutions. After obtaining the hyaluronic acid solution and the gellan gum solution, the two solutions are mixed and subjected to an esterification reaction to obtain the hydrogel. This preparation method has a simple process flow, mild esterification reaction conditions, and readily available raw materials, which is beneficial for mass production and application promotion.

[0012] In one or more embodiments, gellan gum is dissolved in deionized water at 50°C to obtain the gellan gum solution.

[0013] In one or more embodiments, the hyaluronic acid solution and gellan gum solution undergo esterification at 40–60°C for 18–36 h. In some preferred embodiments, the esterification reaction is carried out at 50–55°C for 24–36 h.

[0014] In some embodiments, the content of hyaluronic acid in the hyaluronic acid solution is 0.5~2 wt%; and / or the content of gellan gum in the gellan gum solution is 0.5~2 wt%. Preferably, the content of both hyaluronic acid and gellan gum is 1 wt%.

[0015] In some embodiments, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) was dissolved in an ethanol solution and then added dropwise to a mixed solution of hyaluronic acid and gellan gum.

[0016] In some preferred embodiments, after the esterification reaction is completed, the reaction solution is placed in a dialysis bag and dialyzed in deionized water. The liquid in the dialysis bag is collected and freeze-dried to obtain the hydrogel. In one or more embodiments, the molecular weight cutoff of the dialysis bag is 150-300 kDa, preferably 200-300 kDa. In one or more embodiments, the dialysis time in deionized water is five days.

[0017] In this technical solution, a covalent bond is formed between the hydroxyl groups of hyaluronic acid and the carboxyl groups of gellan gum through an esterification reaction to fix the molecular chain network. Experiments have shown that a higher mass ratio of gellan gum results in a corresponding increase in the number of carboxyl sites that can participate in the reaction, leading to increased cross-linking density and thus increased network rigidity. Simultaneously, a higher cross-linking density also results in a denser hydrogel structure, increased entanglement density between molecular chains, leading to increased viscosity and injection force, and enhanced mechanical strength, which is beneficial for the hydrogel's stability at ocular defects. However, as the gellan gum content increases, the viscosity also increases, while the flowability decreases. Therefore, considering that too low a gellan gum content would lead to reduced mechanical properties and low post-filling stability, while too high a gellan gum content would cause increased injection force, tissue damage, and reduced flowability, this technical solution, in order to balance the hydrogel's flowability, mechanical properties, and injection force, while also considering the impact on the uniformity of the dispersion of the contrast agent, selects a mass ratio of hyaluronic acid to gellan gum of 1:2 to 1:6.

[0018] In some preferred embodiments, the mass ratio of hyaluronic acid to gellan gum is 1:3 to 1:5. More preferably, the mass ratio of hyaluronic acid to gellan gum is 1:4.

[0019] Another object of the present invention is to provide an injectable polysaccharide hydrogel prepared by any of the foregoing preparation methods.

[0020] Another object of the present invention is to provide a hydrogel injection solution prepared based on injectable polysaccharide hydrogel. Specifically, the preparation method includes the following steps:

[0021] The hydrogel is added to deionized water to swell and obtain a hydrogel solution. The hydrogel solution is continuously stirred to remove air bubbles, and then a hydrogel injection solution is obtained.

[0022] In this technical solution, the hydrogel is swollen in deionized water and continuously stirred. After removing air bubbles, it is evenly dispersed in water to obtain a hydrogel injection solution. In some preferred embodiments, the lyophilized hydrogel is swollen in deionized water to obtain a hydrogel solution. This solution is then continuously stirred until no white fibers are present, followed by centrifugation and settling to remove air bubbles, thus obtaining the hydrogel injection solution.

[0023] In a preferred embodiment of the hydrogel injection solution of the present invention, the hydrogel injection solution further contains a contrast agent, and its preparation method specifically includes the following steps:

[0024] The contrast agent was dissolved under light-protected conditions to obtain a contrast agent solution;

[0025] The hydrogel is added to the contrast agent solution to swell and obtain a hydrogel solution. The hydrogel solution is continuously stirred to remove air bubbles, and then a hydrogel injection solution containing contrast agent is obtained.

[0026] In this technical solution, the hydrogel system constructed from hyaluronic acid and gellan gel can effectively carry the contrast agent. Furthermore, experiments have shown that even after adding the contrast agent, the hydrogel injection solution can still maintain the desired viscosity and injection force, which is beneficial for visually observing whether the hydrogel accurately matches the defect in the orbit after implantation.

[0027] Furthermore, in vitro and in vivo experiments have shown that the hydrogel injection solution still exhibits good cell compatibility after being loaded with contrast agents. Moreover, the hydrogel can remain stable in vivo for a long time, and changes in the filler can be observed through the excellent CT imaging properties of the contrast agent. This provides a solution for next-generation materials that combine visual diagnostics with excellent filling and therapeutic performance.

[0028] Furthermore, the mass ratio of the contrast agent to the hydrogel is 1:1.5 to 1:5.

[0029] The most crucial aspect of adding contrast agents to hydrogels is finding a balance between material safety, imaging effectiveness, and material performance. If too little contrast agent is added, the signal intensity or contrast of the hydrogel in the target imaging modality (X-ray / CT) will be insufficient, resulting in blurred imaging, difficulty in clearly distinguishing the boundaries, shape, and location of the hydrogel, or even complete lack of imaging, thus negating the purpose of adding the contrast agent. However, if too much contrast agent is added, it will not only alter the gel properties of the material but also lead to poor dispersibility, resulting in extremely uneven distribution of the contrast agent within the final hydrogel, uneven imaging, and localized areas of excessive brightness or darkness. This severely affects the accuracy of image interpretation and may even introduce toxicity.

[0030] Therefore, in this technical solution, based on tests of rheological properties, mechanical properties, cell compatibility, and animal imaging, the mass ratio of contrast agent to the hydrogel is 1:1.5 to 1:5, so that the contrast agent does not affect the injection force and viscosity of the gel, while having good biocompatibility and imaging capabilities.

[0031] Another object of the present invention is to provide a reagent for filling defects in the orbit, the reagent comprising any of the aforementioned injectable polysaccharide hydrogels, or any of the aforementioned injectable polysaccharide hydrogel injection solutions.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] 1. The hydrogel system composed of hyaluronic acid and gellan gum disclosed in this invention not only has excellent flow properties, mechanical properties and injection force, but also can serve as a high-performance carrier for contrast agents, achieving efficient loading, uniform dispersion and stable retention of contrast agents. It provides a solution for filling materials that combine visualization diagnostics and excellent filling therapy performance, and has broad application value.

[0034] 2. This invention balances the flow properties, mechanical properties and injection force of hydrogel by optimizing the mass ratio of hyaluronic acid and gellan gel, while also taking into account the impact on the uniformity of the dispersion of the contrast agent, thus further improving the performance of hydrogel.

[0035] 3. The hydrogel injection solution of the present invention can still maintain the desired viscosity and injection force after the addition of contrast agent, which is conducive to visually observing whether the hydrogel accurately matches the defect in the orbit after implantation; at the same time, the hydrogel injection solution still has good cell compatibility, and the hydrogel can exist stably in vivo for a long time, and the changes of the filler can be observed through the good CT imaging properties of the contrast agent.

[0036] 4. This invention optimizes the mass ratio of contrast agent to hydrogel in the hydrogel injection solution, so that the contrast agent does not affect the injection force and viscosity of the gel, while having good biocompatibility and imaging capabilities.

[0037] 5. The preparation process of this invention is simple, the esterification reaction conditions are mild, and the raw materials are readily available, which is conducive to mass production and application promotion. Attached Figure Description

[0038] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0039] Figure 1 This is a flowchart illustrating the preparation method of hydrogel in a specific embodiment of the present invention;

[0040] Figure 2 A synthetic route diagram of the hydrogel in a specific embodiment of the present invention is shown;

[0041] Figure 3 The NMR characterization of hyaluronic acid, gellan gum, and hydrogel in specific embodiments of the present invention is shown.

[0042] Figure 4 This is a flowchart illustrating the preparation method of the hydrogel injection solution in a specific embodiment of the present invention.

[0043] Figure 5 This is a bar chart showing the injection force of each hydrogel injection solution in a specific embodiment of the present invention;

[0044] Figure 6 This is a viscosity curve diagram of each hydrogel injection solution in a specific embodiment of the present invention;

[0045] Figure 7 The frequency scan curves of each hydrogel injection solution under 0.1% shear strain are shown in the specific embodiments of the present invention.

[0046] Figure 8 The following are (A) injection force curves and (B) viscosity curves of the group without contrast agent (HG) and the group containing contrast agent (HG+Iohexol) in specific embodiments of the present invention;

[0047] Figure 9 These are fluorescence images of human umbilical vein endothelial cells cultured for 1 day, 3 days, and 5 days in various experimental groups according to a specific embodiment of the present invention.

[0048] Figure 10 The survival rates of human umbilical vein endothelial cells cultured for 1 day, 3 days, and 5 days in each experimental group are shown in specific embodiments of the present invention.

[0049] Figure 11 These are morphological images of the human umbilical vein endothelial cell skeletons cultured for 1 day, 3 days, and 5 days in various experimental groups according to specific embodiments of the present invention.

[0050] Figure 12 The experimental process of removing the globus liposome from the experimental rabbit is shown in a specific embodiment of the present invention;

[0051] Figure 13 The illustration shows cross-sectional images of the orbit in coronal, sagittal, and horizontal planes of an experimental rabbit four weeks after the implantation of hydrogel injection into the orbital defect, according to a specific embodiment of the present invention. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0053] All raw materials used in this invention are not particularly limited in their source; they can be purchased commercially or prepared using conventional methods well-known to those skilled in the art. The purity of all raw materials used in this invention is not particularly limited; however, analytical grade or conventional purity requirements in the field of biomedical polymer materials are preferred. All raw materials used in this invention have common designations and abbreviations in the field, and each designation and abbreviation is clearly defined within its relevant application. Those skilled in the art can obtain these materials from commercial sources or prepare them using conventional methods based on the designation, abbreviation, and corresponding application.

[0054] The present invention does not impose any particular restrictions on the expression of the substituents, and all expressions are well known to those skilled in the art. Based on common sense, those skilled in the art can correctly understand their meaning according to their expression.

[0055] The term "link" as used in this invention can refer to direct linking or indirect linking via other substituents, unless otherwise specified.

[0056] I. Preparation of Hydrogel Injection Solution

[0057]

Example 1

[0058] Hyaluronic acid (HA) was dissolved in deionized water at a mass fraction of 1 wt% at room temperature to obtain a hyaluronic acid solution. Gellan gum (GG) was completely dissolved in deionized water at a mass fraction of 1 wt% at 90 °C to obtain a gellan gum solution, wherein the mass ratio of hyaluronic acid to gellan gum was 1:4. Subsequently, the hyaluronic acid solution and the gellan gum solution were mixed to obtain a mixed solution.

[0059] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) was dissolved in an ethanol solution and then added dropwise to the mixed solution. The reaction was carried out at 50 °C for 24 h until the reaction was complete. The synthetic route is shown below. Figure 2 As shown.

[0060] After the reaction was complete, the liquid was transferred to a dialysis bag with a molecular weight cutoff of 200 kDa and dialyzed with deionized water for five days until all other reagents and unreacted hyaluronic acid were completely removed. Finally, the liquid in the dialysis bag was collected and freeze-dried to obtain a white fibrous material, namely hydrogel H1.

[0061] Figure 3 The NMR characterization of hyaluronic acid, gellan gum, and hydrogel (HG) is shown, demonstrating that hydrogel can be synthesized using the above preparation method.

[0062] Hydrogel injection solutions can be prepared based on hydrogels. Specifically, the lyophilized hydrogel H1 is swollen in deionized water to obtain a hydrogel solution. The solution is stirred continuously until no white fibers are present, then centrifuged at 6000 rpm for 10 minutes and allowed to stand at 4 °C for 12 hours to remove air bubbles, thus obtaining the hydrogel injection solution Inj 1.

[0063]

Example 2

[0064] In this embodiment, the preparation method is basically the same as in Example 1, except that the mass ratio of hyaluronic acid to gellan gum is 1:2 to prepare hydrogel H2, and hydrogel injection solution Inj 2 is prepared based on hydrogel H2.

[0065]

Example 3

[0066] In this embodiment, the preparation method is basically the same as in Example 1, except that the mass ratio of hyaluronic acid to gellan gum is 1:3 to prepare hydrogel H3, and hydrogel injection solution Inj 3 is prepared based on hydrogel H3.

[0067]

Example 4

[0068] In this embodiment, the preparation method is basically the same as in Example 1, except that the mass ratio of hyaluronic acid to gellan gum is 1:5 to prepare hydrogel H4, and hydrogel injection solution Inj 4 is prepared based on hydrogel H4.

[0069]

Example 5

[0070] In this embodiment, the preparation method is basically the same as in Example 1, except that the mass ratio of hyaluronic acid to gellan gum is 1:6 to prepare hydrogel H5, and hydrogel injection solution Inj 5 is prepared based on hydrogel H5.

[0071]

Example 6

[0072] In this embodiment, the preparation method is basically the same as in Example 1, except that the contrast agent iohexol is dissolved in deionized water under light-protected conditions to prepare a contrast agent solution with a concentration of 0.150 mg / mL. The lyophilized hydrogel H1 is then swollen in the contrast agent solution and stirred continuously until the hydrogel H1 and the contrast agent are evenly mixed in the solution. After centrifugation and removal of air bubbles, the hydrogel injection solution Inj 6 is obtained.

[0073]

Example 7

[0074] In this embodiment, the preparation method is basically the same as in Example 1, except that the contrast agent Iohexol is dissolved in deionized water under light-protected conditions to prepare a contrast agent solution with a concentration of 0.219 mg / mL. The lyophilized hydrogel H1 is then swollen in the contrast agent solution, and the mixture is stirred continuously until the hydrogel H1 and the contrast agent are evenly mixed in the solution. After centrifugation and removal of air bubbles, the hydrogel injection solution Inj 7 is obtained.

[0075]

Example 8

[0076] In this embodiment, the preparation method is basically the same as in Example 1, except that the contrast agent Iohexol is dissolved in deionized water under light-protected conditions to prepare a contrast agent solution with a concentration of 0.250 mg / mL. The lyophilized hydrogel H1 is then swollen in the contrast agent solution, and the mixture is stirred continuously until the hydrogel H1 and the contrast agent are evenly mixed in the solution. After centrifugation and removal of air bubbles, the hydrogel injection solution Inj 8 is obtained.

[0077] II. Performance Testing of Hydrogel Injection Solutions

[0078]

Example 9

[0079] In this embodiment, the injection force of the three hydrogel injection solutions from Examples 1, 2, and 5, as well as the gellan gum solution and hyaluronic acid solution, was tested. Specifically, each injection solution was loaded into a syringe (1 mL) for injection testing, and the injection force of each injection solution was measured using a texture analyzer in compression mode at an injection speed of 30 mm / min.

[0080] Experimental results are as follows Figure 5 As shown, the injection force of Inj 1 is 0.617 N, Inj 2 is 0.517 N, and Inj 5 is 1.212 N. This demonstrates that the injection force of the hydrogel injection solution increases with the increase of the gellan gum mass ratio. This is because increasing the amount of gellan gum increases the number of carboxyl sites involved in the reaction, leading to a significant increase in crosslinking density and enhanced network rigidity. This results in a denser hydrogel structure, increasing the injection force and significantly enhancing mechanical strength, which is beneficial for maintaining the stability of the hydrogel at the ocular defect site.

[0081]

Example 10

[0082] In this embodiment, the rheological properties of the three hydrogel injection solutions from Examples 1, 2, and 5, as well as gellan gum solution and hyaluronic acid solution, were tested. Specifically, rheological analysis was performed at 37 °C using a modular intelligent advanced rotational rheometer equipped with a plate-to-plate geometry (50 mm diameter, 0.37 mm gap). 1 mL of sample was loaded onto the plate using a pipette, and excess sample around the plate was removed. The sample was preloaded for 3 minutes before each measurement to eliminate any loading effects and shear history.

[0083] First, in shear mode, the time interval is from 0.1 to 100 seconds. -1 The viscosity change of each hydrogel injection solution was measured by shear rate testing. For example... Figure 6 As shown, the three hydrogel injection solutions prepared in this embodiment all exhibit appropriate viscosity and typical shear-thinning behavior.

[0084] Then, frequency scanning tests were performed at 0.1% shear strain, and the changes in shear modulus (G') and viscous modulus (G") of the samples were measured in the frequency range of 0.1-1 Hz. The frequency scanning curves of hyaluronic acid solution, gellan gum solution, and hydrogel injection solution Inj 1 are shown in the figure. Figure 7As shown, the hydrogel injection solution exhibits viscoelastic properties with an elastic modulus greater than its viscous modulus. Compared with the gellan gum solution, both the elastic modulus and viscous modulus are increased, reflecting that the hydrogel injection solution prepared in this application has excellent rheological properties.

[0085] Furthermore, from Figure 6 It can be seen that as the gellan gum content increases, the viscosity of the hydrogel injection solution also increases, while the flowability decreases accordingly. Therefore, considering that too low a gellan gum content will lead to reduced mechanical properties and low stability after filling, while too high a gellan gum content will cause increased injection force, tissue damage, and reduced flowability, in this application, in order to balance the flowability, mechanical properties, and injection force of the hydrogel, and considering the impact on the uniformity of the dispersion of the contrast agent, the mass ratio of hyaluronic acid to gellan gum is selected as 1:2 to 1:6. In some preferred embodiments, the mass ratio of hyaluronic acid to gellan gum is selected as 1:3 to 1:5. In a more preferred embodiment, the mass ratio of hyaluronic acid to gellan gum is 1:4.

[0086]

Example 11

[0087] In this embodiment, in order to verify whether the addition of contrast agent would have a significant impact on the mechanical properties of the hydrogel, tests were conducted on hydrogel injection solution Inj 1 without contrast agent and hydrogel injection solution Inj 7 with contrast agent.

[0088] Experimental results are as follows Figure 8 As shown, the injection forces of the two hydrogel injection solutions were almost identical, and both exhibited typical shear-thinning behavior typical of gels, with no significant change in viscosity. This indicates that the addition of the contrast agent did not significantly affect the mechanical properties of the hydrogel injection solution. Even after adding the contrast agent, the hydrogel injection solution maintained the desired viscosity and injection force, demonstrating that the hydrogel system provided in this application can effectively carry the contrast agent, facilitating direct observation after implantation to determine whether the hydrogel accurately matches the defect within the orbit.

[0089]

Example 12

[0090] In this embodiment, in vitro cytotoxicity, proliferation and morphology experiments of the hydrogel injection solution were performed.

[0091] In the cytotoxicity assay, human umbilical vein endothelial cells (HUVECs) were cultured in F12K medium containing 10% fetal bovine serum and 100 U / ml penicillin / streptomycin at 37°C in a humidified incubator with 5% CO2. The cells were incubated using the Calcein / PI cell viability / toxicity assay kit (KGA1107-100, Jiangsu Kaiji Biotechnology Co., Ltd.) at 37°C with 5% CO2.

[0092] The experimental group consisted of four groups: the control group (cells + culture medium); the HG group (cells + culture medium + hydrogel injection, with a hydrogel concentration of 100 μg / ml); the Iohexol group (cells + culture medium + contrast agent, with a contrast agent concentration of 437.5 μg / ml); and the HG + Iohexol group (cells + culture medium + hydrogel injection, with both hydrogel and contrast agent concentrations of 100 μg / ml and 437.5 μg / ml).

[0093] The activity of the samples was quantitatively analyzed at 1, 3, and 5 days post-inoculation to assess toxicity. Live cells (green fluorescent label) and dead cells (red fluorescent label) were observed and images were acquired using a fluorescence microscope (IX-73, Olympus, Japan). Figure 9 Fluorescence images of HUVECs cultured for 1, 3, and 5 days are shown. Live cells and dead cells are stained green and red, respectively. The images demonstrate that the hydrogel injection solution exhibits good cell compatibility.

[0094] In the cell proliferation assay, the four experimental groups were configured identically to those in the cytotoxicity assay. Cell viability was quantitatively analyzed at 1, 3, and 5 days post-inoculation to assess toxicity. HUVECs were seeded at 2000 cells / well in 96-well plates, with five parallel wells per concentration group. Cells were cultured for 1, 3, and 5 days before assay. For assay, the old culture medium was discarded, the cells were washed three times with PBS, and 100 μL of assay solution was added to each well. A blank control was included. All procedures were performed in the dark. After incubation for 2 hours, the absorbance at 450 nm was measured using a full-wavelength microplate reader, and cell viability was calculated for each group. The experimental results are as follows: Figure 10 As shown, the absorbance of cells in the HG group on days 3 and 5 of the CCK-8 experiment was higher than that in the control group, the contrast agent group, and the HG+Iohexol group (p<0.5), indicating that the hydrogel injection solution prepared in this application has good cell compatibility and promotes the growth of HUVECs. Furthermore, although the cell viability decreased slightly after adding the contrast agent, it was still higher than that in the contrast agent group, demonstrating not only the excellent cell compatibility of the hydrogel but also its ability to facilitate observation of implantation by loading contrast agents.

[0095] In the cell morphology experiment, the four experimental groups were set up in the same way as in the cytotoxicity experiment. Cultured HUVECs cells were cleaned, washed twice with preheated PBS (37°C), and then covered with fixative containing 4.0% formaldehyde. The cells were fixed at room temperature for 15 min. The fixative was then removed, and the cells were washed twice with PBS for 10 min each time. The cells were then covered with PBS containing 0.1% Triton X-100 and permeabilized at room temperature for 5 min. The cells were washed twice with PBS for 10 min each time. 100 μL of freshly prepared IF555-phalloidin (manufacturer: Wuhan Saiweier Biotechnology Co., Ltd., catalog number: G1249-100T) staining working solution was used to completely cover the cells, and the cells were incubated at room temperature in the dark for 120 min. The cells were washed three times with PBS. Ready-to-use DAPI staining solution (manufacturer: Wuhan Saiweier Biotechnology Co., Ltd., catalog number: G1012) was added to the slide to cover the cells for nuclear staining for 5 min. The cells were washed twice with PBS buffer, and the results were observed under a fluorescence microscope. Figure 11 The cytoskeleton of HUVECs cultured for 1, 3, and 5 days was displayed, with bright fluorescence highlighting actin filaments in the HUVECs. This indicates that the hydrogel injection solution has good cell compatibility and did not affect the normal structure of HUVECs.

[0096]

Example 13

[0097] In this embodiment, animal experiments were conducted based on the hydrogel injection solution.

[0098] Figure 12 The procedure for removing retrobulbar fat from experimental rabbits is shown. Specifically, after adequate general anesthesia, the hair in the area below and around the eyes of the rabbits was shaved, and the area was disinfected with 5% povidone-iodine. At a distance of 2-3 mm from the lower eyelid, the skin was incised with a blade for about 2-3 cm, and the skin was dissected layer by layer along the infraorbital wall to the orbital periosteum. The orbital periosteum was then incised, and about 0.5 ml of retrobulbar fat was completely removed.

[0099] Approximately 0.5 ml of hydrogel solution containing contrast agent was injected into the fat defect using a 5 ml syringe. The orbital periosteum and lower eyelid skin were sutured layer by layer, and Tobradex eye ointment was applied to the incision. The rabbit was euthanized 4 weeks after hydrogel implantation. After euthanasia, the rabbit's head was separated from the body and stored at 0–4°C. A Micro-CT scan was performed within one day. The CT scan parameters were set to 70 kV tube voltage and 100 μA tube current, and the scan was performed using a KKS-MCT-Sharp CT system manufactured in China.

[0100] Figure 13The images show coronal, sagittal, and horizontal cross-sectional views of the orbit on Micro-CT scans four weeks after hydrogel implantation. As shown, four weeks after implantation, hydrogel filling is visible at the retrobulbar fat defect in the left eye's retrobulbar soft tissue space. The contrast agent makes the hydrogel appear high-density, indicating that the contrast-enhanced hydrogel has good CT imaging properties. Furthermore, the hydrogel remains stable as a retrobulbar soft tissue filler four weeks post-implantation, reflecting its good stability in vivo. Therefore, the contrast-enhanced hydrogel injection solution prepared in this application can serve as a stable and effective hydrogel injection solution for ocular filling.

[0101] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an injectable polysaccharide hydrogel, characterized in that, Includes the following steps: Dissolve hyaluronic acid to obtain a hyaluronic acid solution; Dissolve gellan gum to obtain a gellan gum solution; A hydrogel is prepared by mixing the hyaluronic acid solution and the gellan gum solution and then esterifying the hyaluronic acid with the gellan gum. The mass ratio of hyaluronic acid to gellan gum is 1:2 to 1:

6.

2. The method for preparing an injectable polysaccharide hydrogel according to claim 1, characterized in that, The mass ratio of hyaluronic acid to gellan gum is 1:3 to 1:

5.

3. The method for preparing an injectable polysaccharide hydrogel according to claim 1, characterized in that, The hyaluronic acid content in the hyaluronic acid solution is 0.5~2 wt%; and / or The content of gellan gum in the gellan gum solution is 0.5~2 wt%.

4. The method for preparing an injectable polysaccharide hydrogel according to claim 1, characterized in that, The hyaluronic acid solution and gellan gum solution were subjected to an esterification reaction at 40-60°C for 18-36 h.

5. The method for preparing an injectable polysaccharide hydrogel according to claims 1-4, characterized in that, After the esterification reaction is completed, the reaction solution is placed in a dialysis bag and dialyzed in deionized water. The liquid in the dialysis bag is collected and freeze-dried to obtain the hydrogel.

6. An injectable polysaccharide hydrogel, characterized in that, It was prepared using the method for preparing an injectable polysaccharide hydrogel as described in any one of claims 1 to 5.

7. An injectable polysaccharide hydrogel injection solution, characterized in that, The injectable polysaccharide hydrogel described in claim 6 is prepared by means of the following steps: The hydrogel is added to deionized water to swell and obtain a hydrogel solution. The hydrogel solution is continuously stirred to remove air bubbles, and then a hydrogel injection solution is obtained.

8. An injectable polysaccharide hydrogel injection solution, characterized in that, The injectable polysaccharide hydrogel described in claim 6 is prepared by means of the following steps: The contrast agent was dissolved under light-protected conditions to obtain a contrast agent solution. The hydrogel is added to the contrast agent solution to swell and obtain a hydrogel solution. The hydrogel solution is continuously stirred to remove air bubbles, and then a hydrogel injection solution containing contrast agent is obtained.

9. The injectable polysaccharide hydrogel injection solution according to claim 8, characterized in that, The mass ratio of the contrast agent to the hydrogel is 1:1.5 to 1:

5.

10. A reagent for filling defects in the orbit, characterized in that, The reagent includes an injectable polysaccharide hydrogel as described in claim 6, or an injectable polysaccharide hydrogel injection solution as described in any one of claims 7 to 9.

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