Gel having regulatable free water content and morphology, preparation method therefor, and use thereof

Abstract

Disclosed in the present invention are a gel having a regulatable free water content and morphology, a preparation method therefor, and the use thereof. The method comprises: S1, preparing a solution A from a natural polymer material; S2, mixing a small molecule material containing a positively charged polar group with the solution A to obtain a composite solution B; S3, mixing the composite solution B with a regulator to obtain a composite solution C, stirring same, then adding a condensing agent to the reaction system, and carrying out a curing crosslinking reaction; and S4, removing the regulator and the condensing agent to obtain the gel. By adjusting the concentrations of the natural polymer material and the regulator, gels having different morphologies can be prepared; the gel undergoes a physical process to remove hydrogen bond interaction between the gel and free water, so that the content of free water attached to the surface of the gel can be regulated, thereby regulating the physical and chemical properties of the gel. The gel of the present invention has regulatable morphology and controllable elastic modulus, and has many advantages such as good lubrication effect, good biocompatibility and biodegradability, capability of being biologically active, and capabilities of stimulating cell growth and accelerating wound repair.

Description

A gel with adjustable free water content and morphology, its preparation method and application Technical Field

[0001] This invention relates to the field of gel production technology, including multiple technical fields such as medical aesthetic fillers, chromatography media, tissue engineering, joint cavity lubrication, and viscoelastic agents. Specifically, it relates to a gel with controllable free water content and morphology, its preparation method, and its application. Background Technology

[0002] Natural polymer materials, derived from plant and animal extracts, possess excellent biocompatibility and biodegradability. Currently, various natural polymer materials have been prepared into gels, microspheres, aerogels, scaffolds, and other forms. However, most gels or microspheres prepared from plant-derived natural polymers suffer from a lack of bioactivity and regenerative capacity, such as carboxymethyl cellulose gel and sodium hyaluronate gel. Furthermore, while traditional natural polymer gels or microspheres contain a large amount of bound water internally, the free water on their surface is easily lost, and the bound water locked inside also evaporates quickly. Therefore, the application of hydrogels or microspheres is limited, necessitating the development of a gel capable of locking in free water.

[0003] Meanwhile, gels prepared in the same system cannot be converted into microspheres, and microspheres cannot be converted into gel systems. For example, sodium hyaluronate microspheres proposed in Chinese patent CN118141984A prepared a gel with redox properties, but it was impossible to prepare gels with other morphologies without changing the system conditions.

[0004] Summary of the Invention

[0005] The purpose of this invention is to address the problems of easy loss of free water in gels prepared from natural polymer materials, which limits their application, and the inability to control the morphology of gels prepared in the same system. This invention provides a method for preparing gels with controllable free water content and morphology, thereby solving the above problems.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] This invention provides a method for preparing gels with controllable free water content and morphology. The method involves preparing gels with different free water contents and morphologies using the same system, and includes the following steps:

[0008] S1. Prepare solution A from natural polymer materials;

[0009] S2. Mix small molecule materials containing positively charged polar groups into solution A to obtain composite solution B;

[0010] S3. Mix the composite solution B with the regulator to obtain composite solution C, stir the reaction, and then add a condensing agent to the reaction system to carry out a curing and crosslinking reaction.

[0011] S4. After the reaction, remove the regulator and condensing agent to obtain a gel;

[0012] By adjusting the concentration of the natural polymer material and the regulator, gels with different morphologies can be prepared, including gel-like, rod-like, and microsphere-like morphologies.

[0013] The resulting gel is subjected to physical action to break the hydrogen bonds between the gel and free water, thereby controlling the content of free water adhering to the gel surface and regulating the physicochemical properties of the gel (including dynamic viscosity, elastic modulus, etc.).

[0014] Specifically, the preparation method provided by this invention does not have any particular restrictions on the mixing order of the reactants; for example, ① it can be as described above, first mixing the natural polymer material and the polar small molecule material evenly, then mixing with the regulator, and finally adding the condensing agent to carry out the curing and crosslinking reaction; ② it can also be that first mixing the natural polymer material and the regulator, then mixing with the polar small molecule material evenly, and then adding the condensing agent to carry out the curing and crosslinking reaction; ③ or first mixing the regulator and the polar small molecule material evenly, then mixing with the natural polymer material, and then adding the condensing agent to carry out the curing and crosslinking reaction; ④ it can also be that first mixing the natural polymer material, the regulator, and the polar small molecule material evenly, and finally adding the condensing agent to carry out the curing reaction.

[0015] The gels with different morphologies prepared by the method of the present invention mainly include gel-like, rod-like and microspherical gels. These gels have a strong adsorption force with free water, mainly through hydrogen bonding and surface tension to adsorb free water on the gel surface. The physicochemical properties of the gel, such as dynamic viscosity and elastic modulus, are controlled by physical action to control the content of free water adsorbed by the gel.

[0016] Furthermore, a method for preparing a gel with adjustable free water content and morphology: the concentration of natural polymeric material in solution A is 0.5–5.0 wt%; the concentration of polar small molecule material in composite solution B is 0.1–10.0 wt%; the final concentration of regulator in solution C is 0.01–5.0 wt%; and the concentration after adding the condensing agent is 0.5–3.0 wt%.

[0017] Furthermore, a method for preparing a gel with adjustable free water content and morphology: the water contact angle of the natural polymer material is 35-100°, the water contact angle of the regulator is 60-160°, and the difference between the water contact angle of the regulator and the natural polymer material is 20-120°.

[0018] Furthermore, a method for preparing a gel with adjustable free water content and morphology: the natural polymer material is selected from at least one of polysaccharide A or structural protein material;

[0019] The polysaccharide A includes sodium hyaluronate, chitosan, or agarose; the structural protein material includes silk fibroin, collagen, or wool keratin.

[0020] Furthermore, a method for preparing a gel with adjustable free water content and morphology: the regulator is selected from at least one of polyols or polysaccharide B;

[0021] The polyols include polyvinyl alcohol or polyethylene glycol; the polysaccharide B includes carboxymethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, methacrylamide hyaluronic acid, sodium alginate, chitosan or thermosensitive chitosan.

[0022] Furthermore, a method for preparing a gel with adjustable free water content and morphology: the polar small molecule material is a material containing one or more polar groups selected from -OH, -CHO, -COOH, and -RCO.

[0023] Furthermore, a method for preparing a gel with adjustable free water content and morphology: the polar small molecule material is selected from at least one of amino acids or peptides;

[0024] The amino acids include lysine, arginine, polylysine, or polyarginine; the polypeptides include collagen peptides or silk fibroin peptides.

[0025] Furthermore, a method for preparing a gel with adjustable free water content and morphology: the condensing agent is selected from one of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM), N-hydroxysuccinimide, 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 6-chloro-2,4-dimethoxy1,3,5-triazine (CDMT).

[0026] This invention also provides a gel with adjustable free water content and morphology, which is prepared by the above-described method. The dynamic viscosity of the gel is controlled within (1-2) × 10⁻⁶ by physical actions such as pressurization, filtration, or evaporation. 7mPa·s, elastic modulus in the range of 50 to 10000 Pa.

[0027] This invention also provides an application of a gel with adjustable free water content and morphology. The gel prepared by the above method has the following multiple uses:

[0028] ① As a carrier, it can be used for drug delivery, cell delivery, or enzyme delivery in the field of tissue engineering;

[0029] ② Its application as a filler in facial filling within the field of medical aesthetics;

[0030] ③ As a joint lubricant, it is injected into the joint cavity to reduce friction between damaged tissues, and its application in the field of orthopedics;

[0031] ④ When used as a microsphere morphology, it can be used to prepare magnetic microspheres and porous microspheres;

[0032] ⑤ Application as a chromatography medium.

[0033] The beneficial effects of this invention are:

[0034] (1) The preparation method provided by this invention uses the same reaction system. By adjusting the concentration ratio of natural polymer materials and regulators, the difference in hydrophilicity and hydrophobicity between the two can be changed, thereby forming different degrees of hydrogen bonds, surface tension and other forces, thus controlling the gel morphology to be gel-like. By using different hydrophilic and hydrophobic forces, gels with controllable morphology can be prepared, such as gels, rod-shaped gels and microsphere gels. When the gel is prepared into a microsphere morphology, the microspheres use polar small molecule materials to make them have strong hydrophilicity. They form a strong adsorption effect with free water through hydrogen bonding. Physical external force is used to destroy the free water binding force on the surface of the microspheres and between the microspheres, including hydrogen bonds and surface tension. When the physical external force is greater than the force between the microspheres and free water, the free water between the microspheres is preferentially removed, and then the free water on the surface of the microspheres is removed, forming a gradient release of water. In this process, the physicochemical properties of the microspheres, such as dynamic viscosity, elastic modulus and microsphere content, can be precisely controlled, which solves the problem that the physicochemical properties of microspheres prepared in the same system cannot be controlled. Meanwhile, the positively charged surface of the microspheres can adsorb proteins, thereby inducing the migration of monocytes and neutrophils. The microspheres themselves can also control the stiffness of the microspheres by regulating free water, thereby regulating the polarization of macrophages (M1, M2), achieving the purpose of cell proliferation and collagen regeneration, and possessing biological regeneration activity.

[0035] (2) The present invention uses natural polymer materials and polar small molecule materials as the main components for preparing gels, and blends them with regulators. The regulators are also safe and biodegradable polymer materials. The prepared gels can control the morphology and elastic modulus, and have many advantages such as good lubrication, good biocompatibility and biodegradability, biological activity, and can stimulate cell growth and accelerate wound repair.

[0036] (3) This invention can utilize the difference in hydrophilicity and hydrophobicity to control hydrogen bonding forces, thereby preparing gels with different morphologies, including porous gels, rod-shaped gels, and spherical gels. When preparing gels with microsphere morphology, the particle size of the microspheres can be further controlled between 1 and 1000 μm, and the elastic modulus can be adjusted between 50 and 10000 Pa. This invention can utilize physical methods such as filtration, evaporation, and pressurization to break the hydrogen bonding between the gel and free water, thereby controlling the content of free water adhering to the gel surface and regulating the physicochemical properties of the gel, such as dynamic viscosity and elastic modulus.

[0037] (4) This invention can utilize small molecule materials with positively charged polar groups to regulate the amount of charge, thereby inducing protein adhesion, attracting neutrophils and monocytes to release factors, and regulating the polarization of macrophages by regulating the elastic modulus and the amount of positive charge, thereby promoting macrophage adhesion and the release of anti-inflammatory and pro-inflammatory factors, and thus promoting the generation of type I and III collagen and elastin.

[0038] (5) The gel prepared by the present invention can be used as a carrier, such as for carrying cells, drugs, enzymes, etc., and has good prospects in the field of tissue engineering; or, the gel can also be used as a filler for facial injection filling with needles, and has good development prospects in the field of medical aesthetics; or, the gel can also be used for joint lubrication to reduce friction between damaged tissues, and has good application prospects in the field of orthopedics; or, when the gel is in the form of microspheres, it can be used to prepare magnetic microspheres and porous microspheres, and has various applications such as drug carriers, chromatography media, joint cavity lubrication, and facial filling. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 is an optical image of the microsphere gel prepared in Example 1 of the present invention;

[0041] Figure 2 is an optical image of the gel prepared in Example 2 of the present invention;

[0042] Figure 3 is an optical image of the rod-shaped gel prepared in Example 5 of the present invention;

[0043] Figure 4 shows optical images of the gels prepared in Examples 9-13 and Comparative Example 1 of the present invention;

[0044] Figure 5 shows optical images of the gels prepared in Examples 14-18 and Comparative Example 2 of the present invention;

[0045] Figure 6 shows the morphology of the microsphere gels obtained in Example 1 and Comparative Example 2;

[0046] Figure 7 shows the staining optical images and water content change graphs of the microsphere gels obtained in Example 1 and Comparative Example 2.

[0047] Figure 8 shows optical images of the microsphere gels prepared by different material mixing methods in Examples 1 and 19-21;

[0048] Figure 9 shows the fluorescence values ​​of TNF-α and IL-10;

[0049] Figure 10 shows the dissolution of aspirin-loaded microspheres in a simulated body fluid environment;

[0050] Figure 11 shows the live / dead staining images of cells cultured on the surface of the microspheres, i.e., optical images of cell-carrying fluorescence staining on microspheres;

[0051] Figure 12 shows the appearance of rabbit skin and the degradation and retention morphology of its internal gel microspheres 7 days after subcutaneous injection. Detailed Implementation

[0052] 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. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0053] Example 1

[0054] This embodiment 1 provides a method for preparing gels with controllable free water content and morphology. The method involves preparing gels with different free water contents and morphologies using the same system, and includes the following steps:

[0055] S1. Prepare a 3.0 wt% solution A from natural polymer material (sodium hyaluronate) using water;

[0056] S2. Mix a small molecule material containing a positively charged polar group (arginine is selected) into the above solution A, dissolve it completely, and make its concentration 1.0 wt% to obtain composite solution B;

[0057] S3. Mix the above composite solution B with a regulator (carboxymethyl cellulose) to a final concentration of 4.8 wt% to obtain composite solution C. Then, stir at 25°C at a speed of 50 rpm until uniform. Add a condensing agent (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM)) to the reaction system to a final concentration of 2.0 wt%. Continue stirring at 50 rpm for 2 days to cure and crosslink the reaction.

[0058] S4. After the reaction, the regulator and condensing agent were removed by dialysis with pure water to obtain microsphere-shaped gel. The optical image of the obtained microsphere-shaped gel is shown in Figure 1.

[0059] In this process, the hydrogen bonds between the microsphere-shaped gel and free water can be broken through physical means, thereby controlling the content of free water adhering to the gel surface and regulating the physicochemical properties of the gel.

[0060] In Example 1 above, the content of free water adhering to the gel surface was adjusted by different physical actions (such as pressurization, filtration, or evaporation). The physicochemical properties (including dynamic viscosity and elastic modulus) of the microsphere gel were then tested, and the results are shown in Tables 1 and 2 below. In order to break the adsorption between the microsphere gel and free water, it is necessary to use external physical forces to break the hydrogen bonding forces between the microspheres and free water, or to volatilize the free water on the surface of the microspheres. The specific process is as follows:

[0061] (1) Vacuum pump was used to filter the microsphere gel by 10%, 30%, 50%, 70% and 80% water respectively. Then, the dynamic viscosity and elastic modulus were detected by HAKKE MARS III rheometer. The results are shown in Table 1 and Table 2 respectively.

[0062] (2) The microsphere gel was pressurized by an exhaust pump and the microspheres were blocked by a 300-mesh filter. 10%, 30%, 50%, 70% and 80% of the water were filtered out. Then the dynamic viscosity and elastic modulus were detected by a HAKKE MARS III rheometer. The results are shown in Table 1 and Table 2, respectively.

[0063] (3) The free water on the surface of the microsphere gel was evaporated by a vacuum drying oven. 10%, 30%, 50%, 70% and 80% of the water were evaporated. Then the dynamic viscosity and elastic modulus were detected by a HAKKE MARS III rheometer. The results are shown in Table 1 and Table 2, respectively.

[0064] Table 1 shows the dynamic viscosity (mPa·s) of the microsphere gel after different physical methods were used to adjust the free water content attached to the gel surface.

[0065] Table 2 shows the results of elastic modulus (Pa) of the microsphere gel after different physical methods were used to adjust the free water content attached to the gel surface.

[0066] As can be seen from the test results in Tables 1 and 2 above, free water on the gel surface can be removed by methods such as filtration, pressure, and evaporation (vacuum drying). This proves that the gel of the present invention has excellent water retention. At the same time, by controlling the content of free water, the dynamic viscosity, elastic modulus, and other physical properties of the microsphere gel can be further adjusted. The dynamic viscosity is controlled within (1~2)×10⁻⁶. 7 The elastic modulus was controlled between 50 and 10000 Pa; the average particle size of the microsphere gel obtained in Example 1 was 40 to 400 μm.

[0067] Example 2

[0068] This embodiment 2 provides a method for preparing gels with adjustable free water content and morphology. The method involves preparing gels with different free water contents and morphologies using the same system, and includes the following steps:

[0069] S1. Prepare a 3.0 wt% solution A from natural polymer material (sodium hyaluronate) using water;

[0070] S2. Mix a small molecule material containing a positively charged polar group (arginine is selected) into the above solution A, dissolve it completely, and make its concentration 1.0 wt% to obtain composite solution B;

[0071] S3. Mix the above composite solution B with a regulator (carboxymethyl cellulose) to a final concentration of 0.05 wt% to obtain composite solution C. Then, stir at 50 rpm at 25°C until homogeneous. Add a condensing agent (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM)) to the reaction system to a final concentration of 2.0 wt%. Continue stirring at 50 rpm for 2 days to cure and crosslink the reaction.

[0072] S4. After the reaction, the regulator and condensing agent were removed by dialysis with pure water to obtain a gel-like gel. The optical image of the obtained microsphere gel is shown in Figure 2.

[0073] In this process, the hydrogen bonds between the gel and free water are broken through physical means, thereby controlling the content of free water adhering to the gel surface and regulating the physicochemical properties of the gel.

[0074] The difference between Example 2 and Example 1 is that the final concentration of the regulator (carboxymethyl cellulose) in Example 2 is 0.05 wt%, which is different from that in Example 1, and the gel morphology obtained in Example 2 is different from that in Example 1.

[0075] Example 3

[0076] This embodiment 3 provides a method for preparing gels with controllable free water content and morphology. The method involves preparing gels with different free water contents and morphologies using the same system, and includes the following steps:

[0077] S1. Prepare a 3.0 wt% solution A from natural polymer material (sodium hyaluronate) using water;

[0078] S2. Mix a small molecule material containing a positively charged polar group (arginine is selected) into the above solution A, dissolve it completely, and make its concentration 1.0 wt% to obtain composite solution B;

[0079] S3. Mix the above composite solution B with a regulator (carboxymethyl cellulose) to a final concentration of 1.0 wt% to obtain composite solution C. Then, stir at 25°C at a speed of 50 rpm until uniform. Add a condensing agent (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM)) to the reaction system to a final concentration of 2.0 wt%. Continue stirring at 50 rpm for 2 days to cure and crosslink the reaction.

[0080] S4. After the reaction, the regulator and condensing agent are removed by dialysis with pure water to obtain a gel-like gel.

[0081] In this process, the hydrogen bonds between the gel and free water are broken through physical means, thereby controlling the content of free water adhering to the gel surface and regulating the physicochemical properties of the gel.

[0082] The difference between Example 3 and Example 2 is that the final concentration of the regulator (carboxymethyl cellulose) in Example 3 is 1.0 wt%, which is different from that in Example 2, but the gel morphology obtained in Example 3 is the same as that in Example 2.

[0083] Example 4

[0084] Example 4 provides a method for preparing gels with adjustable free water content and morphology. This method involves preparing gels with different free water contents and morphologies using the same system, and includes the following steps:

[0085] S1. Prepare a 3.0 wt% solution A from natural polymer material (sodium hyaluronate) using water;

[0086] S2. Mix a small molecule material containing a positively charged polar group (arginine is selected) into the above solution A, dissolve it completely, and make its concentration 1.0 wt% to obtain composite solution B;

[0087] S3. Mix the above composite solution B with a regulator (carboxymethyl cellulose) to a final concentration of 2.0 wt% to obtain composite solution C. Then, stir at 50 rpm at 25°C until homogeneous. Add a condensing agent (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM)) to the reaction system to a final concentration of 2.0 wt%. Continue stirring at 50 rpm for 2 days to cure and crosslink the reaction.

[0088] S4. After the reaction, the regulator and condensing agent are removed by dialysis with pure water to obtain a rod-shaped gel.

[0089] In this process, the hydrogen bonds between the aforementioned rod-shaped gel and free water are broken through physical means, thereby controlling the content of free water adhering to the gel surface and regulating the physicochemical properties of the gel.

[0090] The difference between Example 4 and Example 3 is that the final concentration of the regulator (carboxymethyl cellulose) in Example 4 is 2.0 wt%, which is different from that in Example 3, and the gel morphology obtained in Example 4 is different from that in Example 3, with the gel obtained in Example 4 being a rod-shaped gel.

[0091] Example 5

[0092] The difference between Example 5 and Example 4 is that the final concentration of the regulator (carboxymethyl cellulose) in Example 5 is 3.0 wt%, which is different from that in Example 4. However, the gel morphology obtained in Example 5 is the same as that in Example 4. The gel obtained in Example 5 is also a rod-shaped gel, and the optical image of the obtained rod-shaped gel is shown in Figure 3.

[0093] Example 6

[0094] The difference between Example 6 and Example 5 is that the final concentration of the regulator (carboxymethyl cellulose) in Example 6 is 3.5 wt%, which is different from that in Example 5, and the gel morphology obtained in Example 6 is different from that in Example 5, with the gel being a microsphere gel.

[0095] Example 7

[0096] The difference between Example 7 and Example 6 is that the final concentration of the regulator (carboxymethyl cellulose) in Example 7 is 10.0 wt%, which is different from that in Example 6. However, the gel morphology obtained in Example 7 is the same as that in Example 6, and the gel obtained in Example 7 is also a microsphere gel.

[0097] Example 8

[0098] The difference between Example 8 and Example 7 is that the final concentration of the regulator (carboxymethyl cellulose) in Example 7 is 12.0 wt%, which is different from Example 7. Also, the gel morphology obtained in Example 8 is different from that in Example 7. In Example 8, the solution is more viscous and will form a gel.

[0099] Examples 2-8 above illustrate that gels with different morphologies can be obtained by changing the concentration of the regulator in the same reaction system. The specific effect of the regulator concentration on the gel morphology can be seen in Table 3 below:

[0100] Table 3 shows the effect of different regulator concentrations on gel morphology.

[0101] Combining the results in Figures 1-3 and Table 3, it can be seen that, without changing the regulator, the difference in hydrophilicity and hydrophobicity between the natural polymer material and the regulator can be altered by adjusting the concentration of the regulator, thereby forming different degrees of hydrogen bonds, surface tension, and other forces. This allows the morphology of the gel to be controlled as gel-like, rod-like, or microsphere-like. If the concentration of the regulator is too high when the two solutions are mixed (Example 8), the overall solution will be too viscous, resulting in a gel-like morphology. If no regulator is added (i.e., the regulator concentration is 0), it will also be gel-like. Therefore, both too high and too low regulator content will result in a gel-like morphology.

[0102] Example 9

[0103] This embodiment 9 provides a method for preparing gels with controllable free water content and morphology. The method involves preparing gels with different free water contents and morphologies using the same system, and includes the following steps:

[0104] S1. Prepare a 3.0 wt% solution A from natural polymer material (chitosan) using water;

[0105] S2. Mix a small molecule material containing a positively charged polar group (arginine is selected) into the above solution A, dissolve it completely, and make its concentration 1.0 wt% to obtain composite solution B;

[0106] S3. Mix the above composite solution B with a regulator (carboxymethyl cellulose) to a final concentration of 4.8 wt% to obtain composite solution C. Then, stir at 25°C at a speed of 50 rpm until uniform. Add a condensing agent (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM)) to the reaction system to a final concentration of 2.0 wt%. Continue stirring at 50 rpm for 2 days to cure and crosslink the reaction.

[0107] S4. After the reaction, the regulator and condensing agent were removed by dialysis with pure water to obtain microsphere-shaped gel. The optical image of the obtained microsphere-shaped gel is shown in Figure 4a.

[0108] The aforementioned microsphere-shaped gel can be physically broken by terminating the hydrogen bonds between the gel and free water, thereby controlling the content of free water adhering to the gel surface and regulating the physicochemical properties of the gel.

[0109] The difference between Example 9 and Example 1 is that the selection of natural polymer materials in Example 9 is different from that in Example 1, but the rest are the same.

[0110] Example 10

[0111] The difference between Example 10 and Example 1 is that the natural polymer material used in Example 10 is agarose, which is different from Example 1. Otherwise, it is the same as Example 1. The optical image of the microsphere gel obtained in Example 10 is shown in Figure 4b.

[0112] Example 11

[0113] The difference between Example 11 and Example 1 is that the natural polymer material used in Example 11 is silk fibroin, which is different from Example 1. Otherwise, it is the same as Example 1. The optical image of the microsphere gel obtained in Example 11 is shown in Figure 4c.

[0114] Example 12

[0115] The difference between Example 12 and Example 1 is that the natural polymer material used in Example 12 is collagen, which is different from Example 1. Otherwise, it is the same as Example 1. The optical image of the microsphere gel obtained in Example 12 is shown in Figure 4d.

[0116] Example 13

[0117] The difference between Example 13 and Example 1 is that the natural polymer material used in Example 13 is wool keratin, which is different from Example 1. The rest is the same as Example 1. The optical image of the microsphere gel obtained in Example 13 is shown in Figure 4e.

[0118] Comparative Example 1

[0119] The difference between Comparative Example 1 and Example 1 is that the natural polymer material used in Comparative Example 1 is lysine, which is different from Example 1. The rest is the same as Example 1. Comparative Example 1 cannot form microsphere gels. The optical image of the gel obtained is shown in Figure 4f.

[0120] As can be seen from the results in Figure 4, similar microsphere gels can be prepared by selecting different natural polymer materials in Examples 9 to 13 above, indicating that these natural polymer materials have certain commonalities. However, Comparative Example 1 could not form microspheres, indicating that the reason for the different morphologies formed between the regulator and the natural polymer material is due to hydrogen bonding and surface tension. These interactions are usually related to hydrophilicity and hydrophobicity. Therefore, it is speculated that the hydrophilicity and hydrophobicity between the natural polymer material selected in this invention and the regulator are related. In contrast, the natural polymer material (lysine) selected in Comparative Example 1 cannot form hydrogen bonds or surface tension interactions with the regulator, so it cannot form microsphere gels. This invention can achieve the preparation of gels with different morphologies by using the selected natural polymer material to form hydrogen bonds, surface tension, and other interactions with the regulator.

[0121] Example 14

[0122] This embodiment 14 provides a method for preparing gels with controllable free water content and morphology. The method involves preparing gels with different free water contents and morphologies using the same system, and includes the following steps:

[0123] S1. Prepare a 3.0 wt% solution A from natural polymer material (sodium hyaluronate) using water;

[0124] S2. Mix a small molecule material containing a positively charged polar group (lysine is selected) into the above solution A, dissolve it completely to make its concentration 1.0 wt%, and obtain composite solution B;

[0125] S3. Mix the above composite solution B with a regulator (carboxymethyl cellulose) to a final concentration of 4.8 wt% to obtain composite solution C. Then, stir at 25°C at a speed of 50 rpm until uniform. Add a condensing agent (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM)) to the reaction system to a final concentration of 2.0 wt%. Continue stirring at 50 rpm for 2 days to cure and crosslink the reaction.

[0126] S4. After the reaction, the regulator and condensing agent were removed by dialysis with pure water to obtain microsphere-shaped gel. The optical image of the obtained microsphere-shaped gel is shown in Figure 5a.

[0127] The aforementioned microsphere-shaped gel can be physically broken by terminating the hydrogen bonds between the gel and free water, thereby controlling the content of free water adhering to the gel surface and regulating the physicochemical properties of the gel.

[0128] The difference between Example 14 and Example 1 is that the selection of polar small molecule materials in Example 14 is different from that in Example 1, but the rest are the same.

[0129] Example 15

[0130] The difference between Example 15 and Example 1 is that the polar small molecule material used in Example 15 is polylysine, which is different from that in Example 1. The rest is the same as in Example 1. The optical image of the microsphere gel obtained in Example 15 is shown in Figure 5b.

[0131] Example 16

[0132] The difference between Example 16 and Example 1 is that the polar small molecule material used in Example 16 is polyarginine, which is different from that in Example 1. The rest is the same as in Example 1. The optical image of the microsphere gel obtained in Example 16 is shown in Figure 5c.

[0133] Example 17

[0134] The difference between Example 17 and Example 1 is that the polar small molecule material used in Example 17 is collagen peptide, which is different from that in Example 1. The rest is the same as in Example 1. The optical image of the microsphere gel obtained in Example 17 is shown in d in Figure 5.

[0135] Example 18

[0136] The difference between Example 18 and Example 1 is that the polar small molecule material used in Example 18 is silk fibroin peptide, which is different from that in Example 1. The rest is the same as in Example 1. The optical image of the microsphere gel obtained in Example 18 is shown in Figure 5e.

[0137] Comparative Example 2

[0138] The difference between Comparative Example 2 and Example 1 is that no polar small molecule material is added in Comparative Example 2, while the rest is the same as in Example 1. Microspherical gels are also present in Comparative Example 2, and its optical image is shown in Figure 5f.

[0139] As can be seen from the optical images in Figure 5, microsphere-shaped gels can be prepared from small molecule materials with different polar groups. However, microspheres also exist in Comparative Example 2, which further illustrates the relationship between hydrophilicity and hydrophobicity in forming microsphere-shaped gels. The small molecule materials with polar groups all contain one or more polar groups such as -OH, -CHO, -COOH, and -RCO. These groups can significantly increase the hydrogen bonding interaction with free water, making their adsorption stronger.

[0140] test:

[0141] To further analyze the properties and mechanism of action of the microsphere gel in Example 1, microspheres prepared in Example 1 and Comparative Example 2 were selected for analysis:

[0142] (1) The microspheres prepared in Example 1 and Comparative Example 2 were freeze-dried respectively. Then, a portion of the microspheres were placed on conductive adhesive and sputtered with gold for 40 seconds. The morphological characteristics were then observed using a Hitachi TM4800 scanning electron microscope. The results are shown in Figure 6 (in Figure 6, a is the morphology of the microspheres in Example 1 under a wide field of view, b is the morphology of a single microsphere in Example 1, c is the cross-sectional morphology of the microspheres in Example 1, and d is the morphology of the microspheres in Comparative Example 2). As can be seen from Figure 6, the surface of the microsphere gel prepared in this invention has barbs-like results, which indicates that a large amount of free water is lost, resulting in so many gaps. In contrast, the surface of the microspheres in Comparative Example 2 is smooth and basically has no pores, indicating that its ability to bind free water is poor. This proves that the microspheres prepared in this invention have a stronger ability to bind free water than microspheres prepared by conventional methods.

[0143] (2) The microspheres prepared in Example 1 and Comparative Example 2 were stained with 0.1 wt% methylene blue solution and their hydrophilicity differences were observed. The results are shown in Figure 7 (Figure 7a is an optical image of the microspheres of Example 1 after staining, and b is an optical image of the microspheres of Comparative Example 2 after staining). It can be seen from Figure 7 that the two microspheres stained with methylene blue are in a whole state in Example 1, indicating that they are tightly bound to free water and have strong integrity, while the microspheres of Comparative Example 2 are very dispersed, basically treating water as a solution and the microspheres as a solvent, so their integrity is poor.

[0144] (3) The microspheres prepared in Example 1 and Comparative Example 2 were soaked in pure water, and then most of the water was filtered off with silk. The water-containing gel was collected and then filtered using a vacuum filter (50 μm filter membrane, aqueous phase) to verify the difference in their hydrophilicity. The results are shown in Figure 7c. From the degree of free water in the two samples, it can be seen that the water content of the microsphere gel in Comparative Example 2 was about 50% lost in about 2 seconds, while there was almost no loss in Example 1. This significant difference also shows that the microsphere gel prepared in this invention is more firmly and tightly bound to free water.

[0145] In summary, the methods of microscopic morphology, staining observation, and water loss detection all demonstrate that the gel prepared by the method of this invention has a better free water binding capacity compared to other gels or microspheres. Free water is more likely to form hydrogen bonds with polar groups, so small molecule materials with polar groups provide super hydrophilicity.

[0146] Example 19

[0147] The difference between Example 19 and Example 1 is that the mixing order of the reactants in Example 19 is different from that in Example 1, while the rest is the same as in Example 1. The mixing method of the materials in Example 19 is as follows: first, the natural polymer material and the regulator are mixed, then the polar small molecule material is mixed evenly, and then a condensing agent is added to carry out a curing and crosslinking reaction. After the reaction, the regulator and the condensing agent are removed by dialysis with pure water, thereby obtaining a microsphere gel. The optical image of the obtained microsphere gel is shown in Figure 8b, and Figure 8a is the optical image of the microsphere gel prepared by the method of Example 1.

[0148] Example 20

[0149] The difference between Example 20 and Example 1 is that the mixing order of the reactants in Example 20 is different from that in Example 1, while the rest is the same as in Example 1. The mixing method of the materials in Example 20 is as follows: first, the regulator and the polar small molecule material are mixed evenly, then mixed with the natural polymer material, and then a condensing agent is added to carry out a curing and cross-linking reaction. After the reaction, the regulator and the condensing agent are removed by dialysis with pure water, thereby obtaining a microsphere gel. The optical image of the obtained microsphere gel is shown in Figure 8c.

[0150] Example 21

[0151] The difference between Example 21 and Example 1 is that the mixing order of the reactants in Example 21 is different from that in Example 1, while the rest is the same as in Example 1. The mixing method of the materials in Example 21 is as follows: first, the natural polymer material, regulator, and polar small molecule material are mixed evenly, and finally, a condensing agent is added to carry out a curing reaction. After the reaction, the regulator and condensing agent are removed by dialysis with pure water to obtain microsphere-shaped gel. The optical image of the obtained microsphere-shaped gel is shown in Figure 8d.

[0152] Based on Examples 1 and 19-21, all four mixing methods can produce ideal microsphere gels.

[0153] Example 22

[0154] The difference between Example 22 and Example 1 is that the stirring rate in Example 22 is 100 rpm, while the rest is the same as in Example 1. The average particle size of the microsphere gel obtained in Example 22 is 40-230 μm, while the average particle size of the microsphere gel obtained in Example 1 is 40-400 μm.

[0155] Example 23

[0156] The difference between Example 23 and Example 1 is that the stirring rate in Example 23 is 500 rpm, while the rest is the same as in Example 1. The average particle size of the microsphere gel obtained in Example 23 is 20-90 μm.

[0157] Example 24

[0158] The difference between Example 24 and Example 1 is that the stirring rate in Example 24 is 5000 rpm, while the rest is the same as in Example 1. The average particle size of the microsphere gel obtained in Example 24 is 5-25 μm.

[0159] Example 25

[0160] The difference between Example 25 and Example 1 is that the stirring rate in Example 25 is 10,000 rpm, while the rest is the same as in Example 1. The average particle size of the microsphere gel obtained in Example 25 is 1 to 10 μm.

[0161] Comparative Example 3

[0162] The difference between Comparative Example 3 and Example 1 is that the stirring rate in Comparative Example 3 is 20 rpm, while the rest is the same as in Example 1. It is difficult to obtain microsphere gels in Comparative Example 3.

[0163] Based on Examples 1, 22-25, and Comparative Example 3, when the stirring speed is 20 rpm, due to the slow movement of the natural polymer material, the amplitude and frequency of the gel sphere movement are too small, making it easy for the spheres to collide and aggregate. The incompletely cross-linked gel sphere aggregates gradually cross-link to form blocky gels, rather than cross-linked gel spheres. However, in Examples 1 and 22-25, gel spheres can be formed at stirring speeds of 50 rpm, 100 rpm, 500 rpm, 5000 rpm, and 10000 rpm, and the microsphere particle size can be controlled within the range of 1.0–1000.0 μm.

[0164] Example 26

[0165] This embodiment 26 provides a method for preparing a gel with adjustable free water content and morphology, which includes the following steps:

[0166] S1. Prepare a 3.0 wt% solution A from natural polymer material (sodium hyaluronate) using water;

[0167] S2. Mix a small molecule material containing positively charged polar groups (polylysine) into the above solution A, dissolve it completely to a concentration of 0.1 wt%, and obtain composite solution B;

[0168] S3. Mix the above composite solution B with a regulator (carboxymethyl cellulose) to a final concentration of 4.8 wt% to obtain composite solution C. Then, stir at 25°C at a speed of 50 rpm until uniform. Add a condensing agent (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM)) to the reaction system to a final concentration of 2.0 wt%. Continue stirring at 50 rpm for 2 days to cure and crosslink the reaction.

[0169] S4. After the reaction, the regulator and condensing agent are removed by dialysis with pure water to obtain a microsphere-shaped gel.

[0170] The difference between Example 26 and Example 1 is that the selection and concentration of the polar small molecule material in Example 26 are different from those in Example 1, while the rest are the same.

[0171] Example 27

[0172] The difference between Example 27 and Example 26 is that the concentration of the polar small molecule material in Example 27 is different from that in Example 26, while the rest are the same. The concentration of the polar small molecule material (polylysine) in Example 27 is 2.0 wt%.

[0173] Example 28

[0174] The difference between Example 28 and Example 26 is that the concentration of the polar small molecule material in Example 28 is different from that in Example 26, while the rest are the same. The concentration of the polar small molecule material (polylysine) in Example 28 is 5.0 wt%.

[0175] Example 29

[0176] The difference between Example 29 and Example 26 is that the concentration of the polar small molecule material in Example 29 is different from that in Example 26, while the rest are the same. The concentration of the polar small molecule material (polylysine) in Example 29 is 6.0 wt%.

[0177] In Examples 26-29, carboxymethyl cellulose was selected as a regulator, sodium hyaluronate as a natural polymer material, and polylysine as a polar small molecule material. The final concentrations of polylysine were adjusted to 0.1 wt%, 2.0 wt%, 5.0 wt%, and 6.0 wt%, respectively. Polylysine is composed of approximately 25-30 lysine molecules. Each lysine molecule contains two amino groups, and the amino groups on the main chain are more active and therefore react more easily. The side chain will expose one amino group. By adding different polylysines in this way, microspheres with different charges can be obtained, thereby achieving the control of positive charge.

[0178] The number of positive charges in the microsphere gels prepared with different polylysine contents in Examples 26-29 is shown in Table 4 below;

[0179] Table 4 shows the number of positive charges in microsphere gels prepared with different polylysine contents.

[0180] test:

[0181] (1) Regulation of gel immunomodulatory activity: The microsphere gel prepared by the method in Example 1 was used, and 10% and 50% water were removed by vacuum filtration to obtain microspheres with two different elastic moduli. Two types of microspheres obtained in Examples 26 and 27 were also used. These four types of microspheres were co-cultured with raw264.7 macrophages. After 24 hours of culture, the cells and microspheres were centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded, and the fluorescence results in the supernatant were detected using a TNF-α kit and an IL-10 kit. The control group was tested with LPS (final concentration 1.0 μg / ml). The experimental results... As shown in Figure 9 (where a is a microsphere with 10% free water filtered, b is a microsphere with 50% free water filtered, c is a microsphere from Example 26 (charge 0.018 mmol / ml), and d is a microsphere from Example 27 (charge 10.288 mmol / ml), it can be seen from Figure 9 that by controlling microspheres with different moduli and charge amounts, the transformation of macrophages into M1 and M2 types can be regulated. By adjusting the elastic modulus, the number of M1 macrophages increases, but ultimately they are M2 type. The higher the charge, the more proteins adsorbed on the surface of the microspheres, the more macrophages, and ultimately the expression of M2 is significantly increased.

[0182] (2) Promoting the production of type I and type III collagen: Thirty Wistar rats were randomly assigned to various experimental groups, namely, a sham control group, experimental group 1 (microspheres prepared according to Example 1 with 10% water content filtered out), experimental group 2 (microspheres prepared according to Example 1 with 50% water content filtered out), experimental group 3 (charged microspheres with a charge of 0.018 mmol / ml obtained in Example 26), experimental group 4 (charged microspheres with a charge of 0.018 mmol / ml obtained in Example 27), and an aging control group of 5 rats in each group. Except for the sham control group (no treatment group), Wistar rats in each experimental group were subcutaneously injected with D-galactose (D-gal) at a dose of 125 mg / kg·d for 6 consecutive weeks to induce a rat skin aging model. On the 18th day after the D-galactose (D-gal) injection, each rat was anesthetized with chloral hydrate (0.25 ml per 100 g body weight), and its buttock area was disinfected. A 3cm circular tattoo area was pre-made on both sides of the rat's buttocks. Then, under general anesthesia, the rats were treated with 0.2ml of microspheres injected weekly for 4 weeks. The experimental groups 1-4 were treated with microspheres injected with 0.2ml each (the aging control group and the sham control group underwent the same surgical procedure but did not receive any of the microsphere injection treatments described in this application). Forty-two days after treatment, all animals were euthanized, and the expression levels of skin wounds and type I collagen, type III collagen, and MMP-1 were compared by histological and quantitative real-time PCR tests, as shown in Table 5 below.

[0183] Table 5 shows the promoting effects of different treatments on the expression levels of different factors.

[0184] The results showed that, compared with the sham control group, aging skin exhibited reduced thickness in both the epidermis and dermis. Furthermore, the number of cell layers in the aging epidermis decreased, and dermal collagen fibers became sparse, elongated, or broken. However, compared with the aging control group, these histological changes were significantly improved after injection of microspheres 1–4 in the experimental groups, with significantly higher epidermal and dermal thickness and collagen fiber density.

[0185] Furthermore, compared to the sham control rats, the expression levels of type I collagen in any other aging group were significantly lower. When filled with microspheres from experimental groups 1–4, the transcriptional level of type III collagen was significantly higher than that in the aging control group, but similar to that in the sham control group. As a potential key regulator of skin aging, MMP-1 information decreased in response to 0.25 ml or 0.5 ml of regenerating silk fibroin solution from mesotherapy, showing a statistically significant difference compared to the aging control group, but no significant difference compared to the sham control rats.

[0186] use:

[0187] (1) Application as a joint lubricant in the field of orthopedics: The microspheres, gels and rod-shaped gel products prepared in Examples 1, 2 and 5 above were used as test samples. The friction coefficient of the samples was detected by atomic force microscopy (AFM). The gels of Examples 1, 2 and 5 were dissolved in PBS to prepare 10.0 mg / ml lubricating solutions. Sodium hyaluronate solution of the same concentration and hydroxyapatite microsphere lubricating solution of the same particle size and concentration (without free water on the surface) were used as control group 1. In AFM operation, a rectangular cantilever beam probe with 10.0 μm polystyrene microspheres was used. A polished silicon wafer, fixed to a glass slide, was fixed on an operating stage equipped with a liquid tank. The contact mode was selected to test the friction force. After calibrating the probe using the thermal calibration method in atmospheric mode, the solution to be measured was injected into the liquid tank. Using the probe under thermally calibrated liquid phase, a set load was applied to the probe in the transverse force mode to conduct the friction test. The friction coefficient was calculated by the ratio of transverse force to load. The results are shown in Table 6 below.

[0188] Table 6 shows the coefficients of friction for lubricants of different concentrations and compositions.

[0189] As can be seen from Table 6, the gels with different morphologies prepared by this invention all have the effect of reducing friction, and their coefficients of friction are all less than 0.02, which have a good lubrication effect. After intra-articular injection, they can reduce the friction between damaged tissues and have good applications in the field of orthopedics. In contrast, the coefficient of friction of the control group is larger, which is related to the adsorption of free water by this invention.

[0190] (2) As a viscoelastic material, it can be compounded with other components for use in intraocular filling: The intraocular filling components include: viscoelastic material (microsphere gel prepared in Example 1) 1.8wt%, boric acid 0.75wt%, borax 0.05wt%, sodium chloride 0.4wt%, dexamethasone 0.1wt%, povidone-iodine 0.1wt%, and the balance being physiological saline.

[0191] (3) As a carrier for drug loading: The sodium hyaluronate microsphere gel prepared in Example 1 above can be used as aspirin-loaded microspheres. The absorbance of the gel microspheres is determined by the ultraviolet absorption intensity at 303±2nm, and the concentration of aspirin is calculated according to the standard curve. The drug loading and encapsulation efficiency are calculated according to formulas (I) and (II), respectively: Drug loading efficiency (%) = (Drug loading / Total mass of microgel) * 100% (I) Encapsulation efficiency (%) = (Drug loading / Drug input) * 100% (II)

[0192] The dissolution of drugs in drug-loaded microsphere gels was characterized by ultraviolet absorption spectroscopy. Aspirin-loaded microspheres were resuspended in 10.0 ml of PBS buffer at a concentration of 10.0 mg / ml to obtain a suspension (as the experimental group). The suspension was then transferred to a 15.0 ml centrifuge tube, and a control group was set up (specifically, aspirin powder with the same total drug loading as the microspheres was directly dissolved in PBS). The control group and the experimental group were placed in a constant temperature shaker at 37℃ and 100 rpm for reaction. At 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 10 h after the start of the reaction, 2.0 ml of the medium was taken out and the same volume of PBS was added. The absorbance was characterized at 303±2 nm using an ultraviolet spectrophotometer to determine the absorbance. Then, the concentration of aspirin was calculated according to the standard curve. The results are shown in Figure 10. As can be seen from Figure 10, the sodium hyaluronate gel microsphere material prepared in Example 1 of this invention can effectively encapsulate the model drug aspirin and release it stably in a simulated body fluid environment. The drug-loaded microspheres did not exhibit obvious initial burst release phenomenon. This is attributed to the differentiated water forms present in the microspheres. The drug on the surface of the microspheres first dissolves in free water and diffuses to the external environment with the free water due to external friction and other effects. Subsequently, the drug inside dissolves into the external environment along with bound water as the microsphere matrix degrades. Through the combined action of free water and bound water, graded controlled release of the drug can be achieved, thereby avoiding the occurrence of initial burst release effect.

[0193] (4) As a carrier for cell loading: The microsphere gel prepared in Example 1 was spread evenly in a laminar flow hood and sterilized for 4 hours with the built-in UV lamp. After sterilization, it was covered with aluminum foil and placed in the laminar flow hood for later use. 0.3-3.0 ml of agarose solution (which was sterilized by autoclaving at 121°C for 30 minutes, with a concentration of 1.0-2.0 wt% and a temperature of 50-80°C) was added to wells with a diameter of 10-50 mm and a height of 0.5-3.0 mm. After cooling, it solidified at the bottom of the wells. 0.5-2.0 ml of the sterilized gel microspheres was transferred into the wells, and then mouse fibroblast L929 cells were loaded at a rate of 3 × 10⁻⁶ cells per well. 4 ~3×10 6 The cells were seeded into the wells at a density of 100:10:1. At the same time, 1.0-3.0 ml of high glucose DMEM complete medium (prepared by mixing high glucose DMEM, fetal bovine serum, and penicillin-streptomycin at a volume ratio of 100:10:1) was added to each well of the plate. The wells were then placed in a 37°C, 5% cell culture incubator for incubation.

[0194] After cells were encapsulated and cultured in the microspheres for 7 days, the cell viability inside the hydrogel was assessed by live / dead staining. After the culture medium was aspirated, 50–1000 μL of Calcein-AM staining working solution and PI staining working solution were added to each well. The cells were incubated in a cell culture incubator in the dark for 1 hour. The live / dead cell images were obtained by observing the cells under a fluorescence microscope at excitation wavelengths of 488 nm (green light) and 561 nm (red light), as shown in Figure 11. As can be seen from Figure 11, after 7 days of in vitro culture, the cells on the surface of the sodium hyaluronate microsphere gel still maintained high viability. Statistical results showed that the survival rate of cells on the surface of all microspheres exceeded 80%, and the adhesion morphology was good with no obvious aggregation. This proves that the microsphere gel material prepared in this invention has good cell affinity and biocompatibility. In addition, the free water component outside the gel microspheres provides a good micro-aqueous environment for cell growth, which facilitates the transport of cell nutrients and signal transduction. The above positive factors combined led to the high survival rate of cells on the surface of all sodium hyaluronate gel microspheres.

[0195] (5) Application as a chromatography medium: ① Wash 100.0g of the sodium hyaluronate microsphere gel prepared in Example 1 with deionized water, then add it to a 250ml reaction flask, add 30.0g of sodium hydroxide solution (10.0mol / l), stir and mix well, then add 0.2g of sodium borohydride, stir for 10 minutes, add 30.0g of epichlorohydrin, heat to 35℃, stir for 4 hours, filter, wash with deionized water, and obtain 100.0g of intermediate microsphere A with a large number of epoxy groups on the surface; ② Add the above 100.0g of intermediate microsphere A to a 250ml reaction flask, add 80.0g of polysaccharide. The (dextran) solution was mixed and stirred at 50°C for 1 hour. Then, 20.0 g of sodium hydroxide solution (10.0 mol / L) and 0.1 g of sodium borohydride were added, and the mixture was stirred at 50°C for 20 hours. The mixture was then filtered and washed with deionized water to obtain intermediate microspheres B modified with a large amount of polysaccharide (dextran). ③ 100.0 g of the above intermediate microspheres B was added to a 250 ml reaction flask, along with 50.0 g of deionized water. The mixture was stirred and mixed, then 90 g of sulfonate ester aglycone and 50.0 g of sodium hydroxide solution (10.0 mol / L) were added. The mixture was heated to 35°C and stirred for 5 hours. The mixture was then filtered and washed with deionized water to obtain a cation exchange medium. Its ion exchange capacity reached 74.6 meq / g, and its protein binding capacity reached 180.3 mg / ml, meeting the requirements for use as a chromatographic medium.

[0196] (6) Application as a filler in the field of medical aesthetics: The gels prepared in Examples 1, 2 and 5 were used as test products, and polycaprolactone microspheres were used as the control group; the microspheres were filled into 1.0ml BD tubes and sterilized by moist heat; four rabbits (weighing 4-6kg, half male and half female) were prepared for skin test experiments. The hair on the surface of the rabbits was removed and the epidermis was wiped with 75% alcohol. Each batch of microspheres was injected into the superficial dermis of the rabbits (0.2ml / point, n=3), so that it macroscopically presents a hemispherical protrusion with a height of about 0.3-0.7mm and a diameter of about 0.5-1.5mm. Then the rabbits were put back into the rabbit cage and allowed to move freely. Seven days after the injection, the experimental rabbits were quickly euthanized by intravenous air injection. Then the epidermis of the injection area was cut open with a knife to check the degradation of the injected part and whether there was any chemical reaction. The purulent phenomenon was observed, and the results are shown in Figure 12. As can be seen from Figure 12, the gels of Examples 1, 2, and 5 showed no obvious inflammatory reaction after 7 days of injection into rabbits. The inflammatory reaction was initially severe from day 1 to 3, and basically subsided from day 3 to 7. As shown in the control group, if inflammation occurred, the tissue on day 7 would exhibit severe redness, swelling, yellowing, and purulent discharge. However, the three morphologies of gels prepared in Examples 1, 2, and 5 of this invention showed no obvious redness, swelling, or lumps on the skin surface after injection, and the gel itself did not exhibit yellowing or purulent discharge, indicating excellent biocompatibility. Furthermore, the gels exhibited significant swelling and support. The good biocompatibility and slow degradation rate suggest that this gel system has the potential to be used as a tissue filler material, suitable for facial filling and application in the medical aesthetics field.

[0197] The above-described preferred embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of the invention. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a gel with controllable free water content and morphology, characterized in that, This method is a way to prepare gels with different free water contents and different morphologies using the same system, and it includes the following steps: S1. Prepare solution A from natural polymer materials; S2. Mix small molecule materials containing positively charged polar groups into solution A to obtain composite solution B; S3. Mix the composite solution B with the regulator to obtain composite solution C, stir the reaction, and then add a condensing agent to the reaction system to carry out a curing and crosslinking reaction. S4. After the reaction, remove the regulator and condensing agent to obtain a gel; By adjusting the concentration of the natural polymer material and the regulator, gels with different morphologies can be prepared, including gel-like, rod-like, and microsphere-like morphologies. In this process, the hydrogen bonds between the gel and free water are broken through physical means, thereby controlling the content of free water adhering to the gel surface and regulating the physicochemical properties of the gel.

2. The method for preparing a gel with adjustable free water content and morphology according to claim 1, characterized in that, The concentration of the natural polymer material in solution A is 0.5–5.0 wt%; the concentration of the polar small molecule material in the composite solution B is 0.1–10.0 wt%; the final concentration of the regulator in solution C is 0.01–5.0 wt%; and the concentration after adding the condensing agent is 0.5–3.0 wt%.

3. A method for preparing a gel with adjustable free water content and morphology according to claim 1 or 2, characterized in that, The water contact angle of the natural polymer material is 35–100°, the water contact angle of the regulator is 60–160°, and the difference between the water contact angle of the regulator and the natural polymer material is 20–120°.

4. A method for preparing a gel with adjustable free water content and morphology according to claim 1 or 3, characterized in that, The natural polymer material is selected from at least one of polysaccharide A or structural protein material; The polysaccharide A includes sodium hyaluronate, chitosan, or agarose; the structural protein material includes silk fibroin, collagen, or wool keratin.

5. A method for preparing a gel with adjustable free water content and morphology according to claim 1 or 3, characterized in that, The regulator is selected from at least one of polyols or polysaccharide B; The polyols include polyvinyl alcohol or polyethylene glycol; the polysaccharide B includes carboxymethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, methacrylamide hyaluronic acid, sodium alginate, chitosan or thermosensitive chitosan.

6. A method for preparing a gel with adjustable free water content and morphology according to claim 1 or 2, characterized in that, The polar small molecule material is a material containing one or more polar groups selected from -OH, -CHO, -COOH, and -RCO.

7. The method for preparing a gel with adjustable free water content and morphology according to claim 6, characterized in that, The polar small molecule material is selected from at least one of amino acids or peptides; The amino acids include lysine, arginine, polylysine, or polyarginine; the polypeptides include collagen peptides or silk fibroin peptides.

8. A method for preparing a gel with adjustable free water content and morphology according to claim 1 or 2, characterized in that, The condensing agent is selected from one of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, N-hydroxysuccinimide, 1,3-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 6-chloro-2,4-dimethoxy1,3,5-triazine.

9. A gel with adjustable free water content and morphology, characterized in that, The gel is prepared by the method described in any one of claims 1 to 8, and the dynamic viscosity of the gel is controlled within (1 to 2) × 10⁻⁶ by physical actions such as pressurization, filtration, or evaporation. 7 mPa·s, elastic modulus in the range of 50 to 10000 Pa.

10. An application of a gel with adjustable free water content and morphology, characterized in that, The gel prepared by the method according to any one of claims 1 to 8 has the following uses: ① As a carrier, it can be used for drug delivery, cell delivery, or enzyme delivery in the field of tissue engineering; ② Its application as a filler in facial filling within the field of medical aesthetics; ③ As a joint lubricant, it is injected into the joint cavity to reduce friction between damaged tissues, and its application in the field of orthopedics; ④ When used as a microsphere morphology, it can be used to prepare magnetic microspheres and porous microspheres; ⑤ Application as a chromatography medium.

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