Preparation method and application of cross-linked carboxymethyl cellulose

Abstract

The invention relates to the technical field of biomedical materials, and provides a preparation method and application of cross-linked carboxymethyl cellulose. The preparation method comprises the following steps: S1, dissolving anhydrous calcium chloride in absolute ethyl alcohol, sterilizing and filtering to obtain a sterile calcium chloride solution; s2, adding the dehydrated and purified sodium carboxymethyl cellulose into a sterile calcium chloride solution, and stirring for reaction; s3, after the reaction is finished, suction filtration is performed, filter residues are collected and washed, residual calcium chloride is removed, then vacuum drying is performed, and cross-linked carboxymethyl cellulose is obtained. The cross-linked carboxymethyl cellulose and sodium carboxymethyl cellulose have high consistency in chemical activity, physiological and biochemical activity, biocompatibility and safety risk, but in physical performance, the cross-linked carboxymethyl cellulose is hydrogel fiber, is insoluble in water, can more remarkably maintain the volume of a filling part, and is longer in duration time, so that the cross-linked carboxymethyl cellulose is more stable in filling. And the effects of instant filling and long-acting filling of the microsphere-containing surface filling agent are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a preparation method and application of cross-linked carboxymethyl cellulose. Background Art

[0002] Facial fillers are key injectable implants that have been widely used in the aesthetic medical field in recent years for facial contouring, soft tissue defect repair, and facial rejuvenation. Typical facial fillers include biopolymers such as hyaluronic acid, polylactic acid, and polycaprolactone, as well as their microsphere-shaped complexes. These fillers, injected subcutaneously or deep within the skin, can enhance facial volume and shape over a period of time.

[0003] In the existing art, many approved facial filler products (such as National Medical Device Registration Nos. 20213130276, 20243132279, 20243131626, and 20213130460) often incorporate sodium carboxymethylcellulose (CMC-Na) as a viscosity modifier (thickener) to ensure stable dispersion of solid particles such as microspheres in solutions or suspensions and prevent sedimentation. This facilitates medical manipulation and controlled shaping after tissue injection. Because CMC-Na is a water-soluble polymer, after injection into soft tissue, it circulates with body fluids and is excreted from the body, causing the filler's physical filling effect to rapidly decrease or even disappear, failing to achieve the desired WYSIWYG effect. This not only affects the filler's long-term cosmetic and restorative properties but also increases the frequency of repeat injections and post-operative maintenance. Therefore, there is an urgent need to develop materials that can enhance the physical filling effect of facial fillers containing microspheres. Summary of the Invention

[0004] In view of this, the present invention proposes a preparation method and application of cross-linked carboxymethyl cellulose that can achieve long-term filling.

[0005] Cross-linked sodium hyaluronate gel containing L-lactic acid-ethylene glycol copolymer microspheres, approved by the National Medical Device Registration No. 20213130460, addresses the issue of maintaining the physical filling effect of microsphere-containing facial fillers by using cross-linked sodium hyaluronate. The inventors of this application have discovered that, in addition to cross-linked sodium hyaluronate, which is widely used in facial fillers, the carboxymethylcellulose calcium described in this invention also exhibits excellent biocompatibility and can effectively maintain the physical filling effect. It can be used as a new material in the production of medical aesthetic fillers.

[0006] The technical solution of the present invention is achieved as follows: In a first aspect, the present invention provides a method for preparing cross-linked carboxymethyl cellulose, comprising the following steps: S1, dissolving anhydrous calcium chloride in anhydrous ethanol, and then sterilizing and filtering to obtain a sterile calcium chloride solution; S2, adding the dehydrated and purified sodium carboxymethyl cellulose to the sterile calcium chloride solution and stirring for 1-3 hours; S3, after the reaction is completed, suction filtration is performed, and the filter residue is collected and rinsed with anhydrous ethanol to remove residual calcium chloride to obtain carboxymethyl cellulose calcium hydrogel, which is then vacuum dried for use.

[0007] Because sodium carboxymethyl cellulose is a highly water-soluble polymer, it circulates with body fluids and is excreted from the body. This causes the filled area to maintain a certain volume for only a short time before quickly decreasing in size or even disappearing completely. At this point, only the polymer microspheres and other particles in the filler remain. These particles primarily work by stimulating collagen regeneration in the body. However, due to the long collagen regeneration cycle, achieving a "what you see is what you get" effect is difficult.

[0008] This invention uses ionic crosslinking to form a dense three-dimensional network of carboxymethyl cellulose molecular chains and divalent calcium ions, transforming the material into a water-insoluble hydrogel material with both strength and elasticity. Once injected into the filler area, this CMC-Ca hydrogel exhibits excellent biocompatibility and instantaneous shaping, providing significant volume replenishment and shaping effects for tissues. This achieves the synergistic advantages of both immediate filling and long-term maintenance, providing a new material and alternative for preparing facial fillers.

[0009] However, currently, sodium carboxymethyl cellulose or calcium carboxymethyl cellulose is commonly used as an excipient for oral tablet compression in the pharmaceutical industry. The quality requirements of the Chinese Pharmacopoeia do not include microorganism and endotoxin items. As a result, calcium carboxymethyl cellulose that meets the Chinese Pharmacopoeia standards cannot be directly used as a raw material for the production of implantable medical devices. The present invention proposes a purification method for calcium carboxymethyl cellulose, which ensures that the microorganism and endotoxin content of calcium carboxymethyl cellulose can meet the requirements for raw materials for implantable devices.

[0010] On the basis of the above technical solution, preferably, in step S2, the dehydration purification method is: sodium carboxymethyl cellulose is gradient purified with volume concentrations of 75%-80%, 85%-90% and anhydrous ethanol in sequence to remove endotoxins step by step and kill microorganisms in the raw materials. Each step is carried out under conditions of heating, stirring and condensation reflux. After the purification is completed, it is vacuum dried to obtain purified sodium carboxymethyl cellulose.

[0011] Before vacuum drying, the sodium carboxymethyl cellulose is sealed in a Tyvek paper-plastic packaging bag to prevent the sodium carboxymethyl cellulose from being contaminated by microorganisms again during the drying process and before use. The vacuum drying parameter is 0.098 Kpa and the vacuum drying is performed for 6-24 hours so that the ethanol residue in the raw material meets the requirements of the Chinese Pharmacopoeia for residual solvents.

[0012] As an injectable implant material that can stay in the body for a long time, CMC-Ca hydrogel needs to be highly valued for its biosafety and purity. Currently commercially available sodium carboxymethyl cellulose raw material oral preparation excipients are not strictly controlled for microorganisms and endotoxins for medical implant purposes, and their microorganism and endotoxin contents often exceed acceptable ranges. If such raw materials are directly used to prepare CMC-Ca hydrogel, endotoxins will be embedded in the hydrogel network and will be difficult to effectively remove in subsequent processes, thereby causing potential immune risks and biosafety hazards after human injection. Therefore, before the preparation of CMC-Ca hydrogel, the present invention performs sufficient endotoxin removal treatment on the sodium carboxymethyl cellulose raw material to obtain pure, safe, low-endotoxin sodium carboxymethyl cellulose, laying the foundation for the biosafety of subsequent products.

[0013] This invention utilizes a step-by-step gradient purification process using anhydrous ethanol to efficiently remove microorganisms and endotoxins from sodium carboxymethylcellulose. The principle is that ethanol concentrations exceeding 60% swell and disperse CMC molecules, loosening their structure and facilitating the release of deep-seated endotoxins. It also prevents dissolution of the sodium carboxymethylcellulose, which would result in a sharp increase in viscosity. Simultaneously, ethanol kills microorganisms while dissolving endotoxins. The graded treatment process, using 75%-80%, 85%-90%, and anhydrous ethanol, prevents CMC dissolution (high ethanol concentrations inhibit swelling and gelation) while also avoiding agglomeration and compaction caused by a single-step dehydration process. The process provides for efficient step-by-step exchange and effective desorption and extraction of impurities. The heating and condensation reflux steps not only enhance microbial killing and endotoxin dissolution efficiency, but also maintain a constant ethanol concentration in the system through the condensation device, preventing ethanol volatilization and abnormal concentration fluctuations, thereby ensuring the effectiveness and safety of the purification process. After treatment by this process, the endotoxin content of CMC raw materials can be greatly reduced, and the purity and safety of the product can be significantly improved, laying a solid foundation for the subsequent preparation of high-quality medical CMC-Ca hydrogel.

[0014] On the basis of the above technical solution, preferably, in the first purification step, in addition to adding an ethanol solution with a volume concentration of 75%-80%, 0.5-1 mol / L hydrochloric acid is also added to adjust the pH value of the ethanol solution to 1-1.5.

[0015] The molecular structure of bacterial endotoxins (primarily lipopolysaccharides) is more easily destroyed in a strongly acidic environment. The low pH adjusted by hydrochloric acid not only destabilizes and dissolves surface-adsorbed endotoxins, but also disrupts electrostatic or hydrogen bonding between endotoxins and polymers that may be embedded within the CMC polymer, thereby significantly improving endotoxin removal efficiency.

[0016] Based on the above technical solution, preferably, in each purification step, the heating temperature is 30-70°C, the stirring speed is 100-500 rpm, and the time is 10-60 min; after each reflux, the mixture is allowed to stand for 0.5-2 h, the supernatant is removed, and the precipitate is retained for the next purification step; in each purification step, the material-liquid ratio of sodium carboxymethyl cellulose to ethanol solution is 1:5-10.

[0017] Based on the above technical solution, preferably, in step S2, the mass volume ratio of sodium carboxymethyl cellulose to calcium chloride solution is 1:3-10, and the mass concentration of the calcium chloride solution is 10%.

[0018] Based on the above technical solution, preferably, the stirring reaction temperature in step S2 is 30-70° C. and the rotation speed is 100-500 rpm.

[0019] In a second aspect, the present invention provides a use of cross-linked carboxymethyl cellulose in an injection filler, wherein the cross-linked carboxymethyl cellulose is prepared by the above-mentioned preparation method.

[0020] On the basis of the above technical solution, preferably, the components of the injection filler include polymer microspheres, carboxymethyl cellulose calcium, a thickener, a pH regulator, an osmotic pressure regulator and water.

[0021] Based on the above technical solution, preferably, based on 100% by mass, the polymer microspheres are 20%-28%, cross-linked carboxymethyl cellulose is 2%-3.5%, thickener is 0.5%-1%, pH regulator is 0.13%-0.2%, osmotic pressure regulator is 1%, and the balance is water.

[0022] Based on the above technical solution, preferably, the polymer microspheres are poly-L-lactic acid microspheres or polycaprolactone microspheres, with an intrinsic viscosity of 0.5-2.0 dL / g (0.25%), a molecular weight distribution coefficient of 1.0-3.0, and a cumulative proportion of microspheres with a particle size distribution in the range of 20-55 μm of not less than 70%.

[0023] On the basis of the above technical solution, preferably, the thickener is one or more of sodium carboxymethyl cellulose, sodium hyaluronate, polyvinyl pyrrolidone, and hydroxypropyl methylcellulose; more preferably, the thickener is sodium carboxymethyl cellulose or sodium hyaluronate.

[0024] On the basis of the above technical solution, preferably, the weight average molecular weight of the sodium carboxymethyl cellulose is 390,000-1,000,000; and the weight average molecular weight of sodium hyaluronate is 1,000,000-2,000,000.

[0025] On the basis of the above technical solution, preferably, the pH adjuster is a conjugate acid-base pair composed of phosphate or citrate; more preferably, the pH adjuster is phosphate.

[0026] On the basis of the above technical solution, preferably, when the pH adjuster is a phosphate, its components are potassium dihydrogen phosphate and disodium hydrogen phosphate, and the ratio thereof is 1:(1-9); more preferably, potassium dihydrogen phosphate: disodium hydrogen phosphate = 1:9 or 1:2.

[0027] Based on the above technical solution, preferably, the injection filler is a liquid injection or a powder injection.

[0028] Based on the above technical solution, preferably, the injection filling site is selected from any one of the face, neck, abdomen, chest, buttocks, thighs, calves, upper arms, and lower arms, or a combination thereof; more preferably, the injection filling site is the face.

[0029] On the basis of the above technical solutions, preferably, the injectable filler is used to improve any one or a combination of facial emaciation, fat atrophy, sunken cheeks, sunken eye sockets, and skin wrinkles in patients.

[0030] The preparation method and application of cross-linked carboxymethyl cellulose of the present invention have the following beneficial effects compared with the prior art: (1) The carboxymethyl cellulose calcium of the present invention is highly consistent with carboxymethyl cellulose sodium in terms of chemical activity, physiological and biochemical activity, biocompatibility, and safety risk. However, in terms of physical properties, carboxymethyl cellulose calcium is a hydrogel fiber (see Figure 1 ), is insoluble in water, has better filling effect, and can achieve long-lasting filling effect.

[0031] (2) Existing sodium carboxymethyl cellulose contains certain microorganisms and endotoxins, which do not meet the quality control standards for raw materials for implantable medical devices. When directly used to prepare CMC-Ca hydrogel, endotoxins will be embedded in the hydrogel and difficult to remove. After injection into the human body, it will cause potential immune risks and biosafety hazards. Therefore, the present invention adopts an ethanol solution step-by-step gradient purification process to remove microorganisms and endotoxins from sodium carboxymethyl cellulose, thereby improving its biosafety.

[0032] (3) In order to efficiently remove microorganisms and endotoxins from sodium carboxymethyl cellulose, the present invention adds hydrochloric acid during the first purification step, which significantly improves the removal effect of bacterial endotoxins.

[0033] (4) The injectable filler of the present invention can maintain the volume of the filled area more significantly and for a longer period of time, thereby improving the immediate filling effect of microsphere-containing facial fillers. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 Figure 3 is the effect diagram of carboxymethylcellulose calcium gel. Figure A is the suspension of carboxymethylcellulose calcium gel particles after swelling, and Figure B is the Congo red staining of carboxymethylcellulose calcium gel particles (4×10 optical microscope field of view).

[0036] Figure 2 This is a physical picture of the injection filler liquid prepared in the present invention.

[0037] Figure 3 It is a pre-filled injection filler.

[0038] Figure 4 This is a diagram of drug administration for animal experiments.

[0039] Figure 5 This is a diagram of the physical volume of the filler 6 months after injection.

[0040] Figure 6 This is the anatomy 6 months after filler injection.

[0041] Figure 7 This is a HE staining picture of the filled site tissue 6 months after injection.

[0042] Figure 8 This is a MASSON staining image of the filled site tissue 6 months after injection.

[0043] Figure 9 This is a picture of picrosirius red staining of the tissue at the filling site 6 months after injection. DETAILED DESCRIPTION

[0044] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0045] The sodium carboxymethylcellulose used in this invention was purchased from Anhui Shanhe Pharmaceutical Excipients Co., Ltd., with a weight-average molecular weight of 881,753; sodium hyaluronate was purchased from Bloomage Biotechnology Co., Ltd., with a weight-average molecular weight of 1,200,000. Polymer microspheres: poly-L-lactic acid microspheres and polycaprolactone microspheres were purchased from Shenzhen Jusheng Biotechnology Co., Ltd., with an intrinsic viscosity of 0.5-2.0 dL / g (0.25%), a molecular weight distribution coefficient of 1.0-3.0, and a cumulative proportion of microspheres with a particle size distribution of 20-55 μm of no less than 70%.

[0046] Example 1 This embodiment provides cross-linked carboxymethyl cellulose, an injection filler, and a preparation method thereof, wherein the preparation method of cross-linked carboxymethyl cellulose comprises the following steps: S11, sodium carboxymethyl cellulose purification: Purification Step 1: Fill a dry-heat sterilized glass flask with 700 mL of 75% ethanol-water solution. Add 0.5 mol / L hydrochloric acid to adjust the pH of the ethanol solution to 1. Add 100 g of sodium carboxymethyl cellulose at a material-to-liquid ratio of 1:7. Place a magnetic stirrer and maintain a magnetic stirrer at 300 rpm. Connect a condenser to the flask (to condense and reflux the ethanol and maintain the ethanol concentration in the solution. Ethanol concentrations below 60% will cause the sodium carboxymethyl cellulose to dissolve and dramatically increase the viscosity of the solution). Heat the flask with a heating mantle to maintain a temperature of 60°C (to increase the solubility of bacterial endotoxins and physically kill bacteria). Stir continuously for 30 minutes. Discontinue magnetic stirring, let the solution stand for 60 minutes, and then pour off the supernatant and retain the precipitate.

[0047] The second step of purification: add 85% ethanol aqueous solution by volume at a material-liquid ratio of 1:7 (gradient dehydration to avoid agglomeration of sodium carboxymethyl cellulose), turn on the magnetic stirring and maintain the magnetic rotation speed at 300 rpm, connect the condenser to the mouth of the flask, turn on the electric heating mantle, heat the flask to maintain the temperature of the material liquid at 60 ° C, continue stirring for 30 minutes, let it stand for 60 minutes after the end, pour out the supernatant, and retain the precipitate.

[0048] The third step is purification: add anhydrous ethanol at a material-liquid ratio of 1:7, turn on the magnetic stirring and maintain the magnetic rotation at 300 rpm, connect the condenser to the mouth of the flask, turn on the electric heating mantle, heat the flask to maintain the temperature of the material liquid in the bottle at 60°C, continue stirring for 30 minutes, let it stand for 60 minutes after the end, pour out the supernatant, and retain the precipitate.

[0049] S12, preparation of cross-linking solution: dissolving anhydrous calcium chloride in anhydrous ethanol at a material-liquid ratio of 1:9, and then sterilizing and filtering through a 0.2 μm sterilizing filter to obtain a sterile calcium chloride solution; S13, Preparation of Carboxymethylcellulose Calcium: Take 10g of dehydrated and purified sodium carboxymethylcellulose precipitate and add 80mL of sterile calcium chloride solution. Turn on magnetic stirring and maintain the magnetic rotation at 300rpm. Connect a condenser to the mouth of the flask and turn on the electric heating mantle to heat the flask. Maintain the temperature of the liquid in the flask at 50°C and continue stirring for 2h. Pour the reaction suspension into a suction filtration device and filter, collecting the filter residue. Then rinse the filter residue with 100mL of anhydrous ethanol 5 times to remove residual calcium chloride.

[0050] Vacuum drying: Place the filter residue after removing calcium chloride into a Tyvek paper-plastic packaging bag and heat-seal it to form a breathable and antibacterial sealed package (to reduce contamination). Vacuum dry at 0.098Kpa for 20 hours to remove the ethanol in the gel fiber.

[0051] Ethanol recovery: collect the supernatant poured out during the preparation process and the filtrate filtered, and use a rotary evaporator to recover the ethanol.

[0052] The injectable filler includes polymer microspheres (poly-L-lactic acid), carboxymethylcellulose calcium (prepared by the above method), carboxymethylcellulose sodium, phosphates (potassium dihydrogen phosphate and disodium hydrogen phosphate), glycerol and water.

[0053] The preparation method of the injectable filler comprises the following steps: S21, weigh 840 g of poly-L-lactic acid microspheres, 60 g of carboxymethylcellulose calcium, 30 g of carboxymethylcellulose sodium, 0.7 g of potassium dihydrogen phosphate, 4.3 g of disodium hydrogen phosphate, 30 g of glycerol, and 2035 g of water; S22, adding the materials weighed in step S1 into the dry heat sterilized material tank through the feed port of the material tank, and sealing the feed port; S23, placing the material tank in the material cup of the VM20000DW planetary mixer, placing a balance tank of the same weight in the corresponding position, and closing the hatch of the planetary mixer; S24, set the stirring program: 250 rpm, stirring for 600 s; 800 rpm, stirring for 120 s.

[0054] S25, take out the material tank, connect the material tank outlet to the filling machine, fill 1mL / branch of material liquid into each pre-filled syringe, add rubber stopper, and obtain the finished product (see Figure 2-3 ).

[0055] Example 2 This embodiment provides cross-linked carboxymethyl cellulose, an injection filler, and a preparation method thereof, wherein the preparation method of cross-linked carboxymethyl cellulose comprises the following steps: S11, sodium carboxymethyl cellulose purification: Purification Step 1: The difference from Example 1 is that the volume concentration of ethanol is 80%, and the pH of the ethanol solution is 1.5. The material-liquid ratio of sodium carboxymethyl cellulose to ethanol solution is 1:10. The temperature of the liquid in the bottle is 70°C, and the magnetic stirring speed is 500 rpm for 40 minutes. After reflux, the mixture is allowed to stand for 0.5 hours, the supernatant is poured off, and the precipitate is retained.

[0056] The second step of purification: The difference from Example 1 is that the volume concentration of ethanol is 90%, the material-liquid ratio of sodium carboxymethyl cellulose and ethanol solution is 1:8, the material liquid temperature in the bottle is 50°C, the magnetic stirring speed is 200 rpm, the time is 50 min, and after the reflux is completed, it is allowed to stand for 1 h, the supernatant is poured out, and the precipitate is retained.

[0057] The third step of purification: The difference from Example 1 is that the material-liquid ratio of sodium carboxymethyl cellulose and ethanol solution is 1:5, the material liquid temperature in the bottle is 30°C, the magnetic stirring speed is 100 rpm, the time is 20 min, and after the reflux is completed, it is allowed to stand for 0.5 h, the supernatant is poured out, and the precipitate is retained.

[0058] S12, same as Example 1.

[0059] S13, Preparation of Carboxymethylcellulose Calcium: Take 10g of dehydrated and purified sodium carboxymethylcellulose precipitate and add 100mL of sterile calcium chloride solution. Turn on magnetic stirring and maintain the magnetic rotation at 500rpm. Connect a condenser to the mouth of the flask and turn on the electric heating mantle to heat the flask. Maintain the temperature of the liquid in the flask at 30°C and continue stirring for 3h. Pour the reaction suspension into a suction filtration device and filter, collecting the filter residue. Then rinse the filter residue with 100mL of anhydrous ethanol 5 times to remove residual calcium chloride.

[0060] Vacuum drying and ethanol recovery are the same as in Example 1.

[0061] The preparation method of the injectable filler differs from that of Example 1 in terms of the raw materials and amounts used, specifically: 600 g of polycaprolactone microspheres, 80 g of carboxymethylcellulose calcium (prepared by the above method), 15 g of polyvinylpyrrolidone, 1 g of potassium dihydrogen phosphate, 3 g of disodium hydrogen phosphate, 30 g of sodium chloride, and 2271 g of water. The remaining ingredients are the same.

[0062] Example 3 This embodiment provides cross-linked carboxymethyl cellulose, an injection filler, and a preparation method thereof, wherein the preparation method of cross-linked carboxymethyl cellulose comprises the following steps: S11, sodium carboxymethyl cellulose purification: Purification Step 1: The difference from Example 1 is that the volume concentration of ethanol is 78%, and the pH of the ethanol solution is 1.3. The material-liquid ratio of sodium carboxymethyl cellulose to ethanol solution is 1:5. The temperature of the liquid in the bottle is 70°C, and the magnetic stirring speed is 500 rpm for 10 minutes. After reflux, the mixture is allowed to stand for 1 hour, the supernatant is poured off, and the precipitate is retained.

[0063] The second step of purification: The difference from Example 1 is that the volume concentration of ethanol is 88%, the material-liquid ratio of sodium carboxymethyl cellulose to ethanol solution is 1:7, the material liquid temperature in the bottle is 50°C, the magnetic stirring speed is 300 rpm, the time is 40 min, and after the reflux is completed, it is allowed to stand for 1.5 h, the supernatant is poured out, and the precipitate is retained.

[0064] The third step of purification: The difference from Example 1 is that the material-liquid ratio of sodium carboxymethyl cellulose and ethanol solution is 1:6, the material liquid temperature in the bottle is 40°C, the magnetic stirring speed is 200 rpm, the time is 50 min, and after the reflux is completed, it is allowed to stand for 2 h, the supernatant is poured out, and the precipitate is retained.

[0065] S12, same as Example 1.

[0066] S13, Preparation of Carboxymethylcellulose Calcium: Take 10g of dehydrated and purified sodium carboxymethylcellulose precipitate and add 30mL of sterile calcium chloride solution. Turn on magnetic stirring and maintain the magnetic rotation at 100rpm. Connect a condenser to the mouth of the flask and turn on the electric heating mantle to heat the flask. Maintain the temperature of the liquid in the flask at 70°C and continue stirring for 1 hour. Pour the reaction suspension into a suction filtration device and filter, collecting the filter residue. Then rinse the filter residue with 100mL of anhydrous ethanol five times to remove residual calcium chloride.

[0067] Vacuum drying and ethanol recovery are the same as in Example 1.

[0068] The preparation method of the injectable filler differs from that of Example 1 in terms of the raw materials and amounts used, specifically: 780 g of polylactic acid microspheres, 90 g of carboxymethylcellulose calcium (prepared by the above method), 20 g of sodium hyaluronate, 0.6 g of potassium dihydrogen phosphate, 5.4 g of disodium hydrogen phosphate, 30 g of glycerol, and 2074 g of water. The remaining ingredients are the same.

[0069] Example 4 The difference between Example 4 and Example 1 is that the amount of carboxymethylcellulose calcium is 105g, the amount of water is 1990g, and the rest of the contents are the same as in Example 1.

[0070] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that no carboxymethylcellulose calcium is added, but the added thickener carboxymethylcellulose sodium is purified by the purification step described in the example, and the rest of the contents are the same as Example 1.

[0071] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that carboxymethyl cellulose calcium is directly prepared using sodium carboxymethyl cellulose and the purification step is omitted. The rest of the contents are the same as Example 1.

[0072] 1. Performance Study 1.1 The molecular weight and distribution of sodium carboxymethyl cellulose, ethanol residue, microorganisms, and endotoxin content before and after purification were analyzed. The results are shown in Table 1.

[0073] The weight-average molecular weight and molecular weight distribution of sodium carboxymethyl cellulose samples were determined by gel permeation chromatography (GPC). Ethanol residue was tested according to the Determination of Residual Solvents, General Chapter 0861 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. Microbiological testing was performed according to the Sterility Test, General Chapter 1101 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. Endotoxin testing was performed according to the Gel Limit Method, General Chapter 1143 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III.

[0074] Table 1 Effect of purification process on the molecular weight of sodium carboxymethyl cellulose

[0075] As shown in Table 1, the purification process using gradient ethanol concentrations (75%-80%, 85%-90%, and 100%) in Example 1 can kill microorganisms in sodium carboxymethylcellulose, achieving sterile raw material quality and significantly reducing endotoxin levels, bringing the sodium carboxymethylcellulose's microbial and endotoxin levels to levels suitable for production of medical and aesthetic filler products. The molecular weight of the sodium carboxymethylcellulose after purification did not significantly decrease, indicating that the purification process did not cause material degradation. Furthermore, the residual ethanol content met the residual solvent limit specified in the Chinese Pharmacopoeia.

[0076] 1.2 Rheometer Testing: Frequency sweep tests were performed on the fillers prepared in Examples 1-4 and Comparative Examples 1-2, comparing their G' (elastic modulus), G'' (viscous modulus), and η* (complex viscosity) data at a frequency of 0.688 Hz. The test temperature was set at 25°C, and the gap between the clamps was 0.5 mm. The extrusion force of the fillers prepared in Examples 1-4 and Comparative Examples 1-2 was measured using a syringe extrusion force tester, using a 27G needle and an extrusion speed of 30 mm / min. The results are shown in Table 2.

[0077] Table 2 Rheological properties and extrusion force test results of fillers

[0078] As shown in Table 2, the addition of calcium carboxymethyl cellulose can significantly increase the physical support effect of the filler. As the content of calcium carboxymethyl cellulose increases, the composite viscosity and elastic modulus of the filler increase, and the pushing force increases.

[0079] 2. Animal Experiments 1. Test product information Table 3 Test product information

[0080] 2. Trial Grouping and Dosing Thirty female Sprague-Dawley rats, 6-8 weeks old, weighing 180-220 g, were randomly selected for the experiment after 12 days of acclimatization. Six groups of four animals were set up according to the test article. The injection site was carefully shaved with a razor the day before injection.

[0081] Test drug administration: Figure 4 As shown, each animal was injected with a test article and a negative control. The test article was injected at four sites (blue), with 0.2 mL injected at each site. Each animal was injected with a negative control site (red) for subsequent histopathological scoring comparison. Samples were collected at four time points: 7 days, 1 month, 3 months, and 6 months.

[0082] 3. Test indicators 3.1 Measurement of filler appearance and morphology In vitro dimensions were measured before dissection at 7 days, 1 month, 3 months, and 6 months, and the implant volume changes (mm 3 ), the volume on the day of injection was 200 ± 0.5 mm 3 , evaluate the filling effect. The results are shown in Table 4 and Figure 5 .

[0083] Table 4 Effect of subcutaneous injection in rats

[0084] As shown in Table 4, compared with Comparative Example 1, the carboxymethyl cellulose calcium prepared in Example 1 of the present invention can significantly extend the filling period when added to the injection filler. Comparison between Example 1 and Example 4 shows that as the amount of carboxymethyl cellulose calcium increases, the filling time of the injection filler increases accordingly.

[0085] Figure 5 It can be seen that the fillers in Examples 1-4 can still be clearly seen 6 months after injection, while the fillers in Comparative Example 1 have completely disappeared and become invisible.

[0086] 3.2 Tissue sampling and observation After administration, the tissues of the rats at the injection site were sampled at 7 days, 1 month, 3 months and 6 months (the sampling area was approximately 2 cm 2), one animal was dissected in each group at each time point, and the filled area and surrounding tissues were cut out according to the injection diagram and fixed on the specimen measurement board. Photos were taken with a SLR camera, which should be fixed on a desktop stand for overhead photography, and the position, angle, and lighting should be kept consistent for each shot.

[0087] 3.3 Histopathology After administration, the tissues of the filled sites were collected at 7 days, 1 month, 3 months and 6 months after administration, and HE staining, MASSON staining and Sirius red staining were performed on the tissues of the filled sites (see Figure 7-9 ) and histopathological examination was performed. The tissue response to the filler after administration was evaluated. The results are shown in Table 5.

[0088] Tissue response = total cell type totals * 2 + total response type totals. Skin Tissue Irritation Scale: 0.0-2.9 indicates no irritation or very mild irritation, 3.0-8.9 indicates mild irritation, 9.0-15.0 indicates moderate irritation, and >15 indicates severe irritation.

[0089] Table 5 Tissue response scoring results

[0090] As shown in Table 3, compared with Comparative Example 2, the filler prepared from the purified sodium hydroxymethyl cellulose in Example 1 removed cellular toxins and significantly reduced the inflammatory response of the tissue, with no irritation or very mild irritation after 6 months.

[0091] Figure 6-9 These are the anatomical and staining images of the filled site tissue at 6 months after administration. Figure 5-8 It can be seen that after the injectable filler prepared in Example 1 of the present invention was subcutaneously injected into the animal, the anatomical diagram clearly showed a full filling effect ( Figure 6 ), the staining also shows a gel-like filler ( Figure 7-9 ); while the physical filling volume of the filler in Comparative Example 1 almost disappears ( Figure 6 ), no gel was visible in the staining images ( Figure 7-9 ).

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing cross-linked carboxymethyl cellulose, characterized in that: The following steps are involved: S1, dissolving anhydrous calcium chloride in anhydrous ethanol, and then sterilizing and filtering to obtain a sterile calcium chloride solution; S2, adding the dehydrated and purified sodium carboxymethyl cellulose to a sterile calcium chloride solution and stirring for 1-3 hours; S3, after the reaction is completed, the filter is filtered, the filter residue is collected and rinsed with anhydrous ethanol to remove residual calcium chloride, and then vacuum dried to obtain cross-linked carboxymethyl cellulose.

2. The method for preparing cross-linked carboxymethyl cellulose according to claim 1, wherein: In step S2, the dehydration purification method is as follows: sodium carboxymethyl cellulose is gradient purified with volume concentrations of 75%-80%, 85%-90% and anhydrous ethanol in sequence, each step is carried out under conditions of heating, stirring and condensation reflux, and after purification, vacuum drying is performed to obtain purified sodium carboxymethyl cellulose.

3. The method for preparing cross-linked carboxymethyl cellulose according to claim 2, wherein: During the first purification step, in addition to adding an ethanol solution with a volume concentration of 75%-80%, 0.5-1 mol / L hydrochloric acid is also added to adjust the pH value of the ethanol solution to 1-1.

5.

4. The method for preparing cross-linked carboxymethyl cellulose according to claim 3, wherein: In each purification step, the heating temperature is 30-70°C, the stirring speed is 100-500 rpm, and the time is 10-60 minutes; after each reflux, the mixture is allowed to stand for 0.5-2 hours, the supernatant is removed, and the precipitate is retained for the next purification step; in each purification step, the material-liquid ratio of sodium carboxymethyl cellulose to ethanol solution is 1:5-10.

5. The method for preparing cross-linked carboxymethyl cellulose according to claim 3, wherein: In step S2, the mass volume ratio of sodium carboxymethyl cellulose to calcium chloride solution is 1:3-10, and the mass concentration of the calcium chloride solution is 10%.

6. The method for preparing cross-linked carboxymethyl cellulose according to claim 3, wherein: The stirring reaction temperature in step S2 is 30-70° C. and the rotation speed is 100-500 rpm.

7. A use of cross-linked carboxymethyl cellulose in an injection filler, characterized in that: The cross-linked carboxymethyl cellulose is prepared by the preparation method according to any one of claims 1 to 6.

8. The use according to claim 7, characterized in that: The components of the injection filler include polymer microspheres, cross-linked carboxymethyl cellulose, a thickener, a pH regulator, an osmotic pressure regulator and water.

9. The use according to claim 8, characterized in that: Based on 100% by mass, the polymer microspheres are 20%-28%, cross-linked carboxymethyl cellulose is 2%-3.5%, a thickener is 0.5%-1%, a pH regulator is 0.13%-0.2%, an osmotic pressure regulator is 1%, and the balance is water.

10. The use according to claim 8, characterized in that: The polymer microspheres are poly (L-lactic acid) microspheres or poly (caprolactone) microspheres; The thickener is one or a combination of sodium carboxymethyl cellulose, sodium hyaluronate, polyvinyl pyrrolidone, and hydroxypropyl methylcellulose; The pH regulator is a conjugate acid-base pair composed of phosphate or citrate; The osmotic pressure regulator is glycerol or sodium chloride.

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