Process for the preparation and use of cross-linked carboxymethylcellulose

Abstract

The application relates to the technical field of biomedical materials, and discloses a preparation method and application of cross-linked carboxymethyl cellulose. The preparation method comprises the following steps: S1, dissolving anhydrous calcium chloride in anhydrous ethanol, filtering to obtain a sterile calcium chloride solution; S2, adding dehydrated and purified sodium carboxymethyl cellulose into the sterile calcium chloride solution, and stirring to react; S3, after the reaction is completed, performing suction filtration, collecting filter residues, washing the filter residues to remove residual calcium chloride, and then vacuum drying the filter residues to obtain cross-linked carboxymethyl cellulose. The cross-linked carboxymethyl cellulose and the sodium carboxymethyl cellulose are highly consistent in chemical activity, physiological and biochemical activity, biocompatibility and safety risk, but the cross-linked carboxymethyl cellulose is a hydrogel fiber and is insoluble in water, can more significantly maintain the volume of a filling site, and has a longer duration, so that the effect of instant filling and long-acting filling of a microsphere-containing facial filler is realized.

Description

TECHNICAL FIELD

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

[0002] Facial fillers are key injection implant materials widely used in facial contour modification, soft tissue defect repair and facial rejuvenation in the field of medical aesthetics in recent years. Typical facial filler types include hyaluronic acid, polylactic acid, polycaprolactone and other biopolymers and microsphere type compounds. These fillers can enhance the volume and improve the shape of the face through subcutaneous or deep injection for a certain period of time.

[0003] In the prior art, many approved facial filler products (such as State Food and Drug Administration Approval 20213130276, 20243132279, 20243131626 and 20213130460) often add sodium carboxymethyl cellulose (CMC-Na) as a viscosity regulator (thickening agent) to ensure the stable dispersion of solid particles such as microspheres in the solution or suspension system and to avoid sedimentation, facilitating medical operation and controlled shaping after tissue injection. Since CMC-Na is a water-soluble polymer, it will be excreted from the body after being injected into soft tissue, resulting in a rapid decrease or disappearance of the physical filling effect of the filler, which does not achieve the ideal effect of seeing is believing. This not only affects the long-term cosmetic repair function of the filler, but also increases the frequency of repeated injection and postoperative maintenance. Therefore, it is urgent to develop a material that can improve the physical filling effect of microsphere-containing facial fillers. SUMMARY

[0004] Therefore, the present application provides a preparation method and application of cross-linked carboxymethyl cellulose that can achieve long-acting filling.

[0005] The cross-linked hyaluronic acid sodium gel containing levorotatory lactic acid-ethylene glycol copolymer microspheres in State Food and Drug Administration Approval 20213130460 solves the problem of maintaining the physical filling effect of microsphere-containing facial fillers by using cross-linked hyaluronic acid sodium. The present inventors found that in addition to the widely used cross-linked hyaluronic acid sodium in facial fillers, the carboxymethyl cellulose calcium described in the present application also has good biocompatibility and can better maintain the physical filling effect. It can be used as a new material for the production of medical fillers.

[0006] The technical solution of the present application is as follows: In a first aspect, the present application provides a preparation method of cross-linked carboxymethyl cellulose, comprising the following steps:

[0007] S1, dissolving anhydrous calcium chloride in anhydrous ethanol, then performing sterilization filtration to obtain a sterile calcium chloride solution;

[0008] S2, the sodium carboxymethyl cellulose after dehydration purification treatment is added into sterile calcium chloride solution, and stirred and reacted for 1-3h;

[0009] S3, after the reaction, filtration is carried out, the filter residue is collected and washed with anhydrous ethanol, and the residual calcium chloride is removed to obtain calcium carboxymethyl cellulose hydrogel, which is then vacuum dried for standby.

[0010] Since the sodium carboxymethyl cellulose is a strong water-soluble polymer, it circulates with the body fluid of the human body and is excreted from the body, so that the filling site maintains a certain volume only in a short time, and soon the volume is reduced or even completely disappears. At this time, only the particles such as high molecular microspheres in the filler remain, which mainly play a role by stimulating the regeneration of collagen in the body. Since the collagen regeneration cycle is long, it is difficult to achieve the effect of what you see is what you get.

[0011] The present application converts the carboxymethyl cellulose into a water-soluble, strong and elastic hydrogel material by ion cross-linking, so that the carboxymethyl cellulose molecular chain forms a dense three-dimensional network structure with divalent calcium ions. The CMC-Ca hydrogel has good biocompatibility after being injected into the filling site, can be shaped immediately, provides significant volume supplement and shaping effect for the tissue, realizes the synergistic advantages of instant filling and long-term maintenance, and provides a new material and new choice for preparing a facial filler.

[0012] However, at present, sodium carboxymethyl cellulose or calcium carboxymethyl cellulose is usually used as a tabletting aid for oral tablets in the pharmaceutical industry, and the quality requirements of Chinese Pharmacopoeia do not include the microbial and endotoxin items, which leads to that the calcium carboxymethyl cellulose meeting the standard of Chinese Pharmacopoeia cannot be directly used as a raw material for producing implantable medical devices. The present application provides a purification method of calcium carboxymethyl cellulose, so that the microorganisms and endotoxins of the calcium carboxymethyl cellulose can meet the requirements of implantable device raw materials.

[0013] On the basis of the above technical scheme, preferably, in step S2, the dehydration purification treatment method is: the sodium carboxymethyl cellulose is sequentially subjected to gradient purification with anhydrous ethanol with a volume concentration of 75%-80%, 85%-90% and anhydrous ethanol, so as to remove endotoxins step by step and kill microorganisms in the raw material, each step is carried out under the conditions of heating, stirring and condensation reflux, after purification, vacuum drying is carried out to obtain purified sodium carboxymethyl cellulose.

[0014] 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 parameters are 0.098 Kpa, and the vacuum drying time is 6-24h, so that the residual ethanol in the raw material meets the requirements of Chinese Pharmacopoeia for residual solvents.

[0015] As an injectable implant material that can stay in the body for a long time, the biosafety and purity of CMC-Ca hydrogel should be highly valued. The carboxymethyl cellulose sodium raw material for oral preparation of the current market does not control the microorganism and endotoxin strictly for medical implantation, and the microorganism and endotoxin content often exceeds the acceptable range. If such raw material is directly used to prepare CMC-Ca hydrogel, the endotoxin will be embedded in the hydrogel network, which is difficult to remove effectively in the subsequent process, thereby causing potential immune risk and biological safety hazard after injection in the human body. Therefore, in the present application, the carboxymethyl cellulose sodium raw material is subjected to sufficient endotoxin removal treatment before the preparation of CMC-Ca hydrogel, so as to obtain pure, safe and low endotoxin content carboxymethyl cellulose sodium, laying a foundation for the biosafety of the subsequent product.

[0016] The present application adopts anhydrous ethanol stepwise gradient purification process, which can efficiently remove the microorganisms and endotoxins in carboxymethyl cellulose sodium. The principle is that ethanol with a concentration of more than 60% can swell and disperse CMC molecules, making the structure loose, which is conducive to the release of deep-layer endotoxins, and at the same time, it will not cause the dissolution of carboxymethyl cellulose sodium, resulting in a sharp increase in viscosity. At the same time, ethanol can kill the microorganisms in the raw material while dissolving the endotoxins therein. Through the "75%-80%, 85%-90% and anhydrous ethanol" step-by-step treatment, it can not only ensure that CMC will not be dissolved (high-concentration ethanol can inhibit its swelling and gelation), but also avoid the agglomeration and hardening caused by single-step dehydration. Step-by-step exchange, effective desorption and extraction of impurities. The heating and condensation reflux link improves the killing effect of microorganisms and the dissolution efficiency of endotoxins, and at the same time, the ethanol concentration is kept constant by the condensation device to prevent ethanol evaporation and abnormal concentration fluctuation, ensuring the effect and safety of the purification operation. After the treatment by the process, the endotoxin content of the CMC raw material can be greatly reduced, and the purity and safety of the product are significantly improved, laying a solid foundation for the subsequent preparation of high-quality medical CMC-Ca hydrogel.

[0017] On the basis of the above technical scheme, preferably, in the first step of purification treatment, 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.

[0018] The molecular structure of bacterial endotoxin (mainly lipopolysaccharide) is more easily destroyed in a strong acidic environment. The low pH value after adjustment by hydrochloric acid not only promotes the dissolution of the endotoxins adsorbed on the surface, but also destroys the electrostatic or hydrogen bond interaction between the endotoxins possibly embedded in the CMC polymer and the polymer, thereby significantly improving the removal efficiency of endotoxins.

[0019] On the basis of the above technical scheme, preferably, the heating temperature is 30-70℃, the stirring speed is 100-500rpm, and the time is 10-60min during each purification treatment; after each reflux is completed, the supernatant is removed, and the precipitate is reserved for the next purification treatment after being placed for 0.5-2h; during each purification treatment, the solid-liquid ratio of sodium carboxymethyl cellulose to ethanol solution is 1:5-10.

[0020] On the basis of the above technical scheme, preferably, the mass-volume ratio of sodium carboxymethyl cellulose to calcium chloride solution in step S2 is 1:3-10, and the mass concentration of the calcium chloride solution is 10%.

[0021] On the basis of the above technical scheme, preferably, the stirring reaction temperature in step S2 is 30-70℃, and the rotation speed is 100-500rpm.

[0022] In a second aspect, the application provides an application of cross-linked carboxymethyl cellulose in an injection filler, wherein the cross-linked carboxymethyl cellulose is prepared by the above preparation method.

[0023] On the basis of the above technical scheme, preferably, the components of the injection filler include high-molecular microspheres, calcium carboxymethyl cellulose, a thickening agent, a pH regulator, an osmotic pressure regulator, and water.

[0024] On the basis of the above technical scheme, preferably, according to 100% by mass, the high-molecular microspheres are 20%-28%, the cross-linked carboxymethyl cellulose is 2%-3.5%, the thickening agent is 0.5%-1%, the pH regulator is 0.13%-0.2%, the osmotic pressure regulator is 1%, and the balance is water.

[0025] On the basis of the above technical scheme, preferably, the high-molecular microspheres are poly-L-lactic acid microspheres or polycaprolactone microspheres, the intrinsic viscosity is 0.5-2.0dL / g (0.25%), the molecular weight distribution coefficient is 1.0-3.0, and the cumulative proportion of microspheres with a particle size distribution in the range of 20-55μm is not less than 70%.

[0026] On the basis of the above technical scheme, preferably, the thickening agent is one or more of sodium carboxymethyl cellulose, sodium hyaluronate, polyvinylpyrrolidone, and hydroxypropyl methyl cellulose; more preferably, the thickening agent is sodium carboxymethyl cellulose or sodium hyaluronate.

[0027] On the basis of the above technical scheme, preferably, the weight-average molecular weight of the sodium carboxymethyl cellulose is 390000-1000000, and the weight-average molecular weight of the sodium hyaluronate is 1000000-2000000.

[0028] On the basis of the above technical scheme, preferably, the pH regulator is a conjugate acid-base pair composed of phosphate or citrate; more preferably, the pH regulator is phosphate.

[0029] On the basis of the above technical scheme, preferably, when the pH regulator is phosphate, the composition is potassium dihydrogen phosphate and disodium hydrogen phosphate, and the ratio is 1: (1-9); more preferably, the ratio of potassium dihydrogen phosphate to disodium hydrogen phosphate is 1:9 or 1:2.

[0030] On the basis of the above technical scheme, preferably, the injection filler is a liquid injection or a powder injection.

[0031] On the basis of the above technical scheme, preferably, the injection filling site is selected from any one or a combination of the face, neck, abdomen, chest, buttocks, thigh, lower leg, upper arm, lower arm; more preferably, the injection filling site is the face.

[0032] On the basis of the above technical scheme, preferably, the injection filler is used to improve any one or a combination of facial emaciation, fat atrophy, cheek sagging, eye socket sagging, and skin wrinkles.

[0033] The preparation method and application of the cross-linked carboxymethyl cellulose of the present application have the following beneficial effects relative to the prior art:

[0034] (1) The carboxymethyl cellulose calcium of the present application has high consistency with carboxymethyl cellulose sodium in chemical activity, physiological and biochemical activity, biocompatibility, and safety risk, but in terms of physical properties, the carboxymethyl cellulose calcium is a hydrogel fiber (see Figure 1 ), which is insoluble in water and has better filling effect, and can achieve long-acting filling effect.

[0035] (2) The existing carboxymethyl cellulose sodium contains certain microorganisms and endotoxins, which do not meet the quality control standards of implantable medical device raw materials. When directly used to prepare CMC-Ca hydrogel, the endotoxins will be embedded in the hydrogel and difficult to remove, which will cause potential immune risks and biological safety hazards after injection into the human body. Therefore, the present application adopts an ethanol solution step-by-step gradient purification process to remove microorganisms and endotoxins in carboxymethyl cellulose sodium and improve its biological safety.

[0036] (3) In order to efficiently remove microorganisms and endotoxins in carboxymethyl cellulose sodium, hydrochloric acid is added in the first step of purification, which significantly improves the removal effect of bacterial endotoxins.

[0037] (4) The injection filler described in the present application can more significantly maintain the volume of the filling site, and has a longer duration, thereby improving the immediate filling effect of the microsphere-containing facial filler. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings described below only show some of the embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0039] Figure 1 Figure A is a picture of carboxymethyl cellulose calcium gel particle suspension after swelling, and figure B is a picture of carboxymethyl cellulose calcium gel particle Congo red staining (4x10 optical microscope field).

[0040] Figure 2 Figure is a picture of the injection filler liquid prepared by the present application.

[0041] Figure 3 Figure is a picture of the injection filler.

[0042] Figure 4 Figure is a picture of the injection filler.

[0043] Figure 5 Figure is a picture of the physical volume of the filler after 6 months of the injection filler.

[0044] Figure 6 Figure is a picture of the dissection after 6 months of the injection filler.

[0045] Figure 7 Figure is a picture of the HE staining of the tissue at the filling site after 6 months of injection.

[0046] Figure 8 Figure is a picture of the MASSON staining of the tissue at the filling site after 6 months of injection.

[0047] Figure 9 Figure is a picture of the SIRUS red staining of the tissue at the filling site after 6 months of injection. DETAILED DESCRIPTION

[0048] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments only show some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0049] The sodium carboxymethyl cellulose used in the application is purchased from Anhui Shanhel Pharmaceutical Auxiliary Co., Ltd., and the weight average molecular weight is 881753; the sodium hyaluronate is purchased from Huaxi Biological Technology Co., Ltd., and the weight average molecular weight is 1200000. The polymer microspheres: the poly-L-lactic acid microspheres and the polycaprolactone microspheres are both purchased from Shenzhen Juxing Biological Technology Co., Ltd., the intrinsic viscosity is in the range of 0.5-2.0 dL / g (0.25%), the molecular weight distribution coefficient is in the range of 1.0-3.0, and the cumulative proportion of the microspheres with a particle size distribution in the range of 20-55 μm is not less than 70%.

[0050] Example 1

[0051] The present embodiment provides a cross-linked carboxymethyl cellulose, an injection filler and a preparation method thereof, wherein the preparation method of the cross-linked carboxymethyl cellulose comprises the following steps:

[0052] S11, purification of sodium carboxymethyl cellulose:

[0053] First step of purification: a dry heat sterilized glass flask is charged with 700 mL of an ethanol aqueous solution with a volume concentration of 75%, 0.5 mol / L hydrochloric acid is added, the pH value of the ethanol solution is adjusted to 1, 100 g of sodium carboxymethyl cellulose is added according to a solid-liquid ratio of 1:7, a magnetic stirring rod is put in, and the magnetic stirring is kept at a speed of 300 rpm. A condenser tube is connected to the mouth of the flask (used for condensing the refluxing ethanol, keeping the concentration of ethanol in the solid-liquid, and the concentration of ethanol in the solid-liquid is lower than 60% which will cause the dissolution of sodium carboxymethyl cellulose and the sharp increase of the viscosity of the solid-liquid), the electric heating jacket is turned on, the flask is heated to keep the temperature of the solid-liquid in the flask at 60℃ (used for improving the solubility of bacterial endotoxin and physically killing bacteria), and the stirring is continuously kept for 30 min. After the end, the magnetic stirring is stopped, the solid-liquid is kept still for 60 min, the supernatant is poured out, and the precipitate is reserved.

[0054] Second step of purification: an ethanol aqueous solution with a volume concentration of 85% is added according to a solid-liquid ratio of 1:7 (gradient dehydration, avoiding the agglomeration of sodium carboxymethyl cellulose), the magnetic stirring is kept at a speed of 300 rpm, a condenser tube is connected to the mouth of the flask, the electric heating jacket is turned on, the flask is heated to keep the temperature of the solid-liquid in the flask at 60℃, and the stirring is continuously kept for 30 min. After the end, the solid-liquid is kept still for 60 min, the supernatant is poured out, and the precipitate is reserved.

[0055] Third step of purification: anhydrous ethanol is added according to a solid-liquid ratio of 1:7, the magnetic stirring is kept at a speed of 300 rpm, a condenser tube is connected to the mouth of the flask, the electric heating jacket is turned on, the flask is heated to keep the temperature of the solid-liquid in the flask at 60℃, and the stirring is continuously kept for 30 min. After the end, the solid-liquid is kept still for 60 min, the supernatant is poured out, and the precipitate is reserved.

[0056] S12, cross-linking liquid preparation: anhydrous calcium chloride was dissolved in anhydrous ethanol at a ratio of 1:9, and then sterilized by filtering through a 0.2 μm sterilization filter to obtain a sterile calcium chloride solution;

[0057] S13, preparation of carboxymethyl cellulose calcium: 10 g of carboxymethyl cellulose sodium precipitate after dehydrated purification treatment was added to 80 mL of sterile calcium chloride solution, and magnetic stirring was started at a speed of 300 rpm. A condenser tube was connected to the mouth of the flask, and an electric heating jacket was turned on to heat the flask, maintaining the temperature of the liquid in the flask at 50°C. The stirring was continued for 2 h. The completed reaction suspension was poured into a filtration device for filtration, and the filtrate was collected. Then, the filtrate was washed with 100 mL of anhydrous ethanol for 5 times to remove residual calcium chloride.

[0058] Vacuum drying: the filtrate after removing calcium chloride was placed in a Teflon paper packaging bag, and a heat seal was formed to form a sealed package that was breathable and bacteria-resistant (reducing contamination). The vacuum drying was performed at 0.098 Kpa for 20 h to remove ethanol in the gel fibers.

[0059] Ethanol recovery: the supernatant poured out during the preparation and the filtrate after filtration were collected, and ethanol was recovered by a rotary evaporator.

[0060] The injection filler includes high molecular microspheres (poly-L-lactic acid), carboxymethyl cellulose calcium (prepared by the above method), carboxymethyl cellulose sodium, phosphate (potassium dihydrogen phosphate and sodium hydrogen phosphate), glycerol and water.

[0061] The preparation method of the injection filler includes the following steps:

[0062] S21, 840 g of poly-L-lactic acid microspheres, 60 g of carboxymethyl cellulose calcium, 30 g of carboxymethyl cellulose sodium, 0.7 g of potassium dihydrogen phosphate, 4.3 g of sodium hydrogen phosphate, 30 g of glycerol and 2035 g of water were weighed;

[0063] S22, the substances weighed in step S1 were poured into a dry heat sterilized tank from the inlet of the tank, and the inlet was sealed;

[0064] S23, the tank was placed in the cup of the tank of the VM20000DW planetary mixer, and a balance tank with the same weight was placed in the corresponding position, and the cover of the planetary mixer was closed;

[0065] S24, the stirring program was set as follows: speed 250 rpm, stirring for 600 s; speed 800 rpm, stirring for 120 s.

[0066] S25, the tank was taken out, and the outlet of the tank was connected to a filling machine. Each pre-filled syringe was filled with 1 mL of the liquid, a rubber plug was added, and a finished product was obtained (see Figures 2-3 ).

[0067] Example 2

[0068] The present embodiment provides a cross-linked carboxymethyl cellulose, an injection filler and a preparation method thereof, wherein the preparation method of the cross-linked carboxymethyl cellulose comprises the following steps:

[0069] S11, purification of sodium carboxymethyl cellulose:

[0070] First step of purification: the difference from Example 1 is that the volume concentration of ethanol is 80%, and the pH value of the ethanol solution is 1.5. The liquid-solid ratio of sodium carboxymethyl cellulose and the ethanol solution is 1:10, the in-bottle liquid temperature is 70℃, the magnetic stirring speed is 500 rpm, the time is 40 min, after the reflux is completed, it is placed for 0.5 h, the supernatant is poured out, and the precipitate is reserved.

[0071] Second step of purification: the difference from Example 1 is that the volume concentration of ethanol is 90%, the liquid-solid ratio of sodium carboxymethyl cellulose and the ethanol solution is 1:8, the in-bottle liquid temperature is 50℃, the magnetic stirring speed is 200 rpm, the time is 50 min, after the reflux is completed, it is placed for 1 h, the supernatant is poured out, and the precipitate is reserved.

[0072] Third step of purification: the difference from Example 1 is that the liquid-solid ratio of sodium carboxymethyl cellulose and the ethanol solution is 1:5, the in-bottle liquid temperature is 30℃, the magnetic stirring speed is 100 rpm, the time is 20 min, after the reflux is completed, it is placed for 0.5 h, the supernatant is poured out, and the precipitate is reserved.

[0073] S12, same as Example 1.

[0074] S13, preparation of calcium carboxymethyl cellulose: 10 g of the precipitate of sodium carboxymethyl cellulose after the dehydration purification treatment is added into 100 mL of a sterile calcium chloride solution, the magnetic stirring is started and kept at a speed of 500 rpm, a condenser tube is connected to the mouth of the flask, the electric heating jacket is started, and the flask is kept at an in-bottle liquid temperature of 30℃, and the stirring is continuously kept for 3 h. The completed reaction suspension is poured into a filtration device for filtration, and the filter residue is collected. Then the filter residue is washed with 100 mL of anhydrous ethanol for 5 times to remove the residual calcium chloride.

[0075] Vacuum drying and ethanol recovery are the same as Example 1.

[0076] The preparation method of the injection filler is different from Example 1 in that the raw materials and the amount are different, specifically: polycaprolactone microspheres 600 g, calcium carboxymethyl cellulose (prepared by the above method) 80 g, polyvinylpyrrolidone 15 g, potassium dihydrogen phosphate 1 g, sodium phosphate dibasic 3 g, sodium chloride 30 g and water 2271 g, and the rest is the same.

[0077] Example 3

[0078] The present embodiment provides a cross-linked carboxymethyl cellulose, an injection filler and a preparation method thereof, wherein the preparation method of the cross-linked carboxymethyl cellulose comprises the following steps:

[0079] S11, purification of sodium carboxymethyl cellulose:

[0080] First step of purification: the difference from example 1 is that the volume concentration of ethanol is 78%, the pH value of the ethanol solution is 1.3, the material-liquid ratio of sodium carboxymethyl cellulose and the ethanol solution is 1:5, the bottle-liquid temperature is 70℃, the magnetic stirring speed is 500rpm, the time is 10min, after the reflux is finished, it is placed for 1h, the supernatant is poured out, and the precipitate is reserved.

[0081] 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 and the ethanol solution is 1:7, the bottle-liquid temperature is 50℃, the magnetic stirring speed is 300rpm, the time is 40min, after the reflux is finished, it is placed for 1.5h, the supernatant is poured out, and the precipitate is reserved.

[0082] Third step of purification: the difference from example 1 is that the material-liquid ratio of sodium carboxymethyl cellulose and the ethanol solution is 1:6, the bottle-liquid temperature is 40℃, the magnetic stirring speed is 200rpm, the time is 50min, after the reflux is finished, it is placed for 2h, the supernatant is poured out, and the precipitate is reserved.

[0083] S12, same as example 1.

[0084] S13, preparation of calcium carboxymethyl cellulose: 10g of the precipitate of sodium carboxymethyl cellulose after the dehydration purification treatment is added into 30mL of sterile calcium chloride solution, the magnetic stirring is started and kept at 100rpm, a condenser is connected to the mouth of the flask, the electric heating jacket is started, the bottle-liquid temperature is kept at 70℃, and the stirring is continued for 1h. The finished suspension is poured into a filtration device for filtration, and the filter residue is collected. Then the filter residue is washed with 100mL of anhydrous ethanol for 5 times to remove the residual calcium chloride.

[0085] Vacuum drying and ethanol recovery are the same as example 1.

[0086] The preparation method of the injection filler is different from example 1 in that the raw materials and the amount are different, specifically: polylactic acid microspheres 780g, calcium carboxymethyl cellulose (prepared by the above method) 90g, sodium hyaluronate 20g, potassium dihydrogen phosphate 0.6g, sodium phosphate dibasic 5.4g, glycerol 30g and water 2074g, and the rest is the same as example 1.

[0087] Example 4

[0088] Example 4 is different from example 1 in that the amount of calcium carboxymethyl cellulose is 105g, the amount of water is 1990g, and the rest is the same as example 1.

[0089] Comparative Example 1

[0090] The difference between Comparative Example 1 and Example 1 is that no carboxymethyl cellulose calcium is added, but the thickening agent carboxymethyl cellulose sodium is purified by the purification step described in the example, and the rest is the same as Example 1.

[0091] Comparative Example 2

[0092] The difference between Comparative Example 2 and Example 1 is that carboxymethyl cellulose calcium is directly prepared from carboxymethyl cellulose sodium, and the purification step is omitted, and the rest is the same as Example 1.

[0093] I. Performance Research

[0094] 1.1 Analyze the molecular weight and its distribution, ethanol residue, microorganisms and endotoxin content of carboxymethyl cellulose sodium before and after purification, and the results are shown in Table 1.

[0095] The weight average molecular weight and molecular weight distribution of carboxymethyl cellulose sodium samples are determined by gel permeation chromatography (GPC). The ethanol residue is detected according to the residual solvent determination method specified in the 2020 edition of the People's Republic of China Pharmacopoeia Part Three 0861. Microorganisms are detected according to the sterility test method in the 2020 edition of the People's Republic of China Pharmacopoeia Part Three 1101; Endotoxin is checked according to the gel limit method in the 2020 edition of the People's Republic of China Pharmacopoeia Part Three 1143 Bacterial Endotoxin Test.

[0096] Table 1 Effect of purification process on molecular weight of carboxymethyl cellulose sodium

[0097]

[0098] As shown in Table 1, using gradient concentration ethanol (75% to 80%, 85% to 90%, 100%) purification treatment can kill microorganisms in carboxymethyl cellulose sodium, reaching the sterile raw material level, and can significantly reduce the endotoxin content, so that the microorganism and endotoxin content of carboxymethyl cellulose sodium meets the production of medical and beauty filling products. The molecular weight of the purified carboxymethyl cellulose sodium does not decrease significantly, indicating that the purification process does not cause degradation of the material, and the ethanol residue content meets the residual solvent limit requirements specified in the Chinese Pharmacopoeia.

[0099] 1.2 Frequency scanning tests were performed on the fillers prepared in Examples 1-4 and Comparative Examples 1-2 using a rheometer. The G' (elastic modulus), G'' (viscous modulus), and η* (composite viscosity) data at 0.688 Hz were compared. The test temperature was set at 25℃, and the clamping distance 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. A 27G injection needle was selected, and the extrusion speed was set to 30 mm / min. The results are shown in Table 2.

[0100] Table 2. Test results of filler rheological properties and extrusion force

[0101]

[0102] Table 2 shows that adding calcium carboxymethyl cellulose can significantly increase the physical support effect of the filler. As the calcium carboxymethyl cellulose content increases, the composite viscosity and elastic modulus of the filler increase, and the pushing force increases.

[0103] II. Animal Experiments

[0104] 1. Test Item Information

[0105] Table 3. Test Item Information

[0106]

[0107] 2. Experimental grouping and drug administration

[0108] Thirty female SD rats, 6-8 weeks old and weighing 180-220 g, were randomly selected for the experiment after a 12-day acclimatization period. Six groups of four animals were formed based on the test sample. Hair at the injection site was carefully removed with a razor the day before injection.

[0109] Test administration: such as Figure 4 As shown, each animal was injected with one test sample and one negative control. The test sample was injected at four sites (blue), with 0.2 mL injected at each site. Each animal was injected with one negative control site (red) for subsequent histopathological scoring comparison. Four sampling time points were set: 7 days, 1 month, 3 months, and 6 months.

[0110] 3. Test Indicators

[0111] 3.1 Measurement of the appearance and morphology of the filler

[0112] External dimensional measurements were performed at 7 days, 1 month, 3 months, and 6 months prior to dissection to calculate changes in implant volume (mm). 3 The volume on the day of injection was 200±0.5mm. 3 The filling effect was evaluated. The results are shown in Table 4 and...Figure 5 .

[0113] Table 4 Subcutaneous injection effect of rats

[0114]

[0115] As shown in Table 4, compared with Comparative Example 1, the carboxymethyl cellulose calcium prepared in Example 1 can significantly prolong the filling period when added to the injection filler; as shown by the comparison between Example 1 and Example 4, with the increase of the amount of carboxymethyl cellulose calcium, the filling time of the injection filler is also increased.

[0116] Figure 5 As can be seen, the fillers in Examples 1-4 can still be seen after 6 months of injection, while Comparative Example 1 completely disappears and becomes invisible.

[0117] 3.2 Tissue sampling and observation

[0118] After administration, the tissues at the injection site of rats in each group were sampled (the sampling area was about 2 cm 2 ) at 7 days, 1 month, 3 months and 6 months, respectively. One animal was dissected at each time point in each group. The filling site and the surrounding tissues should be cut according to the injection administration diagram and fixed on the specimen measurement board. A single-lens reflex camera should be fixed on the table top support and the position, angle and light should be consistent for each shooting.

[0119] 3.3 Histopathology

[0120] After administration, the tissues at the filling site were sampled at 7 days, 1 month, 3 months and 6 months, respectively. The tissues at the filling site were subjected to HE staining, MASSON staining and Sirius red staining (see Figures 7-9 ), and histopathological detection was performed. The reaction of the filling agent to the tissues after administration was evaluated, and the results are shown in Table 5.

[0121] Tissue reaction = total sum of cell types * 2 + total sum of reaction types. Skin tissue irritation level: 0.0-2.9 is no irritation or very slight irritation, 3.0-8.9 is mild irritation, 9.0-15.0 is moderate irritation, and >15 is severe irritation.

[0122] Table 5 Results of tissue reaction score

[0123]

[0124] As shown in Table 3, compared with Comparative Example 2, the filler prepared from the purified hydroxymethyl cellulose sodium in Example 1 can significantly reduce the inflammatory reaction of the tissues by removing the intracellular toxins, and show no irritation or very slight irritation after 6 months.

[0125] Figures 6-9 Anatomical and staining pictures of the tissue of the filling site at the 6th month after administration. Figures 5-8 It can be seen that after the injection of the injection filler prepared by the embodiment 1 of the present application into the animals, the anatomical picture can obviously show the filling effect (Fig. 1) Figure 6 ), and the staining picture also shows the gel-like filler (Fig. 2) Figures 7-9 ); and the physical filling volume of the comparative example 1 almost disappears (Fig. 3) Figure 6 ), and no gel can be seen in the staining picture (Fig. 4) Figures 7-9 .

[0126] The above merely describes the preferred embodiments of the present application, and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A process for the preparation of cross-linked carboxymethylcellulose, characterized in that: The method comprises the following steps: S1, dissolving anhydrous calcium chloride in anhydrous ethanol, and then performing sterilization filtration to obtain a sterile calcium chloride solution; S2, adding the dehydrated and purified carboxymethyl cellulose sodium to the sterile calcium chloride solution, and stirring and reacting for 1-3 hours; the mass-volume ratio of the carboxymethyl cellulose sodium to the calcium chloride solution is 1:3-10, and the mass concentration of the calcium chloride solution is 10%; The dehydrated and purified treatment method is: the carboxymethyl cellulose sodium is sequentially subjected to gradient purification with 75%-80%, 85%-90% and anhydrous ethanol, each step is performed under the conditions of heating stirring and condensation reflux, and after the purification is completed, the purified carboxymethyl cellulose sodium is obtained by vacuum drying; In the first purification treatment, in addition to adding the 75%-80% ethanol solution, 0.5-1 mol / L hydrochloric acid is also added to adjust the pH value of the ethanol solution to 1-1.5; In each purification treatment, the heating temperature is 30-70°C, the stirring speed is 100-500 rpm, and the time is 10-60 min; after each reflux is completed, it is statically placed for 0.5-2 hours, the supernatant is removed, and the precipitate is retained for the next purification treatment; in each purification treatment, the solid-liquid ratio of the carboxymethyl cellulose sodium to the ethanol solution is 1:5-10; S3, after the reaction is completed, suction filtration is performed, the filter residue is collected and washed with anhydrous ethanol to remove the residual calcium chloride, and then vacuum drying is performed to obtain the crosslinked carboxymethyl cellulose.

2. Use of cross-linked carboxymethylcellulose for the manufacture of a injectable filler, characterized in that: The crosslinked carboxymethyl cellulose is prepared by the preparation method of claim 1.

3. Use according to claim 2, wherein: The components of the injection filler include high molecular microspheres, crosslinked carboxymethyl cellulose, a thickening agent, a pH regulator, an osmotic pressure regulator and water.

4. Use according to claim 3, wherein: According to 100% by mass, the high molecular microspheres are 20%-28%, the crosslinked carboxymethyl cellulose is 2%-3.5%, the thickening agent is 0.5%-1%, the pH regulator is 0.13%-0.2%, the osmotic pressure regulator is 1%, and the balance is water.

5. Use according to claim 4, wherein: The high molecular microspheres are poly-L-lactic acid microspheres or polycaprolactone microspheres; The thickening agent is one or a combination of several of carboxymethyl cellulose sodium, sodium hyaluronate, polyvinylpyrrolidone and hydroxypropyl methyl cellulose; The pH regulator is a conjugate acid-base pair composed of a phosphate or a citrate; The osmotic pressure regulator is glycerol or sodium chloride.

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