An injectable filling composition and a method for its preparation

Abstract

The present application relates to a kind of injectable filling composition and its preparation method, the filling composition is crosslinked hyaluronic acid hydrogel, the percentage of the mass of crosslinked hyaluronic acid in its volume in crosslinked hyaluronic acid hydrogel is 0.1-2w / v%;It also includes hydroxyl antioxidant and growth factor;The hydroxyl antioxidant includes at least one of catechin, quinic acid, tannic acid, gallic acid, vitamin E, vitamin B compound;The injectable filling composition of the present application can significantly reduce the reabsorption rate of transplanted adipocyte, improve the quality retention rate of adipose tissue, and at the same time improve the survival rate of autologous transplanted fat.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to an injectable filling composition and a preparation method thereof. BACKGROUND

[0002] The main means for present plastic filling is autologous fat filling and prosthesis filling. Autologous fat filling is to extract the fat of the beauty seeker from the body, separate and purify the active fat cells, and then inject the purified active fat cells into the corresponding part. Autologous fat tissue is a long-standing filling material in plastic surgery. Autologous fat transplantation is not only used for facial and body plastic and cosmetic surgery, but also used to fill the tissue defects caused by burn scar, cleft palate and pharynx, trauma, congenital deformity and tumor surgery, etc. The advantages of this method are no foreign body rejection, small trauma and wide material source. However, its disadvantages are also obvious. First, the transplanted fat cannot guarantee 100% survival rate, and the autologous fat is quickly absorbed by the body; in addition, autologous fat filling surgery needs secondary filling for adjustment, which cannot achieve the expected result at one time and takes a long time; moreover, the liposuction process may also bring additional risks. Another common method is prosthesis implantation filling. The advantages of prosthesis implantation are short operation time, one-time completion, immediate and long-lasting effect, and low requirement for the physical quality of the beauty seeker. However, the prosthesis is a foreign body, which has a certain probability of capsular contracture side effects. In addition, a long period of recuperation is required after prosthesis implantation, otherwise problems such as prosthesis displacement, prosthesis exposure and asymmetry may occur, which brings inconvenience to the life of the beauty seeker and may even need to remove the prosthesis again, causing additional pain. In summary, the existing filling surgery has many defects, which is a heavy burden on the time and economy of the beauty seeker. It is difficult to achieve a relatively favorable balance between safety and effectiveness. Therefore, the present field needs to provide a more effective injectable filling composition to provide a more convenient, fast and safe long-acting filling product for the beauty seeker.

[0003] Based on the above technical problems that autologous fat transplantation cannot guarantee 100% survival rate and common hyaluronic acid fillers degrade quickly and autologous fat is quickly absorbed, the present application provides an injectable filling composition and a preparation method thereof. SUMMARY

[0004] The injectable filling composition of the present application is a cross-linked hyaluronic acid filling composition, which can significantly reduce the reabsorption rate of transplanted fat cells, improve the quality retention rate of adipose tissue, and at the same time improve the survival rate of autologous transplanted fat.

[0005] In order to achieve the above purposes, the present application realizes the following technical scheme: the filling composition is a cross-linked hyaluronic acid hydrogel, the percentage of the mass of cross-linked hyaluronic acid to the volume of the cross-linked hyaluronic acid hydrogel is 0.1-2 w / v%; and further comprises a hydroxyl antioxidant and a growth factor;

[0006] The hydroxyl antioxidant includes at least one of catechin, quinic acid, tannic acid, gallic acid, vitamin E, and vitamin B compound.

[0007] Further, in the cross-linked hyaluronic acid hydrogel, the ratio of the amount of the hyaluronic acid, the growth factor and the hydroxyl antioxidant is 100 g:2000-30000 IU:0.1-5 g, calculated based on the amount of the raw material hyaluronic acid.

[0008] Further, in the cross-linked hyaluronic acid hydrogel, the mass ratio of the cross-linking agent to the hyaluronic acid is (1-15):(1-5), calculated based on the amount of the raw material hyaluronic acid. The modification ratio of the cross-linked hyaluronic acid in the cross-linked hyaluronic acid hydrogel is (2.5±1.0)%, and the cross-linking degree is controlled by the length and condition of the cross-linking reaction (the cross-linking degree is referred to as the modification ratio in the case of a smaller cross-linking degree). In the present application, the cross-linking degree of the hydrogel is smaller. If the cross-linking degree is too high, the hydrogel is harder and more difficult to inject.

[0009] Further, the cross-linking agent is one or more of ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and divinyl sulfone, and preferably is 1,4-butanediol diglycidyl ether. The cross-linking agent is removed during production.

[0010] Further, the hyaluronic acid is hyaluronic acid or a salt thereof with a molecular weight of not less than 500,000 Daltons, and preferably is hyaluronic acid or a salt thereof with a molecular weight of 600-1,000,000 Daltons. The hyaluronic acid salt is, for example, but not limited to, sodium hyaluronate, magnesium hyaluronate, calcium hyaluronate, or zinc hyaluronate. The growth factor is one of rhEGF, PRP, VEGF, and SVF. The hydroxyl antioxidant is a combination of tannic acid and vitamin E.

[0011] The preparation method of the injectable filling composition is characterized by comprising the following steps:

[0012] (1) The hydroxyl antioxidant, the growth factor and the cross-linking agent are added in a proportion in an alkaline aqueous solution with a pH>10, and are fully stirred, dissolved and sterilized, and then the hyaluronic acid is added to react at 25-45°C for 2-24 h to form a cross-linked hyaluronic acid hydrogel block;

[0013] (2) After being taken out, the cross-linked hyaluronic acid hydrogel block is placed in an acidic aqueous solution with a pH<5 at room temperature for 2-24 h, and then is taken out, washed and dialyzed until the concentration of the cross-linked hyaluronic acid hydrogel is ≤20 mg / mL and the pH is in the physiological pH range of the human body, and then is packaged.

[0014] Further, the preparation method further comprises: extruding the product after step 2 under a pressure of 2-8 KG and through a porous filter with a pore size of 50-2000 microns to obtain a micro-particle gel, and filling the micro-particle gel into a sterile container to obtain the injectable filling composition.

[0015] Further, the alkaline aqueous solution is a 0.5-4M sodium hydroxide aqueous solution, and the acidic aqueous solution is a 0.1-2M hydrochloric acid aqueous solution.

[0016] Beneficial technical effects:

[0017] In the preparation of the cross-linked hyaluronic acid hydrogel, the growth factor and the hydroxyl antioxidant are added, and the combination of the two can significantly reduce the reabsorption rate of the autologous transplanted fat cells, improve the quality retention rate of the adipose tissue, prolong the degradation time, maintain the cross-linked state of the hyaluronic acid, and at the same time, improve the survival rate of the autologous transplanted fat, increase and prolong the effective period of filling. It can be applied to face, chest and other fillings. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Hematoxylin-eosin staining results of the adipose tissue of the postoperative control group and the adipose tissue of the test of Example 1 in the first week, the second week and the fourth week; wherein A1, A2 and A3 are the first, second and fourth week staining results of the adipose tissue of the control group, and B1, B2 and B3 are the first, second and fourth week staining results of the adipose tissue of the test of Example 1. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative, but not as any limitation on the present application and its application or use. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0020] Unless otherwise specifically stated, the numerical values set forth in these examples are not limiting of the scope of the present application. Techniques, methods, and materials known to those of ordinary skill in the related art can not be discussed in detail herein, but should be considered within the scope of the disclosure. In all examples shown and discussed herein, any specific values should be interpreted as merely illustrative and not as a limitation. Thus, other examples of the exemplary embodiments can have different values.

[0021] The raw materials used in the injectable filling composition of the present application are as follows:

[0022] "Hyaluronic acid" refers to a linear high molecular weight polysaccharide consisting of N-acetylglucosamine-D-glucuronic acid as a disaccharide unit. For example, but not limited to, hyaluronic acid produced by bacterial fermentation or extracted from tissues, preferably hyaluronic acid dry powder produced by bacterial fermentation. The hyaluronic acid used in the present application is a hyaluronic acid salt, such as but not limited to sodium hyaluronate, magnesium hyaluronate, calcium hyaluronate or zinc hyaluronate, etc. The molecular weight of hyaluronic acid is not less than 500,000 Daltons, preferably 60-100,000 Daltons.

[0023] "growth factor" refers to a cell growth factor that can promote fat growth, and the growth factor used in the present application is selected from rhEGF, PRP, VEGF or SVF.

[0024] "hydroxyl antioxidant" refers to a hydroxyl compound used to capture free radicals, and at least one of catechin, quinic acid, tannic acid, gallic acid, vitamin E, and vitamin B compounds is selected, preferably tannic acid and vitamin E are compounded in a mass ratio of (3-5): 1.

[0025] "crosslinking agent" refers to a compound used to crosslink hyaluronic acid, and the crosslinking agent used in the present application is one of glycidyl ether compounds, divinyl sulfone, and carbimide compounds, preferably the crosslinking agent is 1,4-butanediol diglycidyl ether, and the weight ratio of crosslinking agent to hyaluronic acid is (1-15):(1-5).

[0026] Example 1

[0027] Preparation of an injectable filling composition:

[0028] (1) In a preparation container, add 1000 mL of 0.5 M NaOH solution, then add 100.0 g of butanediol diglycidyl ether, 25000 IU of rhEGF, 1.0 g of hydroxyl antioxidant (tannic acid and vitamin E compounded in a ratio of 4:1), fully stir to completely dissolve and sterilize, add 100.0 g of hyaluronic acid dry powder produced by bacterial fermentation, and react at 35°C for 12 h to form a crosslinked hyaluronic acid hydrogel block;

[0029] (2), take out the crosslinked hyaluronic acid hydrogel block and add it to 0.2 M 5000 mL hydrochloric acid solution, and place it at room temperature for 14 h, pour off the hydrochloric acid aqueous solution, and wash and dialyze using a physiological balanced solution (ensure washing 10 times within 5 days);

[0030] (3) The washed cross-linked hyaluronic acid hydrogel block is placed in an extrusion device, and a 5 kg positive pressure is used to extrude it through a porous filter with a pore size of 500 μm to obtain a microparticle gel. The injectable filling composition is obtained by filling under sterile conditions, wherein the concentration of the cross-linked hyaluronic acid hydrogel is ≤ 20 mg / mL (i.e., the mass percentage of cross-linked hyaluronic acid in the cross-linked hyaluronic acid hydrogel is ≤ 2 w / v %).

[0031] Example 2

[0032] Preparation of an injectable filling composition:

[0033] (1) 1000 mL of 0.2M NaOH solution is added to a preparation container, followed by 120.0 g of butanediol diglycidyl ether, 15000 IU of rhEGF, and 0.5 g of a hydroxyl antioxidant (tannic acid). The mixture is thoroughly stirred to completely dissolve and sterilized, and 100.0 g of hyaluronic acid dry powder produced by bacterial fermentation is added for reaction at 35°C for 10 h to form a cross-linked hyaluronic acid hydrogel block;

[0034] (2) The cross-linked hyaluronic acid hydrogel block is taken out and added to 5000 mL of 0.1M hydrochloric acid solution, and placed at room temperature for 16 h. The hydrochloric acid aqueous solution is poured off, and the cross-linked hyaluronic acid hydrogel block is washed and dialyzed (10 times of washing within 5 days) using a physiological balanced solution;

[0035] (3) The washed cross-linked hyaluronic acid hydrogel block is placed in an extrusion device, and a 5 kg positive pressure is used to extrude it through a porous filter with a pore size of 800 μm to obtain a microparticle gel. The injectable filling composition is obtained by filling under sterile conditions, wherein the concentration of the cross-linked hyaluronic acid hydrogel is ≤ 20 mg / mL (i.e., the mass percentage of cross-linked hyaluronic acid in the cross-linked hyaluronic acid hydrogel is ≤ 2 w / v %).

[0036] Example 3

[0037] Preparation of an injectable filling composition:

[0038] (1) 1000 mL of 0.2M NaOH solution is added to a preparation container, followed by 130.0 g of butanediol diglycidyl ether, 10000 IU of rhEGF, and 0.2 g of a hydroxyl antioxidant (vitamin E). The mixture is thoroughly stirred to completely dissolve and sterilized, and 100.0 g of hyaluronic acid dry powder produced by bacterial fermentation is added for reaction at 35°C for 8 h to form a cross-linked hyaluronic acid hydrogel block;

[0039] (2) The cross-linked hyaluronic acid hydrogel block was taken out and added to 0.1 M 5000 mL hydrochloric acid solution, and was left at room temperature for 20 h. The hydrochloric acid solution was poured out, and the cross-linked hyaluronic acid hydrogel block was washed and dialyzed (10 times of washing within 5 days) using a physiological balanced solution;

[0040] (3) The washed cross-linked hyaluronic acid hydrogel block was placed in an extrusion device, and was extruded through a 400 μm pore size filter using a 5 kg positive pressure to obtain a particulate gel. The particulate gel was filled under sterile conditions to obtain the injectable filling composition of the present example, wherein the concentration of the cross-linked hyaluronic acid hydrogel was ≤ 20 mg / mL (i.e. the percentage of the mass of the cross-linked hyaluronic acid to the volume of the cross-linked hyaluronic acid hydrogel was ≤ 2 w / v %).

[0041] Comparative Example 1

[0042] The filling composition of the present comparative example was prepared in the same manner as in Example 1, except that the hydroxyl antioxidant and the growth factor were not added.

[0043] Comparative Example 2

[0044] The filling composition of the present comparative example was prepared in the same manner as in Example 1, except that the cross-linking agent was not added.

[0045] Comparative Example 3

[0046] The filling composition of the present comparative example was prepared in the same manner as in Example 1, except that the hydroxyl antioxidant was not added.

[0047] Comparative Example 4

[0048] The filling composition of the present comparative example was prepared in the same manner as in Example 1, except that the growth factor was not added.

[0049] Comparative Example 5

[0050] The filling composition of the present comparative example was prepared in the same manner as in Example 1, except that the hydroxyl antioxidant was vitamin C.

[0051] The products of the above examples and comparative examples were applied to animal tests.

[0052] Male SD rats were used as experimental subjects, and were divided into a control group (a physiological saline group) and a test group (the filling compositions of the above examples and comparative examples). The experimental rats were anesthetized with 10% chloral hydrate intraperitoneally, and a circular mark area of about 1.5 cm was made on the back of each rat. The skin of the rat was incised under sterile conditions to expose the inguinal fat pads, and the bilateral inguinal fat pads were completely cut. After the surface envelope and blood vessels were removed, the removed fat tissue was cut into fat particles of about 2 mm in size (about 1 mL per rat) for use.

[0053] The fat cells of the control group were mixed with physiological saline at 1:1, and the fat cells of the test group were mixed with the micro-particle gel filling compositions of the above examples and comparative examples at 1:1, respectively, and each was left for 30 min for standby.

[0054] The back of the rat was disinfected and a small incision was made at the edge of the line. A 1.5 cm diameter subcutaneous pocket was formed by blunt dissection under the skin in the line area. After the treatment, the treated fat tissue and solution mixture were injected into the separated subcutaneous pocket using a 2 mL syringe, respectively. The injection volume was about 2 mL. Finally, the back incision was sutured with a purse string, the incision was disinfected again and aureomycin eye ointment was applied to prevent infection. Autologous fat transplantation was performed on the two groups of rats at the same time, and the specimens were taken out at three time points of 1, 2 and 4 weeks after the operation. The data were analyzed by t test of paired data using Spss 11.0 statistical software.

[0055] Fat tissue mass retention rate: The removed tissue was placed on a sterile operating table, and the fascia covering its surface was removed. The wet weight was determined by weighing with an ultra-precision electronic balance. The fat tissue mass retention rate was calculated as follows: wet weight of the tissue after transplantation / tissue wet weight before transplantation x 100%. The results are shown in Table 1.

[0056] Fat tissue volume retention rate: The weighed fat tissue was placed in a 5 mL graduated cylinder containing 3 mL phosphate buffer, and the height of the rising liquid level in the graduated cylinder was recorded in sequence. The height difference was the volume of the removed fat tissue. The fat tissue volume absorption rate at different time points after transplantation was calculated and recorded as follows: (volume of the tissue before transplantation - volume of the tissue after transplantation) / volume of the tissue before transplantation x 100%. The results are shown in Table 2.

[0057] Histological observation: The removed fat tissue was fixed with 10% formalin for 24 h, fixed with 4% paraformaldehyde, waxed, embedded, and 2 μm thick continuous sections were cut, HE stained, and observed under a light microscope for the morphological structure of the fat tissue. The results are shown in Figure 1 , Figure 1 The hematoxylin-eosin staining results of the fat tissue of the postoperative control group and the test of Example 1 at the first week, the second week and the fourth week, respectively, are shown in A1, A2 and A3, respectively, which are the first, second and fourth week staining results of the fat tissue of the control group, and B1, B2 and B3, respectively, which are the first, second and fourth week staining results of the fat tissue of the test of Example 1.

[0058] Table 1 Fat tissue mass retention rate at 1 week, 2 weeks and 4 weeks after the operation, respectively

[0059]

[0060]

[0061] Table 2 Volume retention rate of adipose tissue at 1 week, 2 weeks, and 4 weeks after surgery, respectively

[0062]

[0063] From Table 1, Table 2, and Figure 1 It can be seen that the present application can significantly reduce the reabsorption rate of transplanted adipocytes, improve the quality retention rate of adipose tissue, and thus improve the survival rate of autologous transplanted fat. In addition, HE staining shows that the control group shows more cords and disordered fat structure. The adipocytes treated by Example 1 have more complete morphology, lower fibrosis degree, and relatively neat arrangement, which further indicates that the filling composition of the present application has the effect of significantly reducing the reabsorption rate of transplanted adipocytes, improving the quality retention rate of adipose tissue, and thus improving the survival rate of autologous transplanted fat.

[0064] Although the vitamin C used in Comparative Example 5 also has good quality and volume retention effect, compared with the product of Example 1 of the present application, the standard deviation of the quality and volume retention rate of the antioxidant prepared by compounding tannic acid and vitamin E in the present application is smaller, which indicates that the quality and volume stability of the product after injection is better.

[0065] The above description is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacement or change according to the technical solution and inventive concept of the present application within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for preparing an injectable filler composition, characterized in that, Includes the following steps: (1) Add hydroxyl antioxidants, growth factors and cross-linking agents to an alkaline aqueous solution with pH > 10 according to the ratio, stir thoroughly to dissolve and sterilize, then add hyaluronic acid and react at 25-45℃ for 2-24h to form cross-linked hyaluronic acid hydrogel blocks. (2) After taking it out, place it in an acidic aqueous solution with pH < 5 at room temperature for 2-24 hours. Then, take out the cross-linked hyaluronic acid hydrogel block, wash and dialyze it until the concentration of the cross-linked hyaluronic acid hydrogel is ≤ 20 mg / mL and the pH is within the range of human physiological pH value before packaging. The filling composition is a cross-linked hyaluronic acid hydrogel, wherein the mass-to-volume percentage of cross-linked hyaluronic acid in the cross-linked hyaluronic acid hydrogel is 0.1-2 w / v%. The hydroxyl antioxidant is a mixture of tannic acid and vitamin E in a mass ratio of 4:

1. The hyaluronic acid is selected with a molecular weight of not less than 500,000 Daltons, or can be replaced with hyaluronic acid salts with a molecular weight of not less than 500,000 Daltons. The growth factor is rhEGF. The cross-linked hyaluronic acid hydrogel contains, based on the amount of hyaluronic acid used as raw material, a ratio of hyaluronic acid, growth factor, and hydroxyl antioxidant of 100g:25000IU:1.0g. In the cross-linked hyaluronic acid hydrogel, the mass ratio of the cross-linking agent to the hyaluronic acid is (1-15):(1-5), calculated based on the amount of hyaluronic acid used as raw material.

2. The method for preparing an injectable filler composition according to claim 1, characterized in that, The crosslinking agent is one or more of the following: ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and divinyl sulfone.

3. The method for preparing an injectable filler composition according to claim 1, characterized in that, The preparation method further includes extruding the product from step (2) under a pressure of 2-8 kg and extruding a porous filter with a pore size of 50 μm-2000 μm to form a microparticle gel, which is then filled under aseptic conditions to obtain an injectable filling composition.

4. The method for preparing an injectable filler composition according to claim 1, characterized in that, The alkaline aqueous solution is a 0.5-4M sodium hydroxide aqueous solution, and the acidic aqueous solution is a 0.1-2M hydrochloric acid aqueous solution.

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