Preparation method of a hyaluronic acid filler for injection

Abstract

The present invention discloses a preparation method of a hyaluronic acid filler for injection. An emulsion with uniform particle size is prepared by microfluidic technology. In the emulsion, hyaluronic acid undergoes a crosslinking reaction to form hyaluronic acid microspheres. Among them, the outer phase in the process of emulsion preparation is an oil phase, and the inner phase is an aqueous phase containing hyaluronic acid. Finally, the hyaluronic acid microspheres and other active ingredients are added to the hyaluronic acid stock solution to obtain the hyaluronic acid filler for injection. After swelling, filling, and sterilizing the hyaluronic acid filler for injection with PBS buffer solution, the finished product of the hyaluronic acid filler for injection is obtained. The present invention adopts microfluidic technology, which can prepare hyaluronic acid microspheres with uniform particle size, improve the quality of the hyaluronic acid filler for injection, has better stability, is not prone to immune reactions, and has a synchronous decomposition and absorption effect, will not show local collapse or protrusion, improves the shaping effect, enhances the treatment effect and satisfaction of patients, and promotes the development of aesthetic medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and specifically refers to a preparation method of an injectable hyaluronic acid filler. Background Art

[0002] Hyaluronic acid (HA) is a natural polysaccharide with good biocompatibility and biodegradability, and is widely used in filling facial wrinkles, enhancing lip shape, restoring facial contour, etc. However, traditional hyaluronic acid fillers have some problems, such as unstable effects, short durability, easy formation of nodules, and pain during injection.

[0003] The biphasic modified hyaluronic acid gel is a new type of filler with good effects and stability. Its main feature is the combination of cross-linked hyaluronic acid gel and uncross-linked hyaluronic acid gel to form a biphasic modified structure. This structure can improve the stability, persistence and shaping effect of the filler, and can reduce pain and discomfort. Therefore, the research and development of the biphasic modified hyaluronic acid gel technology can provide a safer, more effective and sustainable filler choice for the treatment in the fields of facial beauty, medical beauty, etc.

[0004] The existing Chinese patent with the publication number CN111840638B discloses a preparation method of an injectable cross-linked hyaluronic acid filler, which also obtains the injectable cross-linked hyaluronic acid filler by mixing cross-linked hyaluronic acid microspheres and hyaluronic acid gel. However, the hyaluronic acid microspheres are prepared by adding an aqueous phase of hyaluronic acid to an oil phase containing an emulsifier and then through high-speed shear dispersion. The particle size of the cross-linked hyaluronic acid microspheres prepared in this way is difficult to maintain uniform, and the filling dosage cannot be accurately controlled. When the hyaluronic acid filler prepared by using such hyaluronic acid microspheres with uneven particle sizes and hyaluronic acid gel is used, it is easy to have unstable filling effects, and protrusions or depressions are likely to occur at the filling site, thus extremely prone to immune reactions, and its safety is difficult to be effectively guaranteed. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of an injectable hyaluronic acid filler based on microfluidic technology, which can ensure that the particle size of the hyaluronic acid microspheres is uniform and controllable.

[0006] Another purpose of the present invention is to provide an injectable hyaluronic acid filler prepared by the above-mentioned preparation method of the injectable hyaluronic acid filler.

[0007] The present invention is achieved through the following technical solutions: A preparation method of an injectable hyaluronic acid filler, in which emulsions with uniform particle sizes are prepared in a two-phase flow by microfluidic technology, and cross-linking reactions occur to hyaluronic acid in the emulsions to form hyaluronic acid microspheres. Among them, the outer phase in the process of emulsion preparation is the oil phase, and the inner phase is the aqueous phase containing hyaluronic acid. Finally, the hyaluronic acid microspheres and other active ingredients are added to the hyaluronic acid stock solution to obtain the injectable hyaluronic acid filler.

[0008] The working principle of this technical solution is that the half-life of generally uncross-linked hyaluronic acid is relatively short, only 1-2 days. Therefore, in order to extend the degradation time of hyaluronic acid, chemical cross-linking is often used to cross-link hyaluronic acid molecular chains to form a more stable network structure. However, the gel particles formed by cross-linking are not uniform, and even after sieving, there are still edges and corners on the particle surface, which are likely to cause inflammatory reactions. The microsphere gel prepared in the present invention uses microfluidic technology. Each microsphere is an independent cross-linking system, ensuring the consistency of cross-linking conditions while guaranteeing the size. The prepared microspheres have a smooth surface and good uniformity. The length of the degradation time of sodium hyaluronate is closely related to its degree of chemical cross-linking, particle size, injection amount, injection site, and individual differences. The cross-linking agent used in the present invention is 1,4-butanediol diglycidyl ether. Under weak alkaline (pH = 7-9) conditions, the epoxy group preferentially reacts with the primary alcohol of sodium hyaluronate to form an ether bond. The stability of the ether bond is much higher than that of ester and amide bonds. Therefore, the clinical use time of the hyaluronic acid microspheres cross-linked by 1,4-butanediol diglycidyl ether under weak alkaline conditions can reach or exceed half a year.

[0009] In order to better implement the method of the present invention, further, its specific preparation process includes the following steps:

[0010] (1) Prepare an aqueous solution: Add hyaluronic acid or hyaluronate to physiological saline, and add an alkali, a surfactant, and a cross-linking agent, and stir evenly.

[0011] (2) Prepare an oil phase solution, add an emulsifier to the oil phase solution, and stir evenly to fully emulsify it to obtain an emulsion.

[0012] (3) Inject the prepared aqueous solution and oil phase solution into a microfluidic chip mold, control the flow rates and pressures of the aqueous solution and the oil phase solution, and form a hyaluronic acid emulsion with the inner phase being the aqueous phase and the outer phase being the oil phase under the shearing action of the outer phase.

[0013] (4) Adjust the pH of the aqueous phase of the hyaluronic acid emulsion, and let it stand at a certain temperature to cause a cross-linking reaction to form hyaluronic acid microspheres.

[0014] (5) Wash the cross-linked hyaluronic acid microspheres.

[0015] (6) Prepare the hyaluronic acid stock solution. Add the washed hyaluronic acid microspheres and other active ingredients into the hyaluronic acid stock solution and mix evenly to obtain the product.

[0016] To better implement the method of the present invention, further, the molecular weight of the hyaluronic acid or its salt is 900 - 1200 kDa, the diameter of the prepared hyaluronic acid microspheres is 40 - 1200 μm, and the particle size variation coefficient of the prepared hyaluronic acid microspheres is ≤ 10%.

[0017] To better implement the method of the present invention, further, the crosslinking agent added to the aqueous solution is at least one of carbodiimide, divinyl sulfone, ethylene glycol diglycidyl ether, 1,4 - butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, and epichlorohydrin, and the base added to the aqueous solution is at least one of sodium hydroxide and potassium hydroxide.

[0018] To better implement the method of the present invention, further, the surfactant added to the aqueous solution is a composite surfactant of Tween 20 and Tween 80, and the contents of Tween 20 and Tween 80 are the same, both being 1 - 10 wt%.

[0019] To better implement the method of the present invention, further, the oil - phase solution is liquid paraffin, and the emulsifier added is Span 80, and the content of Span 80 is 1 - 10 wt%.

[0020] To better implement the method of the present invention, further, the initial pH value of the aqueous solution prepared in step (1) is 10 - 13. The method for adjusting the pH of the aqueous phase inside the hyaluronic acid microspheres in step (4) is to use a semi - permeable membrane to coat the hyaluronic acid microspheres placed in the oil - phase solution, place them in physiological saline, and add an acid solution to the physiological saline. By using the semi - permeable membrane, the pH inside the aqueous phase inside the hyaluronic acid microspheres is adjusted to 7 - 9, so that the aqueous phase inside the hyaluronic acid microspheres meets the conditions for the cross - linking reaction. The acid solution is 1 wt% hydrochloric acid or an organic acid solution. The cross - linking reaction temperature is 10°C - 60°C, and the cross - linking reaction time is 0.1 - 100 h.

[0021] To better implement the method of the present invention, further, in step (5), wash three times with petroleum ether and physiological saline respectively, and perform filtration treatment using a filter screen.

[0022] To better implement the method of the present invention, further, the other active ingredients in step (6) are at least one of lidocaine hydrochloride, phosphate buffer solution, and physiological saline.

[0023] In order to better implement the method of the present invention, further, the concentration of the prepared hyaluronic acid stock solution in step (6) is 2 mg / ml, and the concentration of the cross-linked hyaluronic acid microspheres is 18 mg / ml.

[0024] A finished product of an injectable hyaluronic acid filler is prepared by the above-mentioned preparation method of the injectable hyaluronic acid filler. Then, the injectable hyaluronic acid filler is swollen with PBS buffer solution, canned, and sterilized to obtain a finished product of the injectable hyaluronic acid filler.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] (1) The injectable hyaluronic acid filler prepared by the present invention, compared with the traditional hyaluronic acid filler, can prepare hyaluronic acid microspheres with uniform particle size and uniform microsphere cross-linking degree due to the use of microfluidic technology. This can improve the quality of the injectable hyaluronic acid, making its stability better, having a longer maintenance time, not easily causing immune reactions, and enabling precise control of the tissue filling dosage.

[0027] (2) For the injectable hyaluronic acid filler prepared by the present invention, since the cross-linking reaction occurs inside the hyaluronic acid microspheres, it can ensure the uniformity of the cross-linking of hyaluronic acid. In this way, after the hyaluronic acid microspheres enter the human body, they have a synchronous decomposition and absorption effect, without local collapse or protrusion, improving the plastic surgery effect.

[0028] (3) The injectable hyaluronic acid provided by the present invention meets the needs of the aesthetic medicine field for safer and more effective injectable filling materials, improves the treatment effect and satisfaction of patients, and promotes the development of aesthetic medicine. Description of the Drawings

[0029] By reading the detailed description of the non-restrictive embodiments with reference to the following drawings, other features, purposes, and advantages of the present invention will become more obvious:

[0030] Figure 1 It is a diagram of the cross-linking situation of hyaluronic acid microspheres in Group A in Example 2 of the present invention;

[0031] Figure 2 It is a diagram of the cross-linking situation of hyaluronic acid microspheres in Group B in Example 2 of the present invention;

[0032] Figure 3 It is a diagram of the cross-linking situation of hyaluronic acid microspheres in Group C in Example 2 of the present invention;

[0033] Figure 4 It is the situation of the combination and cross-linking of hyaluronic acid microspheres at a cross-linking temperature of 37°C in Example 3 of the present invention;

[0034] Figure 5 This shows the situation where hyaluronic acid microspheres merge and crosslink at a crosslinking temperature of 50 °C in Example 3 of the present invention;

[0035] Figure 6 This shows the situation where hyaluronic acid microspheres merge and crosslink at a crosslinking temperature of 55 °C in Example 3 of the present invention. Detailed implementation manners

[0036] The embodiments of the present invention will be described in detail below. The examples are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements with the same or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary and are only used to explain the present invention and should not be construed as limiting the present invention.

[0037] Example 1:

[0038] This example provides a preparation method of an injectable hyaluronic acid filler. A uniform-sized emulsion is prepared in a two-phase flow by microfluidic technology. Crosslinking reaction occurs to hyaluronic acid in the emulsion to form hyaluronic acid microspheres. Among them, the continuous phase in the emulsion preparation process is the oil phase, and the dispersed phase is the aqueous phase containing hyaluronic acid. Finally, the hyaluronic acid microspheres and other active ingredients are added to the hyaluronic acid stock solution to obtain the injectable hyaluronic acid filler.

[0039] The specific preparation process is as follows:

[0040] (1) Prepare the aqueous solution: Add hyaluronic acid or hyaluronate to physiological saline, and add an alkali, a surfactant, and a crosslinking agent, and stir evenly;

[0041] (2) Prepare the oil phase solution, and add an emulsifier to the oil phase solution, and stir evenly to fully emulsify to obtain an emulsion;

[0042] (3) Inject the prepared aqueous solution and oil phase solution into a microfluidic chip mold, control the flow rate and pressure of the aqueous solution and the oil phase solution, and mix the two phases to form a hyaluronic acid emulsion with the aqueous phase as the dispersed phase and the oil phase as the continuous phase;

[0043] (4) Adjust the pH of the aqueous phase of the hyaluronic acid emulsion, and let it stand at a certain temperature to cause a crosslinking reaction to form hyaluronic acid microspheres;

[0044] (5) Wash the crosslinked hyaluronic acid microspheres;

[0045] (6) Prepare the hyaluronic acid stock solution, and add the washed hyaluronic acid microspheres and other active ingredients to the hyaluronic acid stock solution, and mix evenly to obtain.

[0046] Among them, the other active ingredients are at least one of lidocaine hydrochloride, phosphate buffer solution, and physiological saline.

[0047] Its main technical principle is that although a cross-linking agent is added to the prepared aqueous solution, due to the addition of alkali, the pH value is on the high side and does not meet the conditions for the cross-linking reaction to occur. The oil-in-water droplet microfluidic chip mold is used to mix the oil phase and the water phase to form hyaluronic acid microspheres with an outer oil phase and an inner water phase. Then an acid solution is added to adjust the pH value of the hyaluronic acid microsphere environment so that the pH meets the most suitable conditions for the cross-linking reaction to occur, and the cross-linking reaction starts inside the hyaluronic acid microspheres. In this way, the cross-linked hyaluronic acid microspheres can ensure a relatively uniform size particle size distribution, with a diameter difference of ±20 μm, greatly improving the stability of the injectable hyaluronic acid microspheres and significantly enhancing the quality of the finally prepared injectable hyaluronic acid filler. It is ensured that after the hyaluronic acid microspheres enter the human body, they have a synchronous decomposition and absorption effect, and there will be no local collapse or protrusion.

[0048] Example 2:

[0049] On the basis of the above embodiment, this embodiment further discusses and studies the key process steps in the process of preparing hyaluronic acid microspheres using microfluidic technology.

[0050] Generally, in order to prepare the hyaluronic acid aqueous phase, alkali is added to make the prepared aqueous phase have a certain fluidity, so as to better mix with the oil phase to prepare an emulsion and better carry out cross-linking. Due to the addition of alkali, the pH value of the aqueous phase containing hyaluronic acid is generally relatively high. In the process of mixing the oil phase and the water phase by ordinary stirring, there is not much influence. For the hyaluronic acid microspheres prepared by the microfluidic process, since the cross-linking occurs inside the microspheres, the change in pH needs to be considered. In the present invention, a certain mass percentage of hydrochloric acid or organic acid is added to the microfluidic receiving solution to adjust its pH, and then the prepared hyaluronic acid microspheres can cross-link inside. During the cross-linking process of the hyaluronic acid microspheres, the inventor found that the mass percentage of hydrochloric acid or organic acid added has a significant influence on the cross-linking of the hyaluronic acid microspheres. Therefore, the following specific experiments are designed:

[0051] 1. Experimental process: Identical hyaluronic acid microspheres were prepared by microfluidic technology. At this time, the pH of the hyaluronic acid solution in the aqueous phase was about 12. The prepared hyaluronic acid was grouped into Group A, Group B, and Group C. During crosslinking, 0.5 wt% organic acid solution was added to the microfluidic receiving solution of hyaluronic acid microspheres in Group A, 0.5 wt% organic acid solution was added to the microfluidic receiving solution of hyaluronic acid microspheres in Group B, and 1.5 wt% organic acid solution was added to the microfluidic receiving solution of hyaluronic acid microspheres in Group C. By adding organic acids, the pH of hyaluronic acid microspheres in each group was adjusted to 7 - 9. The three groups of hyaluronic acid microspheres were placed in an environment of 37°C. After 5 h, their specific crosslinking conditions were observed.

[0052] 2. Experimental results: After being placed for 5 h, the results were as Figures 1 - 3 shown, Figure 1 , Figure 3 as shown in [reference], during the crosslinking process of hyaluronic acid microspheres with 0.5 wt% organic acid solution added and those with 1.5 wt% organic acid solution added, over-crosslinking also occurred between the microspheres, which was disadvantageous for the preparation of hyaluronic acid microspheres. And Figure 2 shown that adjusting the pH value by adding 1.0 wt% organic acid solution was most beneficial to the crosslinking process of hyaluronic acid microspheres.

[0053] Among them, the viscosity tests of adding organic acids with different concentrations and the retention rates of washed microspheres are shown in Table 1 and Table 2.

[0054] Table 1 Viscosity tests after adding organic acids with different concentrations

[0055] Organic acid concentration (wt%) 0 0.5 1 1.5 Viscosity (mPa·s) 64.684 99.194 176.667 333.207

[0056] Table 2 Retention rates of washed microspheres after adding organic acids with different concentrations

[0057]

[0058] Example 3:

[0059] Based on the above example, this example further discusses and studies the key process steps during the preparation of hyaluronic acid microspheres using microfluidic technology.

[0060] Since the hyaluronic acid microspheres prepared by microfluidic control are prone to coalescence, making the hyaluronic acid microspheres crosslink stably without coalescence is one of the core technical problems to be solved in the preparation of hyaluronic acid microspheres by microfluidic technology. Through the research of the inventor, it is found that the lower the temperature, the more able to avoid the coalescence of hyaluronic acid microspheres. However, too low a temperature will delay the progress of the crosslinking reaction, which is not conducive to the final formation of hyaluronic acid microspheres. Therefore, exploring the crosslinking temperature of hyaluronic acid microspheres is one of the key factors for realizing the preparation of hyaluronic acid microspheres by microfluidic technology. The following experiments are carried out to explore the optimal crosslinking temperature of hyaluronic acid microspheres.

[0061] Experimental process: After preparing the aqueous solution of hyaluronic acid in the internal phase, it is placed in a refrigerator at 4°C. The purpose of refrigeration is to make the internal phase liquid more stable, make the solution distribution more uniform, and have the characteristics of non-Newtonian fluid. In addition, it can also eliminate the generated foam. When in use, after restoring the refrigerated aqueous solution of hyaluronic acid to room temperature, the hyaluronic acid microspheres are prepared by microfluidics. The hyaluronic acid microspheres prepared by microfluidic control are divided into three groups and placed at 37°C, 50°C, and 55°C respectively. After a period of time, observe the crosslinking situation of the microspheres and the coalescence situation between the microspheres.

[0062] After 24 hours, the prepared hyaluronic acid microspheres are observed through an optical microscope, and the results are as Figures 4 - 6 shown. The hyaluronic acid microspheres placed at 37°C show a small amount of coalescence, and the crosslinking phenomenon is obvious. The hyaluronic acid microspheres at 50°C show an obvious crosslinking phenomenon, but obvious microsphere coalescence occurs; while the hyaluronic acid microspheres at 55°C show a large amount of coalescence, and only part of the hyaluronic acid microspheres crosslink. It can be seen that 37°C is the optimal storage temperature for hyaluronic acid microspheres and can achieve a good crosslinking effect. Below this temperature, the temperature is too low, the crosslinking efficiency is too low, and the application value is small.

[0063] Example 4:

[0064] In this example, on the basis of the above example, the key process steps in the process of preparing hyaluronic acid microspheres by microfluidic technology are further discussed and studied.

[0065] In order to make the prepared hyaluronic acid microspheres exist stably, only by adding a specific surfactant can the liquid-oil interface exist stably. In addition, the existence of bubbles will lead to poor fluid stability, which has an adverse effect on the preparation of hyaluronic acid microspheres by microfluidics. Therefore, whether the bubbles generated by adding the surfactant can be removed well to avoid affecting the realization of the microfluidic process is also one of the selection criteria for surfactants.

[0066] Corresponding comparative tests are carried out for various surfactants, as follows:

[0067] 1. Experimental grouping:

[0068] It is divided into a single series surfactant group (Group A), a PVP composite series surfactant group (Group B), and a tween composite series surfactant group (Group C). Each group is further divided according to specific circumstances as follows:

[0069] The single series surfactant group (Group A) includes:

[0070] Group A1: 1 wt% Tween 80;

[0071] Group A2: 2 wt% Tween 80;

[0072] Group A3: 1 wt% sodium dodecyl sulfate;

[0073] Group A4: 2 wt% sodium dodecyl sulfate;

[0074] Group A5: 3 wt% sodium dodecyl sulfate;

[0075] Group A6: 1 - 3 wt% polyvinylpyrrolidone

[0076] Group A7: 5 - 20 wt% polyvinylpyrrolidone.

[0077] The PVP composite series surfactant group (Group B):

[0078] Group B1: 1 - 3 wt% of the composite surfactant of poloxamer F127 and polyvinylpyrrolidone;

[0079] Group B2: 1 - 3 wt% of the composite surfactant of polyvinylpyrrolidone and sodium dodecyl sulfate;

[0080] Group B3: 1 wt% of the composite surfactant of polyvinylpyrrolidone and 1 wt% Tween 80;

[0081] Group B4: 1 wt% of the composite surfactant of polyvinylpyrrolidone and 2 wt% Tween 80.

[0082] The tween composite series surfactant group (Group C):

[0083] Group C1: 1 wt% of the composite surfactant of Tween 20 and 1 wt% Tween 80;

[0084] Group C2: 2 wt% of the composite surfactant of Tween 20 and 2 wt% Tween 80.

[0085] Experimental procedure:

[0086] During the preparation of the aqueous phase, surfactants of different types and contents were added according to the groups. The presence of bubbles in the aqueous phase was observed respectively. Then, for the aqueous phases prepared according to the groups, the same oil phase was used, and hyaluronic acid microspheres in the form of water-in-oil were prepared by microfluidics, and the specific aggregation situation of the prepared hyaluronic acid microspheres was observed.

[0087] The experimental results are as follows:

[0088] In the case of the presence of bubbles in the prepared aqueous phase, as shown in Table 3:

[0089] Table 3 Generation situation of bubbles in the aqueous phase prepared by adding different surfactants

[0090] Group Bubble condition A1 No bubbles generated inside A2 No bubbles generated inside A3 A large number of small bubbles generated inside A4 A large number of small bubbles generated inside A5 A large number of small bubbles generated inside A6 No bubbles generated inside A7 No bubbles generated inside B1 No bubbles generated inside B2 Bubbles generated inside B3 No bubbles generated inside B4 No bubbles generated inside C1 No bubbles generated inside C2 No bubbles generated inside

[0091] In the specific case of preparing hyaluronic acid microspheres by microfluidics, as shown in Table 4:

[0092] Table 4 Situation of hyaluronic acid microspheres prepared by adding different surfactants

[0093] Group Experimental phenomenon A1 Severe hyaluronic acid microsphere aggregation A2 Severe hyaluronic acid microsphere aggregation A3 Partial hyaluronic acid microsphere aggregation A4 Partial hyaluronic acid microsphere aggregation A5 Partial hyaluronic acid microsphere aggregation A6 Severe hyaluronic acid microsphere aggregation A7 Severe hyaluronic acid microsphere aggregation B1 The viscosity of the aqueous phase is too high to prepare hyaluronic acid microspheres B2 The viscosity of the aqueous phase is too high to prepare hyaluronic acid microspheres B3 Aggregation occurs when the diameter of hyaluronic acid microspheres is less than 100 μm B4 The viscosity of the aqueous phase is too high to prepare hyaluronic acid microspheres C1 Aggregation occurs when the diameter of hyaluronic acid microspheres is 150 μm C2 When the diameter of hyaluronic acid microspheres is 120 μm, no aggregation occurs at room temperature for 10 h

[0094] According to the content of Table 1 and Table 2, for the single surfactant series, even if the added mass percentage content is changed, it cannot meet the stability requirements of the aqueous phase for preparing hyaluronic acid microspheres; while the PVP composite series surfactants can be mixed with hyaluronic acid, but the viscosity of the aqueous phase is too high to obtain hyaluronic acid microspheres by microfluidics. The tween composite series surfactants have good bubble conditions in the aqueous phase and good stability after being prepared into microspheres, meeting the technical requirements of the present invention for preparing hyaluronic acid microspheres by microfluidics technology.

[0095] Example 5:

[0096] The injectable hyaluronic acid filler prepared according to the above example in this example was tested for the residual amount of cross-linking agent. The specific test process is as follows:

[0097] Draw the standard curve of the crosslinking agent (1,4-butanediol diglycidyl ether). Prepare a standard stock solution of 1,4-butanediol diglycidyl ether at 80 μg / mL. Appropriately transfer and dilute the stock solution to obtain 1,4-butanediol diglycidyl ether standard solutions at 1.0, 2.0, 4.0, and 8.0 μg / mL. Take 0.1 mL of each concentration standard solution, add 0.05 mL of 125 mmol / L nicotinamide solution and mix. Incubate in a water bath at 37 °C for 2 h. Sequentially add 0.5 mL of 15% acetophenone solution and 1 mol / L potassium hydroxide solution, mix well and then ice-bath for 10 min. Add 2.5 mL of formic acid, incubate in a water bath at 60 °C for 5 min, and cool in an ice bath. Use a fluorescence spectrophotometer to measure its fluorescence value. Fix the emission wavelength at 435 nm and the excitation wavelength at 380 nm, and draw the standard curve of 1,4-butanediol diglycidyl ether.

[0098] Detect the residual amount of 1,4-butanediol diglycidyl ether in the sample. Weigh approximately 50 mg of crosslinked sodium hyaluronate gel microsphere sample precisely according to the method recommended by the standard YY / T 0640 "Crosslinked Sodium Hyaluronate Gel for Surgical Plastic Surgery", add 50 mL of 2.0 U / mL sodium hyaluronidase solution, mix well, and enzymatically degrade at 37 °C for 24 h. Measure the fluorescence value under the above conditions, and substitute the obtained value into the standard curve of 1,4-butanediol diglycidyl ether to obtain the residual concentration of 1,4-butanediol diglycidyl ether.

[0099] The test results are shown in Table 1:

[0100] Table 1 Residual Concentrations of Crosslinking Agents at Different Concentrations

[0101]

[0102] Calculate that the residual amounts of 1,4-butanediol diglycidyl ether are 1.94 μg / mL, 1.74 μg / mL, 1.78 μg / mL, and 1.56 μg / mL respectively. The residual amount of the crosslinking agent of this hyaluronic acid gel is less than 2.0 μg / mL, meeting the usage standard of "Crosslinked Sodium Hyaluronate Gel for Surgical Plastic Surgery".

[0103] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A preparation method of a hyaluronic acid filler for injection, characterized in that, Prepare an emulsion with uniform particle size in a two-phase flow through microfluidic technology. Crosslinking reaction occurs to hyaluronic acid in the emulsion to form hyaluronic acid microspheres. Among them, the outer phase in the emulsion preparation process is the oil phase, and the inner phase is the aqueous phase containing hyaluronic acid. Finally, add the hyaluronic acid microspheres and other active ingredients to the hyaluronic acid stock solution to obtain the injectable hyaluronic acid filler. The surfactant added to the aqueous solution is a composite surfactant of Tween 20 and Tween 80, and the contents of Tween 20 and Tween 80 are the same, both being 1-10 wt%; the oil phase solution is liquid paraffin, and the emulsifier added is Span 80, and the content of Span 80 is 1-10 wt%.

2. The preparation method of a hyaluronic acid filler for injection according to claim 1, wherein, Its specific preparation process includes the following steps: (1) Prepare the aqueous solution: Add hyaluronic acid or hyaluronate into physiological saline, and add an alkali, a surfactant, and a crosslinking agent, and stir evenly; (2) Prepare the oil phase solution, add an emulsifier to the oil phase solution, and stir evenly to fully emulsify it to obtain an emulsion; (3) Inject the prepared aqueous solution and oil phase solution into a microfluidic chip mold, control the flow rate and pressure of the aqueous solution and the oil phase solution, and form a hyaluronic acid emulsion with the inner phase being the aqueous phase and the outer phase being the oil phase under the shearing action of the outer phase; (4) Adjust the pH of the aqueous phase of the hyaluronic acid emulsion, and let it stand at a certain temperature to cause a crosslinking reaction to form hyaluronic acid microspheres; (5) Wash the crosslinked hyaluronic acid microspheres; (6) Prepare the hyaluronic acid stock solution, add the washed hyaluronic acid microspheres and other active ingredients into the hyaluronic acid stock solution, and mix evenly to obtain.

3. The preparation method of a hyaluronic acid filler for injection according to claim 1 or 2, characterized in that, The molecular weight of the hyaluronic acid or its salt is 900-1200 kDa, the diameter of the prepared hyaluronic acid microspheres is 40-1200 μm, and the particle size variation coefficient of the prepared hyaluronic acid microspheres is ≤10%.

4. The preparation method of a hyaluronic acid filler for injection according to claim 1 or 2, characterized in that, The crosslinking agent added to the aqueous solution is at least one of carbodiimide, divinyl sulfone, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, and epichlorohydrin, and the alkali added to the aqueous solution is at least one of sodium hydroxide and potassium hydroxide.

5. The preparation method of a hyaluronic acid filler for injection according to claim 2, wherein For the aqueous solution prepared in step (1), the initial pH value is 10-13. The method for adjusting the pH of the aqueous phase in the hyaluronic acid microspheres in step (4) is to use a semipermeable membrane to coat the hyaluronic acid microspheres placed in the oil phase solution, place it in physiological saline, and add an acid solution to the physiological saline. Use the semipermeable membrane to adjust the pH inside the aqueous phase inside the hyaluronic acid microspheres to 7-9, so that the aqueous phase inside the hyaluronic acid microspheres meets the conditions for the crosslinking reaction. The acid solution is 1 wt% hydrochloric acid or an organic acid solution. The crosslinking reaction temperature is 10°C-60°C, and the crosslinking reaction time is 0.1-100 h.

6. The preparation method of a hyaluronic acid filler for injection according to claim 2 or 5, characterized in that, In step (5), wash three times with petroleum ether and physiological saline respectively, and perform filtration treatment using a filter screen.

7. The preparation method of a hyaluronic acid filler for injection according to claim 2 or 5, characterized in that The other active ingredients in step (6) are at least one of lidocaine hydrochloride, phosphate buffer solution, and physiological saline.

8. The preparation method of a hyaluronic acid filler for injection according to claim 2 or 5, characterized in that, The concentration of the hyaluronic acid stock solution prepared in step (6) is 2 mg / mL, and the concentration of the crosslinked hyaluronic acid microspheres is 18 mg / mL.

9. A finished product of a hyaluronic acid filler for injection, characterized in that, The hyaluronic acid filler for injection is prepared by the preparation method of the hyaluronic acid filler for injection according to any one of claims 1 to 8. Then, the hyaluronic acid filler for injection is swollen, canned, and sterilized with PBS buffer solution to obtain the finished hyaluronic acid filler for injection.

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