Preparation method of lysine cross-linked hyaluronic acid gel as well as gel and application thereof
By directly using dialysate for dialysis treatment, the problems of waste of materials and low efficiency caused by dialysis membrane bags in the prior art are solved, and the quality and production efficiency of cross-linked hyaluronic acid gels are improved, which is suitable for industrial production.
Patent Information
- Application Number
- CN202411388564.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2025-01-07
AI Technical Summary
Disposable dialysis membrane bags are required during the production process of existing crosslinked sodium hyaluronate gels, resulting in waste of materials and low dialysis efficiency, affecting product quality.
Use direct dialysis solution for dialysis, avoid the use of dialysis membrane bags, put the gel after crosslinking reaction directly into the dialysate, and replace the dialysate during the process, and use a mixed solution of phosphate buffer and alcohol solvent as Dialysate.
It saves the use of disposable dialysis membrane bags, improves dialysis efficiency and product quality, and is gentle in preparation and simple in operation, suitable for industrial production.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical cosmetic filling materials, and in particular to a preparation method of a lysine cross-linked hyaluronic acid gel, the gel and application thereof. Background Art
[0002] After cross-linking modification, sodium hyaluronate forms a hydrogel, namely cross-linked sodium hyaluronate gel, which is a special dispersion system in which colloidal particles or polymer molecules are linked to each other to form a spatial network structure, and the gaps in this network structure are filled with liquid. The cross-linked sodium hyaluronate gel has the characteristics of swelling but not dissolving due to the formation of a covalent cross-linked network. Due to the presence of a large number of hydrophilic groups in this hydrogel, it can absorb and store a large amount of water, causing its weight and volume to increase significantly.
[0003] The cross-linked sodium hyaluronate gel has excellent biocompatibility and biodegradability, overcoming the disadvantage of fast degradation of uncross-linked sodium hyaluronate gel. It has the advantages of high mechanical strength, good viscoelasticity, insolubility in water, long degradation time, etc., which greatly improves its application range and performance. It can be widely used in cosmetic fillers, anti-adhesion, hemostasis, ophthalmic viscoelastic agents and other fields.
[0004] In the process of preparing cross-linked sodium hyaluronate gel by cross-linking sodium hyaluronate with chemical cross-linking agents, the gel blocks after the cross-linking reaction need to be dialyzed to purify the excess cross-linking agents in the cross-linking reaction. However, in the existing cross-linked sodium hyaluronate gel production process, dialysis membrane bags are often used for dialysis, such as putting the sodium hyaluronate gel blocks after the cross-linking reaction into the dialysis membrane bag, clamping the two ends of the dialysis membrane bag with clips, and then placing the dialysis membrane bag with the gel blocks in the dialysate, using a stirring paddle or a magnet to continuously stir the dialysate for dialysis, and then replacing the dialysate every once in a while.
[0005] The problems with this dialysis method are: the need to use disposable dialysis membrane bags, resulting in material waste; it takes a long time to load the sodium hyaluronate gel blocks after the cross-linking reaction into the dialysis membrane bag, which increases the processing time; due to the wrapping of the dialysis membrane bag, the dialysis efficiency is low, which greatly affects the quality of the cross-linked sodium hyaluronate gel product after dialysis.
[0006] In view of the problems existing in the existing dialysis methods, the present invention provides a new method for preparing lysine cross-linked hyaluronic acid gel, in which dialysis can be completed without using a dialysis membrane bag in the dialysis process, which not only saves the use of disposable dialysis membrane bags, but also improves the dialysis efficiency and the quality of the lysine cross-linked hyaluronic acid gel product after dialysis. Summary of the invention
[0007] The purpose of the present invention is to overcome the defects of the prior art and provide a new method for preparing lysine cross-linked hyaluronic acid gel, in which dialysis can be completed without using a dialysis membrane bag in the dialysis process, which not only saves the use of disposable dialysis membrane bags, but also improves the dialysis efficiency and the quality of the lysine cross-linked hyaluronic acid gel product after dialysis.
[0008] 11. To achieve the above-mentioned purpose of the invention, the present invention provides a method for preparing a lysine-crosslinked hyaluronic acid gel, comprising the following steps: mixing sodium hyaluronate, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) and lysine, subjecting the mixture to a crosslinking reaction and dialysis treatment to obtain a lysine-crosslinked hyaluronic acid gel; wherein the dialysis treatment uses a dialysate to perform dialysis directly without using a dialysis membrane bag.
[0009] Furthermore, the preparation method includes the following steps: S1 cross-linking reaction: mixing sodium hyaluronate, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) and lysine and a buffer to obtain a gel; S2 dialysis treatment: directly placing the gel obtained in step S1 into a dialysis solution, and replacing the dialysis solution during the process; the dialysis solution contains a phosphate buffer.
[0010] In step S1 of some embodiments, sodium hyaluronate, DMTMM and buffer are mixed and activated to obtain material A; the weight average molecular weight of the sodium hyaluronate is 40 to 220 WDa; the material A is mixed with lysine and subjected to a cross-linking reaction to obtain a gel.
[0011] In step S1 of some embodiments, the weight average molecular weight of the sodium hyaluronate is 70-180 WDa; the mass volume ratio of the sodium hyaluronate to the buffer solution is 30-50 mg / mL; and the mass ratio of the DMTMM to the sodium hyaluronate is (0.74-1.1):1.
[0012] In step S1 of some embodiments, the weight average molecular weight of the sodium hyaluronate is 130 WDa to 180 WDa.
[0013] In step S1 of some embodiments, the mass volume ratio of the sodium hyaluronate to the buffer solution is 35-50 mg / mL.
[0014] In step S1 of some embodiments, the buffer includes at least one of a phosphate buffer, a citrate buffer and a sodium chloride solution.
[0015] In step S1 of some embodiments, the pH value of the buffer solution is 6.2-7.5, for example, 7.
[0016] In step S1 of some embodiments, the osmotic pressure of the buffer solution is 150-300 mOsm / L, for example, 250 mOsm / L.
[0017] In step S1 of some embodiments, the mass ratio of the DMTMM to the sodium hyaluronate is (0.75-0.9):1.
[0018] In step S1 of some embodiments, the activation can be performed under stirring conditions according to conventional practices in the art.
[0019] In step S1 of some embodiments, the activation time is 20 to 30 minutes.
[0020] In step S1 of some embodiments, the activation temperature is room temperature.
[0021] In step S1 of a preferred embodiment, the following steps are specifically included: the sodium hyaluronate and the buffer solution are mixed once, and then mixed with the DMTMM for a second time, and activated to obtain the material A.
[0022] In step S1 of some embodiments, the mass ratio of the lysine to the sodium hyaluronate is 1:(6-16), preferably 1:(8-14), for example 1:10.
[0023] In step S2 of some embodiments, the mass ratio of the dialysate to the gel is (15-25):1; preferably 20:1.
[0024] In step S2 of some embodiments, the dialysate needs to flow during the dialysis process; preferably, a stirring device or a circulating pump is used to ensure the flow of the dialysate;
[0025] In step S2 of some embodiments, the dialysate is replaced 5-10 times, preferably 8 times.
[0026] In step S2 of some embodiments, during the dialysis process, the duration of each dialysis is 1-8 hours, preferably 2-5 hours.
[0027] In step S2 of some embodiments, the dialysate is at least one selected from phosphate buffer, a mixed solution of phosphate buffer and an alcohol solvent; preferably, the alcohol solvent is selected from any one of methanol, ethanol, isopropanol or a combination thereof.
[0028] In step S2 of some embodiments, the mass ratio of the phosphate buffer to the alcohol solvent is (5-9):1, preferably 9:1.
[0029] In step S2 of some embodiments, a mixed solution of phosphate buffer and ethanol is used as the dialysate for the first and second times, and a phosphate buffer is used as the dialysate for the third to eighth times.
[0030] In step S2 of some embodiments, the phosphate buffer comprises dihydrogen phosphate and / or dihydrogen phosphate, preferably disodium hydrogen phosphate and / or sodium dihydrogen phosphate.
[0031] The present invention also provides a lysine cross-linked hyaluronic acid gel, which is prepared by the above-mentioned preparation method.
[0032] The present invention also provides an application of a lysine cross-linked hyaluronic acid gel in the preparation of a product for correcting skin depressions. Preferably, the filling product is used for injection filling of tear grooves, lips, forehead, neck or hands. More preferably, the filling product is used for injection filling of the bridge of the nose or nasolabial groove.
[0033] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.
[0034] Compared with the prior art, the present invention has the following beneficial technical effects:
[0035] 1. In the preparation method of the lysine cross-linked hyaluronic acid gel provided by the present invention, the dialysis process can complete the dialysis without using a dialysis membrane bag, which not only saves the use of disposable dialysis membrane bags, but also improves the dialysis efficiency and the quality of the lysine cross-linked hyaluronic acid gel product after dialysis.
[0036] 2. The preparation method of the lysine-crosslinked hyaluronic acid gel provided by the present invention has mild reaction conditions, is easy to operate, and is easy to industrialize.
[0037] 3. The lysine-crosslinked hyaluronic acid gel provided by the present invention has the characteristics of sufficient crosslinking reaction and good viscoelasticity. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 is a rheological diagram depicting the elastic modulus G' and the viscous modulus G'' of the lysine cross-linked hyaluronic acid gel prepared in Example 2 as a function of the applied strain. DETAILED DESCRIPTION
[0039] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.
[0040] The reagents and raw materials used in the following examples are all commercially available.
[0041] Gel preparation
[0042] Preparation of phosphate buffer: Add 26.7 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and 6.9 g of sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) to every 1 L of water for injection.
[0043] Weigh 15g of sodium hyaluronate (130W) and add it to 300g of phosphate buffer, stir and disperse until completely dissolved. Weigh 11.25g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) and add it to the above sodium hyaluronate solution. After activating the gel for 25 minutes, add lysine solution (1.5g lysine to 30mL phosphate buffer). After adding, seal and place in a 4℃ refrigerator for reaction for 24 hours.
[0044] The agitator used in the above is a Shanghai Si Le Instrument D2010W electric agitator.
[0045] Gel dialysis:
[0046] The gel after the reaction was divided into three parts, and dialyzed according to the description of Example 1, Example 2 and the comparative example respectively. The gel was dialyzed according to the dialysis process described in Table 1 below, and the phosphate in the dialysate was the phosphate buffer prepared in advance.
[0047] Example 1
[0048] The lysine cross-linked hyaluronic acid gel block after the cross-linking reaction is placed in a barrel, and phosphate buffer is added as the dialysate, wherein the mass ratio of the dialysate to the gel is 20:1, and the dialysate is dialyzed for 5 hours with mechanical stirring at a speed of 200 rpm / min, and then a new dialysate is replaced, and the above operation is performed simultaneously for a second dialysis, and the dialysis time is 5 hours; then a new dialysate is replaced, and the above operation is performed simultaneously for a third dialysis, and the dialysis time is 2 hours, and then the dialysis is continued for five times, each dialysis time is 2 hours, and a total of eight dialysis times are completed.
[0049] Example 2
[0050] A phosphate buffer solution and anhydrous ethanol were prepared into a mixed solution of phosphate and ethanol in a mass ratio of 9:1, and the mixed solution was used as a dialysate to dialyze the gel.
[0051] The lysine cross-linked hyaluronic acid gel block after the cross-linking reaction is placed in a barrel, and a pre-prepared mixed solution of phosphate and ethanol is added as the dialysate, wherein the mass ratio of the dialysate to the gel is 20:1. After dialysis for 5 hours with mechanical stirring at a speed of 200 rpm / min, a new mixed solution of phosphate and ethanol is replaced, and the above operation is performed simultaneously for a second dialysis, and the dialysis time is 5 hours; then it is replaced with pure phosphate buffer, and the above operation is performed simultaneously for a third dialysis, and the dialysis time is 2 hours. After that, the pure phosphate buffer is used for five times, and the dialysis time each time is 2 hours, and a total of eight dialysis times are completed.
[0052] Comparative Example
[0053] The lysine cross-linked hyaluronic acid gel block after the cross-linking reaction is loaded into a dialysis membrane bag. The dialysis membrane bag used is Reda Henghui brand model MD-77-14-5M with a molecular weight of 14,000 Daltons. The two ends of the dialysis membrane bag are clamped with clips and placed in a barrel. Phosphate buffer is added as the dialysis fluid, wherein the mass ratio of the dialysis fluid to the gel is 20:1. After dialysis for 5 hours with mechanical stirring at a speed of 200 rpm / min, a new dialysis fluid is replaced. Simultaneously with the above operation, the cross-linked sodium hyaluronate loaded in the dialysis membrane bag is dialyzed for a second time for 5 hours. The new dialysis fluid is replaced again. Simultaneously with the above operation, the third dialysis is performed for 2 hours. After that, the dialysis is continued for five times, each dialysis time is 2 hours, and a total of eight dialysis times are completed.
[0054] Table 1
[0055]
[0056] After dialysis, the dialyzed gels of Example 1, Example 2 and the comparative example were weighed to 20 mg / g using phosphate buffer according to the amount of sodium hyaluronate fed, keeping the content substantially the same. The gels were squeezed and sieved twice using a 200-mesh sieve for granulation, and the granulated samples were dispensed into 20 mL vials, sterilized by wet heat at 121° C. for 15 min, and finally lysine-crosslinked hyaluronic acid gels were obtained.
[0057] Among them, the yield of the lysine cross-linked hyaluronic acid gel obtained in Example 1 was 94%; the yield of the lysine cross-linked hyaluronic acid gel obtained in Example 2 was 95%; and the yield of the lysine cross-linked hyaluronic acid gel obtained in the comparative example was 100%.
[0058] Performance measurement
[0059] The properties of the lysine-crosslinked hyaluronic acid gels prepared in the above Examples 1-2 and the comparative example were measured, specifically including the viscoelasticity of the lysine-crosslinked hyaluronic acid gels, and the residues of DMTMM, N-methylmorpholine, 4,6-dimethyl-1,3,5-triazine-2(1H)-one and ethanol in the lysine-crosslinked hyaluronic acid gels.
[0060] Viscoelasticity: refers to the deformation and flow properties of the cross-linked gel to be tested under the action of external force. Specifically, it is tested using a rheometer in oscillation mode, with a test temperature of 25°C, a rotor spacing of 0.3mm, a starting frequency of 10Hz, an ending frequency of 0.1Hz, a shear strain of 0.5%, and 10 sampling points in each order of magnitude. The elastic modulus G' (Pa), viscous modulus G'' (Pa) and complex viscosity η* (Pa.s) at 1Hz are observed.
[0061] The contents of DMTMM and 4,6-dimethyl-1,3,5-triazine-2(1H)-one in lysine cross-linked hyaluronic acid gel were determined by liquid chromatography, and the contents of N-methylmorpholine and ethanol in lysine cross-linked hyaluronic acid gel were determined by gas chromatography.
[0062] The performance test results of the lysine cross-linked hyaluronic acid gel prepared in Examples 1-2 and Comparative Example are shown in Table 2.
[0063] Table 2
[0064]
[0065] Among them, N-methylmorpholine and 4,6-dimethyl-1,3,5-triazine-2(1H)-one are byproducts in the cross-linking reaction. According to the measurement results in Table 2, Examples 1 and 2 use a direct dialysis process instead of using a dialysis membrane bag for dialysis, but instead obtain a lysine cross-linked hyaluronic acid gel with better viscoelasticity.
[0066] There is no significant difference between the residual DMTMM in Example 1 and the comparative example. However, the residual N-methylmorpholine in the lysine cross-linked hyaluronic acid gel prepared in Example 1 is lower.
[0067] Compared with Example 1 and the comparative example, the DMTMM and N-methylmorpholine residues in Example 2 are lower.
[0068] Generally speaking, the use of dialysis membrane bags for dialysis can avoid the loss of substances. However, for Examples 1 and 2 of the present invention, although no dialysis membrane bags were used for dialysis, the yield was still maintained at a high level, and a cross-linked gel with higher viscoelasticity was obtained. Overall, Examples 1 and 2 of the present invention achieved better results than the comparative example.
[0069] Stability test
[0070] The lysine cross-linked hyaluronic acid gels obtained in Example 1, Example 2 and the comparative example were placed under accelerated stability conditions (40°C ± 2°C / 75% ± 5% RH) for 1 month, and the appearance and viscoelasticity of the gels were observed. The specific measurement results are shown in Table 3.
[0071] Table 3
[0072]
[0073] According to the measurement results in Table 3, the lysine cross-linked hyaluronic acid gel prepared in Example 1 and Example 2 has higher stability, indicating that the dialysis process of the present application can elute the residue more cleanly and thoroughly, and the viscoelasticity is better maintained.
[0074] In summary, the art generally uses a disposable dialysis membrane bag for dialysis, resulting in a waste of materials; it takes a long time to load the sodium hyaluronate gel block after the cross-linking reaction into the dialysis membrane bag, which increases the processing time; due to the wrapping of the dialysis membrane bag, the dialysis efficiency is low, which greatly affects the quality of the gel product after dialysis. However, the dialysis process of the present application does not need to use a dialysis membrane bag to complete dialysis, and maintains a higher yield, which not only saves the use of disposable dialysis membrane bags, but also improves the dialysis efficiency and the quality of the lysine cross-linked hyaluronic acid gel product after dialysis, and is easy to operate and easy to industrialize.
[0075] Finally, it should be noted that in this application, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.
[0076] Although the present application has been disclosed above through the description of the specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed in the present application.
Claims
1. A method for preparing a lysine-crosslinked hyaluronic acid gel, characterized in that: The following steps are involved: Sodium hyaluronate, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and lysine are mixed, and subjected to cross-linking reaction and dialysis treatment to obtain a lysine-cross-linked hyaluronic acid gel; The dialysis treatment uses a dialysate to perform dialysis directly without using a dialysis membrane bag.
2. The preparation method according to claim 1, characterized in that: The following steps are involved: S1 cross-linking reaction: mixing sodium hyaluronate, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and lysine to obtain a gel; S2 dialysis treatment: the gel obtained in step S1 is directly placed in a dialysis solution, and the dialysis solution is replaced during the process; the dialysis solution contains a phosphate buffer.
3. The preparation method according to claim 2, characterized in that: Step S2 satisfies at least one of the following conditions (1) to (4): (1) The mass ratio of the dialysate to the gel is (15-25):1; (2) During the dialysis process, the flow of dialysate must be ensured; (3) The number of times the dialysate is changed is 5-10 times; (4) During the dialysis process, each dialysis session lasts for 1-8 hours.
4. The preparation method according to claim 3, characterized in that: Step S2 satisfies at least one of the following conditions (1) to (4): (1) The mass ratio of the dialysate to the gel is 20:1; (2) using a stirring device or a circulating pump to ensure the flow of the dialysate during the dialysis process; (3) The number of times the dialysate is replaced is 8 times; (4) During the dialysis process, each dialysis session lasts for 2-5 hours.
5. The preparation method according to claim 2, characterized in that: The dialysate is selected from at least one of a phosphate buffer solution and a mixed solution of a phosphate buffer solution and an alcohol solvent; preferably, the alcohol solvent is selected from any one of methanol, ethanol, isopropanol or a combination thereof.
6. The preparation method according to claim 5, characterized in that: The mass ratio of the phosphate buffer to the alcohol solvent is (5-9):1, preferably 9:
1.
7. The preparation method according to claim 6, characterized in that: In step S2, a mixed solution of phosphate buffer and ethanol is used as the dialysate for the first and second times, and a phosphate buffer is used as the dialysate for the third to eighth times.
8. The preparation method according to claim 7, characterized in that: The phosphate buffer comprises dihydrogen phosphate and / or dihydrogen phosphate, preferably disodium hydrogen phosphate and / or sodium dihydrogen phosphate.
9. A lysine-crosslinked hyaluronic acid gel, characterized in that: The gel is prepared by the preparation method described in any one of claims 1 to 8.
10. Use of the lysine cross-linked hyaluronic acid gel according to claim 9 in preparing a product for correcting skin depressions and filling; preferably, the filling product is used for injection filling of tear troughs, lips, forehead, neck or hands.