CN115869250A - Hyaluronic acid or sodium hyaluronate composition for injection and preparation method and application thereof - Google Patents
Hyaluronic acid or sodium hyaluronate composition for injection and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a composition for injection, which is characterized by comprising high molecular weight hyaluronic acid or sodium hyaluronate, medium molecular weight or low molecular weight hyaluronic acid or sodium hyaluronate and a buffer solution, wherein the hyaluronic acid or sodium hyaluronate with different molecular weights is matched to obtain low viscosity, low injection force and longer degradation period, the preparation process is simple, and no cross-linking agent is added, so that the composition has higher safety.
Description
Technical Field
The invention belongs to the fields of biomedicine and medical cosmetology, and particularly relates to a hyaluronic acid or sodium hyaluronate composition for injection, a preparation method thereof and application thereof in the fields of biomedicine and medical cosmetology.
Background
In the past decades, the research on biocompatible polymer materials has been a hot spot in the fields of biomedicine and cosmetology and has made great progress so far, wherein several representative biocompatible polymers and their derivatives, chemical modifiers play a key material basis.
Hyaluronic Acid (HA) is an anionic natural glycosaminoglycan composed of alternating linkages of repeating units D-glucuronic acid and N-acetylglucosamine disaccharides. HA is a non-sulfated glycosaminoglycan. More than 50% of hyaluronic acid is present in skin, lungs and intestines. In addition, it is also present in interstitial tissues such as synovial fluid of joints, cartilage, umbilical cord, and blood vessel wall. Hyaluronic acid synthesized in the human body mainly plays physiological functions of lubrication and buffer action, filling agent and diffusion barrier, free radical removal and the like. Hyaluronic acid products currently used in the market can be extracted from animal tissues (such as cockscomb, vitreous eye, cerebral cartilage, joint fluid) and also fermented from bacteria (such as streptococcus, pseudomonas aeruginosa, etc.). The average molecular weight of HA generally ranges from 5kDa to 10MDa, with typical molecular weight sizes ranging from 2.0MDa to 5.0MDa.
Hyaluronic acid, which is a major constituent of extracellular matrix, is present in skin tissue at about 50% of the age of 20 years and has a water-retaining capacity of 500 times its own weight. With age, the hyaluronic acid content in the skin gradually decreases. By age 60 hyaluronic acid content is reduced to 25% of that of age 20, and the water content of the skin surface is reduced, which causes the stratum corneum to age, the skin to wrinkle and lose elasticity. The molecular weight of hyaluronic acid in skin tissue generally ranges from 50 to 200 kilodaltons. Due to the special properties of the macromolecular size of the hyaluronic acid with high molecular weight, the hyaluronic acid can effectively adjust the water content and osmotic pressure balance of tissues, and can construct a stable extracellular environment through hydration, provide firm binding sites for surrounding cells, collagen, elastin fibers and other components and protect the cells from being damaged by environmental factors. In addition, the hyaluronic acid with high molecular weight can effectively reduce the occurrence probability of skin inflammation after injection. The low molecular weight hyaluronic acid can accelerate the migration and proliferation of cells and promote the generation of new blood vessels and the reconstruction of extracellular matrix by binding with a cell surface CD44 receptor.
The appearance of skin wrinkles is often accompanied by a decrease in skin sagging and elasticity, and the most important cause of the decrease is a decrease in the content of hyaluronic acid, an important constituent of extracellular matrix. In recent years, a great deal of research has been conducted to develop injectable hyaluronic acid solutions or cross-linked hyaluronic acid hydrogels, which improve the appearance of the skin and improve the quality of the skin by means of dermal injection filling.
In recent years, with the intensive research on the functions of HA, HA HAs been widely used in the medical field, such as in the preparation of drug delivery systems and in the treatment of orthopedic and ophthalmic diseases, the prevention of post-operative adhesions, and soft tissue repair, and HAs become a research hotspot in the field of tissue engineering. The natural HA which is easy to degrade can obtain better physical stability and mechanical strength through chemical modification. Chemical crosslinking of HA is typically accomplished by intramolecular crosslinking of HA by small molecule crosslinkers or by post-chemical modification of HA for crosslinking. HA hydrogels obtained by chemical crosslinking have been widely used in the field of tissue engineering.
However, many chemical methods for preparing HA hydrogels have various limitations and safety hazards, such as low efficiency, difficult operation, high cost, etc. for preparing gel by using toxic small molecule cross-linking agents and chemically modifying HA. The cross-linked hyaluronic acid hydrogel effectively provides a filling function due to certain mechanical supporting performance. On the one hand, the use of cross-linking agents makes the hydrogel have certain biological safety risks, on the other hand, the injection of the gel tends to cause pain to the patient during and after the operation, and the pain is further aggravated by the increase of the viscoelasticity of the hydrogel, and the gel is not suitable for filling shallow wrinkles such as neck and forehead due to the higher hardness of the gel. At present, a large number of commercial products improve the injection comfort and the filling effective time by crosslinking and matching non-crosslinked phase hyaluronic acid, but have the defects of complex process, low safety, complex operation, high cost, poor filling effect, easy inflammation and the like. At present, the development of new HA materials with better crosslinking mechanism, or the development of composite materials of HA and other biocompatible polymer compounds, HAs become one of the approaches for solving the existing problems.
The common hyaluronic acid injection in the market at present is a low molecular weight product, has short degradation period in vivo and poor skin filling effect, can only maintain the effect of a short period, and cannot play the activity role of reconstructing extracellular matrix. High molecular weight hyaluronic acid, with a molecular weight exceeding about 100 million daltons, can produce complex interactions with proteins and polysaccharides in the dermal environment and achieve stable mechanical support ability to maintain the smooth appearance of the skin, but too high a molecular weight also causes a large increase in solution viscosity to make it difficult to apply in clinical and medical cosmetic practice.
Profhilo is the first hyaluronic acid product for injection without chemical cross-linking agent for treating skin laxity, and the concentration of hyaluronic acid can reach up to 32mg/mL, so that the goal of skin moisturizing can be achieved, aged and sagging facial tissues can be improved, and the skin state can be maintained within about 28 days, aiming at the following people: 1. the neck line is obvious; 2. apple myoptosis; 3. anti-aging, tightening and lifting; 4. the skin is flaccid. Profhilo hyaluronic acid is produced by fermentation, and is not added with an exogenous cross-linking agent but is cross-linked by heat energy in the production process, and has the advantages of good biocompatibility, low side effect and low risk. Profhilo has high molecular weight and high concentration of 3.2% and can be maintained in human body for a long time, but usually needs to be corrected by secondary injection in one month after primary treatment, and then the correction times are increased according to the specific conditions of the injection part to obtain the effect of inducing autologous collagen secretion, so that the treatment course is long, the injection times are many, and the treatment cost is high.
In conclusion, the development of a hyaluronic acid-based filling product which can maintain an effective degradation period in the human body and can realize practical application in clinical and medical beauty practices has become a practical call for clinicians and users.
Disclosure of Invention
The invention develops a novel injection type hyaluronic acid or sodium hyaluronate product aiming at the defects of high metabolism speed and short filling effect of biocompatible hyaluronic acid or sodium hyaluronate from natural sources or fermentation sources and simultaneously aiming at the defects of residual toxic components of chemically modified hyaluronic acid gel by using an exogenous cross-linking agent, human body toxic reaction, high side effect and high risk, and the problems of long treatment course, multiple injection times, overlarge injection force during use, poor user compliance, high treatment cost and the like of the hyaluronic acid gel from the natural sources.
According to one aspect of the present invention, there is provided an injection composition characterized by containing high molecular weight hyaluronic acid (or sodium hyaluronate), medium molecular weight hyaluronic acid (or sodium hyaluronate) and/or low molecular weight hyaluronic acid (or sodium hyaluronate), and a buffer solution, the high molecular weight hyaluronic acid (or sodium hyaluronate) having a molecular weight of 800kDa to 1800kDa, preferably 800kDa to 1200kDa, the medium molecular weight hyaluronic acid (or sodium hyaluronate) having a molecular weight of 200kDa to 700kDa, the low molecular weight hyaluronic acid (or sodium hyaluronate) having a molecular weight of 80kDa to 120kDa, the weight ratio of the high molecular weight hyaluronic acid (or sodium hyaluronate) to the medium molecular weight hyaluronic acid (or sodium hyaluronate) and/or the low molecular weight hyaluronic acid (or sodium hyaluronate) being (8 to 10): 1, and the concentration of the sum of all hyaluronic acids (or sodium hyaluronate) being 1.0 to 3.5%, preferably 2.8 to 3.3.3%.
According to the invention, the buffer solution is citric acid-sodium citrate-lactic acid buffer solution, which contains 2.0% of sodium citrate, 0.4% of citric acid, 0.01% of lactic acid and has a pH value of 5.0.
According to the present invention, the pharmaceutical composition for injection may further comprise a diluent, a stabilizer, an isotonic regulator, and/or a solubilizing or solubilizing agent, etc., and may further comprise an analgesic such as lidocaine, etc.
According to the invention, the viscosity of the injectable composition of the invention is less than 60000cP, the storage modulus is less than 60Pa and greater than 40Pa, the bolus force is less than 30N, and the in vivo degradation time is more than 6 weeks.
According to another aspect of the present invention, there is provided a method for preparing the above composition for injection, comprising the steps of:
(1) Dissolving high molecular weight hyaluronic acid or sodium hyaluronate in a buffer solution;
(2) Dissolving low molecular weight hyaluronic acid or sodium hyaluronate in a buffer solution;
(3) Mixing, homogenizing and ultrasonically treating the solutions obtained in the steps (1) and (2);
(4) And (5) sterilizing.
Preferably, homogenizing at 11000-13000RPM for 10-20min.
Preferably, the ultrasound is carried out for 10-25min at 800-1600 w.
Preferably, a diluent, a stabilizer, an isotonic regulator, and/or a solubilizing or solubilizing agent, etc., preferably further adding an analgesic such as lidocaine, etc., are added to the solution obtained in step (3) and mixed well.
According to another aspect of the invention, the invention relates to the use of the above-mentioned injectable composition for the preparation of cosmetic and medical products.
The hyaluronic acid or sodium hyaluronate injection with low viscosity and high biological activity is prepared by compounding high molecular weight hyaluronic acid or sodium hyaluronate with medium and/or low molecular weight hyaluronic acid or sodium hyaluronate. In the process that high molecular weight hyaluronic acid or sodium hyaluronate is dissolved in water, macromolecular chains can generate a connection configuration similar to a network through hydrogen bonds and hydrophobic interaction, and on the other hand, the winding among the macromolecular chains also enables the viscosity of the solution to be extremely high. When the solution is homogenized by stirring and sonicated to a certain extent, the interactions between these strands are briefly broken, and the configuration of the hyaluronic acid strands is changed. In the stage after the stirring, homogenizing and ultrasonic treatment, the connection between chains is formed again. By incorporating medium or low molecular weight hyaluronic acid or sodium hyaluronate into the composition, this recombination of hydrogen bonds can be made to occur randomly between chains of different molecular weights and the viscosity of the solution is reduced very significantly. After being injected into subcutaneous tissue, the hyaluronic acid or sodium hyaluronate with medium molecular weight or low molecular weight in the components can be recognized by surrounding cells at first, promote the migration and proliferation of the surrounding cells to a defect part, and accelerate the construction of new extracellular matrix. In between, the high molecular weight hyaluronic acid or sodium hyaluronate component not only plays a mechanical supporting role, but also provides a connection target and a protection role for components such as new cells, collagen, elastin fibers and the like. A series of low-viscosity injections can be obtained by stirring, homogenizing and ultrasonically treating the mixed solution of hyaluronic acid or sodium hyaluronate. Because the molecular weight of hyaluronic acid or sodium hyaluronate is not changed before and after stirring homogenization and ultrasonic treatment, the prepared low-viscosity injection has the advantage of easy injection, and can realize the construction of rapid extracellular matrix and the stable and long-acting skin filling function due to the hyaluronic acid or sodium hyaluronate with different molecular weights.
The high molecular weight hyaluronic acid or sodium hyaluronate and medium and/or low molecular weight hyaluronic acid or sodium hyaluronate composite solution can replace cross-linked sodium hyaluronate gel in physical properties, and has more excellent mechanical properties and biological activities, including similar viscoelasticity, lower injection thrust, capacity of promoting extracellular matrix regeneration and long-acting and safe skin filling effect. Particularly, the production process is simple, no cross-linking agent or catalyst component is added in the preparation process, and the collagen protein-containing hyaluronic acid has better collagen protein stimulation effect and facial blood circulation promotion effect compared with the existing cross-linked hyaluronic acid product. When the hyaluronic acid or sodium hyaluronate compound with different molecular weights is prepared, the selected aqueous solution medium is characterized in that all components are compounded with the biocompatibility of the human tissue filler, and the components do not need to be removed; hyaluronic acid or sodium hyaluronate with different molecular weights is selected as a raw material, no cross-linking agent is added, the components are simple and safe, and the potential toxicity and potential adverse reaction of the cross-linking agent are avoided; the viscosity and the injection force can be effectively reduced without reducing the molecular weight only through controllable stirring homogenization and ultrasonic processes, the operation is simple and easy, the operability of an operator in use is improved, and the compliance of a user in use is also improved; high molecular weight hyaluronic acid or sodium hyaluronate can provide long-acting and stable mechanical supporting capability, and medium and low molecular weight hyaluronic acid or sodium hyaluronate provides excellent bioactivity.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the description of the present invention, and such equivalents also fall within the scope of the invention.
Preparation example 1 preparation of buffer Medium M1 (citric acid-sodium citrate-lactic acid buffer solution)
1. Dissolving 20.0g of sodium citrate in 500mL of water to obtain a sodium citrate aqueous solution A1;
2. dissolving 4.0g of citric acid in 300mL of water to obtain a citric acid aqueous solution B1;
3. dissolving 0.1g of lactic acid in 100mL of water to obtain a lactic acid aqueous solution C;
4. preparing dilute hydrochloric acid solution loaded in an appendix of the 2015 edition Chinese pharmacopoeia;
5. uniformly mixing the sodium citrate aqueous solution A1 obtained in the step 1 and the citric acid aqueous solution B1 obtained in the step 2 in the preparation example to obtain a citric acid buffer solution D1; slowly adding 10mL of lactic acid aqueous solution C in the step 3 of the preparation example into a citric acid buffer solution D1, uniformly mixing, then adding a proper amount of dilute hydrochloric acid in the step 4 of the preparation example dropwise, adjusting the pH value to 5.0, then adding water to a constant volume of 1000mL, and uniformly mixing to obtain a citric acid-sodium citrate-lactic acid buffer solution, which is hereinafter referred to as a buffer medium M1.
Preparation example 2 preparation of buffer Medium M2 (citric acid-sodium citrate buffer solution)
1. Preparing sodium citrate aqueous solution A1, citric acid aqueous solution B1 and dilute hydrochloric acid solution according to steps 1, 2 and 4 in preparation example 1;
2. uniformly mixing the sodium citrate aqueous solution A1 and the citric acid aqueous solution B1 in the step 1 of the preparation example to obtain a citric acid buffer solution D1; then, a proper amount of the dilute hydrochloric acid obtained in the step 1 of the preparation example is dripped, the pH value is adjusted to 5.0, then water is added to the solution to reach a constant volume of 1000mL, and the solution is uniformly mixed to obtain a citric acid-sodium citrate buffer solution, which is hereinafter referred to as a buffer medium M2.
Example 1 preparation of HA Complex solution 1
The preparation method comprises the following steps: weighing 2.70 g of high molecular weight hyaluronic acid (weight average molecular weight is 100 kilodaltons), weighing 0.30 g of low molecular weight hyaluronic acid (weight average molecular weight is 10 kilodaltons), and respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is buffer solution M1 prepared by the method in preparation example 1 to prepare high molecular weight hyaluronic acid solution and low molecular weight hyaluronic acid solution;
the hyaluronic acid solution with high and low molecular weight is placed for 4 hours and stirred to be completely dispersed. After complete dispersion, mixing the high and low molecular weight hyaluronic acid solutions uniformly according to the ratio of 1. Stirring and homogenizing the solution by using an IKA-T25 homogenizer, setting the rotating speed of a cutter head to be 12000rpm respectively, and treating the mixed solution for 15 minutes;
and then, carrying out ultrasonic treatment on the obtained solution for 15 minutes by using an ultrasonic cleaner, wherein the ultrasonic power is set to 1200w, and thus obtaining the HA composite solution 1.
Example 2 preparation of HA Complex solution 2
The preparation method comprises the following steps: weighing 2.50 g of high molecular weight hyaluronic acid (weight average molecular weight is 120 kilodaltons), weighing 0.30 g of low molecular weight hyaluronic acid (weight average molecular weight is 8 kilodaltons), and respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is buffer solution M1 prepared by the method in preparation example 1 to prepare high molecular weight and low molecular weight hyaluronic acid solutions;
the hyaluronic acid solution with high and low molecular weight is placed for 4 hours and stirred to be completely dispersed. After complete dispersion, the high and low molecular weight hyaluronic acid solutions are respectively and uniformly mixed according to the ratio of 1. Stirring and homogenizing the solution by using an IKA-T25 homogenizer, setting the rotating speed of a cutter head to be 11000rpm respectively, and treating the mixed solution for 20 minutes;
and then, carrying out ultrasonic treatment on the obtained solution for 10 minutes by using an ultrasonic cleaner, wherein the ultrasonic power is set to 1600w, and obtaining the HA composite solution 2.
Example 3 preparation of HA Complex solution 3
The preparation method comprises the following steps: weighing 3.00 g of high molecular weight hyaluronic acid (weight average molecular weight is 80 kilodaltons), weighing 0.30 g of low molecular weight hyaluronic acid (weight average molecular weight is 12 kilodaltons), respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is buffer solution M1 prepared by the method in the preparation example 1, and preparing high molecular weight hyaluronic acid solution and low molecular weight hyaluronic acid solution;
the hyaluronic acid solution with high and low molecular weight is placed for 4 hours and stirred to be completely dispersed. After complete dispersion, the high and low molecular weight hyaluronic acid solutions are respectively and uniformly mixed according to the ratio of 1. Stirring and homogenizing the solution by using an IKA-T25 homogenizer, respectively setting the rotating speed of a cutter head to be 13000rpm, and setting the processing time of the mixed solution to be 10 minutes;
and then, carrying out ultrasonic treatment on the obtained solution for 25 minutes by using an ultrasonic cleaner, wherein the ultrasonic power is set to be 800w, and thus obtaining the HA composite solution 3.
Example 4 preparation of HA Complex solution 4
The preparation method comprises the following steps: weighing 2.70 g of high molecular weight hyaluronic acid (weight average molecular weight is 100 kilodaltons), weighing 0.30 g of medium molecular weight hyaluronic acid (weight average molecular weight is 20 kilodaltons), and respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is buffer solution M1 prepared by the method in preparation example 1 to prepare high molecular weight hyaluronic acid solution and medium molecular weight hyaluronic acid solution;
after the high and medium molecular weight hyaluronic acid solution is placed for 4 hours, stirring is carried out to completely disperse the hyaluronic acid solution. After complete dispersion, mixing the high and medium molecular weight hyaluronic acid solutions uniformly according to the ratio of 1. Stirring and homogenizing the solution by using an IKA-T25 homogenizer, setting the rotating speed of a cutter head to be 12000rpm respectively, and setting the processing time of the mixed solution to be 15 minutes;
and then carrying out ultrasonic treatment on the obtained solution for 15 minutes by using an ultrasonic cleaner, wherein the ultrasonic power is set to 1200w, and obtaining the HA composite solution 4.
Example 5 preparation of HA Complex solution 5
The preparation method comprises the following steps: weighing 2.50 g of high molecular weight hyaluronic acid (weight average molecular weight is 120 kilodaltons), weighing 0.30 g of medium molecular weight hyaluronic acid (weight average molecular weight is 50 kilodaltons), respectively dissolving the high and medium molecular weight hyaluronic acid in 50ml of medium solution, wherein the medium solution is buffer solution M1 prepared by the method in preparation example 1, and preparing high and medium molecular weight hyaluronic acid solutions;
after the high and medium molecular weight hyaluronic acid solution is placed for 4 hours, stirring is carried out to completely disperse the hyaluronic acid solution. After complete dispersion, mixing the high and medium molecular weight hyaluronic acid solutions uniformly according to the ratio of 1. Stirring and homogenizing the solution by using an IKA-T25 homogenizer, respectively setting the rotating speed of a cutter head to be 11000rpm, and treating the mixed solution for 20 minutes;
and then, carrying out ultrasonic treatment on the obtained solution for 10 minutes by using an ultrasonic cleaner, wherein the ultrasonic power is set to 1600w, and obtaining the HA composite solution 5.
Example 6 preparation of HA Complex solution 6
The preparation method comprises the following steps: weighing 3.00 g of high molecular weight hyaluronic acid (weight average molecular weight is 80 kilodaltons), weighing 0.30 g of medium molecular weight hyaluronic acid (weight average molecular weight is 70 kilodaltons), respectively dissolving the high and medium molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is buffer solution M1 prepared by the method in preparation example 1, and preparing high and medium molecular weight hyaluronic acid solutions;
the high and medium molecular weight hyaluronic acid solution is left for 4 hours and stirred to be completely dispersed. After complete dispersion, mixing the high and medium molecular weight hyaluronic acid solutions uniformly according to the ratio of 1. Stirring and homogenizing the solution by using an IKA-T25 homogenizer, respectively setting the rotating speed of a cutter head to be 13000rpm, and treating the mixed solution for 10 minutes;
and then, carrying out ultrasonic treatment on the obtained solution for 25 minutes by using an ultrasonic cleaner, wherein the ultrasonic power is set to be 800w, and thus obtaining the HA composite solution 6.
Example 7 preparation of HA Complex solution 7
The preparation method comprises the following steps: weighing 1.50 g of high molecular weight hyaluronic acid (weight average molecular weight is 100 kilodaltons), weighing 1.50 g of low molecular weight hyaluronic acid (weight average molecular weight is 10 kilodaltons), respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is buffer solution M1 prepared by the method in preparation example 1, and preparing high molecular weight hyaluronic acid solution and low molecular weight hyaluronic acid solution;
the subsequent preparation method is the same as that of the example 1, and the HA composite solution 7 is obtained.
Example 8 preparation of HA Complex solution 8
The preparation method comprises the following steps: weighing 2.70 g of high molecular weight hyaluronic acid (weight average molecular weight is 100 kilodaltons), weighing 0.30 g of low molecular weight hyaluronic acid (weight average molecular weight is 10 kilodaltons), and respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is buffer solution M2 prepared by the method in preparation example 2 to prepare high molecular weight hyaluronic acid solution and low molecular weight hyaluronic acid solution;
the subsequent preparation method is the same as that of example 1, and the HA composite solution 8 is obtained.
Example 9 preparation of HA Complex solution 9
The preparation method comprises the following steps: weighing 1.35 g of high molecular weight hyaluronic acid (weight average molecular weight is 100 kilodaltons), weighing 0.15 g of low molecular weight hyaluronic acid (weight average molecular weight is 10 kilodaltons), and respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 100 milliliters of medium solution, wherein the selected medium solution is buffer solution M2 prepared by the method in preparation example 2 to prepare high molecular weight and low molecular weight hyaluronic acid solutions;
the subsequent preparation method is the same as that of example 1, and the HA composite solution 9 is obtained.
Example 10 preparation of HA Complex solution 10
The preparation method comprises the following steps: weighing 2.70 g of high molecular weight hyaluronic acid (weight average molecular weight is 100 kilodaltons), weighing 0.30 g of low molecular weight hyaluronic acid (weight average molecular weight is 10 kilodaltons), respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is buffer solution M1 prepared by the method in the preparation example 1, and preparing high molecular weight hyaluronic acid solution and low molecular weight hyaluronic acid solution;
the hyaluronic acid solution with high and low molecular weight is placed for 4 hours and stirred to be completely dispersed. After complete dispersion, mixing the high and low molecular weight hyaluronic acid solutions uniformly according to the ratio of 1. And (3) stirring and homogenizing the solution by using an IKA-T25 homogenizer, setting the rotation speed of a cutter head to 14000rpm, and setting the treatment time of the mixed solution to 15 minutes to obtain the HA composite solution 10.
Example 11 preparation of HA Complex solution 11
The preparation method comprises the following steps: weighing 2.70 g of high molecular weight hyaluronic acid (weight average molecular weight is 100 kilodaltons), weighing 0.30 g of low molecular weight hyaluronic acid (weight average molecular weight is 10 kilodaltons), respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is buffer solution M1 prepared by the method in the preparation example 1, and preparing high molecular weight hyaluronic acid solution and low molecular weight hyaluronic acid solution;
the hyaluronic acid solution with high and low molecular weight is placed for 4 hours and stirred to be completely dispersed. After complete dispersion, the high and low molecular weight hyaluronic acid solutions are respectively and uniformly mixed according to the ratio of 1. And (3) stirring and homogenizing the solution by using an IKA-T25 homogenizer, setting the rotation speed of a cutter head to 10000rpm respectively, and processing the mixed solution for 15 minutes to obtain the HA composite solution 11.
Example 12 preparation of HA Complex solution 12
The preparation method comprises the following steps: weighing 2.70 g of high molecular weight hyaluronic acid (with the weight average molecular weight of 200 kilodaltons), weighing 0.30 g of low molecular weight hyaluronic acid (with the weight average molecular weight of 10 kilodaltons), and respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is the buffer solution M1 prepared by the method in the preparation example 1 to prepare high molecular weight hyaluronic acid solution and low molecular weight hyaluronic acid solution;
the subsequent preparation method was the same as in example 1, to obtain an HA complex solution 12.
Example 13 preparation of HA Complex solution 13
The preparation method comprises the following steps: weighing 2.70 g of high molecular weight hyaluronic acid (with a weight average molecular weight of 60 kilodaltons), weighing 0.30 g of low molecular weight hyaluronic acid (with a weight average molecular weight of 10 kilodaltons), and respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50ml of medium solution, wherein the medium solution is the buffer solution M1 prepared by the method in the preparation example 1 to prepare high molecular weight hyaluronic acid solution and low molecular weight hyaluronic acid solution;
the subsequent preparation method was the same as in example 1, to obtain an HA composite solution 13.
Example 14 preparation of HA Complex solution 14
The preparation method comprises the following steps: weighing 2.70 g of high molecular weight hyaluronic acid (weight average molecular weight is 100 kilodaltons), weighing 0.30 g of low molecular weight hyaluronic acid (weight average molecular weight is 5 kilodaltons), respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is buffer solution M1 prepared by the method in the preparation example 1, and preparing high molecular weight hyaluronic acid solution and low molecular weight hyaluronic acid solution;
the subsequent preparation method was the same as in example 1, to obtain an HA complex solution 14.
Example 15 preparation of HA Complex solution 15
The preparation method comprises the following steps: weighing 2.25 g of high molecular weight hyaluronic acid (weight average molecular weight is 100 kilodaltons), weighing 0.75 g of medium molecular weight hyaluronic acid (weight average molecular weight is 20 kilodaltons), and respectively dissolving the high molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in 50 milliliters of medium solution, wherein the selected medium solution is buffer solution M1 prepared by the method in preparation example 1 to prepare high molecular weight hyaluronic acid solution and low molecular weight hyaluronic acid solution;
the subsequent preparation method was the same as in example 1, to obtain an HA composite solution 15.
Example 16 measurement of injection force, viscosity, storage modulus of HA Complex solutions 1-15
1. Method for measuring injection force
The injection force was obtained by measuring the syringe handle squeezing force by the following specific measurement method:
A27G needle was attached to the syringe and the syringe handle was advanced at a rate of 32.34mm/min, and the average force of the handle was measured as the force of the injection.
Samples of each of the HA complex solutions prepared in examples 1 to 15 were measured in the above-described manner to obtain the measurement values of the bolus force, and the specific measurement results are shown in Table 1.
2. Viscosity measuring method
The resulting HA composite solution viscosity was tested using a MYR-3000 rotational viscometer with a No. three probe. 30mL of each sample was placed in a 50mL centrifuge tube, the rotational viscometer was turned on and the probe calibrated. And (3) immersing the probe into the sample solution to the scale mark of the probe, starting the rotary probe, and recording data after the viscosity index is stable. Each set of solutions was tested in triplicate.
Samples of each of the HA complex solutions prepared in examples 1 to 15 were measured in the above-described manner to obtain viscosity measurement values, and the specific measurement results are shown in Table 1.
3. Storage modulus measuring method
A200 μ L sample was placed in the center of the parallel jig of the TA-DHR2 rheometer and the sample surface was contacted using a 20mm parallel plate probe to ensure full probe coverage. Detection temperature 25 degrees celsius, shear frequency: 1Hz, and each set of samples was tested for 1 minute.
Samples of each of the HA composite solutions prepared in examples 1 to 15 were measured in the above-described manner to obtain measured values of storage modulus, and the specific measurement results are shown in Table 1.
TABLE 1 injection force, viscosity, storage modulus, in vivo degradation time of HA composite solutions 1-15
Example 17 in vivo degradation Performance assay of HA Complex solutions 1-15 in animals
HA complex solution samples are prepared according to the methods of examples 1-15 respectively, and after the preparation is finished, the degradation cycle of each sample in an animal body is determined and evaluated in an animal body degradation cycle investigation mode, the specific determination data is shown in table 2, and the in-vivo degradation time is shown in table 1.
The method of the in vivo degradation test of animals is as follows:
120 healthy SD rats were screened, and HA complex solutions 1 to 15 prepared in examples 1 to 15 were sampled, and the rats were randomly divided into 15 groups of 8 rats each to which HA complex solution 1 to 15 was administered. The samples were implanted subcutaneously into rats, each of which was implanted with 3 HA complex solutions (200. Mu.l), one PE tube (control), saline (200. Mu.l, control). After implantation, 8 rats per group were sacrificed at 1, 2, 3, 4, 5, 6, 7, 8 weeks, the diameter and height of the implant were measured until the implant disappeared completely, and the volume of the implant was calculated by the spheroid volume (see table below). The end of the degradation cycle was defined as 20% of the sample volume at week 1. No bruise and trauma were observed during the observation period, and no skin bruise, edema and erythema were observed at the implantation site.
The HA composite solution 12 prepared by the method in the embodiment 12 HAs overlarge injection force and viscosity, and the test result for determining the degradation in the animal body HAs no practical significance.
TABLE 2 degradation Performance in animals for HA Complex solutions 1-15
Example 18 in vivo safety Studies in animals
The test method comprises the following steps: 45 healthy rabbits were screened and randomly divided into an HA complex solution group and a physiological saline solution group. Samples were randomly implanted into the dorsal skin of unhaired rabbits at 5 points per sample and 0.2ml per spot. The reaction conditions of each injection site were observed at 24h, 48h and 72h after injection, and the tissue reaction of erythema and edema of each injection site at each observation period was scored according to the intradermal reaction scoring standard.
As a result: under the condition of the experiment, no obvious erythema and edema reaction is observed at the injection part in the rabbit intracutaneous reaction experiment of the experimental sample, and the final scoring difference is less than or equal to 1.0.
The results of examining the HA complex solution samples prepared in examples 1-15 of this patent according to the above test methods show that the HA complex solution samples prepared in examples 1-15 have satisfactory safety.
Claims (10)
1. An injectable composition comprising a high molecular weight hyaluronic acid (or sodium hyaluronate), a medium molecular weight hyaluronic acid (or sodium hyaluronate) and/or a low molecular weight hyaluronic acid (or sodium hyaluronate), and a buffer solution, wherein the high molecular weight hyaluronic acid (or sodium hyaluronate) has a molecular weight of 800kDa to 1800kDa, preferably 800kDa to 1200kDa, the medium molecular weight hyaluronic acid (or sodium hyaluronate) has a molecular weight of 200kDa to 700kDa, and the low molecular weight hyaluronic acid (or sodium hyaluronate) has a molecular weight of 80kDa to 120kDa.
2. The injectable composition of claim 1, wherein the weight ratio of the high molecular weight hyaluronic acid (or sodium hyaluronate) to the medium molecular weight hyaluronic acid (or sodium hyaluronate) and/or the low molecular weight hyaluronic acid (or sodium hyaluronate) is (8-10): 1.
3. Injectable composition according to claim 1 or 2, characterized in that the concentration of the sum of all hyaluronic acid (or sodium hyaluronate) is between 1.0 and 3.5%, preferably between 2.8 and 3.3%.
4. The injectable composition of any one of claims 1-3, wherein the buffer solution is a citric acid-sodium citrate-lactic acid buffer solution comprising sodium citrate 2.0%, citric acid 0.4%, lactic acid 0.01%, and pH 5.0.
5. The injectable composition of any one of claims 1-4, further comprising a diluent, stabilizer, isotonicity modifier, and/or solubilizing agent, and the like, and further comprising an analgesic such as lidocaine and the like.
6. A process for the preparation of an injectable composition according to any of claims 1 to 5, comprising the steps of:
(1) Dissolving high molecular weight hyaluronic acid or sodium hyaluronate in a buffer solution;
(2) Dissolving hyaluronic acid or sodium hyaluronate with medium molecular weight or low molecular weight in a buffer solution;
(3) Mixing, homogenizing and ultrasonically treating the solutions obtained in the steps (1) and (2);
(4) And (5) sterilizing.
7. The method of claim 6, wherein homogenizing is carried out at 11000-13000RPM for 10-20min.
8. The method of claim 6, wherein the ultrasound is performed at 800-1600w for 10-25min.
9. The process according to any one of claims 6 to 8, wherein the solution obtained in step (3) is mixed with a diluent, a stabilizer, an isotonicity adjusting agent, and/or a solubilizing or solubilizing agent, and/or solubilizing agent, and the like, and preferably further an analgesic such as lidocaine and the like.
10. Use of the injectable composition according to any one of claims 1 to 5 for the preparation of cosmetic and medical products.
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