CN115869250A - Hyaluronic acid or sodium hyaluronate composition for injection and preparation method and application thereof - Google Patents

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

A hyaluronic acid or sodium hyaluronate composition for injection and its preparation method and use

technical field

The invention belongs to the fields of biomedicine and medical cosmetology, and in particular relates to a hyaluronic acid or sodium hyaluronate composition for injection, a preparation method thereof, and an application thereof in the fields of biomedicine and medical cosmetology.

Background technique

In the past few decades, people's research on biocompatible polymer materials has become a hot spot in the field of biomedicine and medical cosmetology and has made great progress so far. Several representative biocompatible polymer materials Molecules and their derivatives and chemical modifications play a key role in the material basis.

Hyaluronic acid (Hyaluronic acid, HA) is an anionic natural glycosaminoglycan composed of repeating units of D-glucuronic acid and N-acetylglucosamine disaccharide alternating links. HA is a non-sulfated glycosaminoglycan. More than 50% of hyaluronic acid is found in the skin, lungs and intestines. In addition, it also exists in interstitial tissues such as synovial fluid, cartilage, umbilical cord, and blood vessel wall. The hyaluronic acid synthesized in the human body mainly performs physiological functions such as lubrication and buffering, filler and diffusion barrier, and scavenging free radicals. Hyaluronic acid products currently on the market can be extracted from animal tissues (such as cockscomb, eye vitreous, brain cartilage, joint fluid), or fermented by bacteria (such as Streptococcus, Pseudomonas aeruginosa, etc.). The average molecular weight of HA generally ranges from 5 kDa to 10 MDa, and the common molecular weight range is from 2.0 MDa to 5.0 MDa.

Hyaluronic acid is the main component of the extracellular matrix, about 50% of which exists in the skin tissue at the age of 20, and it has a water retention capacity 500 times its own weight. With the increase of age, the content of hyaluronic acid in the skin gradually decreases. At the age of 60, the content of hyaluronic acid drops to 25% of that at the age of 20. At the same time, the water content of the skin surface also decreases, resulting in aging of the stratum corneum, wrinkles and loss of elasticity of the skin. The approximate molecular weight of hyaluronic acid in skin tissue ranges from 500,000 to 2 million Daltons. High-molecular-weight hyaluronic acid can effectively regulate tissue water content and osmotic pressure balance due to its special properties of large molecular size, and build a stable extracellular environment through hydration, which can provide support for surrounding cells, collagen, and elastin fibers. Such components provide strong binding sites and protect them from environmental factors. In addition, high-molecular-weight hyaluronic acid can also effectively reduce the incidence of skin redness, swelling and inflammation after injection. Low molecular weight hyaluronic acid can accelerate the migration and proliferation of cells and promote the formation of new blood vessels and the reconstruction of extracellular matrix by binding to the CD44 receptor on the cell surface.

The appearance of skin wrinkles is often accompanied by a decrease in skin relaxation and elasticity, and the main reason is the decrease in the content of hyaluronic acid, an important component of the extracellular matrix. In recent years, a lot of research has been devoted to the development of injectable hyaluronic acid solutions or cross-linked hyaluronic acid hydrogels to improve the shape of the skin and improve the quality of the skin through dermal injection filling.

In recent years, with the in-depth research on the function of HA, HA has been widely used in the field of medicine, such as the preparation of drug delivery systems, the treatment of orthopedic diseases and ophthalmic diseases, the prevention of postoperative adhesions, and soft tissue repair. Research hotspots in the field of tissue engineering. The easily degradable natural HA can be chemically modified to obtain better physical stability and mechanical strength. The chemical cross-linking of HA is generally accomplished by intramolecular cross-linking of HA with a small molecule cross-linking agent or by cross-linking after chemical modification of HA. HA hydrogels obtained by chemical cross-linking have been widely used in the field of tissue engineering.

However, many chemical methods for preparing HA hydrogels have many limitations and safety hazards, such as the use of toxic small molecule cross-linking agents to prepare gels, the low efficiency of chemical modification of HA, difficult operation, and high cost. Although the cross-linked hyaluronic acid hydrogel has certain mechanical support properties, it effectively provides the filling function. However, it cannot be ignored that, on the one hand, the use of cross-linking agents makes the hydrogel have certain biological safety hazards; The increase of the viscoelasticity of the gel is further exacerbated, and due to the high hardness of the gel, it is not suitable for filling shallow wrinkles such as the neck and forehead. At present, a large number of commercial products have improved the injection comfort and effective filling time by cross-linking and non-cross-linking hyaluronic acid, but there are still complex processes, low safety, cumbersome operation, high cost, poor filling effect, and easy to cause redness and swelling. Defects such as inflammation. So far, the development of new HA materials with better cross-linking mechanism, or the development of composite materials of HA and other types of biocompatible polymer compounds, has become one of the ways to solve the existing problems.

The common hyaluronic acid injections currently on the market are usually low-molecular-weight products, which have a short degradation cycle in the body, poor skin filling effect, can only maintain the effect of a short cycle, and cannot exert the active effect of rebuilding the extracellular matrix. High-molecular-weight hyaluronic acid with a molecular weight of more than about 1 million Daltons can produce complex interactions with proteins and polysaccharides in the dermal environment, and achieve stable mechanical support to maintain the smooth appearance of the skin, but too high a molecular weight also causes The viscosity of the solution is greatly increased so that it is difficult to apply it in the practice of clinical and medical cosmetology.

Profhilo is the first hyaluronic acid product for injection that does not use chemical cross-linking agents for the treatment of skin laxity. Improve or improve aging and sagging facial tissues, and maintain skin condition within 28 days. The target groups are as follows: 1. Obvious neck lines; 2. Sagging apple muscles; 3. Anti-aging, firming and lifting; 4. sagging skin. Profhilo hyaluronic acid is produced by fermentation. No exogenous cross-linking agent is added in the production process, but it is cross-linked by heat energy. The advantages are good biological adaptability, low side effects and low risk. Although Profhilo has the characteristics of high molecular weight and high concentration of 3.2%, it can be maintained in the human body for a long time, but it usually needs to be corrected by a second injection one month after the initial treatment, and then increase according to the specific conditions of the injection site. Correcting the number of times to obtain the effect of inducing autologous collagen secretion results in a long course of treatment, many injections, and high treatment costs.

In conclusion, the development of a hyaluronic acid-based filling product that can not only maintain an effective degradation cycle in the human body, but also realize the practical application in clinical and medical cosmetic practice has become the actual voice of clinicians and users.

Contents of the invention

The present invention aims at the shortcomings of fast metabolism and short filling effect of biocompatible hyaluronic acid or sodium hyaluronate from natural sources or fermentation sources, and at the same time aims at the residual toxic components of chemically modified hyaluronic acid gels using exogenous cross-linking agents , lead to human toxic reactions, high side effects, and high risks, and overcome the long course of natural source hyaluronic acid gel treatment, many injections, excessive injection force during use, poor user compliance, and high treatment costs To solve these problems, develop a new type of injectable hyaluronic acid or sodium hyaluronate product, which can not only overcome the above-mentioned shortcomings of existing products, but also has high safety, low injection force during injection, and good user compliance. A longer metabolic cycle in the body can achieve excellent local body shaping effects.

According to one aspect of the present invention, the present invention provides a composition for injection, which is characterized in that it contains 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 molecular weight of the high molecular weight hyaluronic acid (or sodium hyaluronate) is 800kDa-1800kDa, preferably 800-1200kDa, the middle molecular weight hyaluronic acid (or sodium hyaluronate) molecular weight is 200kDa-700kDa, the molecular weight of described low molecular weight hyaluronic acid (or sodium hyaluronate) is 80kDa-120kDa, described high molecular weight hyaluronic acid (or sodium hyaluronate) and The weight ratio of medium molecular weight hyaluronic acid (or sodium hyaluronate) and/or low molecular weight hyaluronic acid (or sodium hyaluronate) is (8-10):1, all hyaluronic acid (or sodium hyaluronate) The total concentration is 1.0-3.5%, preferably 2.8-3.3%.

According to the present invention, the buffer solution is a citric acid-sodium citrate-lactic acid buffer solution, containing 2.0% sodium citrate, 0.4% citric acid, 0.01% lactic acid, and a pH of 5.0.

According to the present invention, the pharmaceutical composition for injection may also contain diluents, stabilizers, isotonic regulators, and/or solubilizers or solubilizers, and may further contain analgesics such as lidocaine.

According to the present invention, the viscosity of the injection composition of the present invention is less than 60000cP, the storage modulus is less than 60Pa and greater than 40Pa, the injection force is less than 30N, and the degradation time in vivo exceeds 6 weeks.

According to another aspect of the present invention, the present invention provides a preparation method of the above-mentioned injection composition, comprising the following steps:

(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 ultrasonicating the solutions obtained in steps (1) and (2);

(4) Sterilization.

Preferably, homogenize at 11000-13000RPM for 10-20min.

Preferably, sonicate at 800-1600w for 10-25min.

Preferably, diluents, stabilizers, isotonic regulators, and/or solubilizers or solubilizers are added to the solution obtained in step (3), preferably analgesics such as lidocaine are further added, and mixed.

According to another aspect of the present invention, the present invention relates to the application of the aforementioned composition for injection in the preparation of cosmetic and medical products.

In the present invention, hyaluronic acid or sodium hyaluronate injection with low viscosity and high biological activity is prepared by preparing high molecular weight hyaluronic acid or sodium hyaluronate and neutral/or low molecular weight hyaluronic acid or sodium hyaluronate . In the process of dissolving high molecular weight hyaluronic acid or sodium hyaluronate in water, the macromolecular chains will form a connection configuration similar to a network through hydrogen bonds and hydrophobic interactions. On the other hand, the entanglement between macromolecular chains is also The solution has a very high viscosity. When the solution is stirred homogeneously and ultrasonicated to a certain extent, the interaction between these chains will be temporarily destroyed, and the configuration of the hyaluronic acid chain will also change accordingly. During the post-homogenization and sonication stages, interchain linkages were re-formed. By mixing medium or low molecular weight hyaluronic acid or sodium hyaluronate into the components, this hydrogen bond recombination can randomly occur between chains of different molecular weights, and the viscosity of the solution is significantly reduced. After injection into the subcutaneous tissue, the middle or low molecular weight hyaluronic acid or sodium hyaluronate in the components will first be recognized by surrounding cells, and promote the migration and proliferation of surrounding cells to the defect site, and accelerate the construction of new extracellular matrix . Among them, the high molecular weight hyaluronic acid or sodium hyaluronate component not only plays a role in mechanical support, but also provides connection targets and protection for new cells, collagen, elastin fibers and other components. A series of low-viscosity injections can be obtained by stirring and homogenizing the mixed solution of hyaluronic acid or sodium hyaluronate and ultrasonication. Since the molecular weight of hyaluronic acid or sodium hyaluronate does not change before and after stirring homogenization and ultrasonic treatment, the obtained low-viscosity injection not only has the advantage of being easy to inject, but also has the advantage of containing different molecular weight hyaluronic acid or hyaluronic acid. Sodium enables rapid extracellular matrix construction and stable and long-lasting dermal filler function.

The composite solution of high molecular weight hyaluronic acid or sodium hyaluronate and medium and/or low molecular weight hyaluronic acid or sodium hyaluronate of the present invention can replace cross-linked sodium hyaluronate gel in physical properties, and has more excellent Mechanical properties and biological activity, including similar viscoelasticity, lower injection thrust, ability to promote extracellular matrix regeneration, and long-term and safe skin filling effect. In particular, the production process is simple, and no cross-linking agent or catalyst component is added in the preparation process. Compared with the existing cross-linked hyaluronic acid products, it can provide better collagen stimulation effect and facial blood circulation promotion effect. When the present invention prepares hyaluronic acid or sodium hyaluronate complexes with different molecular weights, the selected aqueous solution medium, wherein each component is compounded with the biocompatibility of the human tissue filler, these components do not need to go through the removal process; Molecular weight hyaluronic acid or sodium hyaluronate is used as raw material without adding any cross-linking agent, the components are simple and safe, and avoid the potential toxicity and potential adverse reactions of cross-linking agents; only through controllable stirring homogenization and ultrasonic process It can effectively reduce the viscosity and injection force without reducing the molecular weight. The operation is simple and easy, which improves the operability of the operator and the compliance of the user during use; high molecular weight hyaluronic acid or sodium hyaluronate It can provide long-term and stable mechanical support ability, and medium and low molecular weight hyaluronic acid or sodium hyaluronate provides excellent biological activity.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the contents of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the present invention.

Preparation Example 1 Preparation of buffer medium M1 (citric acid-sodium citrate-lactic acid buffer solution)

1. Take 20.0 g of sodium citrate and dissolve it in 500 mL of water to obtain sodium citrate aqueous solution A1;

2. Take 4.0g of citric acid and dissolve it in 300mL of water to obtain citric acid aqueous solution B1;

3. Take 0.1g of lactic acid and dissolve it in 100mL of water to obtain lactic acid aqueous solution C;

4. Prepare the dilute hydrochloric acid solution recorded in the appendix of the Chinese Pharmacopoeia in 2015;

5. Mix the sodium citrate aqueous solution A1 in step 1 of this preparation example and the citric acid aqueous solution B1 in step 2 to obtain citric acid buffer solution D1; take 10 mL of lactic acid aqueous solution C in step 3 of this preparation example, and slowly add it dropwise to citric acid In the buffer solution D1, mix evenly, then add dropwise an appropriate amount of dilute hydrochloric acid in step 4 of this preparation example, adjust the pH value to 5.0, then add water to make up to 1000mL, mix well, and obtain a citric acid-sodium citrate-lactic acid buffer solution , hereinafter referred to as the buffer medium M1.

The preparation of preparation example 2 buffer medium M2 (citric acid-sodium citrate buffer solution)

1. Prepare sodium citrate aqueous solution A1, citric acid aqueous solution B1, and dilute hydrochloric acid solution according to steps 1, 2, and 4 of Preparation Example 1;

2. Mix the sodium citrate aqueous solution A1 and the citric acid aqueous solution B1 in step 1 of this preparation example to obtain the citric acid buffer solution D1; then add an appropriate amount of diluted hydrochloric acid in step 1 of this preparation example dropwise to adjust the pH value to 5.0, and then Add water to make up to 1000mL, mix well to obtain citric acid-sodium citrate buffer solution, hereinafter referred to as buffer medium M2.

Example 1 Preparation of HA Composite Solution 1

The preparation method is as follows: weigh 2.70 grams of high-molecular-weight hyaluronic acid (weight-average molecular weight of 1 million Daltons), weigh 0.30 grams of low-molecular-weight hyaluronic acid (weight-average molecular weight of 100,000 Daltons), and mix the above-mentioned high and low The molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was the buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and low molecular weight hyaluronic acid solutions;

After placing the hyaluronic acid solutions with high and low molecular weight for 4 hours, stir them to disperse them completely. After the dispersion was complete, the high and low molecular weight hyaluronic acid solutions were mixed uniformly at a ratio of 1:1 to obtain a mixed solution of high and low molecular weight hyaluronic acid with a final concentration of 3.0%. Use the IKA-T25 homogenizer to stir and homogenize the solution, set the cutter head speed to 12000rpm, and the processing time of the mixed solution is 15 minutes;

Then use an ultrasonic cleaner to ultrasonically treat the obtained solution for 15 minutes, and set the ultrasonic power to 1200w to obtain HA composite solution 1.

Example 2 Preparation of HA Composite Solution 2

The preparation method is as follows: Weigh 2.50 grams of high molecular weight hyaluronic acid (weight average molecular weight of 1.2 million Daltons), weigh 0.30 grams of low molecular weight hyaluronic acid (weight average molecular weight of 80,000 Daltons), and mix the above high and low The molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was the buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and low molecular weight hyaluronic acid solutions;

After placing the hyaluronic acid solutions with high and low molecular weight for 4 hours, stir them to disperse them completely. After the dispersion was complete, the high and low molecular weight hyaluronic acid solutions were mixed uniformly at a ratio of 1:1 to obtain a mixed solution of high and low molecular weight hyaluronic acid with a final concentration of 2.8%. Use the IKA-T25 homogenizer to stir and homogenize the solution, set the cutter head speed to 11000rpm, and the processing time of the mixed solution is 20 minutes;

Then use an ultrasonic cleaner to ultrasonically treat the obtained solution for 10 minutes, and set the ultrasonic power to 1600w to obtain HA composite solution 2.

Example 3 Preparation of HA Composite Solution 3

The preparation method is as follows: weigh 3.00 grams of high molecular weight hyaluronic acid (weight average molecular weight of 800,000 daltons), weigh 0.30 grams of low molecular weight hyaluronic acid (weight average molecular weight of 120,000 daltons), and mix the above high and low The molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was the buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and low molecular weight hyaluronic acid solutions;

After placing the hyaluronic acid solutions with high and low molecular weight for 4 hours, stir them to disperse them completely. After the dispersion was complete, the high and low molecular weight hyaluronic acid solutions were mixed uniformly at a ratio of 1:1 to obtain a mixed solution of high and low molecular weight hyaluronic acid with a final concentration of 3.3%. Use the IKA-T25 homogenizer to stir and homogenize the solution, set the cutter head speed to 13000rpm, and the processing time of the mixed solution is 10 minutes;

Then use an ultrasonic cleaner to ultrasonically treat the obtained solution for 25 minutes, and set the ultrasonic power to 800w to obtain HA composite solution 3.

Example 4 Preparation of HA Composite Solution 4

The preparation method is as follows: weigh 2.70 grams of high-molecular-weight hyaluronic acid (weight-average molecular weight of 1 million Daltons), weigh 0.30 grams of medium-molecular-weight hyaluronic acid (weight-average molecular weight of 200,000 Daltons), and mix the above-mentioned high and low Molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and medium molecular weight hyaluronic acid solutions;

After placing the hyaluronic acid solutions with high and medium molecular weight for 4 hours, stir them to disperse them completely. After the dispersion is complete, the high and medium molecular weight hyaluronic acid solutions are mixed uniformly at a ratio of 1:1 to obtain a mixed solution of high and medium molecular weight hyaluronic acid with a final concentration of 3.0%. Use the IKA-T25 homogenizer to stir and homogenize the solution, set the cutter head speed to 12000rpm, and the processing time of the mixed solution is 15 minutes;

Then use an ultrasonic cleaner to ultrasonically treat the obtained solution for 15 minutes, and set the ultrasonic power to 1200w to obtain HA composite solution 4.

Example 5 Preparation of HA Composite Solution 5

The preparation method is as follows: weigh 2.50 grams of high molecular weight hyaluronic acid (weight-average molecular weight of 1.2 million Daltons), weigh 0.30 grams of medium molecular weight hyaluronic acid (weight-average molecular weight of 500,000 Daltons), and mix the above-mentioned high and medium Molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and medium molecular weight hyaluronic acid solutions;

After placing the hyaluronic acid solutions with high and medium molecular weight for 4 hours, stir them to disperse them completely. After the dispersion is complete, the high and medium molecular weight hyaluronic acid solutions are mixed uniformly at a ratio of 1:1 to obtain a mixed solution of high and medium molecular weight hyaluronic acid with a final concentration of 2.8%. Use the IKA-T25 homogenizer to stir and homogenize the solution, set the cutter head speed to 11000rpm, and the processing time of the mixed solution is 20 minutes;

Then use an ultrasonic cleaner to ultrasonically treat the obtained solution for 10 minutes, and set the ultrasonic power to 1600w to obtain HA composite solution 5.

Example 6 Preparation of HA Composite Solution 6

The preparation method is as follows: Weigh 3.00 grams of high molecular weight hyaluronic acid (weight average molecular weight of 800,000 daltons), weigh 0.30 grams of medium molecular weight hyaluronic acid (weight average molecular weight of 700,000 daltons), and mix the above high and medium Molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and medium molecular weight hyaluronic acid solutions;

After placing the hyaluronic acid solutions with high and medium molecular weight for 4 hours, stir them to disperse them completely. After the dispersion is complete, the high and medium molecular weight hyaluronic acid solutions are mixed uniformly at a ratio of 1:1 to obtain a mixed solution of high and medium molecular weight hyaluronic acid with a final concentration of 3.3%. Use the IKA-T25 homogenizer to stir and homogenize the solution, set the cutter head speed to 13000rpm, and the processing time of the mixed solution is 10 minutes;

Then use an ultrasonic cleaner to ultrasonically treat the obtained solution for 25 minutes, and set the ultrasonic power to 800w to obtain HA composite solution 6.

Example 7 Preparation of HA Composite Solution 7

The preparation method is as follows: weigh 1.50 grams of high-molecular-weight hyaluronic acid (weight-average molecular weight of 1 million Daltons), weigh 1.50 grams of low-molecular-weight hyaluronic acid (weight-average molecular weight of 100,000 Daltons), and mix the above high and low The molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was the buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and low molecular weight hyaluronic acid solutions;

The subsequent preparation method is the same as in Example 1 to obtain HA composite solution 7.

Example 8 Preparation of HA Composite Solution 8

The preparation method is as follows: weigh 2.70 grams of high-molecular-weight hyaluronic acid (weight-average molecular weight of 1 million Daltons), weigh 0.30 grams of low-molecular-weight hyaluronic acid (weight-average molecular weight of 100,000 Daltons), and mix the above-mentioned high and low Molecular weight hyaluronic acid was dissolved in 50 ml of medium solution respectively, and the selected medium solution was the buffer solution M2 prepared by the method in Preparation Example 2 to prepare high and low molecular weight hyaluronic acid solutions;

The subsequent preparation method is the same as in Example 1 to obtain HA composite solution 8.

Example 9 Preparation of HA Composite Solution 9

The preparation method is as follows: weigh 1.35 grams of high-molecular-weight hyaluronic acid (weight-average molecular weight of 1 million Daltons), weigh 0.15 grams of low-molecular-weight hyaluronic acid (weight-average molecular weight of 100,000 Daltons), and Molecular weight hyaluronic acid was respectively dissolved in 100 ml of medium solution, the selected medium solution was the buffer solution M2 prepared by the method in Preparation Example 2, and high and low molecular weight hyaluronic acid solutions were obtained;

The subsequent preparation method is the same as in Example 1 to obtain HA composite solution 9.

Example 10 Preparation of HA Composite Solution 10

The preparation method is as follows: weigh 2.70 grams of high-molecular-weight hyaluronic acid (weight-average molecular weight of 1 million Daltons), weigh 0.30 grams of low-molecular-weight hyaluronic acid (weight-average molecular weight of 100,000 Daltons), and mix the above-mentioned high and low The molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was the buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and low molecular weight hyaluronic acid solutions;

After placing the hyaluronic acid solutions with high and low molecular weight for 4 hours, stir them to disperse them completely. After the dispersion was complete, the high and low molecular weight hyaluronic acid solutions were mixed uniformly at a ratio of 1:1 to obtain a mixed solution of high and low molecular weight hyaluronic acid with a final concentration of 3.0%. The solution was stirred and homogenized using an IKA-T25 homogenizer, and the rotating speed of the cutter head was set at 14000 rpm, and the treatment time of the mixed solution was 15 minutes, and the HA composite solution 10 was obtained.

Example 11 Preparation of HA Composite Solution 11

The preparation method is as follows: weigh 2.70 grams of high-molecular-weight hyaluronic acid (weight-average molecular weight of 1 million Daltons), weigh 0.30 grams of low-molecular-weight hyaluronic acid (weight-average molecular weight of 100,000 Daltons), and mix the above-mentioned high and low The molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was the buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and low molecular weight hyaluronic acid solutions;

After placing the hyaluronic acid solutions with high and low molecular weight for 4 hours, stir them to disperse them completely. After the dispersion was complete, the high and low molecular weight hyaluronic acid solutions were mixed uniformly at a ratio of 1:1 to obtain a mixed solution of high and low molecular weight hyaluronic acid with a final concentration of 3.0%. The solution was stirred and homogenized using an IKA-T25 homogenizer, and the rotation speed of the cutter head was set to 10000 rpm, and the treatment time of the mixed solution was 15 minutes, and the HA composite solution 11 was obtained.

Example 12 Preparation of HA Composite Solution 12

The preparation method is as follows: Weigh 2.70 grams of high molecular weight hyaluronic acid (weight average molecular weight of 2 million Daltons), weigh 0.30 grams of low molecular weight hyaluronic acid (weight average molecular weight of 100,000 Daltons), and mix the above high and low The molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was the buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and low molecular weight hyaluronic acid solutions;

The subsequent preparation method is the same as in Example 1, and HA composite solution 12 is prepared.

Example 13 Preparation of HA Composite Solution 13

The preparation method is as follows: weigh 2.70 grams of high molecular weight hyaluronic acid (weight average molecular weight of 600,000 daltons), weigh 0.30 grams of low molecular weight hyaluronic acid (weight average molecular weight of 100,000 daltons), and mix the above high and low The molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was the buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and low molecular weight hyaluronic acid solutions;

The subsequent preparation method is the same as in Example 1, and HA composite solution 13 is prepared.

Example 14 Preparation of HA Composite Solution 14

The preparation method is as follows: weigh 2.70 grams of high-molecular-weight hyaluronic acid (weight-average molecular weight of 1 million Daltons), weigh 0.30 grams of low-molecular-weight hyaluronic acid (weight-average molecular weight of 50,000 Daltons), and The molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was the buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and low molecular weight hyaluronic acid solutions;

The subsequent preparation method is the same as in Example 1, and HA composite solution 14 is prepared.

Example 15 Preparation of HA Composite Solution 15

The preparation method is as follows: weigh 2.25 grams of high-molecular-weight hyaluronic acid (weight-average molecular weight of 1 million Daltons), weigh 0.75 grams of medium-molecular-weight hyaluronic acid (weight-average molecular weight of 200,000 Daltons), and mix the above high and low The molecular weight hyaluronic acid was respectively dissolved in 50 ml of medium solution, and the selected medium solution was the buffer solution M1 prepared according to the method in Preparation Example 1 to prepare high and low molecular weight hyaluronic acid solutions;

The subsequent preparation method is the same as that in Example 1 to prepare HA composite solution 15.

The injection force, viscosity, storage modulus of embodiment 16 HA composite solution 1-15 are measured

1. Determination of bolus force

The injection force is obtained by measuring the extrusion force of the syringe handle, and the specific measurement method is as follows:

Install a 27G needle on the syringe, advance the syringe handle at a speed of 32.34mm/min, and measure the average extrusion force of the syringe handle, which is the injection force.

The samples of each HA composite solution prepared in Examples 1-15 were measured by the above-mentioned method, and the measured value of the bolus injection force was obtained, and the specific measurement results are shown in Table 1.

2. Viscosity measurement method

Use the MYR-3000 rotational viscometer and No. 3 probe to test the viscosity of the obtained HA composite solution. Take 30mL of each group of samples and place them in a 50mL centrifuge tube, turn on the rotational viscometer and calibrate the probe. Immerse the probe in the sample solution to the scale line of the probe, start the rotating probe, and record the data after the viscosity reading is stable. Each set of solutions was detected three times in parallel.

The samples of each HA composite solution prepared in Examples 1-15 were measured by the above-mentioned method to obtain the viscosity measurement value, and the specific measurement results are shown in Table 1.

3. Storage modulus measurement method

Take a 200 μL sample and place it in the center of the parallel fixture of the TA-DHR2 rheometer, and use a 20mm parallel plate probe to contact the surface of the sample to ensure that the probe is fully covered. The detection temperature is 25 degrees Celsius, the shear frequency is 1 Hz, and the test time for each group of samples is 1 minute.

The samples of the HA composite solutions prepared in Examples 1-15 were measured by the above method to obtain the measured value of the storage modulus. The specific measurement results are shown in Table 1.

Table 1 Injection force, viscosity, storage modulus, degradation time in vivo of HA composite solution 1-15

Example 17 Degradation Performance Determination in Animals of HA Composite Solution 1-15

Prepare the HA composite solution samples according to the method described in Examples 1-15 respectively. After the preparation is completed, measure and evaluate the degradation cycle of each sample in the animal body by examining the degradation cycle in the animal body. The specific measurement data are shown in Table 2. The degradation time in vivo See Table 1.

The method of in vivo degradation test in animals is as follows:

Screen 120 healthy SD rats, take the HA composite solution 1-15 prepared in Example 1-15 as a sample, and divide the rats into 15 groups at random, with 8 rats in each group, and each group is given HA composite solution 1-15 respectively. 15. The samples were implanted subcutaneously in rats, and each rat was implanted with three HA compound solutions (200 μl), one PE tube (control), and normal saline (200 μl, control). After implantation, 8 rats in each group were sacrificed at 1, 2, 3, 4, 5, 6, 7, and 8 weeks, and the diameter and height of the implant were measured until the implant Completely disappeared, and the volume of the implant was obtained by calculating the volume of the partial sphere (see the table below). The end point of the degradation cycle was defined as the sample volume less than 20% of the sample volume at the first week. No abrasions and traumas were found during the observation period, and no skin bruises, edema, and erythema were found on the skin at the implantation site.

Since the injection force and viscosity of the HA composite solution 12 prepared by the method in Example 12 are too large, it is meaningless to measure the degradation test results in animals.

Table 2 Degradation performance test data of HA compound solution 1-15 in animals

Example 18 Safety Research in Animals

The test method is as follows: 45 healthy rabbits are selected and randomly divided into HA compound solution group and normal saline group. The samples were randomly implanted into the skin of the back of rabbits without hair, and each sample was injected at 5 points, and each point was injected with 0.2ml. The reaction of each injection site was observed at 24h, 48h, and 72h after injection, and the tissue reaction of erythema and edema at each injection site was scored in each observation period according to the intradermal reaction scoring standard.

Results: Under the conditions of this experiment, no obvious erythema and edema reactions were observed at the injection site in the intradermal reaction test of the experimental samples, and the final score difference was less than or equal to 1.0.

According to the above test method, the HA composite solution samples prepared in Examples 1-15 of this patent were investigated, and the results showed that the safety of the prepared HA composite solution samples in Examples 1-15 met the requirements.

Claims (10)

1. A composition for injection, characterized in that, contains high molecular weight hyaluronic acid (or sodium hyaluronate), medium molecular weight hyaluronic acid (or sodium hyaluronate) and/or low molecular weight hyaluronic acid (or hyaluronate sodium hyaluronate), and a buffer solution, the molecular weight of the high molecular weight hyaluronic acid (or sodium hyaluronate) is 800kDa-1800kDa, preferably 800-1200kDa, the molecular weight of the middle molecular weight hyaluronic acid (or sodium hyaluronate) The molecular weight is 200kDa-700kDa, and the molecular weight of the low molecular weight hyaluronic acid (or sodium hyaluronate) is 80kDa-120kDa. 2. The composition for injection according to claim 1, wherein the high molecular weight hyaluronic acid (or sodium hyaluronate) and the medium molecular weight hyaluronic acid (or sodium hyaluronate) and/or The weight ratio of low molecular weight hyaluronic acid (or sodium hyaluronate) is (8-10):1. 3. The composition for injection according to claim 1 or 2, characterized in that the total concentration of all hyaluronic acid (or sodium hyaluronate) is 1.0-3.5%, preferably 2.8-3.3%. 4. The composition for injection according to any one of claims 1-3, wherein the buffer solution is a citric acid-sodium citrate-lactic acid buffer solution containing 2.0% sodium citrate, Citric acid is 0.4%, lactic acid is 0.01%, and pH is 5.0. 5. The composition for injection according to any one of claims 1-4, characterized in that, the pharmaceutical composition for injection can also contain diluent, stabilizer, isotonic regulator, and/or Solvents or co-solvents, etc., may further contain analgesics such as lidocaine, etc. 6. The preparation method of the composition for injection as described in any one of claims 1-5, is characterized in that, comprises the steps: (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 ultrasonicating the solutions obtained in steps (1) and (2); (4) Sterilization. 7. The preparation method according to claim 6, characterized in that, homogenize at 11000-13000RPM for 10-20min. 8. The preparation method according to claim 6, characterized in that 10-25min is ultrasonicated at 800-1600w. 9. The preparation method according to any one of claims 6-8, characterized in that, in the solution obtained in step (3), add diluent, stabilizer, isotonic regulator, and/or solubilizing or auxiliary Solvent and the like, preferably an analgesic such as lidocaine and the like are further added and mixed. 10. Use of the injectable composition according to any one of claims 1-5 in the preparation of cosmetic and medical products.