Soluble hyaluronic acid drug-loaded microneedle and preparation method thereof

Abstract

The present invention discloses a soluble hyaluronic acid drug-loaded microneedle and a preparation method thereof. The microneedle includes a needle tip portion and a circular base portion, wherein the needle tip portion is prepared using a low-molecular-weight hyaluronic acid within the range of 5-25w as a matrix; the circular base portion is prepared using a mixture of a high-molecular-weight hyaluronic acid within the range of 25-150w and a low-molecular-weight hyaluronic acid within the range of 5-25w as a matrix; wherein the mass ratio of the high-molecular-weight hyaluronic acid to the low-molecular-weight hyaluronic acid is 3:20-3:100. The soluble hyaluronic acid drug-loaded microneedle is loaded with a growth factor that promotes skin healing and regeneration. By the microneedle preparation technology of this method, the different functions of hyaluronic acid of different molecular weights can be fully utilized, and the target growth factor can be concentrated and distributed in the micro-tip portion of the microneedle, which can effectively increase the local drug concentration, improve the therapeutic effect, save production costs, and has the advantages of high precision, good therapeutic effect, batch stability, and easy preparation cost.

Description

Technical Field

[0001] The present invention belongs to the fields of medical cosmetology and medicine, and specifically relates to a soluble hyaluronic acid drug-loaded microneedle and a preparation method thereof. Background Art

[0002] The skin is the outermost layer of the human body, its primary function being to protect the body from harmful factors such as external irritants, ultraviolet rays, bacteria, and moisture. This barrier function is primarily achieved by two components: the stratum corneum and the sebum membrane. First, the stratum corneum is composed of multiple layers of dead keratinocytes, which continuously shed and regenerate to form a solid barrier. This barrier effectively blocks external irritants and regulates water loss, maintaining skin hydration and preventing dryness and roughness. Furthermore, the stratum corneum contains a large amount of natural moisturizing factors that absorb and retain moisture from the air, keeping the skin moisturized. Second, the sebum membrane, formed from a mixture of oil secreted by the sebaceous glands and sweat from the sweat glands, forms a lubricating protective film that protects the skin from external irritants and reduces water loss. The sebum membrane also contains natural antimicrobial and acidic substances that effectively kill bacteria, fungi, and other pathogens, preventing infection. While the skin blocks external stimuli, it also hinders the penetration of drugs targeting the skin into the body to exert their effects. Therefore, a drug delivery method is needed that can deliver drugs below the epidermis in a minimally invasive or even non-invasive manner.

[0003] The microneedle transdermal drug delivery system is a novel drug delivery technology that uses tiny needles to deliver drugs directly into the deeper layers of the skin, achieving more effective therapeutic effects. Microneedles range in height from several hundred microns. These microneedles can penetrate the stratum corneum, the surface layer of the skin, breaking down the skin's barrier without interfering with nerves or capillaries. This allows for faster and more complete drug delivery to the subcutaneous tissue without causing pain or bleeding. Compared to traditional transdermal drug delivery methods, microneedle transdermal drug delivery offers several advantages: First, microneedle transdermal drug delivery can improve drug penetration and absorption. Microneedles can penetrate the stratum corneum, significantly shortening the time it takes for drugs to penetrate the skin and allowing for more even distribution of drugs in the subcutaneous tissue, thereby improving drug absorption efficiency. Second, while some drugs can easily cause side effects in other parts of the body after oral administration or injection, microneedle transdermal drug delivery can deliver drugs directly to the skin where treatment is needed, reducing the risk of side effects within the body. Third, microneedle transdermal drug delivery can improve drug stability. Some drugs are prone to decomposition, inactivation, or oxidation in the external environment, and are also easily metabolized by the digestive system after entering the body. Loading drugs on microneedles and administering them to specific parts of the body can protect them from these factors, thereby improving the stability of the drugs. Finally, microneedle transdermal drug delivery is simple, safe, and painless. Compared with injections and oral administration, microneedle transdermal drug delivery does not require professional operation, and the needles are extremely fine and will not cause obvious damage to the skin, so patients have a better user experience.

[0004] Hyaluronic acid (also known as hyaluronic acid) is a polysaccharide molecule that exists naturally in human tissues, especially the skin. It plays important physiological functions in the body, such as moisturizing, maintaining extracellular space, regulating osmotic pressure, lubricating, and promoting cell repair. Its greatest advantage lies in its extremely strong moisturizing ability. It can form a protective film on the skin surface to prevent water loss, thereby making the skin more moist, soft and smooth. In addition, hyaluronic acid also has the function of promoting cell regeneration and repair, increasing the elasticity and firmness of the skin. Therefore, hyaluronic acid is also widely used in the field of medical cosmetology. It is injected into the facial skin as a filler and can effectively improve wrinkles, nasolabial folds, lip contours and other problems, making the facial lines clearer and more natural, achieving a younger effect. In general, hyaluronic acid has many advantages such as moisturizing, promoting cell regeneration and repair, and medical cosmetology. It has become one of the most popular materials for moisturizing, improving skin problems and plastic surgery.

[0005] Growth factors are a class of multi-effect polypeptide substances that regulate cell growth and other functions by binding to specific, high-affinity cell membrane receptors. Their main mechanisms of action include promoting cell proliferation and differentiation, promoting collagen and elastic fiber synthesis, and promoting angiogenesis. In addition, growth factors can also enhance the skin's immunity and resistance, and promote wound healing and tissue repair. Although growth factors have great potential in treatment, their application still faces some challenges. First, they have a short half-life, are easily decomposed, and have poor stability. Second, due to their large molecular weight, they are difficult to penetrate the stratum corneum and enter the skin to exert their effects through topical administration. Therefore, it is necessary to explore suitable delivery methods for growth factors to better play their role in skin care and beauty. Summary of the Invention

[0006] The present invention aims to solve the problem of poor biocompatibility such as deposition in the body caused by other polymer materials themselves or cross-linkers after repeated use (Khater Ahmed Saeed AL-Japairaia, Syed Mahmood, Samah Hamed Almurisi, et al. Current trends in polymer microneedle for transdermal drug delivery [J]. International journal of pharmaceuticals, 2020, 587: 119673.), by using hyaluronic acid materials to avoid the immune rejection reaction of the organism itself.

[0007] The purpose of the present invention is to provide a soluble hyaluronic acid drug-loaded microneedle and a preparation method thereof, wherein the microneedle is used for rapid subcutaneous drug delivery.

[0008] The soluble hyaluronic acid drug-loaded microneedles provided by the present invention are conical in shape, including a needle tip and a circular base, with a height of 200-900 μm and a base diameter of 100-500 μm.

[0009] The needle tip portion is prepared using a low molecular weight hyaluronic acid in the range of 5-25w as a matrix; the circular base portion is prepared using a mixture of a high molecular weight hyaluronic acid in the range of 25-150w (specifically 40-100w) and a low molecular weight hyaluronic acid in the range of 5-25w as a matrix; wherein the mass ratio of the high molecular weight hyaluronic acid to the low molecular weight hyaluronic acid can be 3:20-3:100 (specifically 3:40);

[0010] The soluble hyaluronic acid drug-loaded microneedles are loaded with growth factors that promote skin healing and regeneration, including but not limited to a combination of one or more factors selected from bFGF, EGF, VEGF, HGF, PLGF, etc.;

[0011] Preferably, the tip portion of the soluble hyaluronic acid drug-loaded microneedle is loaded with growth factors, and the base portion is loaded with or not loaded with growth factors.

[0012] The soluble hyaluronic acid drug-loaded microneedles provided by the present invention are prepared by a method comprising the following steps:

[0013] 1) Prepare a low molecular weight hyaluronic acid solution containing growth factors;

[0014] 2) Add the prepared solution to the microneedle patch mold and remove bubbles using vacuum negative pressure to completely fill the solution to the needle tip of the mold;

[0015] 3) preparing a mixed solution of low molecular weight hyaluronic acid and high molecular weight hyaluronic acid;

[0016] 4) adding the prepared mixed solution to the microneedle patch mold that has been filled with the needle tip portion in step 2), and removing bubbles by vacuum negative pressure to allow the mixed solution to fill the base portion;

[0017] 5) Drying and demoulding to obtain a soluble hyaluronic acid drug-loaded microneedle patch.

[0018] In step 1) of the above method, the growth factors include but are not limited to a combination of one or more factors selected from bFGF, EGF, VEGF, HGF, PLGF, etc.;

[0019] The solutes in the low molecular weight hyaluronic acid solution containing growth factors include growth factors and low molecular weight hyaluronic acid in the range of 5-25w; the solvent is sterile deionized water, PBS buffer, or a mixture of sterile deionized water and PBS buffer, and the volume ratio of sterile deionized water to PBS buffer in the mixture is 1:1-1:10, specifically 1:5;

[0020] In the low molecular weight hyaluronic acid solution containing growth factors, the concentration of the growth factors is 0-900 μg / mL, the endpoint 0 is not desirable, and specifically can be 50 μg / mL; the concentration of the low molecular weight hyaluronic acid can be 0-200 mg / mL, the endpoint 0 is not desirable, and specifically can be 50 mg / mL;

[0021] In step 3), in the mixed solution, the mass ratio of high molecular weight hyaluronic acid to low molecular weight hyaluronic acid may be 3:20-3:100 (specifically 3:40);

[0022] In the mixed solution, the concentration of high molecular weight hyaluronic acid may be 0-20 mg / mL, specifically 3 mg / mL;

[0023] The mixed solution may further contain growth factors, and the concentration of the growth factors in the mixed solution is 0-600 μg / mL, with endpoint 0 being undesirable, and specifically may be 40 μg / mL;

[0024] Step 5) of the above method may further include covering the microneedle substrate with a sticky material, and the sticky material may specifically be 3M glue.

[0025] The obtained microneedle patch is 40-150mm long and 10-100mm wide. The bottom diameter of a single microneedle is 50-1000μm, the height is 100-1000μm, the needle tip spacing is 100-5000μm, the number of microneedles in a single piece is about 500-10,000 needles, and the shape is similar to a crescent.

[0026] The hyaluronic acid with a molecular weight of 5w-150w used in the present invention is a biological source material. The base uses high molecular weight hyaluronic acid to moisturize the skin surface, and the needle tip uses low molecular weight hyaluronic acid to dissolve and penetrate into the dermis. After entering the dermis through the microneedle, it delivers growth factors to promote cell proliferation and migration.

[0027] The present invention utilizes the distinct functions of hyaluronic acid of varying molecular weights in skin tissue to optimize the optimal ratio of high- and low-molecular-weight hyaluronic acid. The low-molecular-weight hyaluronic acid, located at the needle tip, has enhanced permeability and solubility, penetrating the dermis to deliver the loaded growth factor, dilating capillaries, enhancing blood circulation, and promoting nutrient absorption by the skin. The high-molecular-weight hyaluronic acid, located at the base, forms a moisturizing, breathable membrane on the skin surface, keeping it smooth and moisturized. Hyaluronic acid is dissolved in sterile deionized water or sterile PBS buffer at varying ratios to create soluble hyaluronic acid microneedles with different matrices. By adjusting the dosage, different types of hyaluronic acid microneedles can be produced. These microneedles can encapsulate one or more growth factors and rapidly dissolve after puncturing the skin, achieving targeted release. These microneedles exhibit excellent biocompatibility and degradability. The use of soluble drug-loaded microneedles can effectively improve drug targeting and efficacy, enhancing therapeutic efficacy and safety. Compared to traditional topical or injection treatments, this approach achieves higher local drug concentrations and a longer duration, maximizing the drug's effectiveness. Through this microneedle preparation technology, the target growth factor can be concentrated and distributed at the microtip of the microneedle, which can effectively increase the local drug concentration, improve the therapeutic effect, save production costs, and has the advantages of high precision, good therapeutic effect, stable batches, easy preparation and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the microneedle of the present invention used for transdermal drug delivery.

[0029] Figure 2 This is a photo of the specific morphology of the crescent-shaped water-soluble hyaluronic acid microneedle patch produced in Example 1 of the present invention.

[0030] Figure 3This is a side view of the microneedle array of the microneedles produced in Example 2 of the present invention, taken using an optical microscope.

[0031] Figure 4 This is an image of a microneedle array of microneedles produced in Example 2 of the present invention, taken using a scanning electron microscope (SEM).

[0032] Figure 5 An image of a single microneedle produced in Example 2 of the present invention is taken using a scanning electron microscope (SEM).

[0033] Figure 6 3 is a curve showing the mechanical force-displacement relationship that the three microneedle tips can withstand in Example 3 of the present invention.

[0034] Figure 7 4 is a curve showing the change in bFGF release concentration over time from the microneedle patch in Example 4 of the present invention.

[0035] Figure 8 This is a trypan blue staining photograph of the skin after microneedle application in Example 5 of the present invention.

[0036] Figure 9 This is an image of a frozen section of mouse skin with micropores formed after application of the microneedle patch in Example 5 of the present invention.

[0037] Figure 10 These are photos of mouse skin healing at different times after application of the microneedle patch in Example 6 of the present invention.

[0038] Figure 11 These are optical microscope images of the needle tip dissolution at different time points after the microneedle patch in Example 7 of the present invention was applied to mouse skin (a: 10 min, b: 15 min, c: 20 min).

[0039] Figure 12 Images of frozen sections of mouse skin after application of rhodamine B microneedles in Example 8 of the present invention (a: bright field image, b: fluorescence image). Blue: DAPI, red: RhB. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0041] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0042] Example 1. Preparation of water-soluble hyaluronic acid microneedles.

[0043] First, clean the mold thoroughly and use an ultrasonic cleaner to clean it for 3 hours. Replace the sterile deionized water halfway through. After completion, dry the mold. Accurately take 50mg of hyaluronic acid with a molecular weight of 15w-25w and 1mL of sterile deionized water, mix the two and add them to a beaker, stir magnetically until they are completely dissolved, and centrifuge to remove the bubbles to obtain a 50mg / mL small molecule water-soluble hyaluronic acid solution. Take 400μL of the prepared solution and add it to the mold. Vacuum negative pressure to remove bubbles so that the solution completely fills the mold cavity. Each time the valve is closed, the vacuum negative pressure is increased for about 15s, and then the exhaust hole is opened. Repeat 3 times and then use a pipette to remove larger bubbles. Repeat the cycle until the solution completely enters the needle tip. At the same time, take 15 mg of hyaluronic acid with a molecular weight of 40w-100w and 1 mL of sterile deionized water, and prepare a 15 mg / mL large molecular weight water-soluble hyaluronic acid solution according to the above solution preparation method. Mix it with the small molecular weight water-soluble hyaluronic acid solution in a volume ratio of 1:4 (the volume of the large molecular weight water-soluble hyaluronic acid solution is 1 part and the volume of the small molecular weight water-soluble hyaluronic acid solution is 4 parts) and then take 1.2 mL and add it to the mold to make the solution fully fill the base. After completion, place the mold in a constant temperature drying oven to dry and then demold. Cover the microneedle base with 3M glue or other sticky materials to complete the production of microneedles. The resulting crescent-shaped microneedle patch is 46.61 mm long and 18.09 mm wide. The bottom diameter of a single microneedle is 280 μm, the height is 650 μm, the needle tip spacing is 800 μm, the number of microneedles on a single piece is about 1000 needles, and the shape is similar to a crescent.

[0044] Figure 1 Schematic diagram of microneedles used for transdermal drug delivery.

[0045] Figure 2 The morphology of the prepared water-soluble hyaluronic acid microneedle patch was displayed under natural light.

[0046] Example 2: Preparation of hyaluronic acid microneedles

[0047] Take 50 mg of low-molecule hyaluronic acid and 15 mg of high-molecule hyaluronic acid, dissolve them in 1 mL of PBS buffer respectively, stir magnetically until they are completely dissolved, centrifuge to remove bubbles in the solution, and then repeat the subsequent operations in Example 1.

[0048] Figure 3 This is an optical microscope side view of the prepared microneedle patch.

[0049] Figure 4 and Figure 5 The SEM image of the prepared microneedle patch shows that the obtained microneedle is conical with a height of 650 μm and a bottom diameter of 280 μm.

[0050] Example 3: Microneedle Mechanical Properties Test

[0051] Referring to the preparation method in Example 1, 50 mg of hyaluronic acid with a molecular weight of 15w-25w was dissolved in 1 mL of a mixed solution of PBS and water in a volume ratio of 5:1. All other operations remained unchanged, and microneedles 3 were prepared according to the method in Example 1. The mechanical stress that the microneedles prepared in Examples 1 and 2, as well as microneedle 3, could withstand was tested using a texture analyzer. The microneedle patch was placed horizontally on a platform with the needle tip facing upward. The probe was used to vertically compress the microneedles at a speed of 0.1 mm / s, and the compression force was recorded simultaneously.

[0052] Figure 6 The mechanical force that each microneedle can withstand and the displacement curve show that the mechanical force that the microneedle can withstand for transdermal penetration should be greater than 0.045N. The stress of the microneedle obtained in the present invention is greater than 0.38N when the compression displacement reaches 600μm, indicating that the obtained microneedle can successfully penetrate the skin.

[0053] Example 4: Microneedle patch drug release ability test

[0054] In vitro simulated drug release experiments were conducted using basic fibroblast growth factor (bFGF). 1 mg of bFGF was dissolved in 1 mL of sterile deionized water. 300 μL of the solution was added to 5.7 mL of sterile deionized water and thoroughly mixed. 300 mg of low-molecular-weight hyaluronic acid was then added to create a solution with a concentration of 50 μg / mL bFGF and 50 mg / mL low-molecular-weight hyaluronic acid. A magnetic stirrer was then used to fully dissolve the solution, and the drug-containing solution was then pipetted into the mold. During vacuum degassing, the solution volume was carefully controlled to prevent overflow and inaccurate quantitative determination. After drying and demolding, the bFGF-embedded microneedles were fabricated. After microneedle fabrication, the microneedles were placed in 30 mL of sterile deionized water and placed in a shaker at 150 rpm and 37°C for release. Samples were collected at 1, 2.5, 5, 7.5, 10, 15, 20, 30, 40, 50, and 60 minutes during the release process. The samples were diluted and assayed for bFGF concentration using an ELISA kit according to the manufacturer's instructions. Four control groups were set up. The sample absorbance was measured using a microplate reader. The bFGF release concentration was calculated based on the measured data and the standard curve.

[0055] Figure 7 The release concentration-time curve of the drug was simulated in vitro using bFGF loaded as an example for the microneedle patch. It can be seen that the microneedle has the ability to release the drug rapidly.

[0056] Example 5: Skin puncture ability test

[0057] After anesthesia, the mouse was anesthetized and the back hair was removed with a depilatory cream. The skin surface was wiped clean with normal saline and dried. The microneedle patch prepared in Example 1 was attached to the skin and removed after pressing for 5 minutes. The skin was stained with trypan blue solution for 5 minutes. The mouse was killed by cervical dislocation, and the skin was immediately removed and fixed in 4% paraformaldehyde fixative for 30 minutes. After removal, it was placed in OCT embedding medium and frozen overnight. The tissue was cut into 7 μm thick frozen sections using a freezing microtome for observation.

[0058] Figure 8 This is a photo of the skin after trypan blue staining. The blue dots represent the microneedle puncture sites.

[0059] Figure 9 This is an optical microscope photo of a frozen section of mouse skin, showing that the microneedles can successfully penetrate the epidermis of the skin.

[0060] Example 6: Mouse skin healing experiment

[0061] After anesthesia, the mice were anesthetized and their back hair was removed using a depilatory cream. The skin surface was wiped clean with normal saline and the microneedle patch prepared in Example 1 was attached to the skin. After pressing for 3 minutes, the patch was removed and the healing speed of the micropores in the back skin of the mice and subsequent performance were observed.

[0062] according to Figure 10 After the microneedles were removed after the application, a clear puncture mark remained on the mouse's back skin. This mark gradually diminished over the next 20 minutes, and after 20 minutes, it was no longer visible to the naked eye. The mouse's skin subsequently appeared normal, with no adverse reactions.

[0063] Example 7: Determination of the actual dissolution of microneedles in vivo by applying them to mouse skin

[0064] After anesthesia, the mice were anesthetized and their back hair was removed with a depilatory cream. The skin surface was cleaned with saline. The microneedle patch prepared in Example 1 was placed on the skin and removed after 10, 15, and 20 minutes, respectively. The dissolution of the microneedle tip was observed using an optical microscope.

[0065] Figure 11 The dissolution of the microneedle tip at different time points shows that the needle tip has completely dissolved at 20 minutes.

[0066] Example 8: Skin drug release ability test

[0067] In order to evaluate the drug release and diffusion ability of microneedles after application to the skin, rhodamine B was used to prepare 1 mg / mL RhB and 50 mg / mL15w-25w small molecule water-soluble hyaluronic acid solution, replacing the 50 mg / mL small molecule water-soluble hyaluronic acid solution in Example 1. The microneedles prepared by the method of Example 1 were attached to the back skin of depilated mice and pressed for 20 minutes. Then, frozen sections were prepared according to the method of Example 6 and stained with DAPI. After sealing, the bright field and fluorescence images of the sections were observed using a fluorescence microscope.

[0068] Figure 12 It demonstrates the ability of microneedles to release drugs transdermally. The bright field image shows that the needle tip penetrates the subcutaneous tissue, and the fluorescent image proves that the needle tip dissolves and releases the drug, which can then diffuse into the surrounding skin.

[0069] High molecular weight hyaluronic acid solution at 20mg / ml and above is very viscous and difficult to prepare, while too low a concentration will result in a high water content. During the drying process, the solution must be added repeatedly, making the process more complicated. Therefore, a concentration of 15mg / ml is selected and mixed with a low molecular weight hyaluronic acid solution (50mg / mL). Under the premise of ensuring one-time drying and coverage by high molecular weight hyaluronic acid, the volume ratio of high molecular weight: low molecular weight solution is between 1:2-1:10.

[0070] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A soluble hyaluronic acid drug-loaded microneedle, wherein the soluble hyaluronic acid drug-loaded microneedle is conical in shape, comprising a needle tip portion and a circular base portion, with a height of 200-900 μm and a base portion diameter of 100-500 μm; Its characteristics are: The needle tip is prepared using low molecular weight hyaluronic acid in the range of 5-25w as a matrix; The circular base portion is prepared using a mixture of high molecular weight hyaluronic acid in the range of 25-150w and low molecular weight hyaluronic acid in the range of 5-25w as a matrix; Wherein, the mass ratio of the high molecular weight hyaluronic acid to the low molecular weight hyaluronic acid is 3:20-3:100; The soluble hyaluronic acid drug-loaded microneedles are loaded with growth factors that promote skin healing and regeneration; The growth factor is a combination of one or more of bFGF, EGF, VEGF, HGF, and PLGF.

2. A method for preparing the soluble hyaluronic acid drug-loaded microneedles according to claim 1, comprising the following steps: 1) Prepare 5-25w low molecular weight hyaluronic acid solution containing growth factors; 2) Add the prepared solution to the microneedle patch mold and use vacuum negative pressure to remove bubbles so that the solution is completely filled to the needle tip of the mold; 3) Prepare a mixed solution of low molecular weight hyaluronic acid and high molecular weight hyaluronic acid; 4) Add the prepared mixed solution to the microneedle patch mold that has been filled with the needle tip portion in step 2), and remove bubbles under vacuum to allow the mixed solution to fill the base portion; 5) Drying and demolding to obtain a soluble hyaluronic acid drug-loaded microneedle patch; In step 3), in the mixed solution, the mass ratio of high molecular weight hyaluronic acid to low molecular weight hyaluronic acid is 3:20-3:100; The concentration of high molecular weight hyaluronic acid is 3-20 mg / mL.

3. The method according to claim 2, wherein: The needle tip portion of the soluble hyaluronic acid drug-loaded microneedle is loaded with growth factors, and the base portion is loaded with or not loaded with growth factors.

4. The method according to claim 2, wherein: In step 1), the solutes in the low molecular weight hyaluronic acid solution containing growth factors include growth factors and low molecular weight hyaluronic acid in the range of 5-25w; The solvent is sterile deionized water, PBS buffer or a mixture of sterile deionized water and PBS buffer, wherein the volume ratio of sterile deionized water to PBS buffer in the mixture is 1:1-1:

10.

5. The method according to claim 2, wherein: In the low molecular weight hyaluronic acid solution containing growth factors, the concentration of growth factors is 50-900 μg / mL, and the concentration of low molecular weight hyaluronic acid is 50-200 mg / mL.

6. The method according to claim 2, wherein: In step 3), the mixed solution further contains growth factors, and the concentration of the growth factors in the mixed solution is 40-600 μg / mL.

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