A composition containing PDRN microspheres, a preparation method thereof, and applications

Abstract

The present invention discloses a composition containing PDRN microspheres, a preparation method thereof, and an application, relating to the technical field of repair. The composition containing PDRN microspheres of the present invention is composed of the following components by weight: 1 part - 10 parts of PDRN microspheres, 30 parts - 60 parts of sodium hyaluronate, 0.05 part - 1 part of a cross-linking agent, 0.5 part - 5 parts of bisabolol, and 1 part - 8 parts of trehalose; the molecular weight of the sodium hyaluronate is 300 kDa - 800 kDa. By adding PDRN microspheres and selecting a reasonable ingredient combination, the present invention significantly improves stability and exhibits a synergistic effect in terms of repair.

Description

Technical Field

[0001] The present invention relates to the technical field of repair, and specifically relates to a composition containing PDRN microspheres, a preparation method thereof, and applications thereof. Background Art

[0002] Polydeoxyribonucleotide (PDRN) is a low-molecular-weight DNA derivative of natural origin, with a molecular weight between 50 kDa and 1500 kDa. The most representative molecular weight of PDRN is 80 kDa - 200 kDa, and the peak of the Gaussian distribution is approximately 132 kDa. The base length of PDRN is between 50 bp and 2000 bp. It is a linear polymer of deoxyribonucleotides with phosphodiester bonds. PDRN contains 50% double-stranded deoxyribonucleotides. Two polydeoxyribonucleotides are connected by hydrogen bonds between base pairs to finally form a double helix structure. PDRN is mainly extracted and purified from the sperm cells of salmon or rainbow trout.

[0003] The base composition of PDRN has a similarity of up to 98% with human DNA. Based on this biological effect, its application scope in clinical practice has been gradually expanded, and obvious effects have been shown in tissue repair, wound healing, anti-ischemia, anti-inflammation, etc. In vitro and in vivo experiments have shown that the most relevant mechanism of action of PDRN is as an agonist of adenosine A2A receptor, activating the A2A receptor and producing various physiological effects.

[0004] Chinese Patent CN114642606A discloses a composition with skin barrier repair function, a preparation method thereof, and applications thereof. It includes the following components: high-molecular-weight hyaluronic acid or its salt 0.01% - 0.5%, hydrolyzed hyaluronic acid or its salt 0.1% - 1.0%, silk fibroin 0.01% - 10%, polydeoxyribonucleic acid 0.05% - 1%, hydroxyproline 0.1% - 10%. The high-molecular-weight hyaluronic acid or its salt of this invention can target the stratum corneum of the skin and form a moist and breathable protective film on the skin surface. The extremely low-molecular-weight hydrolyzed hyaluronic acid or its salt can penetrate through the skin and enter the dermis layer of the skin to play the role of stimulating angiogenesis. PDRN, silk fibroin, and hydroxyproline can promote the synthesis of collagen and cell migration. These several components are synergistically combined, having an outstanding effect of promoting wound healing, being able to repair the damaged skin barrier, and achieving the effect of quickly repairing the skin wound surface.

[0005] Chinese Patent CN112932988A discloses an anti-aging composition containing polydeoxyribonucleic acid, including honey and polydeoxyribonucleic acid; the invention also discloses a skin care product, which includes the anti-aging composition containing polydeoxyribonucleic acid and also contains additives acceptable in cosmetics. The anti-aging composition disclosed in the invention and the skin care product including the anti-aging composition enhance the effects of promoting skin cell regeneration and anti-aging through the synergistic effect of honey and polydeoxyribonucleic acid.

[0006] At present, PDRN has the problem of poor stability and still has certain limitations when applied in repair products. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a composition containing PDRN microspheres, its preparation method and application. By adding PDRN microspheres and selecting a reasonable ingredient formulation, the stability is significantly improved, and a synergistic effect is demonstrated in terms of repair.

[0008] To achieve the above-mentioned invention purpose, the technical solution of the present invention is as follows:

[0009] On the one hand, the present invention provides a composition containing PDRN microspheres, which is composed of the following components by weight: 1 part - 10 parts of PDRN microspheres, 30 parts - 60 parts of sodium hyaluronate, 0.05 part - 1 part of cross-linking agent, 0.5 part - 5 parts of bisabolol and 1 part - 8 parts of trehalose.

[0010] Preferably, the composition containing PDRN microspheres is composed of the following components by weight: 3 parts - 8 parts of PDRN microspheres, 35 parts - 55 parts of sodium hyaluronate, 0.1 part - 0.6 part of cross-linking agent, 1 part - 4 parts of bisabolol and 2 parts - 6 parts of trehalose.

[0011] More preferably, the composition containing PDRN microspheres is composed of the following components by weight: 6 parts of PDRN microspheres, 50 parts of sodium hyaluronate, 0.5 part of cross-linking agent, 3 parts of bisabolol and 5 parts of trehalose.

[0012] Preferably, the molecular weight of the sodium hyaluronate is 300 kDa - 800 kDa.

[0013] More preferably, the molecular weight of the sodium hyaluronate is 400 kDa - 600 kDa.

[0014] If the molecular weight of sodium hyaluronate is too large, the strength of the cross-linking system is too high, the use feeling is poor, and it is not conducive to the release of active ingredients; if the molecular weight of sodium hyaluronate is too small, the stability is reduced. Selecting a suitable molecular weight range can ensure the comprehensive performance of the composition.

[0015] Preferably, the crosslinking agent is selected from at least one of divinyl sulfone, 1,4-butanediol diglycidyl ether, carbodiimide, and N-hydroxysuccinimide. Further preferably, it is a combination of carbodiimide and N-hydroxysuccinimide. More preferably, the molar ratio of carbodiimide to N-hydroxysuccinimide is 1-4:1.

[0016] Preferably, the particle size of the PDRN microspheres < 500 μm. In the present invention, there are not many restrictions on the particle size of the PDRN microspheres. The particle size is not a key influencing factor. The current form of PDRN microspheres can achieve a synergistic effect through the above compatibility, improving the repair activity of the product. Considering the current particle size distribution of microsphere products and reducing the impact on the preparation process, it is more preferable to select PDRN microspheres with a particle size of 10 μm - 200 μm.

[0017] On the other hand, the present invention provides a method for preparing the above composition containing PDRN microspheres, comprising the following steps:

[0018] (1) Dissolve the formulated amount of trehalose and bisabolol in a solvent, and then add the formulated amount of PDRN microspheres and sodium hyaluronate to obtain a mixed solution;

[0019] (2) Dissolve the formulated amount of crosslinking agent in a solvent, and gradually add it dropwise to the mixed solution obtained in step (1), perform ice bath treatment, adjust the pH, and raise the temperature for reaction to obtain a reaction product;

[0020] (3) Concentrate the reaction product obtained in step (2) to obtain a composition containing PDRN microspheres.

[0021] Preferably, in step (1), the solvent is a combination of phosphate buffer solution (PBS) and propylene glycol. Further preferably, the pH of the phosphate buffer solution is 5.0 - 6.0, and more preferably 5.5. Further preferably, the volume ratio of the phosphate buffer solution (PBS) to propylene glycol is 1 - 10:1, and most preferably 5:1.

[0022] Preferably, in step (1), in the mixed solution, the mass content of sodium hyaluronate is 1% - 5%, and further preferably 2%.

[0023] Preferably, in step (2), the solvent is propylene glycol.

[0024] Preferably, in step (2), the time of ice bath treatment is 5 min - 30 min, and further preferably 15 min.

[0025] Preferably, in step (2), the adjusting of pH is: adjusting the pH to 5.0 - 6.5, and further preferably 5.5 - 6.0.

[0026] The carboxyl groups can be maximally activated by ice bath treatment at an appropriate time and at an appropriate pH, which helps the cross-linking reaction proceed.

[0027] Preferably, in step (2), the conditions for the temperature-raising reaction are: raising the temperature to 20°C - 35°C and reacting for 2 h - 5 h, and more preferably raising the temperature to 30°C and reacting for 2.5 h.

[0028] Preferably, in step (3), the concentration is ultrafiltration concentration.

[0029] As a specific example of the present invention, the preparation method of the above composition containing PDRN microspheres includes the following steps:

[0030] (1) Dissolve the formulated amount of trehalose and bisabolol in a solvent, and then add the formulated amount of PDRN microspheres and sodium hyaluronate to obtain a mixed solution. In the mixed solution, the mass content of sodium hyaluronate is 1% - 5%;

[0031] (2) Dissolve the formulated amount of cross-linking agent in a small amount of solvent, and dropwise add it to the mixed solution obtained in step (1). Perform ice bath stirring for 5 min - 30 min, adjust the pH to be maintained at 5.5 - 6.0, raise the temperature to 20°C - 35°C and react for 2 h - 5 h. Add ethanolamine to the obtained reactant and stir to quench the reaction to obtain a gel-like reactant;

[0032] (3) Concentrate the gel-like reactant obtained in step (2) with a ceramic ultrafiltration membrane to obtain a composition containing PDRN microspheres.

[0033] Finally, the present invention provides the application of the above composition containing PDRN microspheres in the preparation of repair products.

[0034] Preferably, the repair products include cosmetics and drugs.

[0035] More preferably, the repair is to promote wound healing.

[0036] The beneficial effects of the present invention are as follows:

[0037] (1) The present invention selects PDRN microspheres to replace the conventional PDRN components, which can significantly improve the repair activity of the product and improve the product stability;

[0038] (2) In the present invention, PDRN microspheres are used in combination with sodium hyaluronate, bisabolol, and trehalose, and are prepared into a composition gel under appropriate ratios, which can play a synergistic effect and achieve a very significant improvement in terms of stability and repair activity.

[0039] (3) The present invention selects a suitable preparation method and sodium hyaluronate with a specific molecular weight, and encapsulates PDRN microspheres and bisabolol in the prepared network cross-linked structure, realizing the long-term effect of PDRN and bisabolol on the epidermis, and can play a repair function in a relatively long period of time after use, significantly improving the actual repair effect. Description of the Drawings

[0040] Figure 1 It is a graph showing the test results of the PDRN retention rate of the composition of the present invention. Detailed Embodiments

[0041] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further clarified below in conjunction with specific embodiments. However, the following embodiments are only the preferred embodiments of the present invention, not all of them. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used is all conventional equipment, and the equipment materials used in each embodiment are the same.

[0042] In the following embodiments, the raw material sources are as follows:

[0043] Carbodiimide (specifically dicyclohexylcarbodiimide used in the examples) and N-hydroxysuccinimide are purchased from Guangdong Yuanfeng; bisabolol is purchased from Xi'an Xirun, product number XRHZ-207, purity 99%; trehalose is of Linyuan brand, particle size 100 mesh; centella asiatica extract is purchased from Shaanxi Zhelang, product number HJDT0020, purity > 90%; sodium alginate is purchased from Jiangsu Nuojia, particle size 40 mesh - 60 mesh.

[0044] The PDRN microspheres are prepared by oneself according to the following method, and this preparation method does not limit the present invention:

[0045] (1) Material preparation:

[0046] Prepare a PDRN aqueous solution with a concentration of 20 mg / mL;

[0047] Prepare a poly-L-lactic acid organic solution with a mass-volume concentration of 5% (the organic phase is dichloromethane);

[0048] Prepare a zinc sulfate organic solution with a mass-volume concentration of 5% (the organic phase is dichloromethane);

[0049] Prepare an aqueous solution of polyvinyl alcohol with a mass-volume concentration of 60%.

[0050] The meaning of "mass-volume concentration" is: the ratio of the mass (g) of the solute in the solution to the volume (mL) of the solution.

[0051] (2)Preparation of microspheres:

[0052] Mix the zinc sulfate organic phase solution and the poly-L-lactic acid organic phase solution in a volume ratio of 1:1 to obtain mixed solution 1; under high-speed shearing conditions, inject the PDRN aqueous phase solution, and the volume ratio of the PDRN aqueous phase solution to mixed solution 1 is 1:1 to obtain a water-in-oil emulsion; under high-speed stirring conditions, slowly inject the water-in-oil emulsion into the polyvinyl alcohol aqueous solution, and the volume ratio of the water-in-oil emulsion to the polyvinyl alcohol aqueous solution is 1:1 to obtain a water-in-oil-in-water emulsion; stir at 1000 rpm until microspheres are formed, remove the solvent, wash, and obtain PDRN microspheres. It is measured that the particle size distribution is 40μm - 90μm.

[0053] Example 1

[0054] Formulation: 6 parts of PDRN microspheres, 50 parts of sodium hyaluronate (400 kDa - 600 kDa, purity > 95%), 0.5 part of cross-linking agent (obtained by mixing carbodiimide and N-hydroxysuccinimide in a molar ratio of 2:1), 3 parts of bisabolol, and 5 parts of trehalose.

[0055] Preparation process:

[0056] (1) Dissolve the formulated amount of trehalose with a solvent, which is prepared by mixing PBS (pH 5.5) and propylene glycol in a volume ratio of 5:1, to obtain a trehalose solution, and then add the formulated amounts of PDRN microspheres and sodium hyaluronate, and mix well to obtain a mixed solution; in the mixed solution, the mass content of sodium hyaluronate is 2%;

[0057] (2) Dissolve the formulated amount of cross-linking agent with a small amount of propylene glycol, and add it dropwise to the mixed solution obtained in step (1), perform ice bath stirring for 15 min, adjust the pH to maintain at 5.5, raise the temperature to 30°C and react for 2.5 h, add ethanolamine to the obtained reactant and stir at room temperature for 1 hour to quench the reaction, and obtain a gel-like reactant;

[0058] (3) Concentrate the gel-like reactant obtained in step (2) with a ceramic ultrafiltration membrane (100 kDa) to obtain a composition containing PDRN microspheres.

[0059] Example 2

[0060] Formulation: 10 parts of PDRN microspheres, 60 parts of sodium hyaluronate (300 kDa - 600 kDa, purity > 95%), 1 part of cross-linking agent (carbodiimide), 0.5 part of bisabolol, and 1 part of trehalose.

[0061] The preparation process is the same as that of Example 1.

[0062] Example 3

[0063] Formulation: 1 part of PDRN microspheres, 30 parts of sodium hyaluronate (500 kDa - 800 kDa, purity > 95%), 0.05 part of crosslinking agent (N-hydroxysuccinimide), 5 parts of bisabolol, and 8 parts of trehalose.

[0064] The preparation process is the same as that of Example 1.

[0065] Example 4

[0066] Formulation: 3 parts of PDRN microspheres, 35 parts of sodium hyaluronate (400 kDa - 600 kDa, purity > 95%), 0.1 part of crosslinking agent (obtained by mixing carbodiimide and N-hydroxysuccinimide in a molar ratio of 2:1), 4 parts of bisabolol, and 2 parts of trehalose.

[0067] The preparation process is the same as that of Example 1.

[0068] Example 5

[0069] Formulation: 8 parts of PDRN microspheres, 55 parts of sodium hyaluronate (400 kDa - 600 kDa, purity > 95%), 0.6 part of crosslinking agent (obtained by mixing carbodiimide and N-hydroxysuccinimide in a molar ratio of 2:1), 1 part of bisabolol, and 6 parts of trehalose.

[0070] The preparation process is the same as that of Example 1.

[0071] Example 6

[0072] The formulation is the same as that of Example 1.

[0073] Preparation process:

[0074] (1) Dissolve the formulated amount of trehalose in a solvent, which is prepared by mixing PBS (pH 5.5) and propylene glycol in a volume ratio of 5:1, to obtain a trehalose solution. Then add the formulated amounts of PDRN microspheres and sodium hyaluronate, and mix well to obtain a mixed solution; in the mixed solution, the mass content of sodium hyaluronate is 5%;

[0075] (2) Dissolve the formulated amount of crosslinking agent in a small amount of propylene glycol, and add it dropwise to the mixed solution obtained in step (1). Stir under ice bath for 30 min, adjust the pH to maintain at 6.0, raise the temperature to 35 °C and react for 2 h. Add ethanolamine to the resulting reactant and stir at room temperature for 1 hour to quench the reaction, obtaining a gel-like reactant;

[0076] (3) Concentrate the gel-like reactant obtained in step (2) using a ceramic ultrafiltration membrane (100 kDa) to obtain a composition containing PDRN microspheres.

[0077] Example 7

[0078] The formulation is the same as that of Example 1.

[0079] Preparation process:

[0080] (1) Dissolve the formulated amount of trehalose with a solvent, where the solvent is PBS (pH 5.5) and propylene glycol configured in a volume ratio of 5:1 to obtain a trehalose solution. Then add the formulated amount of PDRN microspheres and sodium hyaluronate, and mix well to obtain a mixed solution; in the mixed solution, the mass content of sodium hyaluronate is 1%;

[0081] (2) Dissolve the formulated amount of cross-linking agent with a small amount of propylene glycol, and gradually add it dropwise to the mixed solution obtained in step (1). Stir in an ice bath for 5 min, adjust the pH to maintain at 5.5, raise the temperature to 20 °C and react for 5 h. Add ethanolamine to the resulting reactant and stir at room temperature for 1 hour to quench the reaction, obtaining a gel-like reactant;

[0082] (3) Concentrate the gel-like reactant obtained in step (2) with a ceramic ultrafiltration membrane (100 kDa) to obtain a composition containing PDRN microspheres.

[0083] Comparative Example 1

[0084] Different from Example 1, the formulation selection is as follows: 6 parts of PDRN microspheres, 50 parts of sodium hyaluronate (400 kDa - 600 kDa, purity > 95%), 0.5 part of cross-linking agent (obtained by mixing carbodiimide and N-hydroxysuccinimide in a molar ratio of 2:1), 3 parts of centella asiatica extract, and 5 parts of trehalose. The rest are the same.

[0085] In Comparative Example 1, centella asiatica extract is used instead of bisabolol.

[0086] Comparative Example 2

[0087] Different from Example 1, the formulation selection is as follows: 15 parts of PDRN microspheres, 50 parts of sodium hyaluronate (400 kDa - 600 kDa, purity > 95%), 0.5 part of cross-linking agent (obtained by mixing carbodiimide and N-hydroxysuccinimide in a molar ratio of 2:1), 0.2 part of bisabolol, and 7 parts of trehalose. The rest are the same.

[0088] The component ratio of Comparative Example 2 is different from that of Example 1.

[0089] Comparative Example 3

[0090] Different from Example 1, the molecular weight of sodium hyaluronate is 1000 kDa - 1200 kDa, and the purity > 95%. The rest are the same.

[0091] Comparative Example 4

[0092] Different from Example 1, the molecular weight of sodium hyaluronate is 100 kDa - 200 kDa, and the purity > 95%. The rest are the same.

[0093] Comparative Example 5

[0094] Different from Example 1, the formulation selection is as follows: 6 parts of PDRN microspheres, 55 parts of sodium hyaluronate (400 kDa - 600 kDa, purity > 95%), 0.5 part of crosslinking agent (obtained by mixing carbodiimide and N-hydroxysuccinimide in a molar ratio of 2:1), and 3 parts of bisabolol. The rest are the same.

[0095] Trehalose was not used in Comparative Example 5.

[0096] Comparative Example 6

[0097] Different from Example 1, the formulation selection is as follows: 6 parts of PDRN microspheres, 50 parts of sodium hyaluronate (400 kDa - 600 kDa), 0.5 part of crosslinking agent (obtained by mixing carbodiimide and N-hydroxysuccinimide in a molar ratio of 2:1), and 5 parts of trehalose. The rest are the same.

[0098] Bisabolol was not used in Comparative Example 6.

[0099] Comparative Example 7

[0100] Different from Example 1, in step (2), ice bath treatment is not carried out. The rest are the same.

[0101] Comparative Example 8

[0102] Different from Example 1, the same amount of PDRN is used as the raw material, and PDRN microspheres are not used. The rest are the same.

[0103] Comparative Example 9

[0104] Different from Example 1, the formulation selection is as follows: 6 parts of PDRN microspheres, 50 parts of sodium hyaluronate (400 kDa - 600 kDa, purity > 95%), 0.5 part of crosslinking agent (obtained by mixing carbodiimide and N-hydroxysuccinimide in a molar ratio of 2:1), 3 parts of bisabolol, and 5 parts of sodium alginate. The rest are the same.

[0105] Sodium alginate was used in Comparative Example 9 instead of trehalose.

[0106] Result Detection

[0107] 1. Stability Detection

[0108] During the wound inflammation stage and the proliferation stage of epidermal cells and fibroblasts, DNA enzymes such as DNase I are released to clear free DNA, and its enzymatic hydrolysis ability will also act on PDRN, causing the decomposition of PDRN and thus affecting the product effect. Therefore, in the present invention, the enzymatic hydrolysis process of PDRN is simulated by DNase I to verify the product stability.

[0109] Take 0.5 g of the compositions prepared in Examples 1 - 7 and Comparative Examples 1 - 9 respectively, add them to 50 mL of water, then add DNase I (the addition amount is 100 u / mL according to enzyme activity), seal, and place them in a constant temperature water bath shaker for enzymatic hydrolysis (enzymatic hydrolysis temperature 37°C, enzymatic hydrolysis time 2 h, stirring speed 100 rpm). After the enzymatic hydrolysis is completed, add 20 mL of EDTA standard solution (concentration 0.01 mol / L) and 1 mL of sodium dodecyl sulfate aqueous solution (mass fraction 10%), stir at 1500 rpm for 20 min to obtain a mixed solution. Place the mixed solution in a centrifuge and centrifuge at 10000 rpm for 20 min. After centrifugation, take the supernatant and measure the PDRN content by the diphenylamine method, denoted as M1. For each group, a blank control group without enzymatic hydrolysis is set, and the PDRN content of the blank control group is measured by the diphenylamine method, denoted as M0.

[0110] Calculate the retention rate according to the following formula:

[0111] Retention rate = M1 / M0 × 100%.

[0112] The results are as Figure 1 shown. Figure 1 Among them, the results of Examples 1 - 7 show that the PDRN microspheres encapsulated by the hydrogel prepared in the present invention can isolate the contact with DNase I, delay the enzymatic hydrolysis of DNase I, and ensure its stable release. In our related previous tests, the retention rate of the prepared pure PDRN microspheres could only reach about 58%. And in Figure 1 the experimental results show that the retention rates of Examples 1 - 7 and Comparative Example 3 are > 88%, indicating a significant improvement in stability. At the same time, the results of Comparative Examples 1 - 9 show that although the hydrogel encapsulation can improve the retention rate of PDRN microspheres, the molecular weight of sodium hyaluronate and the preparation process will also affect the cross-linking process and thus affect the retention rate. At the same time, the addition of bisabolol and trehalose will also significantly improve the stability of PDRN microspheres.

[0113] 2. Detection of repair effect

[0114] Select the examples and comparative examples with a retention rate reaching 70% for further repair effect detection experiments.

[0115] Male SD rats (body weight 220 g - 230 g) are selected as experimental animals. After one week of adaptive feeding, they are randomly grouped with 8 rats in each group. All rats are anesthetized with 10 mg / kg of xylazine and 80 mg / kg of ketamine, the hair on the back is removed, and a 1.5 × 1.5 cm incision is made with a scalpel. 2Full-thickness skin wounds can inhibit the self-healing ability of rats. In the control group, a commercially available sodium hyaluronate gel (Tuoneng Pharmaceutical, with sodium hyaluronate as the main component) was used. In the experimental group, the products of Examples 1-7, Comparative Examples 1-3, Comparative Example 6, and Comparative Example 9 were respectively used and fully applied to the wound at a thickness of 1 mm - 2 mm above the epidermis, and then bandaged with medical transparent tape.

[0116] The wound areas at 0 days, 5 days, and 9 days were respectively counted, and the percentage of the original wound area was calculated. The results are shown in Table 1:

[0117] Table 1

[0118]

[0119] Note: In the table, represents p < 0.05 compared with the control group, represents p < 0.01 compared with the control group.

[0120] On the 9th day after wound treatment, after counting the wound area, the rats were anesthetized and sacrificed, and the wound surface was excised and divided into two groups. One group was used for inflammation diagnosis by labeling macrophages with CD68, and the other group was used to observe the cell growth at the wound surface by KI-67 fluorescence staining. The results are shown in Table 2:

[0121] Table 2

[0122]

[0123] Note: In the table, represents p < 0.05 compared with the control group, represents p < 0.01 compared with the control group.

[0124] The data in Table 1 show that even when only using the commercially available sodium hyaluronate gel, the wound can still heal by itself. When using the products of Examples 1-7 of the present invention, a more obvious repair effect of promoting wound healing is presented, and the repair effect of Example 1 is the best. At the same time, the composition of the present invention has a long-term effect. Therefore, after being applied once, a continuous repair effect is presented within the 9-day experimental period.

[0125] When a wound occurs, the body will trigger a self-defense mechanism, which will in turn lead to inflammation. Early macrophages can remove pollutants and inhibit inflammatory necrotic tissues. However, excessive inflammatory responses will delay wound healing and cause scar formation. Therefore, the control of inflammation during the repair stage is very crucial. By observing the proportion of KI-67 positive cells, the cell proliferation situation during wound healing can be known to a certain extent. The results in Table 2 show that the products prepared in the examples of the present invention can significantly slow down the early inflammatory response and promote cell proliferation, so as to achieve the purpose of promoting repair.

[0126] Although the results of the comparative examples also showed significant differences compared with the control group, there were still obvious gaps compared with the examples. The results of Comparative Example 1, Comparative Example 6 and Comparative Example 9 showed that bisabolol and trehalose are important components, which can synergistically improve the repair effect with PDRN microspheres, and none of them can be missing; the result of Comparative Example 2 showed that selecting raw materials in appropriate proportions is more conducive to wound repair; in Comparative Example 3, sodium hyaluronate with a higher molecular weight was selected, which showed high stability in the stability experiment, but to a certain extent, it was not conducive to the release of PDRN, resulting in a decrease in the repair effect.

[0127] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A composition comprising PDRN microspheres, characterized in that, By weight parts, the raw materials for preparing the composition are composed of the following components: 1 part - 10 parts of PDRN microspheres, 30 parts - 60 parts of sodium hyaluronate, 0.05 part - 1 part of crosslinking agent, 0.5 part - 5 parts of bisabolol, and 1 part - 8 parts of trehalose; the molecular weight of the sodium hyaluronate is 300 kDa - 800 kDa; The preparation method of the composition includes the following steps: (1) Dissolve the formulated amount of trehalose and bisabolol with a solvent, and then add the formulated amount of PDRN microspheres and sodium hyaluronate to obtain a mixed solution; (2) Dissolve the formulated amount of crosslinking agent with a solvent, and add it dropwise to the mixed solution obtained in step (1), perform ice bath treatment, adjust the pH, and raise the temperature for reaction to obtain a reaction product; (3) Concentrate the reaction product obtained in step (2) to obtain a composition containing PDRN microspheres.

2. The composition comprising PDRN microspheres according to claim 1, wherein By weight parts, the raw materials for preparing the composition are composed of the following components: 3 parts - 8 parts of PDRN microspheres, 35 parts - 55 parts of sodium hyaluronate, 0.1 part - 0.6 part of crosslinking agent, 1 part - 4 parts of bisabolol, and 2 parts - 6 parts of trehalose.

3. The composition comprising PDRN microspheres according to claim 2, wherein By weight parts, the raw materials for preparing the composition are composed of the following components: 6 parts of PDRN microspheres, 50 parts of sodium hyaluronate, 0.5 part of crosslinking agent, 3 parts of bisabolol, and 5 parts of trehalose.

4. The composition comprising PDRN microspheres according to claim 1, characterized in that, The molecular weight of the sodium hyaluronate is 400 kDa - 600 kDa.

5. The composition comprising PDRN microspheres according to claim 1, characterized in that, The crosslinking agent is selected from at least one of divinyl sulfone, 1,4-butanediol diglycidyl ether, carbodiimide, and N-hydroxysuccinimide.

6. The composition comprising PDRN microspheres according to claim 5, characterized in that, The crosslinking agent is a combination of carbodiimide and N-hydroxysuccinimide.

7. The composition comprising PDRN microspheres according to claim 6, characterized in that, The molar ratio of the carbodiimide to the N-hydroxysuccinimide is 1 - 4:

1.

8. A method for preparing the composition containing PDRN microspheres according to any one of claims 1-7, characterized in that, It includes the following steps: (1) Dissolve the formulated amount of trehalose and bisabolol with a solvent, and then add the formulated amount of PDRN microspheres and sodium hyaluronate to obtain a mixed solution; (2) Dissolve the formulated amount of crosslinking agent with a solvent, and add it dropwise to the mixed solution obtained in step (1), perform ice bath treatment, adjust the pH, and raise the temperature for reaction to obtain a reaction product; (3) Concentrate the reaction product obtained in step (2) to obtain a composition containing PDRN microspheres.

9. The preparation method according to claim 8, characterized in that, In step (1), in the mixed solution, the mass content of sodium hyaluronate is 1% - 5%.

10. The preparation method according to claim 9, wherein, In step (1), in the mixed solution, the mass content of sodium hyaluronate is 2%.

11. The preparation method according to claim 8, wherein In step (2), the time of the ice bath treatment is 5 min - 30 min.

12. The preparation method according to claim 11, wherein In step (2), the time of the ice bath treatment is 15 min.

13. The preparation method according to claim 8, characterized in that, In step (2), the adjustment of the pH is: adjust the pH to 5.0 - 6.

5.

14. The preparation method according to claim 13, characterized in that, In step (2), the adjustment of the pH is: adjust the pH to 5.5 - 6.

0.

15. The preparation method according to claim 8, characterized in that, In step (2), the conditions for the temperature-raising reaction are: raise the temperature to 20°C - 35°C and react for 2 h - 5 h.

16. The preparation method according to claim 8, characterized in that, It includes the following steps: (1) Dissolve the formulated amount of trehalose and bisabolol with a solvent, and then add the formulated amount of PDRN microspheres and sodium hyaluronate to obtain a mixed solution; in the mixed solution, the mass content of sodium hyaluronate is 1% - 5%; (2) Dissolve the crosslinking agent in an amount according to the formula in a small amount of solvent, and add it dropwise to the mixed solution obtained in step (1). Perform ice bath stirring for 5 min - 30 min, adjust the pH to maintain it at 5.5 - 6.0, raise the temperature to 20°C - 35°C and react for 2 h - 5 h. Add ethanolamine to the obtained reactant, stir to quench the reaction, and obtain a gel-like reactant; (3) Concentrate the gel-like reactant obtained in step (2) with a ceramic ultrafiltration membrane to obtain a composition containing PDRN microspheres.

17. Use of the composition containing PDRN microspheres according to any one of claims 1 - 7 in the preparation of a repair product.

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