In-situ gel forming solution containing polydeoxyribonucleotide as well as preparation method and application of in-situ gel forming solution

Abstract

The invention relates to the technical field of polymer gel, and provides an in-situ gel forming solution containing polydeoxyribonucleotide as well as a preparation method and application of the in-situ gel forming solution. The preparation method comprises the following steps: dissolving sodium hyaluronate, adding EDC, adding NHS, and mixing to obtain an activated sodium hyaluronate solution; dropwise adding a 5-hydroxydopamine solution into the activated sodium hyaluronate solution in a stirring state, and reacting to obtain an intermediate solution; dissolving collagen and polydeoxyribonucleotide, adding the dissolved collagen and polydeoxyribonucleotide into the intermediate solution, mixing, and adding EDC and NHS to obtain an activated solution; dropwise adding a 5-hydroxydopamine solution into the activated solution, and reacting to obtain an in-situ gel forming solution; the invention also provides application of the in-situ gel formation solution in the fields of beauty and plastic surgery. Skin aging caused by light aging can be resisted, generation of fibroblasts, collagen synthesis and angiogenesis at an injection part can be promoted, and good injection performance is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer gel, in particular to an in-situ gel forming solution containing polydeoxyribonucleotides and a preparation method and application thereof. Background Art

[0002] Loss of extracellular matrix is ​​an important cause of skin aging. With age, photoaging and other environmental damage, the loss of collagen and sodium hyaluronate in the skin causes the skin to lose elasticity and luster, become grayish yellow, develop wrinkles, and even damage cell tissues, resulting in aging. By supplementing the extracellular matrix to the dermis or subcutaneous tissue, stimulating collagen production can effectively delay skin anti-aging.

[0003] Sodium hyaluronate is a natural polysaccharide and an important component of the extracellular matrix. Because sodium hyaluronate has many hydrophilic groups, its macromolecular structure can be folded into a network layer by layer to hold water in the gaps, so it has a strong ability to absorb water. Sodium hyaluronate plays a key role in moisturizing, repairing, and nourishing the skin. It is the most important substance for retaining moisture in human tissues. Its content in the skin will decrease with age. If the sodium hyaluronate in the dermal matrix decreases, the skin's bound water content will decrease, making the skin dry, dull, and less elastic, resulting in wrinkles, dull skin tone, and other problems.

[0004] Collagen is an important structural protein in the extracellular space of various connective tissues. It constitutes the extracellular matrix network structure, which plays a role in supporting organs, protecting the body and repairing the skin. 70% of the skin is composed of collagen, which can form a dense elastic network to support the skin and maintain elasticity.

[0005] Therefore, in the prior art, the elasticity of the skin is mostly maintained by injecting sodium hyaluronate and collagen into the skin. However, free sodium hyaluronate and collagen degrade too quickly in the body and have a short maintenance time, which greatly limits their application. Cross-linking polymers is a major strategy in the current injection beauty field. While a high degree of cross-linking can achieve a long retention time in the body and strong mechanical strength, it also means that the pushing force required for intradermal injection is greater, which will cause difficulty in injection in clinical practice and increase patient pain. Summary of the invention

[0006] In order to solve the above problems, the present invention provides an in-situ gel-forming solution containing polydeoxyribonucleotides and a preparation method and application thereof, which can effectively delay the degradation of sodium hyaluronate and collagen, prolong the action time in the body, give full play to the effects of sodium hyaluronate and recombinant humanized collagen, and after being loaded with polydeoxyribonucleotides, can effectively achieve the slow release of polydeoxyribonucleotides, resist skin aging caused by photoaging, promote the production of fibroblasts, collagen synthesis and angiogenesis at the injection site, and have good injection performance.

[0007] The embodiments of the present invention are achieved through the following technical solutions:

[0008] A method for preparing an in-situ gel-forming solution loaded with polydeoxyribonucleotides comprises the following steps:

[0009] S1. Activating sodium hyaluronate: dissolving sodium hyaluronate in water for 5-8 hours; then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stirring for 10-30 minutes, and then adding N-hydroxysuccinimide, stirring for 10-30 minutes to activate the carboxyl groups on the sodium hyaluronate molecular chain to obtain an activated sodium hyaluronate solution;

[0010] S2. Modification of 5-hydroxydopamine: 5-hydroxydopamine is dissolved in water to prepare a 5-hydroxydopamine solution, which is then added dropwise to the activated sodium hyaluronate solution prepared in S1 under stirring, and the reaction is carried out overnight to obtain an intermediate solution;

[0011] S3, preparation of activation solution: dissolving collagen and polydeoxyribonucleotide in water, stirring for 20-45 minutes, adding to the intermediate solution prepared in S2, continuing to stir for 20-45 minutes, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stirring for 20-45 minutes, adding N-hydroxysuccinimide, stirring for 20-45 minutes to obtain an activation solution; preferably, the collagen is recombinant type III humanized collagen;

[0012] S4, in situ gel forming solution: 5-hydroxydopamine is dissolved in water to prepare a 5-hydroxydopamine solution, which is then added dropwise to the activation solution prepared in S3 under stirring. After high-speed stirring, the solution is allowed to stand for 1.5-2.5 hours to obtain an in situ gel forming solution.

[0013] The present invention also provides a post-treatment process of an in-situ gel-forming solution, comprising the following steps: placing the in-situ gel-forming solution obtained in S4 into a dialysis bag for dialysis, and then adding a buffer solution to dilute it to a desired concentration; wherein the dialysis bag is 3500Da, the dialysis time is 48h, and the water is changed 3 to 5 times; the buffer solution comprises any one or more of sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate, the osmotic pressure is 200 to 400mOsmol / kg, and the pH value is 6.5 to 8.0.

[0014] The present invention also provides an in-situ gel-forming solution containing polydeoxyribonucleotides, which is prepared by the above preparation method.

[0015] The present invention also provides an application of an in-situ gel-forming solution containing polydeoxyribonucleotides in the field of beauty and plastic surgery.

[0016] The technical solution of the embodiment of the present invention has at least the following advantages and beneficial effects:

[0017] 1. The present invention mainly utilizes 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups on the molecular chains of sodium hyaluronate and collagen, and then uses 5-hydroxydopamine for modification to form a solution with the characteristic of low injection difficulty, and subsequently collagen and polydeoxyribonucleotide are loaded in the cross-linked gel and activated, and then 5-hydroxydopamine is used for reaction to obtain an in-situ gel-forming solution, that is, after being injected into the human body in the form of a solution, a polymerization reaction occurs under the action of human horseradish peroxidase and hydrogen peroxide, thereby forming a cross-linked gel with a three-dimensional structure in the body.

[0018] 2. The multi-crosslinked gel loaded with polydeoxyribonucleotides prepared by the present invention has good cell adhesion and strong antioxidant effect, can effectively delay the degradation of the gel, prolong the in vivo action time, give full play to the role of sodium hyaluronate and recombinant humanized collagen, and can effectively achieve the slow release of polydeoxyribonucleotides, resist skin aging caused by photoaging, and promote the production of fibroblasts, collagen synthesis and angiogenesis at the injection site BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 is the PDRN release rate of each group of samples within 12 weeks;

[0021] Figure 2 is the effect of each group of samples on the migration of fibroblasts (L929) after incubation for 24 hours;

[0022] Figure 3 This is a graph showing the proliferation effect of each group of samples on fibroblasts (L929) after incubation for 48 hours. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0024] The following is a detailed description of an in-situ gel-forming solution containing polydeoxyribonucleotides and its preparation method and application provided in an embodiment of the present invention.

[0025] A method for preparing an in-situ gel-forming solution containing polydeoxyribonucleotides comprises the following steps:

[0026] S1. Activating sodium hyaluronate: dissolving sodium hyaluronate in water for 5-8 hours; then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stirring for 10-30 minutes, adding N-hydroxysuccinimide, and continuing to stir for 10-30 minutes to obtain an activated sodium hyaluronate solution;

[0027] S2. Modification of 5-hydroxydopamine: 5-hydroxydopamine is dissolved in water to prepare a 5-hydroxydopamine solution, which is then added dropwise to the activated sodium hyaluronate solution prepared in S1 under stirring, and the reaction is carried out overnight to obtain an intermediate solution;

[0028] S3, preparation of activation solution: dissolving collagen and polydeoxyribonucleotide in water, stirring for 20-45 minutes, adding to the intermediate solution prepared in S2, continuing to stir for 20-45 minutes, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stirring for 20-45 minutes, adding N-hydroxysuccinimide, stirring for 20-45 minutes to obtain an activation solution; preferably, the collagen is recombinant type III humanized collagen;

[0029] S4, in situ gel forming solution: 5-hydroxydopamine is dissolved in water to prepare a 5-hydroxydopamine solution, which is then added dropwise to the activation solution prepared in S3 under stirring, and after high-speed stirring, the solution is allowed to stand for 1.5-2.5 hours to obtain an in situ gel forming solution;

[0030] S5, placing the in situ gel forming solution obtained in S4 into a dialysis bag for dialysis, and then adding a buffer solution to dilute to the desired concentration. The molecular weight cutoff of the dialysis bag is 3500Da, the dialysis time is 48h, and the water is changed 3 to 5 times; the buffer solution is composed of any one or more of sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate, the osmotic pressure is 200 to 400mOsmol / kg, and the pH value is 6.5 to 8.0.

[0031] The present invention also provides an in-situ gel-forming solution containing polydeoxyribonucleotides, which is prepared by the above-mentioned preparation method.

[0032] The present invention also provides an application of the in-situ gel-forming solution prepared by the above preparation method in the fields of beauty and plastic surgery.

[0033] The invention mainly utilizes 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl groups on the molecular chains of sodium hyaluronate and collagen (recombinant type III humanized collagen), and then uses 5-hydroxydopamine to modify the formed solution, which has the characteristic of low injection difficulty. Subsequently, collagen and polydeoxyribonucleotide are loaded in the in-situ gel-forming solution and activated, and then 5-hydroxydopamine is used to react to obtain the in-situ gel-forming solution, that is, after being injected into the human body in the form of a solution, a polymerization reaction occurs under the action of human horseradish peroxidase and hydrogen peroxide, thereby forming a cross-linked gel with a three-dimensional structure in the body.

[0034] More specifically, the carboxyl groups of sodium hyaluronate and recombinant humanized type III collagen are first activated with EDC. After the carboxyl groups in the collagen or sodium hyaluronate molecules are activated, a highly active but easily hydrolyzed O-acylisourea intermediate is formed, and then N-hydroxysuccinimide is added to form an active ester that is not easily hydrolyzed. The ester easily reacts with the amino group on 5-hydroxydopamine to form a stable intermediate solution containing amide bonds, thereby enhancing the mechanical strength and connection strength of the intermediate solution of the in situ gel formation solution.

[0035] Although the intermediate solution formed above can improve the mechanical strength and connection strength of the in situ gel-forming solution, it also means that the pushing force required for intradermal injection is greater, which will cause difficulty in injection in clinical practice, increase the pain of patients, and affect the injection filling effect; therefore, the inventor innovatively loads collagen and polydeoxyribonucleotides, activators and adds 5-hydroxydopamine solution again on the basis of the intermediate solution formed, but does not participate in the cross-linking reaction, so that the in situ gel-forming solution is in the form of solution before injection and has good injection performance. After injection, it can undergo polymerization reaction between pyrogallol under the action of human horseradish catalase and hydrogen peroxide to form a gel, thereby exerting a cosmetic effect. That is, the present invention changes the gel form of traditional injection products before injection, and adopts a solution state for injection and forms a gel in vivo, effectively solving the problem of injection difficulty.

[0036] In addition, it should be noted that in the process of loading polydeoxyribonucleotides, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide only serve as activation media and do not participate in cross-linking, and can be removed by subsequent dialysis steps. In addition, the rich polyphenol structure on 5-hydroxydopamine has good cell adhesion properties and strong antioxidant effects, which can make the material difficult to shift and deform at the injection site, play a good antioxidant effect, and make the polydeoxyribonucleotides slowly released at the injection site, constantly stimulating the body to promote the proliferation of fibroblasts, promote collagen proliferation at the injection site, and improve wrinkles.

[0037] Example 1

[0038] This embodiment provides an in situ gel-forming solution containing polydeoxyribonucleotides (PDRN-HA-RHC-III-GA-1), which is prepared by the following method:

[0039] S1. Dissolve 5 g of sodium hyaluronate in 20 mL of water for 6 h, add 46 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stir for 15 min, then add 34 mg of N-hydroxysuccinimide, and continue stirring for 15 min to obtain a sodium hyaluronate solution;

[0040] S2, dissolving 25 mg of 5-hydroxydopamine in 5 mL of water to prepare a 5-hydroxydopamine solution, and then adding it dropwise to the sodium hyaluronate solution under stirring, and reacting overnight;

[0041] S3, dissolving 0.5 g of recombinant humanized type III collagen (RHC-III) and 0.5 g of polydeoxyribonucleotide (PDRN) in 20 mL of water, stirring for 30 min, adding to the solution obtained in S2, continuing to stir for 30 min, adding 92 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stirring for 30 min, adding 68 mg of N-hydroxysuccinimide, stirring for 30 min to obtain an activation solution;

[0042] S4, dissolving 50 mg of 5-hydroxydopamine in 5 mL of water to prepare a 5-hydroxydopamine solution, then adding it dropwise to the activation solution, and reacting for 2 h to obtain an in-situ gel-forming solution;

[0043] S5. The in situ gel-forming solution obtained in S4 was placed in a dialysis bag with a molecular weight cutoff of 3500Da and dialyzed for 48 hours; then, a buffer solution of sodium chloride with an osmotic pressure of 300mOsmol / kgd was added to dilute it to a concentration of 20mg / mL to obtain the final product, which was recorded as PDRN-HA-RHC-III-GA-1.

[0044] Example 2

[0045] An in situ gel-forming solution containing polydeoxyribonucleotides (PDRN-HA-RHC-III-GA-2) is prepared by the following method:

[0046] S1. Dissolve 5 g of sodium hyaluronate in 20 mL of water for 6 h, add 92 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stir for 15 min, then add 68 mg of N-hydroxysuccinimide, and continue stirring for 15 min to obtain a sodium hyaluronate solution;

[0047] S2, dissolving 50 mg of 5-hydroxydopamine in 5 mL of water to prepare a 5-hydroxydopamine solution, and then adding the solution dropwise to the sodium hyaluronate solution under stirring to react overnight;

[0048] S3, dissolving 0.5 g of recombinant type III humanized collagen and 0.5 g of polydeoxyribonucleotide in 20 mL of water, stirring for 30 min, adding to the solution obtained in S2, continuing to stir for 30 min, adding 92 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stirring for 30 min, adding 68 mg of N-hydroxysuccinimide, stirring for 30 min to obtain an activation solution;

[0049] S4, dissolving 50 mg of 5-hydroxydopamine in 5 mL of water to prepare a 5-hydroxydopamine solution, and then adding it dropwise to the above activation solution, reacting for 2 h to obtain an in situ gel-forming solution;

[0050] S5. The in situ gel-forming solution obtained in S4 was placed in a dialysis bag with a molecular weight cutoff of 3500Da and dialyzed for 48 hours; then, a buffer solution of sodium chloride with an osmotic pressure of 300mOsmol / kgd was added to dilute it to a concentration of 20mg / mL to obtain the final product, which was recorded as PDRN-HA-RHC-III-GA-2.

[0051] Comparative Example 1

[0052] A sodium hyaluronate-recombinant humanized type III collagen-5-hydroxydopamine activation solution (PDRN-HA-RHC-III) is prepared by the following method:

[0053] S1. Dissolve 5 g of sodium hyaluronate in 20 mL of water for 6 h, add 46 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stir for 15 min, then add 34 mg of N-hydroxysuccinimide, and continue stirring for 15 min to obtain a sodium hyaluronate solution;

[0054] S2, dissolving 0.5 g of recombinant type III humanized collagen and 0.5 g of polydeoxyribonucleotide in 20 mL of water, stirring for 30 min, adding to the sodium hyaluronate solution obtained in S1, continuing to stir for 30 min, adding 92 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stirring for 30 min, adding 68 mg of N-hydroxysuccinimide, stirring for 30 min to obtain a composite gel;

[0055] S3. Place the composite gel obtained in S2 into a dialysis bag with a molecular weight cutoff of 3500Da and dialyze for 48 hours; then add sodium dihydrogen phosphate with an osmotic pressure of 300mOsmol / kgd to dilute to a concentration of 20mg / mL to obtain the final product, which is recorded as PDRN-HA-RHC-III.

[0056] Comparative Example 2

[0057] A polydeoxyribonucleotide-loaded sodium hyaluronate-5-hydroxydopamine activation solution (PDRN-HA-GA) is prepared by the following method:

[0058] S1. Dissolve 5 g of sodium hyaluronate in 20 mL of water for 6 h, add 46 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stir for 15 min, then add 34 mg of N-hydroxysuccinimide, and continue stirring for 15 min to obtain a sodium hyaluronate solution;

[0059] S2, dissolving 25 mg of 5-hydroxydopamine in 5 mL of water to prepare a 5-hydroxydopamine solution, and then adding it dropwise to the sodium hyaluronate solution under stirring, and reacting overnight;

[0060] S3, dissolving 0.5 g of polydeoxyribonucleotide in 20 mL of water, stirring for 30 min, adding to the solution obtained in S2, and stirring for 30 min to obtain an in situ gel-forming solution;

[0061] S4. The in situ gel-forming solution obtained in S3 was placed in a dialysis bag with a molecular weight cutoff of 3500Da and dialyzed for 48 hours; then sodium hydrogen phosphate with an osmotic pressure of 300mOsmol / kgd was added to dilute it to a concentration of 20mg / mL to obtain the final product, which was recorded as PDRN-HA-GA.

[0062] Comparative Example 3

[0063] A preparation of an activation solution of recombinant type III humanized collagen-5-hydroxydopamine loaded with polydeoxyribonucleotide (PDRN-RHC-III-GA) is prepared by the following method:

[0064] S1. Dissolve 50 mg of 5-hydroxydopamine in 5 mL of water to prepare a 5-hydroxydopamine solution, then add the solution dropwise to the sodium hyaluronate solution under stirring and react overnight;

[0065] S2, dissolving 0.5 g of recombinant type III humanized collagen and 0.5 g of polydeoxyribonucleotide in 20 mL of water, stirring for 30 min, adding to the solution obtained in S1, continuing to stir for 30 min, adding 92 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stirring for 30 min, adding 68 mg of N-hydroxysuccinimide, stirring for 30 min to obtain an activation solution;

[0066] S3, dissolving 30 mg of 5-hydroxydopamine in 5 mL of water to prepare a 5-hydroxydopamine solution, then adding it dropwise to the activation solution, and reacting for 2 h to obtain an in situ gel-forming solution;

[0067] S4. Place the in situ gel-forming solution obtained in S3 into a dialysis bag with a molecular weight cutoff of 3500Da and dialyze for 48 hours; then add disodium hydrogen phosphate with an osmotic pressure of 300mOsmol / kgd to dilute to a concentration of 20mg / mL to obtain the final product, which is recorded as PDRN-RHC-III-GA.

[0068] Experimental Example 1

[0069] Horseradish catalase and hydrogen peroxide were added to the samples prepared in Examples 1-2 and Comparative Examples 1-3 to simulate the in vivo environment, so that the solutions prepared in the above Examples and Comparative Examples formed gels, and then the physicochemical properties of the gels processed from the samples of Examples 1-2 and Comparative Examples 1-3 were tested, specifically including:

[0070] 1. Syringe handle pushing force: The syringe handle pushing force of each group of samples before and after gel formation was tested; the results are shown in Table 1:

[0071] Table 1 - Changes in the pushing force of the syringe handle before and after adding horseradish catalase and hydrogen peroxide to each group of materials

[0072]

[0073] From the results in Table 1, it can be seen that the samples prepared in each embodiment and comparative example have a smaller syringe handle pushing force before adding horseradish catalase and hydrogen peroxide, and the syringe handle pushing force is greatly improved after the gel is formed. It can be seen that the liquid in situ gel-forming solution material is easier to inject, which can reduce the difficulty of injection and the pain of patients; especially the samples of Examples 1-2 of the present invention have a smaller syringe handle pushing force; while in Comparative Example 1 (5-hydroxydopamine is not added, and gel is directly formed) there is no significant change in the syringe handle pushing force before and after the enzyme is added, and the syringe handle pushing force value is large, which can be seen that the in situ gel-forming solution formed by adding 5-hydroxydopamine can form a gel in situ under the catalysis of horseradish catalase and hydrogen peroxide after the material is injected, and the material has good injectability.

[0074] 2. Antioxidant activity test: The DPPH method was used to determine the free radical scavenging ability of each group of samples after gel formation; at the same time, a control group was set up: pH 7.2 phosphate buffer solution. The results are shown in Table 2:

[0075] Table 2-DPPH free radical scavenging rate of each group of samples after gel formation

[0076] Group DPPH free radical scavenging rate (%) Example 1 73.53 Example 2 79.38 Comparative Example 1 7.32 Comparative Example 2 59.48 Comparative Example 3 61.35 Control group 0.03

[0077] From the results in Table 2, it can be seen that the samples of Examples 1-2 of the present invention have significantly better free radical scavenging ability after forming a gel than those of Comparative Examples 1-3 and the control group; in particular, the free radical scavenging rate of the sample of Comparative Example 1 is significantly lower than that of the other groups. It can be seen that the presence of 5-hydroxydopamine allows sodium hyaluronate, recombinant type III humanized collagen and polydeoxyribonucleotides to form an in situ gel-forming solution, and enables the material to have a good free radical scavenging rate, so that it can exhibit a stronger antioxidant effect at the injection site and protect cells from damage.

[0078] 3. Cell adhesion effect: The cell adhesion effect of each group of samples after gel formation was tested; the results are shown in Table 3:

[0079] Table 3 - Cell adhesion effect of each group of samples after gel formation

[0080] Group Cell adhesion percentage (%) Example 1 71.35 Example 2 80.57 Comparative Example 1 10.33 Comparative Example 2 39.54 Comparative Example 3 22.44 Comparison 1.03

[0081] From the results in Table 3, it can be seen that the samples of Examples 1-2 exhibited better cell adhesion performance than the samples of Comparative Examples 1-3, making the injection filling effect better, the injection site accurate, and not easy to cause filling displacement, while having better biocompatibility, safer to use, and better promoting tissue repair and regeneration. The sample of Comparative Example 1 exhibited the worst cell adhesion performance due to the gel formed by not containing 5-hydroxydopamine. It can be seen that 5-hydroxydopamine allows sodium hyaluronate, recombinant type III humanized collagen, and polydeoxyribonucleotides to form an in situ gel-forming solution, and makes the material have better cell adhesion performance, so that the material has a good shaping effect after injection, and is not easy to shift or deform.

[0082] 4. Release effect of polydeoxyribonucleotides: Soak the gel in a phosphate buffer solution of pH 7.0, adjust the pH value of the solution to 7.5-7.6, centrifuge at different times to obtain the supernatant, and use a UV-spectrophotometer to detect the absorbance at A260nm in the supernatant; the results are shown in Figure 1 ;

[0083] Depend on Figure 1 It can be seen that the samples prepared in Comparative Examples 1-3 all showed a certain degree of burst release, and the polydeoxyribonucleotide release rate within 12 weeks was more than 80%, while the samples prepared in Example 1 and Example 2 formed solutions of in situ gel, and the polydeoxyribonucleotides could be released slowly and evenly. In addition, by comparing Example 1 and Example 2, it was found that increasing the degree of activation can make the sustained release effect of the polydeoxyribonucleotide after in situ gel formation better, prolong the in vivo action time, better resist skin aging caused by environmental factors, photoaging, etc., and improve the effect on the skin.

[0084] 5. Fibroblast migration and proliferation effects: The migration effects of each group of samples on fibroblasts (L929) were tested; the results are shown in Figure 2 ; and the proliferation effect of each group of samples on fibroblasts (L929); the results are shown in Figure 3 ;

[0085] Figure 2 and Figure 3The cell migration rate and cell proliferation rate of each group of samples are shown. The materials prepared in Example 1 and Example 2 show higher cell migration rate and cell proliferation rate than those in Comparative Examples 1 to 3. By comparing Example 1 and Example 2, it is found that increasing the degree of activation can increase the cell migration rate and cell proliferation rate.

[0086] In summary, the in-situ gel-forming solution containing polydeoxyribonucleotides prepared by the present invention has good injection performance. After injection, it is polymerized in situ under the action of horseradish catalase and hydrogen peroxide in the human body to form a three-dimensional gel. The gel has good cell adhesion and strong antioxidant effect, and can effectively delay the degradation of the gel, prolong the in vivo action time, give full play to the effects of sodium hyaluronate and recombinant humanized collagen, and can effectively achieve the slow release of polydeoxyribonucleotides, resist skin aging caused by environmental factors, photoaging, etc., and promote the generation of fibroblasts, collagen synthesis and angiogenesis at the injection site.

[0087] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, 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 method for preparing an in situ gel-forming solution containing polydeoxyribonucleotides, characterized in that: The following steps are involved: S1. Activating sodium hyaluronate: After dissolving sodium hyaluronate, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stir for a period of time, add N-hydroxysuccinimide, continue stirring, and obtain an activated sodium hyaluronate solution; S2. Modifying 5-hydroxydopamine: preparing 5-hydroxydopamine into a 5-hydroxydopamine solution, and then adding it dropwise to the activated sodium hyaluronate solution prepared in S1 under stirring, and obtaining an intermediate solution after the reaction; S3, preparation of activation solution: after dissolving collagen and polydeoxyribonucleotide, add them to the intermediate solution prepared in S2, continue stirring for a period of time, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide, stir for a period of time, add N-hydroxysuccinimide, continue stirring for a period of time, and obtain an activation solution; S4, in situ gel forming solution: 5-hydroxydopamine is prepared into a 5-hydroxydopamine solution, which is then added dropwise to the activation solution prepared in S3. After stirring and standing for a period of time, the in situ gel forming solution is obtained.

2. The method for preparing an in situ gel-forming solution containing polydeoxyribonucleotides according to claim 1, characterized in that: The collagen is selected from recombinant type III humanized collagen.

3. The method for preparing an in situ gel-forming solution containing polydeoxyribonucleotides according to claim 1, characterized in that: In S1, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:

1.

4. The method for preparing an in situ gel-forming solution containing polydeoxyribonucleotides according to claim 1, characterized in that: In S2, the mass of the added 5-hydroxydopamine is 1%-5% of the mass of sodium hyaluronate.

5. The method for preparing an in situ gel-forming solution containing polydeoxyribonucleotides according to claim 1, characterized in that: In S3, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:1; and the mass ratio of collagen to polydeoxyribonucleotide is 1:

1.

6. The method for preparing an in situ gel-forming solution containing polydeoxyribonucleotides according to claim 1, characterized in that: In S4, the ratio of the mass of the added 5-hydroxydopamine to the mass of the added 5-hydroxydopamine in S2 is 1-2:

1.

7. The method for preparing an in situ gel-forming solution containing polydeoxyribonucleotides according to claim 1, characterized in that: The method also includes a post-treatment process of the cross-linked gel, which includes the following steps: dialyzing the in-situ gel-forming solution obtained in S4, and then adding a buffer solution to dilute it to a desired concentration.

8. The method for preparing an in situ gel-forming solution containing polydeoxyribonucleotides according to claim 7, characterized in that: The buffer solution is any one or more of sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate; the osmotic pressure of the buffer solution is 200-400 mOsmol / kg, and the pH value is 6.5-8.

0.

9. An in situ gel-forming solution containing polydeoxyribonucleotides, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the in-situ gel-forming solution prepared by the preparation method according to any one of claims 1 to 8 in the fields of beauty and plastic surgery.

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