Compositions of polynucleotides and sodium hyaluronate and methods of making and using the same

Abstract

The application discloses a kind of compositions containing polynucleotide and sodium hyaluronate, its preparation method and application, by weight ratio, the composition includes polynucleotide 0.01-2%, micro-crosslinking sodium hyaluronate 0.01-2%, sodium dihydrogen phosphate 0.003-0.006%, disodium hydrogen phosphate 0.015-0.022%, sodium chloride 0.6-0.95%, injection water as solvent, the polynucleotide is obtained by 2'-o-methylation modification nucleotide monomer polymerization.The application selects the polynucleotide after 2'-o-methylation modification and the synergistic effect of component such as micro-crosslinking sodium hyaluronate, can be applied to intradermal dermis injection filling, improve skin quality.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and more particularly to compositions of polynucleotides and sodium hyaluronate, their preparation methods, and applications. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In the field of non-surgical cosmetic medicine, soft tissue fillers are highly favored by those seeking beauty. By injecting biocompatible fillers into the dermis or subcutaneous tissue of the skin, the volume of soft tissue is increased, thereby achieving the purpose of smoothing wrinkles and shaping contours. An ideal soft tissue filler usually needs to have the following characteristics: (1) long-lasting effect; (2) easy to inject; (3) flexible / plastic. Among many filler materials, hyaluronic acid (HA) is currently the most widely used polysaccharide in clinical practice. As a linear high molecular weight mucopolysaccharide, it is composed of repeating D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units. It exists naturally in the human body and many other organisms. It has the characteristics of no species difference, non-toxicity, non-immunogenicity and excellent biocompatibility. Its safety has been widely verified.

[0004] Polynucleotides (PNs), as important bioactive substances, have shown great potential in cell repair, anti-aging, and healthcare. They are natural biomolecules polymerized from nucleotides, possessing excellent biocompatibility and biodegradability. They not only provide nutrient substrates for cells but have also been shown to have multiple biological effects, including promoting cell proliferation and differentiation, enhancing cell vitality, improving local microcirculation, and stimulating collagen regeneration. However, the performance of traditional polynucleotides still needs improvement.

[0005] However, despite the high safety profile of hyaluronic acid fillers, traditional cross-linked sodium hyaluronate gel still has significant limitations: 1. Limited composition and efficacy: Conventional products contain only cross-linked sodium hyaluronate, relying primarily on its physical filling effect, lacking in bioactivity such as promoting collagen regeneration and improving the cellular microenvironment. 2. Durability and degradation rate: While cross-linking technology extends its retention time in the body, the degradation rate remains relatively fast, requiring frequent injections to maintain the effect. Its long-term anti-aging and wrinkle-reducing effects have not fully met market expectations. 3. Safety risks: To extend degradation time, high concentrations of chemical cross-linking agents are often used in production, increasing the risk of residual cross-linking agents in the product, potentially triggering foreign body reactions or allergic reactions. Summary of the Invention

[0006] In view of this, the present invention provides a composition of polynucleotide and sodium hyaluronate, a preparation method thereof, and its application, to solve the problems of high concentration of crosslinking agent, risk of excessive crosslinking agent residue in the product, single composition, single efficacy, and excessively rapid degradation in vivo, and to improve the anti-aging and wrinkle-removing effect of the composition.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a composition of polynucleotide and sodium hyaluronate, comprising, by weight percentage: 0.01-2% polynucleotide, 0.01-2% micro-crosslinked sodium hyaluronate, 0.003-0.006% sodium dihydrogen phosphate, 0.015-0.022% disodium hydrogen phosphate, 0.6-0.95% sodium chloride, and water for injection as solvent.

[0008] Preferably, the relative molecular mass of the micro-crosslinked sodium hyaluronate is 600,000 to 1,400,000 Daltons.

[0009] Preferably, the micro-crosslinked sodium hyaluronate is made from sodium hyaluronate, sodium hydroxide solution and crosslinking agent.

[0010] Preferably, the sodium hydroxide solution has a mass fraction of 0.5%-3%.

[0011] Preferably, the crosslinking agent is selected from at least one of puerarin, 1,4-butanediol glycidyl ether, divinyl sulfone, polyethylene glycol, genipin, and carbodiimide.

[0012] The composition of polynucleotides and sodium hyaluronate provided by this invention has the following advantages: (1) The cross-linking agent is used in small amounts, is safe and non-toxic, and can also play a synergistic role with polynucleotides in anti-oxidation and anti-aging effects. Moreover, when injected into the dermis, it reduces the swelling and migration of the gel; (2) The product has a good ability to stimulate collagen regeneration. Polynucleotides and puerarin promote the migration, proliferation and angiogenesis of vascular endothelial cells, provide sufficient blood supply and nutrients to the tissue, and can improve the skin condition; (3) The macromolecular three-dimensional scaffold structure can play a certain supporting role when injected into the subcutaneous layer; (4) By slowly degrading and releasing nucleotides, it continuously activates A 2A Receptors continuously stimulate collagen synthesis, maintaining a relatively long-lasting anti-aging and wrinkle-reducing effect.

[0013] Preferably, the polynucleotide is obtained by polymerizing a nucleotide monomer modified with 2'-o-methylation. The 2'-o-methylation modification of the polynucleotide monomer introduces a methyl group at the 2'-o position of the nucleotide, altering its chemical properties and making it more difficult for nucleases to recognize and degrade. This significantly improves the stability of the polynucleotide, resulting in a longer duration of action in vivo and a more sustained effect. Furthermore, this modification can enhance the interaction between the polynucleotide and adenosine A. 2A The binding affinity of the receptor increases the interaction between the polynucleotide and the receptor, improves the stability and specificity of the binding, and enables the polynucleotide to more accurately recognize and bind to the target receptor, activate the p53 signaling pathway, and exert anti-inflammatory and anti-aging effects.

[0014] All polynucleotides were obtained by polymerization of nucleotide monomers modified with 2'-o-methylation. Preparation method: Using polynucleotides as raw materials, 2'-o-methylguanosine was obtained by alkylation of the 2'-hydroxyl group with iodomethane and reaction with adenosine deaminase; the N2-amino group of the purine ring was protected with isobutyryl chloride, and the 5'-hydroxyl group of the sugar ring was protected with 4,4'-dimethoxytriphenylchloromethane to prepare 2'-O-methyl-5'-O-(4,4'-dimethoxytriphenylmethyl)-N2-isobutyrylguanosine.

[0015] In a second aspect, the present invention provides a method for preparing the composition of the polynucleotide and sodium hyaluronate described in the first aspect, comprising the following steps: (1) Dissolve sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride in water for injection to prepare a phosphate-sodium chloride buffer solution; (2) First, dissolve the polynucleotide in a phosphate-sodium chloride buffer solution and continue stirring until a semi-transparent solution is obtained; (3) Add micro-crosslinked sodium hyaluronate to the semi-transparent solution and stir until homogeneous to obtain a polynucleotide-sodium hyaluronate composition; (4) Vacuuming, filling and sterilizing the polynucleotide-sodium hyaluronate composition.

[0016] Preferably, the stirring temperature in step (2) is 40-60 ℃ and the stirring time is 1-2 h.

[0017] Preferably, in step (3), the preparation method of micro-crosslinked sodium hyaluronate is as follows: (3.1) Mix sodium hyaluronate and crosslinking agent evenly, then add sodium hydroxide solution to obtain crosslinking product through crosslinking reaction; (3.2) The cross-linked product was cut into small pieces of gel, dialyzed in a phosphate-sodium chloride buffer solution, and the pH was adjusted to neutral with hydrochloric acid. The gel swelled, sieved and granulated to obtain cross-linked sodium hyaluronate.

[0018] Preferably, in step (3.1), the mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent and water is 10:5:3-7:300.

[0019] Preferably, in step (3.1), the reaction temperature is 20-40 °C and the reaction time is 12-25 h.

[0020] Preferably, in step (3.2), the crosslinking product is cut into 1-2 cm pieces. 3 Small gel blocks; hydrochloric acid concentration of 0.8-1M.

[0021] Preferably, in step (3.2), the gel swells to 9-13 times the weight of the crosslinked product.

[0022] Thirdly, the present invention provides the application of the composition of the polynucleotide and sodium hyaluronate described in the first aspect in improving skin quality.

[0023] Preferably, the improvement in skin quality includes improvement in skin wrinkles, improvement in skin firmness, and improvement in skin elasticity.

[0024] Preferably, the composition of the polynucleotide and sodium hyaluronate is used by injection.

[0025] Fourthly, the present invention provides the application of the composition of the polynucleotide and sodium hyaluronate described in the first aspect in the preparation of anti-inflammatory and repair products.

[0026] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) Polynucleotides promote the migration, proliferation and angiogenesis of vascular endothelial cells, providing sufficient blood supply and nutrients to tissues and improving skin condition. When polynucleotides are used in combination with sodium hyaluronate for intradermal injection filling, the filling and wrinkle-reducing effect of the product is greatly enhanced.

[0027] (2) This invention uses polynucleotides modified with 2'-o-methylation at both ends of the sequence. The 2'-o-methylation modification alters the chemical properties of the nucleotide, making it more difficult for nucleases to recognize and degrade it, thereby significantly improving the stability of the polynucleotide and giving it a longer duration of action in vivo, enabling it to exert its effect more persistently. Furthermore, this modification can also enhance the interaction between the polynucleotide and adenosine A. 2A The increased binding affinity of the receptor enhances its interaction with anti-aging and anti-inflammatory factors, improves the stability and specificity of binding, and enables polynucleotides to more accurately recognize and bind to target receptors, activate the p53 signaling pathway, thereby greatly enhancing the anti-aging, anti-inflammatory and repair effects of polynucleotides and improving bioavailability.

[0028] (3) The crosslinking agent in the polynucleotide and sodium hyaluronate composition provided by the present invention is safe and non-toxic, and the step of washing off the crosslinking agent is eliminated, which greatly reduces the process time and cost. Moreover, the crosslinking agent has anti-inflammatory and antioxidant effects, which synergistically enhance the anti-inflammatory and anti-aging effects of the polynucleotide. Furthermore, all components used in the polynucleotide and sodium hyaluronate composition provided by the present invention are biocompatible, reducing the risks of clinical use. Attached Figure Description

[0029] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0030] Figure 1 This is a flowchart of the preparation method of the polynucleotide-sodium hyaluronate composition of the present invention; Figure 2 This is a microscopic image of cross-linked sodium hyaluronate after sieving in Example 4 of the present invention; Figure 3 This is a microscopic observation of the polynucleotide-sodium hyaluronate composition in Example 4 of the present invention. Detailed Implementation

[0031] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] Polynucleotides were purchased from Ruijiming (Shandong) Biotechnology Co., Ltd. (RJMPN-M, batch number 20240517), and sodium hyaluronate was purchased from Bloomage Biotechnology Co., Ltd. (J101230140).

[0033] Unless otherwise specified, the polynucleotides used in the embodiments and comparative examples of this invention have all been modified with 2'-o-methylation. Preparation method: Take 0.02 g of polynucleotide and add it to 19.98 g of purified water to prepare a 0.1% polynucleotide solution. Take 2 g of the polynucleotide solution and add 5 mL of 10× reaction buffer (containing 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT), 1.6 mL of SAM solution (32 mM), and 2.5 mL of M.SssI enzyme (4 U / μL) at pH=7.9 and 25 ℃. Add nuclease-free water to 50 mL. Then, place the reaction tube in a 37 ℃ incubator or water bath and incubate for 4 hours to overnight (16 hours). Finally, add nuclease-free water to 100 mL of the reaction system, perform phenol / chloroform extraction and ethanol precipitation, dissolve the purified DNA in 20-50 mL of TE buffer, and determine the concentration.

[0034] In the following examples and comparative examples, the phosphate-sodium chloride buffer solution used in the preparation of micro-crosslinked sodium hyaluronate consists of: 0.005% sodium dihydrogen phosphate, 0.022% disodium hydrogen phosphate, 0.7% sodium chloride, and 99.273% water for injection. The above-mentioned amounts do not include the 0.005% sodium dihydrogen phosphate, 0.022% disodium hydrogen phosphate, and 0.7% sodium chloride in the formulation of the polynucleotide and sodium hyaluronate composition, with the remainder being water for injection. Specifically, the amounts of sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride mentioned in the formulation of the polynucleotide and sodium hyaluronate composition, with the remainder being water for injection, refer only to the amount of phosphate-sodium chloride buffer solution used in the preparation method of the polynucleotide and sodium hyaluronate composition, and do not include the phosphate-sodium chloride buffer solution used in the preparation method of micro-crosslinked sodium hyaluronate.

[0035] Unless otherwise specified, the relative molecular mass of the micro-crosslinked sodium hyaluronate described in the embodiments and comparative examples of this invention refers to the relative molecular mass of the sodium hyaluronate used as the raw material for preparing micro-crosslinked sodium hyaluronate.

[0036] Example 1 A composition of a polynucleotide and sodium hyaluronate: by weight percentage, comprising: 2% polynucleotide, 2% micro-crosslinked sodium hyaluronate, 0.005% sodium dihydrogen phosphate, 0.022% disodium hydrogen phosphate, 0.7% sodium chloride, and the balance being water for injection.

[0037] Microcrosslinked sodium hyaluronate is made from sodium hyaluronate, sodium hydroxide solution, and a crosslinking agent. The relative molecular mass of microcrosslinked sodium hyaluronate is 1.4 million Daltons.

[0038] The cross-linking agent is selected from puerarin.

[0039] The sodium hydroxide solution has a mass fraction of 2.5%.

[0040] A method for preparing a composition of polynucleotides and sodium hyaluronate includes the following steps: (1) Dissolve sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride in water for injection to prepare a phosphate-sodium chloride buffer solution; (2) First, dissolve the polynucleotide in a phosphate-sodium chloride buffer solution, stirring at 55 °C for 2 hours. After dissolution, it becomes a semi-transparent solution. (3) Add micro-crosslinked sodium hyaluronate and stir evenly to obtain a polynucleotide-sodium hyaluronate composition, which is milky white and semi-transparent; In step (3), the preparation method of micro-crosslinked sodium hyaluronate is as follows: (3.1) Sodium hyaluronate and crosslinking agent are mixed evenly, and sodium hydroxide solution is added to create an alkaline environment. The reaction temperature is 30 °C and the reaction time is 22 h to obtain the crosslinking product. The mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent and water for injection is 10:5:5:300.

[0041] (3.2) Cut the cross-linked product into 2 cm pieces. 3 Small gel blocks were placed in a phosphate-sodium chloride buffer solution for dialyzing. 1 M hydrochloric acid was added to adjust the pH to neutral. When the gel swelled to a certain weight (10 times the weight of the cross-linked product), it was sieved and granulated to obtain cross-linked sodium hyaluronate.

[0042] (4) Vacuum the polynucleotide-sodium hyaluronate composition to remove air bubbles, then transfer it to a filling machine to fill it into a syringe and sterilize it.

[0043] Example 2 A composition of a polynucleotide and sodium hyaluronate: by weight percentage, comprising: 0.01% polynucleotide, 0.01% microcrosslinked sodium hyaluronate, 0.005% sodium dihydrogen phosphate, 0.022% disodium hydrogen phosphate, 0.7% sodium chloride, and the balance being water for injection.

[0044] Microcrosslinked sodium hyaluronate is made from sodium hyaluronate, sodium hydroxide solution, and a crosslinking agent. The relative molecular mass of microcrosslinked sodium hyaluronate is 1.4 million Daltons.

[0045] The cross-linking agent is selected from puerarin.

[0046] The sodium hydroxide solution has a mass fraction of 2.5%.

[0047] A method for preparing a composition of polynucleotides and sodium hyaluronate includes the following steps: (1) Dissolve sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride in water for injection to prepare a phosphate-sodium chloride buffer solution; (2) First, dissolve the polynucleotide in a phosphate-sodium chloride buffer solution, stir at 55 °C for 2 hours. After dissolution, it will be a semi-transparent solution. (3) Add micro-crosslinked sodium hyaluronate and stir evenly to obtain a polynucleotide-sodium hyaluronate composition, which is milky white and semi-transparent.

[0048] In step (3), the preparation method of micro-crosslinked sodium hyaluronate is as follows: (3.1) Sodium hyaluronate and crosslinking agent are mixed evenly, and sodium hydroxide solution is added to create an alkaline environment. The reaction temperature is 30 °C and the reaction time is 22 h to obtain the crosslinking product. The mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent and water for injection is 10:5:5:300.

[0049] (3.2) Cut the cross-linked product into 2cm pieces. 3 Small gel blocks were placed in a phosphate-sodium chloride buffer solution for dialyzing. 1 M hydrochloric acid was added to adjust the pH to neutral. When the gel swelled to a certain weight (10 times the weight of the cross-linked product), it was sieved and granulated to obtain cross-linked sodium hyaluronate.

[0050] (4) Vacuum the polynucleotide-sodium hyaluronate composition to remove air bubbles, then transfer it to a filling machine to fill it into a syringe and sterilize it.

[0051] Example 3 A composition of a polynucleotide and sodium hyaluronate: by weight percentage, comprising: 2% polynucleotide, 2% micro-crosslinked sodium hyaluronate, 0.005% sodium dihydrogen phosphate, 0.022% disodium hydrogen phosphate, 0.7% sodium chloride, and the balance being water for injection.

[0052] Microcrosslinked sodium hyaluronate is made from sodium hyaluronate, sodium hydroxide solution, and a crosslinking agent. The relative molecular mass of microcrosslinked sodium hyaluronate is 1.4 million Daltons.

[0053] The cross-linking agent is selected from puerarin.

[0054] The sodium hydroxide solution has a mass fraction of 2.5%.

[0055] A method for preparing a composition of polynucleotides and sodium hyaluronate includes the following steps: (1) Dissolve sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride in water for injection to prepare a phosphate-sodium chloride buffer solution; (2) First, dissolve the polynucleotide in a phosphate-sodium chloride buffer solution, stir at 55 °C for 2 hours. After dissolution, it will be a semi-transparent solution. (3) Add micro-crosslinked sodium hyaluronate and stir evenly to obtain a polynucleotide-sodium hyaluronate composition, which is milky white, semi-transparent and viscous.

[0056] In step (3), the preparation method of micro-crosslinked sodium hyaluronate is as follows: (3.1) Sodium hyaluronate and crosslinking agent are mixed evenly, and sodium hydroxide solution is added to create an alkaline environment. The reaction temperature is 30 °C and the reaction time is 22 h to obtain the crosslinking product. The mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent and water for injection is 10:5:3:300.

[0057] (3.2) Cut the cross-linked product into 2cm pieces. 3 Small gel blocks were placed in a phosphate-sodium chloride buffer solution for dialyzing. 1 M hydrochloric acid was added to adjust the pH to neutral. When the gel swelled to a certain weight (10 times the weight of the cross-linked product), it was sieved and granulated to obtain cross-linked sodium hyaluronate.

[0058] (4) Vacuum the polynucleotide-sodium hyaluronate composition to remove air bubbles, then transfer it to a filling machine to fill it into a syringe and sterilize it.

[0059] Example 4 A composition of a polynucleotide and sodium hyaluronate: by weight percentage, comprising: 0.75% polynucleotide, 1.2% micro-crosslinked sodium hyaluronate, 0.005% sodium dihydrogen phosphate, 0.022% disodium hydrogen phosphate, 0.7% sodium chloride, and the balance being water for injection.

[0060] Microcrosslinked sodium hyaluronate is made from sodium hyaluronate, sodium hydroxide solution, and a crosslinking agent. The relative molecular mass of microcrosslinked sodium hyaluronate is 1.4 million Daltons.

[0061] The cross-linking agent is selected from puerarin.

[0062] The sodium hydroxide solution has a mass fraction of 2.5%.

[0063] A method for preparing a composition of polynucleotides and sodium hyaluronate includes the following steps: (1) Dissolve sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride in water for injection to prepare a phosphate-sodium chloride buffer solution; (2) First, dissolve the polynucleotide in a phosphate-sodium chloride buffer solution, stir at 55 °C for 2 hours. After dissolution, it will be a semi-transparent solution. (3) Add micro-crosslinked sodium hyaluronate and stir evenly to obtain a polynucleotide-sodium hyaluronate composition, which is milky white and semi-transparent.

[0064] In step (3), the preparation method of micro-crosslinked sodium hyaluronate is as follows: (3.1) Sodium hyaluronate and crosslinking agent are mixed evenly, and sodium hydroxide solution is added to create an alkaline environment. The reaction temperature is 30 °C and the reaction time is 22 h to obtain the crosslinking product. The mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent and water for injection is 10:5:5:300.

[0065] (3.2) Cut the cross-linked product into 2cm pieces. 3 Small gel blocks were placed in a phosphate-sodium chloride buffer solution for dialyzing. 1 M hydrochloric acid was added to adjust the pH to neutral. When the gel swelled to a certain weight (10 times the weight of the cross-linked product), it was sieved and granulated to obtain cross-linked sodium hyaluronate.

[0066] (4) Vacuum the polynucleotide-sodium hyaluronate composition to remove air bubbles, then transfer it to a filling machine to fill it into a syringe and sterilize it.

[0067] Example 5 A composition of polynucleotides and sodium hyaluronate: By weight percentage, it comprises: 2% polynucleotide, 2% micro-crosslinked sodium hyaluronate, 0.005% sodium dihydrogen phosphate, 0.022% disodium hydrogen phosphate, 0.7% sodium chloride, and the balance being water for injection.

[0068] Microcrosslinked sodium hyaluronate is made from sodium hyaluronate, sodium hydroxide solution, and a crosslinking agent. The relative molecular mass of microcrosslinked sodium hyaluronate is 1.4 million Daltons.

[0069] The crosslinking agent is selected from 1,4-butanediol glycidyl ether.

[0070] The sodium hydroxide solution has a mass fraction of 2.5%.

[0071] A method for preparing a composition of polynucleotides and sodium hyaluronate includes the following steps: (1) Dissolve sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride in water for injection to prepare a phosphate-sodium chloride buffer solution; (2) First, dissolve the polynucleotide in a phosphate-sodium chloride buffer solution, stirring at 55 °C for 2 hours. After dissolution, it becomes a semi-transparent solution. (3) Add micro-crosslinked sodium hyaluronate and stir evenly to obtain a polynucleotide-sodium hyaluronate composition, which is milky white and semi-transparent; In step (3), the preparation method of micro-crosslinked sodium hyaluronate is as follows: (3.1) Sodium hyaluronate and crosslinking agent are mixed evenly, and sodium hydroxide solution is added to create an alkaline environment. The reaction temperature is 30 °C and the reaction time is 22 h to obtain the crosslinking product. The mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent and water for injection is 10:5:5:300.

[0072] (3.2) Cut the cross-linked product into 2 cm pieces. 3 Small gel blocks were dialyzed in a phosphate-sodium chloride buffer solution. 1 M hydrochloric acid was added to adjust the pH to neutral. The buffer solution was changed every two hours. When the gel swelled to a certain weight (10 times the weight of the cross-linked product), it was sieved and granulated to obtain cross-linked sodium hyaluronate.

[0073] (4) Vacuum the polynucleotide-sodium hyaluronate composition to remove air bubbles, then transfer it to a filling machine to fill it into a syringe and sterilize it.

[0074] This embodiment differs from Example 1 in that it uses a different crosslinking agent, and the corresponding steps for preparing micro-crosslinked sodium hyaluronate are adjusted. The reason for changing the buffer solution periodically in this embodiment is to remove residual BDDE. Incomplete removal of BDDE will fail to meet testing standards, leading to product defects and affecting subsequent performance. In Example 1, puerarin was chosen as the crosslinking agent because it can be used as an active ingredient in injectable products. As a natural plant extract, puerarin is non-toxic, safe, and low-cost, and can synergistically enhance anti-aging and anti-inflammatory effects with other ingredients. Therefore, using puerarin as the crosslinking agent eliminates the need for circulating buffer solution to remove excess puerarin, saving steps, reducing manpower and resources, facilitating the preparation of micro-crosslinked sodium hyaluronate, promoting industrial production, and improving the final product's efficacy.

[0075] Example 6 A composition of polynucleotides and sodium hyaluronate: By weight percentage, it comprises: 2% polynucleotide, 2% micro-crosslinked sodium hyaluronate, 0.005% sodium dihydrogen phosphate, 0.022% disodium hydrogen phosphate, 0.7% sodium chloride, and the balance being water for injection.

[0076] Microcrosslinked sodium hyaluronate is made from sodium hyaluronate, sodium hydroxide solution, and a crosslinking agent. The relative molecular mass of microcrosslinked sodium hyaluronate is 1.4 million Daltons.

[0077] The cross-linking agent is selected from puerarin.

[0078] The sodium hydroxide solution has a mass fraction of 2.5%.

[0079] A method for preparing a composition of polynucleotides and sodium hyaluronate includes the following steps: (1) Dissolve sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride in water for injection to prepare a phosphate-sodium chloride buffer solution; (2) First, dissolve the polynucleotide in a phosphate-sodium chloride buffer solution, stirring at 55 °C for 2 hours. After dissolution, it becomes a semi-transparent solution. (3) Add micro-crosslinked sodium hyaluronate and stir evenly to obtain a polynucleotide-sodium hyaluronate composition, which is milky white and semi-transparent.

[0080] In step (3), the preparation method of micro-crosslinked sodium hyaluronate is as follows: (3.1) Sodium hyaluronate and crosslinking agent are mixed evenly, and sodium hydroxide solution is added to create an alkaline environment. The reaction temperature is 30 °C and the reaction time is 22 h to obtain the crosslinking product. The mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent and water for injection is 10:5:5:300.

[0081] (3.2) Cut the cross-linked product into 2 cm pieces. 3 Small gel blocks were dialyzed in a phosphate-sodium chloride buffer solution. 1 M hydrochloric acid was added to adjust the pH to neutral. The buffer solution was changed every two hours. When the gel swelled to a certain weight (10 times the weight of the cross-linked product), it was sieved and granulated to obtain cross-linked sodium hyaluronate.

[0082] (4) Vacuum the polynucleotide-sodium hyaluronate composition to remove air bubbles, then transfer it to a filling machine to fill it into a syringe and sterilize it.

[0083] Comparative Example 1 The specific operation of this comparative example is the same as in Example 1 above, with the following differences: the polynucleotide content is 0.005%, the micro-crosslinked sodium hyaluronate is 0.005%, the sodium dihydrogen phosphate is 0.005%, the disodium hydrogen phosphate is 0.022%, the sodium chloride is 0.7%, and the balance is water for injection. The micro-crosslinked sodium hyaluronate is prepared from sodium hyaluronate, sodium hydroxide solution, and puerarin, with a mass ratio of sodium hyaluronate, sodium hydroxide, puerarin, and water for injection of 10:5:5:300. The relative molecular mass of the micro-crosslinked sodium hyaluronate is 400,000 Daltons.

[0084] Comparative Example 2 The specific operation of this comparative example is the same as in Example 2 above, with the following differences: the polynucleotide content is 2.5%, the micro-crosslinked sodium hyaluronate is 2.5%, sodium dihydrogen phosphate is 0.005%, disodium hydrogen phosphate is 0.022%, sodium chloride is 0.7%, and the balance is water for injection. The micro-crosslinked sodium hyaluronate is prepared from sodium hyaluronate, sodium hydroxide solution, and a crosslinking agent, with a mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent, and water for injection of 10:5:5:300. The relative molecular mass of the micro-crosslinked sodium hyaluronate is 2 million Daltons.

[0085] Comparative Example 3 The specific operation of this comparative example is the same as in Example 3 above, with the following difference: the micro-crosslinked sodium hyaluronate is made from sodium hyaluronate, sodium hydroxide solution, and a crosslinking agent. The crosslinking agent used is 1,4-butanediol glycidyl ether. The mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent, and water for injection is 100:3:0.8:500. The relative molecular mass of the micro-crosslinked sodium hyaluronate is 1.4 million Daltons.

[0086] Comparative Example 4 The specific operation of this comparative example is the same as in Example 4 above, except that the micro-crosslinked sodium hyaluronate is made from sodium hyaluronate, sodium hydroxide solution, and a crosslinking agent, with a mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent, and water for injection of 10:5:10:300. The relative molecular mass of the micro-crosslinked sodium hyaluronate is 1.4 million Daltons. The crosslinking agent is selected from puerarin.

[0087] Comparative Example 5 The specific operation of this comparative example is the same as that of Example 4 above, except that the polynucleotide is polymerized from nucleotide monomers that have not been modified by 2'-o-methylation, and the polynucleotide is used directly.

[0088] Comparative Example 6 The specific operation of this comparative example is the same as that of comparative example 4 above, except that the crosslinking agent is 1,4-butanediol glycidyl ether.

[0089] In step (3), the preparation method of micro-crosslinked sodium hyaluronate is as follows: (3.1) Sodium hyaluronate and crosslinking agent are mixed evenly, and sodium hydroxide solution is added to create an alkaline environment. The reaction temperature is 30 °C and the reaction time is 22 h to obtain the crosslinking product. The mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent and water for injection is 10:5:5:300.

[0090] (3.2) Cut the cross-linked product into 2 cm pieces. 3 Small gel blocks were dialyzed in a phosphate-sodium chloride buffer solution. 1 M hydrochloric acid was added to adjust the pH to neutral. The buffer solution was changed every two hours. When the gel swelled to a certain weight (10 times the weight of the cross-linked product), it was sieved and granulated to obtain cross-linked sodium hyaluronate.

[0091] (4) Vacuum the polynucleotide-sodium hyaluronate composition to remove air bubbles, then transfer it to a filling machine to fill it into a syringe and sterilize it.

[0092] The remaining steps are the same as in Comparative Example 4.

[0093] Comparative Example 7 The comparative example differs from Example 1 in the following ways: 0.005% polynucleotide, 2% micro-crosslinked sodium hyaluronate, 0.005% sodium dihydrogen phosphate, 0.022% disodium hydrogen phosphate, 0.7% sodium chloride, and the balance being water for injection.

[0094] The preparation method is the same as in Example 1.

[0095] Comparative Example 8 The comparative example differs from Example 1 in the following ways: 2.5% polynucleotide, 2% micro-crosslinked sodium hyaluronate, 0.005% sodium dihydrogen phosphate, 0.022% disodium hydrogen phosphate, 0.7% sodium chloride, and the balance being water for injection.

[0096] The preparation method is the same as in Example 1.

[0097] The material quantities of Examples 1-6 and Comparative Examples 1-8 are summarized in Table 1: Table 1. Component types and proportions (%) of Examples 1-6 and Comparative Examples 1-8

[0098] Comparative Example 2, compared to Example 2, increased the molecular weight of polynucleotides, micro-crosslinked sodium hyaluronate, and micro-crosslinked sodium hyaluronate, resulting in excessive product extrusion force, making it unsuitable for clinical use. Comparative Example 6, due to an excessively high amount of the chemical crosslinking agent 1,4-butanediol glycidyl ether, resulted in over-crosslinking, which was difficult to completely wash away, rendering the product unusable. Comparative Examples 2 and 6 also demonstrate that the amount of each component used and the molecular weight of sodium hyaluronate affect the properties of the final product. Only by adhering to the molecular weight and amount of each substance specified in this invention can the product properties be guaranteed and the product yield improved.

[0099] Efficacy test During the trial, patients were divided into experimental groups (Examples 1-6) and control groups (Comparative Examples 1, 3-5, and 7-8). Ten female volunteers aged 40 to 50 were randomly selected from each group. The volunteers underwent the following tests, and the average test results of each group were recorded.

[0100] Volunteer Recruitment Criteria: 1) Has problems such as dry skin, fine lines, dull skin tone, large pores, and pigmentation, and requests facial rejuvenation; 2) Has not undergone other facial treatments within 1 month; 3) Agrees to complete follow-up.

[0101] Those with the following conditions should be excluded: a. Those who have used antihistamines in the past week or immunosuppressants in the past month; b. Those who have used any anti-inflammatory drugs on the test site within the past two months; c. Subjects with clinically unhealed inflammatory skin diseases; d. Patients with insulin-dependent diabetes mellitus; e. Patients with asthma or other chronic respiratory diseases who are currently receiving treatment; f. Those who have received anti-cancer chemotherapy within the past 6 months; g. Patients with immunodeficiency or autoimmune diseases; h. Breastfeeding or pregnant women; i. Patients who have undergone bilateral mastectomy and bilateral axillary lymph node dissection; j. If the determination of the test results is affected by scars, pigmentation, atrophy, port-wine stains or other blemishes at the skin test site; k. Researchers participating in other clinical trials; l. Individuals with highly sensitive constitutions; m. Those who are not volunteers or who cannot complete the prescribed content according to the experimental requirements.

[0102] Injection method: Use a disposable sterile injection needle (nine needles, purchased from Suzhou Hannuoxin Biotechnology Co., Ltd.) and a syringe-assisted propulsion device (BZ-1, purchased from Shaoxing Yannuo Medical Technology Co., Ltd.) to perform facial injections on the patient. The needle length is 0.8-1.2 mm and the injection volume is 2 mL.

[0103] Experimental method: Three consecutive facial injection treatments were performed, with a 4-week interval between the two treatments. The initial values ​​of each indicator were measured before the first injection (T0), and all indicators were measured 2 weeks after the first injection (T1), 2 weeks after the third injection (T2), and 4 weeks after the third injection (T3).

[0104] Experimental Example 1: Detection of Skin Wrinkle Improvement Effect The skin wrinkles of volunteers were detected at various time points using the VISIA CR skin analyzer (where lower values ​​indicated fewer wrinkles), and the rate of change (%) of skin wrinkles was calculated and recorded. The calculation method for the rate of change of skin wrinkles was as follows: Change rate (%) = [(skin wrinkles at each time point after treatment - initial skin wrinkles before treatment) / initial skin wrinkles before treatment] × 100%.

[0105] Table 2. Rate of change in skin wrinkles (%)

[0106] Experimental Example 2: Detection of Skin Tightness Improvement Effect The neck skin tightness of volunteers was measured at various time points using a Corneometer MPA580 skin elastometer (where a smaller value indicates tighter skin). The rate of change (%) of neck skin tightness was calculated and recorded. The rate of change (%) of neck skin tightness was calculated as follows: Rate of change (%) = [(Neck skin tightness at each time point after treatment - Initial neck skin tightness before treatment) / Initial neck skin tightness before treatment] × 100%.

[0107] Table 3. Change rate of skin firmness (%)

[0108] Experimental Example 3: Detection of Skin Elasticity Improvement Effect The skin elasticity of volunteers was measured at various time points using a Corneometer MPA580 skin elasticity meter (a higher measured value indicates better improvement in skin elasticity), and the rate of change (%) of skin elasticity was calculated and recorded. The rate of change (%) of skin elasticity was calculated as follows: Rate of change (%) = [(Skin elasticity at each time point after treatment - Initial skin elasticity before treatment) / Initial skin elasticity before treatment] 100%.

[0109] Parameter description: Reflects the skin's ability to return to its original shape after being stretched or compressed; the higher the measured value, the better the skin elasticity.

[0110] Table 4. Change rate of skin elasticity (%)

[0111] Experimental Example 4: Anti-inflammatory Repair Test RAW264.7 mouse macrophages in the logarithmic growth phase were selected, washed twice with PBS, and immediately incubated overnight in 3 mL of DMEM complete culture medium (37 ℃, 5% CO2) at a rate of 100 μg / mL with DXMS. The experiment included a positive control group, a model group, and a sample group. The positive control group received 100 μg / mL dexamethasone (DXMS), and the model group received 1 μg / mL LPS. This experiment utilized the binding of bacterial lipopolysaccharide (LPS) to macrophage surface antigen recognition receptors to induce the secretion of the inflammatory factor IL-6 from mouse RAW264.7 cells. After 24 h of culture, the cell culture supernatant was collected in EP tubes (Note: the amount of sample collected was determined according to the detection indicators). After collection, the samples used for IL-6 content detection were frozen at -80 ℃. The IL-6 content in the cell culture medium was detected using an ELISA kit-double antibody sandwich method, and the data were compiled as shown in Table 5.

[0112] Table 5 Anti-inflammatory Repair Results Data Table

[0113] As can be seen from Tables 1-5, when the molecular weights of polynucleotides, micro-crosslinked sodium hyaluronate, and the mass ratios of sodium hyaluronate, sodium hydroxide, crosslinking agent, and water for injection are within the scope of protection of this invention, they can achieve good results in improving skin wrinkles, skin firmness, skin elasticity, and anti-inflammatory repair.

[0114] Comparative Example 1, compared to Example 1, reduced the molecular weight of polynucleotides, microcrosslinked sodium hyaluronate, and sodium hyaluronate. In clinical trials, these components were not conducive to anti-aging, wrinkle reduction, and anti-inflammatory repair effects; specifically, wrinkle changes were not significant, skin firmness and elasticity were reduced, and anti-inflammatory repair effects were poor. Comparative Example 2 increased the molecular weight of polynucleotides, microcrosslinked sodium hyaluronate, and sodium hyaluronate, resulting in excessive product extrusion force, making it inconvenient for clinical use. Comparative Example 3 reduced the amount of cross-linking agent used compared to Example 3. However, Comparative Example 3 showed significantly lower anti-wrinkle, anti-aging, and anti-inflammatory repair effects compared to Example 3. Comparative Examples 4 and 6 increased the amount of cross-linking agent used compared to Example 4. Comparative Example 6 suffered from excessive cross-linking due to an excessive amount of the chemical cross-linking agent 1,4-butanediol glycidyl ether, which was difficult to completely wash off, rendering the product unusable. Comparative Example 4 used puerarin, which, although not having the BDDE residue problem, also suffered from excessive cross-linking due to the excessive amount of puerarin, resulting in a significantly worse product effect. Adding too much or too little cross-linking agent is detrimental to the product's effectiveness in actual use. Comparative Example 5 used an unmodified polynucleotide compared to Example 4. While the unmodified polynucleotide had a fast onset of action, it also degraded quickly. The modified polynucleotide, on the other hand, had a fast onset of action, provided stable and sustained anti-aging effects, had high bioavailability, and enhanced skin improvement effects. Therefore, Example 4 showed significant progress in anti-aging, wrinkle removal, and anti-inflammatory repair compared to Example 5. Comparative Example 7 reduced the amount of polynucleotides added compared to Example 1, while Comparative Example 8 increased the amount of polynucleotides added compared to Example 1. The significantly reduced amount of polynucleotides resulted in a very noticeable decrease in product efficacy. However, the increased amount of polynucleotides actually led to a decrease in the effectiveness of anti-aging, wrinkle reduction, and anti-inflammatory repair. This is because excessive addition of long-chain polynucleotides affects solubility and causes precipitation after sterilization, both of which affect the properties of the final product, resulting in poorer efficacy and increased costs. This further illustrates that the amount of polynucleotides added needs to be controlled within a specific range in conjunction with other substances to achieve the best results.

[0115] Example 5, compared to Example 1, uses 1,4-butanediol glycidyl ether as a cross-linking agent. Correspondingly, to reduce BDDE residue, the buffer solution needs to be constantly replaced during dialysis. Example 1, using puerarin, does not have this residue problem, eliminating the need to replace the buffer solution, effectively reducing steps, costs, and efficiency. Examples 1 and 5 differ only in the cross-linking agent; however, in terms of anti-aging, wrinkle reduction, and anti-inflammatory repair, Example 5 is significantly less effective than Example 1. This is partly because puerarin itself is beneficial for improving the anti-aging, wrinkle reduction, and anti-inflammatory repair effects of the product. Furthermore, the presence of BDDE residue in the product and the unique advantages of puerarin as a cross-linking agent are also significant. Puerarin can function as both a cross-linking agent and an active ingredient, a function not possessed by other cross-linking agents. Example 6, compared to Example 1, involves constantly replacing the buffer solution during dialysis to remove excess puerarin. Clinical trials show that after removing excess puerarin, the anti-aging, wrinkle reduction, and anti-inflammatory repair effects of the product in Example 6 are reduced compared to Example 1, further demonstrating that not removing excess puerarin actually enhances the product's effectiveness.

[0116] The above experimental data shows that the specific substances selected in this invention, the amount of each substance added, the synergy between their molecular weights, and the specific preparation method are crucial for the product to achieve its optimal effect. Factors such as molecular weight, the amount of substance added, whether modification is required, and whether residual substances need to be removed all influence the product's efficacy. Within the scope of this invention, the prepared product achieves good results in improving skin wrinkles, skin firmness, skin elasticity, and in anti-inflammatory and repairing effects.

[0117] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composition of polynucleotide and sodium hyaluronate, characterized in that, By weight percentage, it comprises the following components: polynucleotide 0.01-2%, micro-crosslinked sodium hyaluronate 0.01-2%, sodium dihydrogen phosphate 0.003-0.006%, disodium hydrogen phosphate 0.015-0.022%, sodium chloride 0.6-0.95%, and water for injection as solvent; The polynucleotide is obtained by polymerizing nucleotide monomers modified with 2'-o-methylation.

2. The composition according to claim 1, characterized in that, The relative molecular mass of micro-crosslinked sodium hyaluronate is 600,000 to 1,400,000 Daltons.

3. The composition according to claim 1, characterized in that, Micro-crosslinked sodium hyaluronate is made from sodium hyaluronate, sodium hydroxide solution and crosslinking agent; or, the mass fraction of sodium hydroxide solution is 0.5%-3%; or, the crosslinking agent is selected from at least one of puerarin, 1,4-butanediol glycidyl ether, divinyl sulfone, polyethylene glycol, genipin and carbodiimide.

4. The method for preparing the composition of polynucleotide and sodium hyaluronate as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride in water for injection to prepare a phosphate-sodium chloride buffer solution; (2) First, dissolve the polynucleotide in a phosphate-sodium chloride buffer solution and continue stirring until a semi-transparent solution is obtained; (3) Add micro-crosslinked sodium hyaluronate to the semi-transparent solution and stir until homogeneous to obtain a polynucleotide-sodium hyaluronate composition; (4) Vacuuming, filling and sterilizing the polynucleotide-sodium hyaluronate composition.

5. The preparation method according to claim 4, characterized in that, In step (2), the stirring temperature is 40-60 ℃ and the stirring time is 1-2 h.

6. The preparation method according to claim 4, characterized in that, In step (3), the preparation method of micro-crosslinked sodium hyaluronate is as follows: (3.1) Mix sodium hyaluronate and crosslinking agent evenly, then add sodium hydroxide solution to obtain crosslinking product through crosslinking reaction; (3.2) The cross-linked product was cut into small pieces of gel, dialyzed in a phosphate-sodium chloride buffer solution, and the pH was adjusted to neutral by adding hydrochloric acid. The gel swelled, sieved and granulated to obtain cross-linked sodium hyaluronate.

7. The preparation method according to claim 6, characterized in that, In step (3.1), the mass ratio of sodium hyaluronate, sodium hydroxide, crosslinking agent and water is 10:5:3-7:300; and / or, in step (3.1), the reaction temperature is 20-40 ℃ and the reaction time is 12-25 h.

8. The preparation method according to claim 6, characterized in that, In step (3.2), the crosslinking product is cut into 1-2 cm pieces. 3 Small gel blocks; and / or, in step (3.2), the gel swells to 9-13 times the weight of the crosslinked product.

9. The use of the composition of polynucleotide and sodium hyaluronate as described in any one of claims 1 to 3 in improving skin quality; preferably, the improvement in skin quality includes improvement of skin wrinkles, improvement of skin firmness, and improvement of skin elasticity.

10. The use of the composition of polynucleotide and sodium hyaluronate as described in any one of claims 1 to 3 in the preparation of anti-inflammatory and repair products.

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