Recombinant III-type collagen and PDRN compound composition and preparation method thereof

Abstract

The invention discloses a recombinant III-type collagen and PDRN compound composition and a preparation method thereof, and relates to the technical field of biological medicines. The composition is formed by compounding recombinant human III type collagen and polydeoxyribonucleotide according to the mass ratio of 1: 10-10: 1, the weight-average molecular weight of the recombinant human III type collagen and the weight-average molecular weight of the polydeoxyribonucleotide are 50-100 kDa, the ratio can generate synergistic interaction, fibroblast proliferation, migration and collagen secretion are remarkably promoted, and the activity of the composition is superior to that of a single component and commercially available products. The composition can be used for preparing medical instruments, cosmetics or water-light injections for skin repair, rejuvenation or skin improvement. The invention also provides a water-light injection containing the compound composition, and the obtained preparation is stable, clear, transparent, safe and non-irritant.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a recombinant type III collagen and PDRN compound composition and its preparation method. Background Technology

[0002] In recent years, a relatively safe and effective medical aesthetic method has emerged: direct injection of fillers into facial soft tissue to deeply moisturize the skin and improve its condition. Injectable cosmetic techniques have become one of the main procedures in soft tissue cosmetic surgery, and their minimally invasive nature (no surgery, no bleeding), ease of operation, and quick results have made them popular among beauty enthusiasts.

[0003] Collagen is a structural protein, a polysaccharide protein, milky white in color, containing small amounts of galactose and glucose. It is the main component of the extracellular matrix, found in the highest concentration in mammals, forming a dense elastic network in the skin, acting as a scaffold to support it. With age, collagen is gradually lost, leading to the breakage of collagen peptide bonds and the elastic network that support the skin. Its helical network structure is subsequently destroyed, causing skin tissue to oxidize, atrophy, and collapse, resulting in signs of aging such as dryness, wrinkles, sagging, and loss of elasticity. Because collagen has low antigenicity, good biocompatibility, and biodegradability, it can be used in facial contouring and wrinkle repair.

[0004] Polydeoxyribonucleotide (PDRN) is a DNA fragment extracted from salmon sperm with significant biological activity. PDRN is mainly composed of 2000 to 3000 base pairs of deoxyribonucleic acid and can effectively promote cell regeneration and tissue repair. Its mechanisms mainly include activating A2A adenosine receptors, promoting fibroblast proliferation, and improving tissue microcirculation by stimulating angiogenesis. Due to its good biocompatibility and low immunogenicity, PDRN is widely used in skin care, wound healing, and anti-aging. Currently, to fully utilize the skin-improving effects of PDRN, injection is the most common method. Injection methods mainly include intradermal injection and subcutaneous injection. These methods ensure that PDRN reaches the deep layers of the skin directly, exerting its cell-regenerating, anti-inflammatory, and antioxidant effects.

[0005] Currently, skin injection products such as mesotherapy often contain active ingredients like PDRN and collagen to achieve hydration and anti-aging effects. However, existing products have the following problems:

[0006] 1. The wide or unclear molecular weight range of the active ingredients leads to low transdermal absorption efficiency and low bioavailability.

[0007] 2. Different active ingredients are mostly mixed in a simple physical way, lacking synergistic effects, with the effect being only 1+1=2, or even inhibiting each other.

[0008] 3. Poor solution stability; the activity is easily degraded during storage and transportation, affecting the final efficacy.

[0009] Therefore, developing a compound system with high absorption efficiency, good stability, and synergistic effect has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0010] The purpose of this invention is to provide a recombinant type III collagen and PDRN compound composition that has good absorption, high stability and can produce a significant synergistic effect, so as to solve the problems existing in the prior art.

[0011] To address the aforementioned technical problems, this invention provides a recombinant type III collagen and PDRN compound composition, comprising recombinant human type III collagen (rhCol III) and polydeoxyribonucleotide (PDRN), wherein the weight-average molecular weight of the recombinant human type III collagen is 50-100 kDa, and the weight-average molecular weight of the PDRN is 50-100 kDa; the mass ratio of PDRN to the recombinant human type III collagen is 1:10 to 10:1.

[0012] Furthermore, the mass ratio of PDRN to recombinant human type III collagen is preferably 1:2 to 2:1.

[0013] This invention provides the use of a recombinant type III collagen and PDRN compound composition in medical devices or cosmetics for skin repair, skin rejuvenation, or improvement of skin texture.

[0014] This invention provides the use of a recombinant type III collagen and PDRN compound composition in the preparation of a medicament for promoting fibroblast proliferation and / or migration.

[0015] This invention provides the use of a recombinant type III collagen and PDRN compound composition in the preparation of a medicament for promoting the secretion of type I collagen and / or type III collagen.

[0016] This invention provides a skin repair hyaluronic acid injection, comprising a compound composition of recombinant type III collagen and PDRN, and a pharmaceutically or cosmetically acceptable carrier; the concentration of PDRN in the hyaluronic acid injection is 1-20 mg / mL, and the concentration of recombinant human type III collagen in the hyaluronic acid injection is 1-50 mg / mL.

[0017] Furthermore, the carrier includes a local anesthetic, a buffer system, an isotonic regulator, and a stabilizer.

[0018] Furthermore, the local anesthetic is lidocaine hydrochloride, and its concentration in the hyaluronic acid injection is 0.1%-0.3% (w / v).

[0019] Furthermore, the isotonic regulator is sodium chloride.

[0020] Furthermore, the stabilizer is ethylenediaminetetraacetic acid salt, and its concentration in the hyaluronic acid injection is 0.01%-0.05% (w / v).

[0021] Furthermore, the buffer system is a phosphate buffer pair, composed of sodium dihydrogen phosphate and disodium hydrogen phosphate, which maintains the pH value of the hyaluronic acid injection between 6.0 and 7.0.

[0022] This invention provides a method for preparing a skin-repairing hyaluronic acid injection, comprising the following steps:

[0023] S1. Dissolve disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, and disodium ethylenediaminetetraacetate in water for injection, mix and stir until homogeneous, and prepare a solution for later use.

[0024] S2. After heating the S1 solution in a water bath, slowly add PDRN powder and stir until dissolved;

[0025] S3. After cooling the S2 solution, slowly add the recombinant human type III collagen powder and stir until dissolved;

[0026] S4. Add lidocaine hydrochloride powder to solution S3 and stir until dissolved;

[0027] S5. After adjusting the pH, add water for injection and weigh to 100%;

[0028] S6. After filtering and sterilizing the solution, fill it into a pre-filled syringe.

[0029] Furthermore, in step S1, the mixing and stirring are carried out at room temperature at a speed of 200-400 r / min for 20-30 min.

[0030] Furthermore, in step S2, the water bath is heated to 35-45°C, the stirring speed is 200-400 r / min, and the stirring time is 20-30 min.

[0031] Furthermore, in step S3, the temperature is lowered to 20-30℃, the stirring speed is 200-400 r / min, and the stirring time is 20-30 min.

[0032] Furthermore, in step S4, the stirring temperature is room temperature, the stirring speed is 200-400 r / min, and the stirring time is 20-30 min.

[0033] Furthermore, in step S5, sodium hydroxide solution is used to adjust the pH to 6.0-7.0.

[0034] Furthermore, in step S6, the filtration and sterilization method is to use a 0.22μm PES material filter membrane for filtration and sterilization.

[0035] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0036] 1. This invention, through extensive experiments, discovered and verified that a combination of rhCol III within a specific molecular weight range (50-100 kDa) and PDRN at a specific mass ratio (2:1) can produce unexpected synergistic biological effects. As shown in the examples, its effect on promoting fibroblast proliferation (184%) is significantly higher than that of the single-component group (collagen: 126%; PDRN: 138%) and the theoretical summation value (164%), proving that its effect is not simply additive, but a true synergistic amplification. The combination composition of this invention promotes type III collagen secretion at an effect as high as 470.8% of the blank group, an extremely significant effect far exceeding any single component and commercially available products, providing a highly efficient solution for addressing skin aging, loss of elasticity, and other problems.

[0037] 2. The molecular weight range of 50-100kDa defined in this invention not only retains good bioactivity, but is also easily absorbed by the skin through the microchannels created by the water-light injection, which significantly improves bioavailability and avoids the waste of active ingredients.

[0038] 3. This invention effectively solves the technical problem of easy aggregation and degradation of macromolecular active substances in liquid environments through a specific low-temperature dissolution process and the combination of EDTA stabilizer. Accelerated stability tests show that after being placed at 40°C for 3 months, the content of active ingredients and the retention rate of bioactivity are both >95%, which is far superior to the control group with mismatched molecular weights, ensuring that the efficacy of the product remains consistent throughout its shelf life.

[0039] 4. All ingredients are known safe pharmaceutical excipients or bioactive components. The pH value is similar to that of human body fluids, and the osmotic pressure is isotonic with human blood plasma, which greatly reduces the irritation and adverse reaction risk during injection, resulting in high safety. Attached Figure Description

[0040] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0041] Figure 1 This is a graph showing the cell proliferation activity test results of the present invention;

[0042] Figure 2This is a diagram illustrating the collagen secretion-promoting effect of the present invention. Detailed Implementation

[0043] To better understand the above technical solution, the following detailed explanation will be provided through specific implementation methods.

[0044] Example 1: Preparation of PDRN Recombinant Collagen Aqueous Solution for Injection

[0045] Both recombinant type 3 collagen and PDRN were prepared into aqueous solutions using molecular weights in the range of 50-100 kDa.

[0046] Table 1 Experimental Sample Formulation Table

[0047]

[0048] The preparation method of injectable recombinant collagen PDRN hydrating solution includes the following steps:

[0049] S1. Dissolve disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, and disodium ethylenediaminetetraacetate in water for injection and stir at 300 r / min for 25 min to obtain a solution for later use.

[0050] S2. After heating the S1 solution in a 40°C water bath, slowly add PDRN powder and stir at 300 r / min for 25 min.

[0051] S3. After cooling the S2 solution to 25°C, slowly add the recombinant collagen powder and stir at 300 r / min for 25 min.

[0052] S4. Add lidocaine hydrochloride powder to solution S3 and stir at 300 r / min for 25 min.

[0053] S5. Adjust the pH of the solution to the range of 6.0-7.0 with 1M sodium hydroxide, and add water for injection to bring the weight to 100%.

[0054] S6. After sterilizing the solution by filtering it through a 0.22μm PES filter membrane, it is then filled into bottles.

[0055] Comparative Example 1: Preparation of PDRN Aqueous Solution for Injection

[0056] Both recombinant type 3 collagen and PDRN were prepared into aqueous solutions using molecular weights in the range of 50-100 kDa.

[0057] Table 2 Experimental Sample Formulation Table

[0058]

[0059] The preparation method of injectable recombinant collagen PDRN hydrating solution includes the following steps:

[0060] S1. Dissolve disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, and disodium ethylenediaminetetraacetate in water for injection and stir at 300 r / min for 25 min to obtain a solution for later use.

[0061] S2. After heating the S1 solution in a 40°C water bath, slowly add PDRN powder and stir at 300 r / min for 25 min.

[0062] S3. After cooling the S2 solution to 25°C, slowly add the recombinant collagen powder and stir at 300 r / min for 25 min.

[0063] S4. Add lidocaine hydrochloride powder to solution S3 and stir at 300 r / min for 25 min.

[0064] S5. Adjust the pH of the solution to the range of 6.0-7.0 with 1M sodium hydroxide, and add water for injection to bring the weight to 100%.

[0065] S6. After sterilizing the solution by filtering it through a 0.22μm PES filter membrane, it is then filled into bottles.

[0066] Comparative Example 3: Injectable Recombinant Collagen PDRN Aqua Solution

[0067] Table 3 Experimental Sample Formulation Table

[0068]

[0069] The preparation method of injectable recombinant collagen PDRN hydrating solution includes the following steps:

[0070] S1. Dissolve disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, and disodium ethylenediaminetetraacetate in water for injection and stir at 300 r / min for 25 min to obtain a solution for later use.

[0071] S2. After heating the S1 solution in a 40°C water bath, slowly add PDRN powder and stir at 300 r / min for 25 min.

[0072] S3. After cooling the S2 solution to 25°C, slowly add the recombinant collagen powder and stir at 300 r / min for 25 min.

[0073] S4. Add lidocaine hydrochloride powder to solution S3 and stir at 300 r / min for 25 min.

[0074] S5. Adjust the pH of the solution to the range of 6.0-7.0 with 1M sodium hydroxide, and add water for injection to bring the weight to 100%.

[0075] S6. After sterilizing the solution by filtering it through a 0.22μm PES filter membrane, it is then filled into bottles.

[0076] Comparative Example 4: Preparation of blank control aqueous solution

[0077] Table 4 Experimental Sample Formulation Table

[0078]

[0079] The preparation method of injectable recombinant collagen PDRN hydrating solution includes the following steps:

[0080] S1. Dissolve disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, and disodium ethylenediaminetetraacetate in water for injection and stir at 300 r / min for 25 min to obtain a solution for later use.

[0081] S2. Add lidocaine hydrochloride powder to solution S3 and stir at 300 r / min for 25 min.

[0082] S3. Adjust the pH of the solution to the range of 6.0-7.0 with 1M sodium hydroxide, and add water for injection to bring the weight to 100%.

[0083] S4. After sterilization by filtering the solution through a 0.22μm PES filter membrane, it is then filled into bottles.

[0084] Performance testing

[0085] I. In vitro fibroblast proliferation activity assay (MTT method)

[0086] Methods: Human dermal fibroblasts (HDF) were seeded in 96-well plates and cultured for 24 hours. The culture medium was then replaced with fresh medium containing 1% FBS and the above-mentioned samples. After culturing for another 72 hours, the absorbance (OD value) was measured at 570 nm using the standard MTT assay, and the relative cell proliferation rate was calculated (with the blank control group as 100%).

[0087] Table 5. Results of cell proliferation activity assay (n=6, Mean±SD)*

[0088]

[0089] Theoretical superposition value = (B1% + B2% - 100%) = (126 + 138 - 100) = 164%

[0090] like Figure 1As shown, group A1 (rhCol Ⅲ:PDRN 2:1) exhibited the highest cell proliferation activity, significantly outperforming the single-component groups (B1, B2), the theoretical summation (164%), the molecular weight mismatch group (C1, C2), and the commercially available product (E). This strongly demonstrates a synergistic effect (1+1>2) at a specific molecular weight range (50-100kDa) and a specific mass ratio (2:1), rather than a simple additive effect.

[0091] II. Determination of Type I and Type III Collagen Secretion (ELISA Method)

[0092] Methods: HDF cells were treated with each sample for 48 hours, and the cell culture supernatant was collected. The collagen content in the supernatant was detected using a human type I and type III collagen-specific ELISA kit, strictly following the instructions.

[0093] Table 6. Effects on collagen secretion promotion (ng / mL, n=3, Mean±SD)*

[0094]

[0095] like Figure 2 As shown, group A1 significantly outperformed the single-component group and other control groups in promoting the secretion of type I and type III collagen, especially in promoting type III collagen. This further verifies that this compound system not only promotes cell proliferation but also efficiently guides cells to synthesize the crucial extracellular matrix, demonstrating a dual function.

[0096] III. Determination of the Optimal Mass Ratio

[0097] Using PDRN and collagen at fixed concentrations of 50-100 kDa, samples with different mass ratios were prepared for cell proliferation experiments.

[0098] Table 7 Quality ratio screening results (n=6, Mean±SD)*

[0099]

[0100] As shown in Table 7, the synergistic effect reaches its peak when the mass ratio is in the range of 1:2 to 2:1.

[0101] IV. Long-term stability assessment

[0102] Method: Samples from group A1, group C1, and commercially available product E were placed in a 40℃ accelerated stability test chamber. Samples were taken at 0, 1, 2, and 3 months for testing.

[0103] ①HPLC-SEC: Analyzes the molecular weight distribution and degradation of active ingredients;

[0104] ② Appearance and pH value: Check the clarity and pH change of the solution;

[0105] ③ Biological activity: The activity retention rate was detected by the MTT assay.

[0106] Table 8. Results of accelerated stability tests (3 months)

[0107]

[0108] The specific molecular weight combination (50-100kDa) of the present invention exhibits superior stability under accelerated conditions, with less degradation of active ingredients and higher retention of bioactivity.

[0109] V. Efficacy Test of the Hydrating Solution Combining Recombinant Type III Collagen and PDRN

[0110] By recruiting subjects with dry, dehydrated skin, roughness, peeling, dryness, tightness, redness, and other skin problems to use this product, the repair effect of this water-light solution was evaluated by repeatedly measuring the baseline skin values ​​of the test areas. All 20 subjects used the product as required within the specified time, and no adverse reactions occurred.

[0111] Table 9 Results of transdermal water loss test

[0112]

[0113] Note: Improvement rate (%) = (Base value - Test value after N days of product use) / Base value x 100%

[0114] Table 10 Results of Skin Hemoglobin Content Test

[0115]

[0116] Note: Improvement rate (%) = (Base value - Test value after N days of product use) / Base value x 100%

[0117] The test results showed that the transepidermal water loss and skin hemoglobin content of the subjects decreased significantly on day 28 compared with day 0, and >90% of the subjects felt that their skin became more moisturized and softer.

[0118] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No markings in the claims should be construed as limiting the scope of the claims.

Claims

1. A recombinant type III collagen and PDRN compound composition, comprising recombinant human type III collagen (rhCol III) and polydeoxyribonucleotide (PDRN), characterized in that, The recombinant human type III collagen has a weight-average molecular weight of 50-100 kDa, and the PDRN has a weight-average molecular weight of 50-100 kDa. The mass ratio of PDRN to recombinant human type III collagen is 1:10 to 10:

1.

2. The recombinant type III collagen and PDRN compound composition according to claim 1, characterized in that, The preferred mass ratio of PDRN to recombinant human type III collagen is 1:2 to 2:

1.

3. Use of the recombinant type III collagen and PDRN compound composition according to any one of claims 1-2 in medical devices or cosmetics for skin repair, skin rejuvenation or improvement of skin texture.

4. Use of the recombinant type III collagen and PDRN compound composition according to any one of claims 1-2 in the preparation of a medicament for promoting fibroblast proliferation and / or migration.

5. Use of the recombinant type III collagen and PDRN compound composition according to any one of claims 1-2 in the preparation of a medicament for promoting the secretion of type I collagen and / or type III collagen.

6. A skin repair water-light injection, characterized in that, The product comprises a compound composition as described in any one of claims 1-2, and a pharmaceutically or cosmetically acceptable carrier; wherein the concentration of the compound composition PDRN in the mesotherapy injection is 1-20 mg / mL, and the concentration of recombinant human type III collagen in the mesotherapy injection is 1-50 mg / mL.

7. A skin repair water-light injection solution according to claim 6, characterized in that, The carrier includes a local anesthetic, a buffer system, an isotonic regulator, and a stabilizer.

8. A skin repair water-light injection solution according to claim 6, characterized in that, The local anesthetic is lidocaine hydrochloride, with a concentration of 0.1%-0.3% (w / v) in the mesotherapy injection; the isotonicity adjuster is sodium chloride; and the stabilizer is ethylenediaminetetraacetic acid, with a concentration of 0.01%-0.05% (w / v) in the mesotherapy injection.

9. A skin repair water-light injection solution according to claim 6, characterized in that, The buffer system is a phosphate buffer pair, composed of sodium dihydrogen phosphate and disodium hydrogen phosphate, which maintains the pH value of the hyaluronic acid injection between 6.0 and 7.

0.

10. A method for preparing a skin-repairing water-light injection, characterized in that, Includes the following steps: S1. Dissolve disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, and disodium ethylenediaminetetraacetate in water for injection to obtain a solution for later use. S2. After heating the S1 solution in a water bath, slowly add PDRN powder and stir until dissolved; S3. After cooling the S2 solution, slowly add the recombinant human type III collagen powder and stir until dissolved; S4. Add lidocaine hydrochloride powder to solution S3 and stir until dissolved; S5. After adjusting the pH, add water for injection and weigh to 100%; S6. After filtering and sterilizing the solution, fill it into a pre-filled syringe.

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