A recombinant humanized collagen dressing containing sodium polyphosphate and a preparation method and application thereof

Abstract

The application discloses a recombinant humanized collagen dressing containing sodium polyphosphate and a preparation method thereof, and belongs to the technical field of biomedical materials. The dressing is mainly prepared from the following raw materials: recombinant type III collagen, sodium hyaluronate, sodium polyphosphate, glycerol, phenoxyethanol and purified water. The unique physical and chemical and biological properties of sodium polyphosphate are fully tapped, so that a synergistic system is formed with the recombinant type III collagen and the sodium hyaluronate. The dressing can not only significantly improve the moisturizing and water-locking performance of the dressing and prolong the maintenance time of the moist environment of a wound surface, but also efficiently promote the healing of a skin wound through the regulation of cell proliferation, the promotion of cytokine secretion and the acceleration of extracellular matrix deposition. Meanwhile, the dressing is verified by systematic biological evaluation to be free of cytotoxicity, sensitization reaction and acute systemic toxicity, has excellent biocompatibility and high safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a recombinant humanized collagen dressing containing sodium polyphosphate, its preparation method, and its application. Background Technology

[0002] As the largest barrier organ in the human body, the skin is susceptible to mechanical, physical, chemical, and biological damage, as well as disease factors, leading to wounds. These wounds are classified as acute or chronic. Acute wounds follow four stages: hemostasis, inflammatory response, proliferation, and remodeling, and with proper care, they can heal quickly. Chronic wounds, due to metabolic abnormalities and circulatory disorders, have a healing period exceeding four weeks, easily leading to serious complications, causing pain and burden for patients, and increasing the difficulty of clinical treatment. Traditional inert dressings only provide physical coverage of the wound and have drawbacks such as easy adhesion, secondary trauma, and poor moisture retention, which no longer meet clinical needs. With the development of the theory of moist wound healing, bioactive dressings containing natural bioactive ingredients, capable of regulating cell behavior, and promoting wound healing have become a research hotspot and are widely used.

[0003] Collagen is a major component of the skin's extracellular matrix, accounting for 70%–80% of the skin's dry weight. Type III collagen, in particular, can promote fibroblast proliferation, regulate collagen synthesis, and improve wound healing quality. Traditional animal-derived collagen has safety risks such as limited sources, low purity, and a tendency to trigger immune rejection. Recombinant human collagen, prepared through genetic engineering, has an amino acid sequence highly homologous to human collagen, superior biocompatibility, and is free of animal-derived impurities, effectively solving the above problems and becoming one of the core raw materials for wound repair dressings. However, existing recombinant collagen dressings still have significant shortcomings: limited moisturizing properties, easy water loss, and difficulty in adapting to dry wounds or wounds with little exudate; insufficient wound repair efficiency, only providing a scaffold for cell adhesion and unable to actively regulate cytokine secretion and angiogenesis, resulting in poor efficacy for chronic wounds; poor system stability, with collagen molecules easily degrading and agglomerating, and some dressings containing cross-linking agents and penetration enhancers may introduce toxicity or sensitization risks, reducing safety.

[0004] Sodium polyphosphate is a linear inorganic polymer formed by multiple phosphate groups linked by phosphate ester bonds, with the chemical formula Na. n+2 P n O 3n+1 (n represents the degree of polymerization), sodium polyphosphate can be classified into low, medium, and high degrees of polymerization. It possesses excellent physicochemical properties and biological activity, and is widely used in many fields. At the physicochemical level, it can chelate Ca... 2+ Mg 2+It contains metal ions to regulate the ion concentration of the system, and its molecular chains contain a large number of hydrophilic phosphate groups, which can form hydrogen bonds to achieve efficient moisturizing and water-locking. It can also act as a dispersant and stabilizer to improve the system's dispersibility and prevent molecular aggregation and sedimentation. At the bioactivity level, its degradation product is phosphate ions, which can participate in human metabolism without residual toxicity. It can promote the proliferation of fibroblasts and vascular endothelial cells, regulate inflammatory responses, accelerate coagulation, and create favorable conditions for wound healing. Currently, sodium polyphosphate is mainly used in the biomedical field in dentistry, orthopedics, and hemostatic materials. In recombinant collagen wound dressings, it is only added in small amounts as an auxiliary stabilizer, failing to fully utilize its core functions of moisturizing and promoting repair, and not forming a synergistic system with recombinant collagen and sodium hyaluronate, thus failing to solve the pain points of existing dressing technology.

[0005] Currently, recombinant collagen wound dressings on the market generally suffer from five major drawbacks: First, poor moisturizing durability, relying on traditional moisturizing ingredients and unable to maintain a moist wound environment for a long time; second, low wound repair efficiency, lacking components that actively regulate the healing process, resulting in poor efficacy for chronic wounds; third, insufficient system stability, prone to problems such as layering and thinning, and the addition of cross-linking agents increases safety risks; fourth, incomplete biological safety, with some products lacking systematic evaluation or containing potentially harmful ingredients; and fifth, limited application of sodium polyphosphate, failing to play a synergistic role as a core functional component and lacking differentiated advantages. Therefore, there is an urgent need for a high-performance medical dressing that combines moisturizing, repair-promoting, system-stabilizing, and biosafety properties to fill the technological gap and meet the clinical needs for various wound repairs. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a recombinant humanized collagen dressing containing sodium polyphosphate.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a recombinant humanized collagen dressing containing sodium polyphosphate, characterized in that it is composed of the following raw materials in the following mass percentages: Recombinant type III collagen 0.05%~2.0%; Sodium hyaluronate 0.1%~3.0%; Sodium polyphosphate 0.05%~2.0%; Phenoxyethanol 0.3%~1.0%; Glycerin 1.0%~10.0%; The remainder is purified water.

[0010] As a preferred embodiment of the recombinant humanized collagen dressing of the present invention, wherein: the recombinant type III collagen is a humanized recombinant collagen produced by genetic engineering fermentation, with a weight-average molecular weight of 5000~100000 Da, a purity of ≥95%, and no animal-derived impurities.

[0011] As a preferred embodiment of the recombinant humanized collagen dressing of the present invention, the sodium polyphosphate has an average chain length of n≥20, a pH value of 6.5~8.0, and a water solubility of ≥99%.

[0012] As a preferred embodiment of the recombinant humanized collagen dressing of the present invention, wherein the sodium hyaluronate is high molecular weight sodium hyaluronate with a weight-average molecular weight of 1,500,000 Da.

[0013] As a preferred embodiment of the recombinant humanized collagen dressing of the present invention, the purified water is medical-grade purified water with a conductivity ≤1.0 μS / cm.

[0014] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a recombinant humanized collagen dressing containing sodium polyphosphate, characterized by comprising the following steps: Pretreatment: Place purified water in a sterile stirred tank, adjust the temperature to 25~35℃, purge with nitrogen for protection, and set aside. Preparation of recombinant type III collagen solution: Take the prescribed amount of recombinant type III collagen and slowly add it to pretreated purified water. Stir at 500~1500 r / min for 30~60 min until completely dissolved to obtain recombinant type III collagen solution. Preparation of the composite system: Sodium polyphosphate and sodium hyaluronate were added sequentially to the obtained recombinant type III collagen solution. The temperature was maintained at 25~35℃, the rotation speed was adjusted to 800~2000 r / min, and the mixture was stirred for 60~120 min to form a uniform composite dispersion. Introduction of moisturizing and antiseptic system: Add the prescribed amount of glycerol and phenoxyethanol to the obtained composite dispersion, stir at 800~1500 r / min for 30~45 min, stir evenly, and then filter with a filter membrane to remove impurities and bubbles. Sterilization and shaping: The obtained filtrate is placed in a sterile environment, cooled to room temperature, and dispensed into sterile containers to obtain the recombinant humanized collagen dressing containing sodium polyphosphate.

[0015] As a preferred embodiment of the preparation method described in this invention, the nitrogen protection time is 10-20 min to maintain the hypoxic environment of the system and prevent the oxidative degradation of recombinant type III collagen.

[0016] As a preferred embodiment of the preparation method described in this invention, the interval between adding sodium polyphosphate and sodium hyaluronate sequentially is 5-15 min. Sodium polyphosphate is added first to pre-disperse it in the solution, and then sodium hyaluronate is added to avoid clumping and ensure the uniformity of the compound system.

[0017] Another objective of this invention is to overcome the shortcomings of the prior art and provide a recombinant humanized collagen dressing containing sodium polyphosphate, or the application of the recombinant humanized collagen dressing containing sodium polyphosphate prepared by the above-mentioned preparation method in the preparation of cosmetics, medical devices or pharmaceuticals for skin repair, wound healing, post-medical aesthetic care or anti-aging and moisturizing.

[0018] Beneficial effects of this invention: (1) Significantly improved moisturizing durability: Through the three-dimensional water-locking network of sodium polyphosphate and the synergistic moisturizing effect of sodium hyaluronate and glycerin, the wound can maintain a moist environment for a long time, especially suitable for dry wounds and wounds with less exudate, avoiding scab formation and delayed healing, and improving the quality of wound healing.

[0019] (2) High wound repair efficiency and wide range of applications: Sodium polyphosphate can promote wound healing through multiple pathways. It can work synergistically with recombinant type III collagen and sodium hyaluronate to not only accelerate the healing of acute wounds, but also effectively improve the healing microenvironment of chronic wounds, enhance the repair rate of chronic wounds, and shorten the healing cycle. It can be widely used for various acute and chronic wounds and has strong clinical applicability.

[0020] (3) Excellent biological safety with no potential risks: The dressing has been verified by systematic biological evaluation such as cytotoxicity test, sensitization test and acute systemic toxicity test. It has no cytotoxicity, no sensitization reaction and no acute systemic toxicity, and has excellent biocompatibility. No cross-linking agent, penetration enhancer or other potentially harmful ingredients have been added. The degradation products of sodium polyphosphate have no residual toxicity and can be safely applied to various wounds, especially suitable for sensitive skin and long-term use. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1The results are from the cytotoxicity test in Example 2 of this invention.

[0022] Figure 2 This is the microscope comparison result in Example 8 of the present invention.

[0023] Figure 3 This is a comparison of cell migration rates in Example 8 of the present invention. Detailed Implementation

[0024] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0025] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0026] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0027] Raw materials used in this invention: Recombinant type III collagen, Zhejiang Zhuji Juyuan Biotechnology Co., Ltd., Yuelaixin recombinant type III collagen, model SSH01; Sodium hyaluronate, Shandong Zhongshan Biotechnology Co., Ltd., batch number 04HA01250204, molecular weight 1.51×10⁻⁶. 6 Da; Phenoxyethanol, Jiangxi Ipsen Pharmaceutical Co., Ltd.; Glycerin, Hunan Ercon Pharmaceutical Co., Ltd. The preparation method of sodium polyphosphate in this embodiment of the invention refers to the preparation method of Example 1 in CN117025688A.

[0028] The instrument used in this embodiment of the invention is a Merck Millipore Simplicity® UV ultrapure water system, product number SIMS V00 01.

[0029] Example 1 This embodiment provides a method for preparing a recombinant humanized collagen dressing containing sodium polyphosphate, specifically including the following steps: (1) Weigh the raw materials according to the following formula, by mass percentage: 0.30% recombinant type III collagen; 0.30% sodium hyaluronate; 0.30% sodium polyphosphate; 0.40% phenoxyethanol; 1.50% glycerin; the remainder is purified water.

[0030] Measure 97.20g of purified water and transfer it all to a sterile temperature-controlled stirring tank. Turn on the temperature control function and stabilize the temperature of the purified water at 30±2℃. Introduce sterile nitrogen into the purified water for 10 minutes to create a nitrogen-protected atmosphere throughout the process, which will isolate oxygen and prevent collagen from being oxidized and deactivated. Set aside for later use. Maintain the temperature of the mixing tank at 30±2℃, turn on the stirrer, and keep the speed stable at 800r / min to form a stable vortex without violent splashing. Add the weighed 0.30g of recombinant type III collagen slowly in three batches, with a 5-minute interval between each addition, to avoid the protein powder clumping together and failing to dissolve completely due to a single addition. After all materials have been added, maintain a speed of 800 r / min and a temperature of 30±2℃, and continue stirring for 45 minutes. After stirring, check the state of the solution with a light to confirm that the protein is completely dissolved, the solution is clear, without visible flocculent matter or lumps, and thus the recombinant type III collagen solution is obtained.

[0031] (2) Keep the temperature of the mixing tank at 30±2℃. First, add 0.30g of sodium polyphosphate (average chain length 40≤n≤60) to the collagen solution and stir at 800r / min for 20min to confirm that the powder is completely dissolved and the solution is clear. Adjust the stirring speed to 1200r / min, and slowly sprinkle 0.30g of sodium hyaluronate in 4 to 5 batches. Stir for 15 minutes after each addition, and wait until there are no visible particles before adding the next batch. After all the sodium hyaluronate has been added, maintain a speed of 1200 r / min and a temperature of 30±2℃, and continue stirring for 90 min; After stirring, a light inspection was conducted to confirm that the system was a uniform and clear dispersion, free of particles, lumps, and stratification, thus completing the preparation of the composite system.

[0032] (3) Adjust the stirring speed to 1000 r / min, maintain the temperature at 30±2℃, add 1.50 g of glycerin, and stir for 10 min until completely mixed; Add another 0.40g of phenoxyethanol and stir continuously for 35 minutes to ensure that the preservative is completely dissolved and the system is homogeneous and stable. After stirring, accurately weigh the total weight of the system using an electronic balance. If there is a weight deviation due to stirring and evaporation, add the weight to 100.00g with the reserved purified water. After adding the weight, continue stirring for 5 minutes to mix thoroughly. A filtration device with a 0.22μm PES sterile filter membrane was assembled under sterile conditions. After pre-wetting the filter membrane with 10m of sterile purified water, the system solution was slowly poured into the filter and the filtrate was collected into a sterile storage bottle to thoroughly remove trace impurities, insoluble matter and air bubbles.

[0033] (4) Because recombinant type III collagen is sensitive to high temperature, high temperature sterilization will lead to protein degradation and loss of activity. This product adopts a sterile protection process of sterile filtration + sterile filling. The recombinant type III collagen filtrate obtained in step (1) has been terminally sterilized and can be directly used for filling. In the local Class 100 laminar flow environment of the Class 10,000 clean area, the sterile filtrate is dispensed into pre-sterilized sterile vials, and immediately sealed and capped to complete the sterile filling. Each bottle of finished product is inspected by light to confirm that the solution is clear, free of foreign matter, and without leakage, thus obtaining the finished product of sodium polyphosphate recombinant human collagen liquid dressing.

[0034] Example 2 This embodiment describes a cytotoxicity test of the recombinant collagen liquid dressing (polyphosphate) prepared in Example 1, specifically including the following steps: The test sample was a filtered, sterilized, and aseptically filled recombinant collagen liquid dressing (polyphosphate).

[0035] The experimental system used L-929 mouse fibroblasts (CTCC source) recommended by the standard, and selected cells that were passaged for 48-72 hours and were growing vigorously; all instruments and consumables used met the requirements for sterility testing.

[0036] Sample and control preparation: Under aseptic conditions, extract samples (sample:extraction medium) in sealed, inert containers according to the methods and proportions in Table 1. The extraction medium was MEM complete medium containing 10% fetal bovine serum. The extraction process was carried out in a full-temperature shaking incubator. The shaking was continuous at 60 rpm. The control group was prepared under the same conditions.

[0037] The state of the extract showed no visible change before and after leaching, and the extract was used for testing within 24 hours after leaching. The extract was not subjected to pH adjustment, filtration, centrifugation, or dilution before use. There were no changes in particulate matter, color, or clarity in the extract before and after leaching.

[0038] Prepare L-929 single-cell suspension and adjust cell density to 1×10⁻⁶. 5 Cells per mL were seeded into 96-well culture plates and incubated at 37°C in a 5% CO2 incubator for 24-26 hours to form a nearly confluent monolayer. Only cells that showed healthy and uniform growth under a microscope were used for experiments.

[0039] Remove the original culture medium from the wells and add 100 μL of sample extracts of different concentrations (25%, 50%, 75%, and 100% in four gradients) and extracts of each control, with 6 replicates per group (Table 1). Incubate the culture plates in a 37°C, 5% CO2 incubator for 24 ± 0.5 h and record the changes in cell morphology after incubation under a microscope.

[0040] Remove the supernatant from the wells, add 50 μL of freshly prepared 1 mg / mL MTT solution to each well, and incubate in the dark for 2 h; after removing the MTT solution, add 100 μL of isopropanol to each well and shake until the MTT crystals are completely dissolved; use a microplate reader to read the absorbance OD value of each well with 570 nm as the detection wavelength and 650 nm as the reference wavelength.

[0041] Data calculation: Summarize the OD values ​​of each well, calculate the mean ± standard deviation of absorbance for each group, and calculate the cell survival rate for each concentration group according to the formula: Cell survival rate = (OD difference of sample / control group / OD difference of blank control group) × 100%.

[0042] The results of this experiment are as follows Figure 1 As shown, the cell viability was greater than 80%, and there was no cytotoxicity.

[0043] Example 3 This embodiment involves an acute systemic toxicity test on the recombinant collagen liquid dressing (polyphosphate) prepared in Example 1, specifically including the following steps: The test sample was a filtered, sterilized, and aseptically filled recombinant collagen liquid dressing (polyphosphate), and the undiluted solution was used directly in the test; the negative control was 0.9% sodium chloride injection.

[0044] The experimental animals were SPF-grade ICR mice, female, non-pregnant and nulliparous, 3-4 weeks old, with an initial weight of 17-23g, totaling 10 mice, which were acclimatized for 5 days; they were randomly divided into experimental group and negative control group, with 5 mice in each group, and were marked by ear tagging.

[0045] Before the drug administration experiment, mice were identified and weighed. Two groups of mice were administered the drug via intraperitoneal injection: the experimental group was injected with the original sample solution, and the negative control group was injected with 0.9% sodium chloride injection. The dosage for both groups was 50.0 mL / kg body weight. The immediate response of the mice was observed immediately after the injection.

[0046] The systemic toxicity of mice was observed and recorded at four time points: 4h, 24h, 48h, and 72h after administration. The toxicity covered multiple systems, including respiratory, central nervous, muscle movement, cardiovascular, ocular mucosa, skin and hair, and gastrointestinal function. The number of animal deaths was recorded simultaneously.

[0047] The mice were weighed at 24h, 48h and 72h after administration, and the weight changes were recorded.

[0048] Table 1 Clinical observations in mice

[0049] Note: Record the abnormal situation when there is an abnormality, and mark "√" when there is no abnormality.

[0050] Table 2. Changes in mouse body weight (g)

[0051] The experimental results are as follows: Clinical observation: The observation results for each animal are shown in Table 1.

[0052] Weight changes: Table 2 shows the weight changes of mice after exposure to the virus. The weight data of the animals in this experiment were within an acceptable range.

[0053] Deaths: No animals died during the experiment.

[0054] Therefore, the sample meets the requirements and has no systemic toxicity.

[0055] Example 4 This embodiment involves a skin sensitization test on the recombinant collagen liquid dressing (polyphosphate) prepared in Example 1, specifically including the following steps: The test product was a recombinant collagen gel dressing, which was not treated before the test. A negative control group (extraction medium) and a positive control group (2,4-dinitrochlorobenzene, which was tested every 3 months to verify the effectiveness of the test system) were set up simultaneously.

[0056] The experimental animals were SPF-grade Hartley albino male guinea pigs, 6-8 weeks old, with an initial weight of 300-500g, and acclimatized for 7 days; a total of 15 animals were used, including 10 experimental group and 5 negative control group. They were marked by ear tagging. Animal husbandry and welfare met the requirements of GB / T 16886.2-2011.

[0057] Hair was removed from the backs of guinea pigs. The experimental group was injected intradermally with a mixture of the test formulation and Freund's complete adjuvant, while the negative control group was injected simultaneously with the corresponding control and adjuvant, thus completing the first sensitization induction.

[0058] On the 7th day after intradermal induction, the hair at the induction site was removed again. The experimental group was covered with the test product patch for (24±2)h to complete the second induction. The control group underwent the same procedure.

[0059] On day 14 after local induction, test / control patches were applied to the hairless area of ​​the abdomen of guinea pigs that had not been exposed to the test product for 24±2 h to simulate a clinical skin contact scenario.

[0060] At 24 and 48 hours after removal of the stimulation patch, the skin erythema and edema reactions were graded and scored according to the Magnusson and Kligman grading system; the animals' clinical signs and weight changes were monitored throughout the process.

[0061] Table 3 Clinical observations and weight changes in guinea pigs

[0062] Table 4. Allergic Skin Reactions in Guinea Pigs

[0063] Table 5 Skin reactions in the positive control group

[0064] The experimental results are as follows: Clinical observation: Clinical observations and weight changes for each animal are shown in Table 3. No abnormal reactions were observed in any animal other than the expected skin reaction induced by intradermal injection of Freund's complete adjuvant. All animals remained clinically normal throughout the experiment.

[0065] As shown in Table 4, the positive rate of sensitization reaction in the sample group guinea pigs was 0%, and the positive rate of sensitization reaction in the negative control group guinea pigs was 0%.

[0066] The skin reaction scores during the stimulation phase are shown in Table 5.

[0067] Under the conditions of this experiment, the original sample solution did not cause skin sensitization reactions in guinea pigs, and the tested sample met the experimental requirements.

[0068] Example 5 This embodiment involves an intradermal irritation test on the recombinant collagen liquid dressing (polyphosphate) prepared in Example 1, specifically including the following steps: Three healthy, young adult female New Zealand rabbits (non-pregnant and nulliparous) with an initial weight of >2kg were selected as experimental animals. They were acclimatized for 7 days and numbered using ink markings on the inside of their ears.

[0069] Four hours before the experiment, the hair on both sides of the spine on the back of the experimental rabbits was removed to demarcate the injection areas. Sample stock solution was injected intradermally at 0.2 mL at 5 sites on the left side of the spine on the back of each rabbit; negative control solution was injected intradermally at 0.2 mL at 5 sites on the right side of the back, achieving left-right control within the same animal.

[0070] After injection, the erythema and edema reactions at the injection site were observed and recorded at three time points: 24h, 48h, and 72h, according to the standard grading table, and a primary stimulation score of 0-4 was given accordingly.

[0071] The total scores of the three rabbits at three time points and five injection sites were summarized. The average score S1 of the sample group and the average score S2 of the negative control group were calculated respectively. The final score of the sample was S1-S2.

[0072] The final score of the intradermal reaction of the sample group was less than 1.00, indicating that the sample met the test requirements and had no intradermal irritation.

[0073] Example 6 This embodiment involves pyrogen testing of the recombinant collagen liquid dressing (polyphosphate) prepared in Example 1, specifically including the following steps: The test sample was a filtered, sterilized, and aseptically filled recombinant collagen liquid dressing (polyphosphate), which was simultaneously used as an extraction medium with pyrogen-free 0.9% sodium chloride injection. All instruments and consumables used were of pyrogen-free specifications.

[0074] Three healthy, young adult female New Zealand rabbits (non-pregnant and nulliparous) with an initial weight ≥2.0 kg were selected as experimental animals. They were acclimatized for 7 days and numbered using ink markings on the inside of their ears.

[0075] The sample extract was prepared according to GB / T 16886.12-2023. 20.3g of sample was randomly selected and 101.5mL of extract was added at a ratio of 0.2g sample to 1mL medium using 0.9% sodium chloride injection. The extract was then stirred at 37℃ and 60rpm for 72h. The resulting extract was colorless, clear, and free of particulate matter. It was to be used within 24h after preparation, without pH adjustment, filtration, dilution, or other additional treatments before use.

[0076] Before the experiment, the rabbits were fasted and deprived of water, and placed in a fixed box for ≥1 hour. A calibrated temperature probe was inserted, and the body temperature was measured every 30 minutes for two consecutive times. The difference between the two body temperatures was ≤0.2℃ to be considered qualified, and the average of the two measurements was taken as the normal body temperature of the rabbit.

[0077] Within 15 minutes after the normal body temperature was measured, the sample extract, which had been preheated to 37±2℃, was administered via a marginal ear vein in rabbits at a dose of 10 mL / kg.

[0078] After administration, the rabbits' body temperature was measured every 30 minutes for a total of 6 times. The temperature changes of each rabbit were recorded, and the maximum temperature increase of a single rabbit was calculated. The experimental results are shown in Table 6.

[0079] Table 6

[0080] Table 6 shows the pyrogen test results, indicating no deviation from the protocol throughout the experiment. The individual body temperature increases of the three rabbits were 0.10℃, 0.07℃, and 0.13℃, respectively, all below 0.6℃, with a total increase of 0.30℃, far below the compliance threshold of 1.3℃. Therefore, the pyrogen test results of the samples meet the requirements. This result clearly demonstrates that the extract of the test sample does not contain pyrogenic substances and meets the pyrogen safety requirements for medical biomaterials.

[0081] Comparative Example 1 The difference between this comparative example and Example 1 is that sodium polyphosphate in step (1) is omitted, while the remaining steps are the same as in Example 1, to obtain recombinant humanized collagen dressing.

[0082] Comparative Example 2 The difference between this comparative example and Example 1 is that sodium polyphosphate in step (1) is replaced with sodium hexametaphosphate (commercially available, CAS: 10124-56-8), and the remaining steps are the same as in Example 1, to obtain recombinant humanized collagen dressing.

[0083] Comparative Example 3 The difference between this comparative example and Example 1 is that sodium polyphosphate in step (1) is replaced with sodium polyphosphate (commercially available, CAS: 68915-31-1, average chain length n < 20), and the remaining steps are the same as in Example 1, to obtain recombinant humanized collagen dressing.

[0084] Example 7 The difference between this embodiment and Embodiment 1 is that the average chain length of sodium polyphosphate in step (1) is replaced with 60 < n ≤ 80, and the remaining steps are the same as in Embodiment 1, to obtain recombinant humanized collagen dressing.

[0085] Example 8 This embodiment is a comparative experiment on the recombinant humanized collagen dressings prepared in Examples 1 and 7 and Comparative Examples 1-3, and specifically includes the following steps: Revive the frozen target cells, culture them in complete medium to the logarithmic growth phase (70%-80% confluence), and passage them 1-2 times to ensure cell viability ≥90%.

[0086] Preparation of material extract: Following the standard GB / T 16886.12, extract at 37℃ and 5% CO2 for 24h at a ratio of 2g sample + 10mL serum-free DMEM. After sterilization by filtration through a 0.22μm filter membrane, store at 4℃ for no more than 72h.

[0087] Take cells in the logarithmic growth phase, digest them with 0.25% trypsin-EDTA for 2-3 minutes, add complete culture medium to stop digestion, and gently pipette to prepare a single-cell suspension.

[0088] Collect cells by centrifugation at 1000 rpm for 5 min, discard the supernatant, resuspend in complete culture medium, count the cells, and adjust the cell concentration to 2 × 10⁻⁶. 5 cells / mL.

[0089] Add 2 mL of cell suspension to each well of the plate, shake gently, and incubate at 37°C with 5% CO2 for 24-48 hours until the cell confluence reaches 90%-100% and a dense monolayer is formed.

[0090] Aspirate the supernatant from the well and gently rinse twice with sterile PBS to remove floating cells and residual serum.

[0091] Using a sterile 200μL pipette tip, make three parallel scratches (spaced ≥0.5cm) perpendicular to the bottom of the well, ensuring the scratches penetrate the bottom of the well with consistent pressure to avoid damaging the substrate at the bottom of the well.

[0092] Rinse gently with sterile PBS 2-3 times to thoroughly remove cell debris caused by the scratches, and add 2 mL of the corresponding serum-free culture medium or extract according to the group.

[0093] Immediately (0h), place the 6-well plate under an inverted microscope, take a picture with a 4× objective lens, mark 3 fixed fields of view (both ends of the scratch + the middle) for each well and record the position.

[0094] Images were taken from the same marked field of view at 6h, 12h, 24h, and 48h of incubation. After taking a picture at the last time point, the width of the scratches in each field of view was measured using ImageJ software (the average of 3 locations was taken), and the cell migration rate was calculated as: (scratch area at 0h - scratch area at each time point) / scratch area at 0h × 100%.

[0095] One-way ANOVA was performed using GraphPad Prism, and Tukey's test was used for comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0096] The results are as follows Figure 2 , 3 As shown, sodium polyphosphate in Examples 1 and 7 has the effect of promoting cell migration and can promote skin repair.

[0097] The purpose of this invention is to overcome the technical shortcomings of existing recombinant collagen wound dressings, such as poor moisturizing durability, slow wound repair rate, insufficient system stability, incomplete biological safety, and limited application of sodium polyphosphate. This invention provides a sodium polyphosphate recombinant human collagen dressing, its preparation method, and its applications. This dressing uses sodium polyphosphate as its core innovation. Through optimized formulation and preparation process, it achieves a synergistic effect between sodium polyphosphate, recombinant type III collagen, and sodium hyaluronate. It possesses long-lasting moisturizing properties, highly effective wound healing promotion, excellent biocompatibility, and good system stability. Furthermore, the preparation process is simple, suitable for large-scale production, and can be widely used in the repair and treatment of various skin wounds.

[0098] This invention breaks through the limitation of existing technologies in which sodium polyphosphate is only used as an auxiliary stabilizer. For the first time, it is used as a core functional component of wound repair dressings. It fully explores the physicochemical properties and bioactivity of its linear polyphosphate groups, enabling it to simultaneously perform three core functions: long-lasting moisturizing, system stability, and active wound healing promotion. This fills the technological gap in the core functional application of sodium polyphosphate in recombinant collagen dressings.

[0099] This invention precisely constructs a ternary synergistic system of recombinant type III humanized collagen, sodium polyphosphate, and high molecular weight sodium hyaluronate, clearly defining the optimal quality ratio range and strict quality control standards for each component: high-purity recombinant type III humanized collagen serves as the repair core, providing a cell adhesion scaffold and regulating collagen synthesis; sodium polyphosphate with a specific degree of polymerization serves as the functional core, achieving a water-locking and stabilizing system and actively regulating cell behavior and inflammatory responses; and high molecular weight sodium hyaluronate serves as the core of the moisturizing barrier, strengthening water retention and wound protection, systematically solving multiple technical pain points of existing dressings.

[0100] This invention sequentially completes collagen dissolution, complex system construction, and moisturizing and preservative system introduction according to the activity gradient of raw materials. It precisely controls the stirring speed, temperature, and reaction time, and uses a 0.22μm filter membrane for filtration to remove bacteria and impurities. The entire process involves no high-temperature and high-pressure reactions or chemical cross-linking treatments, maximizing the preservation of the spatial structure and physiological functions of bioactive components. The process steps are simple, the parameters are highly controllable, the production cost is low, and it can be directly adapted to large-scale industrial production.

[0101] This invention achieves a comprehensive breakthrough in dressing performance through synergistic optimization of formulation and process: First, it provides long-lasting moisturizing and water-locking, constructing a three-dimensional hydrophilic network to maintain a moist environment on the wound surface for an extended period, perfectly suited for dry wounds with low exudation; Second, it promotes efficient healing throughout the entire healing cycle, regulating cell proliferation, promoting cytokine secretion, and accelerating extracellular matrix deposition and angiogenesis through multiple pathways, catering to both acute and chronic, difficult-to-heal wounds, and significantly shortening the healing cycle; Third, the system is stable over a long period, effectively inhibiting the degradation and aggregation of collagen and sodium hyaluronate through the metal ion chelation and dispersion effects of sodium polyphosphate, preventing product stratification, thinning, and precipitation, and significantly extending shelf life; Fourth, it is biosafe throughout the entire chain, without adding any cross-linking agents, penetration enhancers, or other potentially harmful ingredients. The degradation products of sodium polyphosphate can completely participate in human metabolism without residue. Systematic biological evaluation has verified that it has no cytotoxicity, no sensitization reaction, and no acute systemic toxicity, making it safe for use on sensitive skin and for long-term wound repair.

[0102] The dressing of this invention can be applied directly to various skin wounds, without being limited by the location or shape of the wound. It can be widely used in the repair and treatment of acute wounds caused by mechanical, physical, chemical, and biological factors, as well as chronic, difficult-to-heal wounds caused by metabolic abnormalities, blood circulation disorders, and other factors. It has strong clinical applicability and extremely high clinical translational value and market promotion prospects.

[0103] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A recombinant humanized collagen dressing containing sodium polyphosphate, characterized in that: Composed of the following raw materials by mass percentage: Recombinant type III collagen 0.05%~2.0%; Sodium hyaluronate 0.1%~3.0%; Sodium polyphosphate 0.05%~2.0%; Phenoxyethanol 0.3%~1.0%; Glycerin 1.0%~10.0%; The remainder is purified water.

2. The recombinant humanized collagen dressing as described in claim 1, characterized in that: The recombinant type III collagen has a weight-average molecular weight of 5,000 to 100,000 Da, a purity of ≥95%, and is free of animal-derived impurities.

3. The recombinant humanized collagen dressing as described in claim 2, characterized in that: The sodium polyphosphate has an average chain length of n≥20, a pH value of 6.5~8.0, and a water solubility of ≥99%.

4. The recombinant humanized collagen dressing as described in claim 3, characterized in that: The sodium hyaluronate is a high molecular weight sodium hyaluronate with a weight-average molecular weight of 1,500,000 Da.

5. The recombinant humanized collagen dressing as described in claim 4, characterized in that: The purified water is medical-grade purified water with a conductivity ≤1.0 μS / cm.

6. The method for preparing the recombinant humanized collagen dressing containing sodium polyphosphate as described in any one of claims 1 to 5, characterized in that: include, Place purified water in a sterile stirring tank, adjust the temperature to 25~35℃, purge with nitrogen for protection, and set aside. Take the prescribed amount of recombinant type III collagen and slowly add it to pretreated purified water. Stir until completely dissolved to obtain a recombinant type III collagen solution. Add sodium polyphosphate and sodium hyaluronate sequentially to the obtained recombinant type III collagen solution, maintain the temperature at 25~35℃, adjust the rotation speed to 800~2000 r / min, and stir for 60~120 min to form a uniform composite dispersion. Add the prescribed amounts of glycerol and phenoxyethanol to the obtained composite dispersion, stir at 800~1500 r / min for 30~45 min, and after stirring evenly, filter with a filter membrane to remove impurities and bubbles. The obtained filtrate was placed in a sterile environment, cooled to room temperature, and dispensed into sterile containers to obtain the recombinant humanized collagen dressing containing sodium polyphosphate.

7. The preparation method according to claim 6, characterized in that: The nitrogen protection time is 10-20 minutes.

8. The preparation method according to claim 6, characterized in that: The interval between adding sodium polyphosphate and sodium hyaluronate sequentially is 5 to 15 minutes.

9. The preparation method according to claim 6, characterized in that: The mixture is stirred until completely dissolved to obtain a recombinant type III collagen solution, wherein the stirring speed is 500~1500 r / min and the stirring time is 30~60 min.

10. The use of the sodium polyphosphate-containing recombinant humanized collagen dressing as described in any one of claims 1 to 5 in the preparation of cosmetics, medical devices, or pharmaceuticals for skin repair, wound healing, post-medical aesthetic care, or anti-aging and moisturizing.

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