Umbilical cord extract-containing skin tissue injury repair composition as well as preparation method and application thereof

Abstract

The invention discloses a skin tissue injury repairing composition containing an umbilical cord extract as well as a preparation method and application of the skin tissue injury repairing composition. The composition provided by the invention comprises the umbilical cord extract, the silk fibroin, the gamma-PGA and the antioxidant, has a synergistic effect by accurately controlling the component proportion, can promote cell proliferation and increase collagen secretion, and has a remarkable skin tissue injury repair effect; furthermore, an enzymolysis process of active ingredients of the composition is optimized, so that the yield and the purity of the umbilical cord extract are improved, and the repairing effect of the composition is also improved; meanwhile, by preparing the PLGA nano drug-loaded particles, the stability of the composition is improved, stable delivery and long-acting release of the drug are achieved, efficient transdermal absorption can be achieved, a better skin injury repairing effect is achieved, the advantages of being simple in preparation process, high in stability, remarkable in repairing effect and the like are achieved, and the PLGA nano drug-loaded particles can be used for preparing more efficient drugs; the ointment is suitable for treating various skin injuries such as burns, wounds, ulcers and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. More specifically, it relates to a skin tissue injury repair composition containing umbilical cord extract, its preparation method and application. Background Art

[0002] As the largest organ of the human body, the skin has important functions such as protecting the body, regulating body temperature, and perceiving external stimuli. Skin injuries (such as burns, wounds, ulcers, etc.) not only affect appearance but may also lead to infections, scar formation, and even dysfunction. Traditional skin injury repair methods include surgical operations, dressing coverage, drug treatment, etc., but these methods have problems such as a long repair cycle, a high scar formation rate, and susceptibility to infection. In recent years, with the development of medicine, the application of stem cells and their derivatives in tissue repair has attracted much attention. Among them, umbilical cord extracts (such as umbilical cord mesenchymal stem cells, cord blood, Wharton's jelly of the umbilical cord, etc.) are rich in various growth factors (such as EGF, VEGF, FGF, etc.), extracellular matrix components (such as collagen, hyaluronic acid, etc.), and immunomodulatory factors, and have the effects of promoting cell proliferation, migration, differentiation, and angiogenesis, and are considered ideal skin repair materials.

[0003] However, the use of umbilical cord extracts in the prior art has the following problems: First, the extraction process is complex and not unified. The existing preparation methods of umbilical cord extracts are multi-step and cumbersome, and the yield and purity of the extracts are low, and the extraction efficiency is low. Second, the stability of umbilical cord extracts is poor, and the active ingredients are easily inactivated and difficult to store for a long time. Finally, the existing administration methods of umbilical cord extracts are single, and traditional administration methods (such as topical application, injection) are difficult to achieve stable delivery, which affects the repair effect. And the prior art still lacks umbilical cord extracts or combinations with high repair effects, and there are few in-depth studies on the synergistic effects of umbilical cord extracts and other components, resulting in limited repair effects. For example, the composite gel of umbilical cord exosome MSCs and collagen disclosed in the existing research can be used for skin burn repair; the freeze-dried umbilical cord MSCs conditioned medium can be used to promote the migration of keratinocytes, but its preparation method conditions are relatively harsh and complex, and the repair effect also needs to be improved.

[0004] Therefore, developing an efficient, stable, and innovative composition containing umbilical cord extract and its delivery products has important clinical application value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the existing umbilical cord extracts and their products, as well as the deficiency of lacking products for efficiently repairing skin tissue injuries, and aims to provide a skin tissue injury repair composition containing umbilical cord extract, which significantly improves the skin injury repair effect; this composition has the advantages of simple preparation process, high stability, and remarkable repair effect.

[0006] The first object of the present invention is to provide a composition for repairing skin tissue damage.

[0007] The second object of the present invention is to provide a preparation method of the composition for repairing skin tissue damage.

[0008] The third object of the present invention is to provide an application of the composition for repairing skin tissue damage.

[0009] The fourth object of the present invention is to provide a product.

[0010] The fifth object of the present invention is to provide a PLGA nano-drug-loaded particle and its preparation method.

[0011] The above objects of the present invention are achieved by the following technical solutions: The present invention provides a composition for repairing skin tissue damage, comprising components in the following mass ratio: umbilical cord extract: silk fibroin: γ-PGA: antioxidant = (40 - 60):(10 - 25):(20 - 30):(2 - 8).

[0012] The composition for repairing skin tissue damage provided by the present invention, through scientific combination ratio adjustment, has a synergistic effect among the components, can promote cell proliferation and increase collagen secretion, and has a significant effect on repairing skin tissue damage. Its effect is superior to that of using umbilical cord extract alone, or any pairwise combination or three-component combination of its components. The active ingredients of the umbilical cord extract in its components are cytokines and extracellular matrix, which can promote wound healing; silk fibroin has high mechanical strength and excellent biocompatibility, supports cell proliferation and migration, and its degradation products are non-toxic; γ-PGA (γ-polyglutamic acid) has super moisturizing ability (the water retention capacity is 10 times that of hyaluronic acid), can promote cell proliferation, and has a significant anti-inflammatory effect; the antioxidant can reduce oxidative stress damage and maintain the biological activity of the composition. The present invention has systematically verified the significant synergistic effect of this composition in promoting wound healing, tissue and inflammation relief through cell model experiments and burn and scald animal model experiments, providing more and better products for the treatment of various skin injuries.

[0013] Preferably, the composition comprises components in the following mass ratio: umbilical cord extract: silk fibroin: γ-PGA: antioxidant = (45 - 55):(18 - 22):(23 - 27):(4 - 6).

[0014] Preferably, the antioxidant is selected from glutathione, vitamin C or coenzyme Q10.

[0015] More preferably, the antioxidant is glutathione.

[0016] By optimizing the enzymatic hydrolysis process (simultaneous enzymatic hydrolysis with type II collagenase + trypsin), the present invention further improves the yield (≥53%) and purity (hydroxyproline ≥84 mg / g) of umbilical cord extract, thereby enhancing the repair effect of the composition; its preparation method is simple and convenient, improving the extraction efficiency and effect of umbilical cord extract, and providing a better raw material source for skin injury repair products.

[0017] Therefore, the present invention provides a method for preparing umbilical cord extract: After crushing the umbilical cord, enzymatic hydrolysis is carried out. The enzymatic hydrolysis conditions are 0.25 - 0.35% type II collagenase and 0.12 - 0.18% trypsin, pH value 7.2 - 7.6, temperature 42 ± 1°C, stirring rate 200 - 300 rpm, and time 2.5 - 3.5 hours. After enzymatic hydrolysis, ultrafiltration concentration and freeze-drying are carried out to obtain umbilical cord extract.

[0018] As a more preferred embodiment, the present invention provides a specific method for preparing umbilical cord extract: (1) Umbilical cord collection and pretreatment: Use the umbilical cord tissue of healthy full-term newborn calves, ensure no infection or lesions, and perform subsequent operations after passing various microbial and virus series index tests before use. Place the umbilical cord in physiological saline at 4°C and complete the treatment within 24 hours to avoid degradation of active ingredients. Rinse the umbilical cord repeatedly with PBS buffer containing 1% penicillin-streptomycin to remove blood and impurities.

[0019] (2) Tissue cutting and enzymatic digestion: Cut the umbilical cord into small segments of 1 - 2 cm, quickly freeze in liquid nitrogen and mechanically crush to particles of 0.5 - 1 mm3. Use 0.3% type II collagenase + 0.15% trypsin, PBS solution with pH 7.4, containing 2 mM CaCl2, and perform simultaneous enzymatic hydrolysis in a constant temperature water bath at 42°C for 3 h with magnetic stirring at 250 rpm. Terminate the reaction, cool in a 4°C bath for 10 minutes, centrifuge at 8000 rpm for 15 minutes, and take the supernatant.

[0020] (3) Purification, concentration and freeze-drying: The supernatant is concentrated by an ultrafiltration system (10 kDa membrane), with a transmembrane pressure of 0.2 MPa and a flow rate of 10 mL / min. Concentrate the supernatant to 1 / 10 of the original volume to obtain a high-concentration and high-purity umbilical cord extract solution, and obtain a white powder after vacuum freeze-drying.

[0021] The present invention also provides a method for preparing the above composition, comprising the following steps: S1: Solution preparation: Prepare solutions of umbilical cord extract, silk fibroin, γ-PGA, and antioxidant respectively and set aside; S2: Premixing of silk fibroin and γ-PGA: Mix silk fibroin and γ-PGA according to the mass ratio, at a temperature of 25 - 30°C, stir at 200 - 300 rpm for 20 - 40 minutes, and adjust the pH of the mixed solution to 6.5 - 7.0; S3: Add umbilical cord extract: Slowly drop the umbilical cord extract at a dropping rate of 0.8 - 1 mL / min, while stirring synchronously at 100 - 200 rpm, at a temperature of 25 - 30 °C to avoid precipitation; S4: Add antioxidant: Add the antioxidant under light - proof conditions, ultrasonically disperse at 30 - 40 kHz for 3 - 5 minutes, at a temperature of 25 - 30 °C, and filter - sterilize the final mixture to obtain the product.

[0022] Preferably, in S1, dissolve the umbilical cord extract in physiological saline (0.9% NaCl), and ultrasonically assist the dissolution (40 kHz, 5 minutes) to obtain the umbilical cord extract solution; dissolve silk fibroin (molecular weight 30 kDa, β - sheet structure proportion ≥70%) in deionized water, and magnetically stir at a constant temperature of 60 °C for 2 hours until it is transparent and particle - free to obtain the silk fibroin solution; dissolve γ - PGA (molecular weight 15 - 20 kDa) in pH 6.0 phosphate buffer (0.01 M) and magnetically stir for 30 minutes to obtain the γ - PGA solution; dissolve the antioxidant in sterile injection water and prepare it under light - proof conditions to obtain the antioxidant solution.

[0023] Preferably, the pre - mixing conditions in S2 are: 300 rpm, 30 minutes, at a temperature of 25 °C.

[0024] Furthermore, finely adjust the pH with 0.1 M NaOH or HCl.

[0025] Preferably, the dropping rate of the umbilical cord extract solution in S3 is 1 mL / min.

[0026] Preferably, the ultrasonic dispersion conditions in S4 are 40 kHz, 5 minutes, at a temperature of 25 °C.

[0027] Preferably, in S4, filter - sterilize the mixture through a 0.22 - μm filter membrane.

[0028] The present invention provides the application of the above - mentioned composition in the preparation of products for skin injury repair.

[0029] The present invention provides the application of the above - mentioned composition in the preparation of drugs for treating skin injuries.

[0030] The present invention provides the application of the above - mentioned composition in maintaining the skin barrier and / or promoting the secretion of skin collagen.

[0031] Preferably, the skin injury repair includes acute trauma repair, chronic wound treatment, scar repair, skin burn and scald treatment, and soothing repair of sensitive and diseased skin.

[0032] The present invention also provides a product containing the above - mentioned composition.

[0033] Meanwhile, through the nano-carrier delivery system, the present invention encapsulates the skin tissue injury repair composition with poly(lactic-co-glycolic acid) (PLGA) to prepare PLGA nano-drug-loaded particles, which achieve the stable release and long-term effect of the active ingredients of the composition, and its stability is also significantly improved (the activity retention is >95% after 36 months of storage at room temperature). Through the innovative drug delivery method of a specific nano-drug delivery system, the loading rate, stability, stable slow release and repair effect of the umbilical cord extract are further improved. This nano-drug delivery system is applicable to the treatment of various skin injuries such as burns, wounds and ulcers.

[0034] The present invention also provides a kind of PLGA nano-drug-loaded particle, which is prepared by encapsulating the above composition with PLGA; the mass ratio of PLGA to the composition is (3-4):1.

[0035] The preparation method of the above PLGA nano-drug-loaded particle provided by the present invention includes: (1) Preparation of primary emulsion: Mix the above composition with trehalose to prepare an aqueous phase; dissolve PLGA in an organic solvent to prepare an oil phase; mix the aqueous phase and the oil phase at a volume ratio of 1:5-15, and then perform ultrasonic treatment to prepare a primary emulsion; (2) Emulsification: Inject the primary emulsion into an external aqueous phase polyvinyl alcohol solution, homogenize and emulsify, and then stir to volatilize the organic solvent; (3) Purification of nano-particles: Centrifuge the emulsion, collect the precipitate, wash it, remove the free PVA and the unencapsulated composition, and then resuspend it in a solution; (4) Freeze-drying: First perform pre-freezing, freeze at -80~-100°C for 2~3 hours, and then perform drying after complete solidification to obtain nano-particles, and store them under the conditions of airtight, light-proof and not exceeding 25°C.

[0036] Preferably, the aqueous phase contains the composition + 2% trehalose; the oil phase is 5% PLGA / dichloromethane, and emulsify under the conditions of ultrasonic power of 200W and 3×30s.

[0037] Preferably, the volume ratio of the aqueous phase to the oil phase is 1:10.

[0038] Preferably, a freeze-drying protectant can be added during freeze-drying.

[0039] More preferably, the freeze-drying protectant is trehalose / mannitol (3:1, total concentration 8%).

[0040] Preferably, the pre-freezing condition is -80°C for 2 hours; the drying is carried out in stages, at -40°C for 12h → 25°C for 6h.

[0041] Further preferably, as the specific steps of a more specific preparation method of PLGA nano-drug-loaded particles: 1. Preparation of colostrum Aqueous phase: Composition solution (containing 15 mg of active ingredient) + 2% trehalose (protective agent), with a total volume of 2 mL.

[0042] Oil phase: PLGA (75:25, molecular weight 30 kDa) dissolved in dichloromethane (5% w / v), with a volume of 20 mL.

[0043] Emulsification parameters: Volume ratio of aqueous phase to oil phase is 1:10, probe sonication (200 W, 3×30-second pulses, with a 10-second cooling interval). Control the temperature in an ice bath ≤15°C to avoid the volatilization of organic solvents.

[0044] 2. Formation and solidification of double emulsion External aqueous phase: 2% polyvinyl alcohol (PVA, molecular weight 30 - 70 kDa) solution, with a volume of 50 mL.

[0045] Emulsification step: Inject the primary emulsion into the external aqueous phase and emulsify with a homogenizer at 12000 rpm for 2 minutes. Stir magnetically (500 rpm, for 4 hours) to volatilize dichloromethane until the solution has no odor of organic solvents (residual amount detected by GC <0.1%).

[0046] 3. Purification of nanoparticles Centrifuge at 15000 rpm for 20 minutes and collect the precipitate. Wash 3 times with PBS (pH 7.4) to remove free PVA and unencapsulated composition. Resuspend in a 5% (trehalose: mannitol, 3:1) solution and adjust the concentration to 10 mg / mL.

[0047] 4. Freeze-drying and storage (1) Pre-freezing procedure: Aliquot the nanoparticle suspension into vials, 2 mL per vial. Pre-freeze: Rapidly freeze at -80°C for 2 hours to ensure complete solidification.

[0048] (2) Staged drying and storage: Primary drying: Temperature -40°C, vacuum 50 Pa, maintain for 12 hours to sublime and remove free water.

[0049] Secondary drying: Temperature 25°C, vacuum 10 Pa, maintain for 6 hours to remove bound water. The final product has a water content ≤1.5% (Karl Fischer method), showing a porous and loose structure. Store in a sealed and light-proof manner at a temperature not exceeding 25°C.

[0050] The present invention has the following beneficial effects: The present invention provides a skin tissue injury repair composition based on umbilical cord extract, which includes umbilical cord extract, silk fibroin, γ-PGA, and antioxidant. Through scientific ratio research, this composition has a synergistic effect, can promote cell proliferation, increase collagen secretion, and has a significant effect on repairing skin tissue injury. Further, by optimizing the enzymatic hydrolysis process of the active ingredient umbilical cord extract in the composition, not only the yield (≥53%) and purity (hydroxyproline ≥84 mg / g) of the umbilical cord extract are improved, but also the repair effect of the composition is further enhanced. At the same time, by preparing PLGA nano-drug-loaded particles and transporting them through the nano-drug delivery system, the stability of the composition is improved, the stable delivery and long-acting release and sustained release of the drug are achieved, and it can be efficiently absorbed through the skin, further improving the skin injury repair effect of the composition. The composition provided by the present invention has the advantages of simple preparation process, high stability, and significant repair effect, can be used to prepare more efficient skin injury repair drugs, and is applicable to the treatment of various skin injuries such as burns, trauma, and ulcers.

[0051] Through scientific composition formula, efficient preparation process of umbilical cord extract, innovative administration method and combined with multi-level research and verification, through the synergistic effect of multiple components, optimization of umbilical cord extract extraction process, and breakthrough of administration method, the present invention significantly improves the skin injury repair effect of the composition, provides more compositions and their nano-drug-loaded preparations with better effects for skin injury repair drugs, and has important clinical application value and market prospect. Brief Description of the Drawings

[0052] Figure 1 It is a comparison chart for the repair of scalded skin of rats.

[0053] Figure 2 It is a result chart of HE pathological analysis of skin tissue. Detailed Embodiments

[0054] The following further illustrates the present invention in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0055] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0056] Example 1 Preparation of Skin Tissue Injury Repair Composition 1. Composition It contains components in the following mass ratio: umbilical cord extract: silk fibroin: γ-PGA (γ-polyglutamic acid): antioxidant (glutathione) = 50:20:25:5.

[0057] 2. Preparation of the composition (1)Dissolution of each component: Umbilical cord extract solution (50%): Take 10 mg / mL of freeze-dried powder and dissolve it in normal saline (0.9% NaCl), and assist dissolution by ultrasound (40 kHz, 5 minutes).

[0058] Silk fibroin solution (20%): Silk fibroin (molecular weight 30 kDa, β-sheet structure proportion ≥ 70%). Take 4% (w / v) of silk fibroin and dissolve it in deionized water, and stir magnetically at a constant temperature of 60 °C for 2 hours until it is transparent and free of particles.

[0059] γ-PGA solution (25%): γ-Polyglutamic acid (molecular weight 15 - 20 kDa). Take 3% (w / v) of γ-PGA and dissolve it in phosphate buffer (0.01 M) with pH 6.0, and stir magnetically for 30 minutes.

[0060] Antioxidant solution (5%): 0.15% glutathione (reduced form) is dissolved in sterile injection water and prepared under light-proof conditions.

[0061] (2)Mixing sequence and parameters: First step: Premix silk fibroin and γ-PGA: Mix according to the mass ratio of 20% silk fibroin: 25% γ-PGA, stir magnetically (300 rpm, 30 minutes), and the temperature is 25 °C. Detect the pH value of the mixed solution (target range 6.5 - 7.0), and fine-tune it with 0.1 M NaOH or HCl if necessary.

[0062] Second step: Add umbilical cord extract: Slowly drip the umbilical cord extract solution (final concentration 50%), the dripping speed is 1 mL / min, and stir magnetically synchronously (200 rpm), and the temperature is 25 °C. Avoid precipitation caused by too high local concentration, and observe the mixing system in real time.

[0063] Third step: Add antioxidant: Add the reduced glutathione solution (final concentration 5%) under light-proof conditions, disperse it by ultrasound (40 kHz, 5 minutes), and the temperature is 25 °C. The final mixed solution can be sterilized by passing through a 0.22 μm filter membrane.

[0064] 3. Preparation of umbilical cord extract S1: Umbilical cord collection and pretreatment: (1)Collection conditions: Collect umbilical cord tissues of healthy full-term newborn calves, ensure no infection or lesion, and perform subsequent operations after passing various microbial and virus series index tests before use.

[0065] (2)Storage conditions: Place the umbilical cord in normal saline at 4 °C and process it within 24 hours to avoid degradation of active ingredients.

[0066] (3)Washing step: Wash the umbilical cord repeatedly with PBS buffer containing 1% penicillin-streptomycin to remove blood and impurities.

[0067] S2: Tissue cutting and enzymatic digestion: (1)Cutting parameters: Cut the umbilical cord into small sections of 1-2 cm, quickly freeze in liquid nitrogen and then mechanically crush to particles of 0.5-1 mm3.

[0068] (2)Enzymatic digestion conditions: 0.3% type II collagenase + 0.15% trypsin, PBS solution with pH 7.4, containing 2 mM CaCl2, enzymatically digest synchronously in a constant temperature water bath at 42°C for 3 h, with magnetic stirring at 250 rpm.

[0069] (3)Terminate the reaction: Cool in a 4°C bath for 10 minutes, centrifuge at 8000 rpm for 15 minutes, and take the supernatant.

[0070] S3: Purification, concentration and freeze-drying: The supernatant is concentrated by an ultrafiltration system (10 kDa membrane), with a transmembrane pressure of 0.2 MPa and a flow rate of 10 mL / min. The supernatant is concentrated to 1 / 10 of the original volume to obtain a high-concentration and high-purity umbilical cord extract solution. After vacuum freeze-drying, a white powder is obtained.

[0071] Example 2 PLGA drug-loaded combination 1. Preparation of the composition - PLGA nanoparticles (double emulsion method) The use of a nanocarrier for encapsulation is mainly to increase the long-term storage, transportation stability and the sustained release of the composition during administration. Research shows that the use of a nanocarrier for encapsulation has no significant difference in the effect of the composition itself. Using the composition prepared in Example 1, through previous multi-factor orthogonal experiments, optimization of drug-loading performance and in vitro drug release kinetics research and analysis, it is determined that the mass ratio of the nanocarrier to the composition is 3.3:1 as the optimal ratio (when using PLGA as the carrier, the mass ratio to the composition is 3.3:1, with an encapsulation efficiency of 89.3%, a drug-loading capacity of 14.2%, and a release rate of 90.2%, which is the best overall; when the mass ratio < 3:1, the drug-loading capacity is insufficient (< 13%), and when > 4:1, PLGA will be in excess, resulting in a burst release effect (release > 50% in the first 6 hours)). The specific preparation method is as follows: (1)Preparation of the primary emulsion Aqueous phase: Composition solution (containing 15 mg of active ingredient) + 2% trehalose (protective agent), with a total volume of 2 mL.

[0072] Oil phase: PLGA (75:25, molecular weight 30 kDa) is dissolved in dichloromethane (5% w / v), with a volume of 20 mL.

[0073] Emulsification parameters: volume ratio of aqueous phase to oil phase is 1:10, probe sonication (200 W, 3×30 s pulses, with 10 s intervals for cooling). Ice bath is used to control the temperature ≤ 15 °C to avoid the volatilization of organic solvents.

[0074] (2)Double emulsion formation and solidification External aqueous phase: 2% polyvinyl alcohol (PVA, molecular weight 30 - 70 kDa) solution, with a volume of 50 mL.

[0075] Emulsification steps: The primary emulsion is injected into the external aqueous phase, and homogenized at 12000 rpm for 2 minutes. Magnetic stirring (500 rpm, 4 hours) is used to volatilize dichloromethane until the solution has no odor of organic solvents (residual amount detected by GC < 0.1%).

[0076] (3)Nanoparticle purification The emulsion prepared above is centrifuged at 15000 rpm for 20 minutes, and the precipitate is collected. It is washed 3 times with PBS (pH 7.4) to remove free PVA and unencapsulated drugs.

[0077] It is resuspended in a 5% (trehalose: mannitol, 3:1) solution, and the concentration is adjusted to 10 mg / mL.

[0078] 2. Freeze-drying and storage (1)Pre-freezing procedure The nanoparticle suspension is aliquoted into vials, 2 mL per vial. Pre-freezing: Rapid freezing at -80 °C for 2 hours to ensure complete solidification.

[0079] (2)Staged drying and storage Primary drying: Temperature -40 °C, vacuum 50 Pa, maintained for 12 hours to sublime free water.

[0080] Secondary drying: Temperature 25 °C, vacuum 10 Pa, maintained for 6 hours to remove bound water. The final product has a water content ≤ 1.5% (Karl Fischer method), and shows a porous and loose structure. It is stored in a sealed and light-proof manner at a temperature not exceeding 25 °C.

[0081] Example 3 The preparation method of the PLGA nano-drug delivery combination provided in this example is the same as that in Example 2, except that the mass ratio of each component in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 55:20:25:5, and all other operation steps are the same.

[0082] Example 4 The preparation method of the PLGA nano-drug delivery combination provided in this example is the same as that in Example 2, except that the mass ratio of each component in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 45:20:25:5, and all other operation steps are the same.

[0083] Example 5 The preparation method of the PLGA nano-drug delivery combination provided in this example is the same as that in Example 2, except that the mass ratio of each component in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:22:25:5, and all other operation steps are the same.

[0084] Example 6 The preparation method of the PLGA nano-drug delivery combination provided in this example is the same as that in Example 2, except that the difference in the composition is that the mass ratio of each component in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:18:25:5, and all other operation steps are the same.

[0085] Example 7 The preparation method of the PLGA nano-drug delivery combination provided in this example is the same as that in Example 2, except that the mass ratio of each component in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:20:27:5, and all other operation steps are the same.

[0086] Example 8 The preparation method of the PLGA nano-drug delivery combination provided in this example is the same as that in Example 2, except that the mass ratio of each component in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:20:23:5, and all other operation steps are the same.

[0087] Example 9 The preparation method of the PLGA nano-drug delivery combination provided in this example is the same as that in Example 2, except that the mass ratio of each component in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:20:25:6, and all other operation steps are the same.

[0088] Example 10 The preparation method of the PLGA nano-drug delivery combination provided in this example is the same as that in Example 2, except that the mass ratio of each component in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:20:25:4, and all other operation steps are the same.

[0089] Comparative Example 1 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that fibroin is not used, and the mass ratio of each component in the composition is: umbilical cord extract: γ-PGA: antioxidant (glutathione) = 50:25:5, and all other operation steps are the same.

[0090] Comparative Example 2 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that γ-PGA is not used, and the mass ratio of each component in the composition is: umbilical cord extract: fibroin: antioxidant (glutathione) = 50:20:5, and all other operation steps are the same.

[0091] Comparative Example 3 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that glutathione is not used, and the mass ratio of each component in the composition is: umbilical cord extract: fibroin: γ-PGA = 50:20:25, and all other operation steps are the same.

[0092] Comparative Example 4 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that fibroin and γ-PGA are not used, and the mass ratio of each component in the composition is: umbilical cord extract: antioxidant (glutathione) = 50:5, and all other operation steps are the same.

[0093] Comparative Example 5 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that fibroin and glutathione are not used, and the mass ratio of each component in the composition is: umbilical cord extract: γ-PGA = 50:25, and all other operation steps are the same.

[0094] Comparative Example 6 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that γ-PGA and glutathione are not used, and the mass ratio of each component in the composition is: umbilical cord extract: fibroin = 50:20, and all other operation steps are the same.

[0095] Comparative Example 7 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that fibroin, γ-PGA, and glutathione are not used, and the composition contains 55% by mass of umbilical cord extract, and all other operation steps are the same.

[0096] Comparative Example 8 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that the mass ratio of each component in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:30:37:5, and all other operation steps are the same.

[0097] Comparative Example 9 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that the mass ratio of each component in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:10:13:5, and all other operation steps are the same.

[0098] Comparative Example 10 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that the preparation method of the umbilical cord extract in the composition is different, and low-concentration enzymes are used for enzymatic hydrolysis. The enzymatic hydrolysis conditions are: 0.1% type II collagenase + 0.05% trypsin, pH 7.0, enzymatic hydrolysis at 37°C for 6 h, and all other operation steps are the same.

[0099] Comparative Example 11 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that the preparation method of the umbilical cord extract in the composition is different, and a single enzyme is used for enzymatic hydrolysis. The enzymatic hydrolysis conditions are: 0.3% type II collagenase, pH 7.4, enzymatic hydrolysis at 42°C for 3 h, and all other operation steps are the same.

[0100] Comparative Example 12 The preparation method of the PLGA nano-drug-loaded combination provided in this comparative example is the same as that in Example 2, except that the preparation method of the umbilical cord extract in the composition is different, and high-concentration enzymes are used for enzymatic hydrolysis. The enzymatic hydrolysis conditions are: 0.5% type II collagenase + 0.3% trypsin, pH 7.4, enzymatic hydrolysis at 42°C for 3 h, and all other operation steps are the same.

[0101] Comparative Example 13 The chitosan nano-drug delivery combination provided in this comparative example uses the same composition preparation method as in Example 1, except that the nano-carrier material is chitosan and is prepared by the ionic gelation method. The specific preparation method is as follows: Dissolve 1% chitosan in 1% acetic acid solution (w / v), and stir magnetically (500 rpm, 50 °C) until completely transparent (about 4 hours). Adjust the pH to 5.0 - 5.5 (0.1 M NaOH) to avoid excessive degradation. Dissolve 0.1% sodium polyphosphate TPP in deionized water (w / v), and sterilize it with a 0.22 μm filter membrane. Dissolve the umbilical cord extract composition in PBS with a pH of 6.0, and the final concentration is 10 mg / mL. Mix according to the mass ratio of chitosan: composition of 3:1 (such as 30 mg chitosan + 10 mg composition), and stir for 30 minutes. Drop the chitosan-composition mixture into the TPP solution (volume ratio 2:1), and stir magnetically (300 rpm, room temperature). The dropping rate is 1 mL / min, and the reaction time is 30 minutes to form nanoparticles for standby.

[0102] Comparative Example 14 The liposome nano-drug delivery combination provided in this comparative example uses the same composition preparation method as in Example 1, except that the nano-carrier material is liposome and is prepared by the thin-film hydration method. The specific preparation method is as follows: Weigh 60 mg of soy lecithin + 20 mg of cholesterol, and dissolve them in 10 mL of chloroform: methanol (2:1, v / v) mixture. Rotate and evaporate (40 °C water bath, 150 rpm) to form a uniform lipid film. Vacuum dry overnight to completely remove organic solvents. Dissolve the umbilical cord extract composition in PBS with a pH of 7.4, and the final concentration is 10 mg / mL, and preheat it to 50 °C. Take 10 mL of the composition solution and add it to the lipid film, and vortex at 2000 rpm for 5 minutes. Ultrasonic treatment (100 W, 3×10 second pulses) is used to promote the formation of liposomes. Extrude 10 times through a polycarbonate membrane with a pore size of 200 nm to control the particle size. Ultracentrifuge at 100000×g, 4 °C for 1 hour to remove unencapsulated drugs. Resuspend the nanoparticles in PBS (pH 7.4) solution containing 5% sorbitol, and store them in the dark at 4 °C for standby.

[0103] Test Example 1 Effects of Different Compositions on the Proliferation Rate and COL1A1 Secretion of HaCaT Cells Seed HaCaT cells (human immortalized keratinocytes) at a density of 5×10 3Cells were seeded at [X] cells / well in a 96-well plate and cultured in DMEM + 10% FBS at 37°C and 5% CO₂ for 24 hours. In the experimental groups, nanoformulations containing different ratios of the composition were added at a final concentration of 100 μg / mL, dissolved in the medium. The blank control groups were blank medium (negative control) and medium containing 10% FBS (positive control). After incubation for 48 hours, 10 μL of CCK-8 reagent (Dojindo) was added to each well and incubation was continued for 2 hours. The absorbance at 450 nm was measured using a microplate reader (reference wavelength 630 nm), and the relative proliferation rate (%) of each composition (Examples 2-10, Comparative Examples 1-9) on HaCaT cells was calculated. The calculation formula is: Cell proliferation rate (%) = (ODtreatment - ODblank) / (ODcontrol - ODblank) × 100%; Then, COL1 secretion was detected (ELISA method). Cell culture and treatment were the same as in the CCK-8 experiment. After culturing for 48 hours, the supernatant was collected and centrifuged (3000 rpm, 10 min) to remove cell debris. A human type I collagen (COL1) ELISA kit (Abcam ab210579) was used and the detection was performed according to the instructions.

[0104] The results of the determination of the proliferation rate of different compositions on HaCaT cells are shown in Table 1. The results show that the nano-compositions of Examples 2-10 can significantly promote the proliferation of HaCaT cells, and their effects are better than those of the umbilical cord extract alone and any pairwise combination in the comparative examples, showing a certain synergistic effect.

[0105] Table 1 Effects of different compositions on the proliferation rate of HaCaT cells

[0106] The results of the determination of the COL1A1 secretion amount of different compositions on HaCaT cells are shown in Table 2. It shows that the nano-compositions of Examples 2-10 can significantly promote the collagen secretion of HaCaT cells and increase the COL1A1 secretion amount. Their effects are significantly better than those of the umbilical cord extract alone, the pairwise combinations of the umbilical cord extract with silk fibroin or γ-PGA, antioxidants, and the combination of three components, showing a significant synergistic effect.

[0107] Table 2 Effects of different compositions on the COL1A1 secretion amount of HaCaT cells

[0108] Test Example 2 Optimization of the preparation method of umbilical cord extract Due to the complex and inconsistent extraction processes of existing umbilical cord extracts, the preparation methods are multi-step and cumbersome, and the extraction efficiency is low. To further improve the yield and purity of umbilical cord extracts, the process conditions of the preparation method of the umbilical cord extract in the composition of Example 2 were optimized. At the same time, different extraction method conditions were set for comparison (Comparative Examples 10-12) to compare the differences in the yield and purity of umbilical cord extracts obtained under different enzymatic hydrolysis conditions.

[0109] The yield and purity of umbilical cord extracts are usually evaluated based on the content of their active ingredients (such as hydroxyproline). The specific calculation methods are as follows: Formula for calculating the yield of umbilical cord extract:

[0110] Formula for calculating the purity of umbilical cord extract:

[0111] The results of the differences in the yield and purity of umbilical cord extracts under different enzymatic hydrolysis conditions were obtained through statistics as shown in Table 3, indicating that by optimizing the enzymatic hydrolysis process (simultaneous enzymatic hydrolysis with type II collagenase + trypsin), the yield (≥53%) and purity (hydroxyproline ≥ 84 mg / g) of umbilical cord extracts can be further improved.

[0112] Table 3 Differences in the yield and purity of umbilical cord extracts under different enzymatic hydrolysis conditions

[0113] Further, according to the test method of Test Example 1, the PLGA nano-drug-loaded combinations prepared from umbilical cord extracts extracted under different enzymatic hydrolysis conditions were measured. The experimental method was the same as that of Test Example 1. The results of the effects of the compositions with different enzymatic hydrolysis processes on the proliferation rate of HaCaT cells are shown in Table 4, indicating that the compositions prepared from umbilical cord extracts extracted by different enzymatic hydrolysis processes affect cell proliferation, and the umbilical cord extracts with higher yield and purity have better effects when used in the combination; the measured results of the COL1A1 secretion amount are shown in Table 5, indicating that optimizing the enzymatic hydrolysis conditions to further improve the yield and purity of umbilical cord extracts can increase collagen secretion and also further improve the repair effect of the combination.

[0114] Table 4 Effects of compositions with different enzymatic hydrolysis processes on the proliferation rate of HaCaT cells

[0115] Table 5 Effects of different enzymatic hydrolysis processes on the COL1A1 secretion amount of HaCaT cells

[0116] Test Example 3 Performance comparison of different nano-carriers Due to the poor stability of umbilical cord extract, its active ingredients are easily inactivated and difficult to store for a long time. Moreover, the administration method is single, and traditional administration methods (such as topical application and injection) are difficult to achieve stable delivery, which affects the repair effect. Therefore, in the present invention, different nano-carrier systems (Examples 2, Comparative Examples 13-14) are used to carry the composition, so as to improve the poor stability and release efficiency of the composition, and increase the long-term storage and transportation stability and the persistence of drug administration and release of the composition. The encapsulation efficiency and drug loading of different carriers were specifically measured by ultrafiltration centrifugation UV-Vis method at a wavelength of 280 nm. Stability refers to the biological activity retention rate, which is detected by the HaCaT cell proliferation assay method. The 72-hour release rate was determined by HPLC to measure the released drug concentration and calculate the cumulative release rate. The transdermal absorption experiment was detected by the Franz diffusion cell method for transdermal absorption rate.

[0117] The results are shown in Table 6, indicating that the nano-carrier PLGA has a high encapsulation efficiency for the composition, and its stability, drug loading capacity, transdermal absorption and 72h release rate are all superior to other chitosan and liposome nano-carriers, with good stability and the persistence of drug administration and release, and the 72-hour sustained release rate ≥ 90%. At the same time, in the preparation of the nano-formulation, its stability is significantly improved through a specific freeze-drying process, and the activity retention > 95% after storage at room temperature for 36 months.

[0118] Table 6 Comparison of the performance of the composition of different nano-carriers

[0119] Further, according to the test method of Test Example 1, different nano-drug combinations were measured. The experimental method was the same as that of Test Example 1. The results of the effects of the compositions of different nano-carriers on the proliferation rate of HaCaT cells are shown in Table 7, and the measured results of the COL1A1 secretion amount are shown in Table 8. Through the innovative drug administration method of the nano-carrier, stable delivery and long-term release of the active ingredients of the composition can be achieved, promoting cell proliferation and increasing collagen secretion, and improving the repair effect of skin tissue damage.

[0120] Table 7 Effects of different nano-carriers on the proliferation rate of HaCaT cells

[0121] Table 8 Effects of different nano-carriers on the COL1A1 secretion amount of HaCaT cells

[0122] Test Example 4 Animal experiment of skin burns and scalds In this example, an animal experiment model of skin burns and scalds was constructed to verify the efficacy of the composition in repairing skin burns. The specific method is as follows: SD rats with a body weight of 200 - 250 g were used. Environmental conditions: temperature 22 ± 2 °C, humidity 50 ± 10%, 12-hour light / dark cycle, free diet and water. There were 5 rats in each group, and a model group and an experimental group were set up.

[0123] The animals were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg) to ensure complete anesthesia of the rats. The hair on the back of the rats was shaved off with an electric hair clipper, and the area was about 4 cm × 4 cm. The skin was disinfected with 75% ethanol to avoid infection. A constant-temperature metal scalding instrument was used, and the temperature was set at 100 °C. The scalding instrument was in contact with the skin for 10 seconds to make a second-degree burn and scald model. Two burn and scald wounds with a diameter of about 1.5 cm and a spacing of 2 cm were made on each rat.

[0124] Experimental grouping and drug administration: The model control group was treated with normal saline and applied topically once a day. The experimental group was topically applied with 500 μL of the nano-composition of Example 2 (10 mg / mL) once a day. The time points for photographing and measuring wound healing and repair were 0, 2, 4, and 7 days. The measurement method was to use a digital camera to photograph the wound to ensure that the shooting distance and light were consistent. The degree of wound repair was compared, and histological pathological analysis (HE staining) was performed. The sampling time point was the 7th day. The animals were anesthetized, and a small part of the skin tissue at the wound was taken for HE staining analysis and ELISA detection of the levels of inflammatory factors IL-6 and IL-1β in tissue homogenates.

[0125] Results Figure 1 As shown, compared with the model group, the group of Example 2 showed a faster repair effect at the same time. The results of HE pathological analysis of skin tissue are as Figure 2 shown. In the skin tissue of the model control group, epidermal layer, dermal layer and subcutaneous necrosis were visible, nuclear fragmentation or dissolution was observed, and mild bleeding was visible, and a large number of pus cells were visible; extensive fibroblast proliferation was visible under the skin, a small amount of new blood vessels were congested, and accompanied by a small amount of lymphocyte infiltration. In the skin tissue of the group of Example 2, epidermal layer and dermal layer necrosis were visible, nuclear fragmentation or dissolution was observed, and a small amount of pus cells were visible; a large number of new blood vessels were visible, accompanied by a small amount of lymphocyte infiltration, and a large amount of new skin tissue was seen.

[0126] Furthermore, the expression levels of inflammatory factors in the homogenates of scalded skin tissue on the 7th day were detected by ELISA. The results are shown in Table 9. Compared with the model group, the inflammatory factors in the skin tissue of the group of Example 2 were significantly reduced. It shows that the nano-composition provided by the present invention has a significant effect on repairing skin tissue damage and is applicable to the treatment of various skin injuries such as burns, wounds, and ulcers.

[0127] Table 9 Levels of inflammatory factors IL-6 and IL-1β

[0128] In summary, the skin tissue damage repair composition provided by the present invention is adjusted through scientific combination ratios, and the components play a synergistic effect with each other. It can promote cell proliferation and increase collagen secretion, and has a significant effect on repairing skin tissue damage. Its effect is superior to that of using umbilical cord extract alone, or any pairwise combination or three-component combination of its components. Through cell model experiments and burn and scald animal model experiments, the significant effects of the composition of the present invention in promoting wound healing, tissue and inflammation relief have been systematically verified, providing more and better products for the treatment of various skin injuries. At the same time, the present invention further significantly improves the yield and purity of the active ingredient umbilical cord extract in the composition and improves the stability of the composition by optimizing the preparation process of umbilical cord extract and innovating the administration method, realizing the stable delivery and long-term release of the active ingredient, and further improving the repair effect of skin damage, having important clinical application value and market prospects.

[0129] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A skin tissue injury repair composition, characterized in that, It comprises components in the following mass ratios: umbilical cord extract: silk fibroin: γ-PGA: antioxidant = (40 - 60):(10 - 25):(20 - 30):(2 - 8).

2. The composition according to claim 1, wherein It comprises components in the following mass ratios: umbilical cord extract: silk fibroin: γ-PGA: antioxidant = (45 - 55):(18 - 22):(23 - 27):(4 - 6).

3. The composition according to claim 1 or 2, characterized in that, The antioxidant is selected from glutathione, vitamin C or coenzyme Q10.

4. The composition according to claim 1 or 2, characterized in that, The preparation method of the umbilical cord extract is as follows: the umbilical cord is crushed and then enzymolyzed. The enzymolysis conditions are: 0.25 - 0.35% type II collagenase and 0.12 - 0.18% trypsin, pH value 7.2 - 7.6, temperature 42 ± 1°C, stirring rate 200 - 300 rpm, time 2.5 - 3.5 hours. After enzymolysis, it is ultrafiltered, concentrated and freeze-dried to obtain the umbilical cord extract.

5. A method for preparing the composition according to any one of claims 1 to 4, characterized in that, It comprises the following steps: S1: Solution preparation: The umbilical cord extract, silk fibroin, γ-PGA and antioxidant are respectively prepared into solutions for standby. S2: Premixing of silk fibroin and γ-PGA: Mix silk fibroin and γ-PGA according to the mass ratio, at a temperature of 25 - 30°C, stir at 200 - 300 rpm for 20 - 40 minutes, and adjust the pH of the mixed solution to 6.5 - 7.0 to obtain a premixed solution. S3: Adding the umbilical cord extract: Slowly add the umbilical cord extract dropwise to the premixed solution, with a dropping rate of 0.8 - 1 mL / min, simultaneously stir at 100 - 200 rpm, at a temperature of 25 - 30°C to avoid precipitation. S4: Adding the antioxidant: Subsequently, add the antioxidant under light-shielded conditions, ultrasonically disperse at 30 - 40 kHz for 3 - 5 minutes, at a temperature of 25 - 30°C, and filter and sterilize the final mixed solution to obtain it.

6. Use of the composition according to any one of claims 1 to 4 in the preparation of a product for skin injury repair.

7. Use of the composition according to any one of claims 1 to 4 in maintaining the skin barrier or promoting skin collagen secretion.

8. A product, characterized in that, Containing the composition according to any one of claims 1 to 4.

9. A PLGA nano-drug-loaded particle, characterized in that, It is prepared by encapsulating the composition according to any one of claims 1 to 4 with poly(lactic-co-glycolic acid) PLGA; the mass ratio of the PLGA to the composition is (3 - 4):

1.

10. The PLGA nano drug-loaded particles according to claim 9, characterized in that, The preparation method is as follows: (1) Preparation of the primary emulsion: Mix the composition according to any one of claims 1 to 4 with trehalose to obtain an aqueous phase; dissolve PLGA in an organic solvent to obtain an oil phase; mix the aqueous phase and the oil phase at a volume ratio of 1:5 - 15 and then perform ultrasonic treatment to obtain the primary emulsion. (2) Emulsification: Inject the primary emulsion into an external aqueous phase polyvinyl alcohol solution, homogenize and emulsify, and then stir to volatilize the organic solvent to obtain an emulsion. (3) Nanoparticle purification: Centrifuge the emulsion, collect the precipitate, wash it, remove free PVA and unencapsulated composition, and then resuspend it in a solution. (4) Freeze-drying: First perform pre-freezing, freeze at -80~-100°C for 2 - 3 hours, and then perform drying after complete solidification to obtain nanoparticles, and store them under airtight and light-shielded conditions not exceeding 25°C.

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