A skin tissue damage repair composition containing umbilical cord extract, and its preparation method and application

Abstract

The present invention discloses a skin tissue damage repair composition containing an umbilical cord extract, and its preparation method and application. The composition provided by the present invention includes an umbilical cord extract, silk fibroin, γ-PGA, and an antioxidant. By precisely controlling the ratio of the components, it has a synergistic effect, can promote cell proliferation, increase collagen secretion, and has a significant skin tissue damage repair effect; further, by optimizing the enzymatic hydrolysis process of the active ingredients of the composition, not only the yield and purity of the umbilical cord extract are improved, but also the repair effect of the composition is enhanced; at the same time, by preparing PLGA nano drug-loaded particles, the stability of the composition is improved, stable delivery and long-term release of the drug are achieved, efficient transdermal absorption is achieved, and better skin damage repair effect is achieved. It has the advantages of simple preparation process, high stability, significant repair effect, etc., can be used to prepare more high-efficiency drugs, and is suitable for the treatment of various skin injuries such as burns, wounds, and ulcers.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to a skin tissue damage repair composition containing umbilical cord extract, and a preparation method and application thereof. Background Art

[0002] As the largest organ in the human body, the skin performs crucial functions, including protecting the body, regulating body temperature, and sensing external stimuli. Skin injuries (such as burns, wounds, and ulcers) not only affect appearance but can also lead to infection, scarring, and even functional impairment. Traditional methods for repairing skin injuries include surgery, dressings, and medications. However, these methods suffer from long repair cycles, high scarring rates, and susceptibility to infection. In recent years, with advances in medicine, the application of stem cells and their derivatives in tissue repair has garnered significant attention. Umbilical cord extracts (such as umbilical cord mesenchymal stem cells, umbilical cord blood, and umbilical cord Wharton's jelly) are rich in various growth factors (such as EGF, VEGF, and FGF), extracellular matrix components (such as collagen and hyaluronic acid), and immunomodulatory factors. They promote cell proliferation, migration, differentiation, and angiogenesis, and are therefore considered ideal skin repair materials.

[0003] However, existing techniques using umbilical cord extracts present the following challenges: First, the extraction process is complex and inconsistent. Existing methods for preparing umbilical cord extracts involve multiple, tedious steps, resulting in low yield and purity, and low extraction efficiency. Second, umbilical cord extracts suffer from poor stability, with active ingredients easily inactivated and difficult to preserve long-term. Finally, existing umbilical cord extracts are administered in a limited manner. Traditional methods (such as topical application and injection) struggle to achieve stable delivery, which compromises the repair effect. Furthermore, the existing art still lacks umbilical cord extracts or combinations with highly effective repair effects, and in-depth research on the synergistic effects of umbilical cord extracts with other ingredients is scarce, resulting in limited repair effects. For example, existing research has shown that a composite gel of umbilical cord exosomes, MSCs, and collagen can be used for skin burn repair; freeze-dried umbilical cord MSC-conditioned medium can be used to promote keratinocyte migration, but the preparation methods are relatively demanding and complex, and the repair effect needs to be improved.

[0004] Therefore, developing an efficient, stable and innovative composition containing umbilical cord extract and its delivery product 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 existing umbilical cord extracts and their products, as well as the lack of products that can efficiently repair skin tissue damage. The present invention aims to provide a skin tissue damage repair composition containing umbilical cord extract, which significantly improves the skin damage repair effect; the composition has the advantages of simple preparation process, high stability, and significant 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 method for preparing 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] A fourth object of the present invention is to provide a product.

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

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention provides a skin tissue damage repair composition, comprising the following components in the following mass ratio: umbilical cord extract: silk fibroin: gamma-PGA: antioxidant = (40-60): (10-25): (20-30): (2-8).

[0013] The skin tissue repair composition provided by the present invention features a scientifically formulated combination of components that synergistically enhance each other's effectiveness, promoting cell proliferation and increasing collagen secretion. This composition exhibits significant skin tissue repair efficacy, surpassing umbilical cord extract alone or in any combination of two or three of its components. The active ingredients of umbilical cord extract are cytokines and extracellular matrix, which promote wound healing. Silk fibroin, with its high mechanical strength and excellent biocompatibility, supports cell proliferation and migration, while its degradation products are non-toxic. γ-PGA (γ-polyglutamic acid), with its exceptional moisturizing capacity (10 times that of hyaluronic acid), promotes cell proliferation and exhibits significant anti-inflammatory effects. Antioxidants reduce oxidative stress damage and maintain the composition's biological activity. Experiments in cell models and animal models of burns and scalds systematically validate the composition's significant synergistic effects in promoting wound healing and alleviating tissue and inflammation, providing a more comprehensive and effective product for treating a variety of skin injuries.

[0014] 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).

[0015] Preferably, the antioxidant is selected from glutathione, vitamin C or coenzyme Q10.

[0016] More preferably, the antioxidant is glutathione.

[0017] The present invention further improves the yield (≥53%) and purity (hydroxyproline ≥84 mg / g) of umbilical cord extract by optimizing the enzymatic hydrolysis process (simultaneous enzymatic hydrolysis using collagenase type II and trypsin), thereby improving the repair effect of the composition. The preparation method is simple and convenient, improves the extraction efficiency and effect of umbilical cord extract, and provides a better source of raw materials for skin damage repair products.

[0018] Therefore, the present invention provides a method for preparing an umbilical cord extract: the umbilical cord is crushed and then enzymatically hydrolyzed, wherein the enzymatic hydrolysis conditions are 0.25-0.35% collagenase type II and 0.12-0.18% trypsin, pH 7.2-7.6, temperature 42±1°C, stirring rate 200-300 rpm, and time 2.5-3.5 hours. After enzymatic hydrolysis, the umbilical cord extract is obtained by ultrafiltration concentration and freeze-drying.

[0019] As a more preferred embodiment, the present invention provides a specific method for preparing umbilical cord extract: (1) Umbilical cord collection and pretreatment: Umbilical cord tissue from healthy full-term newborn calves is used to ensure that it is free of infection or pathology. Before use, various microbial and viral indicators are tested and qualified before subsequent operations. The umbilical cord is placed in 4°C normal saline and processed within 24 hours to avoid degradation of the active ingredients. The umbilical cord is repeatedly rinsed with PBS buffer containing 1% penicillin-streptomycin to remove blood and impurities.

[0020] (2) Tissue cutting and enzymatic digestion: The umbilical cord was cut into 1-2 cm segments, quickly frozen in liquid nitrogen, and then mechanically pulverized into 0.5-1 mm³ particles. 0.3% collagenase type II + 0.15% trypsin were used in a PBS solution (pH 7.4) containing 2 mM CaCl₂. Enzymatic digestion was performed in a 42°C constant temperature water bath for 3 h with magnetic stirring at 250 rpm. The reaction was terminated, the tissue was cooled at 4°C for 10 min, and the supernatant was collected by centrifugation at 8000 rpm for 15 min.

[0021] (3) Purification and concentration and freeze-drying: The supernatant was concentrated by ultrafiltration system (10 kDa membrane), with a transmembrane pressure of 0.2 MPa and a flow rate of 10 mL / min. The supernatant was concentrated to 1 / 10 of the original volume to obtain a high-concentration and high-purity umbilical cord extract solution, which was then vacuum-freeze-dried to obtain a white powder.

[0022] The present invention also provides a method for preparing the above composition, comprising the following steps:

[0023] S1: Solution preparation: umbilical cord extract, silk fibroin, γ-PGA, and antioxidant are prepared into solutions for later use;

[0024] S2: Premixing silk fibroin and γ-PGA: Mix silk fibroin and γ-PGA according to the mass ratio, stir at 200-300 rpm for 20-40 minutes at 25-30°C, and adjust the pH of the mixture to 6.5-7.0;

[0025] S3: Add umbilical cord extract: slowly add umbilical cord extract dropwise at a rate of 0.8-1 mL / min, stirring at 100-200 rpm, at a temperature of 25-30°C, to avoid precipitation;

[0026] S4: Add antioxidant: Add antioxidant in dark conditions, ultrasonically disperse at 30-40 kHz for 3-5 minutes at 25-30°C, and filter and sterilize the final mixture.

[0027] Preferably, in S1, the umbilical cord extract is dissolved in physiological saline (0.9% NaCl), and ultrasonic-assisted dissolution (40 kHz, 5 minutes) is performed to obtain an umbilical cord extract solution; silk fibroin (molecular weight 30 kDa, β-pleated structure ≥70%) is dissolved in deionized water, and magnetic stirring is performed at a constant temperature of 60°C for 2 hours until transparent and free of particles, to obtain a silk fibroin solution; γ-PGA (molecular weight 15-20 kDa) is dissolved in pH 6.0 phosphate buffer (0.01 M) and magnetic stirring is performed for 30 minutes to obtain a γ-PGA solution; an antioxidant is dissolved in sterile water for injection and prepared under light-proof conditions to obtain an antioxidant solution.

[0028] Preferably, the premixing conditions in S2 are: 300 rpm, 30 minutes, and a temperature of 25°C.

[0029] Furthermore, the pH was fine-tuned using 0.1 M NaOH or HCl.

[0030] Preferably, the dripping speed of the umbilical cord extract solution in S3 is 1 mL / min.

[0031] Preferably, the ultrasonic dispersion conditions in S4 are 40 kHz, 5 minutes, and a temperature of 25°C.

[0032] Preferably, in S4, the mixed solution is sterilized by filtering it through a 0.22 μm filter membrane.

[0033] The present invention provides application of the composition in preparing products for repairing skin damage.

[0034] The present invention provides application of the above composition in preparing medicine for treating skin damage.

[0035] The present invention provides use of the composition in maintaining skin barrier or promoting skin collagen secretion.

[0036] Preferably, the skin damage repair includes acute trauma repair, chronic wound treatment, scar repair, skin burn and scald treatment, and soothing repair of sensitive and diseased skin.

[0037] The present invention also provides a product containing the above composition.

[0038] At the same time, the present invention uses a nanocarrier delivery system to encapsulate the skin tissue damage repair composition with poly(lactic-co-glycolic acid, PLGA). The prepared PLGA nano-drug-carrying particles achieve stable release and long-lasting effects of the active ingredients of the composition, and their stability is also significantly improved (activity retention is >95% after 36 months of storage at room temperature). Through the innovative administration method of a specific nano-drug-carrying system, the loading rate, stability, stable sustained release and repair effect of the umbilical cord extract are further improved. The nano-drug-carrying system is suitable for the treatment of various skin injuries such as burns, wounds, and ulcers.

[0039] The present invention also provides a PLGA nano drug-loaded particle, which is prepared by encapsulating the above composition in PLGA; the mass ratio of the PLGA to the composition is (3-4):1.

[0040] The present invention provides a method for preparing the above-mentioned PLGA nanoparticles, comprising:

[0041] (1) Preparation of colostrum: The above composition is mixed with trehalose to prepare an aqueous phase; PLGA is dissolved in an organic solvent to prepare an oil phase; the aqueous phase and the oil phase are mixed at a volume ratio of 1:5-15 and then ultrasonicated to prepare colostrum;

[0042] (2) Emulsification: Inject colostrum into the external aqueous phase polyvinyl alcohol solution, homogenize and emulsify, and then stir to volatilize the organic solvent;

[0043] (3) Nanoparticle purification: the emulsion is centrifuged, the precipitate is collected, washed, free PVA and unencapsulated components are removed, and the precipitate is resuspended in a solution;

[0044] (4) Freeze-drying: Pre-freeze the sample at -80~-100℃ for 2~3 hours, dry it after it is completely solidified to obtain nanoparticles, and store it in a sealed, dark place at no more than 25℃.

[0045] Preferably, the aqueous phase contains the composition + 2% trehalose; the oil phase is 5% PLGA / dichloromethane, and is emulsified under ultrasonic conditions of 200W, 3×30s.

[0046] Preferably, the volume ratio of the water phase to the oil phase is 1:10.

[0047] Preferably, a lyoprotectant may be added during freeze-drying.

[0048] More preferably, the freeze-drying protective agent is trehalose / mannitol (3:1, total concentration 8%).

[0049] Preferably, the pre-freezing condition is -80°C for 2 hours; and the drying is carried out in stages, at -40°C for 12 hours → 25°C for 6 hours.

[0050] More preferably, as a more specific method for preparing PLGA nano drug-loaded particles, the specific steps are:

[0051] 1. Colostrum preparation

[0052] Aqueous phase: composition solution (containing 15 mg active ingredient) + 2% trehalose (protective agent), total volume 2 mL.

[0053] Oil phase: PLGA (75:25, molecular weight 30 kDa) dissolved in dichloromethane (5% w / v), volume 20 mL.

[0054] Emulsification parameters: water phase to oil phase volume ratio of 1:10, probe ultrasound (200 W, 3 × 30-second pulses, 10-second cooling intervals). Ice bath temperature controlled ≤15°C to prevent evaporation of organic solvents.

[0055] 2. Emulsion formation and solidification

[0056] External aqueous phase: 2% polyvinyl alcohol (PVA, molecular weight 30-70 kDa) solution, volume 50 mL.

[0057] Emulsification step: Colostrum was injected into the external aqueous phase and emulsified in a homogenizer at 12,000 rpm for 2 minutes. The dichloromethane was evaporated using magnetic stirring (500 rpm, 4 hours) until the solution was free of organic solvent odor (GC residual < 0.1%).

[0058] 3. Nanoparticle purification

[0059] Centrifuge at 15,000 rpm for 20 minutes and collect the precipitate. Wash three times with PBS (pH 7.4) to remove free PVA and unencapsulated components. Resuspend in 5% (trehalose:mannitol, 3:1) solution and adjust the concentration to 10 mg / mL.

[0060] 4. Freeze-dried storage

[0061] (1) Prefreezing procedure: Dispense the nanoparticle suspension into vials, 2 mL per vial. Prefreezing: Quickly freeze at -80°C for 2 hours to ensure complete solidification.

[0062] (2) Drying and storage in stages:

[0063] Primary drying: temperature -40℃, vacuum degree 50 Pa, maintain for 12 hours, sublimate to remove free water.

[0064] Secondary drying: 25°C, vacuum 10 Pa, maintain for 6 hours to remove bound water. The final product should have a moisture content of ≤1.5% (Karl Fischer method) and a porous structure. Store in a sealed container away from light, at a temperature not exceeding 25°C.

[0065] The present invention has the following beneficial effects:

[0066] The present invention provides a skin tissue damage repair composition based on umbilical cord extract. The composition comprises umbilical cord extract, silk fibroin, γ-PGA, and an antioxidant. Through scientifically formulated formulations, the composition exhibits synergistic effects, promoting cell proliferation and increasing collagen secretion, significantly repairing skin tissue damage. Further optimization of the enzymatic hydrolysis process for the active ingredient, umbilical cord extract, increases the yield (≥53%) and purity (hydroxyproline ≥84 mg / g) of the umbilical cord extract, further enhancing the composition's repair efficacy. Furthermore, by preparing PLGA nanoparticles and delivering the drug via a nanoparticle delivery system, the stability of the composition is improved, enabling stable drug delivery and sustained release, as well as efficient transdermal absorption, further enhancing the composition's skin damage repair efficacy. The composition provided by the present invention offers advantages such as a simple preparation process, high stability, and significant repair efficacy. It can be used to prepare a wider range of highly effective skin damage repair drugs and is suitable for treating a variety of skin injuries, including burns, wounds, and ulcers.

[0067] The present invention significantly improves the skin damage repair effect of the composition through a scientific composition formula, an efficient umbilical cord extract preparation process, an innovative administration method, and combined with multi-level research verification, through the synergistic effect of multiple components, the optimization of the umbilical cord extract extraction process, and a breakthrough in the administration method, providing more and better-effective compositions and nano-drug-carrying preparations for skin damage repair drugs, which has important clinical application value and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 A comparison chart of burn repair on rat skin.

[0069] Figure 2 This is the result of HE pathological analysis of skin tissue. DETAILED DESCRIPTION

[0070] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0071] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0072] Example 1 Preparation of a composition for repairing skin tissue damage

[0073] 1. Composition

[0074] Contains the following components in the following mass ratio: umbilical cord extract: silk fibroin: γ-PGA (γ-polyglutamic acid): antioxidant (glutathione) = 50:20:25:5.

[0075] 2. Preparation of the composition

[0076] (1) Dissolution of each component:

[0077] Umbilical cord extract solution (50%): Dissolve 10 mg / mL lyophilized powder in normal saline (0.9% NaCl) using ultrasound (40 kHz, 5 minutes).

[0078] Silk fibroin solution (20%): 4% (w / v) silk fibroin (molecular weight 30 kDa, β-sheet structure ≥70%) was dissolved in deionized water and stirred at 60°C for 2 h until transparent and particle-free.

[0079] γ-PGA solution (25%): γ-polyglutamic acid (molecular weight 15-20 kDa), take 3% (w / v) γ-PGA and dissolve it in pH 6.0 phosphate buffer (0.01 M) and stir magnetically for 30 minutes.

[0080] Antioxidant solution (5%): 0.15% glutathione (reduced form) is dissolved in sterile water for injection and prepared in the dark.

[0081] (2) Mixing order and parameters:

[0082] First, premix silk fibroin and γ-PGA at a mass ratio of 20% silk fibroin to 25% γ-PGA. Stir magnetically (300 rpm, 30 minutes) at 25°C. Check the pH of the mixture (target range 6.5-7.0) and adjust it with 0.1 M NaOH or HCl if necessary.

[0083] In the second step, add the umbilical cord extract: Slowly add the umbilical cord extract solution (50% final concentration) dropwise at a rate of 1 mL / min with simultaneous magnetic stirring (200 rpm) at 25°C. Avoid precipitation caused by excessive local concentration and monitor the mixture in real time.

[0084] In the third step, antioxidants are added: Reduced glutathione solution (5% final concentration) is added in the dark, and ultrasonic dispersion is performed (40 kHz, 5 minutes) at 25°C. The final mixture is sterilized by passing through a 0.22 μm filter.

[0085] 3. Preparation of umbilical cord extract

[0086] S1: Umbilical cord collection and pretreatment:

[0087] (1) Collection conditions: Umbilical cord tissue was collected from healthy full-term newborn calves to ensure that there was no infection or lesions. All microbial and viral indicators were tested and qualified before use before subsequent operations were carried out.

[0088] (2) Storage conditions: Place the umbilical cord in normal saline at 4°C and process within 24 hours to avoid degradation of active ingredients.

[0089] (3) Cleaning step: Rinse the umbilical cord repeatedly with PBS buffer containing 1% penicillin-streptomycin to remove blood and impurities.

[0090] S2: Tissue cutting and enzymatic digestion:

[0091] (1) Cutting parameters: The umbilical cord was cut into small segments of 1-2 cm, quickly frozen in liquid nitrogen, and then mechanically crushed into particles of 0.5-1 mm3.

[0092] (2) Enzymatic hydrolysis conditions: 0.3% collagenase type II + 0.15% trypsin, PBS solution pH 7.4, containing 2 mM CaCl2, synchronous enzymatic hydrolysis in a constant temperature water bath at 42°C for 3 h, and magnetic stirring at 250 rpm.

[0093] (3) Termination of the reaction: cold bath at 4°C for 10 minutes, centrifugation at 8000 rpm for 15 minutes, and collection of the supernatant.

[0094] S3: Purification and concentration freeze-drying:

[0095] The supernatant was concentrated using an ultrafiltration system (10 kDa membrane) at a transmembrane pressure of 0.2 MPa and a flow rate of 10 mL / min. The supernatant was concentrated to 1 / 10 of its original volume to obtain a highly concentrated and pure umbilical cord extract solution. The solution was then freeze-dried to obtain a white powder.

[0096] Example 2 PLGA nano-drug carrier combination

[0097] 1. Composition - Preparation of PLGA Nanoparticles (Double Emulsion Method)

[0098] The main purpose of using nanocarriers for encapsulation is to increase the long-term storage and transportation stability of the composition and the sustainability of drug release. Studies have shown that not using nanocarriers for encapsulation has no significant effect on the effect of the composition itself. Using the composition prepared in Example 1, after preliminary multi-factor orthogonal experiments, drug loading performance optimization and in vitro drug release kinetics analysis, it was determined that the mass ratio of the nanocarrier to the composition was 3.3:1, which was the optimal ratio (when PLGA was used as the carrier, the mass ratio to the composition was 3.3:1, and the encapsulation rate was 89.3%, the drug loading was 14.2%, and the release rate was 90.2%, which was the best overall ratio; when the mass ratio was <3:1, the drug loading was insufficient (<13%), and when it was >4:1, the PLGA would be excessive, resulting in a burst release effect (>50% release in the first 6 hours)). The specific preparation method is as follows:

[0099] (1) Colostrum preparation

[0100] Aqueous phase: composition solution (containing 15 mg of active ingredient) + 2% trehalose (protective agent), total volume 2 mL.

[0101] Oil phase: PLGA (75:25, molecular weight 30 kDa) dissolved in dichloromethane (5% w / v), volume 20 mL.

[0102] Emulsification parameters: water phase to oil phase volume ratio of 1:10, probe ultrasound (200 W, 3 × 30-second pulses, 10-second cooling intervals). Ice bath temperature controlled ≤15°C to prevent evaporation of organic solvents.

[0103] (2) Emulsion formation and solidification

[0104] External aqueous phase: 2% polyvinyl alcohol (PVA, molecular weight 30-70 kDa) solution, volume 50 mL.

[0105] Emulsification step: Colostrum was injected into the external aqueous phase and emulsified in a homogenizer at 12,000 rpm for 2 minutes. The dichloromethane was evaporated using magnetic stirring (500 rpm, 4 hours) until the solution was free of organic solvent odor (GC residual < 0.1%).

[0106] (3) Nanoparticle purification

[0107] The prepared emulsion was centrifuged at 15,000 rpm for 20 minutes, and the precipitate was collected and washed three times with PBS (pH 7.4) to remove free PVA and unencapsulated drug.

[0108] Resuspend in 5% (trehalose:mannitol, 3:1) solution and adjust the concentration to 10 mg / mL.

[0109] 2. Freeze-dried storage

[0110] (1) Pre-freezing procedure

[0111] Aliquot the nanoparticle suspension into 2 mL vials. Prefreeze: Quickly freeze at -80°C for 2 hours to ensure complete solidification.

[0112] (2) Drying and storage in stages

[0113] Primary drying: temperature -40℃, vacuum degree 50 Pa, maintain for 12 hours, sublimate to remove free water.

[0114] Secondary drying: 25°C, vacuum 10 Pa, maintain for 6 hours to remove bound water. The final product has a moisture content of ≤1.5% (Karl Fischer method) and a porous structure. Store in a sealed container away from light at a temperature not exceeding 25°C.

[0115] Example 3

[0116] The preparation method of the PLGA nano-drug carrier combination provided in this embodiment is the same as that in Example 2, except that the mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 55:20:25:5, and all other operating steps are the same.

[0117] Example 4

[0118] The preparation method of the PLGA nano-drug carrier combination provided in this embodiment is the same as that in Example 2, except that the mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 45:20:25:5, and all other operating steps are the same.

[0119] Example 5

[0120] The preparation method of the PLGA nano-drug carrier combination provided in this embodiment is the same as that in Example 2, except that the mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:22:25:5, and all other operating steps are the same.

[0121] Example 6

[0122] The preparation method of the PLGA nano-drug carrier combination provided in this embodiment is the same as that in Example 2, except that the mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:18:25:5, and all other operating steps are the same.

[0123] Example 7

[0124] The preparation method of the PLGA nano-drug carrier combination provided in this embodiment is the same as that in Example 2, except that the mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:20:27:5, and all other operating steps are the same.

[0125] Example 8

[0126] The preparation method of the PLGA nano-drug carrier combination provided in this embodiment is the same as that in Example 2, except that the mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:20:23:5, and all other operating steps are the same.

[0127] Example 9

[0128] The preparation method of the PLGA nano-drug carrier combination provided in this embodiment is the same as that in Example 2, except that the mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:20:25:6, and all other operating steps are the same.

[0129] Example 10

[0130] The preparation method of the PLGA nano-drug carrier combination provided in this embodiment is the same as that in Example 2, except that the mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:20:25:4, and all other operating steps are the same.

[0131] Comparative Example 1

[0132] The preparation method of the PLGA nano-drug carrier combination provided in this comparative example is the same as that in Example 2, except that silk fibroin is not used. The mass ratio of the components in the composition is: umbilical cord extract: γ-PGA: antioxidant (glutathione) = 50:25:5, and all other operating steps are the same.

[0133] Comparative Example 2

[0134] The preparation method of the PLGA nano-drug delivery combination provided in this comparative example is the same as that in Example 2, except that γ-PGA is not used. The mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: antioxidant (glutathione) = 50:20:5, and all other operating steps are the same.

[0135] Comparative Example 3

[0136] The preparation method of the PLGA nano-drug carrier combination provided in this comparative example is the same as that in Example 2, except that glutathione is not used. The mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: γ-PGA = 50:20:25, and all other operating steps are the same.

[0137] Comparative Example 4

[0138] The preparation method of the PLGA nano-drug carrier combination provided in this comparative example is the same as that in Example 2, except that silk fibroin and γ-PGA are not used, and the mass ratio of the components in the composition is: umbilical cord extract: antioxidant (glutathione) = 50:5, and all other operating steps are the same.

[0139] Comparative Example 5

[0140] The preparation method of the PLGA nano-drug carrier combination provided in this comparative example is the same as that in Example 2, except that silk fibroin and glutathione are not used. The mass ratio of the components in the composition is: umbilical cord extract: γ-PGA = 50:25, and all other operating steps are the same.

[0141] Comparative Example 6

[0142] The preparation method of the PLGA nano-drug carrier 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 the components in the composition is: umbilical cord extract: silk fibroin = 50:20, and all other operating steps are the same.

[0143] Comparative Example 7

[0144] The preparation method of the PLGA nano-drug carrier combination provided in this comparative example is the same as that in Example 2, except that silk fibroin, γ-PGA and glutathione are not used, and the composition contains 55% by mass of umbilical cord extract, and all other operating steps are the same.

[0145] Comparative Example 8

[0146] The preparation method of the PLGA nano-drug carrier combination provided in this comparative example is the same as that in Example 2, except that the mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:30:37:5, and all other operating steps are the same.

[0147] Comparative Example 9

[0148] The preparation method of the PLGA nano-drug carrier combination provided in this comparative example is the same as that in Example 2, except that the mass ratio of the components in the composition is: umbilical cord extract: silk fibroin: γ-PGA: antioxidant (glutathione) = 50:10:13:5, and all other operating steps are the same.

[0149] Comparative Example 10

[0150] The preparation method of the PLGA nano-drug carrier 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 enzyme is used for enzymatic hydrolysis, wherein the enzymatic hydrolysis conditions are: 0.1% collagenase type II + 0.05% trypsin, pH 7.0, enzymatic hydrolysis at 37°C for 6 hours, and all other operating steps are the same.

[0151] Comparative Example 11

[0152] The preparation method of the PLGA nano-drug carrier 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, wherein the enzymatic hydrolysis conditions are: 0.3% collagenase type II, pH 7.4, and enzymatic hydrolysis at 42°C for 3 hours. All other operating steps are the same.

[0153] Comparative Example 12

[0154] The preparation method of the PLGA nano-drug carrier 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 enzyme is used for enzymatic hydrolysis, wherein the enzymatic hydrolysis conditions are: 0.5% collagenase type II + 0.3% trypsin, pH 7.4, enzymatic hydrolysis at 42°C for 3h, and all other operating steps are the same.

[0155] Comparative Example 13

[0156] The chitosan nanoparticle drug delivery combination provided in this comparative example was prepared using the same method as in Example 1, except that the nanocarrier material was chitosan, prepared by the ionotropic gel method. The specific preparation method was as follows: 1% chitosan was dissolved in a 1% acetic acid solution (w / v) and stirred magnetically (500 rpm, 50°C) until completely transparent (approximately 4 hours). The pH was adjusted to 5.0-5.5 (0.1 M NaOH) to prevent excessive degradation. 0.1% sodium polyphosphate (TPP) was dissolved in deionized water (w / v) and sterilized with a 0.22 μm filter. The umbilical cord extract composition was dissolved in pH 6.0 PBS to a final concentration of 10 mg / mL. The mixture was mixed at a chitosan:composition ratio of 3:1 (e.g., 30 mg chitosan + 10 mg composition) and stirred for 30 minutes. The chitosan-composition mixture was added dropwise to the TPP solution (2:1 volume ratio) and stirred magnetically (300 rpm, room temperature). The dropwise addition rate was 1 mL / min and the reaction time was 30 minutes to form nanoparticles for later use.

[0157] Comparative Example 14

[0158] The liposome nano-drug delivery composition provided in this comparative example was prepared using the same method as in Example 1, except that the nano-carrier material was liposomes, prepared by the thin film hydration method. Specifically, the following preparation method was used: 60 mg of soybean lecithin and 20 mg of cholesterol were weighed and dissolved in 10 mL of a chloroform:methanol (2:1, v / v) mixture. Rotary evaporation (40°C water bath, 150 rpm) was performed to form a uniform lipid film. The organic solvent was completely removed by vacuum drying overnight. The umbilical cord extract composition was dissolved in pH 7.4 PBS to a final concentration of 10 mg / mL, preheated to 50°C. 10 mL of the composition solution was added to the lipid film and vortexed at 2000 rpm for 5 minutes. Sonication (100 W, 3 × 10-second pulses) was performed to promote liposome formation. The mixture was extruded through a polycarbonate membrane with a pore size of 200 nm 10 times to control particle size. Unencapsulated drug was removed by ultracentrifugation at 100,000 × g at 4°C for 1 hour. The nanoparticles were resuspended in PBS (pH 7.4) containing 5% sorbitol and stored at 4°C in the dark until use.

[0159] Test Example 1 Effects of different compositions on HaCaT cell proliferation rate and COL1A1 secretion

[0160] HaCaT cells (human immortalized keratinocytes) were cultured at a density of 5 × 10 3 Cells / well were seeded in a 96-well plate, the culture medium was DMEM + 10% FBS, and cultured at 37°C and 5% CO2 for 24 hours. The experimental groups were added with nanoformulations containing different ratios of compositions at a final concentration of 100 μg / mL and dissolved in the culture medium. The blank control group consisted of blank culture medium (negative control) and culture medium containing 10% FBS (positive control). After incubation for 48 hours, 10 μL of CCK-8 reagent (Dojindo) was added to each well and incubated for another 2 hours. The absorbance at 450 nm (reference wavelength 630 nm) was detected using a microplate reader, and the relative proliferation rate (%) of each composition (Example 2-10, Comparative Example 1-9) on HaCaT cells was calculated. The calculation formula is:

[0161] Cell proliferation rate (%) = (OD treatment-OD blank) / (OD control-OD blank) × 100%;

[0162] COL1 secretion was then assayed (ELISA). Cell culture and treatment were performed as in the CCK-8 assay. After 48 hours of culture, the supernatant was collected and centrifuged (3000 rpm, 10 minutes) to remove cell debris. Human type I collagen (COL1) ELISA kit (Abcam ab210579) was used according to the manufacturer's instructions.

[0163] The results of the measurement of the proliferation rate of HaCaT cells by different compositions are shown in Table 1. The results show that the nanocomposites 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 combination of two in the comparative example, and have a certain synergistic effect.

[0164] Table 1 Effects of different compositions on the proliferation rate of HaCaT cells

[0165]

[0166] The results of measuring the secretion of COL1A1 in HaCaT cells by different compositions are shown in Table 2, which shows that the nanocompositions of Examples 2-10 can significantly promote collagen secretion in HaCaT cells and increase the secretion of COL1A1. The effects are significantly better than those of using umbilical cord extract alone, and the combination of umbilical cord extract with silk fibroin or γ-PGA and antioxidants, as well as the combination of the three components, showing a significant synergistic effect.

[0167] Table 2 Effects of different compositions on COL1A1 secretion in HaCaT cells

[0168]

[0169] Test Example 2 Optimization of the Preparation Method of Umbilical Cord Extract

[0170] Since the existing extraction process of umbilical cord extract is complex and non-uniform, the preparation method has multiple steps and is cumbersome, and the extraction efficiency is low, in order to further improve the yield and purity of the umbilical cord extract, the process conditions of the preparation method of the umbilical cord extract in the composition of Example 2 were optimized, and different extraction method conditions were set for comparison (Comparative Examples 10-12) to compare the differences in the yield and purity of the umbilical cord extract obtained using different enzymatic hydrolysis conditions.

[0171] The yield and purity of umbilical cord extracts are usually evaluated based on the content of its active ingredients (such as hydroxyproline). The specific calculation method is as follows:

[0172] The calculation formula for the yield of umbilical cord extract is:

[0173]

[0174] Purity calculation formula for umbilical cord extract:

[0175]

[0176] The statistical differences in the yield and purity of umbilical cord extracts under different enzymatic hydrolysis conditions are shown in Table 3, which shows that the yield (≥53%) and purity (hydroxyproline ≥84 mg / g) of umbilical cord extracts can be further improved by optimizing the enzymatic hydrolysis process (collagenase type II + trypsin simultaneous enzymatic hydrolysis).

[0177] Table 3 Differences in yield and purity of umbilical cord extracts under different enzymatic hydrolysis conditions

[0178]

[0179] Further, according to the experimental method of Test Example 1, the PLGA nano-drug carrier combination prepared from umbilical cord extracts extracted under different enzymatic hydrolysis conditions was tested. The experimental method was the same as that of Test Example 1. The results of the effects of the compositions of different enzymatic hydrolysis processes on the proliferation rate of HaCaT cells are shown in Table 4, which shows that the compositions prepared from umbilical cord extracts extracted by different enzymatic hydrolysis processes affect cell proliferation, and the umbilical cord extracts with high yield and purity have better effects when used in combination; the results of the measured COL1A1 secretion are shown in Table 5, which shows that optimizing the enzymatic hydrolysis conditions to further increase the yield and purity of the umbilical cord extract can increase collagen secretion, and can also further improve the repair effect of the combination.

[0180] Table 4 Effects of different enzymatic hydrolysis process combinations on HaCaT cell proliferation rate

[0181]

[0182] Table 5 Effects of different enzymatic hydrolysis processes on COL1A1 secretion in HaCaT cells

[0183]

[0184] Test Example 3 Performance comparison of different nanocarriers

[0185] Due to the poor stability of umbilical cord extract, its active ingredients are easily inactivated, making long-term storage difficult. Furthermore, the limited route of administration makes traditional methods (such as topical application and injection) difficult to achieve stable delivery, thus compromising the repair effect. Therefore, the present invention utilizes different nanocarrier systems (Example 2, Comparative Examples 13-14) to deliver the composition, improving its poor stability and release efficiency, enhancing its long-term storage and transport stability, and improving its sustained release. The encapsulation efficiency and drug loading of the various carriers were measured using ultrafiltration centrifugation UV-Vis analysis at a wavelength of 280 nm. Stability, defined as the retention of bioactivity, was assessed using a HaCaT cell proliferation assay. The 72-hour release rate was determined by HPLC, and the released drug concentration was calculated as the cumulative release rate. Transdermal absorption was determined using a Franz diffusion cell assay.

[0186] The results, shown in Table 6, demonstrate that the PLGA nanocarrier achieves a high encapsulation efficiency for the composition. Its stability, drug loading capacity, transdermal absorption, and 72-hour release rate are superior to those of other chitosan and liposome nanocarriers, demonstrating excellent stability and sustained release, with a 72-hour sustained-release rate of ≥90%. Furthermore, a specific freeze-drying process significantly enhances the stability of the nanoformulation, resulting in >95% activity retention after 36 months of storage at room temperature.

[0187] Table 6 Comparison of performance of different nanocarrier compositions

[0188]

[0189] Different nanocarrier combinations were further tested according to the experimental method of Test Example 1. The experimental method was the same as that of Test Example 1. The effects of different nanocarrier compositions on the proliferation rate of HaCaT cells are shown in Table 7, and the results of the measured COL1A1 secretion are shown in Table 8. Through the innovative administration method of nanocarriers, stable delivery and long-term release of the active ingredients of the composition can be achieved, promoting cell proliferation, increasing collagen secretion, and improving the repair effect of skin tissue damage.

[0190] Table 7 Effects of different nanocarriers on the proliferation rate of HaCaT cells

[0191]

[0192] Table 8 Effects of different nanocarriers on COL1A1 secretion in HaCaT cells

[0193]

[0194] Test Example 4: Skin burn animal experiment

[0195] In this example, an animal model of skin burns was established to verify the efficacy of the composition in repairing skin burns. The specific method is as follows: SD rats weighing 200-250 g were used. Environmental conditions: temperature 22±2°C, humidity 50±10%, 12-hour light / dark cycle, free access to food and water. Each group consisted of 5 rats, divided into a model group and an experimental group.

[0196] Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg / kg) to ensure complete anesthesia. Hair was shaved from the back of the rats using an electric shaver, covering an area approximately 4 cm × 4 cm. The skin was disinfected with 75% ethanol to prevent infection. A second-degree burn model was created using a constant-temperature metal burn instrument set to 100°C. The instrument was placed in contact with the skin for 10 seconds. Two burn wounds, approximately 1.5 cm in diameter and 2 cm apart, were created on each rat.

[0197] 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 nanocomposition of Example 2 (10 mg / mL) once a day. The wound healing and repair were photographed and measured at 0, 2, 4, and 7 days. The measurement method used a digital camera to photograph the wound, ensuring 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 7 days. The animals were anesthetized, and a small part of the skin tissue at the wound was taken for HE staining analysis and tissue homogenate ELISA to detect the levels of inflammatory factors IL-6 and IL-1β.

[0198] result Figure 1 As shown in Figure 2, compared with the model group, the Example 2 group showed a faster repair effect at the same time. Figure 2 As shown, in the model control group, skin tissue showed necrosis of the epidermis, dermis, and subcutaneous layer, nuclear fragmentation or dissolution, mild bleeding, and a large number of pus cells; extensive fibroblast proliferation was seen in the subcutaneous layer, with a small amount of neovascularization and congestion, accompanied by a small amount of lymphocyte infiltration. In the example 2 group, skin tissue showed necrosis of the epidermis and dermis, nuclear fragmentation or dissolution, and a small amount of pus cells; a large number of neovascularization was seen, accompanied by a small amount of lymphocyte infiltration, and more new skin tissue.

[0199] The expression levels of inflammatory factors in the burned skin tissue homogenate on day 7 were further examined by ELISA. The results are shown in Table 9. Compared with the model group, the inflammatory factors in the skin tissue of the Example 2 group were significantly reduced. This indicates that the nanocomposition provided by the present invention has a significant effect on repairing skin tissue damage and is suitable for treating various skin injuries such as burns, wounds, and ulcers.

[0200] Table 9 Levels of inflammatory factors IL-6 and IL-1β

[0201]

[0202] In summary, the skin tissue damage repair composition provided by the present invention has a synergistic effect between the components after scientific combination ratio adjustment, can promote cell proliferation, increase collagen secretion, and has a significant skin tissue damage repair effect, which is better than the umbilical cord extract alone, or any combination of two or three of its components. The system verifies the significant effect of the composition of the present invention in promoting wound healing, tissue and inflammation relief through cell model experiments and burn animal model experiments, providing more and better products for the treatment of various skin injuries. At the same time, the present invention further improves the yield and purity of the active ingredient umbilical cord extract in the composition by optimizing the preparation process of the umbilical cord extract and innovating the mode of administration, and improves the stability of the composition, achieving stable delivery and long-term release of the active ingredient, which can further enhance the repair effect of skin damage and has important clinical application value and market prospects.

[0203] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A PLGA nanoparticle drug-loaded particle, characterized in that: The skin tissue damage repair composition is prepared by encapsulating polylactic acid-glycolic acid copolymer PLGA; the mass ratio of the PLGA to the composition is (3-4):1; The skin tissue damage repair composition is composed of the following components in the following mass ratios: Composition: Umbilical cord extract: Silk fibroin: γ-PGA: Antioxidant = (40-60): (10-25): (20-30): (2-8); The preparation method of the umbilical cord extract comprises the following steps: crushing the umbilical cord and then performing enzymatic hydrolysis; the enzymatic hydrolysis conditions are: 0.25-0.35% collagenase type II and 0.12-0.18% trypsin, pH 7.2-7.6, temperature 42±1°C, stirring speed 200-300 rpm, and time 2.5-3.5 hours; and obtaining the umbilical cord extract by ultrafiltration concentration and freeze-drying after enzymatic hydrolysis.

2. The drug-loaded nanoparticles according to claim 1, characterized in that: The skin tissue damage repair composition is composed of the following components in the following mass ratio: umbilical cord extract: silk fibroin: γ-PGA: antioxidant = (45-55): (18-22): (23-27): (4-6).

3. The drug-loaded nanoparticles according to claim 1 or 2, characterized in that: The antioxidant is selected from glutathione, vitamin C or coenzyme Q10.

4. The method for preparing the drug-loaded nanoparticles according to claim 1 or 2, comprising the steps of: S1: Solution preparation: umbilical cord extract, silk fibroin, γ-PGA, and antioxidant are prepared into solutions for later use; S2: Premixing silk fibroin and γ-PGA: Mix silk fibroin and γ-PGA according to the mass ratio, stir at 200-300 rpm for 20-40 minutes at 25-30°C, and adjust the pH of the mixture to 6.5-7.0 to prepare a premixed solution; S3: Add umbilical cord extract: Slowly add umbilical cord extract to the premixed solution at a rate of 0.8-1 mL / min, stirring at 100-200 rpm, at a temperature of 25-30°C, to avoid precipitation; S4: adding antioxidants: then adding antioxidants in a dark condition, ultrasonically dispersing at 30-40 kHz for 3-5 minutes at a temperature of 25-30°C, and filtering and sterilizing the final mixture.

5. Use of the drug-loaded nanoparticles according to any one of claims 1 to 4 in the preparation of products for repairing skin damage.

6. A product, characterized in that Containing the nano drug-loaded particles according to any one of claims 1 to 4.

7. The method for preparing the drug-loaded nanoparticles according to any one of claims 1 to 4 is: (1) Preparation of colostrum: The skin tissue damage repair composition is mixed with trehalose to prepare an aqueous phase; PLGA is dissolved in an organic solvent to prepare an oil phase; the aqueous phase and the oil phase are mixed at a volume ratio of 1:5-15 and then ultrasonicated to prepare colostrum; (2) Emulsification: Inject colostrum into the external aqueous phase polyvinyl alcohol solution, homogenize and emulsify, and then stir to evaporate the organic solvent to obtain an emulsion; (3) Nanoparticle purification: the emulsion is centrifuged, the precipitate is collected, washed, free polyvinyl alcohol (PVA) and unencapsulated components are removed, and the precipitate is resuspended in a solution; (4) Freeze-drying: Pre-freeze the sample at -80~-100℃ for 2-3 hours, dry it after it is completely solidified to obtain nanoparticles, and store it in a sealed, dark place at no more than 25℃.

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