Composition containing polypeptide exosome and application of composition in preparation of products with skin repairing ability

Abstract

The invention discloses a composition containing a polypeptide exosome and application of the composition in preparation of a product with skin repair ability, and particularly relates to the technical field of biology, the composition comprises polypeptide-exosome composite nanoparticles, and a polypeptide containing a GRGDS motif and an umbilical cord Wharton's jelly exosome are covalently crosslinked; the lipid double-layer coating layer comprises phosphatidylcholine, cholesterol and PEG-2000, and the molar ratio of the phosphatidylcholine to the cholesterol to the PEG-2000 is 5: 3: 2; the synergist is prepared from 0.1 to 5 percent of epicatechin gallate and 2 to 8 percent of recombinant collagen III oligopeptide. The nano-liposome is prepared through microfluidic self-assembly and a film hydration-ultrasonic intercalation method, and can be loaded on a degradable microneedle patch, so that targeted slow release of a wound part is realized. Through the synergistic effect of targeted delivery, oxidation resistance and collagen regeneration, the skin repair efficiency is remarkably improved, and the healing time is shortened.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a polypeptide-containing exosome composition and its application in preparing a product with skin repairing ability. Background Art

[0002] As the body's largest organ, impaired skin integrity (e.g., through trauma, burns, and chronic ulcers) can lead to infection, scarring, and even organ dysfunction. Currently, key challenges in the field of skin repair include low drug delivery efficiency at the site of injury, inhibitory repair processes caused by the inflammatory microenvironment, insufficient extracellular matrix (ECM) regeneration, and the limited sustained-release properties of traditional formulations.

[0003] Mesenchymal stem cell exosomes, rich in growth factors (such as EGF and TGF-β), miRNAs, and lipid mediators, have demonstrated potential for anti-inflammatory, pro-angiogenic, and fibroblast activation in skin repair. However, non-targeted delivery results in rapid clearance of exosomes from the circulation, making them difficult to accumulate at the site of injury. For example, when conventional exosome suspensions are directly applied, less than 10% of exosomes penetrate the epidermis and reach the damaged area, with the majority being blocked by the stratum corneum or lost with body fluids. Furthermore, the poor stability of exosomes (e.g., uneven particle size and aggregation due to protein adsorption) further limits their clinical application.

[0004] Oxidative stress is a key pathological mechanism following skin injury. Excessive reactive oxygen species (ROS) can damage cell membrane lipids, DNA, and the ECM, delaying healing. Epicatechin gallate, a potent antioxidant, can scavenge ROS and inhibit inflammatory factors (such as IL-6 and TNF-α), but its poor water solubility and short in vivo half-life (approximately 2 hours) limit its efficacy. On the other hand, recombinant human collagen type III short peptides can directly activate COL3A1 gene expression in fibroblasts and promote new collagen synthesis. However, when used alone, they have low penetration efficiency and are easily degraded by matrix metalloproteinases (MMPs). Existing technologies have failed to achieve synergistic delivery by simply combining antioxidants with collagen fragments, resulting in a temporal and spatial disconnect between antioxidant and pro-regenerative effects.

[0005] While traditional liposomes (such as phosphatidylcholine-cholesterol systems) possess certain encapsulation capabilities, they lack environmental responsiveness. For example, in the neutral environment of normal tissue, the negative surface charge of liposomes is easily recognized and cleared by serum proteins. However, in the acidic microenvironment of a wound site (pH 5.5-6.5), traditional liposomes are unable to trigger drug release, resulting in "ineffective retention" or "premature burst release." Furthermore, non-PEG-modified liposomes have poor particle size stability (PDI > 0.3) and are prone to aggregation during storage or delivery, reducing encapsulation efficiency.

[0006] Therefore, a polypeptide-containing exosome composition and its application in preparing a product with skin repair ability are proposed. Summary of the Invention

[0007] In order to overcome the above-mentioned defects of the prior art, the present invention provides a polypeptide-containing exosome composition and its application in preparing products with skin repair ability, so as to solve the problems raised in the above-mentioned background technology.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a polypeptide-containing exosome composition, characterized in that it comprises the following components: Peptide-exosome composite nanoparticles are formed by microfluidic self-assembly of a hydrophobic peptide core and surface-covalently modified mesenchymal stem cell exosomes, wherein the peptide sequence contains a repetitive Gly-Arg-Gly-Asp-Ser (GRGDS) motif; The lipid bilayer coating, composed of phosphatidylcholine, cholesterol and polyethylene glycol-2000 in a molar ratio of 5:3:2, is coated on the surface of the composite nanoparticles to form a nanoliposome structure; The synergist comprises 0.1-5% by mass of epicatechin gallate and 2-8% by mass of a recombinant human collagen type III short peptide fragment.

[0009] By covalently cross-linking the hydrophobic peptide core (GRGDS motif) with the exosome surface, the targeting property of the peptide (GRGDS binds to cell surface integrins) and the natural repair signal transmission ability of the exosome are utilized to enhance the directional effect on skin cells.

[0010] Phosphatidylcholine provides biocompatibility, cholesterol enhances membrane stability, and PEG-2000 extends circulation time and imparts pH responsiveness. PEG is shed in acidic environments, exposing positive charges to enhance cellular uptake.

[0011] Epicatechin gallate improves antioxidant capacity and recombinant collagen type III short peptide promotes collagen synthesis to synergistically repair damaged tissues.

[0012] Thus, targeted delivery and sustained release can be achieved through the composite structure, thereby improving transdermal efficiency; antioxidant and collagen regeneration work synergistically to increase the repair speed; pH-responsive release reduces systemic toxicity and local drug concentration is controllable.

[0013] Preferably, the mesenchymal stem cell exosomes are selected from umbilical cord Wharton's jelly-derived exosomes, whose surface expression of CD63 and CD81 markers is higher than 1×10 4 particles / μg, and are purified by low-temperature ultracentrifugation combined with size exclusion chromatography, with a particle size distribution of 80-150 nm.

[0014] Umbilical cord Wharton's jelly-derived exosomes are rich in growth factors and miRNA, promoting cell proliferation and anti-inflammation; and the purity of exosomes is verified by high expression of CD63 / CD81 markers to ensure functional stability; impurities are removed by ultracentrifugation, and size exclusion chromatography accurately separates 80-150nm active exosomes, thereby improving exosome activity and increasing the inhibition rate of inflammatory factors, and ensuring delivery consistency through particle size uniformity.

[0015] Preferably, the sequence of the hydrophobic polypeptide core is Ac-GRGDS-(GVPGV)6-NH2, wherein GVPGV is an elastin repeating unit with a molecular weight of 8-12 kDa, and a palmitoyl modification is introduced at the end by solid phase synthesis.

[0016] Cell adhesion is enhanced through the GRGDS motif, and the GVPGV repeating unit gives the nanoparticles elasticity and adapts to the dynamic microenvironment of the skin; combined with palmitoyl modification, the hydrophobic tail drives self-assembly to form a stable core, thereby enhancing the mechanical strength of the nanoparticles, improving the structural integrity under shear force, and improving cell adhesion efficiency.

[0017] Preferably, the thickness of the lipid bilayer coating is 8-15 nm, and polyethylene glycol-2000 is anchored to the phosphatidylcholine head through an amide bond to form a surface charge inversion structure with pH responsiveness.

[0018] Membrane fluidity is optimized by using a 5:3:2 phospholipid / cholesterol / PEG ratio, and PEG is linked through amide bonds to achieve acid-sensitive cleavage; and when pH < 6.5, PEG falls off, exposing positive charges and enhancing cell membrane fusion, thereby increasing drug release at the inflammatory site and reducing leakage from normal tissues.

[0019] Preferably, the amino acid sequence of the recombinant human collagen type III short peptide fragment is GPPGPPGPRGQOGVMGFO, which is prepared by a Pichia pastoris expression system and loaded into the inner cavity of the nanoliposome in the form of a lyophilized powder.

[0020] By expressing in Pichia pastoris to avoid animal-derived risks, the GPPGPPGPRGQOGVMGFO sequence simulates the natural type III collagen domain and directly activates the fibroblast synthesis pathway; by electroporation, the short peptide is embedded in the liposome cavity to avoid enzymatic degradation, thereby increasing the amount of collagen synthesis, and the freeze-drying process is used to improve the activity retention rate.

[0021] A method for preparing the above-mentioned polypeptide-containing exosome composition comprises the following steps: (1) The hydrophobic peptide was dissolved in tetrahydrofuran and mixed with phosphate buffer at a flow rate of 200-500 μL / min through a microfluidic chip to form nanomicelles with a particle size of 50-80 nm; (2) The purified mesenchymal stem cell exosomes were mixed with the nanomicelles obtained in step (1) at a mass ratio of 1:10, 1-ethyl-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC / NHS) crosslinker was added, the mixture was reacted at 4°C for 12 hours, and the composite nanoparticles were obtained by centrifugation; (3) Phosphatidylcholine, cholesterol, and polyethylene glycol-2000 were dissolved in chloroform, rotary evaporated to form a film, and then hydrated and processed by a high-pressure homogenizer at 800 bar for 5 times to form blank liposomes; (4) mixing the composite nanoparticles of step (2) and the blank liposomes of step (3) in a mass ratio of 1:3, and incubating at 55° C. for 2 hours using a thin film hydration-ultrasonic intercalation method to form a coating structure; (5) Epicatechin gallate and recombinant collagen type III short peptide fragment are dissolved in pH 7.4 buffer and loaded into the nanoliposome cavity of step (4) by electroporation.

[0022] By precisely controlling the peptide / exosome ratio (1:10), uniform nanomicelles are formed; the carboxyl-amino covalent bond ensures the stability of the composite particles; and 800 bar pressure is used to uniformly coat the lipid layer, avoiding phase separation and improving drug loading efficiency.

[0023] Preferably, the ultrasonic intercalation method in step (4) uses a probe-type ultrasonic instrument with a frequency of 40 kHz and a power of 100 W, with pulse treatment every 30 seconds to prevent overheating and damage to the structure, and the total duration is 10 minutes.

[0024] A use of the above-mentioned polypeptide exosome composition in the preparation of a medical device with skin repair ability, wherein the medical device is a degradable microneedle patch, comprising: The basal layer is composed of hyaluronic acid-gelatin composite hydrogel with a thickness of 200-300 μm; The microneedle array has a height of 500-800 μm, a single needle drug loading capacity of 10-15 μg, and the needle tip is embedded with the composition and 0.1% trehalose freeze-drying protective agent.

[0025] The drug-loaded nanoparticles are released by dissolving in the tissue fluid in the hyaluronic acid-gelatin matrix; the 500-800μm height penetrates the stratum corneum, and the trehalose protective agent maintains the freeze-dried activity, thereby improving the transdermal efficiency and shortening the wound healing time.

[0026] Preferably, the microneedle patch dissolves within 30 minutes after contacting tissue fluid, and the released composite nanoparticles form a sustained-release gradient at the skin wound site, and the local concentration of epicatechin gallate is maintained at ≥5 μm within 24 hours.

[0027] Epicatechin gallate is gradually degraded through the epidermis and released with zero-order kinetics; 5μm is the effective antioxidant threshold, which continuously inhibits ROS generation, thereby reducing drug concentration fluctuations within 24 hours and reducing scar area.

[0028] Technical effects and advantages of the present invention: The hydrophobic polypeptide core is bound to the exosomes through covalent bonds, and the targeting ability of GRGDS to cell surface integrins is used to achieve precise positioning of the skin trauma site; the PEG-modified lipid bilayer is negatively charged in normal tissues to avoid nonspecific adsorption. At the inflammatory site, PEG falls off and exposes positive charges, enhancing cellular uptake and drug release, forming a local sustained-release gradient.

[0029] Umbilical cord Wharton's jelly-derived exosomes are rich in growth factors such as EGF, TGF-β, and miR-21, which can promote fibroblast proliferation and inhibit inflammatory response; epicatechin gallate scavenges free radicals through antioxidant action, and recombinant collagen III short peptide directly activates the collagen synthesis pathway. The two work together to accelerate wound healing.

[0030] The lipid bilayer is homogenized under high pressure to form a uniform membrane structure with a thickness of 8-15nm. It is combined with a palmitoyl-modified polypeptide core to enhance the mechanical strength and shear stability of the nanoparticles. The hyaluronic acid-gelatin microneedle patch can be dissolved within 30 minutes, and the trehalose protective agent maintains the activity of the ingredients. It has high transdermal efficiency and no animal-derived risk. DETAILED DESCRIPTION

[0031] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0032] Example 1 This embodiment provides a method for preparing a polypeptide-containing exosome composition, the raw materials of which include: Peptide-exosome composite nanoparticles: hydrophobic peptide core: Ac-GRGDS-(GVPGV)6-NH2 (palmitoyl modified, molecular weight 10kDa) 10 copies, umbilical cord Wharton's jelly exosomes (CD63 / CD81 ≥ 1×10 4 particles / μg, particle size 80nm) 1 portion; Lipid bilayer coating: 5 parts phosphatidylcholine, 3 parts cholesterol, 2 parts polyethylene glycol-2000 (PEG-2000); Synergist: epicatechin gallate 0.5 parts, recombinant collagen III short peptide (GPPGPPGPRGQOGVMGFO) 5 parts.

[0033] The specific preparation steps are: (1) The hydrophobic peptide was dissolved in tetrahydrofuran and mixed with phosphate buffer at a flow rate of 200-500 μL / min through a microfluidic chip to form nanomicelles with a particle size of 50-80 nm; (2) The purified mesenchymal stem cell exosomes were mixed with the nanomicelles obtained in step (1) at a mass ratio of 1:10, 1-ethyl-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC / NHS) crosslinker was added, the mixture was reacted at 4°C for 12 hours, and the composite nanoparticles were obtained by centrifugation; (3) Phosphatidylcholine, cholesterol, and polyethylene glycol-2000 were dissolved in chloroform, rotary evaporated to form a film, and then hydrated and processed by a high-pressure homogenizer at 800 bar for 5 times to form blank liposomes; (4) the composite nanoparticles of step (2) and the blank liposomes of step (3) were mixed in a mass ratio of 1:3, and incubated at 55° C. for 2 hours using a thin film hydration-ultrasonic intercalation method to form a coating structure. The ultrasonic intercalation method used a probe-type ultrasonic instrument with a frequency of 40 kHz and a power of 100 W, with pulse treatment every 30 seconds; (5) Epicatechin gallate and recombinant collagen type III short peptide fragment are dissolved in pH 7.4 buffer solution, and loaded into the nanoliposome cavity of step (4) by electroporation to prepare a polypeptide-containing exosome composition.

[0034] Example 2 This example provides a method for preparing a polypeptide-containing exosome composition. The difference from Example 1 is that the particle size of the umbilical cord Wharton's jelly exosomes is adjusted to 120 nm and screened by fractional centrifugation. The rest is the same as Example 1.

[0035] Example 3 This example provides a method for preparing a polypeptide-containing exosome composition, which differs from Example 1 in that 1 part of epicatechin gallate and 3 parts of recombinant collagen III short peptide are used, and the rest is the same as Example 1.

[0036] Comparative Example 1 This comparative example provides a method for preparing a polypeptide-containing exosome composition, which differs from Example 1 in that the umbilical cord Wharton's jelly exosomes are removed, and the amount of the hydrophobic polypeptide core is increased to 11 parts, and the rest is the same as Example 1.

[0037] Comparative Example 2 This comparative example provides a method for preparing a polypeptide-containing exosome composition. The difference from Example 1 is that the lipid layer only contains 5 parts of phosphatidylcholine and 3 parts of cholesterol (without PEG-2000), and the rest is the same as Example 1.

[0038] The compositions prepared in Examples 1-3 and Comparative Examples 1-2 were tested.

[0039] Physical properties testing Particle size and potential: measured by dynamic light scattering (DLS), with each group of samples measured 3 times and the average value was taken.

[0040] Encapsulation efficiency: After ultrafiltration and centrifugation, the epicatechin gallate content was determined by HPLC using the formula: Encapsulation efficiency (%) = drug amount in liposomes / total drug amount × 100%. The test results are shown in Table 1.

[0041] Table 1: Physical properties test results data table Group Particle size (nm) PDI Zeta potential (mV) Encapsulation efficiency (%) Example 1 110±5 0.18±0.02 -12.5±1.2 89.5±2.1 Example 2 145±8 0.22±0.03 -11.8±1.5 85.3±1.8 Example 3 108±4 0.19±0.01 -13.2±1.0 88.7±1.9 Comparative Example 1 95±3 0.25±0.04 -15.0±2.0 72.0±3.5 Comparative Example 2 120±6 0.30±0.05 -20.0±2.5 78.0±2.8 2. Skin repair function test In vitro experiments: Fibroblast proliferation: OD value was measured by CCK-8 method (cultured for 48 hours); scratch healing rate: the area covered by cell migration was calculated under a microscope after 24 hours.

[0042] In vivo experiment: SD rat skin defect model (5 mm in diameter) was prepared. The composition was applied daily, and the healing rate was calculated after 7 days: healing rate (%) = (initial area - remaining area) / initial area × 100%; the test results are shown in Tables 2 and 3 below.

[0043] Table 2: In vitro cell experiment results data table Group Proliferation rate (OD value) Scratch healing rate (%) Blank group 1.00±0.05 35.0±4.0 Example 1 1.85±0.10 78.5±5.0 Example 2 1.72±0.08 72.0±4.5 Example 3 1.68±0.09 70.0±4.0 Comparative Example 1 1.20±0.06 48.0±3.5 Comparative Example 2 1.45±0.07 55.0±4.0 Table 3: In vivo wound healing results (Day 7) Group Initial area (mm²) Remaining area (mm²) Healing rate (%) Blank group 20.0±1.0 12.5±1.2 37.5±3.0 Example 1 20.0±1.0 3.0±0.5 85.0±2.0 Example 2 20.0±1.0 4.8±0.8 76.0±2.5 Example 3 20.0±1.0 5.2±0.7 74.0±2.3 Comparative Example 1 20.0±1.0 8.5±1.0 57.5±3.5 Comparative Example 2 20.0±1.0 7.0±0.9 65.0±3.0 3. Sustained release performance test In vitro release: In a pH 7.4 buffer solution (37°C with shaking), the epicatechin concentration was regularly measured by HPLC. The test results are shown in Table 4.

[0044] Table 4: Epicatechin concentration and gallate concentration test data time Example 1 Concentration (μm) Example 2 Concentration (μm) Comparative Example 2 Concentration (μm) 0.5 4.2±0.3 3.8±0.2 5.5±0.4 2 6.8±0.5 6.2±0.4 8.0±0.6 12 5.5±0.4 5.0±0.3 3.5±0.3 24 5.2±0.3 4.8±0.2 2.0±0.2 As can be seen from Tables 1-4 above, due to the presence of exosomes, the encapsulation efficiency of Examples 1-3 (85%-89%) was significantly higher than that of Comparative Example 1 (72%), confirming that the covalent cross-linking of exosomes and polypeptides can improve the stability of the nanostructure.

[0045] Comparative Example 1 lacks exosomes, and the fibroblast proliferation rate is only 65% of that in Example 1, indicating that the bioactive components of exosomes (such as growth factors) are the core of the repair function.

[0046] The absolute value of the Zeta potential (-11 to -13 mV) of Examples 1-3 was lower than that of Comparative Example 2 (-20 mV), indicating that the steric hindrance effect of PEG-2000 reduced the nonspecific adsorption of nanoparticles and prolonged the in vivo circulation time, which is consistent with the sustained-release performance of Examples 1-3 in Table 4 (24 h concentration ≥ 5 μM).

[0047] Example 1 (0.5% epicatechin + 5% collagen III) had the highest healing rate (85%), while Example 3 (1% epicatechin + 3% collagen III) had a slightly lower repair efficiency due to the possible cytotoxicity of the epicatechin concentration being too high, indicating that the enhancer must maintain an optimal ratio.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polypeptide-containing exosome composition, characterized in that: Contains the following components: Peptide-exosome composite nanoparticles are formed by microfluidic self-assembly of a hydrophobic peptide core and surface-covalently modified mesenchymal stem cell exosomes, wherein the peptide sequence contains a repetitive Gly-Arg-Gly-Asp-Ser (GRGDS) motif; The lipid bilayer coating, composed of phosphatidylcholine, cholesterol and polyethylene glycol-2000 in a molar ratio of 5:3:2, is coated on the surface of the composite nanoparticles to form a nanoliposome structure; The synergist comprises 0.1-5% by mass of epicatechin gallate and 2-8% by mass of a recombinant human collagen type III short peptide fragment.

2. The polypeptide-containing exosome composition according to claim 1, characterized in that: The mesenchymal stem cell exosomes are selected from umbilical cord Wharton's jelly-derived exosomes, and the expression levels of CD63 and CD81 markers on their surface are higher than 1×10 4 The particles were purified by low-temperature ultracentrifugation combined with size exclusion chromatography, with a particle size distribution of 80-150 nm.

3. The polypeptide-containing exosome composition according to claim 2, characterized in that: The sequence of the hydrophobic polypeptide core is Ac-GRGDS-(GVPGV)6-NH2, wherein GVPGV is an elastin repeating unit with a molecular weight of 8-12 kDa, and a palmitoyl modification is introduced at the end by solid phase synthesis.

4. The polypeptide-containing exosome composition according to claim 1, characterized in that: The thickness of the lipid bilayer coating is 8-15 nm, and polyethylene glycol-2000 is anchored to the phosphatidylcholine head through an amide bond to form a surface charge reversal structure with pH responsiveness.

5. The polypeptide-containing exosome composition according to claim 1, characterized in that: The amino acid sequence of the recombinant human collagen type III short peptide fragment is GPPGPPGPRGQOGVMGFO, which is prepared by a Pichia pastoris expression system and loaded into the inner cavity of the nanoliposome in the form of a freeze-dried powder.

6. A method for preparing the polypeptide-containing exosome composition according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) The hydrophobic peptide was dissolved in tetrahydrofuran and mixed with phosphate buffer at a flow rate of 200-500 μL / min through a microfluidic chip to form nanomicelles with a particle size of 50-80 nm; (2) The purified mesenchymal stem cell exosomes were mixed with the nanomicelles obtained in step (1) at a mass ratio of 1:10, 1-ethyl-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC / NHS) crosslinker was added, the mixture was reacted at 4°C for 12 hours, and the composite nanoparticles were obtained by centrifugation; (3) Phosphatidylcholine, cholesterol, and polyethylene glycol-2000 were dissolved in chloroform, rotary evaporated to form a film, and then hydrated and processed by a high-pressure homogenizer at 800 bar for 5 times to form blank liposomes; (4) mixing the composite nanoparticles of step (2) and the blank liposomes of step (3) in a mass ratio of 1:3, and incubating at 55° C. for 2 hours using a thin film hydration-ultrasonic intercalation method to form a coating structure; (5) Epicatechin gallate and recombinant collagen type III short peptide fragment are dissolved in pH 7.4 buffer and loaded into the nanoliposome cavity of step (4) by electroporation.

7. The method for preparing a polypeptide-containing exosome composition according to claim 6, characterized in that: The ultrasonic intercalation method in step (4) uses a probe-type ultrasonic instrument with a frequency of 40 kHz and a power of 100 W, with pulse treatment every 30 seconds for a total time of 10 minutes.

8. Use of the polypeptide-containing exosome composition according to any one of claims 1 to 5 in preparing a medical device with skin repairing ability, characterized in that: The medical device is a degradable microneedle patch, which includes: The basal layer is composed of hyaluronic acid-gelatin composite hydrogel with a thickness of 200-300 μm; The microneedle array has a height of 500-800 μm, a single needle drug loading capacity of 10-15 μg, and the needle tip is embedded with the composition and 0.1% trehalose freeze-drying protective agent.

9. The use according to claim 8, characterized in that The microneedle patch dissolves within 30 minutes after contacting tissue fluid, and the released composite nanoparticles form a sustained-release gradient at the skin wound site, and the local concentration of epicatechin gallate is maintained at ≥5 μm within 24 hours.