Recombinant human fibronectin composition as well as preparation method and application thereof

Abstract

The invention discloses a recombinant human fibronectin composition as well as a preparation method and application thereof, and belongs to the technical field of cosmetics. According to the composition of the recombinant human fibronectin provided by the invention, an exosome-nanostructure lipid carrier dual-delivery system is adopted to carry out entrapment delivery on a palmitamide modified recombinant human fibronectin fragment, so that the drug loading efficiency can be remarkably improved. Meanwhile, through a double-delivery system, the stability, the anti-flocculation capability and the slow release performance of the FN can be improved, it is ensured that the FN can stably and continuously exert the activity capability, the composition is endowed with the excellent barrier recovery capability, the water loss rate is reduced, and the composition can be widely applied to the technical field of cosmetic preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and more particularly to a recombinant human fibronectin composition, a preparation method thereof, and an application thereof. Background Art

[0002] Recombinant human fibronectin (FN) is an important extracellular matrix glycoprotein, widely present in human tissues. It participates in key physiological processes such as cell adhesion, migration, proliferation, differentiation, and tissue repair. It is widely used in medical repair, medical diagnosis, cell culture, and other fields. Furthermore, as a bioactive protein with cell adhesion, repair, and regeneration capabilities, recombinant human fibronectin is also increasingly being used in the cosmetic and skincare field to repair skin.

[0003] However, its actual application will have the following problems, such as stability: recombinant human fibronectin is sensitive to temperature, pH and enzymes. Therefore, it is unstable under different conditions, during long-term storage, and in compatibility with other matrices when used in cosmetics. It is easy to flocculate, thus affecting its activity. For the application of large molecular weight proteins, their inherent stability and compatibility are extremely important. Therefore, solving the application stability problem of large molecular weight proteins is conducive to the application of this type of raw materials in different scenarios and helps to exert the efficacy of products; Transdermal absorption limitation: recombinant human fibronectin has a large molecular weight (about 440 kDa) and has difficulty passing through the stratum corneum barrier, which affects its biological activity; Activity retention: The RGD (arginine-glycine-aspartic acid) sequence of recombinant human fibronectin is key to its function, but it may be ineffective due to folding changes in the formula.

[0004] There are some prior art studies on fibronectin compositions, such as Chinese invention patent CN 118370698 A, which discloses the use of a fibronectin-containing composition in skin care products and the composition itself. Another example is Chinese invention patent CN 117679323 A, which discloses a recombinant fibronectin composition and its use in skin care products for sensitive skin. However, these patents only address improvements in a single area: for example, CN 118370698 A only improves storage stability, while CN 117679323 A is more suitable for sensitive skin. There is little research on sustained-release transdermal properties, anti-flocculation, and long-term activity retention.

[0005] Therefore, how to provide a recombinant human fibronectin composition with sustained-release transdermal, anti-flocculation and long-term activity retention properties, as well as a preparation method and application thereof, is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0006] In view of this, the present invention provides a recombinant human fibronectin composition, a preparation method and application thereof, and successfully prepares a recombinant human fibronectin composition with sustained-release transdermal, anti-flocculation and long-term activity retention properties, thereby improving the application effect.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: A recombinant human fibronectin composition comprises the following components in weight percentage: 0.01%-1.0% recombinant human fibronectin, 1%-5% human bone marrow mesenchymal stem cell exosomes, 3%-10% nanostructured lipid carrier, 0.5%-2% anti-flocculation stabilizer, 0.1%-1% citric acid-disodium hydrogen phosphate buffer, 8-10% glycerol, 0.1-0.5% hyaluronic acid, and the balance is ultrapure water to 100%.

[0008] As a preferred technical solution, the C-terminus of the recombinant human fibronectin is palmitoylated; the particle size of the human bone marrow mesenchymal stem cell exosomes is 80-150 nm; and the nanostructured lipid carrier is composed of ceramide III, squalene and hydrogenated phosphatidylcholine in a weight ratio of 0.2-1:1-3:0.1-0.5.

[0009] As a preferred technical solution, the anti-flocculation precipitant consists of Poloxamer 188 and trehalose in a weight ratio of 0.5-1:2; the pH of the citric acid-disodium hydrogen phosphate buffer is 6.5-6.8.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned recombinant human fibronectin composition, which is prepared according to the above-mentioned component ratio and specifically comprises the following steps: (1) Modification of recombinant human fibronectin: Take recombinant human fibronectin, dissolve it in PBS solution, then add palmitic acid-NHS ester, react at 20-25℃ in the dark for 3-4 hours, and remove unreacted palmitic acid-NHS ester by semipermeable membrane dialysis to obtain palmitoylated recombinant human fibronectin; (2) Exosome loading: human bone marrow mesenchymal stem cell exosomes were mixed with palmitoylated recombinant human fibronectin, incubated at 35-37°C with gentle shaking for 2-3 h, and ultracentrifuged to prepare recombinant human fibronectin-exosome complexes; (3) Preparation of lipid phase and aqueous phase: Place the nanostructured lipid carrier at 65-75°C and melt mix until transparent to prepare the lipid phase; disperse the recombinant human fibronectin-exosome complex in ultrapure water at 65-75°C to prepare the aqueous phase; (4) High-pressure homogenization: slowly pour the lipid phase from step (3) into the aqueous phase, pre-emulsify with high-speed shearing, and then homogenize under high pressure to obtain a nanostructured lipid carrier suspension; (5) Addition of stabilizer and active ingredient: dissolve the anti-flocculating stabilizer in citric acid-disodium hydrogen phosphate buffer and mix well to prepare a stabilizer solution. The obtained stabilizer solution is mixed with the NLC suspension and homogenized by ultrasonication. Then, glycerol is added and stirred until completely dissolved. (6) Addition of hyaluronic acid: pre-dissolve hyaluronic acid in ultrapure water, let it stand for 3-5 hours to fully hydrate, then add it to the system in step (5), add ultrapure water to make up to 100%, and adjust the pH to 6.8±0.2; filter with a sterile filter membrane, and dispense into a light-proof container to prepare a recombinant human fibronectin composition.

[0011] The beneficial effects of the above step (1) are as follows: palmitoylation modification of the C-terminus can enhance the lipid solubility of recombinant human fibronectin (FN). Palmitoyl chains are long hydrocarbon chains (hydrophobic groups). After modification, the hydrophobicity of the FN surface is enhanced, and the compatibility with skin stratum corneum lipids (such as ceramide and cholesterol) is improved. In addition, the sustained release time can be prolonged. Palmitoylated FN can be slowly released to avoid rapid clearance, improve the stability of FN, and prevent it from being degraded by skin proteases.

[0012] The beneficial effects of the operations of steps (2) and (3) above are as follows: the present invention constructs an exosome-lipid dual delivery system. Exosome loading introduces FN into the exosomes, thereby improving the drug loading efficiency. In addition, CD47 on the surface of the exosomes can prevent monocyte phagocytosis. Nanostructured lipid carrier (NLC) encapsulation can enhance the sustained release of FN and improve the transdermal absorption rate. In addition, ceramide III in the nanostructured paper carrier can synergistically enhance the skin barrier repair with FN and reduce the rate of water loss.

[0013] As a preferred technical solution, the pH of the PBS solution in step (1) is 7.2-7.4; and the molar ratio of the recombinant human fibronectin to palmitic acid-NHS ester is 0.8-1.2:4-6.

[0014] As a preferred technical solution, the speed of the ultracentrifugation in step (2) is 100,000-120,000×g, and the time is 0.8-1.5 h; the amount of ultrapure water in the aqueous phase in step (3) is 5-8% of the weight of water in the formula.

[0015] As a preferred technical solution, the high-speed shearing speed in step (4) is 10,000-15,000 rpm, and the time is 1-2 min; the pressure of the high-pressure homogenization is 800-1200 bar, and the number of cycles is 2-4 times; and the molecular particle size in the NLC suspension is 100-200 nm.

[0016] As a preferred technical solution, the power of the ultrasonic homogenization treatment in step (5) is 50-100 W, and the treatment time is 3-5 min; the amount of ultrapure water used in step (6) is 4-6% of the weight of water in the formula; and the pore size of the sterile filter membrane is 0.22 μm.

[0017] Another object of the present invention is to provide uses of the recombinant human fibronectin composition, including use in preparing cosmetics.

[0018] Preferably, the cosmetics include functional cosmetics with high transdermal absorption rate and sustained release effect.

[0019] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects: (1) The present invention constructs an exosome-lipid dual delivery system with the following technical effects: dual drug loading, increased total drug capacity, improved drug loading rate, and delivery efficiency; exosomes are natural nanovesicles (30-150 nm) secreted by cells, with membrane proteins of parent cells (such as CD47, integrin, etc.) on the surface, which can avoid being cleared by the immune system (such as the mononuclear phagocytic system, MPS), protecting nanostructured lipid carriers (NLC), thereby prolonging circulation time and enhancing tissue accumulation. Exosomes can penetrate the vascular endothelium, intercellular spaces or dense extracellular matrix through endocytosis, membrane fusion or transcytosis, overcome biological barriers, and thus enhance the deep tissue penetration of NLC. In terms of drug release, a single exosome system can quickly release drugs through endocytosis, while a single NLC system plays a sustained release role. When the two are combined, a fast-slow biphasic release can maintain an effective concentration. In terms of barrier repair, the miRNA / protein carried by exosomes activates keratinocyte differentiation, while ceramide III directly fills the gap, replenishes intercellular lipids, and works together with exosomes to repair the "brick-gray" structure, enhancing the skin's barrier repair and reducing the rate of water loss.

[0020] (2) A dynamic stabilization system for anti-flocculation was constructed. Poloxamer 188 is a triblock copolymer (PEO-PPO-PEO). Its hydrophilic polyethylene oxide (PEO) chain stretches in the solution to form a "brush-like" structure, while the hydrophobic polypropylene oxide (PPO) chain can be anchored on the surface of fibronectin FN, preventing the particles from approaching each other through steric hindrance. In addition, trehalose is a non-reducing disaccharide. Its hydroxyl group (-OH) can form a hydrogen bond network with the particle surface or the PEO chain of Poloxamer 188, replacing the original water molecules to form a stable "hydration shell". The hydration shell reduces the free energy of the particle surface and reduces the aggregation driven by hydrophobic interactions. Therefore, Poloxamer 188 and trehalose form a "steric hindrance-hydration layer" dual protection to inhibit FN aggregation. The citric acid buffer system stabilizes the Zeta potential at -25mV to -30mV, which can significantly improve the colloidal stability and anti-flocculation ability of the system. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] The recombinant human fibronectin is published in CN 119060167 A and is titled as: Preparation and Application of Recombinant Human Fibronectin. The sequence of the recombinant human fibronectin is SEQ ID NO.1, the specific sequence is as follows: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDD DDKYVVSVSSVYRGDSSTPLRGRQKTGLDSPYEGQLISIQQYGHQEVTAVPPPTDLRFTNIGPDTMRVTWAPPPSIDITETPSQPNSHPIQWNAPQPSHISKYILRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYVVSVSSVYRGDSSTPLRGRQKTGLDSP; The human bone marrow mesenchymal stem cell exosomes were purchased from Wuhan Warner Biotechnology Co., Ltd.

[0023] Example 1. A recombinant human fibronectin composition comprises the following components in weight percentage: 0.01% recombinant human fibronectin (C-terminally palmitoylated), 1% human bone marrow mesenchymal stem cell exosomes (particle size 80-150 nm), 3% nanostructured lipid carrier (composed of ceramide III, squalene, and hydrogenated lecithin in a weight ratio of 0.2:1:0.1), 0.5% anti-flocculation stabilizer (Poloxamer 188 and trehalose in a weight ratio of 0.5:2), 0.1% citric acid-disodium hydrogen phosphate buffer (pH 6.5), 8% glycerol, and 0.1% hyaluronic acid, with the remainder being ultrapure water to make up 100%.

[0024] The above-mentioned recombinant human fibronectin composition is prepared by the following method: (1) Modification of recombinant human fibronectin: Recombinant human fibronectin was dissolved in PBS solution (7.2), and then palmitic acid-NHS ester was added at a molar ratio of 0.8:4 between recombinant human fibronectin and palmitic acid-NHS ester. The mixture was reacted at 20°C in the dark for 3 h. Unreacted palmitic acid-NHS ester was removed by dialysis with a semipermeable membrane to obtain palmitoylated recombinant human fibronectin. (2) Exosome loading: Human bone marrow mesenchymal stem cell exosomes were mixed with palmitoylated recombinant human fibronectin, incubated with gentle shaking at 35°C for 2 h, and ultracentrifuged at 100,000 × g for 0.8 h to prepare recombinant human fibronectin-exosome complexes; (3) Preparation of lipid phase and aqueous phase: The nanostructured lipid carrier was placed at 65°C and melt-mixed until transparent to prepare a lipid phase; the recombinant human fibronectin-exosome complex was dispersed in ultrapure water (5% of the weight of water in the formula) at 65°C to prepare an aqueous phase; (4) High-pressure homogenization: The lipid phase from step (3) was slowly poured into the aqueous phase, pre-emulsified at 10,000 rpm and high-speed shear for 1 min, and then homogenized at 800 bar for 2 cycles to obtain an NLC suspension (molecular particle size of 100-200 nm); (5) Addition of stabilizer and active ingredient: Dissolve the anti-flocculating stabilizer in citric acid-disodium hydrogen phosphate buffer and mix well to prepare a stabilizer solution. Mix the stabilizer solution with the NLC suspension and homogenize with ultrasonication at 50W for 3 min. Then add glycerol and stir until it is completely dissolved. (6) Addition of hyaluronic acid: pre-dissolve hyaluronic acid in ultrapure water (4% of the weight of water in the formula), let it stand for 3 h to fully hydrate, then add it to the system in step (5), add the remaining ultrapure water to make up to 100%, adjust the pH to 6.8±0.2; filter with a 0.22 μm sterile filter membrane, and dispense into light-proof containers to prepare a recombinant human fibronectin composition.

[0025] Example 2. A recombinant human fibronectin composition comprises the following components in weight percentage: 1.0% recombinant human fibronectin (C-terminally palmitoylated), 5% human bone marrow mesenchymal stem cell exosomes (particle size 80-150 nm), 10% nanostructured lipid carrier (composed of ceramide III, squalene, and hydrogenated lecithin in a weight ratio of 1:3:0.5), 2% anti-flocculation stabilizer (Poloxamer 188 and trehalose in a weight ratio of 1:2), 1% citric acid-disodium hydrogen phosphate buffer (pH 6.5-6.8), 10% glycerol, and 0.5% hyaluronic acid, with the remainder being ultrapure water to make up 100%.

[0026] The above-mentioned recombinant human fibronectin composition is prepared by the following method: (1) Modification of recombinant human fibronectin: Recombinant human fibronectin was dissolved in PBS solution (7.4), and then palmitic acid-NHS ester was added according to the molar ratio of recombinant human fibronectin to palmitic acid-NHS ester of 1.2:6. The mixture was reacted at 23°C in the dark for 4 h. Unreacted palmitic acid-NHS ester was removed by dialysis with a semipermeable membrane to obtain palmitoylated recombinant human fibronectin. (2) Exosome loading: Human bone marrow mesenchymal stem cell exosomes were mixed with palmitoylated recombinant human fibronectin, incubated with gentle shaking at 37°C for 2-3 h, and ultracentrifuged at 120,000 × g for 1.5 h to prepare recombinant human fibronectin-exosome complexes; (3) Preparation of lipid phase and aqueous phase: The nanostructured lipid carrier was placed at 75°C and melt-mixed until transparent to prepare the lipid phase; the recombinant human fibronectin-exosome complex was dispersed in ultrapure water (8% of the weight of water in the formula) at 75°C to prepare the aqueous phase; (4) High-pressure homogenization: The lipid phase from step (3) was slowly poured into the aqueous phase, pre-emulsified at 15,000 rpm and high-speed shear for 2 min, and then high-pressure homogenized at 1,200 bar for 4 cycles to obtain an NLC suspension (molecular particle size of 100-200 nm); (5) Addition of stabilizer and active ingredient: Dissolve the anti-flocculating stabilizer in citric acid-disodium hydrogen phosphate buffer and mix well to prepare a stabilizer solution. Mix the obtained stabilizer solution with the NLC suspension and homogenize with ultrasound at 100W for 5 min. Then add glycerol and stir until completely dissolved. (6) Addition of hyaluronic acid: pre-dissolve hyaluronic acid in ultrapure water (6% of the weight of water in the formula), let it stand for 5 h to fully hydrate, then add it to the system in step (5), add the remaining ultrapure water to make up to 100%, adjust the pH to 6.8±0.2; filter with a 0.22 μm sterile filter membrane, and dispense into light-proof containers to prepare a recombinant human fibronectin composition.

[0027] Example 3. A recombinant human fibronectin composition comprises the following components in weight percentage: 0.05% recombinant human fibronectin (C-terminally palmitoylated), 2% human bone marrow mesenchymal stem cell exosomes (particle size 80-150 nm), 5% nanostructured lipid carrier (composed of ceramide III, squalene, and hydrogenated lecithin in a weight ratio of 0.5:2:0.3), 1% anti-flocculation stabilizer (Poloxamer 188 and trehalose in a weight ratio of 0.7:2), 0.5% citric acid-disodium hydrogen phosphate buffer (pH 6.5-6.8), 9% glycerol, and 0.3% hyaluronic acid, with the balance being ultrapure water to 100%.

[0028] The above-mentioned recombinant human fibronectin composition is prepared by the following method: (1) Modification of recombinant human fibronectin: Take recombinant human fibronectin and dissolve it in PBS solution (7.3). Then add palmitic acid-NHS ester at a molar ratio of 1:5 between recombinant human fibronectin and palmitic acid-NHS ester. React at 25°C in the dark for 3.5 h. Remove unreacted palmitic acid-NHS ester by semipermeable membrane dialysis to obtain palmitoylated recombinant human fibronectin. (2) Exosome loading: Human bone marrow mesenchymal stem cell exosomes were mixed with palmitoylated recombinant human fibronectin, incubated with gentle shaking at 36°C for 2 h, and ultracentrifuged at 100,000 × g for 1 h to prepare recombinant human fibronectin-exosome complexes; (3) Preparation of lipid phase and aqueous phase: The nanostructured lipid carrier was placed at 70°C and melt-mixed until transparent to prepare the lipid phase; the recombinant human fibronectin-exosome complex was dispersed in ultrapure water (6% of the weight of water in the formula) at 70°C to prepare the aqueous phase; (4) High-pressure homogenization: The lipid phase from step (3) was slowly poured into the aqueous phase, pre-emulsified at 12,000 rpm for 2 min, and then homogenized at 1,000 bar for 3 cycles to obtain an NLC suspension (molecular particle size of 100-200 nm). (5) Addition of stabilizers and active ingredients: Dissolve the anti-flocculating stabilizer in citric acid-disodium hydrogen phosphate buffer and mix well to prepare a stabilizer solution. Mix the stabilizer solution with the NLC suspension and homogenize with ultrasonication at 80W for 4 minutes. Then add glycerol and stir until it is completely dissolved. (6) Addition of hyaluronic acid: pre-dissolve hyaluronic acid in ultrapure water (5% of the weight of water in the formula), let it stand for 4 h to fully hydrate, then add it to the system in step (5), add the remaining ultrapure water to make up to 100%, adjust the pH to 6.8±0.2; filter with a 0.22 μm sterile filter membrane, and dispense into light-proof containers to prepare a recombinant human fibronectin composition.

[0029] Comparative Example 1, a recombinant human fibronectin composition, is substantially the same as that of Example 3, except that the recombinant human fibronectin composition does not include a nanostructured lipid carrier.

[0030] The corresponding preparation method also does not involve steps (3) and (4).

[0031] Comparative Example 2, a recombinant human fibronectin composition, has a formula substantially the same as that of Example 3, except that the recombinant human fibronectin composition does not include human bone marrow mesenchymal stem cell exosomes.

[0032] The corresponding preparation method does not involve step (2). In step (3), the recombinant human fibronectin is dispersed in ultrapure water to prepare an aqueous phase, which is then mixed with the lipid phase.

[0033] Comparative Example 3, a recombinant human fibronectin composition, is substantially the same as Example 3, except that the anti-flocculation stabilizer is trehalose only. In the corresponding preparation method, the anti-flocculation stabilizer is trehalose only, and the other steps remain unchanged.

[0034] Comparative Example 4, a recombinant human fibronectin composition, has a formulation substantially identical to that of Example 3, differing only in that the nanostructured lipid carrier comprises squalene and hydrogenated phosphatidylcholine in a weight ratio of 2:0.3. The corresponding preparation method also employs a nanostructured lipid carrier comprised solely of squalene and hydrogenated phosphatidylcholine in a weight ratio of 2:0.3, with all other steps remaining unchanged.

[0035] Comparative Example 5, a recombinant human fibronectin composition, has the same formulation as that of Example 3, except that the recombinant human fibronectin is not palmitoylated.

[0036] In order to verify the effects of the recombinant human fibronectin compositions prepared in different groups of Examples 1-3 and Comparative Examples 1-5, experiments related to FN stability, anti-flocculation, transdermal absorption rate and sustained release were conducted.

[0037] FN stability: A cell adhesion assay was performed to determine the activity of the recombinant human fibronectin compositions prepared in different groups. The compositions were stored at 4°C in the dark and tested for 0, 7, 14, and 30 days. The results are shown in Table 1.

[0038] Table 1 FN activity of recombinant human fibronectin compositions in different groups (%)

[0039] Analysis of the results, as shown in Table 1, shows that Examples 1-3 of the present invention maintained over 90% activity after 30 days of storage at 4°C. However, in Comparative Examples 1-5, FN activity in some groups dropped below 50% after 7 days, and after 14 days of storage, activity in all groups dropped below 40%. Furthermore, this comparison demonstrates that palmitoylation of the nanostructured lipid carrier, human mesenchymal stem cells, and recombinant human fibronectin is crucial for maintaining FN activity. This may be because palmitoylated recombinant human fibronectin facilitates the dual delivery system with exosomes and the nanostructured lipid carrier, effectively protecting FN protein activity.

[0040] Anti-flocculation: Dynamic light scattering (DLS) was used to analyze the anti-flocculation properties, and the polydispersity index (PDI) of different groups was calculated. A PDI < 0.3 was considered a monodisperse system.

[0041] Transdermal absorption rate: Fresh pig ear skin (thickness 0.5 ± 0.1 mm, intact stratum corneum) was obtained, subcutaneous fat was removed, and the skin was rinsed with PBS and stored at -80°C (rewarmed before use). The skin barrier integrity was verified as a resistance value >15 kΩ / cm². The FN concentration was determined by HPLC using a Franz vertical diffusion cell, and the transdermal absorption rate of FN was determined.

[0042] Sustained release determination: Release medium: PBS (pH 7.4) to simulate physiological environment, also containing 0.5% Tween 80 to maintain sink conditions, temperature: 32 ± 0.5°C to simulate skin surface temperature; The dialysis bag method with a molecular weight cutoff of 10 kDa was selected. The samples were boiled in boiling water for 10 min and stored in deionized water at 4°C. When used, the recombinant human fibronectin compositions of each group were placed in the dialysis bag, sealed, and immersed in 50 mL of release medium. The time required for complete release was measured. The experimental results are shown in Table 2.

[0043] Table 2 Anti-flocculation, transdermal absorption rate and sustained release of different groups of fibrin compositions

[0044] Analysis of results: As can be seen from the contents of Table 2, the recombinant human fibronectin composition prepared by the present invention has excellent anti-flocculation specificity, high stability, and an excellent transdermal absorption delivery system, which can improve the in vitro transdermal absorption efficiency and the sustained release time. The sustained release time is extended by more than 4 times compared with the control group, allowing FN to exert its efficacy for a longer period of time, which is a significant improvement compared with Comparative Examples 1-5.

[0045] At the same time, in order to determine the effect of the exosome-lipid dual delivery system of the present invention, the TEWL improvement rate and drug loading efficiency of Example 3 (exosome-liposome dual system), Comparative Example 1 (single exosome delivery system) and Comparative Example 2 (single nanostructured liposome delivery system) were respectively measured, and free FN was set as a blank control group. The experimental results are shown in Table 3.

[0046] Table 3 TEWL improvement rate and drug loading efficiency of different groups of fibrin compositions

[0047] Results analysis shows that from the contents of Table 3, the exosome-liposome dual system of the present invention significantly improved the TEWL improvement rate and drug loading efficiency, which was significantly better than the single exosome delivery system and the single nanostructured liposome delivery system.

[0048] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0049] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A recombinant human fibronectin composition, characterized in that: The composition comprises the following components in weight percentage: 0.01%-1.0% recombinant human fibronectin, 1%-5% human bone marrow mesenchymal stem cell exosomes, 3%-10% nanostructured lipid carrier, 0.5%-2% anti-flocculation stabilizer, 0.1%-1% citric acid-disodium hydrogen phosphate buffer, 8-10% glycerol, 0.1-0.5% hyaluronic acid, and the balance is made up to 100% with ultrapure water.

2. The recombinant human fibronectin composition according to claim 1, characterized in that: The C-terminus of the recombinant human fibronectin is palmitoylated; the particle size of the human bone marrow mesenchymal stem cell exosomes is 80-150 nm; and the nanostructured lipid carrier is composed of ceramide III, squalene, and hydrogenated phosphatidylcholine in a weight ratio of 0.2-1:1-3:0.1-0.

5.

3. The recombinant human fibronectin composition according to claim 1, characterized in that: The anti-flocculation precipitant consists of Poloxamer 188 and trehalose in a weight ratio of 0.5-1:2; and the pH value of the citric acid-disodium hydrogen phosphate buffer is 6.5-6.

8.

4. The method for preparing the recombinant human fibronectin composition according to any one of claims 1 to 3, characterized in that: The preparation is carried out according to the composition ratio of any one of claims 1 to 3, specifically comprising the following steps: (1) Modification of recombinant human fibronectin: dissolve recombinant human fibronectin, then add palmitic acid-NHS ester, react at 20-25℃ in the dark for 3-4 hours, and remove unreacted palmitic acid-NHS ester by semipermeable membrane dialysis to obtain palmitoylated recombinant human fibronectin; (2) Exosome loading: Mix human bone marrow mesenchymal stem cell exosomes with palmitoylated recombinant human fibronectin, incubate with gentle shaking at 35-37°C for 2-3 hours, and ultracentrifuge to prepare recombinant human fibronectin-exosome complexes; (3) Preparation of lipid phase and aqueous phase: Place the nanostructured lipid carrier at 65-75°C and melt mix until transparent to prepare the lipid phase; disperse the recombinant human fibronectin-exosome complex in ultrapure water at 65-75°C to prepare the aqueous phase; (4) High-pressure homogenization: slowly pour the lipid phase from step (3) into the aqueous phase, pre-emulsify with high-speed shearing, and then homogenize under high pressure to obtain a nanostructured lipid carrier suspension; (5) Addition of stabilizer and active ingredient: dissolve the anti-flocculation stabilizer in citric acid-disodium hydrogen phosphate buffer and mix well to prepare a stabilizer solution. The obtained stabilizer solution is mixed with the NLC suspension and homogenized by ultrasonication. Then, glycerol is added and stirred until completely dissolved. (6) Addition of hyaluronic acid: pre-dissolve hyaluronic acid in ultrapure water, let it stand for 3-5 hours to fully hydrate, then add it to the system in step (5), add ultrapure water to make up to 100%, and adjust the pH to 6.8±0.2; filter with a sterile filter membrane, and dispense into a light-proof container to prepare a recombinant human fibronectin composition.

5. The method for preparing the recombinant human fibronectin composition according to claim 4, characterized in that: The pH of the PBS solution in step (1) is 7.2-7.4; the molar ratio of the recombinant human fibronectin to palmitic acid-NHS ester is 0.8-1.2:4-6.

6. The method for preparing the recombinant human fibronectin composition according to claim 4, characterized in that: The speed of the ultracentrifugation in step (2) is 100,000-120,000×g, and the time is 0.8-1.5 h. The amount of ultrapure water in the aqueous phase in step (3) is 5-8% of the weight of water in the formula.

7. The method for preparing the recombinant human fibronectin composition according to claim 4, characterized in that: The high-speed shearing speed in step (4) is 10,000-15,000 rpm, and the time is 1-2 min; the pressure of the high-pressure homogenization is 800-1200 bar, and the number of cycles is 2-4 times; the molecular particle size of the NLC suspension is 100-200 nm.

8. The method for preparing the recombinant human fibronectin composition according to claim 4, characterized in that: The power of the ultrasonic homogenization treatment in step (5) is 50-100W, and the treatment time is 3-5 minutes; the amount of ultrapure water used in step (6) is 4-6% of the weight of water in the formula.

9. Use of the recombinant human fibronectin composition according to claims 1-3, characterized in that: The use includes application in the preparation of cosmetics.

10. The use according to claim 9, characterized in that The cosmetics include functional cosmetics with high transdermal absorption rate and sustained release effect.

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