A recombinant human-derived fibronectin composition, and a method of preparing and using the same

Abstract

The application discloses a recombinant human fibronectin composition and a preparation method and application thereof, and belongs to the technical field of cosmetics. The application provides a recombinant human fibronectin composition, adopts an exosome-nanostructure lipid carrier double delivery system, and carries and delivers a recombinant human fibronectin fragment after palmitamide modification, so that the drug loading efficiency can be significantly improved. Meanwhile, through the double delivery system, the stability, anti-flocculation capacity and slow-release performance of the FN can be improved, so that the FN can stably and continuously exert the active capacity, the composition is endowed with excellent barrier recovery capacity, the water loss rate is reduced, and the composition can be widely applied to the technical field of cosmetic preparation.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more specifically to a recombinant human fibronectin composition, its preparation method, and its application. Background Technology

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

[0003] However, its practical application presents several challenges, including: stability: recombinant human fibronectin is sensitive to temperature, pH, and enzymes, exhibiting instability under different conditions, during long-term storage, and in its compatibility with other matrices in cosmetics. It is prone to flocculation, thus affecting its activity. For the application of large molecular weight proteins, their stability and compatibility are exceptionally important. Therefore, solving the stability problem of large molecular weight proteins is beneficial for the application of this type of raw material in different scenarios and helps the product's efficacy. Transdermal absorption limitations: recombinant human fibronectin has a large molecular weight (approximately 440 kDa), making it difficult to cross the stratum corneum barrier, affecting its biological activity. Activity retention: the RGD (arginine-glycine-aspartic acid) sequence of recombinant human fibronectin is key to its function, but it may become ineffective in formulations due to folding changes.

[0004] There are some existing studies on fibronectin compositions, such as Chinese invention patent CN 118370698 A, which discloses the application of a fibronectin-containing composition in skincare products and the composition thereof. Another example is Chinese invention patent CN117679323 A, which discloses a recombinant fibronectin composition and its application in skincare products for sensitive skin. However, these patents only represent improvements in a single direction. For instance, CN 118370698 A only improves storage stability, and CN117679323 A is more suitable for sensitive skin. There is little research on properties such as sustained-release transdermal absorption, anti-flocculation, and long-lasting activity retention.

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

[0006] In view of this, the present invention provides a recombinant human fibronectin composition, its preparation method and application, 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] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A recombinant human fibronectin composition comprising 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% antiflocculation stabilizer, 0.1%-1% citrate-disodium hydrogen phosphate buffer, 8-10% glycerol, 0.1-0.5% hyaluronic acid, with the balance being ultrapure water to make up to 100%.

[0009] As a preferred technical solution, the C-terminus of the recombinant human fibronectin is modified with palmitoylation; the particle size of the human bone marrow mesenchymal stem cell exosomes is 80-150 nm; the nanostructured lipid carrier is composed of ceramide III, squalene and hydrogenated lecithin in a weight ratio of 0.2-1:1-3:0.1-0.5.

[0010] As a preferred technical solution, the anti-flocculation and precipitation agent is composed of Poloxamer 188 and trehalose in a weight ratio of 0.5-1:2; the pH of the citrate-disodium hydrogen phosphate buffer solution is 6.5-6.8.

[0011] Another object of the present invention is to provide a method for preparing the above-mentioned recombinant human fibronectin composition, wherein the preparation is carried out according to the above-mentioned component ratio, specifically including the following steps:

[0012] (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 h, remove unreacted palmitic acid-NHS ester by semi-permeable membrane dialysis, and obtain palmitoylated recombinant human fibronectin.

[0013] (2) Exosome loading: Human bone marrow mesenchymal stem cell exosomes were mixed with palmitoylated recombinant human fibronectin, and incubated with gentle shaking at 35-37℃ for 2-3 hours. The mixture was then centrifuged at high speed to prepare the recombinant human fibronectin-exosome complex.

[0014] (3) Preparation of lipid phase and aqueous phase: The nanostructured lipid carrier was placed at 65-75℃ and melt-mixed until transparent to prepare the lipid phase; the recombinant human fibronectin-exosome complex was dispersed in ultrapure water at 65-75℃ to prepare the aqueous phase;

[0015] (4) High-pressure homogenization: The lipid phase from step (3) is slowly poured into the aqueous phase, pre-emulsified by high-speed shearing, and then homogenized under high pressure to obtain a nanostructured lipid carrier suspension;

[0016] (5) Addition of stabilizers and active ingredients: Dissolve the anti-flocculation stabilizer in citrate-disodium hydrogen phosphate buffer, mix well, and prepare a stabilizer solution. Mix the obtained stabilizer solution with NLC suspension, sonicate homogenize, and then add glycerol and stir until completely dissolved.

[0017] (6) Addition of hyaluronic acid: Hyaluronic acid is pre-dissolved in ultrapure water, allowed to stand for 3-5 h to fully hydrate, and then added to the system in step (5). Ultrapure water is added to make up to 100%, and the pH is adjusted to 6.8±0.2. The mixture is filtered through a sterile filter membrane, dispensed into a light-proof container, and the recombinant human fibronectin composition is prepared.

[0018] The beneficial effects of step (1) above are as follows: C-terminal palmitoylation modification can enhance the lipophilicity of recombinant human fibronectin (FN). The palmitoylation chain is a long hydrocarbon chain (hydrophobic group). After modification, the hydrophobicity of the FN surface is enhanced, and its compatibility with lipids in the stratum corneum of the skin (such as ceramides and cholesterol) is improved. In addition, it can prolong the sustained release time. Palmitoylated FN can be released slowly, avoiding rapid clearance, improving the stability of FN, and preventing it from being degraded by skin proteases.

[0019] The beneficial effects of steps (2) and (3) above are as follows: This invention constructs an exosome-lipid dual delivery system. Exosome loading introduces FN into the exosomes, thus improving drug loading efficiency. Furthermore, CD47 on the exosome surface can prevent phagocytosis by monocytes. Encapsulation with nanostructured lipid carriers (NLCs) can enhance the sustained-release effect of FN and improve transdermal absorption. In addition, ceramide III in the nanostructured paper carrier can synergistically enhance skin barrier repair with FN, reducing the rate of moisture loss.

[0020] As a preferred technical solution, 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.

[0021] As a preferred technical solution, the speed of ultracentrifugation in step (2) is 100,000-120,000×g, and the time is 0.8-1.5h; the amount of ultrapure water used in the aqueous phase in step (3) is 5-8% of the weight of water in the formula.

[0022] As a preferred technical solution, the high-speed shearing speed in step (4) is 10000-15000 rpm and the time is 1-2 min; the pressure of high-pressure homogenization is 800-1200 bar and the number of cycles is 2-4; the molecular particle size in the NLC suspension is 100-200 nm.

[0023] As a preferred technical solution, the power of the ultrasonic homogenization treatment in step (5) is 50-100W and the treatment time is 3-5min; 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 0.22μm.

[0024] Another object of the present invention is to provide the use of recombinant human fibronectin compositions, including applications in the preparation of cosmetics.

[0025] Preferably, the cosmetics include functional cosmetics with high transdermal absorption rate and sustained-release effect.

[0026] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) This invention constructs an exosome-lipid dual delivery system, which has 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 the mother cell (such as CD47, integrins, etc.) on their surface, which can avoid being cleared by the immune system (such as the mononuclear phagocytic system, MPS), protect the nanostructured lipid carrier (NLC), thereby prolonging the circulation time and enhancing tissue accumulation. Exosomes can penetrate vascular endothelium, intercellular spaces, or dense extracellular matrix through endocytosis, membrane fusion, or transcytosis, overcoming biological barriers, thereby enhancing the deep tissue penetration of NLC. In terms of drug release, the exosome system alone can rapidly release drugs through endocytosis, while the NLC system alone plays a sustained-release role. When the two are combined, the 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 replenishes the deficiency and supplements intercellular lipids, working together with exosomes to repair the "brick-and-mortar" structure, enhance skin barrier repair, and reduce the rate of moisture loss.

[0028] (2) An anti-flocculation dynamic stabilization system was constructed. Poloxamer 188 is a triblock copolymer (PEO-PPO-PEO). Its hydrophilic polyethylene oxide (PEO) segments extend in solution to form a "brush-like" structure, while the hydrophobic polypropylene oxide (PPO) segments can anchor on the surface of fibronectin (FN), preventing particles from approaching each other through steric hindrance. In addition, trehalose is a non-reducing disaccharide, and its hydroxyl groups (-OH) can form a hydrogen bond network with the particle surface or the PEO chains of Poloxamer 188, replacing the original water molecules and forming a stable "hydration shell". The hydration shell reduces the free energy of the particle surface and reduces aggregation driven by hydrophobic interactions. Therefore, Poloxamer 188 and trehalose form a "steric hindrance-hydration layer" dual protection, inhibiting 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 Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] The recombinant human fibronectin mentioned is described in publication number CN 119060167 A, entitled "Preparation and Application of Recombinant Human Fibronectin," with the sequence SEQ ID. NO.1, the specific sequence is as follows: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDD DDKYVVSVSSVYRGDSSTPLRGRQKTGLDSPYEGQLISIQQYGHQEVTAVPPPTDLRFTNIGPDTMRVTWAPPPSIDITETPSQPNSHPIQWNAPQPSHISKYILRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYVVSVSSVYRGDSSTPLRGRQKTGLDSP;

[0031] The human bone marrow mesenchymal stem cell exosomes were purchased from Wuhan Huanna Biotechnology Co., Ltd.

[0032] Example 1: A recombinant human fibronectin composition comprising the following components by weight percentage: 0.01% recombinant human fibronectin (C-terminus 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% antiflocculation stabilizer (Poloxamer 188 and trehalose in a weight ratio of 0.5:2), 0.1% citrate-disodium hydrogen phosphate buffer (pH 6.5), 8% glycerol, 0.1% hyaluronic acid, and the balance being ultrapure water to make up to 100%.

[0033] The above-mentioned recombinant human fibronectin composition was prepared by the following method:

[0034] (1) Modification of recombinant human fibronectin: Take recombinant human fibronectin, dissolve it in PBS solution (7.2), and then add palmitic acid-NHS ester at a molar ratio of 0.8:4. React at 20°C in the dark for 3 hours. Remove unreacted palmitic acid-NHS ester by semipermeable membrane dialysis to obtain palmitoylated recombinant human fibronectin.

[0035] (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 then centrifuged at 100000×g for 0.8 h to prepare recombinant human fibronectin-exosome complex;

[0036] (3) Preparation of lipid phase and aqueous phase: The nanostructured lipid carrier was placed at 65°C and melt-mixed until transparent to prepare the lipid phase; the recombinant human fibronectin-exosome complex was dispersed in ultrapure water (5% by weight of water in the formula) at 65°C to prepare the aqueous phase;

[0037] (4) High pressure homogenization: The lipid phase from step (3) is slowly poured into the aqueous phase, pre-emulsified by high-speed shearing at 10,000 rpm for 1 min, and then high pressure homogenized at 800 bar for 2 cycles to obtain NLC suspension (molecular particle size of 100-200 nm).

[0038] (5) Addition of stabilizers and active ingredients: Dissolve the anti-flocculation stabilizer in citrate-disodium hydrogen phosphate buffer, mix well, and prepare a stabilizer solution. Mix the obtained stabilizer solution with NLC suspension, sonicate at 50W for 3 min, and then add glycerol and stir until completely dissolved.

[0039] (6) Addition of hyaluronic acid: Hyaluronic acid was pre-dissolved in ultrapure water (4% of the weight of water in the formula), allowed to stand for 3 h to fully hydrate, and then added to the system in step (5). The remaining ultrapure water was added to make up 100%, and the pH was adjusted to 6.8±0.2. The mixture was filtered through a 0.22μm sterile filter membrane, dispensed into light-proof containers, and the recombinant human fibronectin composition was prepared.

[0040] Example 2: A recombinant human fibronectin composition comprising the following components by weight percentage: 1.0% recombinant human fibronectin (C-terminus 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% antiflocculation stabilizer (Poloxamer 188 and trehalose in a weight ratio of 1:2), 1% citrate-disodium hydrogen phosphate buffer (pH 6.5-6.8), 10% glycerol, 0.5% hyaluronic acid, and the balance being ultrapure water to make up to 100%.

[0041] The above-mentioned recombinant human fibronectin composition was prepared by the following method:

[0042] (1) Modification of recombinant human fibronectin: Recombinant human fibronectin was dissolved in PBS solution (7.4), and then palmitic acid-NHS ester was added at a molar ratio of 1.2:6. The reaction was carried out at 23°C in the dark for 4 hours. Unreacted palmitic acid-NHS ester was removed by semi-permeable membrane dialysis to obtain palmitoylated recombinant human fibronectin.

[0043] (2) Exosome loading: Human bone marrow mesenchymal stem cell exosomes were mixed with palmitoylated recombinant human fibronectin, and incubated with gentle shaking at 37°C for 2-3 h. The mixture was then centrifuged at 120000×g for 1.5 h to prepare the recombinant human fibronectin-exosome complex.

[0044] (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% by weight of water in the formula) at 75°C to prepare the aqueous phase;

[0045] (4) High pressure homogenization: The lipid phase from step (3) is slowly poured into the aqueous phase, pre-emulsified by high-speed shearing at 15000 rpm for 2 min, and then high pressure homogenized at 1200 bar for 4 cycles to obtain NLC suspension (molecular particle size of 100-200 nm).

[0046] (5) Addition of stabilizers and active ingredients: Dissolve the anti-flocculation stabilizer in citrate-disodium hydrogen phosphate buffer, mix well, and prepare a stabilizer solution. Mix the obtained stabilizer solution with NLC suspension, sonicate at 100W for 5 min, then add glycerol and stir until completely dissolved.

[0047] (6) Addition of hyaluronic acid: Hyaluronic acid was pre-dissolved in ultrapure water (6% of the weight of water in the formula), allowed to stand for 5 h to fully hydrate, and then added to the system in step (5). The remaining ultrapure water was added to make up 100%, and the pH was adjusted to 6.8±0.2. The mixture was filtered through a 0.22μm sterile filter membrane, dispensed into light-proof containers, and the recombinant human fibronectin composition was prepared.

[0048] Example 3: A recombinant human fibronectin composition comprising the following components by weight percentage: 0.05% recombinant human fibronectin (C-terminus 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% antiflocculation stabilizer (Poloxamer 188 and trehalose in a weight ratio of 0.7:2), 0.5% citrate-disodium hydrogen phosphate buffer (pH 6.5-6.8), 9% glycerol, 0.3% hyaluronic acid, and the balance being ultrapure water to make up to 100%.

[0049] The above-mentioned recombinant human fibronectin composition was prepared by the following method:

[0050] (1) Modification of recombinant human fibronectin: Recombinant human fibronectin was dissolved in PBS solution (7.3), and then palmitic acid-NHS ester was added at a molar ratio of 1:5 between recombinant human fibronectin and palmitic acid-NHS ester. The reaction was carried out at 25°C in the dark for 3.5 h. Unreacted palmitic acid-NHS ester was removed by semi-permeable membrane dialysis to obtain palmitoylated recombinant human fibronectin.

[0051] (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 then centrifuged at 100,000 × g for 1 h to prepare recombinant human fibronectin-exosome complex.

[0052] (3) Preparation of lipid phase and aqueous phase: The nanostructured lipid carrier was placed at 70℃ and melt-mixed until transparent to prepare the lipid phase; the recombinant human fibronectin-exosome complex was dispersed in ultrapure water (6% by weight of water in the formula) at 70℃ to prepare the aqueous phase;

[0053] (4) High pressure homogenization: The lipid phase from step (3) is slowly poured into the aqueous phase, pre-emulsified by high-speed shearing at 12000 rpm for 2 min, and then high pressure homogenized at 1000 bar for 3 cycles to obtain NLC suspension (molecular particle size of 100-200 nm).

[0054] (5) Addition of stabilizers and active ingredients: Dissolve the anti-flocculation stabilizer in citrate-disodium hydrogen phosphate buffer, mix well, and prepare a stabilizer solution. Mix the obtained stabilizer solution with NLC suspension, sonicate at 80W for 4 min, and then add glycerol and stir until completely dissolved.

[0055] (6) Addition of hyaluronic acid: Hyaluronic acid was pre-dissolved in ultrapure water (5% of the weight of water in the formula), allowed to stand for 4 h to fully hydrate, and then added to the system in step (5). The remaining ultrapure water was added to make up 100%, and the pH was adjusted to 6.8±0.2. The mixture was filtered through a 0.22μm sterile filter membrane, dispensed into light-proof containers, and the recombinant human fibronectin composition was prepared.

[0056] Comparative Example 1: A recombinant human fibronectin composition, which is basically the same as the formulation in Example 3, except that the recombinant human fibronectin composition does not include a nanostructured lipid carrier.

[0057] The corresponding preparation method does not involve steps (3) and (4).

[0058] Comparative Example 2, a recombinant human fibronectin composition, is basically the same as the formulation in Example 3, except that the recombinant human fibronectin composition does not include human bone marrow mesenchymal stem cell exosomes.

[0059] The corresponding preparation method does not involve step (2). In step (3), recombinant human fibronectin is dispersed in ultrapure water to prepare an aqueous phase, which is then mixed with the lipid phase.

[0060] Comparative Example 3, a recombinant human fibronectin composition, is essentially the same as the formulation in Example 3, except that the antiflocculation stabilizer is only trehalose. In the corresponding preparation method, the antiflocculation stabilizer is only trehalose, and all other steps remain unchanged.

[0061] Comparative Example 4: A recombinant human fibronectin composition, essentially the same as the formulation in Example 3, except that the nanostructured lipid carrier is composed of squalene and hydrogenated lecithin in a weight ratio of 2:0.3. In the corresponding preparation method, the nanostructured lipid carrier is also composed only of squalene and hydrogenated lecithin in a weight ratio of 2:0.3, with all other steps remaining unchanged.

[0062] Comparative Example 5: A recombinant human fibronectin composition, which is the same as the formulation of Example 3, except that the recombinant human fibronectin is not palmitoylated.

[0063] To verify the effects of the recombinant human fibronectin compositions prepared in different groups of Examples 1-3 and Comparative Examples 1-5, experiments were conducted on FN stability, anti-flocculation, transdermal absorption rate, and sustained release.

[0064] FN stability: The activity of recombinant human fibronectin compositions prepared in different groups was determined by cell adhesion assay after storage at 4℃ in the dark for 0, 7, 14 and 30 days. The experimental results are shown in Table 1.

[0065] Table 1. FN activity (%) of different groups of recombinant human fibronectin compositions

[0066]

[0067] The results, as shown in Table 1, indicate that Examples 1-3 of this invention retained over 90% activity after 30 days of storage at 4°C, while in Comparative Examples 1-5, some groups showed FN activity below 50% after 7 days, and all groups showed activity below 40% after 14 days. Furthermore, the comparison revealed that the palmitoylation of the nanostructured lipid carrier, human mesenchymal stem cells, and recombinant human fibronectin was crucial for maintaining FN activity. This may be because palmitoylated recombinant human fibronectin more easily forms a dual delivery system with exosomes and the nanostructured lipid carrier, effectively protecting the protein activity of FN.

[0068] Anti-flocculation property: Dynamic light scattering (DLS) was used to analyze the anti-flocculation property, and the polydispersity index (PDI) of different groups was calculated. A PDI < 0.3 was considered a monodisperse system.

[0069] Transdermal absorption rate:

[0070] Fresh pig ear skin (thickness 0.5±0.1 mm, intact stratum corneum) was taken, subcutaneous fat was removed, rinsed with PBS and stored at -80℃ (warmed before use). Skin barrier integrity was verified: resistivity >15 kΩ / cm². The concentration of FN was detected by HPLC in a Franz vertical diffusion cell, and the transdermal absorption rate of FN was determined.

[0071] Sustained-release assay:

[0072] Release medium: PBS (pH 7.4) to simulate the physiological environment, and also contains 0.5% Tween 80 to maintain the leak conditions; temperature: 32±0.5℃ to simulate the skin surface temperature.

[0073] The dialysis bag method with a molecular weight cutoff of 10 kDa was selected. The mixture was boiled in water for 10 min and stored in deionized water at 4°C. When using the mixture, the recombinant human fibronectin composition of each group was 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.

[0074] Table 2. Antiflocculation, transdermal absorption rate and sustained release of different groups of fibrin compositions

[0075]

[0076] Results Analysis: As shown in Table 2, the recombinant human fibronectin composition prepared by this invention has excellent anti-flocculation properties, high stability, and an excellent transdermal absorption and delivery system, which can improve the in vitro transdermal absorption efficiency and increase 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. This is a significant improvement compared with comparative examples 1-5.

[0077] Meanwhile, in order to determine the effect of the exosome-liposome 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 nanostructure liposome delivery system) were measured respectively. Free FN was set as a blank control group. The experimental results are shown in Table 3.

[0078] Table 3. TEWL improvement rate and drug loading efficiency of different groups of fibrin compositions

[0079]

[0080] The results analysis, as shown in Table 3, indicate that the exosome-liposome dual system of this invention significantly improves the TEWL improvement rate and drug loading efficiency, and is significantly superior to the exosome delivery system alone and the nanostructured liposome delivery system alone.

[0081] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0082] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A recombinant human fibronectin composition, characterized in that, Includes the following components by weight percentage: The composition includes 0.01%-1.0% palmitoylated recombinant human fibronectin, 1%-5% human bone marrow mesenchymal stem cell exosomes, 3%-10% nanostructured lipid carriers, 0.5%-2% anti-flocculation stabilizer, 0.1%-1% citrate-disodium hydrogen phosphate buffer, 8-10% glycerol, 0.1-0.5% hyaluronic acid, with the balance being ultrapure water to make up to 100%; the amino acid sequence of the palmitoylated recombinant human fibronectin is SEQ ID NO.

1. The exosomes of the human bone marrow mesenchymal stem cells have a particle size of 80-150 nm; the nanostructured lipid carrier is composed of ceramide III, squalene and hydrogenated lecithin in a weight ratio of 0.2-1:1-3:0.1-0.

5. The anti-flocculation and precipitation agent is composed of Poloxamer 188 and trehalose in a weight ratio of 0.5-1:2; the pH of the citrate-disodium hydrogen phosphate buffer solution is 6.5-6.8; The preparation method of the above-mentioned recombinant human fibronectin composition specifically includes the following steps: (1) Preparation of palmitoylated recombinant human fibronectin: Recombinant human fibronectin was dissolved in PBS solution, and then palmitic acid-NHS ester was added. The reaction was carried out at 20-25℃ in the dark for 3-4 h. Unreacted palmitic acid-NHS ester was removed by semi-permeable 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, and incubated with gentle shaking at 35-37℃ for 2-3 hours. The mixture was then centrifuged at high speed to prepare the palmitoylated recombinant human fibronectin-exosome complex. (3) Preparation of lipid phase and aqueous phase: The nanostructured lipid carrier was placed at 65-75℃ and melt-mixed until transparent to prepare the lipid phase; the palmitoylated recombinant human fibronectin-exosome complex was dispersed in ultrapure water at 65-75℃ to prepare the aqueous phase; (4) High-pressure homogenization: The lipid phase from step (3) is slowly poured into the aqueous phase, pre-emulsified by high-speed shearing, and then homogenized under high pressure to obtain a nanostructured lipid carrier suspension; (5) Addition of stabilizers and active ingredients: Dissolve the anti-flocculation stabilizer in citrate-disodium hydrogen phosphate buffer, mix well, and prepare a stabilizer solution. Mix the obtained stabilizer solution with the nanostructured lipid carrier suspension, sonicate homogenize, and then add glycerol and stir until completely dissolved. (6) Addition of hyaluronic acid: Hyaluronic acid is pre-dissolved in ultrapure water, allowed to stand for 3-5 h to fully hydrate, and then added to the system in step (5). Ultrapure water is added to make up to 100%, and the pH is adjusted to 6.8±0.

2. The mixture is filtered through a sterile filter membrane, dispensed into a light-proof container, and the recombinant human fibronectin composition is prepared.

2. The recombinant human fibronectin composition according to claim 1, 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.

3. The recombinant human fibronectin composition according to claim 1, characterized in that, In step (2), the speed of ultracentrifugation is 100,000-120,000×g and the time is 0.8-1.5h; in step (3), the amount of ultrapure water used in the aqueous phase is 5-8% of the weight of water in the formula.

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

5. The recombinant human fibronectin composition according to claim 1, characterized in that, The ultrasonic homogenization process in step (5) has a power of 50-100W and a processing time of 3-5min; 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 0.22μm.

6. The use of the recombinant human fibronectin composition according to claim 1 in the preparation of cosmetics.

7. The use according to claim 6, characterized in that, The cosmetics mentioned include functional cosmetics with high transdermal absorption rate and sustained-release effect.

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