Search

CN-120478254-B - Recombinant human fibronectin composition and preparation method and application thereof

CN120478254BCN 120478254 BCN120478254 BCN 120478254BCN-120478254-B

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 composition of recombinant human fibronectin, which adopts an exosome-nanostructure lipid carrier dual delivery system to carry out entrapment delivery on a palmitoamide modified recombinant human fibronectin fragment, so that the drug loading efficiency can be remarkably improved. Meanwhile, the stability, deflocculating capacity and slow release performance of FN can be improved through the double delivery system, the FN can be ensured to stably and continuously exert the activity capacity of FN, the excellent barrier recovery capacity of the composition is endowed, the water loss rate is reduced, and the FN can be widely applied to the technical field of cosmetic preparation.

Inventors

  • CHEN YITANG
  • LI BEIBEI
  • Bao Kangna
  • ZHANG JIAHUI
  • LU QIHONG

Assignees

  • 杭州三言生物科技有限公司

Dates

Publication Date
20260508
Application Date
20250529

Claims (7)

  1. 1. The recombinant human fibronectin composition is characterized by comprising, by weight, 0.01% -1.0% of palmitoylation recombinant human fibronectin, 1% -5% of human bone marrow mesenchymal stem cell exosomes, 3% -10% of nanostructure lipid carriers, 0.5% -2% of deflocculating stabilizers, 0.1% -1% of citric acid-disodium hydrogen phosphate buffer solution, 8-10% of glycerol, 0.1-0.5% of hyaluronic acid and the balance of ultrapure water, wherein the amino acid sequence of the palmitoylation recombinant human fibronectin is SEQ ID NO.1; the particle size of the human bone marrow mesenchymal stem cell exosome is 80-150nm, and the nanostructure lipid carrier consists of ceramide III, squalene and hydrogenated lecithin in the weight ratio of 0.2-1:1-3:0.1-0.5; the deflocculating precipitant consists of Poloxamer 188 and trehalose in a weight ratio of 0.5-1:2, wherein the pH value of the citric acid-disodium hydrogen phosphate buffer solution is 6.5-6.8; the preparation method of the recombinant human fibronectin composition specifically comprises the following steps: (1) Preparing palmitoylation recombinant human fibronectin, namely dissolving recombinant human fibronectin in PBS solution, adding palmitic acid-NHS ester, carrying out light-shielding reaction at 20-25 ℃ for 3-4 h, and removing unreacted palmitic acid-NHS ester by a semipermeable membrane dialysis method to obtain palmitoylation recombinant human fibronectin; (2) Exosome loading, namely mixing human bone marrow mesenchymal stem cell exosome with palmitoylation recombinant human fibronectin, slightly oscillating and incubating for 2-3h at 35-37 ℃, and performing ultracentrifugation to prepare a palmitoylation recombinant human fibronectin-exosome compound; (3) Preparing lipid phase and water phase, namely placing the nanostructure lipid carrier at 65-75 ℃ and melting and mixing the nanostructure lipid carrier until the nanostructure lipid carrier is transparent to prepare the lipid phase; (4) Slowly pouring the lipid phase in the step (3) into an aqueous phase, pre-emulsifying by high-speed shearing, and homogenizing at high pressure to obtain a nanostructure lipid carrier suspension; (5) Dissolving deflocculating stabilizer in citric acid-disodium hydrogen phosphate buffer solution, mixing to obtain stabilizer solution, mixing the stabilizer solution with nanostructure lipid carrier suspension, ultrasonic homogenizing, adding glycerol, and stirring to dissolve completely; (6) Adding hyaluronic acid, namely pre-dissolving the hyaluronic acid into ultrapure water, standing for 3-5 h for full hydration, then adding the mixture into the system of the step (5), adding the ultrapure water to complement 100%, adjusting the pH value to 6.8+/-0.2, filtering by using a sterile filter membrane, and sub-packaging into a light-proof container to prepare the recombinant human fibronectin composition.
  2. 2. The recombinant human fibronectin composition according to claim 1, wherein the PBS solution of step (1) has a pH of 7.2-7.4, and the molar ratio of recombinant human fibronectin to palmitic acid-NHS ester is 0.8-1.2:4-6.
  3. 3. The recombinant human-derived fibronectin composition according to claim 1, wherein the ultracentrifugation in step (2) is performed at a rotational speed of 100000 to 120000×g for 0.8 to 1.5 hours, and the amount of ultrapure water in the aqueous phase in step (3) is 5 to 8% by weight of water in the formulation.
  4. 4. The method for preparing a recombinant human fibronectin composition according to claim 1, wherein the high-speed shearing speed in step (4) is 10000-15000rpm, the time is 1-2min, the high-pressure homogenizing pressure is 800-1200bar, the cycle number is 2-4, and the molecular particle size in the nano-structured lipid carrier suspension is 100-200nm.
  5. 5. The recombinant human fibronectin composition according to claim 1, wherein the power of the ultrasonic homogenization treatment in step (5) is 50-100W, the treatment time is 3-5min, the amount of ultrapure water in step (6) is 4-6% of the weight of water in the formulation, and the pore size of the sterile filter membrane is 0.22 μm.
  6. 6. Use of the recombinant human fibronectin composition of claim 1 in the preparation of a cosmetic.
  7. 7. The use according to claim 6, wherein the cosmetic comprises an efficacy cosmetic having high transdermal absorption and sustained release effects.

Description

Recombinant human fibronectin composition and preparation method and application thereof Technical Field The invention relates to the technical field of cosmetics, in particular to a recombinant human fibronectin composition, a preparation method and application thereof. Background Recombinant human Fibronectin (FN) is an important extracellular matrix glycoprotein, widely exists in human tissues, participates in critical physiological processes such as cell adhesion, migration, proliferation, differentiation and tissue repair, and is widely applied to the fields of medical repair, medical diagnosis, cell culture and the like. In addition, recombinant human fibronectin, which is a bioactive protein with cell adhesion, repair and regeneration functions, is also gradually applied to the field of beauty and skin care so as to achieve the purpose of repairing skin. However, the practical use thereof has problems such as stability that recombinant human fibronectin is sensitive to temperature, pH and enzymes, and thus has a certain instability in various conditions, long-term storage and compatibility with other substrates in cosmetic use, and is easily flocculated to affect activity. Aiming at the application of macromolecular proteins, the stability and the compatibility of the macromolecular proteins are abnormal, so that the problem of the application stability of the macromolecular proteins is solved, the application of the raw materials in different scenes is facilitated, the efficacy of the products is exerted, the transdermal absorption is limited, the molecular weight of the recombinant human fibronectin is large (about 440 kDa), the biological activity of the recombinant human fibronectin is hardly influenced by the barrier of the stratum corneum, and the activity is kept, the RGD (arginine-glycine-aspartic acid) sequence of the recombinant human fibronectin is the key function of the recombinant human fibronectin, but the recombinant human fibronectin possibly fails due to folding change in the formula. There are also some related studies in the prior art, such as chinese patent invention of CN 118370698A, which discloses the use of a fibronectin-containing composition in skin care products and its composition. For example, CN 117679323A discloses a recombinant fibronectin composition and its application in sensitive skin care products, but the above patent is only improvement in one direction, for example, CN 118370698A only improves storage stability, CN 117679323A is more suitable for sensitive skin, and there is little research on the property of sustained release transdermal, deflocculating and long-acting activity retention. Therefore, how to provide a recombinant human fibronectin composition with sustained-release transdermal, deflocculating and long-acting activity maintaining characteristics, and a preparation method and application thereof are technical problems which are needed to be solved by the technicians in the field. Disclosure of Invention In view of the above, the invention provides a recombinant human fibronectin composition, a preparation method and application thereof, and the recombinant human fibronectin composition with sustained-release transdermal, deflocculating and long-acting activity maintaining characteristics is successfully prepared, so that the application effect is improved. In order to achieve the above purpose, the present invention adopts the following technical scheme: A recombinant human fibronectin composition comprises, by weight, 0.01% -1.0% of recombinant human fibronectin, 1% -5% of human bone marrow mesenchymal stem cell exosomes, 3% -10% of nanostructure lipid carriers, 0.5% -2% of deflocculating stabilizers, 0.1% -1% of citric acid-disodium hydrogen phosphate buffer solution, 8-10% of glycerol, 0.1-0.5% of hyaluronic acid, and the balance of ultrapure water for supplementing 100%. As a preferable technical scheme, the C end of the recombinant human fibronectin is modified by palmitoylation, the particle size of the human bone marrow mesenchymal stem cell exosome is 80-150nm, and the nanostructure lipid carrier consists of ceramide III, squalene and hydrogenated lecithin in a weight ratio of 0.2-1:1-3:0.1-0.5. As a preferable technical scheme, the deflocculating 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 solution is 6.5-6.8. Still another object of the present invention is to provide a method for preparing the recombinant human fibronectin composition, which comprises the following steps: (1) The recombinant human fibronectin is modified by dissolving the recombinant human fibronectin in PBS solution, then adding palmitic acid-NHS ester, carrying out light-proof reaction at 20-25 ℃ for 3-4 h, and removing unreacted palmitic acid-NHS ester by a semipermeable membrane dialysis method to obtain palmitoyl recombinant human fibronectin; (2) Exosome l