Oil-soluble recombinant collagen bionic nano-liposome delivery system as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of collagen skin care products, and provides an oil-soluble recombinant collagen bionic nano-liposome delivery system and a preparation method and application thereof.The preparation method comprises the steps that A, a buffer solution with the pH being 3.8-4.2 is added into a lipid film, oscillation and ultrasonic treatment are conducted, and blank liposome is obtained; b, dissolving the integrin alpha6beta4 transmembrane protein by using a buffer solution with the pH value of 7.2-7.4; adding the transmembrane protein solution into blank lipidosome, oscillating and incubating at 37 DEG C, and performing ultrafiltration and centrifugation to obtain lipidosome suspension; c, dropwise adding the recombinant collagen solution into the liposome suspension, stirring at a constant temperature of 40 + / -2 DEG C, and performing ultrasonic treatment to obtain a collagen liposome suspension; and D, homogenizing the collagen liposome suspension at high pressure, and performing ultrafiltration purification to obtain the purified collagen liposome suspension. Through the technical scheme, the problems of low collagen encapsulation efficiency and low transdermal efficiency in the prior art are solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of collagen skin care products, and relates to an oil-soluble recombinant collagen biomimetic nano-liposome delivery system, a preparation method thereof, and an application thereof. Background Art

[0002] Collagen itself has a relatively large molecular weight, presents a triple helix structure in terms of structure, has a relatively large spatial conformation, and shows a trend of gradually increasing molecular particle size within the pH range acceptable for skin physiology. This greatly restricts its application in the R & D and manufacturing of cosmetics and in the market application. How to promote the transdermal absorption of collagen has become a difficult point restricting the application of collagen.

[0003] Existing recombinant collagen products mostly adopt water-soluble formulations or simple emulsification systems. For example: hydrogel carriers: dissolving recombinant collagen in a hydrophilic matrix (such as hyaluronic acid), but it cannot effectively deliver recombinant collagen, and the transdermal absorption rate is low. Ordinary cream systems: dispersing collagen through emulsifiers, but lacking a protection mechanism, easily leading to the aggregation and inactivation of collagen, and unable to achieve targeted delivery.

[0004] Liposomes are microvesicles formed by encapsulating active ingredients within a lipid bilayer. Water-soluble active ingredients are encapsulated in the internal aqueous vesicles, and lipophilic active ingredients are encapsulated in the lipid bilayer. The phospholipid structure in liposomes has a high similarity to human skin lipids, and has advantages such as high biocompatibility, good biodegradability, and the ability to promote the transdermal absorption of drugs. Therefore, liposomes are often used as carriers to encapsulate active ingredients, such as collagen, to promote the penetration of active ingredients through the stratum corneum and form a reservoir of active ingredients to slowly release the active ingredients.

[0005] The main preparation methods of liposomes include thin film evaporation method, ethanol / ether injection method, supercritical fluid method, reverse evaporation method, etc. However, there are still some problems at present: the thin film dispersion method is suitable for embedding lipophilic components, but has problems of low encapsulation efficiency and uneven particle size; the reverse evaporation method is suitable for water-soluble components, and forms liposomes by removing solvents after mixing the organic phase and the aqueous phase, but has a low encapsulation efficiency for collagen, and its stability is significantly affected by the ionic strength.

[0006] Therefore, there are the following defects in the preparation of collagen liposomes: (1) insufficient encapsulation efficiency; (2) low transdermal efficiency: the surface modification of liposomes is not optimized, resulting in insufficient transdermal depth (only reaching below 10 μm of the stratum corneum); in addition, after encapsulation, the liposomes have too large a size (>200 nm), resulting in too high a follicle retention rate, affecting the skin absorption effect.

[0007] In summary, how to improve the encapsulation efficiency and transdermal efficiency of collagen liposomes is the current research focus. Summary of the Invention

[0008] The present invention provides an oil-soluble recombinant collagen biomimetic nano-liposome delivery system, its preparation method and application, which solve the problems of low encapsulation rate and low transdermal efficiency of collagen in the prior art.

[0009] The technical solution of the present invention is realized as follows: Technical theme one A preparation method of an oil-soluble recombinant collagen biomimetic nano-liposome delivery system includes the following steps: A. Dissolve lipids in a solvent, stir, and remove the solvent to prepare a lipid film; add a buffer solution with a pH of 3.8 - 4.2 to the lipid film, shake it to hydrate and swell, and then obtain blank liposomes through probe sonication; the lipids include 70 - 85 wt% soybean lecithin and 15 - 30 wt% cholesterol; B. Dissolve integrin α6β4 transmembrane protein in a buffer solution with a pH of 7.2 - 7.4 to prepare a transmembrane protein solution with a concentration of 0.5 - 1.5 mg / mL; if the concentration of the transmembrane protein solution is not within this range, the transdermal effect of the liposomes will decline; Add the transmembrane protein solution to the blank liposomes obtained in step A, incubate at 37°C with shaking, and ultrafiltrate and centrifuge to obtain a liposome suspension; among them, the mass ratio of integrin α6β4 transmembrane protein to lipids is 0.5 - 5:1000, that is, 0.05% - 0.5% w / w; if the amount of transmembrane protein used is small, it will lead to insufficient targeting efficiency, a significant decrease in transdermal depth (<100 μm), a decrease in transdermal efficiency, and at the same time a decrease in the insertion rate: the proportion of unbound protein increases, and the targeting modification is incomplete after ultrafiltration; if the amount of transmembrane protein used is large, it will increase the risk of liposome aggregation, and the excessive insertion of protein will damage the lipid bilayer structure, resulting in an increase in particle size (>200 nm), and at the same time cost waste. The transmembrane protein is expensive, and excessive use is not beneficial to the improvement of the effect; C. Dropwise add the recombinant collagen solution to the liposome suspension obtained in step B, stir at a constant temperature of 40 ± 2°C, and perform sonication to obtain a collagen liposome suspension, where the mass ratio of collagen to lipids is 1:8 - 12; if the amount of collagen is too small, it will lead to insufficient drug loading and a decrease in transdermal amount; if it is too much, it will lead to a decrease in the encapsulation rate; D. Subject the collagen liposome suspension obtained in step C to high-pressure homogenization and ultrafiltration purification to obtain a purified collagen liposome suspension.

[0010] Preferably, in step A: the buffer solution is citric acid buffer solution or acetate buffer solution, and the citric acid buffer solution contains 150 mM NaCl; the dosage relationship between the buffer solution and the lipids is 8 - 12 ml: 1000 mg.

[0011] Preferably, in step A: the lipid comprises 80-85 wt% soy lecithin and 15-20 wt% cholesterol, preferably soy lecithin and cholesterol with a mass ratio of 800-850:150-200; more preferably 82.1 wt% soy lecithin and 17.9 wt% cholesterol, i.e., soy lecithin and cholesterol with a mass ratio of 821:179. If not within this range, when the amount of soy lecithin is too small, the membrane rigidity is too high, making it difficult for collagen to embed and reducing the transdermal efficiency. When the amount of soy lecithin is too large, the membrane fluidity is too high, resulting in easy leakage during storage (decreased stability) and a decrease in the long-term activity retention rate.

[0012] Preferably, in step A: the solvent is chloroform and methanol with a volume ratio of 1.8-2.2:1, or the solvent is a mixture of ethanol and ether.

[0013] Preferably, in step A: the amount of the solvent and the lipid is 90-110 ml:1000 mg.

[0014] Preferably, in step A: the conditions for stirring include: a stirring rate of 450-550 rpm, 25 ± 2 °C, and a stirring time of 25-35 min.

[0015] Preferably, in step A: the step of removing the solvent includes rotary evaporation and then vacuum drying. The conditions for the rotary evaporation include: a temperature of 38-42 °C, a rotation speed of 150-250 rpm, and a vacuum degree of -0.1 MPa.

[0016] Preferably, in step A: the conditions for vacuum drying include: a vacuum degree of -0.1 MPa, a vacuum drying time of 20-26 h, and a vacuum drying temperature of 25 °C.

[0017] Preferably, in step A: the oscillation rotation speed is 180-220 rpm, the time is 20-40 min, and the temperature is 60-70 °C.

[0018] Preferably, in step A: the conditions for probe sonication include: a power of 180-220 W, 5 s pulses / 5 s intervals, and 18-22 cycles.

[0019] Preferably, in step B: the mass ratio of the integrin α6β4 transmembrane protein to the lipid is 1-3:1000.

[0020] Preferably, in step B: the mass ratio of the integrin α6β4 transmembrane protein to the lipid is 1:1000.

[0021] Preferably, in step B: the conditions for oscillatory incubation include: a rotation speed of 120-180 rpm and a time of 1.5-2.5 h.

[0022] Preferably, in step B: the cut-off molecular weight for ultrafiltration centrifugation is 90 - 110 kDa, the centrifugal force is 9000 - 11000 × g, and the centrifugation time is 25 - 35 min.

[0023] Preferably, in step B: the buffer solution is a buffer solution commonly used in biochemical experiments such as PBS buffer solution or TBS buffer solution; more preferably, for example, the PBS buffer solution contains 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4; the TBS buffer solution contains: Tris-HCl buffer solution, NaCl, pH 7.2 - 7.4.

[0024] Preferably, in step C: the preparation of the recombinant collagen solution includes: dissolving the recombinant collagen in a solvent to prepare a recombinant collagen solution with a concentration of 50 ± 2.5 mg / mL; if the concentration of the recombinant collagen solution is not within this range, it will affect the transdermal effect of the liposomes.

[0025] Preferably, in step C: the concentration of the recombinant collagen solution is 50 mg / mL.

[0026] Preferably, in step C: the solvent is absolute ethanol.

[0027] Preferably, the collagen is recombinant collagen, more preferably recombinant humanized collagen, such as type III recombinant humanized collagen.

[0028] Preferably, in step C: the ultrasonic treatment conditions include: power 180 - 220 W, pulse mode 5 s / 5 s, total treatment time 8 - 12 min.

[0029] Preferably, in step C: the conditions for constant temperature stirring include: stirring rate 250 - 350 rpm, stirring time 20 - 40 min.

[0030] Preferably, in step D: the steps of high-pressure homogenization include: pre-cooling the liposome suspension to 3 - 5 °C, and circulatingly treating it 3 times with a high-pressure homogenizer, with a homogenization pressure of 800 - 1200 bar; Preferably, in step D: the steps of ultrafiltration purification include: cut-off molecular weight of 90 - 110 kDa, centrifugal force of 9000 - 11000 × g, and centrifugation time of 15 - 25 min.

[0031] Preferably, the method further includes step E, and step E includes: freeze-drying the purified collagen liposome suspension obtained in step D to obtain the product.

[0032] Preferably, the freeze-drying step in step E includes: adding a cryoprotectant to the purified collagen liposome suspension obtained in step D, and stirring until dissolved; then pre-freezing at -70~-90°C for 4-6 h; placing it in a freeze dryer, and setting the conditions for primary drying to include: temperature -45~-55°C, pressure 0.01 mBar, time 10-14 h; the conditions for secondary drying to include: temperature -25~-35°C, pressure 0.005 mBar, time 10-14 h; Preferably, the dosage relationship between the cryoprotectant and the purified collagen liposome suspension in step E is 2-8 g: 100 ml.

[0033] Preferably, the cryoprotectant is trehalose, mannitol, sucrose or a trehalose-glycerol composite system.

[0034] More preferably, the cryoprotectant is trehalose.

[0035] Preferably, the stirring rate in step E is 180-220 rpm, at 25±2°C, for 20-40 min.

[0036] Technical theme two The present invention also provides an oil-soluble recombinant collagen biomimetic nano-liposome delivery system, which is prepared based on the preparation method of the oil-soluble recombinant collagen biomimetic nano-liposome delivery system as described above.

[0037] Technical theme three The present invention also provides the application of the above oil-soluble recombinant collagen biomimetic nano-liposome delivery system in the preparation of cosmetics.

[0038] Preferably, the cosmetics include aqueous solutions, emulsions, sprays or facial masks.

[0039] The working principle and beneficial effects of the present invention are as follows: 1. The oil-soluble recombinant collagen biomimetic nano-liposome delivery system provided by the present invention adopts a biomimetic liposome bilayer structure, integrates transmembrane proteins (such as integrin α6β4), embeds recombinant collagen in the hydrophobic core of the lipid bilayer, and its hydrophobic group is coupled with phosphatidylethanolamine through a covalent bond to form a stable encapsulation structure. The transmembrane protein and the lipid bilayer are combined through hydrophobic interaction and hydrogen bond to form a biomimetic targeting interface. Integrin α6β4 guides the liposome to penetrate the skin directionally, improving the delivery efficiency and transdermal absorption rate of collagen, while enhancing stability, and the biomimetic structure reduces oxidative leakage.

[0040] 2. The preparation method of the oil-soluble recombinant collagen biomimetic nano-liposome delivery system provided by the present invention. In the step of directional insertion of membrane proteins, the self-assembly of proteins is driven by the pH gradient inside and outside the liposome, avoiding the damage to the activity caused by chemical coupling; ultrafiltration centrifugation is used to remove unbound proteins, ensuring the targeting efficiency; In the present invention, gradient ultrasonic drug loading promotes the embedding of collagen into the hydrophobic core, avoiding the destruction of the collagen structure by high temperature; In the present invention, high-pressure homogenization with pre-cooling treatment reduces the particle size dispersion, making the particle size uniform. The small size (<150 nm) reduces retention and improves the transdermal efficiency; In the freeze-drying process of the present invention, the use of a freeze-drying protectant can effectively protect the freeze-drying effect of liposomes and improve the stability of liposomes. The freeze-drying protectant is preferably trehalose. Compared with other freeze-drying protectants, trehalose enables liposomes to have better stability and maintain the integrity after reconstitution. In the freeze-drying process, ultra-low temperature pre-freezing forms uniform ice crystals, reducing structural collapse. The test results show good long-term stability; The preparation method provided by the present invention has process universality and is applicable to industrial production (batch stability CV < 5%).

[0041] 3. The oil-soluble recombinant collagen biomimetic nano-liposome provided by the present invention can be used in skin care products and cosmetics, such as essence water, lotion, paste, cream and other products, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

[0043] Figure 1 It is a schematic diagram of the liposome structure of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0046] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention. The production processes, experimental methods, or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art without special instructions, and their names and / or abbreviations are all conventional names in the art and are very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps according to the names and apply the corresponding equipment and implement them under conventional conditions or conditions recommended by the manufacturer.

[0047] There are no special restrictions on the sources of various instruments, equipment, raw materials, or reagents used in the embodiments of the present invention. They are all conventional products that can be obtained through regular commercial channels and can also be prepared according to the conventional methods well-known to those skilled in the art.

[0048] In the following examples and comparative examples, the total phospholipid content of the soy lecithin used is ≥90%; that is, the total proportion of phospholipid components in the soy lecithin is ≥90% (the rest are impurities such as triglycerides and free fatty acids); among them: the content of phosphatidylcholine (PC) is ≥50% (accounting for the total proportion of phospholipids); the peroxide value (POV) ≤5 meq / kg, and the acid value (AV) ≤5 mg KOH / g.

[0049] Both the integrin α6β4 transmembrane protein and collagen are commercially available products. Among them, the collagen used is type III recombinant humanized collagen, and the integrin α6β4 transmembrane protein purchased is human recombinant integrin α6β4. The present invention does not have special requirements for the molecular weight of collagen. It can be used in the range of 3 kDa - 30 kDa, but is not limited to the above range. The present invention preferably uses 3 - 20 kDa, and further preferably 10 - 20 kDa. In the following examples and comparative examples, 17 kDa is specifically used.

[0050] Example 1 A preparation method of an oil-soluble recombinant collagen biomimetic nanoliposome delivery system includes the following steps: A. Preparation of lipid film: Mix 700 mg of soy lecithin with 300 mg of cholesterol and dissolve them in 100 mL of a chloroform-methanol mixed solvent (the volume ratio of chloroform to methanol in the mixed solvent is 2:1). Stir magnetically until completely dissolved. Transfer it to a rotary evaporator and evaporate to form a uniform lipid film, then dry it under vacuum to ensure that the solvent residue is <10 ppm (detected by GC as 8.1 ppm). Among them, the stirring conditions include: stirring rate 500 rpm, 25 °C, stirring time 30 min; the operating conditions of the rotary evaporator are: temperature 40 °C, rotation speed 200 rpm, vacuum degree -0.1 MPa; the vacuum drying conditions include: vacuum degree -0.1 MPa, vacuum drying time 24 h, vacuum drying temperature 25 °C. In the present invention, the chloroform-methanol mixed solvent can also adopt other embodiments, such as using ethanol-ether. If other embodiments are adopted for soy lecithin in the present invention, the encapsulation efficiency and transdermal efficiency of liposomes will decrease. For example, in parallel experiments, when soy lecithin is replaced with equimolar hydrogenated soy phosphatide, egg yolk lecithin or synthetic phospholipid, the encapsulation efficiency, transdermal efficiency and storage stability of liposomes will all be affected: due to the too high membrane rigidity of hydrogenated soy phosphatide, it is difficult to embed collagen, and the encapsulation efficiency decreases by 6.8%; due to the reduced binding efficiency of the targeting protein of hydrogenated soy phosphatide → the transdermal depth decreases by 20% (150 μm → 120 μm); due to the high phase transition temperature (55 °C) of synthetic phospholipid, the hydrophobic core has poor sealing at room temperature, and the encapsulation efficiency decreases by 13.5%; the membrane fluidity of synthetic phospholipid is too high, it is easy to leak during storage, and the transdermal depth decreases by 33% (150 μm → 100 μm); egg yolk lecithin contains unsaturated fatty acids (mainly linoleic acid), and the active retention rate decreases by 2.3%.

[0051] Preparation of blank liposomes: Add 10 mL of pH 4.0 citrate buffer to the lipid film and shake it in a 65 °C water bath to hydrate and swell the lipid. The rotation speed of the shaking is 200 rpm and the time is 30 min. Perform probe sonication to obtain blank liposomes (the particle size detected by DLS is 123 nm and the PDI is 0.22). Among them, the citrate buffer contains 150 mM NaCl. The conditions of the probe sonication include: power 200 W, 5 s pulse / 5 s interval, 20 cycles. If other embodiments are adopted for probe sonication in the present invention, such as using continuous sonication (100 W, 1 min) or high-pressure homogenization (500 bar, 3 cycles), compared with probe pulse sonication, continuous sonication causes high heat (50 - 60 °C) and structural damage due to continuous sound energy input, resulting in a decrease in the encapsulation efficiency of liposomes and an out-of-control increase in particle size to 255.1 nm; while high-pressure homogenization (500 bar, 3 cycles): the pressure of 500 bar is insufficient, resulting in insufficient compression of liposomes and a 13.2% decrease in the encapsulation efficiency.

[0052] B. Oriented insertion of membrane proteins: Dissolve the integrin α6β4 transmembrane protein in phosphate buffer solution at pH 7.4 to prepare a transmembrane protein solution with a concentration of 1 mg / mL; Add the transmembrane protein solution to the blank liposomes obtained in step A, incubate with shaking at 37 °C, and use the pH gradient inside and outside the liposomes to drive the insertion of membrane proteins into the lipid bilayer. Then, perform ultrafiltration centrifugation to remove unbound proteins (the removal rate was verified by SDS-PAGE to be 96.1%) to obtain a liposome suspension. Fluorescently label integrin α6β4 (FITC-labeled), and measure the fluorescence intensity of the liposome suspension using a fluorescence spectrophotometer to calculate the insertion efficiency of 97.3%. Among them, the mass ratio of integrin α6β4 transmembrane protein to lipid is 1:1000; the conditions for incubation with shaking include: rotation speed of 150 rpm and time of 2 h; the cut-off molecular weight for ultrafiltration centrifugation is 100 kDa, the centrifugal force is 10,000×g, and the centrifugation time is 30 min; C. Loading of collagen with drugs: Dissolve recombinant collagen in absolute ethanol to prepare a recombinant collagen solution with a concentration of 50 mg / mL. Dropwise add the recombinant collagen solution to the liposome suspension obtained in step B, stir at a constant temperature of 40 °C, and perform probe sonication to promote the embedding of collagen into the hydrophobic core of the lipid to obtain a collagen liposome suspension (the encapsulation efficiency was determined by HPLC to be 91.3%). Among them, the ultrasonic power is 200 W, the pulse mode is 5 s / 5 s, and the total treatment time is 10 min; the conditions for constant-temperature stirring include: stirring rate of 300 rpm and stirring time of 30 min; the mass ratio of collagen to lipid is 1:10; D. High-pressure homogenization: Pre-cool the collagen liposome suspension obtained in step C to 4 °C, and perform cyclic treatment 3 times using a high-pressure homogenizer (at a pressure of 1000 bar). Then, transfer the collagen liposome suspension to an ultrafiltration centrifugation tube to remove unencapsulated collagen and small molecule impurities (the content of free protein was determined by the BCA method to be 4.1%) to obtain a purified collagen liposome suspension. Dynamic light scattering (DLS) shows that the average particle size is 142.5 nm and the PDI is 0.21; the specific parameters for ultrafiltration include: cut-off molecular weight of 100 kDa, centrifugal force of 10,000×g, and centrifugation time of 20 min.

[0053] Example 2 A preparation method of an oil-soluble recombinant collagen biomimetic nano-liposome delivery system, comprising the following steps: A. Preparation of lipid film: Mix 821 mg of soy lecithin with 179 mg of cholesterol and dissolve in 90 mL of a chloroform-methanol mixed solvent (the volume ratio of chloroform to methanol in the mixed solvent is 2.2:1), and stir magnetically until completely dissolved; transfer to a rotary evaporator and evaporate to form a uniform lipid film, and dry under vacuum to ensure that the solvent residue is <10 ppm (detected by GC as 6.5 ppm); among them, the stirring conditions include: stirring rate 450 rpm, 25 °C, stirring time 35 min; the working conditions of the rotary evaporator are: temperature 38 °C, rotation speed 250 rpm, vacuum degree -0.1 MPa; the vacuum drying conditions include: vacuum degree -0.1 MPa, vacuum drying time 20 h, vacuum drying temperature 25 °C; Preparation of blank liposomes: Add 12 mL of pH 4.0 citrate buffer solution to the lipid film, shake in a water bath at 60 °C to hydrate and swell the lipids. The rotation speed of the shaking is 120 rpm and the time is 40 min; probe sonication is performed to obtain blank liposomes (the particle size detected by DLS is 125 nm and the PDI is 0.26); among them, the citrate buffer solution contains 150 mM NaCl; the conditions of the probe sonication include: power 180 W, 5 s pulse / 5 s interval, 22 cycles; B. Oriented insertion of membrane proteins: Dissolve the integrin α6β4 transmembrane protein in a pH 7.4 phosphate buffer solution to prepare a transmembrane protein solution of 0.5 mg / mL; Add the transmembrane protein solution to the blank liposomes obtained in step A, incubate with shaking at 37 °C, and use the pH gradient inside and outside the liposomes to drive the insertion of the membrane protein into the bilayer lipid. Ultrafiltration and centrifugation are used to remove the unbound protein (the removal rate verified by SDS-PAGE is 97.1%) to obtain a liposome suspension; fluorescently labeled integrin α6β4 (FITC-labeled) is used, and the fluorescence intensity of the liposome suspension is measured by a fluorescence spectrophotometer to calculate the insertion efficiency ≥97.8%; among them, the mass ratio of the integrin α6β4 transmembrane protein to the lipid is 5:1000; the conditions of the incubation with shaking include: rotation speed 120 rpm, time 2.5 h; the cut-off molecular weight of the ultrafiltration and centrifugation is 90 kDa, the centrifugal force is 11,000×g, and the centrifugation time is 25 min; C. Loading of collagen with drugs: Dissolve recombinant collagen in absolute ethanol to prepare a recombinant collagen solution with a concentration of 52.5 mg / mL; dropwise add the recombinant collagen solution to the liposome suspension obtained in step B, stir at a constant temperature of 40 °C, and perform probe sonication to promote the embedding of collagen into the hydrophobic core of the lipid, obtaining a collagen liposome suspension (the encapsulation efficiency measured by HPLC is 92.5%). Among them, the ultrasonic power is 220 W, the pulse mode is 5 s / 5 s, and the total treatment time is 8 min; the conditions for constant temperature stirring include: stirring rate 250 rpm, stirring time 40 min; the mass ratio of collagen to lipid is 1:12; D. High-pressure homogenization: Precool the collagen liposome suspension obtained in step C to 3 °C, and cycle it 3 times using a high-pressure homogenizer (at a pressure of 1000 bar). Then transfer the collagen liposome suspension to an ultrafiltration centrifugal tube to remove unencapsulated collagen and small molecule impurities (the content of free protein measured by the BCA method is 5.5%) to obtain a purified collagen liposome suspension. Dynamic light scattering (DLS) shows an average particle size of 142 nm and a PDI of 0.19; the specific parameters of ultrafiltration include: the molecular weight cut-off is 110 kDa, the centrifugal force is 11000×g, and the centrifugation time is 15 min.

[0054] Example 3 A preparation method of an oil-soluble recombinant collagen biomimetic nano-liposome delivery system includes the following steps: A. Preparation of lipid film: Mix 821 mg of soy lecithin and 179 mg of cholesterol, dissolve them in 110 mL of a chloroform-methanol mixed solvent (the volume ratio of chloroform to methanol in the mixed solvent is 1.8:1), and stir magnetically until completely dissolved; transfer to a rotary evaporator to evaporate to form a uniform lipid film, and perform vacuum drying to ensure that the solvent residue is <10 ppm (detected by GC as 7.0 ppm); among them, the conditions for stirring include: stirring rate 550 rpm, 25 °C, stirring time 25 min; the working conditions of the rotary evaporator are: temperature 42 °C, rotation speed 150 rpm, vacuum degree -0.1 MPa; the conditions for vacuum drying include: vacuum degree -0.1 MPa, vacuum drying time 26 h, vacuum drying temperature 25 °C; Preparation of blank liposomes: Add 8 mL of pH 4.0 citrate buffer to the lipid film, shake in a water bath at 65 °C to hydrate and swell the lipid. The rotation speed of the shaking is 180 rpm and the time is 20 min; perform probe sonication to obtain blank liposomes (the particle size detected by DLS is 112 nm and the PDI is 0.25); among them, the citrate buffer contains 150 mM NaCl; the conditions for the probe sonication include: power 220 W, 5 s pulse / 5 s interval, 18 cycles; B. Oriented insertion of membrane protein: Dissolve the integrin α6β4 transmembrane protein in phosphate buffer solution at pH 7.2 to prepare a transmembrane protein solution with a concentration of 5 mg / mL; Add the transmembrane protein solution to the blank liposomes obtained in step A, incubate with shaking at 37 °C, and use the pH gradient inside and outside the liposomes to drive the insertion of membrane proteins into the lipid bilayer. Then, perform ultrafiltration centrifugation to remove unbound proteins (the removal rate was verified by SDS-PAGE to be 95.5%) to obtain a liposome suspension. Fluorescently label integrin α6β4 (FITC-labeled), and measure the fluorescence intensity of the liposome suspension using a fluorescence spectrophotometer to calculate the insertion efficiency of 96.12%. Among them, the mass ratio of integrin α6β4 transmembrane protein to lipid is 0.5:1000; the conditions for incubation with shaking include: rotation speed of 180 rpm and time of 1.5 h; the cut-off molecular weight for ultrafiltration centrifugation is 110 kDa, the centrifugal force is 9000×g, and the centrifugation time is 35 min; C. Loading the drug with collagen: Dissolve recombinant collagen in absolute ethanol to prepare a recombinant collagen solution with a concentration of 47.5 mg / mL. Dropwise add the recombinant collagen solution to the liposome suspension obtained in step B, stir at a constant temperature of 40 °C, and perform probe sonication to promote the embedding of collagen into the hydrophobic core of the lipid to obtain a collagen liposome suspension (the encapsulation efficiency was determined by HPLC to be 91.12%). Among them, the ultrasonic power is 180 W, the pulse mode is 5 s / 5 s, and the total treatment time is 12 min; the conditions for constant temperature stirring include: stirring rate of 350 rpm and stirring time of 20 min; the mass ratio of collagen to lipid is 1:8; D. High-pressure homogenization: Pre-cool the collagen liposome suspension obtained in step C to 5 °C, and perform cyclic treatment 3 times using a high-pressure homogenizer (at a pressure of 1000 bar). Dynamic light scattering (DLS) shows that the average particle size is 144.7 nm and the PDI is 0.20. Then, transfer the collagen liposome suspension to an ultrafiltration centrifugal tube to remove unencapsulated collagen and small molecule impurities (the content of free protein was determined by the BCA method to be 4.8%) to obtain a purified collagen liposome suspension; the specific parameters for ultrafiltration include: cut-off molecular weight of 90 kDa, centrifugal force of 9000×g, and centrifugation time of 25 min.

[0055] Example 4 A preparation method of an oil-soluble recombinant collagen biomimetic nano-liposome delivery system, comprising the following steps: Trehalose was added to the purified collagen liposome suspension obtained in step D of Example 1 and stirred until dissolved; then it was pre-frozen at -80 °C for 5 h; then it was placed in a freeze dryer, and the conditions for primary drying were set as follows: temperature -50 °C, pressure 0.01 mBar, time 12 h; the conditions for secondary drying were as follows: temperature -30 °C, pressure 0.005 mBar, time 12 h; a white lyophilized powder was obtained; after reconstitution, the encapsulation efficiency (90.5% by HPLC), particle size distribution (PDI was 0.23), and morphological integrity (the structure was verified to be intact by TEM) were detected; the product was dispensed into 2 mL sterile vials, filled with nitrogen and sealed, and labeled with the batch number, production date, and storage conditions (protected from light at 4 °C, validity period 24 months); The dosage relationship between trehalose and the purified collagen liposome suspension was 5 g: 100 ml; the stirring rate was 200 rpm, at 25 °C, for 30 min. In the present invention, the lyoprotectant trehalose can also be used in other embodiments, such as mannitol, sucrose, or a trehalose-glycerol composite system, but these methods are not as effective as trehalose in protecting the freeze-drying effect. After replacing trehalose with an equal mass of lyoprotectant in parallel experiments, the test results showed that: The product freeze-dried with mannitol: The reconstituted encapsulation efficiency was 85.35%, the particle size distribution PDI was 0.31, the liposome part of the lyophilized powder was ruptured, the transdermal depth (24 h) was 120 μm, the cumulative transdermal amount (24 h) was 25.5 μg / cm², and the activity retention rate was 87.53%.

[0056] The product freeze-dried with sucrose: The reconstituted encapsulation efficiency was 80.21%, the particle size distribution PDI was 0.35, the lyophilized powder was significantly aggregated, the transdermal depth (24 h) was 100 μm, the cumulative transdermal amount (24 h) was 18.7 μg / cm², and the activity retention rate was 83.26%.

[0057] The product freeze-dried with a trehalose-glycerol composite system (mass ratio 3:2): The reconstituted encapsulation efficiency was 86.27%, the particle size distribution PDI was 0.28, the lyophilized powder was slightly collapsed, the transdermal depth (24 h) was 125 μm, the cumulative transdermal amount (24 h) was 27.3 μg / cm², and the activity retention rate was 90.55%.

[0058] Example 5 A preparation method of an oil-soluble recombinant collagen biomimetic nano-liposome delivery system, comprising the following steps: Add trehalose to the purified collagen liposome suspension obtained in Step D of Example 2, and stir until dissolved; then pre-freeze at -90°C for 4 h; then place it in a freeze dryer, and set the conditions for primary drying to include: temperature -55°C, pressure 0.01 mBar, time 10 h; the conditions for secondary drying to include: temperature -35°C, pressure 0.005 mBar, time 10 h; obtain a white lyophilized powder; after reconstitution, detect the encapsulation efficiency (HPLC is 90.23%), particle size distribution (PDI is 0.28), and morphological integrity (TEM verifies the complete structure); dispense the product into 2 mL sterile vials, fill with nitrogen and seal, and label with the batch number, production date, and storage conditions (4°C in the dark, validity period 24 months); The dosage relationship between trehalose and the purified collagen liposome suspension is 8 g:100 ml; the stirring rate is 180 rpm, 25°C, and the time is 40 min.

[0059] Example 6 A preparation method of an oil-soluble recombinant collagen biomimetic nano-liposome delivery system, comprising the following steps: Add trehalose to the purified collagen liposome suspension obtained in Step D of Example 3, and stir until dissolved; then pre-freeze at -70°C for 6 h; then place it in a freeze dryer, and set the conditions for primary drying to include: temperature -45°C, pressure 0.01 mBar, time 14 h; the conditions for secondary drying to include: temperature -25°C, pressure 0.005 mBar, time 14 h; obtain a white lyophilized powder; after reconstitution, detect the encapsulation efficiency (HPLC is 90.01%), particle size distribution (PDI is 0.29), and morphological integrity (TEM verifies the complete structure); dispense the product into 2 mL sterile vials, fill with nitrogen and seal, and label with the batch number, production date, and storage conditions (4°C in the dark, validity period 24 months); The dosage relationship between trehalose and the purified collagen liposome suspension is 2 g:100 ml; the stirring rate is 220 rpm, 25°C, and the time is 20 min.

[0060] Comparative Example 1 A preparation method of a collagen biomimetic nano-liposome, comprising the following steps: A. Preparation of lipid film: Mix 700 mg of soy lecithin with 300 mg of cholesterol, dissolve them in 100 mL of a chloroform-methanol mixed solvent (the volume ratio of chloroform to methanol in the mixed solvent is 2:1), and stir magnetically until completely dissolved; transfer to a rotary evaporator and evaporate until a uniform lipid film is formed, then dry under vacuum to ensure that the solvent residue is <10 ppm; among them, the stirring conditions include: stirring rate 500 rpm, 25 °C, stirring time 30 min; the operating conditions of the rotary evaporator are: temperature 40 °C, rotation speed 200 rpm, vacuum degree -0.1 MPa; the vacuum drying conditions include: vacuum degree -0.1 MPa, vacuum drying time 24 h, vacuum drying temperature 25 °C; Preparation of blank liposomes: Add 10 mL of pH 4.0 citrate buffer to the lipid film, shake in a water bath at 65 °C to hydrate and swell the lipids. The rotation speed of the shaking is 200 rpm and the time is 30 min; perform probe sonication to obtain blank liposomes; among them, the citrate buffer contains 150 mM NaCl; the conditions of the probe sonication include: power 200 W, 5 s pulse / 5 s interval, 20 cycles; B. Loading of collagen with drugs: Dissolve recombinant collagen in absolute ethanol to prepare a 50 mg / mL recombinant collagen solution; dropwise add the recombinant collagen solution to the blank liposomes obtained in step A, stir at a constant temperature of 40 °C, and perform probe sonication to promote the embedding of collagen into the hydrophobic core of the lipid to obtain a collagen liposome suspension (the encapsulation efficiency measured by HPLC is 62.33%). Among them, the ultrasonic power is 200 W, the pulse mode is 5 s / 5 s, and the total treatment time is 10 min; the conditions of the constant temperature stirring include: stirring rate 300 rpm, stirring time 30 min; the mass ratio of collagen to lipid is 1:10; Then transfer the collagen liposome suspension to an ultrafiltration centrifugal tube to remove unencapsulated collagen and small molecule impurities to obtain a purified collagen liposome suspension; dynamic light scattering (DLS) shows that the average particle size is 250.1 nm and the PDI is 0.45. The specific parameters of ultrafiltration include: molecular weight cut-off 100 kDa, centrifugal force 10,000×g, centrifugation time 20 min.

[0061] Comparative Example 2 A method for preparing a collagen biomimetic nano-liposome, comprising the following steps: A. Preparation of lipid film: Mix 700 mg of soy lecithin with 300 mg of cholesterol and dissolve in 100 mL of a chloroform-methanol mixed solvent (the volume ratio of chloroform to methanol in the mixed solvent is 2:1). Stir magnetically until completely dissolved; transfer to a rotary evaporator and evaporate to form a uniform lipid film, then vacuum dry to ensure that the solvent residue is <10 ppm. Among them, the stirring conditions include: stirring rate 500 rpm, 25 °C, stirring time 30 min; the working conditions of the rotary evaporator are: temperature 40 °C, rotation speed 200 rpm, vacuum degree -0.1 MPa; the vacuum drying conditions include: vacuum degree -0.1 MPa, vacuum drying time 24 h, vacuum drying temperature 25 °C. Preparation of blank liposomes: Add 10 mL of pH 4.0 citrate buffer to the lipid film and shake in a water bath at 65 °C to hydrate and swell the lipids. The rotation speed of the shaking is 200 rpm and the time is 30 min; perform probe sonication to obtain blank liposomes. Among them, the citrate buffer contains 150 mM NaCl. The conditions of the probe sonication include: power 200 W, 5 s pulse / 5 s interval, 20 cycles. B. Loading of collagen with drugs: Dissolve recombinant collagen in absolute ethanol to prepare a 50 mg / mL recombinant collagen solution; add the recombinant collagen solution dropwise to the blank liposomes obtained in step A, stir at a constant temperature of 40 °C, and perform probe sonication to promote the embedding of collagen into the hydrophobic core of the lipid to obtain a collagen liposome suspension (the encapsulation efficiency measured by HPLC is 77.11%). Among them, the ultrasonic power is 200 W, the pulse mode is 5 s / 5 s, and the total treatment time is 10 min. The conditions of the constant temperature stirring include: stirring rate 300 rpm, stirring time 30 min; the mass ratio of collagen to lipid is 1:10. C. High-pressure homogenization: Pre-cool the collagen liposome suspension obtained in step B to 4 °C and cycle it 3 times using a high-pressure homogenizer (at a pressure of 1000 bar). Then transfer the collagen liposome suspension to an ultrafiltration centrifugal tube to remove unencapsulated collagen and small molecule impurities to obtain a purified collagen liposome suspension. Dynamic light scattering (DLS) shows that the average particle size is 153.5 nm and the PDI is 0.35. The specific parameters of ultrafiltration include: molecular weight cut-off 100 kDa, centrifugal force 10,000×g, centrifugation time 20 min.

[0062] Comparative Example 3 A method for preparing a collagen biomimetic nano-liposome, comprising the following steps: A. Preparation of lipid film: Mix 700 mg of soy lecithin with 300 mg of cholesterol and dissolve them in 100 mL of a chloroform-methanol mixed solvent (the volume ratio of chloroform to methanol in the mixed solvent is 2:1). Stir magnetically until completely dissolved; transfer to a rotary evaporator and evaporate to form a uniform lipid film, then dry under vacuum to ensure that the solvent residue is <10 ppm; among them, the stirring conditions include: stirring rate 500 rpm, 25 °C, stirring time 30 min; the operating conditions of the rotary evaporator are: temperature 40 °C, rotation speed 200 rpm, vacuum degree -0.1 MPa; the vacuum drying conditions include: vacuum degree -0.1 MPa, vacuum drying time 24 h, vacuum drying temperature 25 °C; Preparation of blank liposomes: Add 10 mL of pH 4.0 citrate buffer to the lipid film, shake in a water bath at 65 °C to hydrate and swell the lipids. The rotation speed of the shaking is 200 rpm and the time is 30 min; perform probe sonication to obtain blank liposomes; among them, the citrate buffer contains 150 mM NaCl; the conditions of the probe sonication include: power 200 W, 5 s pulse / 5 s interval, 20 cycles; B. Oriented insertion of membrane proteins: Dissolve integrin α6β4 transmembrane protein in pH 7.4 phosphate buffer solution to prepare a transmembrane protein solution with a concentration of 1 mg / mL; Add the transmembrane protein solution to the blank liposomes obtained in step A, incubate with shaking at 37 °C, and use the pH gradient inside and outside the liposomes to drive the insertion of membrane proteins into the lipid bilayer. Ultrafiltration and centrifugation are used to remove unbound proteins to obtain a liposome suspension; Fluorescently labeled integrin α6β4 (FITC-labeled) is used, and the fluorescence intensity of the liposome suspension is measured by a fluorescence spectrophotometer. The calculated insertion efficiency is 97.3%; among them, the mass ratio of integrin α6β4 transmembrane protein to lipid is 1:1000; the conditions of incubation with shaking include: rotation speed 150 rpm, time 2 h; the cut-off molecular weight of ultrafiltration and centrifugation is 100 kDa, the centrifugal force is 10,000×g, and the centrifugation time is 30 min; C. Loading of collagen with drugs: Dissolve recombinant collagen in absolute ethanol to prepare a recombinant collagen solution with a concentration of 50 mg / mL; add the recombinant collagen solution dropwise to the liposome suspension obtained in step B, stir at a constant temperature of 40 °C, and perform probe sonication to promote the embedding of collagen into the hydrophobic core of the lipid to obtain a collagen liposome suspension (the encapsulation efficiency measured by HPLC is 75.63%); among them, the ultrasonic power is 200 W, the pulse mode is 5 s / 5 s, and the total treatment time is 10 min; the conditions of constant temperature stirring include: stirring rate 300 rpm, stirring time 30 min; the mass ratio of collagen to lipid is 1:10; D. Transfer the collagen liposome suspension obtained in step C to an ultrafiltration centrifugal tube to remove unencapsulated collagen and small molecule impurities, obtaining a purified collagen liposome suspension. Dynamic light scattering (DLS) shows that the average particle size is 252.3 nm and the PDI is 0.47. The specific parameters of ultrafiltration include: the molecular weight cut-off is 100 kDa, the centrifugal force is 10,000×g, and the centrifugation time is 20 min.

[0063] Comparative Example 4 Ordinary cream carrier A method for preparing a cream, comprising the following steps: Step 1: Preparation of the oil phase Weigh the oil phase components: Recombinant collagen: 5.0 g (calculated at a concentration of 50 mg / mL, need to be dissolved in 100 mL of solvent); Squalane: 15.0 g; Ceteareth-20: 3.0 g; Mix the oil phase components: Add recombinant collagen, squalane, and ceteareth-20 to a beaker; heat to 70 ± 2 °C and stir magnetically (300 rpm, 10 min) until completely dissolved to form a uniform oil phase. Detection standard: Visually no particles, light transmittance > 95%.

[0064] Step 2: Preparation of the aqueous phase Weigh the aqueous phase components: Glycerol: 10.0 g; Phenoxyethanol: 0.5 g; Deionized water: 66.5 g; Mix the aqueous phase components: Add glycerol and phenoxyethanol to deionized water; heat to 70 ± 2 °C and stir magnetically (300 rpm, 5 min) until homogeneous. Detection standard: pH 5.5 - 6.5 (measured by pH meter).

[0065] Step 3: Emulsification process Mix the oil phase and the aqueous phase: Slowly pour the oil phase into the aqueous phase while maintaining a constant temperature of 70 °C; High-pressure homogenization emulsification (8000 rpm, 5 min) to form a primary emulsion. Detection standard: The emulsion particle size D50 < 10 μm (laser particle size analyzer); Cooling emulsification: Cool down to 45 °C and continue homogenization (5000 rpm, 3 min); Detection standard: Viscosity 3000 - 5000 cP (rheometer); Naturally cool to room temperature and stir (200 rpm) until the cream texture is uniform.

[0066] Step 4: Filling and storage Sub-packaging: Fill the cream into a sterile aluminum tube, seal it in the dark; Storage conditions: Store in a cool place below 25°C. Shelf life: 12 months.

[0067] Comparative Example 5 Blank control group: Pure recombinant collagen without liposome encapsulation. The transdermal performance of recombinant collagen was directly measured. The transdermal depth (24 h) was 20 μm, the cumulative transdermal amount (24 h) was 4.5 μg / cm², and the collagen activity was 78%.

[0068] Test Example 1. The average particle size, PDI, and transdermal depth of the purified collagen liposome suspensions prepared in Examples 1 - 3 and Comparative Examples 1 - 3 were measured. The measurement results are shown in Table 1.

[0069] Table 1 2. After reconstituting the freeze-dried products prepared in Examples 4 - 6, the encapsulation efficiency, PDI, morphology, transdermal depth, cumulative transdermal amount, and activity retention rate were measured. The results are shown in Table 2.

[0070] Table 2 3. The collagen activity retention rate, transdermal depth, and moisturizing duration of the cream products of Example 1 and Comparative Example 4 were measured. The results are shown in Table 3. The measurement method is as follows: Table 3 The specific measurement methods for the insertion efficiency, encapsulation efficiency, average particle size, PDI, transdermal depth, and cumulative transdermal amount of the membrane protein in the present invention are as follows: Measurement of the insertion efficiency of the membrane protein: The fluorescence labeling quantification method was used. Specifically, fluorescein isothiocyanate (FITC) was covalently labeled on the amino group of the membrane protein. The unbound fluorescein was separated by ultrafiltration centrifugation (10 kDa cut-off molecular weight, 4000×g×15 min). The fluorescence intensity of the binding was measured using a fluorescence spectrophotometer (excitation / emission wavelength 495 / 520 nm). The actual binding amount was calculated by combining with the standard curve (FITC concentration 0.01 - 1 μg / mL, R²>0.99). Finally, the insertion efficiency was defined as the percentage of the actual binding amount to the theoretical maximum binding amount (1:1 stoichiometric ratio according to 15 amino groups per molecule). The operation was carried out in the dark throughout the experiment. The specificity of the labeling was verified by SDS-PAGE fluorescence imaging, and the CV value of the parallel experiment was <5%.

[0071] Encapsulation efficiency: HPLC (High Performance Liquid Chromatography) determination reference: General Rules 0512 "High Performance Liquid Chromatography" in the Fourth Part of the Chinese Pharmacopoeia (2020 Edition), "Determination of the prohibited substance triclosan in cosmetics - High performance liquid chromatography" in GB / T 30939 - 2014. The content of free and total collagen was determined by HPLC to calculate the encapsulation efficiency.

[0072] Average particle size and PDI: Measured by dynamic light scattering (DLS) according to the standard method ISO22412:2017: Particle size analysis - Dynamic light scattering method.

[0073] Determination of transdermal depth and permeation amount: Franz diffusion cell (porcine skin model), referring to General Chapter 0931 "Determination of Release of Transdermal Patches" in the Fourth Part of Chinese Pharmacopoeia (2020 Edition) and OECD TG428: Guidelines for In Vitro Skin Permeation Testing.

[0074] The specific method is as follows: 1. Porcine skin treatment: Take fresh porcine ear skin (thickness 0.8 ± 0.1 mm), remove hair and then peel off subcutaneous fat, wash with PBS, and store at -20 °C for later use.

[0075] 2. Experimental setup: Effective diffusion area of the diffusion cell: 1.8 cm²; Receptor fluid: PBS with pH 7.4 (containing 0.01% NaN3 for antibacterial); Temperature: 32 ± 2 °C (simulating the surface temperature of human skin); Sampling time points: 1, 2, 4, 8, 12, 24 h.

[0076] 3. Calculation of transdermal amount: The collagen concentration in the receptor fluid is determined by HPLC, and the cumulative transdermal amount (Qn) is calculated according to the following formula: Qn = Cn × V + ∑i = 1n - 1 C i × V i where Cn is the concentration at the nth sampling, V is the volume of the receptor pool (5 mL), and V i is the volume of each sampling (0.5 mL).

[0077] 4. Verification of transdermal depth: Use a confocal microscope (fluorescently labeled collagen) to observe the sections and measure the penetration depth of the fluorescent signal.

[0078] The reconstitution of the freeze-dried product in the present invention includes the following steps: Step 1: Pre-reconstitution treatment Equilibration temperature: Preheat the lyophilized powder and the reconstitution solution (PBS: Phosphate Buffered Saline) to 25 ± 2 °C to avoid rupture of liposomes caused by sudden temperature changes.

[0079] Step 2: Stepwise reconstitution Initial wetting: Add 10% of the volume of the reconstitution solution to the lyophilized vial (e.g., if a total of 10 mL is required, add 1 mL first); Gently rotate the vial and let it stand for 5 min to allow the lyophilized powder to initially absorb water and swell.

[0080] Gradual dilution: Add the remaining reconstitution solution in 3 portions (30% each time subsequently), with a 2-min interval between each addition, and vortex (1000 rpm, 10 s) to promote dispersion; Total reconstitution time: ≤15 min.

[0081] Step 3: Homogenization Magnetic stirring: Transfer the reconstitution solution to a sterile glass bottle and stir magnetically at 25°C (200 rpm, 10 min) to ensure uniform dispersion of the liposomes; Avoid high-speed stirring (>500 rpm) that may cause structural damage due to shear force.

[0082] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an oil-soluble recombinant collagen bionic nanoliposome delivery system, characterized in that: The steps include: A. dissolving lipid in a solvent, stirring, and removing the solvent to prepare a lipid film; adding a buffer solution with a pH of 3.8-4.2 to the lipid film, shaking it to hydrate and swell it, and then subjecting it to ultrasonic treatment with a probe to obtain blank liposomes; the lipid comprises 70-85wt% soybean lecithin and 15-30wt% cholesterol; B. Dissolving the integrin α6β4 transmembrane protein in a pH 7.2-7.4 buffer solution to prepare a 0.5-1.5 mg / mL transmembrane protein solution; Add the transmembrane protein solution to the blank liposomes obtained in step A, incubate at 37°C with shaking, and ultrafiltrate and centrifuge to obtain a liposome suspension; wherein the mass ratio of integrin α6β4 transmembrane protein to lipid is 0.5-5:1000; C. Add the recombinant collagen solution dropwise to the liposome suspension obtained in step B, stir at a constant temperature of 40±2° C., and perform ultrasonic treatment to obtain a collagen liposome suspension, wherein the mass ratio of the recombinant collagen to the lipid is 1:8-12; D. The collagen liposome suspension obtained in step C is homogenized under high pressure and purified by ultrafiltration to obtain a purified collagen liposome suspension.

2. The method for preparing an oil-soluble recombinant collagen bionic nanoliposome delivery system according to claim 1, characterized in that: In step A: The solvent is chloroform and methanol in a volume ratio of 1.8-2.2:1, or the solvent is a mixture of ethanol and ether; The amount of solvent and lipid used is 90-110ml:1000mg; The stirring conditions include: stirring rate 450-550 rpm, 25±2°C, stirring time 25-35 min; The step of removing the solvent includes rotary evaporation and then vacuum drying, wherein the conditions of the rotary evaporation include: temperature 38-42°C, rotation speed 150-250rpm, vacuum degree -0.1MPa; the conditions of vacuum drying include: vacuum degree -0.1MPa, vacuum drying time 20-26h, vacuum drying temperature 25°C; The shaking speed is 180-220 rpm, the time is 20-40 min, and the temperature is 60-70 ° C; The conditions of the probe ultrasonic treatment included: power 180-220 W, 5 s pulse / 5 s interval, 18-22 cycles.

3. The method for preparing an oil-soluble recombinant collagen bionic nanoliposome delivery system according to claim 1, characterized in that: In the step B: The shaking incubation conditions include: rotation speed 120-180 rpm, time 1.5-2.5 h; The molecular weight cutoff of ultrafiltration centrifugation is 90-110kDa, the centrifugal force is 9000-11000×g, and the centrifugation time is 25-35min.

4. The method for preparing an oil-soluble recombinant collagen bionic nanoliposome delivery system according to claim 1, characterized in that: In the step C: The preparation of the recombinant collagen solution includes: dissolving the recombinant collagen in a solvent to prepare a recombinant collagen solution with a concentration of 50±2.5 mg / mL; Ultrasonic treatment conditions included: power 180-220 W, pulse mode 5 s / 5 s, total treatment time 8-12 min; The conditions for constant temperature stirring include: stirring rate 250-350 rpm, stirring time 20-40 min.

5. The method for preparing an oil-soluble recombinant collagen bionic nanoliposome delivery system according to claim 1, characterized in that: In the step D: the high-pressure homogenization step comprises: precooling the liposome suspension to 3-5°C, and using a high-pressure homogenizer for 3 cycles, with a homogenization pressure of 800-1200 bar; The ultrafiltration purification steps include: molecular weight cut-off of 90-110 kDa, centrifugal force of 9000-11000×g, and centrifugal time of 15-25 min.

6. The method for preparing an oil-soluble recombinant collagen bionic nanoliposome delivery system according to claim 1, characterized in that: The method further comprises step E, which comprises: freeze-drying the purified collagen liposome suspension obtained in step D to obtain a freeze-dried product.

7. The method for preparing an oil-soluble recombinant collagen bionic nanoliposome delivery system according to claim 6, characterized in that: The freeze-drying step in step E comprises: adding a freeze-drying protective agent to the purified collagen liposome suspension obtained in step D, stirring until dissolved; then pre-freezing at -70~-90°C for 4-6 hours; placing in a freeze dryer, setting the primary drying conditions to include: temperature -45~-55°C, pressure 0.01mBar, time 10-14h; secondary drying conditions include: temperature -25~-35°C, pressure 0.005mBar, time 10-14h; The dosage of the lyophilization protective agent and the purified collagen liposome suspension is 2-8 g: 100 ml; The stirring rate is 180-220 rpm, 25±2° C., and the time is 20-40 min.

8. An oil-soluble recombinant collagen bionic nanoliposome delivery system, characterized in that: The oil-soluble recombinant collagen bionic nanoliposome delivery system is prepared based on the preparation method of the oil-soluble recombinant collagen bionic nanoliposome delivery system according to any one of claims 1 to 7.

9. Use of the oil-soluble recombinant collagen bionic nanoliposome delivery system as claimed in claim 8 in the preparation of cosmetics.

10. The use according to claim 9, characterized in that The cosmetics include water-based agents, emulsions, sprays or facial masks.

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