Multi-structure carrier of recombinant III-type humanized collagen microspheres as well as preparation method and application of multi-structure carrier

Abstract

The invention discloses a multi-structure carrier of recombinant III-type humanized collagen microspheres as well as a preparation method and application of the multi-structure carrier. The multi-structure carrier comprises an inner phase, a middle phase and an outer phase, the recombinant III-type humanized collagen microspheres and hydrogenated lecithin form a chimera which is located in the inner phase; the middle phase is composed of three kinds of grease similar to skin structures, namely phytosterol ester, squalane and polyglycerol-3 diisostearate; the external phase is a sodium hyaluronate aqueous solution. Through W / O / W double emulsification design, recombinant III-type humanized collagen microspheres and hydrogenated lecithin form a chimera and then are wrapped heavily, so that the environmental stability, biocompatibility and transdermal performance of the recombinant III-type humanized collagen microspheres are improved, and the recombinant III-type humanized collagen microspheres have immediate moisturizing and slow-release effects and can be applied to preparation of a skin care product. The effects of long-acting slow release, efficient transdermal and three-dimensional repair of the recombinant III-type humanized collagen microspheres are achieved, and the recombinant III-type humanized collagen microspheres are suitable for high-end cosmetics or medical instruments.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a recombinant type III humanized collagen microsphere multiple structure carrier and a preparation method and application thereof. BACKGROUND

[0002] The recombinant type III human collagen microsphere is a microsphere type protein with a cavity structure formed by folding and circularizing a linear recombinant type III human collagen, as shown in the formula. Figure 1 The space structure of the protein microsphere is changed due to circularization, and the particle size of the protein microsphere is only 7-8 nanometers, which can better pass through the skin barrier. However, it is found in the research and development work that the existing recombinant type III human collagen microsphere has the problem of poor stability in application. The main reason is that the space structure is easily affected by environmental factors such as temperature, light and oxygen, and degrades, resulting in reduced efficacy. In addition, the existing recombinant type III human collagen microsphere has the characteristic of strong water solubility. When absorbed through the skin, it loses a lot when passing through the skin barrier, especially for products with a molecular weight of more than 5000 Da, it is difficult to penetrate the dense stratum corneum, and the bioavailability is difficult to achieve the expected value, which limits its use in skin care products to a certain extent. Therefore, increasing the stability and effective transdermal rate of the recombinant type III human collagen microsphere has become the focus of research and development work. In addition, the main chain of the recombinant type III human collagen microsphere is composed of repeated glycine-proline-hydroxyproline (Gly-Pro-Hyp) tripeptide units, and a large number of hydrophilic groups (hydroxyl group -OH, amino group -NH2, carboxyl group -COOH) are exposed on the surface. Therefore, the recombinant type III human collagen microsphere is insoluble in oil and unstable in oil-containing products, and an emulsifying agent needs to be introduced to help it disperse uniformly. However, the introduction of the emulsifying agent not only increases the complexity of the formula, but also has the risk of causing irritation or allergic reactions. Moreover, the circularized recombinant type III human collagen microsphere has a strong aggregation tendency due to its small particle size, and ordinary emulsion dispersants cannot keep it stable in the solution system for a long time.

[0003] In the prior art, the active ingredients are formed into a package structure in the form of water-in-oil (O / W) or oil-in-water (W / O), which can effectively improve the stability of the active ingredients and improve water solubility. In particular, multiple emulsions, including water-in-oil-in-water (W / O / W) and oil-in-water-in-oil (O / W / O) systems, have the characteristics of high dispersion and different particle sizes, and have the characteristics of multiple protection, promoting absorption, and strong compatibility for the components wrapped in the inner phase, and have also received widespread attention in the industry. For example, Chinese patent application document CN118845520A proposes a double emulsion droplet stably wrapping collagen peptide and a preparation method and application thereof, dissolving collagen peptide in deionized water as an inner aqueous phase, dissolving EM90 in dimethyl silicone oil as an intermediate oil phase, and dissolving Tween 80 in deionized water as an outer phase; then generating water-in-oil-in-water double emulsion droplets wrapped with collagen peptide through microfluidic technology; so that the collagen peptide remains stable at ambient temperature during storage and transportation, and better realizes the moisturizing effect of cosmetics. Another Chinese patent application document CN119745723A proposes a collagen liposome and a moisturizing and anti-aging composition and a preparation method and application thereof, mixing collagen with choline phospholipid and a solvent, then performing normal pressure homogenization and pressure homogenization, so that the collagen is wrapped in the bilayer formed by the choline phospholipid, to overcome the skin barrier and improve the efficiency of collagen penetrating the skin. However, the microfluidic wrapping technology requires too high a requirement, needs precise control of the injection flow of the three-phase solution, and the normal pressure homogenization and pressure homogenization are stacked, which is expensive in equipment cost and low in production efficiency, and is not conducive to popularization and production. In addition, the current wrapping method is for linear space structure collagen, and for recombinant human type III collagen microspheres, direct dissolution in deionized water will accelerate its degradation; and if liposomes are used for direct wrapping, due to the cyclization structure of collagen, not only the encapsulation rate is low, but also the binding force between the liposomes and the protein microspheres is weak, the wrapping is not firm, and the expected function is easily lost.

[0004] Therefore, how to improve the stability and effective transdermal rate of recombinant humanized type III collagen microspheres, and improve its use efficiency, is still a problem to be solved. SUMMARY

[0005] In view of the problems that the recombinant type III human collagen microspheres are easily degraded by environmental factors such as temperature, light and oxygen, resulting in reduced efficacy, and when absorbed transdermally, a large amount of the product is lost in crossing the skin barrier, especially the product with a molecular weight of more than 5000 Da, which is difficult to penetrate the dense stratum corneum, and the bioavailability is difficult to achieve the expected problem, the present application provides a recombinant type III humanized collagen microsphere multi-structure carrier and a preparation method and application thereof, which utilizes the three-dimensional spatial structure of the recombinant type III humanized collagen microspheres, combines the chimeric body with hydrogenated lecithin, then is wrapped with similar skin oil, and finally is wrapped with water phase, thereby significantly improving the stability and effective transdermal rate in high temperature, light and oxidation environment. The specific technical solutions are as follows: Firstly, the present application provides a recombinant type III humanized collagen microsphere multi-structure carrier, wherein the recombinant type III human collagen microspheres are microspheres of collagen with a cavity structure formed by folding and cyclizing linear recombinant type III humanized collagen; the carrier comprises an inner phase, a middle phase and an outer phase; the chimeric body of the recombinant type III humanized collagen microspheres and hydrogenated lecithin is located in the inner phase; the middle phase is composed of three kinds of skin structure similar oils, i.e., phytosterol ester, squalane and polyglycerol-3 diisostearate; and the outer phase is a sodium hyaluronate aqueous solution.

[0006] The recombinant type III humanized collagen microsphere multi-structure carrier, the molecular weight of the recombinant type III humanized collagen microspheres in the inner phase is 5000-10000 Da, and the mass ratio of the recombinant type III humanized collagen microspheres to hydrogenated lecithin is (0.5-2):(20-30).

[0007] The recombinant type III humanized collagen microsphere multi-structure carrier, the mass ratio of the three oils in the middle phase is (2.5-4.5):(3-8):(10-20).

[0008] The recombinant type III humanized collagen microsphere multi-structure carrier, the molecular weight of the sodium hyaluronate in the outer phase is 100w-180w Da; and the components of the sodium hyaluronate aqueous solution include water, sodium hyaluronate, panthenol, glycerol and xanthan gum, and the mass ratio is (93.1-96.9):(0.5-0.9):(0.5-1.5):(2-4):(0.1-0.5).

[0009] The recombinant type III humanized collagen microsphere multi-structure carrier, the mass ratio of the inner phase, the middle phase and the outer phase is (0.2-2):(1-3):(5-8.8); the particle size distribution range of the inner phase of the multi-structure carrier is 50-400 nm, and the particle size distribution range of the middle phase is 1-4 μm.

[0010] Secondly, the application provides a preparation method of the aforementioned multi-structure carrier of the recombinant humanized type III collagen microspheres, comprising the following steps: 1) preparing an inner phase: dissolving the recombinant humanized type III collagen microspheres in a phosphate buffer solution to obtain a recombinant humanized type III collagen microsphere aqueous solution; then pouring the recombinant humanized type III collagen microsphere aqueous solution into a hydrogenated lecithin-glycerol solution to make the recombinant humanized type III collagen microspheres flocculate with the hydrogenated lecithin, forming a recombinant humanized type III collagen microsphere suspension; then ultrasonic dispersion, and then centrifuging to collect the suspension to obtain a chimeric body formed by the recombinant humanized type III collagen microspheres and the hydrogenated lecithin, as the inner phase, for standby; 2) preparing a middle phase: uniformly mixing phytosterol ester, squalane and polyglycerol-3 diisostearate according to a mass ratio to obtain the middle phase, for standby; 3) preparing an outer phase: uniformly mixing water, sodium hyaluronate, panthenol, glycerol and xanthan gum according to a mass ratio to obtain the outer phase, for standby; 4) primary wrapping: adding the inner phase prepared in step 1) into the middle phase prepared in step 2) and shearing to disperse to form a W / O emulsion; 5) secondary wrapping: heating the outer phase prepared in step 3) to 30-50 DEG C, and under the condition of stirring at 1000-1500 r / min, pouring the W / O emulsion formed by the primary wrapping in step 4) into the outer phase to perform secondary wrapping, to obtain a W / O / W multi-structure emulsion, namely the multi-structure carrier.

[0011] As a preferred technical scheme, in step 1): The concentration of the recombinant humanized type III collagen microsphere aqueous solution is 2-4 mg / mL; The hydrogenated lecithin-glycerol solution is prepared by shearing mixing hydrogenated lecithin and glycerol at a mass ratio of (1-2):(8-9), the shearing dispersion speed is 3000-4000 r / min, and the dispersion time is 15-25 min; The concentration of the recombinant humanized type III collagen microsphere suspension is 1-3 mg / mL; The ultrasonic dispersion condition is 240-260 W, and the dispersion time is 15-20 min; The centrifugation condition is 8000-10000 r / min, and the centrifugation time is 10-15 min; The particle size distribution of the obtained chimeric body is in the range of 50-400 nm.

[0012] As a preferred technical scheme, in step 4), the mass ratio of the inner phase to the middle phase in the primary encapsulation is: inner phase: middle phase = (0.2-2):(1-3); the process of the primary encapsulation is: the temperature of the inner phase and the middle phase is both raised to 30-50 DEG C, the middle phase is under the homogenization condition of 2500-3500 rpm, the inner phase is poured into the middle phase at a speed of 5-7 mL / min, after the addition is completed, shearing is carried out at 8000-10000 r / min for 10-30 min, and a W / O emulsion is formed.

[0013] As a preferred technical scheme, in step 5), the volume ratio of the W / O emulsion formed by the primary encapsulation to the outer phase in the secondary encapsulation is (1.2-5):(5-8.8); the process of the secondary encapsulation is: the temperature of the outer phase is raised to 30-50 DEG C, the outer phase is under the stirring condition of homogenization 1000-1500 r / min, the W / O emulsion formed by the primary encapsulation is poured into the outer phase at a speed of 5-7 mL / min, the secondary encapsulation is carried out, after the addition is completed, stirring is continued for 25 min, and the temperature is reduced to room temperature, and a W / O / W multiple structure emulsion is obtained, which is the carrier of the application.

[0014] In addition, the application also provides an application of the multiple structure carrier of the recombinant humanized collagen type III microspheres, which is directly used as a cosmetic or a medical device product, or is used as one of key effective components for preparing a cosmetic or a medical device product.

[0015] The application has the following beneficial effects: 1) The application combines the three-dimensional structure characteristics of the recombinant humanized collagen type III microspheres and the characteristics of the skin, embeds the recombinant humanized collagen type III microspheres with strong water solubility and hydrogenated lecithin into a chimeric structure as an inner phase, then encapsulates the inner phase in a middle phase composed of oil similar to the structure of the skin, the hydrogenated lecithin and the oil layer in the middle phase can form a complete encapsulation environment for the recombinant humanized collagen type III microspheres, effectively isolate the outside oxygen, and improve the oxidation stability; the oil similar to the structure of the skin in the middle phase helps to improve the penetration rate of the recombinant humanized collagen type III microspheres through the stratum corneum; and the outer phase of the carrier is a sodium hyaluronate aqueous solution, which can better protect the protein microspheres, improve the stability of the protein microspheres under high temperature and light, and also enable the protein microspheres to be better absorbed by the skin, play the biological activity of collagen, and have better effects in reducing wrinkle area and improving facial firmness.

[0016] 2) The core of the carrier of the present application is a chimeric body formed by recombinant type III humanized collagen microspheres and hydrogenated lecithin; the principle of the combination of the two is that after the recombinant type III humanized collagen microspheres are dissolved in an aqueous solution, the hydrophobic amino acids are excluded and gathered in the cavity structure formed by cyclization to form a hydrophobic region; the aqueous solution of the recombinant type III humanized collagen microspheres is poured into a hydrogenated lecithin-glycerol solution, and a small amount of the recombinant type III humanized collagen microspheres is quickly surrounded by hydrogenated lecithin; and the hydrogenated lecithin has a two-part structure of a hydrophilic head and a hydrophobic tail, under the action of non-covalent bonds, the hydrophobic tail of the hydrogenated lecithin spontaneously approaches or penetrates into the cavity structure of the recombinant type III humanized collagen microspheres to form a chimeric body. Moreover, the isoelectric point (pI) of the recombinant type III humanized collagen microspheres is between 7.0 and 9.0, and the surface will have a weak positive charge, while the hydrophilic head of the hydrogenated lecithin has a negative charge, and the electrostatic attraction between the positive and negative charges promotes the aggregation of lecithin on the surface of the collagen microspheres, which is conducive to the assembly of the chimeric body and enables the chimeric body to be stably dispersed and wrapped in the oil phase.

[0017] 3) In the carrier of the present application, the embedding of hydrogenated lecithin enables the recombinant type III humanized collagen microspheres to be stably wrapped as a middle phase of the oil phase, realize dispersion in the external phase to present a multiple structure emulsion of W / O / W, fully ensure the activity stability of the recombinant type III humanized collagen microspheres, and avoid oxidative decomposition. Moreover, the particle size of the middle phase formed by the dispersion of the chimeric body in the oil phase is between 1 µm and 4 µm, which is greater than most sunlight wavelengths and can reflect most sunlight, effectively reducing the influence of sunlight on the recombinant type III humanized collagen microspheres and improving the photothermal stability thereof. Experiments show that the carrier of the present application has very good collagen retention rate in a high-temperature environment of 45℃ and a light box with an illumination of 4000 lux, which plays an important role in maintaining the biological activity of collagen.

[0018] 4) The preparation method of the present application is simple, does not require special equipment and complex process, has a low preparation threshold, and requires simple and easily available materials, which is more simple and practical than the prior art and is more conducive to widening the application range of the recombinant type III humanized collagen microspheres.

[0019] 5) The present application has wide application prospects in the fields of biotechnology and daily chemical technology, and can promote the research and development and expansion of recombinant type III humanized collagen microspheres in the field of external use, such as cosmetics and medical dressings, and promote better development in related fields. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 is a schematic diagram of the structure of the recombinant type III humanized collagen microspheres in the internal phase of the multiple structure carrier of the recombinant type III humanized collagen microspheres; Figure 2A schematic diagram of the chimeric structure of the recombinant humanized collagen type III microspheres and hydrogenated lecithin in the internal phase of the multiple structure carrier of the recombinant humanized collagen type III microspheres of the present application (in the figure: yellow is hydrogenated lecithin, and blue is the recombinant humanized collagen type III microspheres); Figure 3 A freeze electron microscope photograph of the chimeric structure of the recombinant humanized collagen type III microspheres and hydrogenated lecithin in the internal phase of the multiple structure carrier of the recombinant humanized collagen type III microspheres of the present application, diluted 250 times; Figure 4 A microscopic detection photograph of the multiple structure carrier of the recombinant humanized collagen type III microspheres prepared in Example 1 of the present application; Figure 5 A skin photograph of the subject No. 3 in Example 2 of the present application before and after using the emulsion prepared in Example 1 and Comparative Example 2 for 28 days. DETAILED DESCRIPTION

[0021] In order to solve the problems that the recombinant human collagen type III microspheres are easily degraded by environmental factors such as temperature, light, and oxygen when applied, resulting in reduced efficacy; and when absorbed transdermally, a large amount is lost in crossing the skin barrier, especially for products with a molecular weight exceeding 5000 Da, which are difficult to penetrate the dense stratum corneum of the skin, and the bioavailability is difficult to achieve the expected results, the present application provides a recombinant humanized collagen type III microsphere multiple structure carrier and a preparation method and application thereof. The three-dimensional spatial structure of the recombinant humanized collagen type III microspheres is combined with hydrogenated lecithin to form a chimeric structure, which has a structure as shown in Figure 2 , and then is wrapped with similar skin oils, and finally is wrapped with an aqueous phase, thereby achieving the purpose of significantly improving its stability in high temperature, light, and oxidation environments and effective transdermal rate.

[0022] The application provides a multiple structure carrier of a recombinant humanized collagen type III microsphere, the recombinant humanized collagen type III microsphere is a microsphere type collagen with a cavity structure formed by folding and circularizing linear recombinant humanized collagen type III; the carrier comprises an inner phase, a middle phase and an outer phase; the recombinant humanized collagen type III microsphere and hydrogenated lecithin form a chimera in the inner phase; the middle phase is composed of skin structure similar oil; the outer phase is a sodium hyaluronate aqueous solution; the mass ratio of the inner phase, the middle phase and the outer phase is (0.2-2):(1-3):(5-8.8). The principle of the chimera formed by the recombinant humanized collagen type III microsphere and hydrogenated lecithin is that after the recombinant humanized collagen type III microsphere is dissolved in an aqueous solution, the hydrophobic amino acids thereof are repelled and gathered in the cavity structure formed by circularization to form a hydrophobic region; the recombinant humanized collagen type III microsphere aqueous solution is poured into a hydrogenated lecithin-glycerol solution, and a small amount of the recombinant humanized collagen type III microsphere is quickly surrounded by hydrogenated lecithin; moreover, the isoelectric point (pI) of the recombinant humanized collagen type III microsphere is between 7.0 and 9.0, the surface thereof has weak positive electricity, the hydrophilic head group of hydrogenated lecithin is negatively charged, and the electrostatic attraction between the positive and negative charges promotes the aggregation of lecithin to the surface of the collagen microsphere; and hydrogenated lecithin has two part structures of a hydrophilic head and a hydrophobic tail, under the action of non-covalent bonds, the hydrophobic tail of hydrogenated lecithin spontaneously approaches or penetrates into the cavity structure of the recombinant humanized collagen type III microsphere to assemble into a chimera.

[0023] The embedding of hydrogenated lecithin is beneficial to the assembly of the chimera and enables the chimera to be stably dispersed in the oil phase, so that the recombinant humanized collagen type III microsphere can be fully and stably wrapped in the middle phase, effectively isolated from external oxygen and improved in oxidation stability; meanwhile, the skin structure similar oil in the middle phase helps to improve the penetration rate of the recombinant humanized collagen type III microsphere through the stratum corneum of the skin; and the particle size distribution range of the inner phase of the multiple structure carrier is between 50 and 400 nm, and the particle size distribution range of the middle phase is between 1 and 4 microns, the particle size is greater than most sunlight wavelengths, can reflect most sunlight, can effectively reduce the influence of sunlight on the recombinant humanized collagen type III microsphere and improve the photothermal stability thereof. Moreover, the outer phase of the carrier is a sodium hyaluronate aqueous solution, which can better protect the protein microsphere, improve the stability thereof under high temperature and light, enable the protein microsphere to be better absorbed by the skin, play the biological activity of collagen and have better effects in reducing wrinkle area and improving facial firmness.

[0024] The multiple structure carrier of the recombinant humanized collagen type III microsphere of the application, the molecular weight of the recombinant humanized collagen type III microsphere in the inner phase is 5000-10000 Da, and the mass ratio of the recombinant humanized collagen type III microsphere and hydrogenated lecithin is 0.5-2:20-30; the recombinant humanized collagen type III microsphere can be fully wrapped.

[0025] The oil similar to the skin structure in the middle phase is selected from phytosteryl esters, squalane and polyglyceryl-3 diisostearate, and the mass ratio of the three is (2.5-4.5):(3-8):(10-20). Among them, polyglyceryl-3 diisostearate is the core hydrophilic-lipophilic balance agent, squalane and phytosteryl esters adjust the viscosity of the system, balance the interfacial tension and enhance the penetration and absorption of the multi-structure carrier on the skin; polyglyceryl-3 diisostearate is a polyglyceryl ester emulsifier, which contains multiple hydrophilic glycerol groups and hydrophobic isostearic acid chains, and the HLB value (hydrophilic-lipophilic balance value) is about 12-14, which can quickly reduce the oil-water interfacial tension, and has good compatibility with hydrogenated lecithin, and the two cross arrangement forms a protective structure to stabilize the chimeric body of the inner phase. Moreover, the viscosity of squalane (about 30-40 mPa·s) and phytosteryl esters (about 80-120 mPa·s) is moderate, which can form a stable middle phase oil layer with the auxiliary carrier, enhance the wrapping force on the inner phase, balance the interfacial tension, reduce oil droplet aggregation and fusion, and avoid the system from becoming thin or stratified; in addition, polyglyceryl-3 diisostearate, squalane and phytosteryl esters are low-irritation and high-biocompatibility ingredients, and in the intercellular lipid, the three kinds of oil similar compounds can improve the skin feel and enhance the absorption effect of the carrier on the skin.

[0026] The molecular weight of the sodium hyaluronate in the outer phase is 100-180 wDa; the components of the sodium hyaluronate aqueous solution include water, sodium hyaluronate, panthenol, glycerol and xanthan gum, and the mass ratio is (93.1-96.9):(0.5-0.9):(0.5-1.5):(2-4):(0.1-0.5). Among them, panthenol is an interfacial stabilizing balance agent, glycerol is an outer phase water-locking agent, and xanthan gum is a thickening agent. Panthenol molecules contain hydroxyl groups (-OH) and amide groups (-CONH-), have weak surface activity, can be adsorbed on the oil-water interface, assist polyglyceryl-3 diisostearate in forming an interfacial film in the middle phase-outer phase, and reduce the aggregation and fusion of oil droplets; glycerol contains three hydroxyl groups (-OH), can form dense hydrogen bonds with water molecules and other hydrophilic ingredients (such as panthenol and hyaluronic acid) in the formula, convert free water into bound water, reduce the change in system viscosity caused by water evaporation, and avoid water loss from causing oil droplets to aggregate; and xanthan gum can quickly dissolve in water and form a high-viscosity three-dimensional network structure, and can crosslink with sodium hyaluronate, limit the movement of oil droplets through the network structure, keep the oil droplets uniform in size, and improve long-term stability.

[0027] The application provides a preparation method of the aforementioned recombinant humanized type III collagen microsphere multi-structure carrier, which comprises the following steps: 1) Preparation of the inner phase: dissolve the recombinant humanized type III collagen microspheres in a phosphate buffer to obtain a recombinant humanized type III collagen microsphere aqueous solution; then pour the recombinant humanized type III collagen microsphere aqueous solution into a hydrogenated lecithin-glycerol solution to make the recombinant humanized type III collagen microspheres flocculate with the hydrogenated lecithin, forming a recombinant humanized type III collagen microsphere suspension; then ultrasonic dispersion, and then centrifugal collection of the suspension to obtain the chimeric body formed by the recombinant humanized type III collagen microspheres and the hydrogenated lecithin, which is used as the inner phase; the concentration of the recombinant humanized type III collagen microsphere aqueous solution is 2-4 mg / mL; the hydrogenated lecithin-glycerol solution is obtained by shearing and mixing hydrogenated lecithin and glycerol at a mass ratio of (1-2):(8-9), the shearing and dispersion speed is 3000-4000 r / min, and the dispersion time is 15-25 min; the concentration of the recombinant humanized type III collagen microsphere suspension is 1-3 mg / mL; the ultrasonic dispersion conditions are 240-260 W for 15-20 min; the centrifugal conditions are 8000-10000 r / min for 10-15 min; and the obtained chimeric body has a particle size distribution in the range of 50-400 nm.

[0028] 2) Preparation of the middle phase: uniformly mix phytosterol esters, squalane and polyglycerol-3 diisostearate according to the mass ratio to obtain the middle phase; 3) Preparation of the outer phase: uniformly mix water, sodium hyaluronate, panthenol, glycerol and xanthan gum according to the mass ratio to obtain the outer phase; 4) First encapsulation: add the inner phase prepared in step 1) to the middle phase prepared in step 2) according to the mass ratio, and shear and disperse to form a W / O emulsion; the mass ratio of the inner phase to the middle phase is: inner phase: middle phase = (0.2-2):(1-3); the first encapsulation process is as follows: the temperature of the inner phase and the middle phase is increased to 30-50℃, the middle phase is subjected to homogenization at 2500-3500 rpm, the inner phase is poured into the middle phase at a speed of 5-7 mL / min, after the addition is completed, the shearing speed is 8000-10000 r / min for 10-30 min to form a W / O emulsion. The key to the preparation of this step is to uniformly disperse the dispersed phase in the continuous phase, i.e. to fully encapsulate the inner phase with the middle phase. If the addition speed is too fast, a large amount of inner phase components will be instantaneously aggregated in the local area, the emulsifier cannot be adsorbed to the newly formed phase interface in time, and problems such as oil droplet / water droplet aggregation, delamination or flocculation are likely to occur; in the present application, the inner phase is poured into the middle phase at a speed of 5-7 mL / min, which enables the dispersed phase to contact the continuous phase and the emulsifier at a small flux, so that the emulsifier can be fully spread on the two-phase interface to form a stable interface film.

[0029] 5) Secondary encapsulation: the outer phase prepared in step 3) is heated to 30-50℃, and the W / O emulsion formed in step 4) is poured into the outer phase under stirring at 1000-1500 r / min to perform secondary encapsulation, thereby obtaining a W / O / W multiple structure emulsion, i.e. the carrier of the present application. The volume ratio of the W / O emulsion formed in the primary encapsulation to the outer phase is (1.2-5):(5-8.8); the secondary encapsulation process is as follows: the temperature of the outer phase is raised to 30-50℃, and the W / O emulsion formed in the primary encapsulation is poured into the outer phase at a speed of 5-7 mL / min under homogenization at 1000-1500 r / min, so that the two phases are fully emulsified and local aggregation is avoided, i.e. the primary encapsulation material is fully secondarily encapsulated by the outer phase; after the addition is completed, the stirring is continued for 25 min, and the temperature is lowered to room temperature, thereby obtaining a W / O / W multiple structure emulsion, i.e. the carrier of the present application.

[0030] The multiple structure carrier of the recombinant humanized collagen type III microspheres of the present application can be directly used as a cosmetic or medical device product, or as one of the key effective components for preparing a cosmetic or medical device product.

[0031] In order to make the object, technical scheme and advantages of the present application more clear, the technical scheme of the present application will be described clearly and completely below in combination with examples.

[0032] Example 1 This embodiment is to prepare a multiple structure carrier of recombinant humanized collagen type III microspheres, which is prepared as follows: 1) Preparation of an inner phase: 0.3 g of recombinant humanized collagen type III microspheres with a molecular weight of 8000 Da is dissolved in a 0.1 mol / L (pH=7.3) phosphate buffer to obtain a 3 mg / mL aqueous solution of recombinant humanized collagen type III microspheres; hydrogenated lecithin and glycerol are weighed by a balance and mixed at a mass ratio of 9:1, and sheared at 3500 r / min for 20 min to obtain a hydrogenated lecithin-glycerol solution. The prepared aqueous solution of recombinant humanized collagen type III microspheres is directly poured into the hydrogenated lecithin-glycerol solution, so that the hydrogenated lecithin is embedded into the cavity structure of the recombinant humanized collagen type III microspheres to produce flocculation, thereby obtaining a 2 mg / mL suspension of recombinant humanized collagen type III microspheres; then the suspension is ultrasonically dispersed at a power of 250 W for 18 min, and centrifuged at a speed of 9000 r / min for 12 min, and the embedding body is collected, as shown in FIG. 1, which is used as the inner phase. Figure 3 1) Preparation of an inner phase: 0.3 g of recombinant humanized collagen type III microspheres with a molecular weight of 8000 Da is dissolved in a 0.1 mol / L (pH=7.3) phosphate buffer to obtain a 3 mg / mL aqueous solution of recombinant humanized collagen type III microspheres; hydrogenated lecithin and glycerol are weighed by a balance and mixed at a mass ratio of 9:1, and sheared at 3500 r / min for 20 min to obtain a hydrogenated lecithin-glycerol solution. The prepared aqueous solution of recombinant humanized collagen type III microspheres is directly poured into the hydrogenated lecithin-glycerol solution, so that the hydrogenated lecithin is embedded into the cavity structure of the recombinant humanized collagen type III microspheres to produce flocculation, thereby obtaining a 2 mg / mL suspension of recombinant humanized collagen type III microspheres; then the suspension is ultrasonically dispersed at a power of 250 W for 18 min, and centrifuged at a speed of 9000 r / min for 12 min, and the embedding body is collected, as shown in FIG. 1, which is used as the inner phase.

[0033] 2) Preparation of middle phase: using a balance to weigh phytosterol esters, squalane and polyglycerol-3 diisostearate, mixed uniformly in a ratio of 3.5:5:1.5, stirred at 100 rpm, and mixed uniformly for 5 min, and the obtained mixed oil liquid was used as the middle phase.

[0034] 3) Preparation of outer phase: using a balance to weigh water, sodium hyaluronate, panthenol, glycerol, xanthan gum, mixed uniformly in a ratio of 95:0.7:1:3:0.3, stirred at 200 rpm for 25 min, and mixed uniformly, and the obtained sodium hyaluronate aqueous solution was used as the outer phase.

[0035] 4) Primary wrapping: the inner phase and the middle phase were both raised to 40℃, the middle phase was under homogenization at 3000 rpm, the inner phase was poured into the middle phase at a speed of 6 mL / min, after the addition was completed, shearing was carried out at 8500 r / min for 20 min, and a W / O emulsion was formed.

[0036] Secondary wrapping: the outer phase was raised to 40℃, and was under stirring at a homogenization speed of 1200 r / min, the W / O emulsion formed by the primary wrapping was poured into the outer phase at a speed of 6 mL / min, secondary wrapping was carried out, after the addition was completed, stirring was continued for 25 min, and then the temperature was lowered to room temperature, and a W / O / W multiple structure emulsion was obtained. The multiple structure emulsion was the carrier of the application, and the microstructure thereof is shown in Figure 4

[0037] Comparative Example 1 The multiple structure carrier of the recombinant humanized type III collagen microspheres prepared in this example is different from that of Example 1 in that the recombinant humanized type III collagen microspheres are not combined with hydrogenated lecithin to form chimeras. That is, the preparation process is: the recombinant humanized type III collagen microspheres are dissolved in a phosphate buffer and then directly added into the inner phase. The related specific process parameters are the same as those of Example 1.

[0038] Comparative Example 2 The multiple structure carrier of the recombinant humanized type III collagen microspheres prepared in this example is different from that of Example 1 in that the chimeras formed by combining the recombinant humanized type III collagen microspheres with hydrogenated lecithin are not subjected to secondary wrapping after being added into the middle phase, that is, there is no outer phase, which is equivalent to the W / O emulsion in Example 1.

[0039] Comparative Example 3 ​The multiple structure carrier of the recombinant humanized type III collagen microspheres prepared in this example is different from that of Example 1 in that the chimeric body formed by the combination of the recombinant humanized type III collagen microspheres and hydrogenated lecithin is not subjected to primary wrapping, and the solution of the recombinant humanized type III collagen microspheres is directly added to the sodium hyaluronate solution in the external phase. Except for the omitted step, the remaining process parameters are the same as those of Example 1.

[0040] Example 2 This example is to investigate the stability of the multiple structure carrier of the recombinant humanized type III collagen microspheres prepared in Example 1 in high temperature, light and oxidation environment. The test method is as follows: The multiple structure emulsions obtained in Example 1 and Comparative Examples 1-3 are respectively placed in a 45°C oven and a 4000 lux light box, and after 30 days, the collagen content is detected by the Folin phenol method.

[0041] The main reagents used in the test include: (1) Alkaline copper reagent (prepared immediately before use): 50 mL of 0.1 mol / L NaOH solution, 50 mL of 10% CuSO4·5H2O solution, and 50 mL of 2% potassium sodium tartrate solution are mixed uniformly in a volume ratio of 1:1:1, and used within 1 hour after preparation. Folin phenol reagent: directly purchase commercial 1 mol / L Folin phenol reagent, and dilute with distilled water before use at a ratio of 1:1, and shake well for use.

[0042] (2) Preparation of protein standard solution: accurately weigh about 50 mg of bovine serum albumin control product, dissolve in 250 mL of water (about 200 μg / mL). Accurately transfer 1.0 mL, 2.0 mL, 4.0 mL, 5.0 mL, and 10.0 mL of the above standard solution to 100 mL (concentrations of 2 μg / mL, 4 μg / mL, 8 μg / mL, 10 μg / mL, and 20 μg / mL).

[0043] (3) Sample treatment and test steps: accurately transfer 1.0 mL of sample, add water to 100 mL and mix uniformly. Add 1 mL of corresponding standard solution or sample solution in a test tube. Add 1 mL of alkaline copper reagent to each tube, shake immediately, and stand at room temperature for 10 minutes (this step is to break the peptide bond and form a blue complex with Cu 2+The diluted Folin-phenol reagent 0.5 mL was quickly added to each tube, mixed gently (avoiding the generation of bubbles), and immediately placed in a 37°C constant temperature water bath for reaction for 30 minutes. After the reaction, the test tube was taken out and cooled to room temperature, and the absorbance values (A750) of the standard sample tube and the sample tube were determined in turn at a wavelength of 750 nm. The data were recorded, and a standard curve was prepared according to the data of the standard sample tube. The collagen content was calculated, and the retention rate of collagen was calculated according to the data of the collagen content before and after the stability test (the formula is: collagen content retention rate = collagen content after 1 month under specific conditions / initial collagen content * 100%). The results are shown in Table 1.

[0044] Table 1: Stability test results of the multi-structure carrier under high temperature, light and oxidation environment

[0045] As can be seen from the results in Table 1, the R 2 of the standard curve at the initial and 1-month time points is greater than 0.995, indicating that the absorbance and protein content have an excellent linear relationship, and the protein content and protein retention rate can be calculated by the standard curve. The multi-structure carrier of the present application has excellent protective effect on the recombinant type III humanized collagen microspheres under high temperature and light conditions. Specifically, in the preparation process, in the absence of the preparation of the inner phase inclusion body, the collagen content under high temperature and light conditions is reduced by 60.1% and 72.8%, respectively; in the preparation process, in the absence of the preparation of the primary wrapping, the collagen content under high temperature and light conditions is reduced by 72.4% and 74.1%, respectively; in the preparation process, in the absence of the preparation of the secondary wrapping, the collagen content under high temperature and light conditions is reduced by 38.9% and 47.1%, respectively; it is shown that the preparation of the inclusion body, the preparation process of the primary wrapping and the secondary wrapping are necessary, and play an important role in maintaining the biological activity of collagen.

[0046] Example 2 This example is to investigate the effective transdermal rate and safety of the multi-structure carrier of the recombinant type III humanized collagen microspheres prepared in Example 1.

[0047] 1) The effective transdermal rate test uses the human efficacy test method, which is as follows: Ten subjects were recruited to test two samples from Example 1 and 1-3, respectively, on the left and right sides of the face. Before and after 28 days of continuous use of the samples, the skin elasticity R2 value and tightness F3 value of the subjects were tested using a Cutometer®2mm MPA580, and the data of the subjects using the same sample were averaged to evaluate the tightness of the face. Before and after 28 days of continuous use of the samples, the area of under-eye wrinkles of the subjects was analyzed and recorded using the PIMIA image analysis system, and the area of wrinkles on the face was evaluated. The results are shown in Table 2 and Figure 5

[0048] Table 2: Test results of effective transdermal rate of multi-structure carrier

[0049] As can be seen from the results in Table 2, the multi-structure carrier of the recombinant type III humanized collagen microspheres has high transdermal rate and efficacy. Specifically, in the preparation process, compared with Example 1, the change rate of the skin elasticity R2 value of the subjects' face decreased by 17.03%, the change rate of the tightness F3 value decreased by 25.52%, and the change rate of the under-eye wrinkle area decreased by 14.30% in the absence of the preparation of the inner phase chimera. In the preparation process, compared with Example 1, the change rate of the skin elasticity R2 value of the subjects' face decreased by 12.46%, the change rate of the tightness F3 value decreased by 14.08%, and the change rate of the under-eye wrinkle area decreased by 9.10% in the absence of the preparation of the primary package. In the preparation process, compared with Example 1, the change rate of the skin elasticity R2 value of the subjects' face decreased by 9.38%, the change rate of the tightness F3 value decreased by 15.67%, and the change rate of the under-eye wrinkle area decreased by 9.49% in the absence of the preparation of the secondary package. This shows that the recombinant type III humanized collagen microspheres have good transdermal absorption capacity, exert the biological activity of collagen, and have good effects in reducing wrinkle area and improving facial tightness.

[0050] 2) Safety test using patch test method, as follows: FinnChambers®AQUA waterproof patch tester was selected, and pure water was used as a blank control for closed patch test. About 0.020 mL of the test substance was placed in the patch tester, and a low-sensitization adhesive tape was applied to the back of the subject. After 24 hours, the test substance was removed. Skin reactions were observed at 0.5, 24, and 48 hours after removal, and the results were recorded according to the skin reaction grading standard in the Cosmetic Safety Technical Specification (2015 edition), as shown in Table 3.

[0051] Table 3: Test results of safety of multi-structure carrier ​

[0052] From the results of Table 3, it can be seen that the patch test of the multiplex structure carrier prepared in the examples and the comparative examples of the present application was performed on 31 people, and the number of people with 0 level reaction was 31, and different preparation schemes had no effect on the safety of the present application. In summary, the multiplex structure carrier prepared in the present application is non-irritating, and can be directly used as a skin care product or as one of the key effective ingredients in a skin care product in the field of cosmetics and medical devices.

[0053] It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and the present application can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting from any point of view. Furthermore, it should be understood that although the present specification is described in terms of embodiments, it does not contain only one technical solution, and the description manner of the specification is only for the sake of clarity, and those skilled in the art should regard the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A multiple structure carrier of a recombinant humanized type III collagen microsphere, the recombinant humanized type III collagen microsphere being a microspheric collagen having a cavity structure formed by folding a linear recombinant humanized type III collagen and then circularizing it; the carrier comprising an inner phase, a middle phase and an outer phase; characterized in that: The chimeric body of the recombinant humanized type III collagen microspheres and hydrogenated lecithin is located in the inner phase; the middle phase is composed of three skin structure similar oils of phytosteryl esters, squalane and polyglyceryl-3 diisostearate; and the outer phase is a sodium hyaluronate aqueous solution. ​ 2. The recombinant type III humanized collagen microsphere multiple structure carrier of claim 1, characterized by: In the inner phase, the molecular weight of the recombinant humanized type III collagen microspheres is 5000-10000 Da, and the mass ratio of the recombinant humanized type III collagen microspheres to hydrogenated lecithin is (0.5-2):(20-30).

3. The recombinant humanized type III collagen microsphere multiple structure carrier of claim 2, characterized in that: In the middle phase, the mass ratio of the three of phytosteryl esters, squalane and polyglyceryl-3 diisostearate is (2.5-4.5):(3-8):(10-20).

4. The recombinant humanized type III collagen microsphere multiple structure carrier of claim 3, characterized in that: In the outer phase, the molecular weight of the sodium hyaluronate is 100w-180w Da. The components of the sodium hyaluronate aqueous solution include water, sodium hyaluronate, panthenol, glycerol and xanthan gum, and the mass ratio is (93.1-96.9):(0.5-0.9):(0.5-1.5):(2-4):(0.1-0.5).

5. The recombinant humanized type III collagen microsphere multiple structure carrier of claim 4, characterized in that: The mass ratio of the inner phase, the middle phase and the outer phase is (0.2-2):(1-3):(5-8.8); the particle size distribution range of the inner phase of the multi-structure carrier is 50-400 nm, and the particle size distribution range of the middle phase is 1-4 μm.

6. A method for preparing the multi-structure carrier of the recombinant humanized type III collagen microspheres according to any one of claims 1-5, characterized by: The method comprises the following steps: 1) preparing the inner phase: dissolving the recombinant humanized type III collagen microspheres in a phosphate buffer to obtain a recombinant humanized type III collagen microsphere aqueous solution; then pouring the recombinant humanized type III collagen microsphere aqueous solution into a hydrogenated lecithin-glycerol solution to make the recombinant humanized type III collagen microspheres flocculate with the hydrogenated lecithin, forming a recombinant humanized type III collagen microsphere suspension; then ultrasonic dispersion, and centrifugal collection of the suspension to obtain the chimeric body of the recombinant humanized type III collagen microspheres and hydrogenated lecithin as the inner phase for standby; 2) preparing the middle phase: uniformly mixing phytosteryl esters, squalane and polyglyceryl-3 diisostearate according to the mass ratio to obtain the middle phase for standby; 3) preparing the outer phase: uniformly mixing water, sodium hyaluronate, panthenol, glycerol and xanthan gum according to the mass ratio to obtain the outer phase for standby; 4) primary wrapping: adding the inner phase prepared in step 1) into the middle phase prepared in step 2) according to the mass ratio, and shearing and dispersing to form a W / O emulsion; 5) secondary wrapping: heating the outer phase prepared in step 3) to 30-50 DEG C, and under the stirring condition of 1000-1500 r / min, pouring the W / O emulsion formed in step 4) into the outer phase to perform secondary wrapping, and obtaining a W / O / W multi-structure emulsion, which is the multi-structure carrier.

7. The method of claim 6, wherein: In step 1), the concentration of the recombinant humanized type III collagen microsphere aqueous solution is 2-4 mg / mL; the hydrogenated lecithin-glycerol solution is prepared by shearing and mixing hydrogenated lecithin and glycerol according to the mass ratio of (1-2):(8-9), the shearing and dispersing speed is 3000-4000 r / min, and the dispersing time is 15-25 min; the concentration of the recombinant humanized type III collagen microsphere suspension is 1-3 mg / mL; The ultrasonic dispersion condition is 240-260 W, and the dispersion time is 15-20 min. The centrifugal condition is 8000-10000 r / min, and the centrifugal time is 10-15 min. The obtained chimeric particle size distribution is in the range of 50-400 nm.

8. The method of claim 6, wherein: In step 4, the mass ratio of the primary encapsulation, the inner phase and the middle phase is: inner phase: middle phase=(0.2-2):(1-3). The primary encapsulation process is as follows: the temperature of the inner phase and the middle phase is increased to 30-50 ℃, the middle phase is under homogenization condition of 2500-3500 rpm, the inner phase is poured into the middle phase at a speed of 5-7 mL / min, after the addition is completed, shearing is carried out at 8000-10000 r / min for 10-30 min, and a W / O emulsion is formed.

9. The method of claim 6, wherein: In step 5, the volume ratio of the secondary encapsulation, the W / O emulsion formed by the primary encapsulation and the outer phase is (1.2-5):(5-8.8). The secondary encapsulation process is as follows: the temperature of the outer phase is increased to 30-50 ℃, the outer phase is under stirring condition of homogenization 1000-1500 r / min, the W / O emulsion formed by the primary encapsulation is poured into the outer phase at a speed of 5-7 mL / min, secondary encapsulation is carried out, after the addition is completed, stirring is continued for 25 min, and the temperature is decreased to room temperature, and a W / O / W multi-structure emulsion is obtained, which is the multi-structure carrier.

10. Use of the multi-structure carrier of the recombinant humanized type III collagen microspheres according to any one of claims 1-5, characterized in that: It is directly used as a cosmetic or medical device product, or used as one of the key effective components for preparing a cosmetic or medical device product. It is directly used as a cosmetic or medical device product, or used as one of the key effective components for preparing a cosmetic or medical device product.

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