Human-like nano microcapsule preparation as well as preparation method and application thereof

Abstract

The invention discloses a human-like nano microcapsule preparation as well as a preparation method and application thereof, and belongs to the technical field of biology. The human body nano microcapsule preparation is prepared by adding a freeze-drying protective agent component into an oil phase component, a water phase component, a targeting component, a modification component and an auxiliary component and then freeze-drying, the oil phase component consists of lipid and a fat-soluble active substance in a mass ratio of 1: (0.05-0.1); the water-phase component is composed of water or a water-soluble active substance solution; the targeting component is composed of cell-penetrating peptide, and the mass ratio of the cell-penetrating peptide to the lipid is 1: (15-25); the modification component is composed of an aqueous solution of plant polysaccharide, and the mass ratio of the plant polysaccharide to lipid is (0.25-1.5): 1; and the auxiliary component consists of grease and a penetration enhancer in a mass ratio of (100-2000): 1. The human body nano microcapsule preparation is simple in preparation method, safe and non-toxic, and transdermal slow release of drugs or active substances can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a human-like nano-microcapsule preparation and a preparation method and application thereof. Background Art

[0002] The delivery of drugs or active substances to specific areas of the skin to achieve the purpose of treatment or improvement of function and properties has received widespread attention in the field of skin disease treatment and cosmetics. This mode of action is the least invasive and more user-friendly, however, due to the presence of the stratum corneum (the main barrier to substances), it is challenging to penetrate the skin. The stratum corneum is a sublayer of proteins and lipids arranged in a manner known as "brick and mortar", which makes it less permeable to foreign substances. In order to improve this transdermal channel, many methods have been proposed, such as physical methods including epidermal erosion, skin puncture devices using probes, high-frequency oscillating needle beams, microneedle arrays and high-speed dry powder injection, and chemical methods including the use of penetration enhancers and excipients. The former is prone to cause pain and skin irritation, while the latter is not very effective.

[0003] The development of nanotechnology has provided a new technical means for this purpose. Encapsulating such active molecules in nanoscale colloidal systems with specific physicochemical properties (<500nm) can compensate for high degradation, low half-life and the ability to penetrate physical barriers, further enhancing the permeability of active molecules to the skin, and the utilization rate of skin selective absorption of effective ingredients is also increased. Among these nanocarriers, lipid nano delivery systems represented by liposomes and solid lipid nanoparticles are highly similar to human cell membrane components and structures, providing protection for encapsulating hydrophilic and lipophilic active substances, targeted and controlled delivery, while minimizing their toxicity to other tissues, and allowing higher skin penetration and time residence, and have been widely used. However, the above-mentioned single nanocarrier as a delivery system still has certain defects. For example, a single liposome is a thermodynamically unstable colloidal system, which tends to coagulate and flocculate when stored for a long time. The shelf life of the product prepared by it is generally only about half a year, while solid lipid nanoparticles have added a certain rigid structure, which is more suitable for industrial production, but the safety of nanoparticle preparation materials is an important factor restricting its development. The development of non-toxic and long-term storage high-performance delivery carriers is an urgent problem to be solved. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a human-like nano-microcapsule preparation and its preparation method and application, so as to solve the problems that the current methods of improving transdermal channels are highly irritating, difficult to achieve expected results and lack of safety.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention discloses a human-like nano-microcapsule preparation, which is obtained by freeze-drying an oil phase component, a water phase component, a targeting component, a modifying component and an auxiliary component by adding a freeze-drying protective agent component;

[0007] Among them, the oil phase component is composed of lipids and fat-soluble active substances in a mass ratio of 1: (0.05-0.1); the aqueous phase component includes water or a water-soluble active substance solution; the targeting component includes a cell-penetrating peptide, and the mass ratio of the cell-penetrating peptide to the lipid is 1: (15-25); the modifying component includes an aqueous solution of plant polysaccharides, and the mass ratio of the plant polysaccharides to the lipid is (0.25-1.5): 1; the auxiliary component is composed of oils and permeation enhancers in a mass ratio of (100-2000): 1.

[0008] Preferably, the fat-soluble active substance is vitamin E, vitamin D, β-carotene, astaxanthin or curcumin.

[0009] Preferably, the lipid comprises phospholipids and cholesterol in a mass ratio of (1-6):1.

[0010] Further preferably, the phospholipids are selected from at least two of soybean lecithin, hydrogenated phosphatidylcholine, rhamnolipid, diphosphatidylglycerol, phosphatidylinositol, phosphatidylserine and phosphatidic acid.

[0011] Preferably, the water-soluble active substance is vitamin B6 or purslane extract.

[0012] Preferably, the cell-penetrating peptide is selected from one of TAT peptide, PEN peptide, SPACE peptide, polyarginine peptide and biological short peptide TD.

[0013] Preferably, the plant polysaccharide is selected from at least one of β-oat glucan, fucoidan, gastrodia elata polysaccharide, aloe polysaccharide and tremella polysaccharide.

[0014] Preferably, the oil is selected from at least two of ω-3 fatty acids, camellia oil, grape seed oil, olive oil, castor oil, caprylic / capric triglyceride and polydimethylsiloxane; the penetration enhancer is selected from at least one of oleic acid, β-caryophyllene, menthone, geraniol and 1,8-cineole; the mass ratio of the penetration enhancer to the lipid is (0.001-0.005):1.

[0015] The present invention also discloses a method for preparing the above-mentioned human-like nano-microcapsule preparation, which comprises preparing oil phase components and water phase components into liposomes as the primary structure by combined shearing, ultrasound and supercritical carbon dioxide method; incubating the targeting component with the primary structure so that the targeting component is modified on the surface of the primary structure to form a secondary structure; slowly injecting the secondary structure into the modified component solution under stirring, and adding trehalose as a lyophilization protective agent to perform freeze drying to obtain a tertiary structure; dispersing the tertiary structure into the auxiliary components, stirring evenly to form a quaternary structure, and forming the final human-like nano-microcapsule preparation.

[0016] The invention also discloses the use of the human-like nano-microcapsule preparation in delivering drugs or active substances to skin parts.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The human-like nano-microcapsule preparation provided by the present invention has the advantage of better loading various drugs or active molecules and achieving transdermal sustained release through the complementary quaternary structure.

[0019] First, the composite structure formed by the outermost layer of oil and penetration enhancer is conducive to forming a functional oil film on the skin surface, protecting the internal structure and preventing leakage. At the same time, with the assistance of the penetration enhancer, the internal structure is further penetrated into the subcutaneous layer and the skin barrier function is enhanced. In addition, oil is an excellent emollient ingredient. The triglyceride component in oil has the effect of improving the closure of the skin surface and reducing the TEWL of the epidermis. The unsaponifiable matter in oil also has a good anti-inflammatory effect.

[0020] Secondly, the modification of the sub-outer layer of plant polysaccharide shell further plays a role. The structure of plant polysaccharides usually contains a large number of hydroxyl groups, carboxyl groups and other polar groups. The hydrogen bonding between them makes them have good hydration ability and can penetrate into the bottom layer of the skin through the intercellular gap, that is, the lipid connection between cells. In addition, the molecular chains of plant polysaccharides are intertwined with each other and connected with water molecules by hydrogen bonds to form a dense network, which can form a uniform film on the surface of the skin, reduce the evaporation of water on the surface of the skin, and play a strong role in water retention. On the other hand, plant polysaccharides are very sensitive to immune regulation and can promote the repair of damaged skin barriers by stimulating immune responses. For example, for β-oat glucan, it not only has good hydration ability, but also can trigger a series of cytokine responses. There is an immune network composed of many Langerhans cells under the stratum corneum of the skin. The tentacles of Langerhans cells can reach the stratum corneum cells and are receptors for β-oat glucan. After β-oat glucan binds to the cell membrane receptor, its planar helical structure undergoes a series of stereochemical changes, which activates macrophages and produces a series of cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), angiogenic factor (AF), etc. These cytokines further act on the surface of the skin to improve the appearance of the skin.

[0021] Thirdly, the cell-penetrating peptides in the second inner layer are further modified, so that the liposomes loaded with active molecules in the innermost layer can efficiently penetrate the barrier of the stratum corneum and enter the dermis tissue, thereby improving the transdermal absorption of active ingredients.

[0022] Fourthly, the innermost primary structure - liposomes, forms an exogenous lipid bilayer membrane, which changes the structure between corneocytes. The hydrophobic internal arrangement in the lipid bilayer is disordered, which is conducive to the fat-soluble active substances to enter the intercellular space through diffusion and capillary action, so that the wetting and hydration of the stratum corneum are enhanced. In addition, one of the important raw materials for liposomes - phospholipids can fuse with the lipids of the stratum corneum, causing the composition and structure of the stratum corneum to change. This exchange and fusion between lipids can pass through the gaps between lipid particles, which is more conducive to the entry of drugs or active molecules loaded by liposomes into the skin.

[0023] In summary, this type of human nano-microcapsule preparation has good skin penetration ability, can be smoothly absorbed through the epidermis and epidermal glands, and can effectively load hydrophobic molecules and heat-resistant functional ingredients (such as fat-soluble vitamins, proteins, growth factors, etc.) and maintain their activity, thereby achieving the expected effect. In addition, this type of human nano-microcapsule preparation avoids the use of preservatives, reduces the use of organic solvents, is safer, does not require complex processes and equipment, and is energy-saving and efficient.

[0024] The present invention provides a method for preparing a human-like nano-microcapsule preparation, wherein the raw materials selected are low-cost and have good biocompatibility, can be used on a large scale, and the multi-level structure can realize the effective loading of multiple active molecules. The preparation process conditions are mild, and the preparation can be stored for a long time, and is expected to be industrialized. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the structure of a human-like nano-microcapsule preparation;

[0026] Figure 2 for Figure 1 A magnified view of the middle A area;

[0027] Figure 3 This is a schematic diagram of the transdermal experimental device;

[0028] Figure 4 This is a statistical chart of the cumulative skin permeability of human-like nano-microcapsule preparations;

[0029] Figure 5 This is a statistical diagram of the deposition amount of human-like nano-microcapsule preparations in different cortical layers;

[0030] Figure 6 This is a fluorescence confocal image of the skin after the human-like nano-microcapsule preparation was applied; A is the microcapsule preparation group, and B is the control group;

[0031] Figure 7 This is a statistical graph of the fluorescence intensity accumulated in the skin after application of the human-like nanocapsule preparation.

[0032] Among them: 1-diffusion cell; 2-analyte; 3-receiving cell; 4-analysis sampling port. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0034] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0035] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0036] The human-like nano-microcapsule preparation provided by the present invention is prepared from an oil phase component, an aqueous phase component, a targeting component, and a modifying component, and then a certain freeze-drying protective agent component is added and freeze-dried, and then embedded in an auxiliary component to obtain the preparation.

[0037] The oil phase component is composed of lipids and fat-soluble active substances in a mass ratio of 1: (0.05-0.1), and the mass ratio is preferably 1: 0.08; the fat-soluble active substances include but are not limited to vitamin E, vitamin D, β-carotene, astaxanthin and curcumin. The lipids include phospholipids and cholesterol in a mass ratio of (1-6): 1, and the phospholipids are selected from two or more of soybean lecithin, hydrogenated lecithin, rhamnolipid, diphosphatidylglycerol, phosphatidylinositol, phosphatidylserine and phosphatidic acid; the mass ratio of phospholipids to lipids is (0.5-0.85): 1.

[0038] The aqueous phase component includes water or a water-soluble active substance solution; the water-soluble active substance includes but is not limited to vitamin B6 and purslane extract.

[0039] The targeting component includes a cell-penetrating peptide, which is selected from one of TAT peptide, PEN peptide, SPACE peptide (amino acid sequence see SEQ ID NO.1 in Table 1), polyarginine peptide and biological short peptide TD (amino acid sequence see SEQ ID NO.2 in Table 1), preferably SPACE peptide; the mass ratio of the cell-penetrating peptide to lipid is 1: (15-25), preferably 1:20.

[0040] Table 1 Amino Acid Sequence Listing

[0041]

[0042] The modifying component includes an aqueous solution of plant polysaccharides, and the plant polysaccharides are selected from at least one of β-oat glucan, fucoidan, gastrodia polysaccharide, aloe polysaccharide and yam polysaccharide; preferably β-oat glucan, and the mass ratio of the plant polysaccharide to lipid is (0.25-1.5):1.

[0043] The auxiliary components are composed of oil and permeation enhancer in a mass ratio of (100-2000):1; the oil is selected from two or more of ω-3 fatty acids, camellia oil, grape seed oil, olive oil, castor oil, caprylic / capric triglyceride and polydimethylsiloxane; the permeation enhancer is selected from at least one of oleic acid, β-caryophyllene, menthone, geraniol and 1,8-cineole; the mass ratio of the permeation enhancer to the lipid is (0.001-0.005):1.

[0044] See also Figure 1 and Figure 2 The preparation method of the human-like nano-microcapsule preparation provided by the present invention comprises the following specific steps:

[0045] 1. The oil phase components and the water phase components are prepared into liposomes as the primary structure by combined shearing, ultrasound, and supercritical carbon dioxide method;

[0046] 2. Incubate the targeting component with the primary structure to modify the targeting component on the surface of the primary structure to form a secondary structure;

[0047] 3. The secondary structure is slowly injected into the modified component solution under stirring, and 10% (w / w) trehalose is added to the secondary structure prepared above as a lyophilization protective agent for freeze drying to prepare the final tertiary structure;

[0048] 4. Disperse the tertiary structure into the auxiliary components and stir evenly to form a quaternary structure, thereby obtaining a human-like nano-microcapsule preparation.

[0049] Example 1

[0050] 1. Preparation of curcumin human nanocapsule preparation

[0051] (1) Primary structure preparation

[0052] According to mass, 456.8mg soybean lecithin, 152.3mg hydrogenated lecithin, 300mg cholesterol and 72.73mg curcumin are weighed and mixed, and are placed in a mortar and ground into a cake-like shape, and 30mL of phosphate buffer (PBS, 0.2mol / L, pH value=6.0) is added in batches, and then the phosphate buffer is continued to be ground to uniformly added in an autoclave. Subsequently, the temperature is adjusted to 45 DEG C, and shear rate is set to 5000rpm / min, and supercritical carbon dioxide is passed through while maintaining the shear state, until the pressure of the reactor reaches 20Mpa, and ultrasound is turned on, and ultrasonic power is 45W, and ultrasound is turned on and the rear processing is continued for 40min, and heating, shearing and ultrasound are subsequently closed successively, and carbon dioxide is released. Then the phosphate buffer of 100mL is added, and ultrasonic treatment is continued for 40min, and curcumin liposome solution can be obtained.

[0053] (2) Secondary structure preparation

[0054] The SPACE peptide solution was incubated with the above primary structure to modify the SPACE peptide on the surface of the primary structure. The specific method was as follows: according to the mass ratio of SPACE peptide to lipid of 1:20, 45.46 mg of SPACE peptide was weighed and dissolved in ultrapure water solution. After mixing the above liposomes with the SPACE peptide solution, ice bath sonication was performed in an ultrasonic crusher. The ultrasonic conditions were: ultrasonic power of 100 W, ultrasonication for 3S with an interval of 6S, for a total of 3 minutes.

[0055] (3) Preparation of tertiary structure

[0056] Weigh 454.55 mg of β-oat glucan and disperse it in water to prepare a solution with a mass concentration of 20 mg / mL. Then add this solution dropwise to the above-mentioned liposome solution modified with SPACE peptide at room temperature, stir for 30 minutes, and incubate at 4°C overnight to obtain the final tertiary structure.

[0057] Add 10% (w / w) trehalose as a freeze-drying protective agent to the prepared tertiary structure, mix well, take the mixture sample and freeze it at -80°C for 2 hours, and then put it into a freeze dryer ( The freeze-drying was carried out at -50°C, 0.1 mbar, and 48 h.

[0058] (4) Preparation of quaternary structure

[0059] According to the mass ratio of penetration enhancer to lipid (0.001-0.005):1, and the mass ratio of oil to penetration enhancer (100-2000):1, the corresponding auxiliary components are weighed, and the above-mentioned tertiary structure is embedded in the auxiliary components to form a quaternary structure for preparing microcapsule preparations for use.

[0060] The specific method is as follows: the tertiary structure is dissolved in ultrapure water as the water phase, 1.8 mg of menthone is added, and then placed in an ultrasonic cleaner for ultrasonic dispersion in a mixed oil of 630 mg of camellia oil and 270 mg of polydimethylsiloxane, the ultrasonic temperature is controlled at 55°C, and the ultrasonic time is 60 minutes to obtain a curcumin human nano-microcapsule preparation.

[0061] 2. In vitro skin penetration and deposition effect verification

[0062] See also Figure 3 The ex vivo drug permeation of this type of human nanocapsule preparation on the skin was evaluated using a Franz diffusion cell. The volume of the receiving cell 3 was 6.5 mL. The transdermal diffusion area was 2.8 cm 2 . The skin of adult female Wistar rats weighing 180-220 g was selected and excised for penetration studies. The rats were killed by overdose of ether anesthesia and the hair was shaved from the back. The underlying fat and subcutaneous tissue were carefully separated from the excised skin using a scalpel. The skin was then cleaned, wrapped in aluminum foil, and frozen at -80°C for subsequent use. The skin was soaked in phosphate buffer (pH = 7.4, constant temperature 25°C) for 60 minutes before the experiment. The skin was then trimmed into a circular specimen with a diameter of approximately 2 cm and a thickness of approximately 1.7 mm, and installed between the two compartments of the Franz cell, at the location of the analyte 2, with its stratum corneum (SC) side facing the diffusion cell 1. The receiving cell 3 was filled with a phosphate buffer medium with a pH value of 7 until it was in close contact with the skin and all bubbles were expelled. The microcapsule preparation was then prepared into a 1 mg / mL solution (the control group used a curcumin solution containing the same concentration), 0.25 mL was added to the diffusion cell 1, and then stirred continuously, the temperature was set to 37±0.5°C, and the stirring speed was 200 rpm / min. Then 0.2 mL was sampled from the analysis sampling port 4 in the receiving cell 3 at periodic intervals of 4, 6, 8, 12 and 24 hours, and after each sampling interval, an equal volume of fresh medium was added to the receiving cell 3. The removed sample was placed in an ELISA instrument to measure the ultraviolet absorbance value of the sample, thereby calculating the penetration efficiency.

[0063] Permeability PE = (absorbance of curcumin standard solution - absorbance of PBS) / (absorbance of sample - absorbance of PBS) × 100%

[0064] The cumulative amount of drug permeated through the skin is plotted as a function of time. The skin permeation flux and the cumulative percentage of permeation per unit area of ​​skin are calculated from the relationship between cumulative drug permeation and time.

[0065] The results are as follows Figure 4The cumulative skin permeability statistics of curcumin nanocapsule preparations at different time points showed that when the experimental time was short (4h), the skin permeability of the nanocapsule preparation group was significantly higher than that of the control group (about 2.3 times), and as time went on, the skin permeability of the nanocapsule preparation group was significantly higher than that of the control group, indicating that the nanocapsule preparation has a good transdermal sustained-release effect.

[0066] At the end of the experiment, the test object 2 (i.e., skin) was removed from the Franz diffusion cell 1, and then wiped with a paper towel to remove the remaining preparation on the surface, and the SC layer was separated from the skin using the tape stripping method. That is, the SC was stripped 15 times in a row using transparent tape. The tape was immersed in a glass bottle containing 10 mL of methanol and shaken for 24 hours. Then, the vial was ultrasonically treated for 15 minutes. The collected methanol was centrifuged at 10,000 rpm and 25°C for 15 minutes, and then filtered through a 0.22 μm nylon syringe filter. The remaining epidermis and dermis were then scored with a knife to expose deeper tissues to facilitate drug extraction. The drugs in each layer were then extracted in the same manner as described for the SC, and detected using high performance liquid chromatography. The results are shown in Figure 5 The deposition amount of the microcapsule preparation in the stratum corneum and deep skin was higher than that of the control group, especially in the deep skin, the content of the microcapsule preparation was higher (about 3.3 times that of the control group), indicating that the microcapsule preparation is more conducive to the delivery of active substances to the deep skin.

[0067] In addition, skin samples were observed to visually display the accumulation of curcumin in the skin in vitro. Balb / c mice were used for the experiment. During the experiment, the microcapsule preparation was applied to the Balb / c mice in the experimental group at a concentration of 0.2 mg / mL and a dosage of 0.5 mL. The control group was administered a curcumin solution containing the same concentration. After 24 hours, the mouse skin was removed, the area receiving local treatment was carefully cut off, the residual liquid on the surface was absorbed with filter paper, and it was placed in a 4% paraformaldehyde solution for fixation for 24 hours. Then, after the skin tissue was fixed, paraffin sections (thickness of about 7 μ degrees perpendicular to the skin surface) were made, and the prepared sections were placed on a glass slide under a laser confocal microscope (CLSM, Carl Zeiss 710, Zeiss, Germany) to observe the tissue morphology and fluorescence distribution. The fluorescence distribution range and fluorescence distribution intensity can be analyzed and calculated by Release Version 4.0SP2 image analysis software. The fluorescence area and intensity under a high-power field of view (100 times) were measured and then converted by standard values ​​to obtain the pixel value of the fluorescence depth permeated into the skin per unit length, and the size of the pixel value was used to semi-quantitatively calculate the penetration intensity of the preparation.

[0068] The results are as follows Figure 6As can be seen from the figure, after 24 hours of transdermal experiment, fluorescence distribution was found in all layers of tissues in the microcapsule preparation group and the control group, but the intensity of the fluorescence distribution was quite different, and the fluorescence intensity of the microcapsule preparation group was stronger. At the same time, through the statistical results of fluorescence intensity, such as Figure 7 It can be seen that the fluorescence intensity of the microcapsule preparation group (122.57±2.17) was significantly higher than that of the control group (40.58±3.35), and the difference was statistically significant.

[0069] Example 2

[0070] 1. Preparation of Vitamin E Human Nanocapsule Preparation

[0071] (1) Primary structure preparation

[0072] According to the mass, 225 mg hydrogenated lecithin, 225 mg rhamnolipid, 150 mg cholesterol, and 60 mg vitamin E were weighed and mixed, placed in a mortar and ground into a cake, and 30 mL of phosphate buffer (PBS, 0.2 mol / L, pH = 6.0) was added in batches, and then added to a high-pressure reactor after grinding until uniform. The temperature was then adjusted to 45 ° C, and the shear rate was set to 5000 rpm / min to keep supercritical carbon dioxide gas in the shear state until the pressure of the reactor reached 20 MPa, and ultrasound was turned on, the ultrasonic power was 45 W, and the ultrasound was turned on and continued to process for 40 min, and then heating, shearing and ultrasound were turned off in turn to release carbon dioxide gas. Then 100 mL of phosphate buffer was added, and the vitamin E liposome solution was obtained by continuing ultrasonic treatment for 40 min.

[0073] (2) Secondary structure preparation

[0074] The SPACE peptide solution was incubated with the above primary structure to modify the surface of the primary structure with the SPACE peptide. The specific method was as follows: according to the mass ratio of SPACE peptide to lipid of 1:18, 33.34 mg of SPACE peptide was weighed and dissolved in an ultrapure aqueous solution. After mixing the above liposome solution with the SPACE peptide solution, ice bath sonication was performed in an ultrasonic crusher. The ultrasonic conditions were: ultrasonic power of 100 W, 3S of ultrasound with an interval of 6S, for a total of 3 minutes.

[0075] (3) Preparation of tertiary structure

[0076] Weigh 900 mg of fucoidan and disperse it in water to prepare a solution with a mass concentration of 20 mg / mL. Then add the solution dropwise to the liposome solution modified with the SPACE peptide at room temperature, stir for 30 minutes, and incubate at 4°C overnight to obtain the pretreated tertiary structure.

[0077] 10% (w / w) trehalose was added to the prepared tertiary structure as a freeze-drying protective agent and mixed evenly. The mixture was frozen at -80°C for 2 hours and then placed in a freeze dryer ( The product was freeze-dried at -50 °C, 0.1 mbar, and 48 h to obtain the final tertiary structure.

[0078] (4) Preparation of quaternary structure

[0079] The above tertiary structure is embedded in the auxiliary component to form a quaternary structure, which is used to prepare a microcapsule preparation for subsequent use. The specific method is as follows: the above tertiary structure is dissolved in ultrapure water as the water phase, 3.0 mg of β-caryophyllene is added, and then placed in an ultrasonic cleaner for ultrasonic dispersion in a mixed oil of 1800 mg of ω-3 fatty acids, 900 mg of polydimethylsiloxane and 300 mg of caprylic / capric triglyceride, the ultrasonic temperature is controlled to be 55°C, and the ultrasonic time is 60 minutes to obtain a vitamin E human nano-microcapsule preparation.

[0080] Example 3

[0081] 1. Preparation of curcumin human nanocapsule preparation

[0082] (1) Primary structure preparation

[0083] According to mass, 270mg soybean lecithin, 180mg phosphatidylserine, 75mg cholesterol, 26.25mg curcumin are weighed and mixed, and are placed in a mortar and ground into a cake-like shape, and 30mL of phosphate buffer (PBS, 0.2mol / L, pH value=6.0) is added in batches, and then added to an autoclave after continuing to grind to uniformity. Subsequently, the temperature is adjusted to 45°C, and shear rate is set to be 5000rpm / min to keep supercritical carbon dioxide gas being passed under shearing state, until the pressure of the reactor reaches 20Mpa, and ultrasound is turned on, and ultrasonic power is 45W, and ultrasound is turned on and continued to process 40min, and heating, shearing and ultrasound are then turned off successively, releasing carbon dioxide gas. Then 100mL of phosphate buffer is added, and curcumin liposome solution can be obtained by continuing ultrasonic treatment for 40min.

[0084] (2) Secondary structure preparation

[0085] The SPACE peptide solution was incubated with the above primary structure to modify the surface of the primary structure with the SPACE peptide. The specific method was as follows: according to the mass ratio of SPACE peptide to lipid being 1:25, 21.0 mg of SPACE peptide was weighed and dissolved in an ultrapure aqueous solution, and the above liposome solution and the SPACE peptide solution were mixed and then subjected to ice bath ultrasonication in an ultrasonic crusher. The ultrasonic conditions were: ultrasonic power of 100 W, ultrasonication for 3S with an interval of 6S, for a total of 3 min.

[0086] (3) Preparation of tertiary structure

[0087] 393.75 mg of β-oat glucan was weighed and dispersed in water to prepare a solution with a mass concentration of 20 mg / mL. The solution was then added dropwise to the above-mentioned liposome solution modified with the SPACE peptide at room temperature, stirred for 30 minutes, and incubated at 4°C overnight to obtain the pretreated tertiary structure.

[0088] 10% (w / w) trehalose was added to the prepared tertiary structure as a freeze-drying protective agent and mixed evenly. The mixture was frozen at -80°C for 2 hours and then placed in a freeze dryer ( The product was freeze-dried at -50 °C, 0.1 mbar, and 48 h to obtain the final tertiary structure.

[0089] (4) Preparation of quaternary structure

[0090] The tertiary structure is embedded in the auxiliary component to form a quaternary structure, which is used to prepare a microcapsule preparation for use. The specific method is as follows: the tertiary structure is dissolved in ultrapure water as the water phase, 1.575 mg of geraniol is added, and then placed in an ultrasonic cleaner for ultrasonic dispersion in a mixed oil of 1417.5 mg of olive oil, 708.75 mg of polydimethylsiloxane and 236.25 mg of caprylic / capric triglyceride, the ultrasonic temperature is controlled to be 55°C, and the ultrasonic time is 60 minutes to obtain a curcumin human nano-microcapsule preparation.

[0091] Example 4

[0092] 1. Preparation of curcumin human nanocapsule preparation

[0093] (1) Primary structure preparation

[0094] According to the mass, 225mg soybean lecithin, 168.75mg rhamnolipid, 56.25mg diphosphatidylinositol, 112.5mg cholesterol, 39.375mg curcumin were weighed and mixed, and then placed in a mortar and ground into a cake shape. 30mL of phosphate buffer (PBS, 0.2mol / L, pH value = 6.0) was added in batches, and then added to a high pressure reactor after continuing to grind to uniformity. The temperature was then adjusted to 45°C, and the shear rate was set to 5000rpm / min to keep supercritical carbon dioxide gas in the shear state, until the pressure of the reactor reached 20Mpa, and ultrasound was turned on, and the ultrasonic power was 45W. After the ultrasound was turned on, it was continued to process 40min, and then heating, shearing and ultrasound were turned off in sequence to release carbon dioxide gas. Then 100mL of phosphate buffer was added, and the curcumin liposome solution was obtained by continuing ultrasonic treatment for 40min.

[0095] (2) Secondary structure preparation

[0096] The SPACE peptide solution was incubated with the above primary structure to modify the surface of the primary structure with the SPACE peptide. The specific method was as follows: according to the mass ratio of SPACE peptide to lipid of 1:15, 37.5 mg of SPACE peptide was weighed and dissolved in an ultrapure aqueous solution, and the above liposome solution and the SPACE peptide solution were mixed and then subjected to ice bath ultrasonication in an ultrasonic crusher. The ultrasonic conditions were: ultrasonic power of 100 W, ultrasonication for 3S with an interval of 6S, for a total of 3 min.

[0097] (3) Preparation of tertiary structure

[0098] 140.625 mg of β-oat glucan was weighed and dispersed in water to prepare a solution with a mass concentration of 20 mg / mL. The solution was then added dropwise to the above-mentioned liposome solution modified with the SPACE peptide at room temperature, stirred for 30 minutes, and incubated at 4°C overnight to obtain the pretreated tertiary structure.

[0099] 10% (w / w) trehalose was added to the prepared tertiary structure as a freeze-drying protective agent and mixed evenly. The mixture was frozen at -80°C for 2 hours and then placed in a freeze dryer ( The product was freeze-dried at -50 °C, 0.1 mbar, and 48 h to obtain the final tertiary structure.

[0100] (4) Preparation of quaternary structure

[0101] The above tertiary structure is embedded in the auxiliary component to form a quaternary structure for use in preparing microcapsule preparations. The specific method is as follows: the above tertiary structure is dissolved in ultrapure water as the water phase, 2.25 mg of 1,8-cineole is added, and then placed in an ultrasonic cleaner for ultrasonic dispersion in a mixture of 2250 mg of camellia oil, 900 mg of grape seed oil and 1350 mg of polydimethylsiloxane, the ultrasonic temperature is controlled to be 55°C, and the ultrasonic time is 60 minutes to obtain a curcumin human nano-microcapsule preparation.

[0102] Example 5

[0103] 1. Preparation of Astaxanthin Human Nanocapsule Preparation

[0104] (1) Primary structure preparation

[0105] Weigh 245 mg soybean lecithin, 125 mg rhamnolipid, 100 mg phosphatidic acid, 100 mg diphosphatidylinositol, 100 mg cholesterol and 33.5 mg astaxanthin by mass, mix well, grind into cakes in a mortar, add 30 mL of vitamin B6 in batches, continue grinding until uniform, and add to the autoclave. Then adjust the temperature to 45 ° C, set the shear rate to 5000 rpm / min, keep supercritical carbon dioxide gas in the shear state, until the pressure of the reactor reaches 20 MPa, turn on ultrasound, the ultrasonic power is 45 W, and continue to process for 40 min after the ultrasound is turned on, then turn off heating, shearing and ultrasound in turn to release carbon dioxide gas. Then add 100 mL of phosphate buffer and continue ultrasonic treatment for 40 min to obtain astaxanthin liposome solution.

[0106] (2) Secondary structure preparation

[0107] The TAT peptide solution was incubated with the above primary structure to modify the surface of the primary structure with TAT peptide. The specific method was as follows: according to the mass ratio of TAT peptide to lipid of 1:18, 37.2 mg of TAT peptide was weighed and dissolved in ultrapure water solution, and the above liposome solution and TAT peptide solution were mixed and then subjected to ice bath ultrasonication in an ultrasonic crusher. The ultrasonic conditions were: ultrasonic power of 100 W, ultrasonication for 3S with an interval of 6S, for a total of 3min.

[0108] (3) Preparation of tertiary structure

[0109] 158.33 mg of Gastrodia elata polysaccharide, 100 mg of aloe polysaccharide and 300 mg of Tremella fuciformis polysaccharide were weighed and dispersed in water to prepare a solution with a mass concentration of 20 mg / mL. The solution was then added dropwise to the above-mentioned liposome solution modified with TAT peptide at room temperature, stirred for 30 minutes, and incubated at 4°C overnight to obtain the pretreated tertiary structure.

[0110] 10% (w / w) trehalose was added to the prepared tertiary structure as a freeze-drying protective agent and mixed evenly. The mixture was frozen at -80°C for 2 hours and then placed in a freeze dryer ( The product was freeze-dried at -50 °C, 0.1 mbar, and 48 h to obtain the final tertiary structure.

[0111] (4) Preparation of quaternary structure

[0112] The above tertiary structure is embedded in the auxiliary component to form a quaternary structure for use in preparing microcapsule preparations. The specific method is as follows: the above tertiary structure is dissolved in ultrapure water as the water phase, 2 mg of oleic acid is added, and then placed in an ultrasonic cleaner for ultrasonic dispersion in a mixture of 90 mg of camellia oil, 30 mg of grape seed oil and 80 mg of castor oil, the ultrasonic temperature is controlled to be 55°C, and the ultrasonic time is 60 minutes to obtain an astaxanthin human nano-microcapsule preparation.

[0113] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A human-like nano-microcapsule preparation, It is characterized in that The oil phase component, the water phase component, the targeting component, the modifying component and the auxiliary component are freeze-dried by adding a freeze-drying protective agent component; Among them, the oil phase component is composed of lipids and fat-soluble active substances in a mass ratio of 1: (0.05-0.1); the aqueous phase component includes water or a water-soluble active substance solution; the targeting component includes a cell-penetrating peptide, and the mass ratio of the cell-penetrating peptide to the lipid is 1: (15-25); the modifying component includes an aqueous solution of plant polysaccharides, and the mass ratio of the plant polysaccharides to the lipid is (0.25-1.5): 1; the auxiliary component is composed of oils and permeation enhancers in a mass ratio of (100-2000):

1.

2. The human-like nano-microcapsule preparation according to claim 1, It is characterized in that The fat-soluble active substance is vitamin E, vitamin D, beta-carotene, astaxanthin or curcumin.

3. The human-like nano-microcapsule preparation according to claim 1, It is characterized in that The lipids include phospholipids and cholesterol in a mass ratio of (1-6):

1.

4. The human-like nano-microcapsule preparation according to claim 3, It is characterized in that The phospholipids are selected from at least two of soybean lecithin, hydrogenated phosphatidylcholine, rhamnolipid, diphosphatidylglycerol, phosphatidylinositol, phosphatidylserine and phosphatidic acid.

5. The human-like nano-microcapsule preparation according to claim 1, It is characterized in that The water-soluble active substance is vitamin B6 or purslane extract.

6. The human-like nano-microcapsule preparation according to claim 1, It is characterized in that The cell-penetrating peptide is selected from one of TAT peptide, PEN peptide, SPACE peptide, polyarginine peptide and biological short peptide TD.

7. The human-like nano-microcapsule preparation according to claim 1, It is characterized in that The plant polysaccharide is selected from at least one of β-oat glucan, fucoidan, gastrodia elata polysaccharide, aloe polysaccharide and tremella polysaccharide.

8. The human-like nano-microcapsule preparation according to claim 1, It is characterized in that The oil is selected from at least two of ω-3 fatty acids, camellia oil, grape seed oil, olive oil, castor oil, caprylic / capric triglyceride and polydimethylsiloxane; the penetration enhancer is selected from at least one of oleic acid, β-caryophyllene, menthone, geraniol and 1,8-cineole; the mass ratio of the penetration enhancer to the lipid is (0.001-0.005):

1.

9. A method for preparing a human-like nano-microcapsule preparation according to any one of claims 1 to 8, It is characterized in that The oil phase component and the water phase component are prepared into liposomes by combined shearing, ultrasound, and supercritical carbon dioxide method as the primary structure; the targeting component is co-incubated with the primary structure so that the targeting component is modified on the surface of the primary structure to form a secondary structure; The secondary structure is slowly injected into the modified component solution under stirring, and trehalose is added as a lyophilization protectant for freeze drying to obtain the tertiary structure; The tertiary structure is dispersed into the auxiliary components and stirred evenly to form a quaternary structure, thereby obtaining a human-like nano-microcapsule preparation.

10. Use of the human-like nano-microcapsule preparation according to any one of claims 1 to 8 in delivering drugs or active substances to skin sites.

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