Glaucocalyxin A nano transdermal delivery system for skin anti-inflammation and preparation method of glaucocalyxin A nano transdermal delivery system

Abstract

The invention provides a glaucocalyxin A nano transdermal delivery system for skin inflammation resistance, which is of a capsule composite structure of protein, glaucocalyxin A and a polymer shell layer, the protein has an affinity effect on skin, glaucocalyxin A is loaded in the protein, the polymer layer coats the surface of the protein, the size of the glaucocalyxin A nano transdermal delivery system is 30-80nm, and the size of the polymer layer is 30-80nm. And the thickness of the polymer shell layer is 10-35 nm. The surface property of the polymer shell can be regulated and controlled according to the depth requirement of transdermal delivery, so that protein breaks through the skin barrier and is efficiently delivered to the deep position of the skin, the glaucocalyxin A is slowly released, the skin inflammation is relieved in a targeted mode, and the defect that the treatment effect is poor due to the non-ideal transdermal effect when glaucocalyxin A is used for treating the skin inflammation is overcome.

Description

Technical Field

[0001] The invention belongs to a nano transdermal delivery system, and in particular relates to a bluecalyx A nano transdermal delivery system for skin anti-inflammatory and a preparation method thereof. Background Art

[0002] Bluecalyx A is a diterpenoid compound with an enantiomeric 15-oxo-16-kaurene skeleton structure. It has multiple pharmacological effects such as anti-inflammatory, antioxidant and immunosuppressive, and has high biocompatibility. Therefore, bluecalyx A has recently attracted much attention as a skin anti-inflammatory active substance. However, the low solubility of bluecalyx A limits its application. In practical applications, when related preparations containing bluecalyx A are applied to the skin for anti-inflammatory treatment, the therapeutic effect is usually poor. This shows that the bioavailability of bluecalyx A when used transdermally is low, and the main reason is that its efficiency of transdermal delivery is too low.

[0003] For the treatment of skin inflammatory diseases, transdermal delivery has the advantages of being convenient and non-invasive, avoiding the extreme pH environment of the gastrointestinal tract, and local targeted delivery, so it is the most commonly used method of administration. However, the existence of the skin barrier seriously hinders the penetration of active ingredients. The structure of the skin is a complex multi-layer structure, and different layers contain different cells and substances, forming an efficient skin barrier including a physical barrier, a chemical barrier, and an immune barrier, which protects the organism from the invasion of foreign substances and excessive loss of water in daily life. For active substances delivered transdermally, simply increasing the concentration of the active substance cannot help it pass through the skin barrier. On the contrary, excessively high concentrations may irritate the skin. In addition, most active ingredients have more hydrophobic structures and have low solubility in traditional transdermal preparations, which limits the effect of transdermal delivery.

[0004] At present, clinical or commercial transdermal delivery technologies are very limited, mainly based on penetration enhancers and liposome loading. Common penetration enhancers include ethanol, isopropanol, ethyl acetate, lauric acid, dimethyl sulfoxide, and surfactants. Their working principle is to temporarily change the skin structure and destroy its barrier function, allowing active ingredients to enter the deep layer of the skin. Therefore, when using penetration enhancers, there is often a greater risk of skin irritation and allergies. [2] In addition, most penetration enhancers (such as ethanol) may also affect the activity of some small molecule components. Liposome loading has been a hot research area in recent years. However, the nanostructures formed by liposome loading of active substances, such as nanovesicles or nanocapsules, are thermodynamically metastable, and the stability of their microemulsions and nanoemulsions is difficult to meet the requirements of long-term use. [3] In addition, the size of the nanosystem prepared by directly encapsulating small molecules as the core is difficult to control, the transdermal effect is not ideal, and the types of small molecules applicable to this method are limited by the encapsulation materials.

[0005] CN116327745A discloses the use of bluecalyx A in the preparation of a drug for treating endotoxemia, comprising bluecalyx A and conventional pharmaceutical excipients. The drug composition can be administered transdermally, but the patent does not explain what the transdermal drug delivery system is, nor does it verify the effect of transdermal drug delivery. Summary of the invention

[0006] In order to solve the defect that blue calyx A treats skin inflammation due to unsatisfactory transdermal effect and low therapeutic effect, the present invention uses protein nanogel with polymer shell as the carrier of blue calyx A, wherein the protein is used as a nano template, and its outer layer forms a nano-sized protein nanogel after wrapping the polymer, and the blue calyx A is loaded by hydrophilic-hydrophobic interaction inside, so as to construct a high-efficiency transdermal delivery system of blue calyx A. After blue calyx A is loaded in nanocapsules, it is efficiently transdermally transported to the deep layer of the skin and released in a controlled manner, so as to achieve accurate relief and treatment of the inflammation site. Since protein nanocapsules significantly improve the transdermal efficiency of blue calyx A, the transdermal delivery system can realize effective improvement of skin problems using active ingredients at a relatively low concentration. In addition, by selecting suitable protein molecules, the protein template itself also has activities including antioxidant function, so as to play other effects such as anti-oxidation and reducing excess oil secretion, which is conducive to the skin maintaining a better physiological state.

[0007] Specifically, the present invention provides the following technical solutions to solve the above technical problems:

[0008] A nano-transdermal delivery system of bluecalyx A for skin anti-inflammatory is a capsule composite structure of protein-bluecalyx A-polymer shell layer. The protein has affinity to the skin, bluecalyx A is loaded in the protein, and the polymer layer is coated on the surface of the protein. The bluecalyx A nano-transdermal delivery system has a size of 30-80nm and a polymer shell layer thickness of 10-35nm.

[0009] In this delivery system, the positive charge on the surface of the polymer shell helps the transdermal delivery of the blue calyx A inside the nanocapsule. On the one hand, the positive charge on the surface of the polymer shell interacts with the negatively charged lipid components that make up the skin barrier, promoting the nanocapsule as a whole to pass through the skin barrier and reach the inflammatory site deep in the skin. On the other hand, after the nanocapsule arrives near the inflammatory cells, its surface positive charge shows a higher affinity with the negatively charged cell membrane, promoting the cell's uptake of the nanocapsule, so that the blue calyx A in the capsule is successfully delivered into the cell for anti-inflammatory effects.

[0010] In addition to improving the transdermal ability of protein nanogels, the polymer shell also has the function of protecting protein structure and maintaining catalytic activity. Therefore, the thickness regulation of the shell is very important in this system. According to the formula, the permeability of protein nanogels is related to the particle size. Too thin or too thick a shell is not conducive to the deep delivery of nanogels; and the protective effect of the polymer shell on protein activity is also related to the thickness of the shell. Too thin a shell has poor ability to resist drastic changes in ambient temperature and pH; and the premise for the enzyme to play a catalytic role is to contact with the substrate, and too thick a shell will affect the transmission of the substrate, thereby hindering the active protein from playing a catalytic role. In the present invention, a series of sizes of catalase nanogels loaded with blue calyx A were prepared, and the effects of shells of different thicknesses on transdermal depth and protein or enzyme activity protection were investigated. The gel size suitable for the treatment of skin inflammation was comprehensively selected to be between 30 and 80 nm, and the corresponding polymer shell thickness was 10 to 35 nm.

[0011] The present invention uses a polymer to modify and coat the protein surface, and then uses the modified protein as a nanocarrier to load bluecalyx A, thereby preventing the active protein and bluecalyx A from being degraded and inactivated during the transdermal delivery process, and effectively delivering the protein loaded with bluecalyx A to the dermis of the skin. In skin verification treatment, the drug delivery target is located in the dermis, and the anti-inflammatory effect of bluecalyx A can be effectively exerted. The protein of the present invention plays the dual role of a bluecalyx A carrier and improving skin activity.

[0012] Furthermore, the surface Zeta potential of the nano transdermal delivery system is +4 to +6 mV. The inventors found that the surface of the delivery system is positively charged, which is more conducive to achieving a deeper penetration depth.

[0013] Furthermore, the protein is selected from at least one of glutathione, horseradish peroxidase, superoxide dismutase, soybean polypeptide, lysozyme, papain, bromelain, lipase, polypeptide, and collagen, and the size of the protein is 5-10 nm. The present invention can select different or compound proteins for different skin problems, such as glutathione for uneven skin pigmentation, glucose oxidase for skin glycation, horseradish peroxidase and superoxide dismutase for skin oxidation, polypeptide and collagen for skin aging and wrinkle problems, soybean polypeptide, lysozyme, papain and bromelain for skin inflammation, and lipase for excessive skin oil secretion.

[0014] Furthermore, the mass ratio of bluecalyx acetalin to the protein carrier is 1:2-20, preferably 1:2-5.

[0015] Furthermore, the polymer is coated on the surface of the protein by in-situ polymerization, hydrogen bonding or electrostatic interaction; the polymer obtained by the in-situ polymerization method, its monomer is selected from vinyl pyrrolidone, acrylamide, N-acrylsuccinimide, 2-methacryloyloxyethyl phosphorylcholine, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide, acrylate; the acrylate is selected from at least one of methyl (meth)acrylate, ethyl (meth)acrylate, hydroxyethyl (meth)acrylate, propyl (meth)acrylate, and butyl (meth)acrylate; the polymer is coated on the surface of the protein by electrostatic interaction The surface of the protein is coated with a positively charged polymer or a negatively charged polymer, wherein the positively charged polyelectrolyte is selected from at least one of poly(propyleneamine hydrochloride), poly(L-lysine), polyethyleneimine, poly(L-histidine), poly(N,N-dimethylaminoethyl methacrylate), poly(methacrylamide propyltrimethylammonium chloride), and natural or synthetic polysaccharides such as chitosan; the negatively charged polymer is selected from at least one of polyacrylic acid, polystyrene sulfonate, alginate, hyaluronic acid, heparan sulfate, chondroitin sulfate, dextran sulfate, polymethacrylic acid, carboxymethyl cellulose, polyaspartic acid and polyglutamic acid.

[0016] Preferably, the polymer is a carbon-carbon unsaturated double bond monomer coated on the protein surface in an in-situ polymerization manner. The polymer raw material includes a surface modification monomer, a functional monomer, a positively charged monomer, a crosslinker and an initiator; or the polymer raw material includes a surface modification monomer, an amphoteric monomer, a crosslinker and an initiator.

[0017] The surface modification monomer is selected from at least one of N-acrylsuccinimide, N-(3-aminopropyl)methacrylamide hydrochloride, and maleimide. The surface modification monomer is used to modify the monomer on the surface of the protein through interactions such as electrostatic adsorption and chemical bonding. The functional monomer is selected from at least one of vinylpyrrolidone, acrylamide, 2-methacryloyloxyethyl phosphorylcholine, and 2-hydroxyethyl acrylate. The positively charged monomer is selected from 2-(dimethylamino)ethyl methacrylate, (3-acrylamidopropyl)trimethylammonium chloride hydrochloride The invention discloses a method for preparing a protein-enhancing agent comprising: preparing a protein-enhancing agent comprising: a first step of ...

[0018] Furthermore, the mass of the surface modification monomer is 2%-30% of the protein mass, such as 2.5%, 12.5%, and 20%; when the polymer raw materials obtained by in situ polymerization are surface modification monomers, functional monomers, positively charged monomers, and cross-linking agents, the molar ratio of protein, functional monomer, and positively charged monomer is 1:11000-52000:1000-6000, and the amount of cross-linking agent is 5-10% of the molar weight of the functional monomer; preferably, the molar ratio of protein, functional monomer, and positively charged monomer is 1:11000-25000:1000-3000, and the molar ratio of functional monomer to positively charged monomer is 1:0.1-0.15, and more preferably 1:0.1-0.121. When the polymer raw materials obtained by in-situ polymerization are surface modification monomers, amphoteric monomers and crosslinking agents, the molar ratio of protein, amphoteric monomers and crosslinking agents is 1:7000-1500:700-1200; preferably 1:7000-10000:700-1000.

[0019] The second object of the present invention is to provide a method for preparing the above-mentioned nano transdermal delivery system of blue calyx A for skin anti-inflammatory, comprising the following steps:

[0020] (S1) adding a surface modification monomer solution to a protein buffer solution to obtain a surface modified protein solution;

[0021] (S2) adding a solution of cyanocalyxin A to the surface-modified protein solution obtained in step (S1), wherein cyanocalyxin A is adsorbed on the protein to obtain a surface-modified protein solution loaded with cyanocalyxin A;

[0022] (S3) adding functional monomers, positively charged monomers, crosslinking agents, and initiators to the surface-modified protein solution loaded with blue calyx A obtained in step (S2), or adding amphoteric monomers, crosslinking agents, and initiators to initiate in situ polymerization to obtain a blue calyx A nano transdermal delivery system.

[0023] Furthermore, in step (S1), the buffer solution is a phosphate buffer solution, specifically a PBS buffer solution with a pH of 7.4-7.6, and the protein concentration in the protein solution is 1-5 mg / mL; the solvent of the surface modification monomer solution is at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, and the concentration is 1-5wt%.

[0024] Furthermore, in step (S2), the solvent of the bluecalyx A solution is preferably the same solvent as the surface modification monomer solution, that is, at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, and the concentration of the bluecalyx A solution is 1-5 mg / mL, such as 2 mg / mL, 3 mg / mL, and 4 mg / mL. The amount of the bluecalyx A solution is such that the mass ratio of protein to bluecalyx A in the protein solution is 1-10:1, preferably 2-5:1.

[0025] Furthermore, in step (S3), when the monomers are functional monomers and positively charged monomers, the molar ratio of protein, functional monomer, and positively charged monomer is 1:11000-25000:1000-3000, and the amount of crosslinker is 5-10% of the molar amount of functional monomer; or the monomers are amphoteric monomers, and the molar ratio of protein, amphoteric monomer, and crosslinker is 1:7000-10000:700-1000; the initiator is a water-soluble initiator, specifically selected from at least one of ammonium persulfate, sodium persulfate, and potassium persulfate, and preferably, an auxiliary initiator is also added, such as sodium bisulfite and tetramethylethylenediamine. 2-5 mg of initiator and 6-10 mg of auxiliary initiator are added per milligram of protein; preferably, 3.5-4 mg of initiator and 7.5-8 mg of auxiliary initiator are added per milligram of protein; the initiated polymerization is room temperature polymerization, and the polymerization time is 6-12 hours, preferably 6-8 hours.

[0026] The third object of the present invention is to provide the use of the above-mentioned bluecalyx A nano transdermal delivery system in the preparation of drugs for preventing or treating local skin inflammation caused by psoriasis, atopic dermatitis, inflammatory vitiligo, etc.

[0027] The excellent effects of the present invention are:

[0028] First, we constructed polymer-coated core-shell nanocapsules to solve the problem that the treatment effect of bluecalyx A in treating skin inflammation is low due to poor transdermal effect. Protein nanocapsules with polymer shells can efficiently penetrate the skin's physical barrier and transport the bluecalyx A-loaded protein with physiological activity to the dermis, which is the treatment target of skin inflammation, by enhancing intercellular permeation, and exerting an accurate and efficient anti-inflammatory effect.

[0029] 2. The polymer shell in this delivery system provides protection for the protein and bluecalyx A, avoiding protein inactivation due to changes in temperature and pH conditions during the delivery process, and preventing bluecalyx A from being phagocytosed by macrophages before reaching the site of inflammation, which helps the active protein and bluecalyx A to exert their effects after arriving at the site of inflammation.

[0030] 3. The protein of the present invention has both the function of drug carrier and beneficial physiological activity. Different proteins can be selected for different skin problems, or compound proteins can be used to solve comprehensive skin problems.

[0031] Fourth, the nano-delivery system constructed by the present invention has a high delivery efficiency for bluecalyx A and high biocompatibility. For the same degree of skin inflammation, compared with traditional transdermal preparations, the system requires less bluecalyx A in preparation and less dosage during administration, so the irritation to the inflamed area is also smaller. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the fitting curve of the penetration depth D and the particle size d of the catalase nanogel with positive surface potential in Example 1 at 24 hours.

[0033] Figure 2 is the relative activity of nCAT or catalase of different sizes.

[0034] Figure 3 This is a transmission electron microscopy photograph of polyphosphorylcholine-catalase nanocapsules (PMPC-nCAT@GLA) loaded with blue calyx acetylcholine.

[0035] Figure 4 This is a transmission electron microscopy photograph of polysulfonate betaine-collagen nanocapsules (PSPE-nC@GLA) loaded with blue calyx acetylcholine.

[0036] Figure 5 The figure shows the penetration of the nanocapsules of Example 3 and Example 4 at different times. DETAILED DESCRIPTION

[0037] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be described in detail below. The following examples are convenient for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified.

[0038] Example 1 Preparation method of polyphosphorylcholine-catalase nanocapsules:

[0039] To 2 mL of 1 mg / mL PBS buffer solution (pH = 7.4) of catalase (CAT), add a dimethyl sulfoxide solution (mass concentration 1 wt%) containing 0.16 mg of N-acrylsuccinimide, mix thoroughly and react for 2 h to obtain catalase (aCAT) with a polymerizable double bond modified on the surface;

[0040] aCAT, 2-methacryloyloxyethyl phosphorylcholine (MPC), N-(3-aminopropyl) methacrylamide hydrochloride (APM) and N,N'-methylenebisacrylamide (BIS) were mixed according to the given molar ratio, and the protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer, and then an initiator was added according to 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per mg of protein, and in situ free radical polymerization was initiated at room temperature to obtain polyphosphorylcholine-catalase nanocapsules (PMPC-nCAT). The preparation conditions and the measured final size and surface zeta potential are shown in Table 1.

[0041] Table 1 Preparation conditions and final sizes and surface potentials of catalase nanogels of a series of sizes

[0042]

[0043]

[0044] It can be seen from Table 1 that increasing the amount ratio of monomer to protein and increasing the polymerization time will increase the particle size of the obtained catalase nanocapsules (PMPC-nCAT), and increasing the proportion of APM in the monomer will make the Zeta potential of PMPC-nCAT more positive.

[0045] Application Example 1: Transdermal experiment of catalase nanogels with a series of sizes

[0046] 4 mL of catalase nanogel PMPC-nCAT of different sizes in Example 1 with a concentration of 1 mg / mL was mixed with 1 mL of fluorescein isothiocyanate with a concentration of 1 mg / mL and reacted overnight, and dialyzed overnight in a dialysis bag with a molecular weight cutoff of 14000 Da to remove unreacted small molecules to obtain fluorescently labeled PMPC-nCAT. Fresh pigskin was washed with PBS, and the stratum corneum was fixed upward in a transdermal diffusion instrument and kept warm at 37°C. 0.5 mL of fluorescently labeled catalase nanogel solution with a concentration of 1 mg / mL was dripped on the pigskin tissue, and frozen sections were performed after 24 hours. The penetration of the nanogel was observed under a confocal microscope, and the results of the statistical penetration depth are shown in Table 2.

[0047] Table 2 24-hour skin penetration depth of catalase nanogels of various sizes

[0048] Sample name nCAT-20- nCAT-50- nCAT-100- nCAT-150- nCAT-200- Transdermal penetration depth (μm) 81.3±11.9 88.1±5.7 69.4±13.5 71.2±8.0 49.1±5.8 Sample name nCAT-20+ nCAT-50+ nCAT-100+ nCAT-150+ nCAT-200+ Transdermal penetration depth (μm) 133.1±5.8 163.0±13.7 121.9±6.3 101.1±11.5 44.3±4.9

[0049] By comparing the penetration depths of catalase nanogels of different sizes with positive surface potentials (+4mV to +6mV), it was found that when the particle size was between 20nm and 150nm, the protein nanogel could penetrate the epidermis and reach the vicinity of the dermis within 24 hours, and as the gel particle size increased, the penetration depth first increased and then decreased; while catalase nanogels larger than 200nm mostly stayed in the stratum corneum above the epidermis. The penetration depths of nCAT-20+, nCAT-50+, nCAT-100+, and nCAT-150+ were fitted. Figure 1 is the fitting curve of the penetration depth D and particle size d of the catalase nanogel with positive surface potential in Example 1 at 24 hours. The fitted formula is as follows:

[0050] D=a1+m*(a2*d+a3*d 2 +a4*d 3 ),d∈[20,150], where the unit of D is μm and the unit of d is nm.

[0051] The parameters m, a1, a2, a3, and a4 in the formula are related to the polymer outer layer material and the type of protein carrier; among them, a2, a3, and a4 are also related to the size of the nanocapsule, which means that nanocapsules of different sizes have polymer shells of different thicknesses, which changes the degree of influence of the protein and polymer materials on transdermal penetration. When the protein type is catalase, the formula can be written as:

[0052] D=95.9+2.33d-0.0301d 2 +6.95E-5d 3 ,d∈[20,150], where the unit of D is μm and the unit of d is nm.

[0053] The expression can be used to preliminarily predict the penetration capacity of catalase nanogels with a particle size of 20-150nm and a positive surface potential. In the treatment of skin inflammation, the drug delivery target is located in the dermis. According to the formula, the catalase nanogel with a size of about 30-80nm finally penetrates to the upper layer of the dermis, which meets the delivery requirements. Taking into account the therapeutic effect, the size of the blue calyx A transdermal delivery system based on protein nanogel of the present invention is preferably 30-80nm.

[0054] Application Example 2 Series Size Catalase Nanogel Activity Retention

[0055] The catalytic activity of nCAT was determined using an ultraviolet spectrophotometer. 3000 μL of PBS buffer at pH 7 was added to the reference pool; 2900 μL of 0.036% hydrogen peroxide solution and 100 μL of nCAT solution or untreated catalase solution were added to the sample pool, and the sample was placed in an ultraviolet spectrophotometer. The time required for the sample absorbance at 240 nm to change from 0.45 to 0.40 was recorded, indicating the relative activity of catalase. The results are shown in Figure 2. Figure 2 As shown in Table 3. This indicates that for catalase nanogels with a size greater than 100 nm, the polymer shell is too thick, which affects the contact between catalase and substrate, or the polymerization conditions cause the loss of catalase activity. The activity loss of catalase nanogels with a size less than 100 nm is within an acceptable range.

[0056] Table 3 Relative activities of catalase of different sizes

[0057]

[0058] Application Example 3 Thermal Stability Test of Series Size Catalase Nanogels

[0059] The catalytic activity of nCAT was determined by ultraviolet spectrophotometer. 3000 μL of PBS buffer with a pH of 7 was added to the reference cell; 2900 μL of 0.036% hydrogen peroxide solution and 100 μL of nCAT solution or original catalase solution after different heat treatments were added to the sample cell, and then placed in the ultraviolet spectrophotometer. The time required for the sample absorbance at 240 nm to change from 0.45 to 0.40 was recorded, indicating the relative activity of catalase. The results are shown in Table 4.

[0060] Table 4 Relative activity of series nCAT

[0061]

[0062] It was found that the original catalase was significantly inactivated at 60°C, while nCAT-20+, nCAT-50+ and nCAT-100+ could still retain more than 30% of their activity after treatment at 60°C for 1 hour, indicating the good thermal protection effect of the polymer shell.

[0063] Application Example 4 Hydrolysis Stability Test of Series Size Catalase Nanogels

[0064] The catalytic activity of nCAT was determined by UV spectrophotometer. The original catalase or nCAT was diluted with PBS to a final concentration of 0.1 mg / mL, and trypsin was added at a final concentration of 50 μg / mL, and incubated at 37°C for 2 h. 3000 μL of PBS buffer with a pH of 7 was added to the reference cell; 2900 μL of a hydrogen peroxide solution with a mass fraction of 0.036% and 100 μL of nCAT solution treated with trypsin or the original catalase solution were added to the sample cell, and placed in a UV spectrophotometer, and the time required for the sample absorbance at 240 nm to change from 0.45 to 0.40 was recorded, indicating the relative activity of catalase. The results are shown in Table 5.

[0065] Table 5 Relative activity of series nCAT

[0066]

[0067] Example 2 Preparation of polyphosphorylcholine-catalase nanogel loaded with cyanocalyxin

[0068] 0.16 mg of N-acrylsuccinimide was added to 2 mL of a 1 mg / mL catalase (CAT) solution, wherein the N-acrylsuccinimide was dissolved in dimethyl sulfoxide, and the mixture was mixed thoroughly and reacted for 2 h to obtain a catalase (aCAT) with a polymerizable double bond modified on the surface.

[0069] To 2 mL of 1 mg / mL catalase solution with surface double bonds modified, 0.5 mL of 2 mg / mL dimethyl sulfoxide solution of cyanobacterium acetylases was added dropwise with stirring at 300 r / min, and the catalase loaded with cyanobacterium acetylases ...

[0070] aCAT@GLA, 2-methacryloyloxyethyl phosphorylcholine, N-(3-aminopropyl) methacrylamide hydrochloride and N,N'-methylenebisacrylamide were mixed in a molar ratio of 1:17000:2000:1700, and the protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, initiator was added at a ratio of 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per mg of protein, and in situ free radical polymerization was initiated at room temperature to obtain polyphosphorylcholine-catalase nanocapsules loaded with blue calyx methyl (PMPC-nCAT@GLA).

[0071] The obtained complex solution was ultrafiltered in an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa to obtain a purified high-concentration mother solution with a concentration of 6.5 mg / mL, which was stored in a refrigerator.

[0072] Example 3 Preparation of polysulfonated betaine-collagen nanocapsules loaded with blue calyx A

[0073] 0.1 mg of N-acrylsuccinimide was added to 2 mL of 1 mg / mL collagen (C) solution, wherein N-acrylsuccinimide was dissolved in dimethyl sulfoxide, and the mixture was mixed thoroughly and reacted for 2 h to obtain catalase (aC) with a surface modified with polymerizable double bonds.

[0074] To 2 mL of 1 mg / mL collagen solution with surface double bonds modified, 0.35 mL of 2 mg / mL dimethyl sulfoxide solution of aC@GLA was added dropwise while stirring at 300 r / min. After thorough mixing, aC@GLA-loaded collagen (aC@GLA) was obtained.

[0075] aC@GLA, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonatepropyl)ammonium hydroxide and N,N'-methylenebisacrylamide were mixed in a molar ratio of 1:7000:700, and the protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine were added to the initiator system per mg of protein, and in situ free radical polymerization was initiated at room temperature. After 4 hours of reaction, polysulfonated betaine-collagen nanocapsules loaded with blue calyx acetylcholine (PSPE-nC@GLA) were obtained.

[0076] The obtained complex solution was ultrafiltered in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3000 Da to obtain a purified high-concentration mother solution with a concentration of 5.4 mg / mL, which was stored in a refrigerator.

[0077] Application Example 5

[0078] Characterization of nanocapsule size by dynamic light scattering (DLS): The protein nanogel solution with a concentration of 1 mg / mL of Example 2 and Example 3 was added to the sample pool, and the particle size and Zeta potential of the complex in the system were tested at room temperature using a Malvern Nano Zs90 nanoparticle size potential meter. The results are summarized in Table 6.

[0079] Table 6 Particle size and Zeta potential of polymer-protein nanocapsules

[0080] Sample name Particle size distribution center (nm) Zeta potential (mV) CAT 10 -3.8±0.5 PMPC-nCAT@GLA 44 +4.1±2.0 Collegan(C) 5 -6.9±1.7 PSPE-nC@GLA 30 -5.9±4.7

[0081] The morphology of nanocapsules was characterized by transmission electron microscopy (TEM): The morphology and dispersion of polyphosphorylcholine-catalase nanocapsules loaded with blue calyx acetone (PMPC-nCAT@GLA) were observed under transmission electron microscopy. Figure 3 The morphology and dispersion of polysulfonated betaine-collagen nanocapsules (PSPE-nC@GLA) loaded with blue calyx acetone are shown in Figure 2. Figure 4 .

[0082] Application Example 6

[0083] 2 mL of the protein nanocapsules of Example 2 and Example 3 with a concentration of 5 mg / mL were placed in a dialysis bag with a molecular weight cutoff of 3000, and the dialysis bag was immersed in a phosphate buffer with a pH of 7, and the whole system was oscillated at 37° C. The sustained release time was 48 h. Within 48 h, samples were taken regularly to measure the concentration of blue calyx A under an ultraviolet spectrophotometer, and the release of blue calyx A in the nanosystem was calculated. The results are summarized in Table 7.

[0084] Table 7 Release of small molecule drugs by the composite system

[0085]

[0086] It can be seen from the data in Table 7 that the protein nanogel slowly releases the functional small molecules within 24 hours, which is beneficial for the functional small molecules to play a role after reaching the deep layers of the skin, thereby improving the utilization rate.

[0087] Application Example 7: Verification of the anti-inflammatory activity of bluecalyx protein nanogel

[0088] The NO concentration of inflammatory cells was detected using the Griess kit. HaCaT cells (1*10 5 / well), after 12h cells were completely attached, the original culture medium was sucked away, 100μL serum-free culture medium was added to all groups, 10μL serum-free culture medium was added to the negative control group, 10μL PBS buffer was added to the positive control group, 10μL of 0.01mg / mL nanogel solution was added to the low concentration experimental group, 10μL of 0.02mg / mL nanogel solution was added to the medium concentration experimental group, and 10μL of 0.04mg / mL nanogel solution was added to the high concentration experimental group. After 2h of co-incubation, the original culture medium was sucked away from all groups, 110μL fresh culture medium was added to the negative control group, and 100μL fresh culture medium and 10μL of 10ng / mL interleukin-1β were added to the other groups. After 24h of co-incubation, the determination was carried out according to the instructions of the Griess kit. The results are statistically shown in Table 8. It was found that PMPC-nCAT@GLA and PSPE-nC@GLA both had good anti-inflammatory ability, and this ability increased with the increase of concentration. The positive charge on the surface of PMPC-nCAT@GLA makes it more efficiently internalized by cells, resulting in a larger amount of bluecalyx acetylcholine being delivered into cells. At the same concentration, it has better anti-inflammatory ability than the negatively charged PSPE-nC@GLA.

[0089] Table 8 The intracellular anti-inflammatory ability of PMPC-nCAT@GLA and PSPE-nC@GLA increases with the concentration

[0090]

[0091] Application Example 8 Antioxidant Capacity Test of Polyphosphorylcholine-Catalase Nanocapsules (PMPC-nCAT@GLA)

[0092] ROS kit was used to detect the level of ROS in human epidermal HaCaT cells. HaCaT cells (1*10 4 / well), after 12h of complete cell attachment, the cells were irradiated with UVB ultraviolet irradiator to induce internal ROS production, and cells not treated with ultraviolet radiation were left as negative controls. After irradiation, the original culture medium was aspirated from all wells and 100 μL of serum-free culture medium was added (the culture medium was not added with serum to avoid the effect of serum on the cell uptake of the complex during the subsequent drug addition process), 10 μL of PBS buffer was added to the positive control group, 10 μL of 0.01 mg / mL nanogel solution was added to the low concentration experimental group, 10 μL of 0.02 mg / mL nanogel solution was added to the medium concentration experimental group, and 10 μL of 0.04 mg / mL nanogel solution was added to the high concentration experimental group. After incubation for 24h, the original culture medium was aspirated from all cells and 100 μL of serum-free culture medium and 10 μL of DCFH-DA culture solution were added, and incubated at 37°C in the dark for 20min. The cells were then washed three times with PBS, and the fluorescence intensity of the cell DCF was detected using an enzyme marker. The results are statistically shown in Table 9.

[0093] Table 9 Antioxidant capacity test of PMPC-nCAT@GLA

[0094]

[0095] It can be seen from the data in Table 9 that polyphosphorylcholine-catalase nanocapsules have good antioxidant capacity, and this capacity increases with increasing concentration.

[0096] Application Example 9: Transdermal Experiment

[0097] The protein nanocapsule solution was mixed with excess FITC and reacted overnight, and the unreacted small molecules were removed by dialysis to obtain fluorescently labeled protein nanocapsules. Fresh pig skin was washed with PBS, fixed with the stratum corneum facing up in a transdermal diffusion instrument, and kept warm at 37°C. 0.5 mL of fluorescently labeled protein nanocapsule solution was dripped on the pig skin tissue, and frozen sections were taken at 24h, 4h, and 1h. The penetration of the nanocapsules was observed under a confocal microscope, as shown in Figure 2. Figure 5 The statistical penetration depth results are in Table 10. Compared with PSPE-nC@GLA with negative surface potential, PMPC-nCAT@GLA with positive surface potential has a deeper penetration depth within 24 h.

[0098] Table 10 Transdermal penetration of polymer-protein complexes

[0099]

Claims

1. A nano transdermal delivery system of bluecalyx A for skin anti-inflammatory, characterized in that: The capsule has a composite structure of protein-coenzyme A-polymer shell. The protein has affinity to the skin, coenzyme A is loaded in the protein, and the polymer layer is coated on the surface of the protein. The coenzyme A nano transdermal delivery system has a size of 30-80nm and a polymer shell thickness of 10-35nm.

2. The nano transdermal delivery system of bluecalyx A for skin anti-inflammatory according to claim 1, characterized in that: The surface Zeta potential of the nano transdermal delivery system is +4 to +6 mV.

3. The nano transdermal delivery system of bluecalyx A for skin anti-inflammatory according to claim 1, characterized in that: The protein is selected from at least one of glutathione, horseradish peroxidase, superoxide dismutase, soybean polypeptide, lysozyme, papain, bromelain, lipase, polypeptide and collagen, and the size of the protein is 5-10 nm.

4. The nano transdermal delivery system of bluecalyx A for skin anti-inflammatory according to claim 1, characterized in that: The mass ratio of bluecalyx acetalin A to the protein carrier is 1:2-20, preferably 1:2-5.

5. The nano transdermal delivery system of bluecalyx A for skin anti-inflammatory according to claim 1, characterized in that: The polymer is coated on the surface of the protein by in situ polymerization, hydrogen bonding or electrostatic interaction; The polymer obtained by the in-situ polymerization method has a monomer selected from vinyl pyrrolidone, acrylamide, N-acrylsuccinimide, 2-methacryloyloxyethyl phosphorylcholine, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide, and acrylate; the acrylate is selected from at least one of methyl (meth)acrylate, ethyl (meth)acrylate, hydroxyethyl (meth)acrylate, propyl (meth)acrylate, and butyl (meth)acrylate; the polymer is coated on the surface of the protein by electrostatic action and is a positively charged polymer or or a negatively charged polymer, wherein the positively charged polyelectrolyte is selected from at least one of poly(propyleneamine hydrochloride), poly(L-lysine), polyethyleneimine, poly(L-histidine), poly(N,N-dimethylaminoethyl methacrylate), poly(methacrylamide propyltrimethylammonium chloride), and natural or synthetic polysaccharides such as chitosan; and the negatively charged polymer is selected from at least one of polyacrylic acid, polystyrene sulfonate, alginate, hyaluronic acid, heparan sulfate, chondroitin sulfate, dextran sulfate, polymethacrylic acid, carboxymethyl cellulose, polyaspartic acid and polyglutamic acid.

6. The nano transdermal delivery system of bluecalyx A for skin anti-inflammatory according to claim 1, characterized in that: The polymer is a carbon-carbon unsaturated double bond monomer coated on the protein surface in an in-situ polymerization manner; the polymer raw material includes a surface modification monomer, a functional monomer, a positively charged monomer, a cross-linking agent and an initiator; or the polymer raw material includes a surface modification monomer, an amphoteric monomer, a cross-linking agent and an initiator; Preferably, the surface modification monomer is selected from at least one of N-acrylsuccinimide, N-(3-aminopropyl)methacrylamide hydrochloride, and maleimide; the functional monomer is selected from at least one of vinylpyrrolidone, acrylamide, 2-methacryloyloxyethyl phosphorylcholine, and 2-hydroxyethyl acrylate; the positive charge monomer is selected from at least one of 2-(dimethylamino)ethyl methacrylate, (3-acrylamidopropyl)trimethylammonium chloride hydrochloride, and N-(3-aminopropyl)methacrylamide hydrochloride; the crosslinker is selected from at least one of N,N'-methylenebisacrylamide, dimethacrylate glycerol, and 1,3-diglycerol alcohol diacrylate; the amphoteric monomer is [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl)ammonium hydroxide; the initiator is a water-soluble initiator, specifically selected from at least one of ammonium persulfate, sodium persulfate, and potassium persulfate; Preferably, a co-initiator is also added, such as sodium bisulfite, tetramethylethylenediamine; More preferably, 2-5 mg initiator and 6-10 mg co-initiator are added per mg protein; preferably, 3.5-4 mg initiator and 7.5-8 mg co-initiator are added per mg protein.

7. The nano transdermal delivery system of bluecalyx A for skin anti-inflammatory according to claim 6, characterized in that: The mass of the surface modification monomer is 2%-30% of the mass of the protein; when the polymer raw materials obtained by in-situ polymerization are surface modification monomers, functional monomers, positively charged monomers, and crosslinking agents, the molar ratio of protein, functional monomer, and positively charged monomer is 1:11000-52000:1000-6000, and the amount of the crosslinking agent is 5-10% of the molar weight of the functional monomer; preferably, the molar ratio of protein, functional monomer, and positively charged monomer is 1:11000-25000:1000-3000, and the molar ratio of functional monomer to positively charged monomer is 1:0.1-0.15, more preferably 1:0.1-0.121; When the polymer raw materials obtained by in-situ polymerization are surface modification monomers, amphoteric monomers and crosslinking agents, the molar ratio of protein, amphoteric monomers and crosslinking agents is 1:7000-1500:700-1200; preferably 1:7000-10000:700-1000.

8. The method for preparing the nano transdermal delivery system of bluecalyx A for skin anti-inflammatory according to any one of claims 1 to 7, characterized in that: The following steps are involved: (S1) adding a surface modification monomer solution to a protein buffer solution to obtain a surface modified protein solution; (S2) adding a solution of cyanocalyxin A to the surface-modified protein solution obtained in step (S1), wherein cyanocalyxin A is adsorbed on the protein to obtain a surface-modified protein solution loaded with cyanocalyxin A; (S3) adding functional monomers, positively charged monomers, crosslinking agents, and initiators to the surface-modified protein solution loaded with blue calyx A obtained in step (S2), or adding amphoteric monomers, crosslinking agents, and initiators to initiate in situ polymerization to obtain a blue calyx A nano transdermal delivery system.

9. The preparation method according to claim 8, characterized in that: In step (S1), the buffer solution is a phosphate buffer solution, specifically a PBS buffer solution with a pH of 7.4-7.6, and the protein concentration in the protein solution is 1-5 mg / mL; the solvent of the surface modification monomer solution is at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, and the concentration is 1-5 wt%; and / or In step (S2), the solvent of the bluecalyx A solution is selected from at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, and the concentration of the bluecalyx A solution is 1-5 mg / mL; the amount of the bluecalyx A solution is such that the mass ratio of protein to bluecalyx A in the protein solution is 1-10:1, preferably 2-5:1; and / or In step (S3), the molar ratio of protein, functional monomer and positively charged monomer is 1:11000-25000:1000-3000, and the amount of crosslinking agent is 5-10% of the molar amount of functional monomer; or the molar ratio of protein, amphoteric monomer and crosslinking agent is 1:7000-10000:700-1000.

10. Use of the bluecalyx A nano transdermal delivery system for skin anti-inflammatory according to any one of claims 1 to 7 in the preparation of a drug for preventing or treating local skin inflammation; preferably, the local skin inflammation includes psoriasis, atopic dermatitis, and inflammatory vitiligo.

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