A transdermal nano-transdermal delivery system for skin anti-inflammatory purposes and its preparation method

Abstract

This invention provides a transdermal nanoparticle delivery system for skin anti-inflammation, comprising a protein-blue calyx methyl ether-polymer shell capsule composite structure. The protein has an affinity for the skin, blue calyx methyl ether is loaded within the protein, and the polymer layer coats the surface of the protein. The blue calyx methyl ether nanoparticle transdermal delivery system has a size of 30-80 nm, and the polymer shell thickness is 10-35 nm. The surface properties of the polymer shell can be controlled according to the required transdermal delivery depth, enabling the protein to penetrate the skin barrier and be efficiently delivered deep into the skin, where the blue calyx methyl ether is then slowly released, targeting and alleviating skin inflammation. This addresses the drawback of low therapeutic efficacy of blue calyx methyl ether in treating skin inflammation due to unsatisfactory transdermal effects.

Description

Technical Field

[0001] This invention pertains to nano-transdermal delivery systems, specifically relating to a blue calyx methyl ether nano-transdermal delivery system for skin anti-inflammatory purposes and its preparation method. Background Technology

[0002] Kauryl kauriin is a diterpenoid compound with an enantiomeric 15-oxo-16-kauriene skeleton. It possesses various pharmacological effects, including anti-inflammatory, antioxidant, and immunosuppressive properties, and exhibits high biocompatibility. Therefore, kuryl kauriin has recently attracted considerable attention as a skin anti-inflammatory active ingredient. However, the low solubility of kuryl kauriin limits its application. In practical applications, the therapeutic effect of applying kuryl kauriin-containing preparations to the skin for anti-inflammatory treatment is usually poor. This indicates low bioavailability of kuryl kauriin via transdermal administration, primarily due to its low transdermal delivery efficiency.

[0003] For the treatment of inflammatory skin diseases, transdermal delivery offers advantages such as convenience, non-invasiveness, avoidance of the extreme pH environment of the gastrointestinal tract, and localized targeted delivery, making it the most commonly used method of drug administration. However, the presence of the skin barrier severely hinders the penetration of active ingredients. The skin has a complex, multi-layered structure, with different layers containing different cells and substances, forming a highly efficient skin barrier that includes physical, chemical, and immune barriers. In daily life, this barrier protects the body from the invasion of foreign substances and excessive water loss. For transdermal delivered active substances, simply increasing the concentration does not help them penetrate the skin barrier; on the contrary, excessively high concentrations may irritate the skin. Furthermore, most active ingredients have numerous hydrophobic structures, resulting in low solubility in traditional transdermal formulations, thus limiting the effectiveness of transdermal delivery.

[0004] Currently, transdermal delivery technologies available for clinical or commercial use are very limited, primarily relying on penetration enhancers and liposome loading. Common penetration enhancers include ethanol, isopropanol, ethyl acetate, lauric acid, dimethyl sulfoxide, and surfactants. Their mechanism of action involves temporarily altering the skin structure and disrupting its barrier function, allowing active ingredients to penetrate deeper into the skin. Therefore, the use of penetration enhancers often carries a significant risk of skin irritation and allergic reactions. [2] Furthermore, most penetration enhancers (such as ethanol) may also affect the activity of some small molecule components. Liposome loading has become a hot research area in recent years; however, the nanostructures formed by loading active substances onto liposomes, such as nanovesicles or nanocapsules, are thermodynamically metastable, and the stability of their microemulsions and nanoemulsions is difficult to meet the requirements for long-term use. [3] Furthermore, the size of nanoscale systems prepared by directly encapsulating small molecules as the core is difficult to control, resulting in unsatisfactory transdermal effects. Moreover, the types of small molecules applicable to this method are limited by the encapsulation materials.

[0005] CN116327745A discloses the use of blue calyx methyl ester in the preparation of a drug for treating endotoxemia, including blue calyx methyl ester and conventional pharmaceutical excipients. The drug composition can be administered transdermally, but the patent does not clarify what the transdermal drug delivery system is, nor does it verify the effectiveness of transdermal drug delivery. Summary of the Invention

[0006] To address the shortcomings of blue calyx methyl methacrylate (BMS) in treating skin inflammation due to its poor transdermal efficacy, this invention utilizes a protein nanogel with a polymer shell as a carrier for BMS. The protein within the nanogel acts as a nanotemplate, and the outer layer encapsulates the polymer to form a nano-sized protein nanogel. BMS is loaded internally through hydrophilic-hydrophobic interactions, constructing a highly efficient transdermal delivery system for BMS. Once loaded into nanocapsules, BMS is efficiently transdermally transported to the deep layers of the skin and released in a controlled manner, achieving precise relief and treatment of inflammation. Because the protein nanocapsules significantly enhance the transdermal efficiency of BMS, this transdermal delivery system can effectively improve skin problems using relatively low concentrations of active ingredients. Furthermore, by selecting suitable protein molecules, the protein template itself possesses activities including antioxidant functions, thereby exerting other effects such as antioxidation and reducing excess sebum secretion, which is beneficial for maintaining a better physiological state of the skin.

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

[0008] A transdermal delivery system for skin anti-inflammatory purposes is a capsule composite structure consisting of a protein, a blue calyx methyl ester, and a polymer shell. The protein has an affinity for the skin, the blue calyx methyl ester is loaded in the protein, and the polymer layer coats the surface of the protein. The transdermal delivery system for the blue calyx methyl ester has a size of 30-80 nm and a polymer shell thickness of 10-35 nm.

[0009] In this delivery system, the positive charge on the surface of the polymer shell facilitates the transdermal delivery of cyanin within the nanocapsules. On one hand, the positive charge on the polymer shell interacts with the negatively charged lipid components that make up the skin barrier, promoting the nanocapsules' penetration through the skin barrier to reach the inflamed site deep within the skin. On the other hand, once the nanocapsules reach the vicinity of inflammatory cells, their positive surface charge exhibits a higher affinity for the negatively charged cell membrane, promoting cellular uptake of the nanocapsules and enabling the successful delivery of cyanin into the cells for its anti-inflammatory effect.

[0010] Besides enhancing the transdermal permeability of protein nanogels, the polymer shell also protects the protein structure and maintains catalytic activity. Therefore, controlling the shell thickness is crucial in this system. According to the formula, the permeability of protein nanogels is related to their particle size; shells that are too thin or too thick are detrimental to deep delivery of the nanogels. The protective effect of the polymer shell on protein activity is also related to its thickness; a thin shell is less resistant to drastic temperature and pH changes. Furthermore, enzymes require contact with the substrate to exert their catalytic activity; an excessively thick shell can impede substrate transport, thus hindering the catalytic activity of the active protein. In this invention, catalase nanogels loaded with blue calyx methyl ether were prepared at various sizes. The effects of different shell thicknesses on transdermal depth and the protection of protein or enzyme activity were investigated. A gel size of 30 to 80 nm, with a corresponding polymer shell thickness of 10 to 35 nm, was selected as suitable for treating skin inflammation.

[0011] This invention employs polymer modification and coating of the protein surface, then uses the modified protein as a nanocarrier to load blue sepal methylphenidate. This avoids the degradation and inactivation of the active protein and blue sepal methylphenidate during transdermal delivery, while effectively delivering the blue sepal-loaded protein to the dermis. In skin validation treatment, the drug delivery target is located in the dermis, effectively exerting the anti-inflammatory effect of blue sepal methylphenidate. The protein of this invention acts as a blue sepal methylphenidate carrier and has the dual function of improving skin activity.

[0012] Furthermore, the surface zeta potential of the nanotransdermal delivery system is +4 to +6 mV. The inventors have discovered that a positively charged surface on the delivery system is more conducive to achieving deeper penetration depths.

[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 protein size is 5-10 nm. This invention allows for the selection of different or combined proteins to address various skin problems. For example, glutathione can be used for uneven skin pigmentation; glucose oxidase can be used for skin glycation; horseradish peroxidase and superoxide dismutase can be used for skin oxidation; polypeptides and collagen can be used for skin aging and wrinkles; soybean polypeptide, lysozyme, papain, and bromelain can be used for skin inflammation; and lipase can be used for excessive sebum secretion.

[0014] Furthermore, the mass ratio of blue calyx methyl ester to protein carrier is 1:2-20, preferably 1:2-5.

[0015] Further, the polymer is coated onto the surface of the protein via in-situ polymerization, hydrogen bonding, or electrostatic interaction; the polymer obtained by in-situ polymerization has monomers selected from vinylpyrrolidone, acrylamide, N-propenylsuccinimide, 2-methacryloyloxyethylphosphocholine, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide, and acrylates; the acrylates are selected from at least one of methyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate, propyl methacrylate, and butyl methacrylate; the polymer is coated onto the surface of the protein via electrostatic interaction. The polymer coating the surface of the protein is either a positively charged or a negatively charged polymer. The positively charged polyelectrolyte is selected from at least one of poly(acrylamide hydrochloride), poly(L-lysine), polyethyleneimine, poly(L-histidine), N,N-dimethylaminoethyl methacrylate, polymethacrylamide 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-bonded monomer coated onto the protein surface via in-situ polymerization. The polymer raw materials include surface-modifying monomers, functional monomers, positively charged monomers, crosslinking agents, and initiators; or the polymer raw materials include surface-modifying monomers, amphoteric monomers, crosslinking agents, and initiators.

[0017] The surface-modifying monomer is selected from at least one of N-propenylsuccinimide, N-(3-aminopropyl)methacrylamide hydrochloride, and maleimide. The surface-modifying monomer modifies the protein surface through interactions such as electrostatic adsorption and chemical bonding. The functional monomer is selected from at least one of vinylpyrrolidone, acrylamide, 2-methacryloyloxyethyl phosphocholine, and 2-hydroxyethyl acrylate. The positively charged monomer is selected from 2-(dimethylamino)methacrylate ethyl ester and (3-acrylamidopropyl)trimethylammonium chloride hydrochloride. The initiator is selected from at least one of the following: a salt, N-(3-aminopropyl)methacrylamide hydrochloride; the crosslinking agent is selected from at least one of N,N'-methylenebisacrylamide, glyceryl dimethacrylate, and glyceryl 1,3-diglyceryl alcohol diacrylate; the amphoteric monomer is [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonylpropyl)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, such as sodium bisulfite or tetramethylethylenediamine, is also added. 2-5 mg of initiator and 6-10 mg of co-initiator are added per milligram of protein; preferably, 3.5-4 mg of initiator and 7.5-8 mg of co-initiator are added per milligram of protein.

[0018] Further, the mass of the surface-modifying monomer is 2%-30% of the protein mass, for example, 2.5%, 12.5%, or 20%; when the polymer raw material obtained by in-situ polymerization is a surface-modifying monomer, a functional monomer, a positively charged monomer, and a crosslinking agent, the molar ratio of protein, functional monomer, and positively charged monomer is 1:11000-52000:1000-6000, and the amount of crosslinking agent is 5-10% of the molar mass 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-modified 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 objective of this invention is to provide a method for preparing the above-mentioned transdermal nano-transdermal delivery system for skin anti-inflammatory purposes, comprising the following steps:

[0020] (S1) Add surface-modified monomer solution to protein buffer solution to obtain surface-modified protein solution;

[0021] (S2) Add blue calyx methyl ester solution to the surface-modified protein solution obtained in step (S1). Blue calyx methyl ester is adsorbed into the protein to obtain a surface-modified protein solution loaded with blue calyx methyl ester.

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

[0023] Further, 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-modified monomer solution is at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, with a concentration of 1-5 wt%.

[0024] Further, in step (S2), the solvent for the blue calyx methyl ether solution is preferably a solvent similar to that of the surface-modifying monomer solution, i.e., at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol. The concentration of the blue calyx methyl ether solution is 1-5 mg / mL, such as 2 mg / mL, 3 mg / mL, or 4 mg / mL. The amount of blue calyx methyl ether solution used is such that the mass ratio of protein to blue calyx methyl ether in the protein solution is 1-10:1, preferably 2-5:1.

[0025] Further, in step (S3), when the monomer is a functional monomer and a positively charged monomer, 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 monomer is an amphoteric monomer, and the molar ratio of protein, amphoteric monomer, and crosslinking agent 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. Preferably, a co-initiator, such as sodium bisulfite or tetramethylethylenediamine, is also added. 2-5 mg of initiator and 6-10 mg of co-initiator are added per milligram of protein; preferably, 3.5-4 mg of initiator and 7.5-8 mg of co-initiator are added per milligram of protein; the initiation polymerization is room temperature polymerization, and the polymerization time is 6-12 hours, preferably 6-8 hours.

[0026] A third objective of this invention is to provide the use of the above-mentioned blue calyx methylphenidate nanotransdermal delivery system in the preparation of drugs for the prevention or treatment of local skin inflammation caused by diseases including psoriasis, atopic dermatitis, and inflammatory vitiligo.

[0027] The superior effects of this invention are as follows:

[0028] I. A polymer-coated core-shell nanocapsule structure was constructed to address the low efficacy of blue calyx extract in treating skin inflammation due to poor transdermal absorption. The protein nanocapsule with its polymer shell can efficiently penetrate the skin's physical barrier, enhancing intercellular permeability to transport physiologically active blue calyx extract-loaded proteins to the dermis, the therapeutic target site for skin inflammation, thus precisely and effectively exerting its anti-inflammatory effect.

[0029] Second, the polymer shell in this delivery system provides protection for the protein and blue calyx methyl esterase, preventing protein inactivation due to changes in temperature and pH during delivery, and preventing blue calyx methyl esterase from being phagocytosed by macrophages before reaching the site of inflammation. This helps both the active protein and blue calyx methyl esterase to function after reaching the site of inflammation.

[0030] Third, the proteins of this invention function as both drug carriers and beneficial physiological activities. Different proteins, or combinations thereof, can be selected to address various skin problems.

[0031] Fourth, the nanodelivery system constructed in this invention exhibits high delivery efficiency for blue calyx methylphenidate and high biocompatibility. For the same degree of skin inflammation, compared with traditional transdermal formulations, this system requires less blue calyx methylphenidate during preparation and a smaller dosage during administration, thus causing less irritation to the inflamed area. Attached Figure Description

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

[0033] Figure 2 It represents the relative activity of nCAT-active catalases of different sizes.

[0034] Figure 3 This is a transmission electron microscope image of polyphosphoric acid choline-catalase nanocapsules (PMPC-nCAT@GLA) loaded with blue calyx methyl ether.

[0035] Figure 4 This is a transmission electron microscope image of polysulfonated betaine-collagen nanocapsules (PSPE-nC@GLA) loaded with blue calyx methyl ester.

[0036] Figure 5 The images show the permeation of the nanocapsules in Examples 3 and 4 at different times. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. The following embodiments are provided to better understand this invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods.

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

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

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

[0041] Table 1 shows the preparation conditions, final size, and surface potential of catalase nanogels of various sizes.

[0042]

[0043]

[0044] As shown in Table 1, increasing the monomer-to-protein ratio and increasing the polymerization time will increase the particle size of the resulting catalase nanocapsules (PMPC-nCAT). 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 a Series of Catalase Nanogels

[0046] 4 mL of 1 mg / mL catalase nanogels of different sizes from Example 1 (PMPC-nCAT) were mixed with 1 mL of 1 mg / mL fluorescein isothiocyanate and reacted overnight. The mixture was then dialyzed overnight in a dialysis bag with a molecular weight cutoff of 14000 Da to remove unreacted small molecules, yielding fluorescently labeled PMPC-nCAT. Fresh pig skin was washed with PBS, and the stratum corneum was fixed upwards in a transdermal diffusion apparatus and incubated at 37°C. 0.5 mL of a 1 mg / mL fluorescently labeled catalase nanogel solution was dropped onto the pig skin tissue. After 24 hours, frozen sections were prepared, and the penetration of the nanogels was observed under a confocal microscope. The penetration depth results are shown in Table 2.

[0047] Table 2 shows the transdermal depth of catalase nanogels of various sizes after 24 hours.

[0048] Sample Name nCAT-20- nCAT-50- nCAT-100- nCAT-150- nCAT-200- Transdermal 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 depth (μm) 133.1±5.8 163.0±13.7 121.9±6.3 101.1±11.5 44.3±4.9

[0049] 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 nanogels could penetrate through the epidermis and reach near the dermis within 24 hours, and the penetration depth first increased and then decreased with increasing gel particle size; while catalase nanogels larger than 200nm mostly remained in the stratum corneum of the upper epidermis. The penetration depths of nCAT-20+, nCAT-50+, nCAT-100+, and nCAT-150+ were fitted. Figure 1 This is a fitted curve of the penetration depth D versus particle size d of the catalase nanogel with a positive surface potential from Example 1 after 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, representing that nanocapsules of different sizes have polymer shells of different thicknesses, thus changing the degree of influence of the protein and polymer materials on transdermal absorption. When the protein 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] This expression can be used to preliminarily predict the permeability of catalase nanogels with positive surface potentials in the particle size range of 20-150 nm. In the treatment of skin inflammation, the drug delivery target is located in the dermis. According to the formula, catalase nanogels with a size of approximately 30-80 nm can ultimately penetrate to the upper dermis, meeting the delivery requirements. Considering the therapeutic effect, the preferred size of the blue calyx methyl methacrylate transdermal delivery system based on protein nanogels of this invention is 30-80 nm.

[0054] Application Example 2: Series of catalase nanogels retain activity

[0055] The catalytic activity of nCAT was determined using a UV spectrophotometer. 3000 μL of pH 7 PBS buffer was added to the reference cell; 2900 μL of 0.036% hydrogen peroxide solution and 100 μL of nCAT solution or untreated catalase solution were added to the sample cell. The samples were then placed in the UV spectrophotometer, and the time required for the absorbance at 240 nm to increase from 0.45 to 0.40 was recorded, representing the relative activity of catalase. Results are shown below. Figure 2 As shown in Table 3, the excessively thick polymer shell of catalase nanogels with a size greater than 100 nm can affect the contact between catalase and the substrate, or the polymerization conditions may cause a 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 with different nCAT sizes

[0057]

[0058] Application Example 3: Thermal Stability Testing of Series-Sized Catalase Nanogels

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

[0060] Table 4 shows the relative activity of nCAT series.

[0061]

[0062] The original catalase was found to be significantly inactivated at 60℃, while nCAT-20+, nCAT-50+ and nCAT-100+ retained more than 30% of their activity after treatment at 60℃ for 1 hour, indicating that the polymer shell has a good thermal protection effect.

[0063] Application Example 4: Hydrolysis Stability Test of Series-Sized Catalase Nanogels

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

[0065] Table 5 shows the relative activity of nCAT series.

[0066]

[0067] Example 2: Preparation of polyphosphocholine-catalase nanogels loaded with cyanin

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

[0069] To 2 mL of a catalase solution with a surface double bond modified at a concentration of 1 mg / mL, 0.5 mL of a dimethyl sulfoxide solution with a concentration of 2 mg / mL of cyanobacterium oxycarboxylate was added dropwise while stirring at 300 r / min. After thorough mixing, catalase loaded with cyanobacterium oxycarboxylate (aCAT@GLA) was obtained.

[0070] aCAT@GLA, 2-methacryloyloxyethyl phosphocholine, N-(3-aminopropyl)methacrylamide hydrochloride, and N,N'-methylenebisacrylamide were mixed in a molar ratio of 1:17000:2000:1700. The protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, an initiator was added at a ratio of 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per milligram of protein. In situ free radical polymerization was initiated at room temperature to obtain polyphosphocholine-catalase nanocapsules loaded with blue calyx acetylcholine (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 liquor with a concentration of 6.5 mg / mL, which was then refrigerated.

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

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

[0074] Add 0.35 mL of a 2 mg / mL dimethyl sulfoxide solution of cyanobacterium methyl ester to a 2 mL solution of collagen with surface double bond modification while stirring at 300 rpm. After thorough mixing, collagen loaded with cyanobacterium methyl ester (aC@GLA) is obtained.

[0075] aC@GLA, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propionic acid)ammonium hydroxide, and N,N'-methylenebisacrylamide were mixed in a molar ratio of 1:7000:700. 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 per milligram of protein were added to the initiation system. In situ free radical polymerization was initiated at room temperature. After 4 h of reaction, polysulfonated betaine-collagen nanocapsules (PSPE-nC@GLA) loaded with blue calyx methyl ester 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 high-concentration mother liquor with a purified concentration of 5.4 mg / mL, which was then refrigerated.

[0077] Application Example 5

[0078] The size of the nanocapsules was characterized using dynamic light scattering (DLS): Protein nanogel solutions with a concentration of 1 mg / mL were added to the sample cell in Examples 2 and 3, and the particle size and zeta potential of the complexes within the system were measured at room temperature using a Malvern Nano Zs90 nanoparticle size potentiometer. 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 the nanocapsules was characterized using transmission electron microscopy (TEM): The morphology and dispersion of polyphosphocholine-catalase nanocapsules (PMPC-nCAT@GLA) loaded with cyanopylene were observed under TEM. The results are as follows: Figure 3 The morphology and dispersion of polysulfonated betaine-collagen nanocapsules (PSPE-nC@GLA) loaded with blue calyx methyl ester are shown in the figure below. Figure 4 .

[0082] Application Example 6

[0083] Two mL of protein nanocapsules (5 mg / mL) from Examples 2 and 3 were placed in a dialysis bag with a molecular weight cutoff of 3000. The dialysis bag was then immersed in phosphate buffer solution at pH 7, and the entire system was shaken at 37°C. The release time was 48 h. During this 48 h periodically, samples were taken and the concentration of cyanobacterium methyl ether was measured using a UV spectrophotometer to calculate the release of cyanobacterium methyl ether from the nanosystem. The results are summarized in Table 7.

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

[0085]

[0086] As can be seen from the data in Table 7, the protein nanogel slowly releases functional small molecules within 24 hours, which is beneficial for the functional small molecules to exert their effects after reaching the deep layers of the skin, thus improving their utilization rate.

[0087] Application Example 7: Verification of the anti-inflammatory properties of blue calyx protein nanogels

[0088] NO concentration in inflammatory cells was detected using the Griess assay kit. HaCaT cells (1*10⁻⁶) were seeded in 24-well plates. 5Cells were incubated (number per well) for 12 hours until complete cell adhesion. Afterward, the original culture medium was removed, and 100 μL of serum-free culture medium was added to all groups. The negative control group received 10 μL of serum-free culture medium, and the positive control group received 10 μL of PBS buffer. The low-concentration experimental group received 10 μL of 0.01 mg / mL nanogel solution, the medium-concentration experimental group received 10 μL of 0.02 mg / mL nanogel solution, and the high-concentration experimental group received 10 μL of 0.04 mg / mL nanogel solution. After co-incubation for 2 hours, the original culture medium was removed from all groups. The negative control group received 110 μL of fresh culture medium, and the remaining groups received 100 μL of fresh culture medium and 10 μL of 10 ng / mL interleukin-1β. After co-incubation for 24 hours, the results were measured according to the Griess kit instructions. The results are shown in Table 8. Both PMPC-nCAT@GLA and PSPE-nC@GLA showed good anti-inflammatory activity, and this activity increased with increasing concentration. The positive charge on the surface of PMPC-nCAT@GLA makes it more efficient at being endocytosed by cells, resulting in a greater amount of blue calyx methyl ester delivered into the cells. At the same concentration, it has better anti-inflammatory ability than the negatively charged PSPE-nC@GLA.

[0089] Table 8. Intracellular anti-inflammatory capacity of PMPC-nCAT@GLA and PSPE-nC@GLA cells increases with increasing concentration.

[0090]

[0091] Application Example 8: Antioxidant Capacity Test of Polyphosphoric Choline-Catase Nanocapsules (PMPC-nCAT@GLA)

[0092] ROS levels in human epidermal HaCaT cells were detected using a ROS kit. HaCaT cells (1*102) were seeded in 96-well plates. 4Cells were incubated at 12 h per well. After complete cell adhesion, the cells were irradiated with UVB to induce internal ROS production. Untreated cells served as a negative control. After irradiation, the original culture medium was aspirated from all wells, and 100 μL of serum-free medium was added (serum was omitted to avoid affecting cell uptake of the complex during subsequent drug administration). The positive control group received 10 μL of PBS buffer, the low-concentration experimental group received 10 μL of 0.01 mg / mL nanogel solution, the medium-concentration experimental group received 10 μL of 0.02 mg / mL nanogel solution, and the high-concentration experimental group received 10 μL of 0.04 mg / mL nanogel solution. After 24 h of incubation, the original culture medium was aspirated from all wells, and 100 μL of serum-free medium and 10 μL of DCFH-DA medium were added. The cells were incubated at 37°C in the dark for 20 min. The cells were then washed three times with PBS, and the fluorescence intensity of the DCF in the cells was detected using an ELISA reader. The results are summarized in Table 9.

[0093] Table 9. Antioxidant Capacity Test of PMPC-nCAT@GLA

[0094]

[0095] The data in Table 9 show that polyphosphocholine-catalase nanocapsules have good antioxidant capacity, and this capacity increases with increasing concentration.

[0096] Application Example 9: Transdermal Test

[0097] The protein nanocapsule solution was mixed with excess FITC and reacted overnight. Unreacted small molecules were removed by dialysis to obtain fluorescently labeled protein nanocapsules. Fresh pig skin was washed with PBS and fixed with the stratum corneum facing upward in a transdermal diffusion apparatus and incubated at 37°C. 0.5 mL of the fluorescently labeled protein nanocapsule solution was dropped onto the pig skin tissue. Frozen sections were prepared at 24 h, 4 h, and 1 h, and the permeation of the nanocapsules was observed under a confocal microscope. Figure 5 The statistical penetration depth results are shown in Table 10. Compared with PSPE-nC@GLA, which has a negative surface potential, PMPC-nCAT@GLA, which has a positive surface potential, has a deeper penetration depth over 24 hours.

[0098] Table 10 Transdermal Properties of Polymer-Protein Complexes

[0099]

Claims

1. A transdermal nano-transdermal delivery system for skin anti-inflammatory purposes, characterized in that, The capsule has a protein-blue calyx methyl ether-polymer shell structure. The protein has an affinity for the skin, blue calyx methyl ether is loaded in the protein, and the polymer layer coats the surface of the protein. The blue calyx methyl ether nanotransdermal delivery system has a size of 30-80 nm and the polymer shell thickness is 10-35 nm. The protein is selected from at least one of catalase and collagen. The preparation method of the blue calyx methyl methacrylate nanoparticle transdermal delivery system for skin anti-inflammatory purposes includes the following steps: (S1) Add surface-modified monomer solution to protein buffer solution to obtain surface-modified protein solution; (S2) Add blue calyx methyl ester solution to the surface-modified protein solution obtained in step (S1). Blue calyx methyl ester is adsorbed into the protein to obtain a surface-modified protein solution loaded with blue calyx methyl ester. (S3) Add a functional monomer, a positively charged monomer, a crosslinking agent, and an initiator to the surface-modified protein solution loaded with blue calyx methyl ether obtained in step (S2); or add an amphoteric monomer, a crosslinking agent, and an initiator to initiate in-situ polymerization to obtain a blue calyx methyl ether nanotransdermal delivery system. The surface-modifying monomer is N-propenylsuccinimide; the functional monomer is 2-methacryloyloxyethyl phosphocholine; the positively charged monomer is N-(3-aminopropyl)methacrylamide hydrochloride; the crosslinking agent is N,N'-methylenebisacrylamide; and the amphoteric monomer is [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide.

2. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 1, characterized in that, The surface zeta potential of the nanotransdermal delivery system is +4 to +6 mV.

3. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 1, characterized in that, The protein is selected from at least one of catalase and collagen, and the protein size is 5-10 nm.

4. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 1, characterized in that, The mass ratio of blue calyx methyl esterase to protein carrier is 1:2-20.

5. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 4, characterized in that, The mass ratio of blue calyx methyl esterase to protein carrier is 1:2-5.

6. The blue calyx methyl methacrylate nanoparticle transdermal delivery system for skin anti-inflammatory purposes according to claim 1, characterized in that, The initiator is a water-soluble initiator.

7. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 6, characterized in that, The water-soluble initiator is selected from at least one of ammonium persulfate, sodium persulfate, and potassium persulfate.

8. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 6, characterized in that, When adding the initiator, a co-initiator is also added.

9. The blue calyx methyl methacrylate nanoparticle transdermal delivery system for skin anti-inflammatory purposes according to claim 8, characterized in that, The co-initiator is selected from sodium bisulfite and tetramethylethylenediamine.

10. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 8, characterized in that, Add 2-5 mg of initiator and 6-10 mg of co-initiator per milligram of protein.

11. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 8, characterized in that, Add 3.5-4 mg of initiator and 7.5-8 mg of co-initiator per milligram of protein.

12. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 1, characterized in that, The mass of the surface-modifying monomer is 2%-30% of the protein mass; 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 mass of the functional monomer.

13. The blue calyx methyl methacrylate nanoparticle transdermal delivery system for skin anti-inflammatory purposes according to claim 12, characterized in that, 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.

14. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 13, characterized in that, The molar ratio of functional monomers to positively charged monomers is 1:0.1-0.

121.

15. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 12, characterized in that, The molar ratio of protein, amphoteric monomer, and cross-linking agent is 1:7000-10000:700-1000.

16. The blue calyx methylphenidate nanotransdermal delivery system for skin anti-inflammatory purposes according to claim 1, 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 for the surface-modifying monomer solution is at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, with a concentration of 1-5 wt%; and / or In step (S2), the solvent for the blue calyx methyl sulfoxide solution is selected from at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, and the concentration of the blue calyx methyl sulfoxide solution is 1-5 mg / mL.

17. Use of the blue calyx quinone nanotransdermal delivery system for skin anti-inflammatory purposes according to any one of claims 1-16 in the preparation of medicaments for the prevention or treatment of local skin inflammation.

18. The use according to claim 17, characterized in that, The localized skin inflammations include psoriasis, atopic dermatitis, and inflammatory vitiligo.

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