Tyrosinase-based NANO transdermal delivery system for treating vitiligo

Abstract

A tyrosinase-based transdermal delivery system for treating vitiligo is of a core-shell structure, wherein the core is tyrosinase, and the shell is a polymer shell layer. Double bonds are introduced into tyrosinase, and the surface of the protein is coated with a polymer protective layer by means of an in-situ polymerization reaction, so that the activity of tyrosinase can be effectively protected, and transdermal delivery of tyrosinase can be promoted. Moreover, by means of regulating polymerization conditions of the polymer shell, the targeted delivery of a tyrosinase nanogel to the melanocytes deep in the skin and hair follicles can be controlled, so that the in-situ synthesis of melanin is promoted. The transdermal delivery system can also load an active small molecule with an anti-inflammatory function in the center of tyrosinase, and the small molecule is released after being delivered along with the tyrosinase nanogel to an inflammatory region of vitiligo in a targeted manner, so that inflammation is relieved, and melanocytes are protected from damage.

Description

A tyrosinase-based nanotransdermal delivery system for the treatment of vitiligo Technical Field

[0001] The present invention belongs to the technical field of drug transdermal delivery systems, and in particular relates to a tyrosinase-based nano transdermal delivery system for treating vitiligo. Background Art

[0002] Vitiligo is a common pigmentary skin disease characterized by a localized loss of functional melanocytes. This disease affects over 2% of the global population, severely impacting both the physical and psychological well-being of patients. Its etiology is complex, and a rapid and effective cure for vitiligo remains under investigation. Several factors have been linked to the onset of vitiligo, including oxidative stress, the production of inflammatory mediators, and autoimmune responses. Clinical and commercial drug treatments have been developed to promote localized pigmentation in patients with vitiligo, targeting different pathogenic mechanisms. Reducing the damage to functional melanocytes in the affected area is currently the first-line treatment. For melanocytes attacked in the affected area by autoimmune responses, topical transdermal delivery of corticosteroids and calcineurin inhibitors can reduce melanocyte damage through local immune regulation. However, treatment with immunosuppressants can be associated with systemic adverse reactions and carries a high risk of symptom recurrence after discontinuation. Oxidative stress-induced damage to melanocytes in the affected area is a key pathogenic mechanism. Topical delivery of small molecule antioxidants or catalase and superoxide dismutase is also a treatment strategy, but clinical trials have shown low patient universality. Anti-inflammatory drugs typically demonstrate their ability to promote pigmentation when used in conjunction with other treatments, such as phototherapy, placing high demands on the equipment and instruments used. In addition to reducing melanocyte damage, directly promoting melanin synthesis within melanocytes is another therapeutic approach to improving pigmentation in the affected area; the delivery of tyrosinase activators is a mainstream approach. The tyrosinase catalytic step is a key step in the melanin synthesis process, and tyrosinase activators promote in situ melanin production by increasing tyrosinase activity within melanocytes in the affected area. However, activators have certain concentration requirements, and excessively high concentrations can produce side effects.

[0003] Compared with oral administration and intravenous injection, local transdermal administration has the advantages of being convenient and non-invasive, maintaining drug activity, and targeted delivery. It is especially suitable for the management of diseases such as vitiligo that require local, long-term, and multiple administrations. Therefore, drug treatment for vitiligo usually adopts transdermal delivery. However, the multi-layered and complex structure of the skin creates a high-efficiency barrier effect including physical barriers, chemical barriers, and immune barriers. Whether it is a small molecule drug or a large molecule drug, it faces the problem of low penetration efficiency and inability to reach the basal layer of the skin and the melanocytes at the bottom of the hair follicles to exert its effect when administered transdermally. Due to the efficient barrier effect of the skin, simply increasing the drug concentration is not very helpful in promoting penetration, but may cause skin irritation or allergies. In addition, most active drugs are hydrophobic substances with low solubility in traditional transdermal preparations, which seriously limits the therapeutic effect of the drug.

[0004] In the field of vitiligo treatment, the delivery technologies used in topical drug delivery preparations that have been put into clinical use are mainly penetration enhancers (such as ethanol), liposome preparations, and microneedles. However, penetration enhancers promote drug penetration by temporarily destroying the skin barrier, which has a greater irritation effect on the skin and has the risk of exacerbating the condition of the affected area for inflammatory vitiligo. After the liposomes encapsulate the drug, the size is generally greater than 200nm, and the preparations formed are in a metastable state, which is difficult to meet the requirements of long-term use and the delivery effect is not ideal. In addition, these two technologies are mainly used in the delivery of small molecule drugs such as immunosuppressants. The transdermal efficiency of active macromolecular drugs such as enzymes is not only limited, but the presence of some penetration enhancers may also cause enzyme inactivation, thereby losing the therapeutic ability for vitiligo. Microneedle technology uses a needle tip with a certain mechanical strength to puncture the skin to form a channel, thereby weakening the barrier during transdermal drug delivery. However, the treatment of vitiligo requires a long cycle and multiple administrations. Frequent use of microneedles will cause damage to the local skin and even cause inflammation. In addition, the depigmentation symptoms of vitiligo come from the damage and loss of melanocytes. Therefore, the transdermal delivery target of vitiligo is the melanocytes located in the basal layer of the skin and deep in the hair follicles. However, the existing transdermal delivery technology cannot control the transdermal depth, resulting in low treatment efficiency.

[0005] In summary, the existing treatments for vitiligo have the following disadvantages: small molecule drugs target different mechanisms, are not universal and have side effects; large molecule transdermal technology is inefficient and has side effects.

[0006] Summary of the Invention

[0007] In order to solve the defects of the existing vitiligo treatment solutions in the art, such as low universality, large side effects, low local administration efficiency and inability to target delivery, the present invention proposes a tyrosinase-based transdermal delivery system to promote in situ pigmentation. The system uses modified tyrosinase gel as a carrier and active component, and forms a tyrosinase nanogel with a polymer shell through in situ polymerization. The polymer shell can protect the activity of tyrosinase, and the surface properties and size of the polymer shell help the nanogel to efficiently penetrate the skin barrier and be delivered to the hair follicles and deep skin, thereby promoting melanin production. In addition, tyrosinase itself can be used as a carrier of anti-inflammatory small molecules. The nanogel can transport tyrosinase and its loaded small molecule active ingredients transdermally to the hair follicles and deep skin as a whole, thereby synergistically exerting the therapeutic effect of vitiligo from the two aspects of treating local inflammation and promoting in situ melanin synthesis.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] A tyrosinase-based transdermal delivery system for treating vitiligo has a core-shell structure, wherein the core is tyrosinase and the shell is a polymer shell layer.

[0010] Furthermore, the polymer can be a polymer material that is coated on the outer layer of tyrosinase through a polymerization reaction and is selected from acrylamide copolymers, vinylpyrrolidone copolymers, polyols, polylactic acid, phosphorylcholine polymers, betaine polymers, and chitosan. The polymer can be obtained by polymerizing monomers with carbon-carbon unsaturated double bonds, such as polyacrylamide and polyvinylpyrrolidone, or by polycondensation or ring-opening polymerization of monomers with different functional groups. Alternatively, a natural polymer can be coated on the surface of tyrosinase through electrostatic attraction, hydrogen bonding, intermolecular interactions, crosslinking, and the like. Alternatively, the polymer material wrapped around the outer layer of tyrosinase through electrostatic interaction is a positively or negatively charged polyelectrolyte, 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), polymethacrylamidopropyltrimethylammonium chloride, and natural or synthetic polysaccharides; and the negatively charged polyelectrolyte is selected from at least one of polyacrylic acid, polystyrene sulfonate, alginate, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, polymethacrylic acid, oxidized cellulose, carboxymethyl cellulose, polyaspartic acid, and polyglutamic acid.

[0011] Correspondingly, the polymer material wrapped in the outer layer of tyrosinase through polymerization reaction, its monomer is selected from at least one of vinyl pyrrolidone, acrylamide, N-propylene succinimide, 2-methacryloyloxyethyl phosphorylcholine, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide, vinyl pyrrolidone, polyethylene glycol methyl ether acrylate, ethylene glycol, propylene glycol, butylene glycol, and lactide.

[0012] Furthermore, in the core-shell structure, the core, i.e., the tyrosinase loaded with a small molecule having anti-inflammatory activity, has a size of 2-15 nm, preferably 5-10 nm; the polymer shell, which serves as the shell, has a thickness of 1-100 nm, preferably 5-50 nm, and more preferably 7-20 nm. Furthermore, the surface zeta potential of the tyrosinase-based transdermal delivery system is -7 mV to +7 mV, preferably a positive potential, such as +4 mV to +6 mV.

[0013] The penetration depth of the tyrosinase-based transdermal delivery system is related to the size of the nanogel, D = 97.3 + 2.05d - 0.0239d, when its surface Zeta potential is +4 to +6mV. 2 +7.08E-5d 3 ,d∈[20,150], where the unit of D is μm and the unit of d is nm.

[0014] Furthermore, in the core-shell structure, the core is a tyrosinase loaded with anti-inflammatory active small molecules, and the anti-inflammatory active small molecules are loaded inside the tyrosinase through hydrophilic and hydrophobic interactions, specifically including but not limited to at least one of cyanocalyxin, vitamin E, lycopene, astaxanthin, hesperidin, and glycyrrhizin.

[0015] The mass ratio of the anti-inflammatory active small molecule to tyrosinase is 1:1-1000, and the ratio varies depending on the different anti-inflammatory active small molecules. For example, the mass ratio of the anti-inflammatory active small molecule to tyrosinase is 1:2-10, and another example is 1:2-5.

[0016] Tyrosinase promotes melanin synthesis in melanocytes in the skin and hair follicles, requiring two conditions: it must be able to successfully penetrate the skin barrier and reach the depth of melanocytes; and it must maintain sufficient catalytic activity to catalyze the synthesis of melanin from the substrate. The polymer shell in this system performs two functions: promoting transdermal penetration and protecting tyrosinase activity, making shell thickness control particularly important. Our research shows that the permeability of protein nanogels is related to particle size; a shell that is too thin or too thick is not conducive to deep delivery of the nanogel. The protective effect of the polymer shell on tyrosinase activity comes from the shell's protection of the protein structure. A shell that is too thin has poor resistance to drastic changes in ambient temperature and pH. Tyrosinase requires contact with the substrate for catalysis, and an overly thick shell impairs substrate transport, thereby hindering tyrosinase activity. In this study, tyrosinase nanogels of various sizes were prepared, and the effects of shell thicknesses on transdermal penetration and tyrosinase activity protection were investigated. A polymer shell thickness of 7 to 20 nm was selected as the optimal thickness for vitiligo treatment.

[0017] The protein core of the tyrosinase nanogel has a hydrophobic structure. Therefore, for inflammatory vitiligo, anti-inflammatory functional small molecules can be loaded inside the tyrosinase, exerting a synergistic therapeutic effect on inflammatory vitiligo by alleviating the inflammatory response in the affected area and promoting the in situ production of melanin. In this system, the tyrosinase nanogel acts as a highly efficient transdermal delivery vehicle, delivering the anti-inflammatory small molecules by loading them inside the tyrosinase. By optimizing the design of the polymer shell of the tyrosinase nanogel and controlling the transdermal depth of the nanogel, the small molecules and the nanogel are targeted and delivered to the site of inflammation, thereby achieving a higher anti-inflammatory effect using a lower concentration of anti-inflammatory small molecules.

[0018] A second object of the present invention is to provide a method for preparing the above-mentioned tyrosinase-based transdermal delivery system, comprising the following steps:

[0019] (1) mixing a tyrosinase solution and a solution containing a small molecule having a polymerizable carbon-carbon double bond to obtain a tyrosinase with a surface modified with a polymerizable double bond;

[0020] (2) A tyrosinase solution with a surface-modified polymerizable double bond, a monomer, and a cross-linking agent are mixed, an initiator is added, and a polymerization reaction is initiated to obtain an active ingredient transdermal delivery system based on a protein carrier.

[0021] Furthermore, in step (1), the small molecule containing a polymerizable carbon-carbon double bond is selected from at least one of N-propylene succinimide and maleimide; the mass ratio of tyrosinase to the small molecule containing a polymerizable carbon-carbon double bond is 5-20:1, preferably 5-10:1. The solvent in the solution containing the small molecule containing a polymerizable carbon-carbon double bond is at least one of dimethyl sulfoxide and ethanol, and the mass concentration of the small molecule containing a polymerizable carbon-carbon double bond in the solution is 1-5wt%.

[0022] Furthermore, in step (1), the solvent of the tyrosinase solution is a phosphate buffer solution or an organic solvent; the phosphate buffer solution is NaH2PO4-Na2HPO4, with a pH of 5-8; the organic solvent is selected from at least one of ethanol, acetone, and dimethyl sulfoxide; and the concentration of protein in the protein solution is 1-100 mg / L, preferably 1-10 mg / L.

[0023] Furthermore, in step (2), the monomer is selected from at least one of acrylamide, 2-methacryloyloxyethyl phosphorylcholine, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide), N-(3-aminopropyl) methacrylamide hydrochloride, vinyl pyrrolidone, and polyethylene glycol methyl ether acrylate; preferably N-(3-aminopropyl) methacrylamide hydrochloride; the cross-linking agent is selected from at least one of N,N'-methylenebisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate, and glycerol dimethacrylate; the initiator is selected from at least one of sodium persulfate, potassium persulfate, and ammonium persulfate; the amount of initiator added is 1-5 mg of initiator per mg of tyrosine in the system; preferably, a co-initiator is also added, such as at least one of sodium bisulfite, potassium bisulfite, and tetramethylethylenediamine, and the mass ratio of the initiator to the co-initiator is 1:1-2.

[0024] Preferably, the monomer includes a certain amount of N-(3-aminopropyl) methacrylamide hydrochloride. The type of monomer determines the surface electrical properties of the polymer shell. Using N-(3-aminopropyl) methacrylamide hydrochloride as a monomer, the resulting polymer surface has a positive charge, which is conducive to interaction with lipid components in the skin, weakening the lipid bilayer's barrier effect on protein nanogels, and promoting the intercellular penetration of protein nanogels. Preferably, the monomer includes 5-20 mol% of N-(3-aminopropyl) methacrylamide hydrochloride, preferably 10-15 mol% of N-(3-aminopropyl) methacrylamide hydrochloride.

[0025] Furthermore, the molar ratio of the surface-modified polymerizable double bond tyrosinase, the monomer, and the cross-linking agent is 1:100-100000:10-10000, preferably 1:2000-20000:200-2000.

[0026] Furthermore, when the initiator is added for polymerization, the concentration of tyrosinase in the reaction system is 1-5 mg / mL, preferably 1-2 mg / mL.

[0027] In a preferred technical solution of the present invention, in step (2), the monomer is a compound of acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride, and the cross-linking agent is N,N'-methylenebisacrylamide; and the feeding ratio of tyrosinase with surface-modified polymerizable double bonds, acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, and N,N'-methylenebisacrylamide is a molar ratio of 1:4500-6600:600-800:450-660.

[0028] Furthermore, in step (2), the polymerization reaction is carried out at room temperature for 2-10 hours, such as 4-6 hours.

[0029] Preferably, for the system of tyrosinase-loaded anti-inflammatory active small molecules, the preparation method of the tyrosinase-based transdermal delivery system comprises the following steps:

[0030] (1) mixing a tyrosinase solution and a solution containing a small molecule having a polymerizable carbon-carbon double bond to obtain a tyrosinase with a surface modified with a polymerizable double bond;

[0031] (2) adding an anti-inflammatory active small molecule solution to the tyrosinase solution with a surface-modified polymerizable double bond, and mixing them uniformly to obtain a tyrosinase solution loaded with an anti-inflammatory active small molecule;

[0032] (3) A tyrosinase solution loaded with anti-inflammatory active small molecules, a monomer, and a cross-linking agent are mixed, an initiator is added, and a polymerization reaction is initiated to obtain an active ingredient transdermal delivery system based on a protein carrier.

[0033] That is, the step of loading tyrosinase with anti-inflammatory active small molecules is added. The addition of anti-inflammatory active small molecules synergizes with tyrosinase to further enhance the therapeutic effect on vitiligo.

[0034] In the anti-inflammatory active small molecule solution, the solvent is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, tetrahydrofuran, ethanol, and isopropanol, and the concentration of the anti-inflammatory active small molecule is 0.1-10 mg / L. The mass ratio of the anti-inflammatory active small molecule and the surface-modified polymerizable double bond tyrosinase (calculated as tyrosinase) is 1:1-100. The ratio of the anti-inflammatory active small molecule and tyrosinase mainly depends on the loading amount of tyrosinase on the anti-inflammatory active small molecule, and the ratio varies depending on different anti-inflammatory active small molecules. For example, the mass ratio of the anti-inflammatory active small molecule and tyrosinase is 1:2-10, for example 1:2-5.

[0035] The third object of the present invention is to provide the use of the above-mentioned tyrosinase-based transdermal delivery system in the preparation of a drug for treating vitiligo, specifically for transdermal administration to the affected area, such as in the form of ointment, patch, gel dressing, etc.

[0036] The present invention achieves the following beneficial effects through the above technical solution:

[0037] First, the present invention modifies tyrosinase with a polymerizable double-bond compound, initiating an in situ polymerization reaction on the protein surface. This results in a biocompatible, non-irritating or minimally irritating polymer layer coating the protein surface. This polymer layer effectively protects tyrosinase activity and promotes transdermal delivery of the enzyme. Furthermore, by regulating the polymerization conditions of the polymer shell (including feed and reaction time), the tyrosinase nanogel can be targeted to melanocytes deep within the skin and hair follicles, promoting in situ melanin synthesis.

[0038] 2. The transdermal delivery system of the present invention can also load active small molecules with anti-inflammatory function in the tyrosinase center. The small molecules are delivered to the inflammatory area of ​​vitiligo along with the tyrosinase nanogel and then released, thereby alleviating inflammation and protecting melanocytes from damage.

[0039] 3. The preparation method of the transdermal delivery system of the present invention is simple, has a high yield, and can be industrialized and produced on a large scale.

[0040] Fourth, the raw materials used in the present invention are all bio-friendly and can be used as pharmaceutical reagents, and therefore can be used in transdermal delivery systems of drugs and other ingredients beneficial to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 is a fitting curve of the penetration depth D and the particle size d of tyrosinase nanogel with positive surface potential at 24 hours;

[0042] Figure 2 shows the relative activities of nTYR tyrosinases of various sizes;

[0043] FIG3 is a TEM image of PAAm-nTYR@GLA obtained in Example 2;

[0044] FIG4 is an EM image of PMPC-nTYR@GLA obtained in Example 3;

[0045] Figure 5 shows the depigmentation of the back of mice during the progression of vitiligo;

[0046] Figure 6 shows the staining results of mouse skin sections. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to specific examples, but the present invention is not limited to the contents of the specification. All reagents used are commercially available reagents in the art.

[0048] Example 1

[0049] To 2 mL of 1 mg / mL tyrosinase solution, 40 mg of 1 wt% N-propylene succinimide solution (a small molecule with a polymerizable carbon-carbon double bond) was added. The solvent was dimethyl sulfoxide, and the mixture was thoroughly mixed and reacted for 2 h to obtain tyrosinase (aTYR) with a surface modified with a polymerizable double bond.

[0050] aTYR, acrylamide (monomer), N-(3-aminopropyl)methacrylamide hydrochloride (monomer), and N,N'-methylenebisacrylamide (cross-linker) were mixed according to the specified molar ratios. The protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. An initiator (3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per mg of protein) was then added. In situ free radical polymerization was initiated at room temperature. After the reaction time was specified, polyacrylamide-tyrosinase nanogels (nTYR) of various sizes were obtained. The preparation conditions and the measured final sizes and surface zeta potentials are shown in Table 1.

[0051] Table 1 Preparation conditions, final sizes and surface potentials of tyrosinase nanogels of various sizes

[0052] As can be seen from Table 1, increasing the ratio of monomer to protein and increasing the polymerization time will increase the particle size of the obtained polyacrylamide-tyrosinase nanogel (nTYR), and increasing the proportion of APM in the monomer will make the Zeta potential of nTYR more positive.

[0053] Application Example 1: Transdermal experiment of tyrosinase nanogels of various sizes

[0054] 4 mL of 1 mg / mL tyrosinase nanogels of varying sizes from Example 1 were mixed with 1 mL of 1 mg / mL fluorescein isothiocyanate and reacted overnight. The mixture was dialyzed overnight in a 14,000 Da dialysis bag to remove unreacted small molecules, yielding fluorescently labeled nTYR. Fresh pigskin was washed with PBS and mounted, stratum corneum facing upward, in a transdermal diffusion apparatus and incubated at 37°C. 0.5 mL of a 1 mg / mL fluorescently labeled tyrosinase nanogel solution was dripped onto the pigskin tissue. After 24 hours, cryosections were performed and the nanogel penetration observed under a confocal microscope. The penetration depth is statistically reported in Table 2.

[0055] Table 2 24-hour skin penetration depth of tyrosinase nanogels of various sizes

[0056] By comparing the penetration depths of tyrosinase nanogels of different sizes with positive surface potential (about +5mV), it was found that when the particle size was between 20nm and 150nm, the protein nanogels were able to 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 tyrosinase nanogels larger than 200nm mostly stayed in the stratum corneum above the epidermis. The penetration depths of nTYR-20+, nTYR-50+, nTYR-100+, and nTYR-150+ were fitted. Figure 1 is a fitting curve of the penetration depth D and particle size d of tyrosinase nanogels with positive surface potential at 24 hours. The fitted formula is as follows: D = 97.3 + 2.05d - 0.0239d 2 +7.08E-5d 3 ,d∈[20,150], where the unit of D is μm and the unit of d is nm.

[0057] This expression can be used to preliminarily predict the penetration capacity of tyrosinase nanogels with a positive surface potential within a particle size range of 20-150 nm. In the treatment of vitiligo, the drug delivery targets are located at the junction of the epidermis and dermis, as well as melanocytes deep within the hair follicles. According to the formula, tyrosinase nanogels with sizes of approximately 20-45 nm and 80-130 nm ultimately penetrate to a depth near the location of melanin, meeting delivery requirements. Taking into account the therapeutic effect, the tyrosinase-based transdermal delivery system of the present invention, also known as the tyrosinase nanogel, is preferably sized between 20-45 nm.

[0058] Application Example 2: Activity retention of tyrosinase nanogels in a series of sizes

[0059] The catalytic activity of nTYR was determined using a UV spectrophotometer. To the reference cell, 1600 μL of pH 6 PBS buffer and 1400 μL of 1 g / L L-tyrosine solution were added. To the sample cell, 1550 μL of pH 6 PBS buffer and 1400 μL of 1 g / L L-tyrosine solution were added. Finally, 50 μL of nTYR solution or untreated tyrosinase solution was added. The cells were placed in a UV spectrophotometer, and the absorbance of the sample cell at 317 nm was recorded over time for the first 20 minutes. The slope of the fitted line represents the relative tyrosinase activity. The results are shown in Figure 2 and Table 3. This indicates that the polymer shell of tyrosinase nanogels larger than 50 nm may be too thick, impairing the contact between tyrosinase and substrate, or that the polymerization conditions may have caused a loss of tyrosinase activity. The activity loss of tyrosinase nanogels smaller than 50 nm was within an acceptable range.

[0060] Table 3 Relative activities of tyrosinase enzymes of different sizes of nTYR

[0061] Application Example 3 Thermal Stability Test of Tyrosinase Nanogels of Various Sizes

[0062] The catalytic activity of nTYR was determined using a UV spectrophotometer. 1600 μL of pH 6 PBS buffer and 1400 μL of 1 g / L L-tyrosine solution were added to the reference cell. 1550 μL of pH 6 PBS buffer and 1400 μL of 1 g / L L-tyrosine solution were added to the sample cell. Finally, 50 μL of nTYR solution after different heat treatments or original tyrosinase solution were added. The cells were placed in a UV spectrophotometer. The absorbance of the sample cell at 317 nm was recorded over time for the first 20 minutes. The slope of the fitted line represents the relative tyrosinase activity. The activity retention of the enzyme after different heat treatments relative to the untreated state was calculated, and the results are shown in Table 4.

[0063] Table 4 Relative activity of series nTYR

[0064] It was found that the original tyrosinase was significantly inactivated at 60°C, while nTYR-20 and nTYR-50 retained 40% of their activity after treatment at 60°C for 1 hour, indicating the good thermal protection effect of the polymer shell.

[0065] Application Example 4: Acid-base stability test of tyrosinase nanogels of various sizes

[0066] The catalytic activity of nTYR was determined using a UV spectrophotometer. 1600 μL of PBS buffer at a specified pH and 1400 μL of 1 g / L L-tyrosine solution were added to the reference cell. 1550 μL of PBS buffer at a specified pH and 1400 μL of 1 g / L L-tyrosine solution were added to the sample cell. Finally, 50 μL of nTYR solution after various heat treatments or original tyrosinase solution were added. The cells were placed in a UV spectrophotometer. The absorbance of the sample cell at 317 nm was recorded over time for the first 20 minutes. The slope of the fitted line represented the relative tyrosinase activity. The enzyme activity retention at different pH values ​​relative to pH 6 was calculated. The results are shown in Table 5.

[0067] Table 5 Relative activity of series nTYR

[0068] It was found that the original tyrosinase had good activity only in a weakly acidic environment (pH around 6), while nTYR-20 and nTYR-50 could exert efficient catalytic effects in a wider pH range, indicating that the polymer shell provides nTYR with better acid and alkali resistance.

[0069] Example 2 Preparation of tyrosinase nanogel loaded with blue calyx A:

[0070] (1) To 2 mL of a 1 mg / mL tyrosinase solution, 40 mg of a 1 wt% N-propylene succinimide solution (solvent: dimethyl sulfoxide) was added, and the mixture was thoroughly mixed and reacted for 2 h to obtain a tyrosinase (aTYR) surface-modified with a polymerizable double bond.

[0071] (2) Take 2 mL of a 1 mg / mL surface double bond-modified tyrosinase solution, add 0.5 mL of a 2 mg / mL DMSO solution of cyanocalyxin A dropwise while stirring at 300 r / min, and mix thoroughly to obtain cyanocalyxin A-loaded tyrosinase (aTYR@GLA).

[0072] (3) aTYR@GLA, acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride and N,N'-methylenebisacrylamide were mixed at a molar ratio of 1:6000:600:600, and 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 mg of protein. In situ free radical polymerization was initiated at room temperature. After 4 h of reaction, polyacrylamide-tyrosinase nanogel loaded with blue calyx methyl was obtained (PAAm-nTYR@GLA).

[0073] 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 6.1 mg / mL, which was refrigerated for storage.

[0074] Example 3 Preparation of polyphosphorylcholine-tyrosinase nanogel loaded with cyanocalyxin A:

[0075] (1) To 2 mL of a 1 mg / mL tyrosinase solution, 40 mg of a 1 wt% N-propylene succinimide solution (solvent: dimethyl sulfoxide) was added, and the mixture was thoroughly mixed and reacted for 2 h to obtain a tyrosinase (aTYR) surface-modified with a polymerizable double bond.

[0076] (2) Take 2 mL of a 1 mg / mL surface double bond-modified tyrosinase solution, add 0.5 mL of a 2 mg / mL DMSO solution of cyanocalyxin A dropwise while stirring at 300 r / min, and mix thoroughly to obtain cyanocalyxin A-loaded tyrosinase (aTYR@GLA).

[0077] (3) aTYR@GLA, 2-methacryloyloxyethyl phosphorylcholine, and N,N'-methylenebisacrylamide were mixed in a mass ratio of 1:40:4, and 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 mg of protein. In situ free radical polymerization was initiated at room temperature. After 4 h of reaction, polyphosphorylcholine-tyrosinase nanogel loaded with blue calyx methyl was obtained (PMPC-nTYR@GLA).

[0078] 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 5.7 mg / mL, which was refrigerated for storage.

[0079] Dynamic light scattering (DLS) was used to characterize the size of the nanogels. A 1 mg / mL solution of the protein nanogels from Examples 2 and 3 was added to a sample cell. The particle size of the complexes was measured at room temperature using a Malvern Nano Zs90 nanometer. The results are summarized in Table 6.

[0080] Table 6 Tyrosinase nanogel particle size and Zeta potential

[0081] Transmission electron microscopy (TEM) was used to characterize the morphology of the nanogels. The morphology and dispersion of PAAm-nTYR@GLA under TEM are shown in Figure 3. The morphology and dispersion of PMPC-nTYR@GLA under TEM are shown in Figure 4. It can be seen that the particle size of the tyrosinase nanogels is consistent with the DLS results, and the overall shape is nearly spherical, indicating good dispersion.

[0082] Application Example 5

[0083] 2 mL of the 5 mg / mL tyrosinase nanogel prepared in Examples 2 and 3 was placed in a 3000 molecular weight cutoff dialysis bag. The bag was then immersed in a pH 6 phosphate buffer solution, and the entire system was shaken at 37°C. The sustained release period was 48 hours. Samples were taken regularly over the 48 hours and the concentration of the loaded small molecule drug was measured using a UV spectrophotometer. The sustained release of the small molecule drug from the composite system was calculated. The results are summarized in Table 7.

[0084] Table 7 Release of small molecule drugs by tyrosinase nanogel

[0085] Application Example 6: Verification of the effectiveness of functional small molecule ingredients

[0086] Determination of the anti-inflammatory ability of polyacrylamide-tyrosinase nanogel (PAAm-nTYR@GLA) of Example 2 and polyphosphocholine-tyrosinase nanogel (PMPC-nTYR@GLA) of Example 3: The NO concentration of inflammatory cells was detected using the Griess kit. HaCaT cells (1*10 5 After 12 hours of complete cell attachment, the original culture medium was removed and 100 μL of serum-free culture medium was added to all groups, 10 μL of serum-free culture medium was added to the negative control group, and 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 2 hours of co-incubation, the original culture medium was removed from all groups, 110 μL of fresh culture medium was added to the negative control group, and 100 μL of fresh culture medium and 10 μL of 10 ng / mL interleukin-1β were added to the remaining groups. After 24 hours of co-incubation, the assay was performed according to the Griess kit instructions. The results are summarized in Table 8. It was found that both PMPC-nTYR@GLA and PAAm-nTYR@GLA had good anti-inflammatory properties, and this property increased with increasing concentration. The positive charge on the surface of PAAm-nTYR@GLA makes it more efficient in cellular endocytosis, and it has better anti-inflammatory ability than the negatively charged PMPC-nTYR@GLA at the same concentration.

[0087] Table 8 The intracellular anti-inflammatory ability of tyrosinase nanogels increases with increasing concentration

[0088] Application Example 7 Melanin-producing Cell Experiment

[0089] The effect of polyphosphorylcholine-tyrosinase nanogel (PMPC-nTYR@GLA) on melanin synthesis in mouse melanoma cells B16F10 was determined by inoculating B16F10 cells (1*10 5After 12 hours of complete cell attachment, the original culture medium was removed from all groups and 100 μL of serum-free culture medium was added. The control group added 10 μL of PBS buffer, the low-concentration experimental group added 10 μL of 0.01 mg / mL nanogel solution, the medium-concentration experimental group added 10 μL of 0.02 mg / mL nanogel solution, and the high-concentration experimental group added 10 μL of 0.04 mg / mL nanogel solution. After 48 hours of co-incubation, cells from all groups were collected and dissolved in 1 mL of NaOH (1 mol / L, containing 10% DMSO) solution, ultrasonically disrupted for 30 minutes, in an 80°C water bath for 30 minutes, and centrifuged at 3000 r / min for 15 minutes. The supernatant was taken and the absorbance was measured at 450 nm. The results are shown in Table 9. It was found that PMPC-nTYR@GLA has a good ability to promote melanin production, and this ability increases with increasing concentration.

[0090] Table 9 The ability of tyrosinase nanogel to promote melanin production increases with increasing concentration

[0091] Application Example 8: Transdermal Animal Experiment on Vitiligo Model

[0092] The ability of PAAm-nTYR@GLA and PMPC-nTYR@GLA transdermal delivery to promote pigmentation in Examples 2 and 3 was investigated using a vitiligo model mouse model. Mice were randomly divided into 5 groups, and the back was uniformly depilated, one of which was used as a normal control, and the remaining 4 groups (positive control, PBS control group, PAAm-nTYR@GLA group and PMPC-nTYR@GLA group) were used for vitiligo modeling. After successful modeling, the corresponding drug or PBS buffer (positive control group) was applied once every two days in the depilatory area, and the back hair was collected and photographed before each administration to record the overall pigmentation. After 20 days of continuous administration, the skin of the back of the mouse was taken, and the overall pigmentation of the back of the mouse was observed to characterize the vitiligo morbidity, as shown in Figure 5. It was found that the untreated positive control group had a significantly worsened vitiligo morbidity in the back after 20 days, while the group treated with tyrosinase nanogel had a weaker worsening of vitiligo symptoms in the back than the PBS group, and the therapeutic effect of the PAAm-nTYR@GLA group with positive surface charge was better than the PMPC-nTYR@GLA group with negative surface potential.

[0093] Table 10 Statistics on the growth of overall decolorization color difference and decolorization area

[0094] Skin tissue sections were taken to observe hair follicle development and pigmentation, with the results shown in Figure 6 and Table 11. The positive control and PBS groups showed fewer hair follicles and poor development, with no pigmented hair shafts within the follicles. In the group treated with tyrosinase nanogel, hair follicle development improved compared to the PBS group. A comparison of the PAAm-nTYR@GLA and PMPC-nTYR@GLA groups revealed more pronounced pigmentation within the hair follicles in the PAAm-nTYR@GLA group. This suggests that the positive surface charge of PAAm-nTYR@GLA enhances transdermal delivery, enabling targeted delivery of tyrosinase to melanocytes, promoting melanin production and significantly improving vitiligo symptoms.

[0095] Table 11 Statistics of hair follicle length and number

Claims

1. A tyrosinase-based transdermal delivery system for treating vitiligo, characterized in that: It is a core-shell structure, the core is tyrosinase or tyrosinase adsorbed with small molecules with anti-inflammatory activity, and the shell is a polymer shell layer.

2. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that: The polymer is a polymer material wrapped around the outer layer of tyrosinase through a polymerization reaction, including at least one of acrylamide copolymer, vinylpyrrolidone copolymer, polyol, polylactic acid, phosphorylcholine polymer, betaine polymer, and chitosan, wherein the polymerization reaction is obtained by polymerization of monomers with carbon-carbon unsaturated double bonds, or condensation polymerization or ring-opening polymerization of monomers with different functional groups; Alternatively, the polymer material wrapped around the outer layer of tyrosinase by electrostatic interaction is a positively or negatively charged polyelectrolyte, 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; and the negatively charged polyelectrolyte is selected from at least one of polyacrylic acid, polystyrene sulfonate, alginate, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, polymethacrylic acid, oxidized cellulose, carboxymethyl cellulose, polyaspartic acid and polyglutamic acid.

3. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 2, characterized in that: The polymer material wrapped around the outer layer of tyrosinase by polymerization reaction has a monomer selected from at least one of vinyl pyrrolidone, acrylamide, 2-methacryloyloxyethyl phosphorylcholine, ethylene glycol, propylene glycol, butylene glycol and lactide.

4. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that: In the core-shell structure, the size of the core, i.e., the tyrosinase loaded with small molecules having anti-inflammatory activity, is 5-15 nm; the polymer shell layer, which is the shell, has a thickness of 1-100 nm.

5. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that: In the core-shell structure, the size of the core, i.e., the tyrosinase loaded with small molecules having anti-inflammatory activity, is 5-10 nm; the thickness of the polymer shell layer, which is the shell, is 5-50 nm.

6. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that: The surface zeta potential of the tyrosinase-based transdermal delivery system is +4 mV to +6 mV.

7. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that: In the core-shell structure, the anti-inflammatory active small molecules are loaded inside the tyrosinase through hydrophilic and hydrophobic interactions, including at least one of cyanocalyxin A, vitamin E, lycopene, astaxanthin, hesperidin, and glabridin; The mass ratio of the anti-inflammatory active small molecule to tyrosinase is 1:1-1000.

8. A method for regulating the transdermal penetration depth of the transdermal delivery system of tyrosinase according to claim 6, characterized in that: The surface Zeta potential is +4 to +6 mV, and the penetration depth is regulated by adjusting the size d of the transdermal delivery system. The penetration depth D and the size d of the transdermal delivery system satisfy the following formula: D=97.3+2.05d-0.0239d 2 +7.08E-5d 3 ,d∈[20,150], where D is in μm and d is in nm; the method is for non-therapeutic / diagnostic purposes.

9. The method for preparing the tyrosinase-based transdermal delivery system according to claim 1, characterized in that: The following steps are involved: (1) mixing a tyrosinase solution and a solution containing a small molecule having a polymerizable carbon-carbon double bond to obtain a tyrosinase with a surface modified with a polymerizable double bond; (2) A tyrosinase solution with a surface-modified polymerizable double bond, a monomer, and a cross-linking agent are mixed, an initiator is added, and a polymerization reaction is initiated to obtain an active ingredient transdermal delivery system based on a protein carrier.

10. The preparation method according to claim 9, characterized in that: In step (1), the small molecule containing a polymerizable carbon-carbon double bond is selected from at least one of N-propylene succinimide and maleimide; and the mass ratio of tyrosinase to the small molecule containing a polymerizable carbon-carbon double bond is 5-20:

1.

11. The preparation method according to claim 10, characterized in that: In step (1), the mass ratio of tyrosinase to small molecules containing polymerizable carbon-carbon double bonds is 5-10:1, the solvent of the tyrosinase solution is a phosphate buffer solution or an organic solvent; the solute of the phosphate buffer solution is NaH2PO4-Na2HPO4, and the pH is 5-8; the organic solvent is selected from at least one of ethanol, acetone, and dimethyl sulfoxide; and the concentration of protein in the protein solution is 1-100 mg / L.

12. The preparation method according to claim 9, characterized in that: In step (2), the monomer is selected from at least one of acrylamide, 2-methacryloyloxyethyl phosphorylcholine, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide), N-(3-aminopropyl) methacrylamide hydrochloride, vinyl pyrrolidone, and polyethylene glycol methyl ether acrylate; the cross-linking agent is selected from at least one of N,N'-methylenebisacrylamide, ethylene glycol diacrylate, polyethylene glycol diacrylate, and glycerol dimethacrylate; the initiator is selected from at least one of sodium persulfate, potassium persulfate, and ammonium persulfate; and the amount of initiator added is 1-5 mg of initiator per mg of tyrosine in the system.

13. The preparation method according to claim 2, characterized in that: The small molecule containing a polymerizable carbon-carbon double bond is selected from at least one of N-propylene succinimide and maleimide; the mass ratio of tyrosinase to the small molecule containing a polymerizable carbon-carbon double bond is 5-20:1; In step (2), the monomer is N-(3-aminopropyl) methacrylamide hydrochloride; at least one co-initiator selected from sodium bisulfite, potassium bisulfite, and tetramethylethylenediamine is added, and the mass ratio of the initiator to the co-initiator is 1:1-2.

14. The preparation method according to claim 9, characterized in that: In step (2), the monomers include 5-20 mol% of N-(3-aminopropyl)methacrylamide hydrochloride.

15. The preparation method according to claim 9, characterized in that: In step (2), the molar ratio of tyrosinase with surface-modified polymerizable double bonds, monomer, and cross-linking agent is 1:2000-20000:200-2000.

16. The preparation method according to claim 9, characterized in that: In step (2), the monomer is a compound of acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride, and the cross-linking agent is N,N'-methylenebisacrylamide; and the feed ratio of tyrosinase with surface-modified polymerizable double bonds, acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, and N,N'-methylenebisacrylamide is 1:4500-6600:600-800:450-660.

17. The preparation method according to claim 9, characterized in that: In step (2), the polymerization reaction is carried out at room temperature for 2-10 hours.

18. The method for preparing the tyrosinase-based transdermal delivery system according to claim 9, characterized in that: The following steps are involved: (1) mixing a tyrosinase solution and a solution containing a small molecule having a polymerizable carbon-carbon double bond to obtain a tyrosinase with a surface modified with a polymerizable double bond; (2) adding an anti-inflammatory small molecule solution to the tyrosinase solution with surface-modified polymerizable double bonds, and mixing them evenly to obtain a tyrosinase solution loaded with anti-inflammatory small molecules; (3) A tyrosinase solution loaded with anti-inflammatory active small molecules, a monomer, and a cross-linking agent are mixed, an initiator is added, and a polymerization reaction is initiated to obtain an active ingredient transdermal delivery system based on a protein carrier.

19. The preparation method according to claim 18, characterized in that: In the anti-inflammatory active small molecule solution, the solvent is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, tetrahydrofuran, ethanol, and isopropanol, and the concentration of the anti-inflammatory active small molecule is 0.1-10 mg / mL; the mass ratio of the anti-inflammatory active small molecule to the surface-modified polymerizable double bond tyrosinase (calculated as tyrosinase) is 1:1-100.

20. Use of the tyrosinase-based transdermal delivery system according to claim 1 in the preparation of a drug for treating vitiligo.

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