Tyrosinase-based nano transdermal delivery system for treating leucoderma

Abstract

The invention provides a tyrosinase-based transdermal delivery system for treating leucoderma, which is of a core-shell structure, the core is tyrosinase, and the shell is a polymer shell layer. According to the invention, double bonds are formed on tyrosinase, and the surface of protein is coated with a polymer protection layer through an in-situ polymerization reaction, so that the activity of the tyrosinase can be effectively protected, and the transdermal delivery of the tyrosinase can be promoted; and the tyrosinase nanogel can be controlled to be delivered to melanocytes deep in skin and hair follicles in a targeted manner by regulating and controlling polymerization conditions of a polymer shell, so that in-situ synthesis of melanin is promoted. According to the transdermal delivery system, active small molecules with an anti-inflammatory function can be loaded at the center of the tyrosinase, and the small molecules are delivered to an inflammatory area of vitiligo in a targeted manner along with the tyrosinase nanogel and then are released, so that inflammation is relieved, and melanocytes are protected from being damaged.

Description

Technical Field

[0001] The 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 pigmented skin disease characterized by the loss of local functional melanocytes. This type of disease affects more than 2% of the world's population, causing serious impacts on the patient's physical and mental health, and its etiology is relatively complex, and a rapid and effective cure for vitiligo is still under study. Several causes have been proven to be associated with the onset of vitiligo, including oxidative stress, the production of inflammatory mediators, and autoimmune responses. In response to different pathogenic mechanisms, clinical and commercial drug treatments have been developed to promote local pigmentation in patients with vitiligo. Reducing damage to functional melanocytes in the affected area is currently the first-line treatment. In response to the attack of melanocytes in the affected area in an autoimmune response, local transdermal delivery of corticosteroids and calcineurin inhibitors can reduce melanocyte damage through local immunomodulation. However, treatment with immunosuppressants may cause systemic adverse reactions, and there is a higher risk of recurrence of symptoms after discontinuation of the drug. [1] Oxidative stress is an important pathogenic mechanism that causes the destruction of melanocytes in the affected area. Local delivery of small molecule antioxidants or catalase and superoxide dismutase is also a treatment strategy, but this method has shown low patient universality in clinical trials. Anti-inflammatory drugs are usually used in combination with other treatments such as phototherapy to show the ability to promote pigmentation, and they have high requirements for equipment and instruments. [2] . In addition to reducing melanocyte damage, directly promoting melanin synthesis in melanocytes is another treatment idea to improve pigmentation in the affected area; among them, delivering tyrosinase activators is the mainstream method. The catalytic step of tyrosinase is a key step in the melanin synthesis process. Tyrosinase activators promote in situ melanin production by increasing tyrosinase activity in melanocytes in the affected area. However, activators have certain requirements for the concentration of use, and too high a concentration of activators will produce side effects.

[0003] Compared with oral administration and intravenous injection, local transdermal administration has the advantages of convenience, non-invasiveness, 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 for administration. However, the multi-layer complex structure of the skin is composed of efficient barrier functions including physical barriers, chemical barriers, and immune barriers. Whether it is a small molecule drug or a macromolecule 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 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 will cause skin irritation or allergies. In addition, most active drugs are hydrophobic substances, and their solubility in traditional transdermal preparations is low, 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, the penetration enhancer promotes drug penetration by temporarily destroying the skin barrier, which has a greater irritating effect on the skin, and has a greater risk of exacerbating the condition of the affected area for inflammatory vitiligo. After the liposome encapsulates the drug, the size is generally greater than 200nm, and the preparation formed is in a metastable state, which is difficult to meet the requirements for 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, etc., and 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 ability to treat 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 period and multiple administrations. Frequent use of microneedles will cause local skin damage 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 has low efficiency and side effects. Summary of the invention

[0006] In order to solve the defects of the prior art in treating vitiligo, 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 for promoting 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 layers, thereby promoting melanin production. In addition, tyrosinase itself can be used as a carrier of anti-inflammatory small molecules, and the nanogel can transport tyrosinase and its loaded small molecule active ingredients through the skin as a whole to the hair follicles and deep skin layers, thereby synergistically exerting the therapeutic effect of vitiligo from the two aspects of treating local inflammation and promoting in situ melanin synthesis.

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

[0008] 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.

[0009] Furthermore, the polymer is selected from acrylamide copolymer, vinylpyrrolidone copolymer, polyol, polylactic acid, phosphorylcholine polymer, betaine polymer, chitosan. The polymer can be obtained by polymerization of 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; or it can be a natural polymer coated on the surface of tyrosinase by electrostatic attraction, hydrogen bonding, intermolecular interaction, cross-linking, etc.

[0010] 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-acrylsuccinimide, 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.

[0011] 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 such as chitosan; 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.

[0012] Furthermore, in the core-shell structure, the size of the core, i.e., the tyrosinase loaded with small molecules having anti-inflammatory activity, is 2-15 nm, preferably 5-10 nm; the thickness of the polymer shell layer as the shell is 1-100 nm, preferably 5-50 nm, 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 when its surface Zeta potential is +4 to +6 mV. 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.

[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 glabridin.

[0015] The mass ratio of the anti-inflammatory active small molecule to tyrosinase is 1:1-1000, and the ratio varies according to 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 for example, 1:2-5.

[0016] Tyrosinase promotes melanin synthesis in melanocytes in the skin and hair follicles and needs to meet two conditions: it can smoothly penetrate the skin barrier and penetrate to the depth of melanocytes; it can maintain sufficient catalytic activity and catalyze the synthesis of melanin by substrate. The polymer shell has two functions in this system, namely promoting transdermal effect and protecting tyrosinase activity, so the thickness regulation of the shell is particularly important. According to our research, the permeability of protein nanogel is related to the particle size. Too thin or too thick shell is not conducive to the deep delivery of nanogel; the protective effect of polymer shell on tyrosinase activity comes from the protection of protein structure by shell, and the ability of too thin shell to resist drastic changes in ambient temperature and pH is poor; and the premise for tyrosinase to play a catalytic role is to contact with the substrate, and too thick shell affects the transmission of the substrate, thereby hindering tyrosinase from playing an active role. In the present invention, a series of tyrosinase nanogels of different sizes are prepared, and the effects of shells of different thicknesses on transdermal depth and tyrosinase activity protection are investigated, and the thickness of the polymer shell suitable for vitiligo treatment is 7 to 20nm.

[0017] The protein core of tyrosinase nanogel has a hydrophobic structure. Therefore, for inflammatory vitiligo, anti-inflammatory functional small molecules can be loaded inside tyrosinase to play a synergistic therapeutic role in inflammatory vitiligo from the two aspects of reducing the inflammatory response of the affected area and promoting the in situ production of melanin. In this system, tyrosinase nanogel is used as an efficient transdermal delivery carrier, and anti-inflammatory small molecules are loaded inside tyrosinase for overall delivery. By optimizing the design of the polymer shell of tyrosinase nanogel and controlling the transdermal depth of nanogel, small molecules and nanogel are targeted and delivered to the location of inflammation, thereby achieving a higher anti-inflammatory effect using lower concentrations of anti-inflammatory small molecules.

[0018] The 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. In the solution containing the small molecule containing a polymerizable carbon-carbon double bond, the solvent 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 NaH 2 PO 4 -Na 2 HPO 4 , pH is 5-8; the organic solvent is selected from at least one of ethanol, acetone, and dimethyl sulfoxide; 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 carries a positive charge, which is conducive to interacting with the lipid components in the skin, weakening the barrier effect of the lipid bilayer on the protein nanogel, and promoting the permeation of the protein nanogel cell pathway. 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 feed 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 with 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 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;

[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 small molecules is added. The addition of anti-inflammatory 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 (measured in tyrosinase) is 1:1-100. The ratio of the anti-inflammatory active small molecule to tyrosinase mainly depends on the loading amount of tyrosinase on the anti-inflammatory active small molecule, and the ratio varies according to different anti-inflammatory active small molecules. For example, the mass ratio of the anti-inflammatory active small molecule to 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, transdermal administration to the affected area, such as applying ointment, patch, gel dressing, etc.

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

[0037] 1. The present invention modifies a polymerizable double bond compound on tyrosinase, initiates a polymerization reaction in situ on the protein surface through a polymerization reaction, and coats the protein surface with a polymer protective layer, which is bio-friendly and has no or little irritation to the skin. The polymer layer can effectively protect the activity of tyrosinase and promote the transdermal delivery of tyrosinase, and by regulating the polymerization conditions of the polymer shell (including feeding and reaction time, etc.), the targeted delivery of tyrosinase nanogel to melanocytes deep in the skin and hair follicles can be controlled to promote the in situ synthesis of melanin.

[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 relieving 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 mass-produced.

[0040] Fourth, the raw materials used in the present invention are all bio-friendly and can be used as drug 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] Figure 1 It is the fitting curve of the penetration depth D and particle size d of tyrosinase nanogel with positive surface potential at 24 hours.

[0042] Figure 2 is the relative activity of tyrosinase activity of nTYR of different sizes.

[0043] Figure 3This is the TEM image of PAAm-nTYR@GLA obtained in Example 2.

[0044] Figure 4 This is the EM image of PMPC-nTYR@GLA obtained in Example 3.

[0045] Figure 5 This is the depigmentation on the back of mice during the development stage of vitiligo.

[0046] Figure 6 This is the staining result of mouse skin sections. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with specific examples, but 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 was added, the solvent was dimethyl sulfoxide, and the mixture was thoroughly mixed and reacted for 2 h to obtain tyrosinase (aTYR) with surface modified with polymerizable double bonds.

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

[0051] Table 1 Preparation conditions and final sizes and surface potentials of tyrosinase nanogels of a series of sizes

[0052]

[0053]

[0054] It can be seen from Table 1 that increasing the amount ratio of monomer to protein and increasing the polymerization time will increase the particle size of the obtained polyacrylamide-tyrosinase nanogel (nTYR), and increasing the proportion of APM in the monomer will make the Zeta potential of nTYR more positive.

[0055] Application Example 1: Transdermal experiment of tyrosinase nanogels of various sizes

[0056] 4 mL of 1 mg / mL tyrosinase nanogel nTYR of different sizes in Example 1 was mixed with 1 mL of 1 mg / mL fluorescein isothiocyanate and reacted overnight, and dialyzed overnight in a dialysis bag with a molecular weight cutoff of 14000 Da to remove unreacted small molecules to obtain fluorescently labeled nTYR. Fresh pigskin was washed with PBS, and the stratum corneum was fixed upward in a transdermal diffusion instrument and kept warm at 37°C. 0.5 mL of 1 mg / mL fluorescently labeled tyrosinase nanogel solution was dripped on the pigskin tissue, and frozen sections were performed after 24 hours. The penetration of the nanogel was observed under a confocal microscope, and the results of the statistical penetration depth are shown in Table 2.

[0057] Table 2 24-hour skin penetration depth of tyrosinase nanogels of various sizes

[0058] Sample name nTYR-20- nTYR-50- nTYR-100- nTYR-150- nTYR-200- Transdermal penetration depth (μm) 79.3±13.1 85.4±9.9 77.6±11.5 69.8±10.2 56.1±7.9 Sample name nTYR-20+ nTYR-50+ nTYR-100+ nTYR-150+ nTYR-200+ Transdermal penetration depth (μm) 128.5±7.8 159.5±11.1 113.6±9.9 99.7±8.5 58.4±6.3

[0059] 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 nanogel could penetrate the epidermis and reach the vicinity of the dermis within 24 hours, and as the gel particle size increased, the penetration depth first increased and then decreased; while 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 the fitting curve of the penetration depth D and particle size d of tyrosinase nanogel with positive surface potential at 24 hours. The fitting formula is as follows:

[0060] 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.

[0061] The expression can be used to preliminarily predict the penetration ability of tyrosinase nanogels with a particle size of 20-150nm and a positive surface potential. In the treatment of vitiligo, the drug delivery target is located at the junction of the epidermis and the dermis and the melanocytes deep in the hair follicles. According to the formula, the final penetration depth of tyrosinase nanogels with a size of about 20-45nm and 80-130nm is near the location of melanin, which meets the delivery requirements. Taking into account the therapeutic effect, the tyrosinase-based transdermal delivery system of the present invention, which can also be referred to as tyrosinase nanogel, is preferably 20-45nm in size.

[0062] Application Example 2 Series Size Tyrosinase Nanogel Activity Retention

[0063] The catalytic activity of nTYR was determined by UV spectrophotometer. 1600 μL of pH 6 PBS buffer and 1400 μL of 1 g / L L-tyrosine solution were added to the reference pool; 1550 μL of pH 6 PBS buffer and 1400 μL of 1 g / L L-tyrosine solution were added to the sample pool, and finally 50 μL of nTYR solution or untreated tyrosinase solution was added. The sample pool was placed in a UV spectrophotometer, and the absorbance of the sample pool at 317 nm was recorded over time for the first 20 minutes. The slope of the fitted straight line was taken to represent the relative activity of tyrosinase. The results are shown in Figure 2. Figure 2 As shown in Table 3. This indicates that the polymer shell of tyrosinase nanogels larger than 50 nm is too thick, which will affect the contact between tyrosinase and substrate, or the polymerization conditions cause the loss of tyrosinase activity. The activity loss of tyrosinase nanogels smaller than 50 nm is within an acceptable range.

[0064] Table 3 Relative activities of tyrosinase enzymes of different sizes of nTYR

[0065]

[0066] Application Example 3 Thermal Stability Test of Series-Sized Tyrosinase Nanogels

[0067] The catalytic activity of nTYR was determined by ultraviolet spectrophotometer. 1600 μL of PBS buffer with a pH of 6 and 1400 μL of L-tyrosine solution with a concentration of 1 g / L were added to the reference pool; 1550 μL of PBS buffer with a pH of 6 and 1400 μL of L-tyrosine solution with a concentration of 1 g / L were added to the sample pool, and finally 50 μL of nTYR solution or original tyrosinase solution after different heat treatments were added, and then placed in an ultraviolet spectrophotometer, and the absorbance of the sample pool at 317 nm was recorded over time for the first 20 minutes. The slope of the fitted straight line was taken to represent the relative activity of tyrosinase. The activity retention of the enzyme after different heat treatments was calculated relative to that without heat treatment, and the results are shown in Table 4.

[0068] Table 4 Relative activity of series nTYR

[0069]

[0070]

[0071] 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 being treated at 60°C for 1 hour, indicating the good thermal protection effect of the polymer shell.

[0072] Application Example 4 Acid-base stability test of tyrosinase nanogels of series size

[0073] The catalytic activity of nTYR was determined by ultraviolet 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 pool; 1550 μL of PBS buffer at a specified pH and 1400 μL of 1 g / L L-tyrosine solution were added to the sample pool, and finally 50 μL of nTYR solution or original tyrosinase solution after different heat treatments was added, and the sample pool was placed in an ultraviolet spectrophotometer, and the absorbance of the sample pool at 317 nm was recorded over time for the first 20 minutes. The slope of the fitted straight line was taken to represent the relative activity of tyrosinase, and the retention of the activity of the enzyme at different pH relative to pH = 6 was calculated. The results are shown in Table 5.

[0074] Table 5 Relative activity of series nTYR

[0075]

[0076] 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 provided nTYR with better acid and alkali resistance.

[0077] Example 2 Preparation of tyrosinase nanogel loaded with blue calyx acetone:

[0078] (1) To 2 mL of a 1 mg / mL tyrosinase solution, 40 mg of a 1 wt % N-acrylsuccinimide solution was added, the solvent was dimethyl sulfoxide, and the mixture was thoroughly mixed and reacted for 2 h to obtain a tyrosinase (aTYR) with a surface modified with polymerizable double bonds.

[0079] (2) Take 2 mL of a 1 mg / mL tyrosinase solution with double bonds modified on the surface, add 0.5 mL of a 2 mg / mL DMSO solution of cyanocysteine ​​A to it while stirring at 300 r / min, and mix thoroughly to obtain tyrosinase loaded with cyanocysteine ​​A (aTYR@GLA).

[0080] (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 to initiate in situ free radical polymerization at room temperature. After 4 h of reaction, polyacrylamide-tyrosinase nanogel loaded with blue calyx methyl was obtained (PAAm-nTYR@GLA).

[0081] 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 stored in a refrigerator.

[0082] Example 3 Preparation of polyphosphorylcholine-tyrosinase nanogel loaded with blue calyx acetum:

[0083] (1) To 2 mL of a 1 mg / mL tyrosinase solution, 40 mg of a 1 wt % N-acrylsuccinimide solution was added, the solvent was dimethyl sulfoxide, and the mixture was thoroughly mixed and reacted for 2 h to obtain a tyrosinase (aTYR) with a surface modified with polymerizable double bonds.

[0084] (2) Take 2 mL of a 1 mg / mL tyrosinase solution with double bonds modified on the surface, add 0.5 mL of a 2 mg / mL DMSO solution of cyanocysteine ​​A to it while stirring at 300 r / min, and mix thoroughly to obtain tyrosinase loaded with cyanocysteine ​​A (aTYR@GLA).

[0085] (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 to initiate in situ free radical polymerization at room temperature. After 4 h of reaction, polyphosphorylcholine-tyrosinase nanogel loaded with blue calyx methyl was obtained (PMPC-nTYR@GLA).

[0086] 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 stored in a refrigerator.

[0087] Characterization of nanogel size by dynamic light scattering (DLS): The protein nanogel solution of Example 2 and Example 3 with a concentration of 1 mg / mL was added to the sample pool, and the particle size of the complex in the system was tested by a Malvern Nano Zs90 nanoparticle size potentiometer at room temperature. The results are summarized in Table 6.

[0088] Table 6 Tyrosinase nanogel particle size and Zeta potential

[0089]

[0090]

[0091] The morphology and dispersion of PAAm-nTYR@GLA were characterized by transmission electron microscopy (TEM). Figure 3 The morphology and dispersion of PMPC-nTYR@GLA under transmission electron microscopy are shown in the following figure. Figure 4 It can be seen that the particle size of tyrosinase nanogel is consistent with the DLS result, and the overall shape is close to spherical, with good dispersibility.

[0092] Application Example 5

[0093] 2 mL of 5 mg / mL tyrosinase nanogel prepared in Example 2 and Example 3 was placed in a dialysis bag with a molecular weight cutoff of 3000, and the dialysis bag was immersed in a phosphate buffer with a pH of 6, and the entire system was oscillated at 37°C. The sustained release time was 48 hours. Within 48 hours, samples were taken regularly to measure the concentration of loaded small molecule drugs under an ultraviolet spectrophotometer, and the sustained release of small molecule drugs by the composite system was calculated. The results are summarized in Table 7.

[0094] Table 7 Release of small molecule drugs by tyrosinase nanogel

[0095]

[0096] Application Example 6: Verification of the effectiveness of functional small molecule ingredients

[0097] Anti-inflammatory ability of polyacrylamide-tyrosinase nanogel (PAAm-nTYR@GLA) of Example 2 and polyphosphorylcholine-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 / well), after 12h cells were completely attached, the original culture medium was sucked away, 100μL serum-free culture medium was added to all groups, 10μL serum-free culture medium was added to the negative control group, 10μL PBS buffer was added to the positive control group, 10μL of 0.01mg / mL nanogel solution was added to the low concentration experimental group, 10μL of 0.02mg / mL nanogel solution was added to the medium concentration experimental group, and 10μL of 0.04mg / mL nanogel solution was added to the high concentration experimental group. After 2h of co-incubation, the original culture medium was sucked away from all groups, 110μL fresh culture medium was added to the negative control group, and 100μL fresh culture medium and 10μL of 10ng / mL interleukin-1β were added to the other groups. After 24h of co-incubation, the determination was performed according to the instructions of the Griess kit. The results are statistically shown in Table 8. It was found that PMPC-nTYR@GLA and PAAm-nTYR@GLA both had good anti-inflammatory ability, and this ability increased with the increase of concentration. The positive charge on the surface of PAAm-nTYR@GLA makes it more efficient in cellular internalization, and it has better anti-inflammatory ability than the negatively charged PMPC-nTYR@GLA at the same concentration.

[0098] Table 8 The intracellular anti-inflammatory ability of tyrosinase nanogel increases with increasing concentration

[0099]

[0100] Application Example 7 Melanin-producing Cell Experiment

[0101] The effect of polyphosphorylcholine-tyrosinase nanogel (PMPC-nTYR@GLA) of Example 3 on the melanin synthesis of mouse melanoma cells B16F10 was determined: B16F10 cells (1*10 5 / well), after 12h cells were completely attached, all groups sucked away the original culture medium and added 100μL serum-free culture medium, the control group added 10μL PBS buffer, the low concentration experimental group added 10μL of 0.01mg / mL nanogel solution, the medium concentration experimental group added 10μL of 0.02mg / mL nanogel solution, and the high concentration experimental group added 10μL of 0.04mg / mL nanogel solution. After 48h of co-incubation, all groups collected cells and dissolved them in 1mL NaOH (1mol / L, containing 10% DMSO) solution, ultrasonically disrupted for 30min, 80℃ water bath for 30min, centrifuged at 3000r / min for 15min, and the supernatant was taken to measure the absorbance at 450nm. 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 the increase of concentration.

[0102] Table 9 The ability of tyrosinase nanogel to promote melanin production increases with increasing concentration

[0103]

[0104]

[0105] Application Example 8: Transdermal Animal Experiment on Vitiligo Model

[0106] The vitiligo model mouse model was used to investigate the ability of PAAm-nTYR@GLA and PMPC-nTYR@GLA in Examples 2 and 3 to promote pigment production through transdermal delivery. The mice were randomly divided into 5 groups, and the backs were uniformly depilated. One group 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 to the depilated area once every two days. Before each administration, the back hair was collected and photographed to record the overall pigmentation. After 20 days of continuous administration, the back skin of the mice was taken, and the overall pigmentation of the back of the mice was observed to characterize the incidence of vitiligo. The results are as follows Figure 5 It was found that the symptoms of vitiligo on the back of the untreated positive control group were significantly worse after 20 days, while the symptoms of vitiligo on the back of the group treated with tyrosinase nanogel were weaker than those of the PBS group, and the therapeutic effect of the PAAm-nTYR@GLA group with positive surface charge was better than that of the PMPC-nTYR@GLA group with negative surface potential.

[0107] Table 10 Statistics on the growth of overall decolorization color difference and decolorization area

[0108] Experimental Group Increase in color difference value Growth rate of decolorization area ratio Positive control group 22.6±5.3 89.4±6.2 PBS 17.8±8.3 88.1±8.2 PMPC-nTYR@GLA 9.4±3.8 61.5±4.1 PAAm-nTYR@GLA 5.9±1.7 35.8±9.5

[0109] Skin tissue sections were taken to observe hair follicle development and pigmentation. Figure 6 As shown in Table 11. It was found that the number of hair follicles in the positive control group and the PBS group was small, the development was poor, and no pigmented hair shafts were found in the hair follicles. In the group treated with tyrosinase nanogel, the development of hair follicles was better than that in the PBS group; and by comparing the PAAm-nTYR@GLA and PMPC-nTYR@GLA groups, it was found that the pigmentation of the hair shafts in the hair follicles in the PAAm-nTYR@GLA group was more obvious. This shows that the better transdermal delivery ability brought by the positive charge on the surface of PAAm-nTYR@GLA can target tyrosinase to melanocytes, promote melanin production, and have a good improvement effect on vitiligo symptoms.

[0110] Table 11 Statistics of hair follicle length and number

[0111]

[0112]

Claims

1. A tyrosinase-based transdermal delivery system for treating vitiligo, characterized in that: It is a core-shell structure, the core is tyrosinase, 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 includes at least one of acrylamide copolymer, vinylpyrrolidone copolymer, polyol, polylactic acid, phosphorylcholine polymer, betaine polymer and chitosan; the polymer is polymerized by monomers with carbon-carbon unsaturated double bonds, such as polyacrylamide and polyvinylpyrrolidone, or obtained by condensation polymerization or ring-opening polymerization of monomers with different functional groups; or the natural polymer is coated on the surface of tyrosinase by electrostatic attraction, hydrogen bonding, intermolecular action, cross-linking and the like.

3. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 2, characterized in that: The polymer material wrapped in the outer layer of tyrosinase by polymerization reaction, wherein the monomer is selected from at least one of vinyl pyrrolidone, acrylamide, N-acrylsuccinimide, 2-methacryloyloxyethyl phosphorylcholine, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonatepropyl)ammonium hydroxide, vinyl pyrrolidone, polyethylene glycol methyl ether acrylate, ethylene glycol, propylene glycol, butylene glycol, and lactide; 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 such as chitosan; 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.

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 2-15 nm, preferably 5-10 nm; the thickness of the polymer shell layer, i.e., the shell, is 1-100 nm, preferably 5-50 nm, more preferably 7-20 nm.

5. 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 from -7 mV to +7 mV, preferably a positive potential, such as +4 mV to +6 mV.

6. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that: In the core-shell structure, the core is a tyrosinase loaded with an anti-inflammatory small molecule, and the anti-inflammatory small molecule is loaded inside the tyrosinase through hydrophilic and hydrophobic interactions, including but not limited to at least one of cyanocalyxin, vitamin E, lycopene, astaxanthin, hesperidin, and glabridin; Preferably, the mass ratio of the anti-inflammatory active small molecule to tyrosinase is 1:1-1000.

7. A method for regulating the transdermal penetration depth of the transdermal delivery system of tyrosinase according to claim 5, characterized in that: When its surface Zeta potential is +4 to +6 mV, 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.

8. The method for preparing the tyrosinase-based transdermal delivery system according to any one of claims 1 to 5, 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.

9. The preparation method according to claim 8, 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; the mass ratio of tyrosinase to the small molecule containing a polymerizable carbon-carbon double bond is 5-20:1, preferably 5-10:1; 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, 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, preferably 1-10 mg / L.

10. The preparation method according to claim 8, 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; preferably N-(3-aminopropyl) methacrylamide hydrochloride; the crosslinking 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.

11. The preparation method according to claim 8, characterized in that: In step (2), the monomer includes N-(3-aminopropyl)methacrylamide hydrochloride. The type of monomer determines the surface electrical properties of the polymer shell; Preferably, the monomer comprises 5-20 mol% of N-(3-aminopropyl)methacrylamide hydrochloride; More preferably, the monomer comprises 10-15 mol % of N-(3-aminopropyl)methacrylamide hydrochloride.

12. The preparation method according to claim 8, characterized in that: In step (2), the molar ratio of tyrosinase with surface-modified polymerizable double bonds, monomer, and cross-linking agent is 1:100-100000:10-10000, preferably 1:2000-20000:200-2000.

13. The preparation method according to claim 8, 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.

14. The preparation method according to claim 8, characterized in that: In step (2), the polymerization reaction is carried out at room temperature for 2-10 hours, preferably 4-6 hours.

15. The method for preparing the tyrosinase-based transdermal delivery system according to claim 6, 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.

16. The preparation method according to claim 15, 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 / L; 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.

17. Use of the tyrosinase-based transdermal delivery system according to any one of claims 1 to 6 in the preparation of a drug for treating vitiligo.

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