A tyrosinase-based nano-transdermal delivery system for treating vitiligo

Abstract

The application provides a tyrosinase-based transdermal delivery system for treating vitiligo, which is a core-shell structure, the core is tyrosinase, and the shell is a polymer shell layer. The application coats a polymer protective layer on the surface of the tyrosinase through in-situ polymerization reaction on the double bond of the tyrosinase, can effectively protect the activity of the tyrosinase and promote the transdermal delivery of the tyrosinase, and can control the targeted delivery of the tyrosinase nanogel to melanocytes in the deep part of the skin and hair follicle by adjusting the polymerization conditions of the polymer shell, so as to promote the in-situ synthesis of melanin. The transdermal delivery system of the application can also load active small molecules with anti-inflammatory function in the center of the tyrosinase, and the small molecules are released after being targeted and delivered to the inflammation area of vitiligo along with the tyrosinase nanogel, so as to relieve inflammation and protect melanocytes from damage.

Description

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 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 confirmed to be associated with the onset of vitiligo, including oxidative stress, the production of inflammatory mediators, and autoimmune responses. Targeted 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 attacks on melanocytes in the affected area in autoimmune responses, local transdermal delivery of corticosteroids and calcineurin inhibitors can reduce melanocyte damage through local immune regulation. 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-induced damage to melanocytes in the affected area is an important pathogenic mechanism. 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 their ability to promote pigmentation, which places high demands on equipment and instruments. [2] In addition to reducing melanocyte damage, directly promoting melanin synthesis in melanocytes is another treatment approach 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 usage concentration, and excessively high concentrations of activators will produce side effects.

[0003] Compared with oral administration and intravenous injection, local transdermal administration has the advantages of convenience, non-invasiveness, maintenance of drug activity, targeted delivery, etc., and is particularly suitable for the management of vitiligo, which requires local, long-term, and multiple drug administration. Therefore, the drug treatment of vitiligo usually adopts the mode of transdermal delivery for drug administration. However, the skin has a high barrier effect composed of multiple complex structures, including physical, chemical, and immune barriers. Whether it is a small molecule drug or a large molecule drug, it faces the problems of low penetration efficiency, inability to reach the skin basal layer and hair follicle bottom melanocytes to exert the effect. Due to the high barrier effect of the skin, simply increasing the drug concentration is of little help in promoting penetration, but it may cause skin irritation or allergy. In addition, most active drugs are hydrophobic substances, and their solubility in traditional transdermal preparations is low, which severely limits the therapeutic effect of the drugs.

[0004] In the field of vitiligo treatment, the delivery technologies used in the clinically used topical drug preparations 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 irritating effect on the skin and may exacerbate the condition of inflammatory vitiligo. The size of drugs wrapped in liposomes is generally greater than 200 nm, and the formed preparation is 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 applied to the delivery of small molecule drugs such as immunosuppressants, and the transdermal efficiency of active macromolecular drugs such as enzymes is limited, and the presence of some penetration enhancers may cause the inactivation of enzymes, thereby losing the ability to treat vitiligo. Microneedle technology uses needle tips with certain mechanical strength to pierce the skin and form pores, thereby weakening the skin barrier during transdermal drug delivery. However, the treatment of vitiligo requires a long period of time and multiple drug administrations, and frequent use of microneedles will cause damage to the local skin and even inflammation. In addition, the depigmentation symptoms of vitiligo are caused by the damage and loss of melanocytes, so the target of transdermal delivery of vitiligo is the melanocytes located in the basal layer of the skin and the deep part of the hair follicle. However, the existing transdermal delivery technology cannot control the transdermal depth, resulting in low treatment efficiency.

[0005] In summary, the existing technology for treating vitiligo has the following disadvantages: small molecule drugs targeting different mechanisms have low universality and side effects; large molecule transdermal technology has low efficiency and side effects SUMMARY

[0006] 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.

[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 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, the polymer can be coated on the surface of tyrosinase through electrostatic attraction, hydrogen bonding, intermolecular interactions, cross-linking, and the like.

[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-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.

[0011] The polymeric material that coats the tyrosinase on the outside is a polyelectrolyte with positive or negative charge, wherein the positive polyelectrolyte is at least one of poly(acrylamide hydrochloride), poly(L-lysine), poly(ethyleneimine), poly(L-histidine), poly(N,N-dimethylaminoethyl methacrylate), poly(methacrylamidopropyl trimethylammonium chloride), and natural or synthetic polysaccharides such as chitosan; the negative polyelectrolyte is at least one of poly(acrylic acid), polystyrene sulfonate, alginate, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, poly(methacrylic acid), oxidized cellulose, carboxymethylcellulose, polyaspartic acid, and polyglutamic acid.

[0012] Further, in the core-shell structure, the size of the core, i.e. the tyrosinase loaded with anti-inflammatory active small molecules, is 2-15 nm, preferably 5-10 nm; the thickness of the polymeric shell layer as the shell is 1-100 nm, preferably 5-50 nm, more preferably 7-20 nm. Further, the surface Zeta potential of the tyrosinase-based transdermal delivery system is -7 mV to +7 mV, preferably positive, such as +4 mV to +6 mV.

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

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

[0015] The mass ratio of the anti-inflammatory active small molecules to the tyrosinase is 1:1-1000, and the ratio varies depending on different anti-inflammatory active small molecules. For example, the mass ratio of the anti-inflammatory active small molecules to the tyrosinase is 1:2-10, such as 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] Figure 1 This 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 with different nTYR activity.

[0043] Figure 3 This 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 in the development stage of vitiligo.

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

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

[0048] Example 1

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

[0050] aTYR, acrylamide, N-(3-aminopropyl) methacrylamide hydrochloride, and N,N'-methylenebisacrylamide were mixed according to the given mass ratio, the protein concentration was diluted to 1 mg / mL with a pH 7.4 phosphate buffer, then 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per mg of protein were added as an initiator, and in-situ radical polymerization was initiated at room temperature. After a specified reaction time, a series of size polyacrylamide-tyrosinase nanogels (nTYR) 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 size and surface potential of a series of size tyrosinase nanogels

[0052]

[0053]

[0054] As can be seen from Table 1, increasing the amount of monomer relative to the amount of protein and increasing the polymerization time will make the particle size of the obtained polyacrylamide-tyrosinase nanogel (nTYR) larger, 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 a series of size tyrosinase nanogels

[0056] 4 mL of tyrosinase nanogel nTYR of different sizes in Example 1 with a concentration of 1 mg / mL was mixed with 1 mL of fluorescein isothiocyanate with a concentration of 1 mg / mL and reacted overnight, and then dialyzed overnight in a dialysis bag with a molecular weight cut-off 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 upwards in a transdermal diffusion instrument and incubated at 37°C. 0.5 mL of a solution of fluorescently labeled tyrosinase nanogel with a concentration of 1 mg / mL was added above the pigskin tissue, and after 24 hours, frozen sections were prepared, and the penetration of the nanogel was observed under a confocal microscope. The penetration depth results 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 depth (pm) 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 depth (pm) 128.5±7.8 159.5±11.1 113.6±9.9 99.7±8.5 58.4±6.3

[0059] Comparing the penetration depths of tyrosinase nanogels of different sizes, all with positive surface potentials (approximately +5 mV), revealed that within 24 hours, the protein nanogels, ranging in size from 20 nm to 150 nm, were able to penetrate the epidermis and reach the dermis. Furthermore, the penetration depth initially increased and then decreased with increasing gel particle size. Tyrosinase nanogels larger than 200 nm largely remained 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] 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.

[0062] Application Example 2: Activity retention of tyrosinase nanogels in a series of sizes

[0063] The catalytic activity of nTYR was determined using an ultraviolet 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 or untreated tyrosinase solution was added and placed in an ultraviolet 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 was used to represent the relative activity of tyrosinase. The results are shown in Figure 2. Figure 2As shown in Table 3, it is shown that for tyrosinase nanogels larger than 50 nm, the polymer shell is too thick, which affects 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 Tyrosinase Nanogels of Various Sizes

[0067] 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.

[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 treatment 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 various sizes

[0073] The catalytic activity of nTYR was determined by UV spectrophotometer. 1600 μL of PBS buffer with a specified pH and 1400 μL of L-tyrosine solution with a concentration of 1 g / L were added to the reference cell; 1550 μL of PBS buffer with a specified pH and 1400 μL of L-tyrosine solution with a concentration of 1 g / L were added to the sample cell, and finally 50 μL of nTYR solution after different heat treatments or the original tyrosinase solution was added. The sample cell was placed in the UV spectrophotometer, and the absorbance at 317 nm was recorded for 20 minutes. The slope of the fitted straight line represented the relative activity of tyrosinase, and the enzyme activity retention at different pH values relative to pH = 6 was calculated. The results are shown in Table 5.

[0074] Table 5 Relative activity of nTYR series

[0075]

[0076] It was found that the original tyrosinase had good activity only in weakly acidic (pH around 6) environment, while nTYR-20 and nTYR-50 could exert high catalytic effect in a wide pH range, indicating that the polymer shell provided good acid and alkali resistance for nTYR.

[0077] Example 2 Preparation of tyrosinase nanogel loaded with glaucocalyxin B:

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

[0079] (2) 2 mL of tyrosinase solution with a surface modified double bond was taken, and 0.5 mL of 2 mg / mL glaucocalyxin B DMSO solution was added dropwise to the solution under stirring at 300 r / min. After thorough mixing, tyrosinase loaded with glaucocalyxin B (aTYR@GLA) was obtained.

[0080] (3) aTYR@GLA, acrylamide, N-(3-aminopropyl) methacrylamide hydrochloride, and N,N'-methylenebisacrylamide were mixed according to the molar ratio of 1:6000:600:600. 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 corresponding to 1 mg of protein were added as an initiator. In situ radical polymerization was initiated at room temperature, and after 4 h of reaction, polyacrylamide-tyrosinase nanogel loaded with glaucocalyxin B (PAAm-nTYR@GLA) was obtained.

[0081] The obtained complex solution was subjected to ultrafiltration treatment in an ultrafiltration centrifuge tube with a molecular weight cut-off of 30 kDa to obtain a high-concentration mother liquor after purification with a concentration of 6.1 mg / mL, which was stored in a refrigerator.

[0082] Example 3 Preparation of aescinate-loaded polyphosphocholine-tyrosinase nanogel:

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

[0084] (2) 2 mL of a tyrosinase solution with a surface modified double bond was taken, and 0.5 mL of a 2 mg / mL GLA DMSO solution was added dropwise to the solution under stirring at 300 r / min, and aTYR@GLA was obtained after fully mixing.

[0085] (3) aTYR@GLA, 2-methacryloyloxyethylphosphocholine, and N,N'-methylenebisacrylamide were mixed in a mass ratio of 1:40:4, the protein concentration was diluted to 1 mg / mL with a pH 7.4 phosphate buffer, and then 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine were added per milligram of protein as an initiator, and in-situ radical polymerization was initiated at room temperature. After 4 h of reaction, aescinate-loaded polyphosphocholine-tyrosinase nanogel (PMPC-nTYR@GLA) was obtained.

[0086] The obtained complex solution was subjected to ultrafiltration treatment in an ultrafiltration centrifuge tube with a molecular weight cut-off of 30 kDa to obtain a high-concentration mother liquor after purification with a concentration of 5.7 mg / mL, which was stored in a refrigerator.

[0087] The size of the nanogel was characterized 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 a sample cell, and the particle size of the complex in the system was tested at room temperature using a Malvern Nano Zs90 nanoparticle size potential instrument. The results are shown in Table 6.

[0088] Table 6 Particle size and zeta potential of tyrosinase nanogel

[0089]

[0090]

[0091] TEM was used to characterize the morphology of the nanogels: The morphology and dispersion of PAAm-nTYR@GLA under transmission electron microscope, the results are as follows Figure 3 The morphology and dispersion of PMPC-nTYR@GLA under transmission electron microscope, the results are as follows Figure 4 It can be seen that the particle size of tyrosinase nanogel is consistent with the DLS results, and the overall shape is close to spherical, and the dispersion is good.

[0092] Application Example 5

[0093] Take 2 mL of the tyrosinase nanogel prepared in Example 2 and Example 3 with a concentration of 5 mg / mL and put it into a dialysis bag with a molecular weight cut-off of 3000, immerse the dialysis bag in a phosphate buffer with a pH of 6, and the whole system is oscillated at 37°C. The release time is 48 h. Within 48 h, take samples at regular intervals and measure the concentration of loaded small molecule drugs under ultraviolet spectrophotometer, and calculate the release of the composite system to small molecule drugs. The results are shown in Table 7.

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

[0095]

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

[0097] Inflammatory capacity determination 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 by Griess kit. HaCaT cells (1*10 5After 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.

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

[0099]

[0100] Application Example 7 Melanin-producing Cell Experiment

[0101] 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 5 After 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.

[0102] Table 9 Tyrosinase nanogel melanogenesis-promoting ability increases with increasing concentration

[0103]

[0104]

[0105] Example 8 Vitiligo model transdermal animal experiment

[0106] The ability of PAAm-nTYR@GLA and PMPC-nTYR@GLA in Examples 2 and 3 to promote pigment production was investigated using a vitiligo model mouse model. The mice were randomly divided into 5 groups, and the back was uniformly depilated. One group served as a normal control, and the remaining 4 groups (positive control, PBS control group, PAAm-nTYR@GLA group, and PMPC-nTYR@GLA group) were subjected to vitiligo modeling. After successful modeling, the corresponding drug or PBS buffer (positive control group) was applied to the depilated area every two days. The back hair was collected and photographed before each administration to record the overall pigmentation. After 20 days of continuous administration, the mouse back skin was taken, and the overall pigmentation of the mouse back was observed to characterize the vitiligo incidence, as shown in Figure 5 It was found that the positive control group without treatment had a significant deterioration in back vitiligo after 20 days, while the tyrosinase nanogel-treated group had a weaker deterioration in back vitiligo than the PBS group, and the PAAm-nTYR@GLA group with a positive surface charge had a better treatment effect than the PMPC-nTYR@GLA group with a negative surface potential.

[0107] Table 10 Overall depigmentation color difference increase and depigmentation area increase statistics

[0108] Experimental group Decolorization color difference value growth Decolorization area proportion growth rate 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, as shown in Figure 6 and Table 11. It was found that in the positive control group and the PBS group, the number of hair follicles was small, and the development was poor, and no pigmented hair shaft was found in the hair follicles. In the tyrosinase nanogel-treated group, the development of the hair follicles was better than that of the PBS group; and comparing the PAAm-nTYR@GLA and PMPC-nTYR@GLA groups, it was found that the pigmentation of the hair shaft in the PAAm-nTYR@GLA group was more obvious. This indicates that the better transdermal delivery ability of the positive charge on the surface of PAAm-nTYR@GLA can target the delivery of tyrosinase to melanocytes, promote melanin production, and have a better improvement effect on vitiligo symptoms.

[0110] Table 11 Hair follicle length and number statistics

[0111]

[0112]

Claims

1. A tyrosinase-based transdermal delivery system for treating vitiligo, characterized in that: It has a core-shell structure, wherein the core is tyrosinase loaded with an anti-inflammatory active small molecule, the anti-inflammatory active small molecule is cyanocalyxin A, and the shell is a polymer shell layer; The preparation method of the tyrosinase-based transdermal delivery system for treating vitiligo comprises the following steps: (1) mixing a tyrosinase solution and a solution containing a polymerizable carbon-carbon double bond small molecule to obtain a tyrosinase with a surface modified with a polymerizable double bond; the small molecule containing the polymerizable carbon-carbon double bond is N-propylene succinimide; (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; (3) A tyrosinase solution loaded with an anti-inflammatory active small molecule, 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; the monomer is a mixture of acrylamide and 2-methacryloyloxyethyl phosphorylcholine, or the monomer is N-(3-aminopropyl) methacrylamide hydrochloride, and the cross-linking agent is selected from N,N'-methylenebisacrylamide.

2. 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; the thickness of the polymer shell layer, i.e., the shell, is 1-100 nm.

3. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 2, characterized in that The size of the tyrosinase loaded with small molecules having anti-inflammatory activity is 5-10 nm, and the thickness of the polymer shell layer as the shell is 5-50 nm.

4. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 2, characterized in that The thickness of the polymer shell layer as the shell is 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.

6. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 5, characterized in that The surface Zeta potential of the tyrosinase-based transdermal delivery system is positive.

7. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 5, characterized in that The surface zeta potential of the tyrosinase-based transdermal delivery system is +4 mV to +6 mV.

8. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that In step (2), the mass ratio of the anti-inflammatory active small molecule to tyrosinase is 1:1-1000.

9. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that In step (1), the mass ratio of tyrosinase to the small molecule containing a polymerizable carbon-carbon double bond is 5-20:

1.

10. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that In step (1), the mass ratio of tyrosinase to the small molecule containing a polymerizable carbon-carbon double bond is 5-10:

1.

11. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that: 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.

12. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that The protein concentration in the protein solution is 1-10 mg / L.

13. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that In step (3), 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.

14. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 13, characterized in that: In step (3), a co-initiator is also added, and the co-initiator is selected from 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.

15. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that In step (3), the monomer includes 5-20 mol% of N-(3-aminopropyl)methacrylamide hydrochloride.

16. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 15, characterized in that: The monomers include 10-15 mol % of N-(3-aminopropyl)methacrylamide hydrochloride.

17. The preparation method according to claim 1, characterized in that In step (3), the molar ratio of the surface-modified polymerizable double bond tyrosinase, the monomer, and the cross-linking agent is 1:100-100000:10-10000.

18. The preparation method according to claim 17, characterized in that: The molar ratio of the surface-modified polymerizable double bond tyrosinase, the monomer, and the cross-linking agent is 1:2000-20000:200-2000.

19. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that In step (3), the monomer is a compound of acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride; 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.

20. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that In step (3), the polymerization reaction is carried out at room temperature for 2-10 hours.

21. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 20, characterized in that: In step (3), the polymerization reaction is carried out at room temperature for 4-6 hours.

22. The tyrosinase-based transdermal delivery system for treating vitiligo according to claim 1, characterized in that In step (2), 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 tyrosinase with surface-modified polymerizable double bonds is 1:1-100, and the mass of the tyrosinase with surface-modified polymerizable double bonds is calculated based on tyrosinase.

23. Use of the tyrosinase-based transdermal delivery system according to any one of claims 1 to 22 in the preparation of a drug for treating vitiligo.

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