A superoxide dismutase-based nano-transdermal delivery system and a preparation method thereof

Abstract

The application provides a kind of nano transdermal delivery system based on superoxide dismutase, which is a capsule composite structure of small molecule active ingredient-superoxide dismutase-polymer shell layer, the small molecule active ingredient is loaded in superoxide dismutase, and the surface of superoxide dismutase is coated with a polymer shell layer.The diameter of the active ingredient transdermal delivery system based on superoxide dismutase is 20-100 nm, and the thickness of the polymer shell layer is 7.5-52.5 nm.The polymer shell layer in the delivery system of the application provides protection for superoxide dismutase and small molecule functional substances with antioxidant effect.The capsule composite structure is in the form of nanogel, which not only promotes its penetration depth, but also reduces its irritation to the skin.The raw materials of the application are biologically friendly, the preparation method is simple, the yield is high, and it can be produced on a large scale.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nano-transdermal delivery system, and particularly relates to a nano-transdermal delivery system based on superoxide dismutase and a preparation method thereof. BACKGROUND

[0002] Superoxide dismutase is a metal enzyme widely distributed in organisms, which plays an antioxidant role by scavenging superoxide anion free radicals, and protects organisms from oxidative stress damage, so it is concerned as an efficient antioxidant substance for skin. However, in practical application, direct application of superoxide dismutase on the surface of the skin for antioxidant management or treatment often cannot achieve good results, which indicates that the transdermal delivery efficiency is not high, and the bioavailability of superoxide dismutase is low.

[0003] Transdermal delivery has advantages including convenient non-invasive, local targeting, avoiding extreme pH environment in the gastrointestinal tract, etc., and is the most commonly used administration method in skin problem management. Oxidative stress is the cause of various skin problems including skin inflammation, excessive secretion of skin oil, dull skin color, wrinkle formation, etc., so the delivery of antioxidant substances to the skin is beneficial to solving various skin problems. However, the multi-layer complex structure of the skin causes high barrier problems including physical barrier, chemical barrier, and immune barrier, and the active substances delivered transdermally are often isolated outside the stratum corneum, the outermost layer of the skin, and are difficult to reach the deep layer and play a role. Blindly increasing the concentration of active substances is of little help to promote penetration, and may even cause irritation to the skin. In addition, for different skin problems, the depth of oxidative stress is different, which may involve the epidermis, the junction between the epidermis and the dermis, and the dermis matrix, so the depth of transdermal delivery is particularly important.

[0004] The existing transdermal delivery technology in clinical or commercial use is still in the early stage of development, and the most commonly used is chemical penetration enhancer and liposome loading. Common penetration enhancers such as ethanol have certain influence on the biological activity of the delivered substances, and due to the principle of promoting penetration by changing the structure of the skin to weaken the barrier effect, there is also a greater risk of skin irritation and allergy during use. The vesicle structure formed by liposome encapsulation generally has a diameter greater than 200 nm, and the system is in a thermodynamic metastable state, which is difficult to meet the requirements of long-term use and storage. SUMMARY

[0005] In order to solve the defects of low delivery efficiency and uncontrollable delivery depth in the existing skin antioxidant transdermal technology, the present application uses superoxide dismutase molecules as a nano template to construct a high-efficiency transdermal delivery system by polymer wrapping, and simultaneously uses the hydrophobic structure inside the superoxide dismutase to load functional small molecules for co-delivery, so as to realize the synergistic effect of various physiological functions including antioxidant. The system uses a modified superoxide dismutase nanogel as a carrier, and the functional small molecules are loaded in the nanogel and then are efficiently transported to the deep layer of the skin and are controlled to be released, so as to play a synergistic therapeutic effect on various problems such as skin oxidation and inflammation. In the system, the superoxide dismutase itself has antioxidant effect and can also be used as a carrier for functional small molecules; the polymer shell on the outer layer of the superoxide dismutase has a customizable diameter and surface potential, which not only helps the internal superoxide dismutase and functional small molecules to penetrate the skin barrier, but also protects the stability of the superoxide dismutase and the functional small molecules, which is helpful for long-term use and storage of the preparation. The excellent penetration efficiency, controllable surface properties and diameter of the nanogel make the delivery depth of the transdermal delivery system controllable, and the superoxide dismutase and small molecule active ingredients target the skin oxidative stress and play a role, so that a lower concentration of active ingredients can achieve comprehensive improvement of various skin problems. In addition, the nanogel can effectively avoid the stimulation of some active ingredients to the skin, and has wider applicability to people with different skin conditions. The loading method is universal for small molecule components with hydrophobic structure.

[0006] Specifically, the present application provides the following technical solutions to solve the above technical problems:

[0007] A nano transdermal delivery system based on superoxide dismutase, characterized in that the nano transdermal delivery system has a capsule composite structure of small molecule active ingredient-superoxide dismutase-polymer shell layer, and the diameter is 20-100 nm, wherein the small molecule active ingredient is loaded in the superoxide dismutase, the surface of the superoxide dismutase is coated with a polymer shell layer, and the thickness of the polymer shell layer is 7.5-52.5 nm.

[0008] Further, the active ingredient transdermal delivery system based on superoxide dismutase has a diameter of 30-80 nm and a polymer shell layer thickness of 10-35 nm.

[0009] Further, the surface Zeta potential of the transdermal delivery system is +4 to +7 mV. The inventors found that the surface charge of the nano transdermal delivery system based on superoxide dismutase is positive, which is more conducive to the depth and delivery efficiency of transdermal delivery and stability.

[0010] The small molecule active ingredient is selected from carotenoids, hesperidin, vitamin C, vitamin E, resveratrol, lycopene, retinol nicotinamide, ascorbic acid, alpha-arbutin or derivatives of the foregoing. The present application can select different small molecule active ingredients for different antioxidant problems, such as carotenoids, hesperidin, vitamin C, vitamin E, etc. for skin oxidation problems, resveratrol, lycopene, etc. for skin inflammation problems, retinol and its derivatives for skin photoaging and wrinkle problems, and nicotinamide, ascorbic acid, alpha-arbutin, etc. for skin color and spot problems caused by excessive pigmentation.

[0011] Further, the mass ratio of the small molecule active ingredient to the superoxide dismutase is 1:1-100, preferably 1:1-5. The functional small molecule is dissolved in water or an organic solvent to prepare a functional small molecule solution, and the functional small molecule solution is added to the surface-modified superoxide dismutase solution. The small molecule is attached to the core of the protein molecule by electrostatic interaction, hydrophilic-hydrophobic interaction, hydrogen bonding, cross-linking, etc. to obtain a superoxide dismutase solution loaded with small molecules.

[0012] In the delivery system of the present application, a polymer that is beneficial to the formation of a hydration layer on the outer layer of the nanogel is selected, including monomers that are beneficial to the formation of hydrogen bonds, monomers that have amphiphilic properties and can naturally escape immune recognition, and monomers with positive charges and monomers with negative charges can be selected for copolymerization to form a positive dipole balance inside the polymer, thereby forming a strong hydration layer, such as phosphocholine polymer, betaine polymer, etc. A positively charged polymer can also be selected to be wrapped around the superoxide dismutase molecule by electrostatic interaction, such as chitosan, etc.

[0013] The polymer shell not only helps the internal superoxide dismutase and functional small molecules to penetrate the skin barrier, but also protects the stability of the superoxide dismutase and functional small molecules, and helps long-term use and storage of the preparation, so the thickness regulation of the shell layer is very important in the system. The penetration performance of the nano-transdermal delivery system is related to the particle size, and the shell layer that is too thin or too thick is not conducive to the deep delivery of the nanogel; and the protection of the polymer shell layer on the activity of the active protein is also related to the thickness of the shell layer, and the shell layer that is too thin has poor ability to resist severe environmental temperature and pH changes; and the premise of the catalytic action of the enzyme is to contact with the substrate, and the shell layer that is too thick will affect the transmission of the substrate, thereby hindering the catalytic action of the active protein. In the present application, a series of different diameters and thicknesses of the shell layer are prepared and investigated for the influence on the transdermal depth and the protection of the protein or enzyme activity, and the results show that the gel suitable for the treatment of skin inflammation has a diameter of 20-100 nm, preferably 30-80 nm, and since the diameter of the superoxide dismutase is about 5 nm, the thickness of the polymer shell layer should be controlled in the range of 7.5-47.5 nm, preferably 10-35 nm. The control of the thickness of the polymer shell layer can be mainly controlled by the ratio of monomers, polymerization time and polymerization conditions.

[0014] Further, the polymer shell is coated on the surface of the protein by in-situ polymerization, hydrogen bonding and / or electrostatic interaction; the polymer obtained by in-situ polymerization is selected from at least one of N-acrylsuccinimide, N-(3-aminopropyl)methacrylamide hydrochloride, maleimide, vinylpyrrolidone, acrylamide, polyethylene glycol methyl ether acrylate, N-acrylsuccinimide, 2-methacryloyloxyethylphosphocholine, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 2-hydroxyethyl acrylate, acrylate, 2-(dimethylamino)ethyl methacrylate, (3-acrylamidopropyl)trimethylammonium chloride hydrochloride, aminopropyl methacrylamide, N,N'-methylenebisacrylamide, glycerol dimethacrylate, glycerol 1,3-diglycerol diacrylate; preferably, the acrylate is selected from at least one of (meth)acrylate methyl ester, (meth)acrylate ethyl ester, (meth)acrylate hydroxyethyl ester, (meth)acrylate propyl ester, (meth)acrylate butyl ester; the polymer is coated on the surface of the protein by electrostatic interaction, and is a positively or negatively charged polymer, wherein the positively charged polymer is selected from at least one of poly(acrylamide hydrochloride), poly(L-lysine), polyethyleneimine, poly(L-histidine), poly(N,N-dimethylaminoethyl methacrylate), poly(methacrylamidopropyl trimethylammonium chloride), and natural or synthetic polysaccharides such as chitosan; the negatively charged polymer is selected from at least one of poly(acrylic acid), polystyrene sulfonate, alginate, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, polymethacrylic acid, oxidized cellulose, carboxymethyl cellulose, polyaspartic acid and polyglutamic acid.

[0015] Preferably, the polymer is coated on the surface of the protein in the form of carbon-carbon unsaturated double bond monomers polymerized in situ. The polymer raw materials include surface modification monomers, functional monomers, cross-linking agents and initiators, and preferably, positive charge monomers are also added; the surface modification monomers are selected from at least one of N-propenyl succinimide, N-(3-aminopropyl) methacrylamide hydrochloride and maleimide, and the surface modification monomers are modified on the surface of the protein by electrostatic adsorption, chemical bonding and other interactions; the functional monomers are selected from at least one of vinyl pyrrolidone, acrylamide, polyethylene glycol methyl ether acrylate, 2-methacryloyloxyethyl phosphocholine and 2-hydroxyethyl acrylate; the positive charge monomers are selected from at least one of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide, 2-(dimethylamino)ethyl methacrylate, (3-acrylamidopropyl) trimethylammonium chloride hydrochloride and aminopropyl methacrylamide hydrochloride; the cross-linking agents are selected from at least one of N,N'-methylene bisacrylamide, glycerol dimethacrylate and glycerol 1,3-diglycerol diacrylate; and the initiator is a water-soluble initiator, and is specifically selected from at least one of ammonium persulfate, sodium persulfate and potassium persulfate, and preferably, a co-initiator such as sodium bisulfite and tetramethyl ethylenediamine is also added; 2-5 mg of the initiator and 6-10 mg of the co-initiator are added per milligram of the protein; and preferably, 3.5-4 mg of the initiator and 7.5-8 mg of the co-initiator are added per milligram of the protein.

[0016] After the superoxide dismutase is modified by the surface modification monomers, the surface has polymerizable carbon-carbon unsaturated double bonds. In a specific embodiment of the present application, the surface modification method comprises mixing the superoxide dismutase with a buffer solution or an organic solvent to prepare a superoxide dismutase solution, and adding surface modification monomers to the superoxide dismutase solution, so that the surface modification monomers are attached to the surface of the protein molecules by electrostatic interaction, covalent cross-linking or other methods, to obtain a surface-modified protein molecule solution.

[0017] Further, the mass ratio of the superoxide dismutase to the surface modification monomers is 1-10:1, and preferably 2-5:1; the molar ratio of the superoxide dismutase to the functional monomers is 1:4500-25000, and the molar ratio of the functional monomers to the cross-linking agents is 1:0.05-0.1; and if the polymer raw materials also include positive charge monomers, the molar ratio of the functional monomers to the positive charge monomers is 1:0.1-0.12.

[0018] Further, the molar ratio of the superoxide dismutase to the functional monomers is 1:6000-10000.

[0019] The second object of the present application is to provide a preparation method of the above-mentioned superoxide dismutase-based nano-transdermal delivery system, which is one of the following methods:

[0020] Method 1: in-situ polymerization method

[0021] (S1) adding a surface modification monomer solution to a buffer solution of superoxide dismutase to obtain a surface-modified protein solution;

[0022] (S2) adding a small molecule active ingredient solution to the surface-modified protein solution obtained in step (S1), and adsorbing the small molecule active ingredient in the superoxide dismutase to obtain a surface-modified superoxide dismutase solution loaded with a small molecule active ingredient;

[0023] (S3) adding a functional monomer, a positively charged electric monomer, a crosslinking agent and an initiator to the surface-modified superoxide dismutase solution loaded with a small molecule active ingredient obtained in step (S2), and initiating in-situ polymerization to obtain a superoxide dismutase-based nanotransdermal delivery system;

[0024] Method 2: electrostatic adsorption method:

[0025] adding a polymer solution with positive or negative charge to a superoxide dismutase solution at 50-70°C (such as 60°C) and stirring, adding a small molecule active ingredient solution at 50-70°C, continuing to stir, adding a salt solution under ice water bath conditions, and reacting for 30-60 min, and obtaining a superoxide dismutase-based nanotransdermal delivery system by ultrafiltration centrifugation.

[0026] Further, in method 1, in step (S1), the buffer solution is a phosphate buffer solution, specifically a PBS buffer solution with pH of 7.4-7.6, and the protein concentration in the protein solution is 1-5 mg / mL; the solvent of the surface modification monomer solution is at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, and the concentration is 1-5 wt%; and / or

[0027] In step (S2), the solvent of the small molecule active ingredient solution is preferably the same solvent as the surface modification monomer solution, i.e., at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, and the concentration of the small molecule active ingredient solution is 1-5 mg / mL, such as 2 mg / mL, 3 mg / mL, or 4 mg / mL. The amount of the small molecule active ingredient solution is such that the mass ratio of superoxide dismutase to small molecule active ingredient is 1-5:1.

[0028] Further, the molar ratio of superoxide dismutase, functional monomer, positive charge monomer, crosslinking agent is 1:4500-10000:600-1000:500-1000; the initiator is a water-soluble initiator, and is specifically at least one selected from the group consisting of ammonium persulfate, sodium persulfate and potassium persulfate, preferably, an assistant initiator such as sodium bisulfite, tetramethyl ethylenediamine is further added; 2-5 mg of initiator and 6-10 mg of assistant initiator are added per milligram of protein; preferably, 3.5-4 mg of initiator and 7.5-8 mg of assistant initiator are added per milligram of protein; the polymerization is room temperature polymerization, and the polymerization time is 6-12 h, preferably 6-8 h.

[0029] Further, in the method 2, the concentration of the polymer solution with positive charge or negative charge, the concentration of the superoxide dismutase solution, and the concentration of the small molecule active ingredient aqueous solution are independently 1-10 mg / mL; when the polymer is positively charged, the salt solution is a salt solution capable of providing anions to induce self-assembly of the positively charged polymer, such as a sodium tripolyphosphate solution; when the polymer is negatively charged, the salt solution is a salt solution capable of providing cations to induce self-assembly of the negatively charged polymer, such as a calcium chloride solution; the concentration of the salt solution is 0.5-5 mg / mL. Further, the mass ratio of superoxide dismutase, polymer with positive charge or negative charge, small molecule active ingredient, and salt in the salt solution is 1:0.5-1:0.05-0.1:0.05-0.1, such as 12:6:1:1.

[0030] Further, in the method 2, the molecular weight cut-off of the ultrafiltration is 3-5 kDa.

[0031] The third object of the present application is to provide the use of the above-mentioned superoxide dismutase-based nano-transdermal delivery system in the preparation of preventing or treating oxidative stress diseases such as skin photoaging, chloasma and eczema.

[0032] The excellent effects of the present application are:

[0033] Firstly, the present application modifies the surface of superoxide dismutase with polymerizable monomers, and then loads small molecule active ingredients, to construct a polymer-coated core-shell nano-capsule with a certain positive charge on the surface by in-situ polymerization and other methods, to solve the defect that the transdermal effect of superoxide dismutase is not ideal, resulting in low antioxidant effect. The superoxide dismutase-based nano-transdermal delivery system of the present application has a polymer shell and can efficiently penetrate the skin physical barrier.

[0034] Secondly, the application initiates polymerization in situ on the surface of superoxide dismutase through polymerization reaction, and a polymer protective layer is coated on the surface of the enzyme, the polymer protective layer is biocompatible, has little or no irritation to the skin, and during transdermal delivery, the surface properties and thickness of the polymer protective layer can be regulated to effectively regulate the transdermal depth of the delivery system, so that the delivery system can effectively transdermally reach the position where superoxide dismutase and active ingredients need to play a role, and the skin problem can be solved in a targeted manner. The defects of poor delivery efficiency of the previous protein-based delivery system are significantly improved.

[0035] Thirdly, in the delivery system, the small-molecule active ingredients are loaded in superoxide dismutase, and the surface of the superoxide dismutase is coated with a polymer shell layer to form a capsule composite structure, and the capsule composite structure is in the form of nanogel, so that the polymer shell provides protection for superoxide dismutase and small-molecule functional substances with antioxidant effect, the nanogel not only promotes the penetration depth, but also reduces the irritation to the skin, and is suitable for a wider population.

[0036] Fourthly, the raw materials used in the application are biocompatible and can be used as drug reagents, so the transdermal delivery system can be used for the transdermal delivery of beneficial ingredients for the human body such as drugs. The preparation method of the transdermal delivery system of the application is simple, the yield is high, and the system can be produced on a large scale in industry. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is the fitting curve of the penetration depth D of the superoxide dismutase nanogel with positive surface potential and the particle size d at 24 hours.

[0038] Figure 2 is a curve graph of the relative activity of superoxide dismutase with incubation time.

[0039] Figure 3 is the morphology and dispersion of chitosan-super oxide dismutase nanogel (CS-nSOD@VC) in Example 2.

[0040] Figure 4 is the morphology and dispersion of retinol-loaded polyacrylamide-super oxide dismutase nanogel (PAAm-nSOD@R) in Example 3.

[0041] Figure 5 is the morphology and dispersion of polyethylene glycol-super oxide dismutase nanogel (PEG-nSOD@NAA) in Example 4.

[0042] Figure 6 is the penetration of nanogel at 1h, 4h and 24h. DETAILED DESCRIPTION

[0043] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. The following examples facilitate better understanding of the present application, but do not limit the present application. The experimental methods in the following examples are all conventional methods, unless otherwise specified.

[0044] Example 1 Preparation of polyacrylamide-super oxide dismutase nanogel

[0045] Dissolve 2 mg of super oxide dismutase in 2 mL of PBS buffer with pH 7.4 to obtain a 1 mg / mL SOD solution. Add a solution containing 0.4 mg of N-propenyl succinimidyl dimethyl sulfoxide (1% by mass concentration) to 2 mL of the 1 mg / mL SOD solution, mix thoroughly and react for 2 h to obtain super oxide dismutase (aSOD) with polymerizable double bonds on the surface.

[0046] Mix aSOD, acrylamide (AAm), aminopropyl methacrylamide hydrochloride (APM) and N,N'-methylenebisacrylamide (BIS) according to the specified mass ratio, dilute the protein concentration to 1 mg / mL with pH 7.4 phosphate buffer, then add 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per mg of protein as an initiator, initiate in-situ radical polymerization at room temperature, and obtain polyacrylamide-super oxide dismutase nanogel (nSOD) of different diameters after reacting for a specified time. The preparation conditions and the measured final diameters and surface zeta potentials are shown in Table 1.

[0047] Table 1 Preparation conditions and final diameters and surface potentials of series-diameter super oxide dismutase nanogel

[0048]

[0049] 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-super oxide dismutase nanogel (nSOD) larger, and increasing the proportion of APM in the monomer will make the Zeta potential of nSOD more positive.

[0050] Example 1 Transdermal experiment of series-diameter super oxide dismutase nanogel

[0051] The fluorescein-labeled nSOD was obtained by mixing 4 mL of the nSOD nanogels of different diameters in Example 1 at a concentration of 1 mg / mL with 1 mL of fluorescein isothiocyanate at a concentration of 1 mg / mL, and reacting overnight, and removing the unreacted small molecules by dialysis overnight in a dialysis bag with a molecular weight cut-off of 14000 Da. After the fresh pigskin was washed with PBS, the stratum corneum was fixed with the stratum corneum facing up in the transdermal diffusion instrument, and incubated at 37°C. 0.5 mL of the fluorescein-labeled nSOD nanogel solution at a concentration of 1 mg / mL was added on the pigskin tissue, and after 24 hours, the penetration of the nSOD nanogels was observed under a confocal microscope after cryosectioning, and the penetration depth results are shown in Table 2.

[0052] Table 2 Penetration depth of nSOD nanogels of different diameters in 24 hours

[0053]

[0054] The penetration depth of nSOD nanogels of different diameters with positive surface potential (about +5 mV) was found to be that when the particle size was 20 nm to 150 nm, the protein nanogels could penetrate through the epidermis to the vicinity of the dermis in 24 hours, and as the particle size of the nanogels increased, the penetration depth first increased and then decreased; and the nSOD nanogels with a particle size greater than 200 nm were mostly retained in the upper stratum corneum of the epidermis. The penetration depth of nSOD-20+, nSOD-50+, nSOD-100+, and nSOD-150+ was fitted. Figure 1 is the fitting curve of the penetration depth D of nSOD nanogels with positive surface potential and the particle size d in 24 hours. The formula after fitting is as follows:

[0055] D = a1 + m * (a2 * d + a3 * d 2 +a4 * d 3 ), d ∈ [20, 150], wherein the unit of D is μm and the unit of d is nm.

[0056] The parameters m, a1, a2, a3, and a4 in the formula are related to the outer polymer material and the type of protein carrier; wherein a2, a3, and a4 are also related to the diameter of the nanocapsule, and represent that nanocapsules of different diameters have different thicknesses of the polymer shell, so that the influence degree of the protein and the polymer material on transdermal penetration changes. When the type of protein is superoxide dismutase, the formula can be written as:

[0057] D = 100.3 + 2.75d - 0.0297d 2 + 7.13E-5d 3 , d ∈ [20, 150], wherein the unit of D is μm and the unit of d is nm.

[0058] The permeation ability of the superoxide dismutase nanogels with positive surface potential in the range of 20-150 nm in diameter can be preliminarily predicted using the expression. The delivery requirement of skin antioxidant is distributed in the depth range from epidermis to dermis, in which the oxidative stress caused by reactive oxygen species (ROS) in the dermal matrix is a common skin problem, leading to wrinkle formation and abnormal pigmentation. According to the formula, the delivery system required for such skin problems has a diameter of 20 to 100 nm.

[0059] Example 2 Activity retention of series of superoxide dismutase nanogels with different diameters

[0060] The activity of superoxide dismutase was determined by ultraviolet spectrophotometer. 3 mL of Tris-HCl buffer with a concentration of 50 mmol / mL and a pH of 8 (containing 1 mmol / mL of EDTA) was added to the reference cell, 2.4 mL of Tris-HCl buffer with a concentration of 50 mmol / mL and a pH of 8 (containing 1 mmol / mL of EDTA) was added to the sample cell, 0.3 mL of pyrogallol with a concentration of 0.2 mmol / mL, and 0.3 mL of original superoxide dismutase or enzyme nanogel solution. The sample cell was placed in the ultraviolet spectrophotometer, and the change in absorbance at 420 nm was recorded for the first 10 minutes. The slope of the fitted straight line represents the relative activity of superoxide dismutase. The results are shown in Table 3. It is shown that the superoxide dismutase nanogels with a diameter greater than 150 nm have a polymer shell that is too thick to affect the contact between superoxide dismutase and the substrate, or the polymerization conditions cause a loss of superoxide dismutase activity. The activity loss of superoxide dismutase nanogels with a diameter less than 100 nm is within an acceptable range.

[0061] Table 3 Relative activity of nSOD with different diameters

[0062]

[0063] Example 3 Stability test of series of superoxide dismutase nanogels with different diameters

[0064] The activity of superoxide dismutase was determined by UV spectrophotometer. 1 mL of original superoxide dismutase or enzyme nanogel solution with a concentration of 1 mg / mL was incubated with 1 mL of pepsin with a concentration of 0.1 mg / mL in a glycine-hydrochloric acid buffer with a pH of 2.0 at 37°C. After incubation, the sample was taken out and immediately placed on ice. In the reference cell, 3 mL of Tris-HCl buffer with a pH of 8 and a concentration of 50 mmol / mL (containing 1 mmol / mL of EDTA) was added, and in the sample cell, 2.4 mL of Tris-HCl buffer with a pH of 8 and a concentration of 50 mmol / mL (containing 1 mmol / mL of EDTA), 0.3 mL of o-phenanthroline with a concentration of 0.2 mmol / mL, and 0.3 mL of the treated original superoxide dismutase or enzyme nanogel solution with a concentration of 0.5 mg / mL were added, and the UV spectrophotometer was used to record the change in absorbance at 420 nm for the first 10 minutes. The slope of the fitted straight line represents the relative activity of superoxide dismutase. The results are shown in Figure 2 It was found that the original superoxide dismutase was significantly inactivated under the action of protease, while nSOD-20+, nSOD-50+, and nSOD-100+ still had 75% of the activity retained after 30 minutes of treatment, indicating the good protective effect of the polymer shell.

[0065] Example 2 Preparation of superoxide dismutase nanogel loaded with vitamin C

[0066] 6 mL of chitosan solution with a concentration of 1 mg / mL was ultrasonicated at a power of 70 W for 10 min, and was left to stand to prevent excessive heat. Under the condition of a 60°C water bath, a superoxide dismutase solution was added, and the concentration of superoxide dismutase in the solution was adjusted to 2 mg / mL, and the reaction was continued for 5 min. To the mixed solution, 0.5 mL of vitamin C aqueous solution with a concentration of 2 mg / mL was added dropwise under the condition of 400 r / min and a 60°C water bath, and the stirring was continued for 5 min. Finally, 1 mL of sodium tripolyphosphate solution with a concentration of 1 mg / mL was added dropwise under the condition of an ice water bath and 400 r / min, and the reaction was continued for 30 min to obtain chitosan-super oxide dismutase nanogel loaded with vitamin C (CS-nSOD@VC).

[0067] The obtained complex solution was subjected to ultrafiltration treatment in an ultrafiltration centrifuge tube with a molecular weight cut-off of 3 kDa to obtain a high-concentration mother liquor with a concentration of 5.9 mg / mL, which was stored in a cold storage.

[0068] Example 3 Preparation method of superoxide dismutase nanogel loaded with retinol

[0069] To 2 mL of superoxide dismutase solution with a concentration of 1 mg / mL, 0.45 mg of N-propenyl succinimide dissolved in dimethyl sulfoxide was added, and the mixture was fully mixed and reacted for 2 h to obtain superoxide dismutase (aSOD) with a surface modified polymerizable double bond.

[0070] 2 mL of superoxide dismutase solution with a surface modified double bond was taken, and 0.16 mL of 5 mg / mL retinol ethanol solution was added dropwise under stirring at 400 r / min, and after fully mixing, superoxide dismutase loaded with retinol (aSOD@R) was obtained.

[0071] aSOD@R, acrylamide, N-(3-aminopropyl) methacrylamide hydrochloride, and N,N'-methylene bisacrylamide were mixed in a molar ratio of 1:7000:1000:700, the protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer, and 3.8 mg of ammonium persulfate and 7.6 mg of tetramethyl ethylenediamine per milligram of protein were added as an initiator, and in-situ radical polymerization was initiated at room temperature. After reacting for 4 h, polyacrylamide-super oxide dismutase nanogel loaded with retinol (PAAm-nSOD@R) was obtained.

[0072] The obtained complex solution was subjected to ultrafiltration treatment in an ultrafiltration centrifuge tube with a molecular weight cut-off of 3 kDa to obtain a high-concentration mother liquor with a concentration of 6.2 mg / mL, which was stored in a cold storage.

[0073] Example 4 Preparation method of superoxide dismutase nanogel loaded with nicotinamide

[0074] To 2 mL of superoxide dismutase solution with a concentration of 1 mg / mL, 0.38 mg of N-propenyl succinimide dissolved in dimethyl sulfoxide was added, and the mixture was fully mixed and reacted for 2 h to obtain superoxide dismutase (aSOD) with a surface modified polymerizable double bond.

[0075] 2 mL of superoxide dismutase solution with a surface modified double bond was taken, and 0.16 mL of 5 mg / mL retinol ethanol solution was added dropwise under stirring at 400 r / min, and after fully mixing, superoxide dismutase loaded with retinol (aSOD@R) was obtained.

[0076] The aSOD@NAA, polyethylene glycol methyl ether acrylate and N,N'-methylene bisacrylamide were mixed in a ratio of 1:6600:600 by mass, 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 tetramethyl ethylenediamine were added per milligram of protein as an initiator, and radical polymerization was initiated in situ at room temperature. After 4 h of reaction, the nicotinamide-loaded polyethylene glycol-superoxide dismutase nanogel (PEG-nSOD@NAA) was obtained.

[0077] The obtained complex solution was subjected to ultrafiltration treatment in an ultrafiltration centrifuge tube with a molecular weight cut-off of 3 kDa, and a high-concentration mother liquor with a concentration of 5.5 mg / mL after purification was obtained and stored in a cold storage.

[0078] The diameter and surface potential of the nanogel were characterized by dynamic light scattering (DLS): the protein nanogel solutions of Examples 2, 3 and 4 with a concentration of 1 mg / mL were added to the sample cell, and the particle size and Zeta potential of the nanogel in the system were tested at room temperature using a Malvern Nano Zs90 nanoparticle size potential instrument. The results are shown in Table 4.

[0079] Table 4 Particle size and Zeta potential of superoxide dismutase nanogel

[0080]

[0081]

[0082] The morphology of the nanogel was characterized by transmission electron microscopy (TEM): the morphology and dispersion of the chitosan-super oxide dismutase nanogel (CS-nSOD@VC) were observed under a transmission electron microscope, and the results are shown in Figure 3 The morphology and dispersion of the polyacrylamide-super oxide dismutase nanogel (PAAm-nSOD@R) were observed, and the results are shown in Figure 4 The morphology and dispersion of the polyethylene glycol-super oxide dismutase nanogel (PEG-nSOD@NAA) were observed, and the results are shown in Figure 5 .

[0083] Application Example 4 Release of active ingredients

[0084] Take 2 mL of superoxide dismutase nanogel with a concentration of 5 mg / mL 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 oscillate the entire system at 37°C. The release time is 48 h. Within 48 h, sample at regular intervals to measure the concentration of loaded small molecule drugs under ultraviolet spectrophotometry, and calculate the release of the complex system to small molecule drugs. The results are statistically summarized in Table 5. The superoxide dismutase nanogel can slowly release small molecules within 24 hours, which is conducive to the sustained release of active small molecules when reaching the deep layer of the skin during transdermal use, thereby efficiently exerting physiological effects.

[0085] Table 5 Release of superoxide dismutase nanogel to small molecule drugs

[0086]

[0087] Application Example 6 Stimulus attenuation effect of nanogel on active ingredients

[0088] Chitosan-super oxide dismutase nanogel (CS-nSOD@VC) attenuation effect on vitamin C stimulation determination: The final concentration of vitamin C in the system is unified, and the pH values of the chitosan-super oxide dismutase nanogel (CS-nSOD@VC) solution loaded with vitamin C and the vitamin C aqueous solution are measured by a pH meter to characterize the overall stimulation degree. The statistical results are shown in Table 6. The results show that under the same concentration of vitamin C, the acidity of the vitamin C system wrapped by the super oxide dismutase nanogel is significantly weaker than that of the free vitamin C aqueous solution, and too strong acidity will lead to stimulation to the skin. It is indicated that the super oxide dismutase nanogel can effectively reduce the stimulation of vitamin C while delivering vitamin C, and is suitable for a wider population.

[0089] Table 6 Attenuation effect of super oxide dismutase nanogel on stimulation of vitamin C

[0090]

[0091] Application Example 7 Protection effect of nanogel on active ingredients

[0092] Polyacrylamide-super oxide dismutase nanogel (PAAm-nSOD@R) protection effect on retinol determination: The retinol-loaded enzyme nanogel solution and free retinol solution were placed at room temperature for 1-7 days, and the absorbance at 325 nm was measured by ultraviolet spectrophotometry to characterize the retinol content, and the ratio of unoxidized retinol content to the initial embedding amount was calculated. The statistical results are shown in Table 7.

[0093] Table 7 Retinol retention

[0094]

[0095]

[0096] As can be seen from the data in Table 7, the retinol can be significantly reduced by the superoxide dismutase nanogel delivery system, i.e. PAAm-nSOD@R solution, of the application.

[0097] Example 8 Characterization of antioxidant performance of nanogel

[0098] The ROS kit was used to detect the ROS level in human epidermal cells HaCaT. HaCaT cells (1*10 4 After 12 h of complete cell adhesion, the cells were irradiated by a UVB ultraviolet radiation instrument to induce the production of internal ROS, and cells without ultraviolet radiation treatment were used as negative controls. After the irradiation treatment, the original culture medium in all the wells was removed and 100 μL of serum-free culture medium was added (the culture medium did not contain serum in order to avoid the influence of serum on the uptake of the complex by the cells in the subsequent drug addition process). The positive control group was added with 10 μL of PBS buffer, the low concentration experimental group was added with 10 μL of complex solution with a concentration of 0.01 mg / mL, the medium concentration experimental group was added with 10 μL of complex solution with a concentration of 0.02 mg / mL, and the high concentration experimental group was added with 10 μL of complex solution with a concentration of 0.04 mg / mL. After 24 h of incubation, the original culture medium of all the cells was removed and 100 μL of serum-free culture medium and 10 μL of DCFH-DA culture solution were added, and the mixture was incubated at 37°C in the dark for 20 min. Then the cells were washed with PBS three times, and the fluorescence intensity of DCF was detected by a microplate reader. The results are shown in Table 8.

[0099] Table 8 Antioxidant capacity of superoxide dismutase nanogel in cells increases with increasing concentration

[0100]

[0101]

[0102] It can be seen that CS-nSOD@VC and PAAm-nSOD@R exhibit better antioxidant capacity than PEG-nSOD@NAA, which may be due to the positive charge on the surface of the nanocapsule, which makes it easier to enter the cells and play a physiological role. The antioxidant effect of CS-nSOD@VC is stronger than that of PAAm-nSOD@R, which may be due to the excellent antioxidant property of vitamin C itself, which plays a synergistic antioxidant function together with superoxide dismutase.

[0103] Example 9 Transdermal experiment

[0104] The superoxide dismutase nanogel solution was mixed with excess FITC overnight, and the unreacted small molecules were removed by dialysis to obtain a fluorescently labeled complex. Fresh pigskin was washed with PBS and the stratum corneum was fixed upside down in a transdermal diffusion instrument and incubated at 37°C. 0.5 mL of the fluorescently labeled complex solution was added on top of the pigskin tissue, and frozen sections were taken at 24 h, 4 h and 1 h time points to observe the penetration of the nanogel under a confocal microscope, as shown in Figure 6 The results of the penetration depth are shown in Table 9. The results show that the superoxide dismutase nanogel can effectively penetrate the skin barrier to reach the deep skin and play an antioxidant role at 24 hours.

[0105] Table 9 Transdermal penetration of superoxide dismutase nanogel

[0106]

Claims

1. A superoxide dismutase based nanotransdermal delivery system, characterized in that, The nano-transdermal delivery system has a capsule composite structure of a small molecule active ingredient-super oxide dismutase-polymer shell layer, and a diameter of 20-100 nm, wherein the small molecule active ingredient is loaded in the super oxide dismutase, the surface of the super oxide dismutase is coated by the polymer shell layer, and the thickness of the polymer shell layer is 7.5-52.5 nm; the small molecule active ingredient is selected from carotenoids, hesperidin, vitamin C, vitamin E, resveratrol, lycopene, retinol nicotinamide, ascorbic acid or alpha-arbutin. The super oxide dismutase-based nano-transdermal delivery system is obtained by a preparation method comprising the following steps: Method 1: in-situ polymerization (S1) adding a surface modification monomer solution to a buffer solution of super oxide dismutase to obtain a surface-modified protein solution; (S2) adding a small molecule active ingredient solution to the surface-modified protein solution obtained in step (S1), so that the small molecule active ingredient is adsorbed in the super oxide dismutase to obtain a surface-modified super oxide dismutase solution loaded with the small molecule active ingredient; (S3) adding a functional monomer, a positive charge monomer, a crosslinking agent and an initiator to the surface-modified super oxide dismutase solution loaded with the small molecule active ingredient obtained in step (S2) to initiate in-situ polymerization, thereby obtaining the super oxide dismutase-based nano-transdermal delivery system; The surface modification monomer is selected from at least one of N-acrylsuccinimide and N-(3-aminopropyl) methacrylamide hydrochloride; the functional monomer is selected from acrylamide or polyethylene glycol methyl ether acrylate; and the crosslinking agent is N,N'-methylene bisacrylamide. Method 2: electrostatic adsorption adding a polymer solution with positive charges to a super oxide dismutase solution at 50-70 ℃, stirring, adding a small molecule active ingredient solution at 50-70 ℃, continuing to stir, adding a salt solution under ice water bath conditions, the salt solution being a sodium tripolyphosphate solution, reacting, ultrafiltration centrifugation, thereby obtaining the super oxide dismutase-based nano-transdermal delivery system; and the polymer with positive charges is chitosan.

2. The superoxide dismutase-based nanotransdermal delivery system according to claim 1, wherein, The super oxide dismutase-based active ingredient transdermal delivery system has a diameter of 30-80 nm and a polymer shell layer thickness of 10-35 nm.

3. The superoxide dismutase based nano-transdermal delivery system according to claim 1, wherein, The surface Zeta potential of the transdermal delivery system is +4 to +7 mV.

4. The superoxide dismutase based nano-transdermal delivery system according to claim 1, wherein, The mass ratio of the small molecule active ingredient to the super oxide dismutase is 1:1-100.

5. The superoxide dismutase-based nanotransdermal delivery system according to claim 4, wherein, The mass ratio of the small molecule active ingredient to the super oxide dismutase is 1:1-5.

6. The superoxide dismutase based nano-transdermal delivery system according to claim 1, wherein, The initiator is a water-soluble initiator.

7. The superoxide dismutase-based nanotransdermal delivery system according to claim 6, wherein, The water-soluble initiator is selected from at least one of ammonium persulfate, sodium persulfate and potassium persulfate.

8. The superoxide dismutase based nano-transdermal delivery system according to claim 6, wherein, An assistant initiator is further added.

9. The superoxide dismutase-based nanotransdermal delivery system according to claim 8, wherein, The assistant initiator is selected from sodium bisulfite and tetramethyl ethylenediamine.

10. The superoxide dismutase based nano-transdermal delivery system according to claim 6, wherein, The mass ratio of the super oxide dismutase to the surface modification monomer is 1-10:1; the molar ratio of the super oxide dismutase to the functional monomer is 1:4500-25000; the molar ratio of the functional monomer to the crosslinking agent is 1:0.05-0.1; and the molar ratio of the functional monomer to the positive charge monomer is 1:0.1-0.

12.

11. The superoxide dismutase-based nanotransdermal delivery system according to claim 10, wherein, The mass ratio of the super oxide dismutase to the surface modification monomer is 2-5:

1.

12. The superoxide dismutase-based nanotransdermal delivery system according to claim 10, wherein, The molar ratio of superoxide dismutase to functional monomer is 1:6000-10000.

13. The superoxide dismutase based nano-transdermal delivery system according to claim 1, wherein, In method 1, in step (S1), the buffer solution is a phosphate buffer solution, the solvent of the surface modification monomer solution is at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, and the concentration is 1-5 wt%; and / or In step (S2), the solvent of the small molecule active ingredient solution is at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, and the concentration of the small molecule active ingredient solution is 1-5 mg / mL; the polymerization is room temperature polymerization, and the polymerization time is 6-12 h. In method 2, the concentration of the positively charged polymer solution, the concentration of the superoxide dismutase solution, and the concentration of the small molecule active ingredient aqueous solution are independently 1-10 mg / mL, and the concentration of the sodium tripolyphosphate solution is 0.5-5 mg / mL; the molecular weight cut-off of the ultrafiltration is 3-5 kDa.

14. The superoxide dismutase-based nanotransdermal delivery system according to claim 13, wherein, The mass ratio of superoxide dismutase, positively charged polymer, small molecule active ingredient, and salt in the salt solution is 1:0.5-1:0.05-0.1:0.05-0.

1.

15. Use of the superoxide dismutase-based nanotransdermal delivery system according to any one of claims 1-14 in the preparation of a medicament for preventing or treating oxidative stress-related diseases.

16. Use according to claim 15, characterized in that, The oxidative stress-related diseases include skin photoaging or chloasma eczema. The oxidative stress-related diseases include skin photoaging or chloasma eczema.

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