Nano transdermal delivery system based on superoxide dismutase and preparation method thereof

Abstract

The invention provides a nano transdermal delivery system based on superoxide dismutase, the nano transdermal delivery system is of a capsule composite structure of a small molecule active component-superoxide dismutase-polymer shell layer, the small molecule active component is loaded in superoxide dismutase, the surface of the superoxide dismutase is coated by the polymer shell layer, and the superoxide dismutase is loaded in the capsule composite structure. The diameter of the superoxide dismutase-based active ingredient transdermal delivery system is 20-100 nm, and the thickness of the polymer shell layer is 7.5-52.5 nm. In the delivery system, the polymer shell layer provides a protection effect for the superoxide dismutase and the micromolecular functional substances with the anti-oxidation effect, the capsule composite structure is in a nanogel form, and the nanogel not only promotes the penetration depth of the capsule, but also reduces the irritation effect of the capsule on the skin. The raw materials are biologically friendly, the preparation method is simple, the yield is high, and industrial large-scale production can be realized.

Description

Technical Field

[0001] The invention belongs to a nano transdermal delivery system, and in particular relates to a nano transdermal delivery system based on superoxide dismutase and a preparation method thereof. Background Art

[0002] Superoxide dismutase is a metalloenzyme widely distributed in organisms. It plays an antioxidant role by scavenging superoxide anion free radicals, protecting organisms from oxidative stress damage, and thus has attracted attention as a highly effective antioxidant for the skin. However, in practical applications, direct application of superoxide dismutase on the skin surface for antioxidant management or treatment often does not produce good results, which indicates that the transdermal delivery efficiency is not high and the bioavailability of superoxide dismutase is low.

[0003] Transdermal delivery has the advantages of being convenient and non-invasive, locally targeted, and avoiding the extreme pH environment of the gastrointestinal tract. It is the most commonly used method of drug administration in the management of skin problems. Oxidative stress is the cause of a variety of skin problems including skin inflammation, excessive skin oil secretion, dull skin tone, wrinkles, etc. Therefore, the delivery of antioxidants to the skin is beneficial for solving a variety of skin problems. However, the multi-layered complex structure of the skin creates high-efficiency barrier problems including physical barriers, chemical barriers, and immune barriers. The active substances delivered transdermally are often isolated outside the stratum corneum of the outermost layer of the skin, making it difficult to reach the deep layer and exert their effects. Simply increasing the concentration of active substances is not very helpful in promoting penetration, but may cause irritation to the skin. In addition, for different skin problems, the depth of oxidative stress is different, and the depth may involve the epidermis, the junction of the epidermis and the dermis, and the dermis matrix, so the depth of transdermal delivery is particularly important.

[0004] The existing clinical or commercial transdermal delivery technology is still in its early stages of development, and the most commonly used ones are chemical permeation enhancers and liposome loading. Common permeation enhancers such as ethanol not only have a certain effect on the biological activity of the delivered substance, but also face greater skin irritation and allergy risks during use because their permeation-enhancing principle is to change the skin structure and weaken the barrier function. The diameter of the vesicle structure formed by liposome encapsulation is generally greater than 200nm, and the system is in a thermodynamically metastable state, which is difficult to meet the requirements of long-term use and storage. Summary of the invention

[0005] In order to solve the defects of low delivery efficiency and uncontrollable delivery depth in the existing skin antioxidant transdermal technology, the present invention uses superoxide dismutase molecules as nano templates, constructs an efficient transdermal delivery system through polymer encapsulation, and uses the hydrophobic structure inside the superoxide dismutase to load functional small molecules for co-delivery, so as to achieve synergistic effects of various physiological functions including antioxidant. The system uses modified superoxide dismutase nanogel as a carrier. After the functional small molecules are loaded in the nanogel, they are efficiently transdermally transported to the deep layer of the skin and released in a controlled manner, playing a synergistic therapeutic role on various problems such as skin oxidation and inflammation. In this system, superoxide dismutase itself has an antioxidant effect and can also be used as a carrier of functional small molecules; the polymer shell of the outer layer of superoxide dismutase has a customizable diameter and surface potential, which not only helps the internal superoxide dismutase and functional small molecules to pass through the skin barrier, but also protects the stability of superoxide dismutase and functional small molecules, which is conducive to the long-term use and storage of the preparation. The excellent penetration efficiency, controllable surface properties and diameter of nanogels make the delivery depth of the transdermal delivery system controllable. Superoxide dismutase and small molecule active ingredients target the oxidative stress of the skin and exert their effects, so that a comprehensive improvement of various skin problems can be achieved with lower concentrations of active ingredients. In addition, nanogels can effectively avoid the irritation of some active ingredients to the skin, and are more applicable to people with different skin conditions. This loading method is universal for small molecule ingredients with hydrophobic structures.

[0006] Specifically, the present invention 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 20-100nm, wherein the small molecule active ingredient is loaded in the superoxide dismutase, the surface of the superoxide dismutase is coated by the polymer shell layer, and the thickness of the polymer shell layer is 7.5-52.5nm.

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

[0009] Furthermore, 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, delivery efficiency and stability of transdermal delivery.

[0010] The small molecule active ingredients are selected from carotenoids, hesperidin, vitamin C, vitamin E, resveratrol, lycopene, retinol nicotinamide, ascorbic acid, α-arbutin or derivatives of the foregoing substances. The present invention can select different small molecule active ingredients for different anti-oxidation 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 wrinkles, and nicotinamide, ascorbic acid, α-arbutin, etc. for skin color and spot problems caused by excessive pigmentation.

[0011] Furthermore, 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 protein molecule core 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 invention, polymers that are conducive to forming a hydration layer on the outer layer of the nanogel are selected, including monomers that are conducive to forming hydrogen bonds, monomers that are amphiphilic and can naturally escape immune recognition, and positively charged monomers and negatively charged monomers can also be selected to copolymerize together to form a positive dipole balance inside the polymer, thereby forming a strong hydration layer, such as phosphorylcholine polymers, betaine polymers, etc. Positively charged polymers can also be selected to wrap around superoxide dismutase molecules through electrostatic interactions, such as chitosan, etc.

[0013] The polymer shell not only helps the internal superoxide dismutase and functional small molecules to pass through the skin barrier, but also protects the stability of superoxide dismutase and functional small molecules, which is helpful for the long-term use and storage of the preparation. Therefore, the thickness regulation of the shell layer is very important in this system. The permeability of the nano transdermal delivery system is related to the particle size. Too thin or too thick a shell layer is not conducive to the deep delivery of nano gels; and the protective effect of the polymer shell layer on the activity of active proteins is also related to the thickness of the shell layer. Too thin a shell layer has poor ability to resist drastic changes in ambient temperature and pH; and the premise for the enzyme to play a catalytic role is to contact with the substrate, and too thick a shell layer will affect the transmission of the substrate, thereby hindering the active protein from playing a catalytic role. In the present invention, a series of diameters and shell layers of different thicknesses are prepared and investigated for the effects of transdermal depth and protein or enzyme activity protection. It is found that the diameter of the gel suitable for the treatment of skin inflammation is 20-100nm, preferably 30-80nm. Since the diameter of superoxide dismutase is about 5nm, the thickness of the polymer shell layer should be controlled at 7.5-47.5nm, preferably 10-35nm. The thickness of the polymer shell can be controlled mainly 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 the in-situ polymerization method, the monomer 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-methacryloyloxyethyl phosphorylcholine, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl)ammonium hydroxide, 2-hydroxyethyl acrylate, acrylate, 2-(dimethylamino)ethyl methacrylate, (3-acrylamidopropyl)trimethylammonium chloride hydrochloride, aminopropyl methacrylamide, N,N'-methylenebisacrylamide, dimethacrylate glyceryl, 1,3-diglycerol alcohol diacrylate; Preferably, the acrylate is selected from at least one of methyl (meth)acrylate, ethyl (meth)acrylate, hydroxyethyl (meth)acrylate, propyl (meth)acrylate, and butyl (meth)acrylate; the polymer is coated on the surface of the protein by electrostatic action and is a positively or negatively charged polymer, wherein the positively charged polymer is selected from at least one of poly(propyleneamine hydrochloride), poly(L-lysine), polyethyleneimine, poly(L-histidine), polymethacrylic acid N,N-dimethylaminoethyl ester, polymethacrylamide propyltrimethylammonium chloride, and natural or synthetic polysaccharides such as chitosan; the negatively charged polymer is selected from at least one of polyacrylic acid, polystyrene sulfonate, alginate, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, polymethacrylic acid, oxidized cellulose, carboxymethyl cellulose, polyaspartic acid, and polyglutamic acid.

[0015] Preferably, the polymer is a carbon-carbon unsaturated double bond monomer that is coated on the protein surface in situ polymerization. The polymer raw materials include surface modification monomers, functional monomers, crosslinking agents and initiators. Preferably, positively charged monomers are also added; the surface modification monomer is selected from at least one of N-propylene succinimide, N-(3-aminopropyl) methacrylamide hydrochloride, and maleimide. The role of the surface modification monomer is to modify the monomer on the surface of the protein through interactions such as electrostatic adsorption and chemical bonding; the functional monomer is selected from at least one of vinyl pyrrolidone, acrylamide, polyethylene glycol methyl ether acrylate, 2-methacryloyloxyethyl phosphorylcholine, and 2-hydroxyethyl acrylate; the positively charged monomer is selected from [ At least one of 2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide, 2-(dimethylamino)ethyl methacrylate, (3-acrylamidopropyl)trimethylammonium chloride hydrochloride, and aminopropyl methacrylamide hydrochloride; the crosslinking agent is selected from at least one of N,N'-methylenebisacrylamide, dimethacrylate glycerol, and 1,3-diglycerol alcohol diacrylate; the initiator is a water-soluble initiator, specifically selected from at least one of ammonium persulfate, sodium persulfate, and potassium persulfate. Preferably, a co-initiator is also added, such as sodium bisulfite and tetramethylethylenediamine. 2-5 mg of initiator and 6-10 mg of co-initiator are added per mg of protein; preferably, 3.5-4 mg of initiator and 7.5-8 mg of co-initiator are added per mg of protein.

[0016] After the superoxide dismutase is modified by the surface modification monomer, the surface has a polymerizable carbon-carbon unsaturated double bond. In a specific embodiment of the present invention, the surface modification method includes mixing the superoxide dismutase with a buffer solution or an organic solvent to prepare a superoxide dismutase solution, adding the surface modification monomer to the superoxide dismutase solution, and the surface modification monomer can be attached to the surface of the protein molecule by electrostatic interaction, covalent crosslinking, etc., to obtain a surface modified protein molecule solution.

[0017] Furthermore, the mass ratio of superoxide dismutase to surface modification monomer is 1-10:1, preferably 2-5:1; the molar ratio of superoxide dismutase to functional monomer is 1:4500-25000, and the molar ratio of functional monomer to cross-linking agent is 1:0.05-0.1; if the polymer raw material also includes a positively charged monomer, the molar ratio of the functional monomer to the positively charged monomer is 1:0.1-0.12.

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

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

[0020] Method 1: In-situ polymerization

[0021] (S1) adding a surface-modified 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), wherein the small molecule active ingredient is adsorbed on the superoxide dismutase, thereby obtaining a surface-modified superoxide dismutase solution loaded with the small molecule active ingredient;

[0023] (S3) adding functional monomers, positively charged monomers, crosslinking agents and initiators to the surface-modified superoxide dismutase solution loaded with small molecule active ingredients obtained in step (S2) to initiate in situ polymerization to obtain a nano-transdermal delivery system based on superoxide dismutase;

[0024] Method 2: Electrostatic adsorption method:

[0025] Add superoxide dismutase solution to a positively or negatively charged polymer solution at 50-70°C (e.g. 60°C) and stir, add small molecule active ingredient solution at 50-70°C, continue stirring, add saline solution in an ice water bath, react for 30-60 minutes, and ultrafilter and centrifuge to obtain a nano transdermal delivery system based on superoxide dismutase.

[0026] Further, in method 1, in step (S1), the buffer solution is a phosphate buffer solution, specifically a PBS buffer solution with a pH of 7.4-7.6, and the protein concentration in the protein solution is 1-5 mg / mL; the solvent of the surface modification monomer solution is at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, and the concentration is 1-5wt%; 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, that 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, such as 2 mg / mL, 3 mg / mL, and 4 mg / mL. The amount of the small molecule active ingredient solution is such that the mass ratio of superoxide dismutase to the small molecule active ingredient is 1-5:1.

[0028] Furthermore, the molar ratio of superoxide dismutase, functional monomer, positively charged monomer and crosslinking agent is 1:4500-10000:600-1000:500-1000; the initiator is a water-soluble initiator, specifically selected from at least one of ammonium persulfate, sodium persulfate and potassium persulfate, preferably, a co-initiator is also added, such as sodium bisulfite and tetramethylethylenediamine. 2-5 mg of initiator and 6-10 mg of co-initiator are added per milligram of protein; preferably, 3.5-4 mg of initiator and 7.5-8 mg of co-initiator are added per milligram of protein; the initiated polymerization is room temperature polymerization, and the polymerization time is 6-12 hours, preferably 6-8 hours.

[0029] Furthermore, in method 2, the concentration of the positively or negatively 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. When the polymer is positively charged, the salt solution is a salt solution that can provide anions to induce the 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 that can provide cations to induce the 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. Furthermore, the mass ratio of superoxide dismutase, the positively or negatively charged polymer, the small molecule active ingredient, and the 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] Furthermore, in method 2, the molecular weight cut-off of ultrafiltration is 3-5 kDa.

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

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

[0033] 1. The present invention first modifies a polymerizable monomer on the surface of superoxide dismutase, then loads a small molecule active ingredient, and constructs a core-shell structure nanocapsule with a polymer coating having a certain positive charge on the surface by means including in situ polymerization, so as to solve the defect that the superoxide dismutase transdermal effect is not ideal and leads to a low antioxidant effect. The nano transdermal delivery system based on superoxide dismutase of the present invention has a polymer shell and can efficiently penetrate the skin physical barrier.

[0034] Second, the present invention initiates polymerization reaction in situ on the surface of superoxide dismutase through polymerization reaction, and coats the surface of the enzyme with a polymer protective layer, which is bio-friendly and has no or little irritation to the skin. During the transdermal delivery process, by regulating the surface properties and thickness of the polymer protective layer, the transdermal depth of the delivery system can be effectively regulated, so that the delivery system as a whole effectively penetrates the skin to the position where superoxide dismutase and active ingredients need to play a role, and solves skin problems in a targeted manner. The defect of poor delivery efficiency of previous protein-based delivery systems has been significantly improved.

[0035] 3. In this delivery system, small molecule active ingredients are loaded in superoxide dismutase, and the surface of superoxide dismutase is coated by a polymer shell to form a capsule composite structure, which is in the form of nanogel. Therefore, the polymer shell provides protection for superoxide dismutase and small molecule functional substances with antioxidant effects. The nanogel not only promotes its penetration depth, but also reduces its irritation to the skin, and is suitable for a wider range of people.

[0036] Fourth, the raw materials used in the present invention are all bio-friendly and can be used as drug reagents, so they can be used in transdermal delivery systems of drugs and other ingredients beneficial to the human body. The preparation method of the transdermal delivery system of the present invention is simple, has a high yield, and can be industrialized and mass-produced. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

[0043] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be described in detail below. The following examples are convenient for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified.

[0044] Example 1 Preparation of polyacrylamide-superoxide dismutase nanogel

[0045] 2 mg of superoxide dismutase was dissolved in 2 mL of PBS buffer at pH 7.4 to obtain a 1 mg / mL SOD solution. A solution of dimethyl sulfoxide containing 0.4 mg of N-propylene succinimide (mass concentration 1%) was added to 2 mL of the 1 mg / mL SOD solution, and the mixture was thoroughly mixed and reacted for 2 h to obtain a superoxide dismutase (aSOD) with a polymerizable double bond modified on the surface.

[0046] aSOD, acrylamide (AAm), aminopropyl methacrylamide hydrochloride (APM) and N,N'-methylenebisacrylamide (BIS) were mixed according to the specified molar ratio, and the protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine were added to the initiator according to each mg of protein. In situ free radical polymerization was initiated at room temperature. After the specified reaction time, polyacrylamide-superoxide dismutase nanogels (nSOD) of different diameters were obtained. The preparation conditions and the measured final diameter and surface zeta potential are shown in Table 1.

[0047] Table 1 Preparation conditions and final diameters and surface potentials of superoxide dismutase nanogels with a series of diameters

[0048]

[0049] It can be seen from Table 1 that increasing the amount ratio of monomer to protein and increasing the polymerization time will increase the particle size of the obtained polyacrylamide-superoxide dismutase nanogel (nSOD), and increasing the proportion of APM in the monomer will make the Zeta potential of nSOD more positive.

[0050] Application Example 1 Series of diameter superoxide dismutase nanogel transdermal experiments

[0051] 4mL of the superoxide dismutase nanogel nSOD of different diameters in Example 1 with a concentration of 1mg / mL was mixed with 1mL of fluorescein isothiocyanate with a concentration of 1mg / mL and reacted overnight, and dialyzed overnight in a dialysis bag with a molecular weight cutoff of 14000Da to remove unreacted small molecules to obtain fluorescently labeled nSOD. After fresh pigskin was washed with PBS, the stratum corneum was fixed upward in a transdermal diffusion instrument and kept warm at 37°C. 0.5mL of fluorescently labeled superoxide dismutase nanogel solution with a concentration of 1mg / mL was dripped onto the pigskin tissue, and frozen sections were performed after 24 hours. The penetration of the nanogel was observed under a confocal microscope, and the results of the statistical penetration depth are shown in Table 2.

[0052] Table 2 24-hour skin penetration depth of superoxide dismutase nanogels with different diameters

[0053]

[0054] By comparing the penetration depths of superoxide dismutase nanogels of different diameters with positive surface potential (about +5mV), it was found that when the particle size was between 20nm and 150nm, the protein nanogel could penetrate the epidermis and reach the vicinity of the dermis within 24 hours, and as the gel particle size increased, the penetration depth first increased and then decreased; while superoxide dismutase nanogels larger than 200nm mostly stayed in the stratum corneum above the epidermis. The penetration depths of nSOD-20+, nSOD-50+, nSOD-100+, and nSOD-150+ were fitted. Figure 1 is the fitting curve of the penetration depth D and particle size d of the superoxide dismutase nanogel with positive surface potential at 24 hours. The fitting formula is as follows:

[0055] D=a 1 +m*(a 2 *d+a 3 *d 2 +a 4 *d 3 ),d∈[20,150], where the unit of D is μm and the unit of d is nm.

[0056] The parameters m and a in the formula 1 , a 2 , a 3 , a 4 It is related to the polymer outer layer material and the type of protein carrier; among them, a 2 , a 3 , a 4 It is also related to the diameter of the nanocapsule, which means that nanocapsules of different diameters have polymer shells of different thicknesses, which changes the degree of influence of the protein and polymer materials on transdermal ...

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

[0058] This expression can be used to preliminarily predict the permeability of superoxide dismutase nanogels with a particle size range of 20-150nm and a positive surface potential. The delivery requirements of skin antioxidants are distributed in the depth range from the epidermis to the dermis. Among them, oxidative stress caused by reactive oxygen species ROS in the dermal matrix is ​​a common skin problem, which leads to wrinkles and abnormal pigmentation. According to the formula, the diameter of the delivery system required for such skin problems is preferably 20 to 100nm.

[0059] Application Example 2 Series of Diameter Superoxide Dismutase Nanogel Activity Retention

[0060] Ultraviolet spectrophotometer was used to measure the activity of superoxide dismutase. 3 mL of Tris-HCl buffer (containing 1 mmol / mL of EDTA) with a pH of 8 and a concentration of 50 mmol / mL was added to the reference pool, 2.4 mL of Tris-HCl buffer (containing 1 mmol / mL of EDTA) with a concentration of 50 mmol / mL was added to the sample pool, 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 were added, and the sample pool was placed in an ultraviolet spectrophotometer, and the change of the absorbance of the sample pool at 420 nm was recorded in the first 10 minutes. The slope of the fitted straight line was taken to represent the relative activity of superoxide dismutase. The results are shown in Table 3. It is shown that the superoxide dismutase nanogel with a diameter greater than 150 nm has a polymer shell that is too thick and will affect the contact of superoxide dismutase with the substrate, or the polymerization conditions cause the loss of superoxide dismutase activity. The activity loss of superoxide dismutase nanogels with a diameter less than 100 nm was within an acceptable range.

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

[0062]

[0063] Application Example 3 Series Diameter Superoxide Dismutase Nanogel Stability Test

[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 at 37°C in glycine-HCl buffer with a pH of 2.0 for different time points. After incubation, the samples were removed and immediately placed on ice. Add 3 mL of 50 mmol / mL Tris-HCl buffer (containing 1 mmol / mL EDTA) at pH 8 to the reference pool, add 2.4 mL of 50 mmol / mL Tris-HCl buffer (containing 1 mmol / mL EDTA) at pH 8 to the sample pool, 0.3 mL of 0.2 mmol / mL pyrogallol, and 0.3 mL of treated 0.5 mg / mL original superoxide dismutase or enzyme nanogel solution, put it in a UV spectrophotometer, record the change in absorbance of the sample pool at 420 nm in the first 10 minutes, and take the slope of the fitted straight line to represent the relative activity of superoxide dismutase. The results are as follows: Figure 2 It was found that the original superoxide dismutase was significantly inactivated by the protease, while nSOD-20+, nSOD-50+, and nSOD-100+ still retained 75% of their activity 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] Take 6mL of chitosan solution with a concentration of 1mg / mL and ultrasonicate it at 70W power for 10min, and let it stand to prevent excessive heat. Under 60℃ water bath conditions, add superoxide dismutase solution, and adjust the superoxide dismutase concentration in the solution to 2mg / mL, and react for 5min. To the mixed solution, add 0.5mL of vitamin C aqueous solution with a concentration of 2mg / mL under 400r / min and 60℃ water bath conditions, and continue stirring for 5min. Finally, under ice water bath and 400r / min conditions, add 1mL of sodium tripolyphosphate solution with a concentration of 1mg / mL dropwise, and react for 30min to obtain chitosan-superoxide dismutase nanogel loaded with vitamin C (CS-nSOD@VC).

[0067] The obtained complex solution was ultrafiltered in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa to obtain a purified high-concentration mother solution with a concentration of 5.9 mg / mL, which was stored in a refrigerator.

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

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

[0070] Take 2 mL of a 1 mg / mL surface double-bond modified superoxide dismutase solution, add 0.16 mL of a 5 mg / mL retinol ethanol solution dropwise thereto while stirring at 400 r / min, and mix thoroughly to obtain retinol-loaded superoxide dismutase (aSOD@R).

[0071] aSOD@R, acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride and N,N'-methylenebisacrylamide were mixed in a molar ratio of 1:7000:1000:700, and the protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine were added as initiators per mg of protein, and in situ free radical polymerization was initiated at room temperature. After 4 hours of reaction, retinol-loaded polyacrylamide-superoxide dismutase nanogel (PAAm-nSOD@R) was obtained.

[0072] The obtained complex solution was ultrafiltered in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa to obtain a purified high-concentration mother solution with a concentration of 6.2 mg / mL, which was stored in a refrigerator.

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

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

[0075] Take 2 mL of a 1 mg / mL surface double-bond modified superoxide dismutase solution, add 0.65 mL of a 1.8 mg / mL nicotinamide ethanol solution dropwise thereto while stirring at 450 r / min, and mix thoroughly to obtain superoxide dismutase loaded with nicotinamide (aSOD@NAA).

[0076] aSOD@NAA, polyethylene glycol methyl ether acrylate and N,N'-methylenebisacrylamide were mixed in a molar ratio of 1:6600:600, and the protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine were added to the initiator per mg of protein, and in situ free radical polymerization was initiated at room temperature. After 4 hours of reaction, nicotinamide-loaded polyethylene glycol-superoxide dismutase nanogel (PEG-nSOD@NAA) was obtained.

[0077] The obtained complex solution was ultrafiltered in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa to obtain a purified high-concentration mother solution with a concentration of 5.5 mg / mL, which was stored in a refrigerator.

[0078] Characterization of nanogel diameter and surface potential by dynamic light scattering (DLS): The protein nanogel solution with a concentration of 1 mg / mL of Examples 2, 3, and 4 was added to the sample pool, 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 meter. The results are summarized in Table 4.

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

[0080]

[0081]

[0082] The morphology and dispersion of chitosan-superoxide dismutase nanogel (CS-nSOD@VC) were observed under a transmission electron microscope. Figure 3 The morphology and dispersion of polyacrylamide-superoxide dismutase nanogel (PAAm-nSOD@R) were observed. Figure 4 The morphology and dispersion of polyethylene glycol-superoxide dismutase nanogel (PEG-nSOD@NAA) were observed. Figure 5 .

[0083] Application Example 4: Sustained Release of Active Ingredients

[0084] Take 2mL of superoxide dismutase nanogel with a concentration of 5mg / mL and put it into a dialysis bag with a molecular weight cutoff of 3000, immerse the dialysis bag in a phosphate buffer with a pH of 6, and oscillate the entire system at 37°C. The sustained release time is 48h. Within 48h, samples are taken regularly to measure the concentration of loaded small molecule drugs under an ultraviolet spectrophotometer, and the sustained release of small molecule drugs by the composite system is calculated. The results are statistically shown in Table 5. Superoxide dismutase nanogel can slowly release small molecules within 24 hours, which is conducive to the continuous release of active small molecules after reaching the deep layer of the skin during transdermal use, thereby efficiently exerting physiological effects.

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

[0086]

[0087] Application Example 6: Nanogel's Stimulation Reduction Effect on Active Ingredients

[0088] Chitosan-superoxide dismutase nanogel (CS-nSOD@VC) was used to reduce the irritation of vitamin C: The final concentration of vitamin C in the system was unified, and the pH value of the chitosan-superoxide dismutase nanogel (CS-nSOD@VC) solution loaded with vitamin C and the vitamin C aqueous solution was measured using a pH meter to characterize the overall irritation level. The statistical results are in Table 6. The results show that under the same vitamin C concentration, the acidity of the vitamin C system encapsulated by superoxide dismutase nanogel is significantly weaker than that of the free vitamin C aqueous solution, and too strong acidity can cause irritation to the skin. This shows that superoxide dismutase nanogel can effectively reduce the irritation of vitamin C while delivering it, and is suitable for a wider range of people.

[0089] Table 6 Superoxide dismutase nanogel reduces the stimulating effect of vitamin C

[0090]

[0091] Application Example 7: Protective Effect of Nanogel on Active Ingredients

[0092] Determination of the protective effect of polyacrylamide-superoxide dismutase nanogel (PAAm-nSOD@R) on retinol: The enzyme nanogel solution loaded with retinol and the free retinol solution were stored at room temperature for 1-7 days, and the absorbance of the sample at 325nm was measured by UV-spectrophotometer to characterize the retinol content, and the ratio of the unoxidized retinol content to the initial embedding amount was calculated. The statistical results are in Table 7.

[0093] Table 7 Retinol Retention

[0094]

[0095]

[0096] It can be seen from the data in Table 7 that the deterioration of retinol can be significantly reduced through the superoxide dismutase nanogel delivery system of the present invention, namely, the PAAm-nSOD@R solution.

[0097] Application Example 8 Characterization of Antioxidant Properties of Nanogel

[0098] ROS kit was used to detect the level of ROS in human epidermal HaCaT cells. HaCaT cells (1*10 4 / well), after 12h of complete cell attachment, the cells were irradiated with UVB ultraviolet irradiator to induce internal ROS production, and cells not treated with ultraviolet radiation were left as negative controls. After irradiation, the original culture medium was removed from all wells and 100μL serum-free culture medium was added (the culture medium was not added with serum to avoid the effect of serum on the cell uptake of the complex during the subsequent drug addition process), 10μL PBS buffer was added to the positive control group, 10μL 0.01mg / mL complex solution was added to the low concentration experimental group, 10μL 0.02mg / mL complex solution was added to the medium concentration experimental group, and 10μL 0.04mg / mL complex solution was added to the high concentration experimental group. After incubation for 24h, the original culture medium was removed from all cells and 100μL serum-free culture medium and 10μL DCFH-DA culture solution were added, and incubated at 37°C in the dark for 20min. The cells were then washed three times with PBS, and the fluorescence intensity of the cell DCF was detected using an enzyme marker. The results are statistically shown in Table 8.

[0099] Table 8 The intracellular antioxidant capacity of superoxide dismutase nanogel increases with increasing concentration

[0100]

[0101]

[0102] It can be seen that CS-nSOD@VC and PAAm-nSOD@R showed 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 cell to play a physiological role. The antioxidant effect of CS-nSOD@VC is stronger than that of PAAm-nSOD@R, which may be because vitamin C itself has excellent antioxidant properties and plays a synergistic antioxidant function with superoxide dismutase.

[0103] Application Example 9: Transdermal Experiment

[0104] The superoxide dismutase nanogel solution was mixed with excess FITC and reacted overnight, and the unreacted small molecules were removed by dialysis to obtain a fluorescently labeled complex. Fresh pig skin was washed with PBS, fixed with the stratum corneum facing up in a transdermal diffusion instrument, and kept warm at 37°C. 0.5 mL of the fluorescently labeled complex solution was dripped on the pig skin tissue, and frozen sections were taken at 24h, 4h, and 1h. The penetration of the nanogel was observed under a confocal microscope, as shown in Figure 2. Figure 6 The results of the statistical penetration depth are shown in Table 9. The results showed that the superoxide dismutase nanogel could effectively penetrate the skin barrier and reach deep into the skin at 24 hours to exert an antioxidant effect.

[0105] Table 9 Superoxide dismutase nanogel transdermal status

[0106]

Claims

1. 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 has a diameter of 20-100 nm, wherein the small molecule active ingredient is loaded in the superoxide dismutase, the surface of the superoxide dismutase is coated by the polymer shell layer, and the thickness of the polymer shell layer is 7.5-52.5 nm.

2. The nano transdermal delivery system based on superoxide dismutase according to claim 1, characterized in that: The superoxide dismutase-based active ingredient transdermal delivery system has a diameter of 30-80 nm and a polymer shell thickness of 10-35 nm.

3. The nano transdermal delivery system based on superoxide dismutase according to claim 1, characterized in that: The surface zeta potential of the transdermal delivery system is +4 to +7 mV.

4. The nano transdermal delivery system based on superoxide dismutase according to claim 1, characterized in that: The small molecule active ingredient is selected from carotenoids, hesperidin, vitamin C, vitamin E, resveratrol, lycopene, retinol niacinamide, ascorbic acid, α-arbutin or derivatives of the aforementioned substances.

5. The nano transdermal delivery system based on superoxide dismutase according to claim 1, characterized in that: The mass ratio of the small molecule active ingredient to superoxide dismutase is 1:1-100, preferably 1:1-5.

6. The nano transdermal delivery system based on superoxide dismutase according to claim 1, characterized in that: 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 the in-situ polymerization method, the monomer 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-methacryloyloxyethyl phosphorylcholine, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl)ammonium hydroxide, 2-hydroxyethyl acrylate, acrylate, 2-(dimethylamino)ethyl methacrylate, (3-acrylamidopropyl)trimethylammonium chloride hydrochloride, aminopropyl methacrylamide, N,N'-methylenebisacrylamide, glyceryl dimethacrylate, glyceryl 1,3-diglycerol alcohol diacrylate; Preferably, the acrylic acid ester is selected from at least one of methyl (meth)acrylate, ethyl (meth)acrylate, hydroxyethyl (meth)acrylate, propyl (meth)acrylate, and butyl (meth)acrylate; The polymer is coated on the surface of the protein by electrostatic action and is a positively or negatively charged polymer, wherein the positively charged polymer is selected from at least one of poly(propyleneamine hydrochloride), poly(L-lysine), polyethyleneimine, poly(L-histidine), poly(N,N-dimethylaminoethyl methacrylate), poly(methacrylamide propyltrimethylammonium chloride), and natural or synthetic polysaccharides such as chitosan; the negatively charged polymer is selected from at least one of polyacrylic acid, polystyrene sulfonate, alginate, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dextran sulfate, polymethacrylic acid, oxidized cellulose, carboxymethyl cellulose, polyaspartic acid and polyglutamic acid.

7. The nano transdermal delivery system based on superoxide dismutase according to claim 6, characterized in that: The polymer is a carbon-carbon unsaturated double bond monomer that is coated on the protein surface in an in-situ polymerization manner; the polymer raw material includes a surface modification monomer, a functional monomer, a crosslinking agent and an initiator, and preferably, a positively charged monomer is also added; the surface modification monomer is selected from at least one of N-acrylsuccinimide, N-(3-aminopropyl) methacrylamide hydrochloride, and maleimide; the functional monomer is selected from at least one of vinyl pyrrolidone, acrylamide, polyethylene glycol methyl ether acrylate, 2-methacryloyloxyethyl phosphorylcholine, and 2-hydroxyethyl acrylate; the positively charged monomer is selected from [2-(methyl The crosslinking agent is selected from at least one of N,N'-methylenebisacrylamide, glyceryl dimethacrylate, and glyceryl 1,3-diglycerol alcohol diacrylate; the initiator is a water-soluble initiator, specifically selected from at least one of ammonium persulfate, sodium persulfate, and potassium persulfate. Preferably, a co-initiator is also added, such as sodium bisulfite and tetramethylethylenediamine; Furthermore, the mass ratio of superoxide dismutase to surface modification monomer is 1-10:1, preferably 2-5:1; the molar ratio of superoxide dismutase to functional monomer is 1:4500-25000, and the molar ratio of functional monomer to crosslinking agent is 1:0.05-0.1; if the polymer raw material also includes a positively charged monomer, the molar ratio of the functional monomer to the positively charged monomer is 1:0.1-0.12; Furthermore, the molar ratio of superoxide dismutase to functional monomer is 1:6000-10000.

8. The method for preparing the nano transdermal delivery system based on superoxide dismutase according to any one of claims 1 to 7, characterized in that: One of the following methods: Method 1: In-situ polymerization (S1) adding a surface-modified monomer solution to a buffer solution of superoxide 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), wherein the small molecule active ingredient is adsorbed on the superoxide dismutase, thereby obtaining a surface-modified superoxide dismutase solution loaded with the small molecule active ingredient; (S3) adding functional monomers, positively charged monomers, crosslinking agents and initiators to the surface-modified superoxide dismutase solution loaded with small molecule active ingredients obtained in step (S2) to initiate in situ polymerization to obtain a nano-transdermal delivery system based on superoxide dismutase; Method 2: Electrostatic adsorption method: Add superoxide dismutase solution to a positively or negatively charged polymer solution at 50-70°C and stir, add small molecule active ingredient solution at 50-70°C and continue stirring, add salt solution in an ice water bath, react, and ultrafilter and centrifuge to obtain a nano transdermal delivery system based on superoxide dismutase.

9. The preparation method according to claim 8, characterized in that: In method 1, in step (S1), the buffer solution is a phosphate buffer solution, and the solvent of the surface modification monomer solution is at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol, with a concentration of 1-5wt%; and / or In step (S2), the solvent of the small molecule active ingredient solution is selected from 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 initiation polymerization is room temperature polymerization, and the polymerization time is 6-12 hours, preferably 6-8 hours; In method 2, the concentration of the positively or negatively 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; when the polymer is positively charged, the salt solution is a salt solution that can provide anions to induce self-assembly of the positively charged polymer, preferably a sodium tripolyphosphate solution; when the polymer is negatively charged, the salt solution is a salt solution that can provide cations to induce self-assembly of the negatively charged polymer, preferably a calcium chloride solution; the salt solution concentration is 0.5-5 mg / mL; the ultrafiltration molecular weight cutoff is 3-5 kDa; Furthermore, the mass ratio of superoxide dismutase, the polymer with positive or negative charge, the small molecule active ingredient, and the salt in the salt solution is 1:0.5-1:0.05-0.1:0.05-0.

1.

10. Use of the superoxide dismutase-based nano transdermal delivery system according to any one of claims 1 to 7 in the preparation of a method for preventing or treating oxidative stress diseases; Preferably, the oxidative stress diseases include skin photoaging or chloasma eczema.

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