Superoxide dismutase-based nanoscale transdermal delivery system and preparation method therefor

Abstract

Provided is a superoxide dismutase-based nanoscale transdermal delivery system, which is a capsule composite structure consisting of a small molecule active ingredient, superoxide dismutase, and a polymer coating. The small molecule active ingredient is loaded in the superoxide dismutase, and the surface of the superoxide dismutase is coated with the polymer coating. The superoxide dismutase-based active ingredient transdermal delivery system has a diameter of 20-100 nm, and the polymer coating has a thickness of 7.5-52.5 nm. In the delivery system, the polymer coating protects the superoxide dismutase and the small molecule functional substance having an antioxidant effect, and the capsule composite structure is in the form of nanogel. The nanogel not only promotes the penetration depth of the delivery system, but also mitigates the stimulation of the delivery system to the skin. The delivery system features biofriendly starting materials, simple preparation method, and high yield, and thus can be produced in large scale industrially.

Description

A nano-transdermal delivery system based on superoxide dismutase and its preparation method Technical Field

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

[0002] Superoxide dismutase (SOD) is a metalloenzyme widely distributed throughout the body. It exerts antioxidant effects by scavenging superoxide anion radicals, protecting organisms from oxidative stress damage. Consequently, it has attracted attention as a highly effective antioxidant for the skin. However, in practical applications, direct application of SOD to the skin for antioxidant management or treatment often yields ineffective results, suggesting inefficient transdermal delivery and low SOD bioavailability.

[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, and wrinkles. Therefore, the delivery of antioxidants to the skin is beneficial for solving a variety of skin problems. However, the multi-layered and complex structure of the skin creates high-efficiency barrier problems including physical barriers, chemical barriers, and immune barriers. Active substances delivered transdermally are often isolated outside the stratum corneum, the outermost layer of the skin, and are difficult to reach deep layers 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. The depth may involve the epidermis, the junction of the epidermis and dermis, and the dermis matrix. Therefore, the depth of transdermal delivery is particularly important.

[0004] Existing clinical and commercial transdermal delivery technologies are still in their early stages of development, with chemical permeation enhancers and liposome loading being the most commonly used. Common permeation enhancers, such as ethanol, not only affect the biological activity of the delivered substance but also pose a significant risk of skin irritation and allergies during use, as their permeation-enhancing mechanism alters the skin's structure and weakens the barrier function. The vesicle structure formed by liposome encapsulation is generally larger than 200 nm in diameter, leaving the system in a thermodynamically metastable state, making it difficult to meet the requirements of long-term use and storage.

[0005] Summary of the Invention

[0006] In order to solve the defects of low delivery efficiency and uncontrollable delivery depth in existing skin antioxidant transdermal technology, the present invention uses superoxide dismutase molecules as nano-templates and constructs an efficient transdermal delivery system through polymer encapsulation. At the same time, the hydrophobic structure inside the superoxide dismutase is used to load functional small molecules for co-delivery, achieving synergistic effects of multiple physiological functions including antioxidant. The system uses modified superoxide dismutase nanogels as carriers. After the functional small molecules are loaded in the nanogels, they are efficiently transdermally transported to the deep layers of the skin and released in a controlled manner, exerting a synergistic therapeutic effect on various problems such as skin oxidation and inflammation. In this system, superoxide dismutase itself has an antioxidant effect and can also serve as a carrier of functional small molecules; the polymer shell of the superoxide dismutase outer layer has a customizable diameter and surface potential, which not only helps the superoxide dismutase and functional small molecules inside to pass through the skin barrier, but also protects the stability of the superoxide dismutase and functional small molecules, which is conducive to the long-term use and storage of the preparation. The nanogel's excellent penetration efficiency and controllable surface properties and diameter allow for controlled transdermal delivery depth. Superoxide dismutase and small molecule active ingredients are targeted to areas of oxidative stress in the skin to exert their effects, enabling comprehensive improvements to various skin concerns with lower concentrations of active ingredients. Furthermore, the nanogel effectively mitigates the skin irritation of some active ingredients, broadening its applicability to individuals with diverse skin conditions. This loading method is universally applicable to small molecule ingredients with hydrophobic structures.

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

[0008] A nano-transdermal delivery system based on superoxide dismutase is 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 with the polymer shell layer, and the thickness of the polymer shell layer is 7.5-52.5 nm.

[0009] Furthermore, 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.

[0010] 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.

[0011] The small molecule active ingredient is selected from carotenoids, hesperidin, vitamin C, vitamin E, resveratrol, lycopene, retinol, niacinamide, ascorbic acid, α-arbutin, or derivatives thereof. The present invention can address different antioxidant issues by selecting different small molecule active ingredients. For example, carotenoids, hesperidin, vitamin C, vitamin E, etc. can be used to address skin oxidation issues; resveratrol, lycopene, etc. can be used to address skin inflammation issues; retinol and its derivatives can be used to address skin photoaging and wrinkles; and niacinamide, ascorbic acid, α-arbutin, etc. can be used to address skin tone and spots caused by hyperpigmentation.

[0012] Furthermore, the mass ratio of the small molecule active ingredient to 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, which is then added to the surface-modified superoxide dismutase solution. The small molecule is attached to the protein molecule core through electrostatic interactions, hydrophilic-hydrophobic interactions, hydrogen bonds, cross-linking, etc., to obtain a small molecule-loaded superoxide dismutase solution.

[0013] In the delivery system of the present invention, polymers are selected that facilitate the formation of a hydration layer on the outer layer of the nanogel. These include monomers that facilitate hydrogen bonding, amphiphilic monomers that naturally evade immune recognition, and copolymers of positively and negatively charged monomers to create a positive dipole balance within the polymer, thereby forming a strong hydration layer, such as phosphorylcholine polymers and betaine polymers. Positively charged polymers, such as chitosan, can also be used to wrap around superoxide dismutase molecules through electrostatic interactions.

[0014] The polymer shell not only helps the superoxide dismutase and functional small molecules inside to penetrate the skin barrier, but also protects the stability of superoxide dismutase and functional small molecules, which helps the long-term use and storage of the preparation. Therefore, the thickness control of the shell is very important in this system. The permeability of the nanotransdermal delivery system is related to the particle size. A shell that is too thin or too thick is not conducive to the deep delivery of the nanogel. The protective effect of the polymer shell on the activity of the active protein is also related to the shell thickness. A too thin shell has a poor ability to withstand severe changes in ambient temperature and pH. The prerequisite for the enzyme to exert its catalytic effect is contact with the substrate. An overly thick shell will affect the transmission of the substrate, thereby hindering the active protein from exerting its catalytic effect. In the present invention, a series of diameters and the effects of shells of different thicknesses on the transdermal depth and the protection of protein or enzyme activity were prepared and investigated. The results showed 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 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.

[0015] Furthermore, 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 comprises a monomer selected from at least one of N-propylene succinimide, N-(3-aminopropyl) methacrylamide hydrochloride, maleimide, vinyl pyrrolidone, acrylamide, polyethylene glycol methyl ether acrylate, N-propylene succinimide, 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, glycerol dimethacrylate, and glycerol 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 interaction 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), polymethacrylamidopropyltrimethylammonium 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.

[0016] 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, a positively charged monomer is 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, glycerol dimethacrylate, and glycerol 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 such as sodium bisulfite or tetramethylethylenediamine is also added. 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.

[0017] After modification with a surface modification monomer, superoxide dismutase has polymerizable carbon-carbon unsaturated double bonds on its surface. In one embodiment of the present invention, the surface modification method comprises mixing superoxide dismutase with a buffer solution or an organic solvent to prepare a superoxide dismutase solution, and then adding the surface modification monomer to the superoxide dismutase solution. The surface modification monomer can attach to the surface of the protein molecule through electrostatic interaction, covalent crosslinking, or other means to obtain a surface-modified protein molecule solution.

[0018] 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.

[0019] Furthermore, the molar ratio of superoxide dismutase to functional monomer is 1:6000-10000.

[0020] 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:

[0021] Method 1: In situ polymerization

[0022] (S1) adding a surface-modified monomer solution to a superoxide dismutase buffer solution to obtain a surface-modified protein solution;

[0023] (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;

[0024] (S3) adding a functional monomer, a positively charged monomer, a crosslinker, and an initiator to the surface-modified superoxide dismutase solution loaded with a small molecule active ingredient obtained in step (S2) to initiate in situ polymerization to obtain a nano-transdermal delivery system based on superoxide dismutase;

[0025] Method 2: Electrostatic adsorption method:

[0026] A superoxide dismutase solution is added to a positively or negatively charged polymer solution at 50-70°C (e.g., 60°C) and stirred. A small molecule active ingredient solution is added at 50-70°C and stirred continuously. A saline solution is added in an ice-water bath and reacted for 30-60 minutes. The mixture is then ultrafiltered and centrifuged to obtain a superoxide dismutase-based nanotransdermal delivery system.

[0027] Furthermore, 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 1-5 wt%; and / or

[0028] 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. The concentration of the small molecule active ingredient solution is 1-5 mg / mL, for example, 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 the small molecule active ingredient is 1-5:1.

[0029] Furthermore, the molar ratio of superoxide dismutase, functional monomer, positively charged monomer, and crosslinker 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, such as sodium bisulfite or tetramethylethylenediamine, is also added. 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 performed at room temperature for 6-12 hours, preferably 6-8 hours.

[0030] Furthermore, in method 2, the concentrations of the positively or negatively charged polymer solution, the superoxide dismutase solution, and the aqueous solution of the small molecule active ingredient are independently 1-10 mg / mL. When the polymer is positively charged, the salt solution is a salt solution that can provide anions to trigger 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 trigger the self-assembly of the negatively charged polymer, such as a calcium chloride solution; and the salt solution concentration 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.

[0031] Furthermore, in method 2, the molecular weight cut-off of ultrafiltration is 3-5 kDa.

[0032] 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.

[0033] The excellent effects of the present invention are:

[0034] First, the present invention first modifies the surface of superoxide dismutase with a polymerizable monomer, then loads it with a small molecule active ingredient. Through methods including in situ polymerization, a core-shell nanocapsule with a polymer coating and a positively charged surface is constructed. This addresses the problem of suboptimal superoxide dismutase transdermal performance, which leads to low antioxidant efficacy. The present superoxide dismutase-based nanotransdermal delivery system, with its polymer shell, can efficiently penetrate the skin's physical barrier.

[0035] Second, the present invention initiates an in situ polymerization reaction on the surface of superoxide dismutase through polymerization, coating the enzyme with a polymer protective layer. This polymer protective layer is biofriendly and non-irritating or minimally irritating to the skin. During transdermal delivery, by regulating the surface properties and thickness of the polymer protective layer, the transdermal depth of the delivery system can be effectively controlled, allowing the entire delivery system to effectively penetrate the skin to the location where superoxide dismutase and active ingredients are needed to exert their effects, thereby specifically addressing skin problems. This significantly improves the poor delivery efficiency of previous protein-based delivery systems.

[0036] 3. In this delivery system, small molecule active ingredients are loaded into superoxide dismutase, and the surface of superoxide dismutase is coated with a polymer shell to form a capsule composite structure. The capsule composite structure is in the form of a nanogel. Thus, the polymer shell provides protection for superoxide dismutase and small molecule functional substances with antioxidant effects. The nanogel not only promotes their penetration depth but also reduces their irritation to the skin, making it suitable for a wider range of people.

[0037] Fourth, the raw materials used in the present invention are all bio-friendly and can be used as pharmaceutical reagents, and thus 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

[0038] FIG1 is a fitting curve of the penetration depth D and the particle size d of the superoxide dismutase nanogel with a positive surface potential at 24 hours;

[0039] FIG2 is a graph showing the relative activity of superoxide dismutase versus incubation time;

[0040] FIG3 is the morphology and dispersion of chitosan-superoxide dismutase nanogel (CS-nSOD@VC) in Example 2;

[0041] FIG4 shows the morphology and dispersion of retinol-loaded polyacrylamide-superoxide dismutase nanogel (PAAm-nSOD@R) in Example 3;

[0042] FIG5 is the morphology and dispersion of polyethylene glycol-superoxide dismutase nanogel (PEG-nSOD@NAA) in Example 4;

[0043] Figure 6 shows the permeation of the nanogel at 1 h, 4 h, and 24 h. DETAILED DESCRIPTION

[0044] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions 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 all conventional methods unless otherwise specified.

[0045] Example 1 Preparation of polyacrylamide-superoxide dismutase nanogel

[0046] Dissolve 2 mg of superoxide dismutase in 2 mL of PBS buffer (pH 7.4) to obtain a 1 mg / mL SOD solution. Add 0.4 mg of N-propylene succinimide in dimethyl sulfoxide (1% by mass) to the 2 mL of 1 mg / mL SOD solution. Mix thoroughly and react for 2 hours to obtain a superoxide dismutase (aSOD) surface-modified with polymerizable double bonds.

[0047] aSOD, acrylamide (AAm), aminopropylmethacrylamide hydrochloride (APM), and N,N'-methylenebisacrylamide (BIS) were mixed at the specified molar ratios and diluted to 1 mg / mL with pH 7.4 phosphate buffer. An initiator (3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per mg of protein) was then added. In situ free radical polymerization was initiated at room temperature. After the specified reaction time, polyacrylamide-superoxide dismutase nanogels (nSOD) of varying diameters were obtained. The preparation conditions, final diameters, and surface zeta potentials are shown in Table 1.

[0048] Table 1 Preparation conditions, final diameters and surface potentials of superoxide dismutase nanogels with a series of diameters

[0049] As can be seen from Table 1, 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: Transdermal Experiment of Superoxide Dismutase Nanogels with a Series of Diameters

[0051] 4 mL of 1 mg / mL superoxide dismutase nanogels (nSOD) of varying diameters from Example 1 were mixed with 1 mL of 1 mg / mL fluorescein isothiocyanate and reacted overnight. The mixture was dialyzed overnight in a 14,000 Da dialysis tubing to remove unreacted small molecules, yielding fluorescently labeled nSOD. Fresh pigskin was washed with PBS and fixed, with the stratum corneum facing upward, in a transdermal diffusion apparatus incubated at 37°C. 0.5 mL of a 1 mg / mL fluorescently labeled superoxide dismutase nanogel solution was dripped onto the pigskin tissue. After 24 hours, frozen sections were taken and the nanogel penetration observed under a confocal microscope. The penetration depth is statistically reported in Table 2.

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

[0053] By comparing the penetration depths of superoxide dismutase nanogels of different diameters with positive surface potentials (about +5mV), it was found that when the particle size was between 20nm and 150nm, the protein nanogel was able to penetrate the epidermis and reach the vicinity of the dermis within 24 hours, and as the gel particle size increased, the penetration depth first increased and then decreased; while 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 a fitting curve of the penetration depth D and particle size d of superoxide dismutase nanogels with positive surface potential at 24 hours. The fitted formula is as follows:

[0054] D=a1+m*(a2*d+a3*d 2 +a4*d 3 ),d∈[20,150], where the unit of D is μm and the unit of d is nm.

[0055] The parameters m, a1, a2, a3, and a4 in the formula are related to the polymer outer layer material and the type of protein carrier. Among them, a2, a3, and a4 are also related to the diameter of the nanocapsule, meaning 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 transdermal delivery. When the protein type is superoxide dismutase, the formula can be written as:

[0056] 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.

[0057] This expression can be used to preliminarily predict the penetration capacity of superoxide dismutase nanogels with a particle size range of 20-150 nm and a positive surface potential. Skin antioxidant delivery requirements are distributed throughout the epidermis and dermis. Oxidative stress caused by reactive oxygen species (ROS) in the dermal matrix is ​​a common skin problem, leading to wrinkles and abnormal pigmentation. According to the formula, the preferred delivery system diameter for this skin problem is 20 to 100 nm.

[0058] Application Example 2 Series Diameter Superoxide Dismutase Nanogel Activity Retention

[0059] Superoxide dismutase activity was measured using an ultraviolet spectrophotometer. A 3 mL Tris-HCl buffer solution (containing 1 mmol / mL of EDTA) at a pH of 8 was added to a reference cell. A 2.4 mL Tris-HCl buffer solution (containing 1 mmol / mL of EDTA) at a 50 mmol / mL concentration was added to a sample cell. The solution was then mixed with 0.3 mL of pyrogallol at a concentration of 0.2 mmol / mL and 0.3 mL of the original superoxide dismutase or enzyme nanogel solution. The sample cell was then placed in an ultraviolet spectrophotometer. The absorbance of the sample cell at 420 nm was recorded for the first 10 minutes. The slope of the fitted straight line was used to represent the relative activity of superoxide dismutase. The results are shown in Table 3. This indicates that for superoxide dismutase nanogels with diameters greater than 150 nm, excessively thick polymer shells can affect the contact of superoxide dismutase with substrates, or that polymerization conditions can lead to a loss of superoxide dismutase activity. The activity loss of superoxide dismutase nanogels with a diameter less than 100 nm was within an acceptable range.

[0060] Table 3 Relative activities of nSOD with different diameters

[0061] Application Example 3: Stability Test of Superoxide Dismutase Nanogels with a Series of Diameters

[0062] Superoxide dismutase activity was measured using a UV spectrophotometer. 1 mL of 1 mg / mL pristine superoxide dismutase or enzyme nanogel solution was incubated with 1 mL of 0.1 mg / mL pepsin in glycine-HCl buffer (pH 2.0) at 37°C for various time points. After incubation, the samples were removed and immediately placed on ice. 3 mL of 50 mmol / mL Tris-HCl buffer (pH 8) (containing 1 mmol / mL EDTA) was added to the reference cell. 2.4 mL of 50 mmol / mL Tris-HCl buffer (pH 8) (containing 1 mmol / mL EDTA), 0.3 mL of 0.2 mmol / mL pyrogallol, and 0.3 mL of a 0.5 mg / mL treated original superoxide dismutase (SOD) or enzyme nanogel solution were added to the sample cell. The cells were placed in a UV spectrophotometer, and the absorbance change at 420 nm was recorded for the first 10 minutes. The slope of the fitted line represents the relative SOD activity. The results are shown in Figure 2. It was found that the original SOD was significantly inactivated by the protease, while nSOD-20+, nSOD-50+, and nSOD-100+ retained 75% of their activity after 30 minutes of treatment, demonstrating the effective protection of the polymer shell.

[0063] Example 2 Preparation of Vitamin C-Loaded Superoxide Dismutase Nanogel

[0064] 6 mL of a 1 mg / mL chitosan solution was ultrasonicated at 70 W for 10 minutes and allowed to stand to prevent excessive heat generation. Superoxide dismutase solution was added in a 60°C water bath, and the superoxide dismutase concentration was adjusted to 2 mg / mL. The mixture was reacted for 5 minutes. To the mixed solution, 0.5 mL of a 2 mg / mL aqueous solution of vitamin C was added dropwise at 400 rpm in a 60°C water bath, and stirring was continued for 5 minutes. Finally, 1 mL of a 1 mg / mL sodium tripolyphosphate solution was added dropwise in an ice-water bath at 400 rpm. After 30 minutes of reaction, vitamin C-loaded chitosan-superoxide dismutase nanogel (CS-nSOD@VC) was obtained.

[0065] 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 refrigerated for storage.

[0066] Example 3 Preparation Method of Retinol-Loaded Superoxide Dismutase Nanogel

[0067] To 2 mL of a 1 mg / mL superoxide dismutase solution, 0.45 mg of N-propylene succinimide was added, wherein N-propylene succinimide was dissolved in dimethyl sulfoxide, and the mixture was thoroughly mixed and reacted for 2 h to obtain a superoxide dismutase (aSOD) with a polymerizable double bond modified on its surface.

[0068] 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 to it while stirring at 400 rpm. After thorough mixing, retinol-loaded superoxide dismutase (aSOD@R) is obtained.

[0069] aSOD@R, acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, and N,N'-methylenebisacrylamide were mixed at 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, an initiator was added at a ratio of 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per mg of protein. In situ free radical polymerization was initiated at room temperature, and after 4 h of reaction, retinol-loaded polyacrylamide-superoxide dismutase nanogel (PAAm-nSOD@R) was obtained.

[0070] 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 refrigerated for storage.

[0071] Example 4 Preparation Method of Nicotinamide-Loaded Superoxide Dismutase Nanogel

[0072] 0.38 mg of N-acrylsuccinimide was added to 2 mL of a 1 mg / mL superoxide dismutase solution, wherein N-acrylsuccinimide was dissolved in dimethyl sulfoxide, and the mixture was thoroughly mixed and reacted for 2 h to obtain a superoxide dismutase (aSOD) with a polymerizable double bond modified on its surface.

[0073] 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 to it while stirring at 450 r / min, and mix thoroughly to obtain nicotinamide-loaded superoxide dismutase (aSOD@NAA).

[0074] aSOD@NAA, polyethylene glycol methyl ether acrylate, and N,N'-methylenebisacrylamide were mixed at a molar ratio of 1:6600:600, and the protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, an initiator was added at a ratio of 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per mg of protein. In situ free radical polymerization was initiated at room temperature, and after 4 h of reaction, nicotinamide-loaded polyethylene glycol-superoxide dismutase nanogel (PEG-nSOD@NAA) was obtained.

[0075] 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 refrigerated for storage.

[0076] Dynamic light scattering (DLS) was used to characterize the nanogel diameter and surface potential: 1 mg / mL protein nanogel solutions from Examples 2, 3, and 4 were added to a sample cell. The particle size and zeta potential of the nanogels were measured at room temperature using a Malvern Nano Zs90 nanometer. The results are summarized in Table 4.

[0077] Table 4 Particle size and Zeta potential of superoxide dismutase nanogel

[0078] Transmission electron microscopy (TEM) was used to characterize the morphology of the nanogels. The morphology and dispersion of chitosan-superoxide dismutase nanogels (CS-nSOD@VC) were observed under a transmission electron microscope, as shown in Figure 3. The morphology and dispersion of polyacrylamide-superoxide dismutase nanogels (PAAm-nSOD@R) were observed, as shown in Figure 4. The morphology and dispersion of polyethylene glycol-superoxide dismutase nanogels (PEG-nSOD@NAA) were observed, as shown in Figure 5.

[0079] Application Example 4: Sustained Release of Active Ingredients

[0080] 2 mL of 5 mg / mL superoxide dismutase nanogel was placed in a 3000 molecular weight cutoff dialysis bag, which was then immersed in a pH 6 phosphate buffer. The entire system was shaken at 37°C. The sustained release period was 48 hours. Samples were taken periodically over 48 hours and the concentration of the loaded small molecule drug was measured using a UV spectrophotometer. The sustained release of the small molecule drug from the composite system was calculated. The results are summarized in Table 5. The superoxide dismutase nanogel can slowly release small molecules over 24 hours, facilitating transdermal use, where the active small molecule is continuously released after reaching the deep layers of the skin, thereby effectively exerting its physiological effects.

[0081] Table 5 Release of small molecule drugs by superoxide dismutase nanogels

[0082] Application Example 6: Nanogel's Stimulation Reduction Effect on Active Ingredients

[0083] The effect of chitosan-superoxide dismutase nanogel (CS-nSOD@VC) on reducing the irritation of vitamin C was determined: The final vitamin C concentration within the system was uniform, and the pH values ​​of the vitamin C-loaded chitosan-superoxide dismutase nanogel (CS-nSOD@VC) solution and the vitamin C aqueous solution were measured using a pH meter to characterize the overall irritation level. The statistical results are shown in Table 6. The results show that, at the same vitamin C concentration, the vitamin C system encapsulated by the superoxide dismutase nanogel is significantly less acidic than the free vitamin C aqueous solution. Excessive acidity can cause skin irritation. This suggests that the superoxide dismutase nanogel can effectively reduce the irritation of vitamin C while delivering it, making it suitable for a wider range of patients.

[0084] Table 6 Superoxide dismutase nanogel reduces the stimulating effect of vitamin C

[0085] Application Example 7: Protective Effect of Nanogel on Active Ingredients

[0086] The protective effect of polyacrylamide-superoxide dismutase nanogel (PAAm-nSOD@R) on retinol was determined by storing retinol-loaded enzyme nanogel solutions and free retinol solutions at room temperature for 1-7 days. The absorbance of the samples at 325 nm was measured using a UV spectrophotometer to characterize the retinol content. The ratio of unoxidized retinol to the initial encapsulated amount was calculated. The statistical results are shown in Table 7.

[0087] Table 7 Retinol retention

[0088] It can be seen from the data in Table 7 that the superoxide dismutase nanogel delivery system of the present invention, namely the PAAm-nSOD@R solution, can significantly reduce the deterioration of retinol.

[0089] Application Example 8 Characterization of the Antioxidant Properties of Nanogels

[0090] The ROS level in human epidermal HaCaT cells was detected using a ROS kit. HaCaT cells (1*10 4Cells were irradiated with a UVB irradiator for 12 hours to induce ROS production. Untreated cells were retained as negative controls. Following irradiation, the culture medium was removed from all wells and 100 μL of serum-free medium was added (serum was omitted to prevent serum from affecting cellular uptake of the complex during subsequent drug additions). The positive control group received 10 μL of PBS buffer, the low-concentration group received 10 μL of a 0.01 mg / mL complex solution, the medium-concentration group received 10 μL of a 0.02 mg / mL complex solution, and the high-concentration group received 10 μL of a 0.04 mg / mL complex solution. After 24 hours of incubation, the culture medium was removed from all wells and replaced with 100 μL of serum-free medium and 10 μL of DCFH-DA culture medium. The cells were incubated at 37°C in the dark for 20 minutes. The cells were then washed three times with PBS, and the fluorescence intensity of DCF in the cells was detected using an enzyme-labeled instrument. The results are shown in Table 8.

[0091] Table 8 The intracellular antioxidant capacity of superoxide dismutase nanogels increases with increasing concentration

[0092] As can be seen, CS-nSOD@VC and PAAm-nSOD@R exhibited superior antioxidant capacity compared to PEG-nSOD@NAA, likely due to the positive surface charge of the nanocapsules, which facilitates their entry into cells and exerts its physiological effects. The stronger antioxidant effect of CS-nSOD@VC than that of PAAm-nSOD@R may be due to the excellent antioxidant properties of vitamin C itself, which works synergistically with superoxide dismutase to achieve antioxidant effects.

[0093] Application Example 9: Transdermal Experiment

[0094] The superoxide dismutase nanogel solution was mixed with an excess of FITC and allowed to react overnight. Unreacted small molecules were removed by dialysis to obtain a fluorescently labeled complex. Fresh pig skin was washed with PBS and fixed with the stratum corneum facing up in a transdermal diffusion apparatus and kept warm at 37°C. 0.5 mL of the fluorescently labeled complex solution was dripped onto the pig skin tissue. Frozen sections were taken at 24 hours, 4 hours, and 1 hour, and the penetration of the nanogel was observed under a confocal microscope, as shown in Figure 6. The results of the penetration depth are statistically shown in Table 9. The results show that the superoxide dismutase nanogel can effectively penetrate the skin barrier and reach deep into the skin after 24 hours, exerting an antioxidant effect.

[0095] Table 9 Superoxide dismutase nanogel transdermal status

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 above 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.

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

7. 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 through in situ polymerization, hydrogen bonding and / or electrostatic interaction.

8. The nano transdermal delivery system based on superoxide dismutase according to claim 7, characterized in that The polymer obtained by the in-situ polymerization method comprises at least one monomer selected from N-propylene succinimide, N-(3-aminopropyl) methacrylamide hydrochloride, maleimide, vinyl pyrrolidone, acrylamide, polyethylene glycol methyl ether acrylate, N-propylene succinimide, 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, glycerol dimethacrylate, and glycerol 1,3-diglycerol alcohol diacrylate.

9. The nano transdermal delivery system based on superoxide dismutase according to claim 8, characterized in that 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.

10. The nano transdermal delivery system based on superoxide dismutase according to claim 7, characterized in that: The polymer is coated on the surface of the protein through electrostatic interaction 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), polymethacrylamidopropyltrimethylammonium 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.

11. The nano transdermal delivery system based on superoxide dismutase according to claim 7, 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 materials include a surface modification monomer, a functional monomer, a crosslinking agent and an initiator; the surface modification monomer is selected from at least one of N-propylene succinimide, 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 At least one selected from [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide, 2-(dimethylamino)ethyl methacrylate, (3-acrylamidopropyl)trimethylammonium chloride hydrochloride, and aminopropyl methacrylamide hydrochloride; the cross-linking agent is selected from at least one selected from N,N'-methylenebisacrylamide, glycerol dimethacrylate, and glycerol 1,3-diglycerol alcohol diacrylate; the initiator is a water-soluble initiator, specifically selected from at least one selected from ammonium persulfate, sodium persulfate, and potassium persulfate.

12. The nano transdermal delivery system based on superoxide dismutase according to claim 11, characterized in that The polymer is a carbon-carbon unsaturated double bond monomer coated on the protein surface in an in-situ polymerization manner; the polymer raw materials include surface modification monomers, functional monomers, cross-linking agents, initiators and positively charged monomers.

13. The nano transdermal delivery system based on superoxide dismutase according to claim 11, characterized in that The mass ratio of superoxide dismutase to surface modification monomer is 1-10:1; 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.

14. The nano transdermal delivery system based on superoxide dismutase according to claim 12, characterized in that: The mass ratio of superoxide dismutase to surface modification monomer is 1-10:1; the molar ratio of superoxide dismutase to functional monomer is 1:4500-25000; the molar ratio of functional monomer to cross-linking agent is 1:0.05-0.1; and the molar ratio of functional monomer to positively charged monomer is 1:0.1-0.

12.

15. The method for preparing the nano transdermal delivery system based on superoxide dismutase according to claim 10, characterized in that: The preparation method is an electrostatic adsorption method: A superoxide dismutase solution is added to a positively or negatively charged polymer solution at 50-70°C and stirred. A small molecule active ingredient solution is added at 50-70°C and stirred continuously. A saline solution is added in an ice-water bath, reacted, and ultrafiltered and centrifuged to obtain a superoxide dismutase-based nanotransdermal delivery system.

16. The method for preparing the nano transdermal delivery system based on superoxide dismutase according to claim 11, characterized in that: The preparation method is an in-situ polymerization method: (S1) adding a surface-modified monomer solution to a superoxide dismutase buffer solution 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 a functional monomer, a positively charged 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) to initiate in situ polymerization to obtain a nano-transdermal delivery system based on superoxide dismutase.

17. The preparation method according to claim 16, characterized in that 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 1-5 wt%; 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.

18. The preparation method according to claim 15, characterized in that In method 2, the concentrations of the positively or negatively charged polymer solution, the superoxide dismutase solution, and the aqueous solution of the small molecule active ingredient 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; 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; the salt solution concentration is 0.5-5 mg / mL; and the molecular weight cut-off of the ultrafiltration is 3-5 kDa. 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.

19. Use of the superoxide dismutase-based nano transdermal delivery system according to claim 1 in the preparation of a method for preventing or treating oxidative stress diseases.

20. The use according to claim 19, characterized in that The oxidative stress diseases include skin photoaging or chloasma eczema.

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