Double-drug-loading cationic mixed micelle as well as preparation method and application thereof

Abstract

The invention discloses a double-drug-loading cationic mixed micelle as well as a preparation method and application thereof, and belongs to the field of pharmaceutics. The micelle is composed of a cationic polymer PEI-stearamide (PSA), an amphiphilic polymer DSPE-PEG2000, a hydrophobic drug resveratrol (RES) and a hydrophilic drug catalase (CAT). The preparation method comprises the following steps: 1) synthesizing PSA; the preparation method comprises 1, dissolving RES, PSA and DSPE-PEG2000 in an organic phase, injecting the obtained solution into a water phase, and carrying out rotary evaporation to obtain RES-loaded micelle (RES / PSA-PEG), and 3, adding a CAT solution to the micelle solution in a dropwise manner to obtain co-loaded micelle (RES / PSA-PEG / CAT). The micelle can effectively penetrate a corneal barrier and target a retinal pigment epithelial layer and a choroidal membrane.Through eye drop administration, RES anti-oxidation and anti-inflammatory effects and CAT hydrogen peroxide removal effects can be synergistically exerted, and an AMD curative effect can be substantially enhanced.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutics and relates to a double-drug-loaded cationic mixed micelle of resveratrol and catalase, and a preparation method and application thereof. Background Art

[0002] Age-related macular degeneration (AMD) is one of the leading causes of vision loss and even loss in middle-aged and elderly people worldwide. It falls under the category of "blurred vision" in Traditional Chinese Medicine (TCM). AMD can be divided into dry and wet forms based on fundus morphology. Its pathogenesis is complex, primarily related to oxidative damage to retinal cells and choroidal neovascularization (CNV). Currently, Western medicine treatments for AMD primarily include complement pathway inhibitors, visual cycle modulators, antioxidant therapy, laser therapy, and gene therapy. Existing therapeutic agents, such as complement inhibitors and anti-vascular endothelial growth factor (VEGF) inhibitors, require multiple doses, are expensive, have poor patient compliance, and are prone to complications such as infection, bleeding, and vascular embolism. Therefore, the discovery of multi-target therapeutics and efficient and safe drug delivery methods are urgent challenges in the field of pharmaceutical formulations for AMD treatment.

[0003] Resveratrol (RES) is a polyphenolic compound primarily found in the traditional Chinese medicines Polygonum cuspidatum and Veratrum truncatum. It exhibits antioxidant and anti-inflammatory properties. Studies have shown that resveratrol can reduce levels of the oxidative stress lipid malondialdehyde (MDA) in the eyes of mice, enhance the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in RPE cells, reduce the expression and secretion of pro-inflammatory cytokines such as IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1), inhibit the chemotactic migration of immune cells to sites of inflammation, and protect photoreceptor cells from damage, thereby treating dry AMD.

[0004] Catalase (CAT) is an antioxidant enzyme whose primary function is to catalyze the decomposition of hydrogen peroxide into water and oxygen, thereby reducing the accumulation of reactive oxygen species (ROS) and maintaining the oxidation-antioxidation balance. In age-related macular degeneration, oxidative stress is a key pathogenic factor. Catalase, due to its efficient hydrogen peroxide scavenging ability, has significant potential in antioxidant therapy, helping to inhibit tissue damage and inflammatory responses caused by increased ROS levels.

[0005] The poor water solubility and low bioavailability of RES, and the poor in vitro stability and low biomembrane permeability of CAT, greatly limit their application in the treatment of ocular diseases. Therefore, the development of suitable drug carriers is of great significance for the clinical application of both.

[0006] Currently, there are three ways to administer drugs to the eye: periocular injection, intraocular injection, and topical administration. Among them, intraocular injection is the main way to administer large molecule drugs for the treatment of retinal diseases. Although this method of administration effectively bypasses multiple barriers of the eye to obtain effective therapeutic concentrations in the posterior segment of the eye, this invasive method of administration leads to poor patient compliance, and injection administration has potential risks and complications. In contrast, topical eye drops are widely popular for their non-invasive treatment and convenience. In order to use topical eye drops to treat eye diseases, it is necessary to break through multiple eye barriers (corneal barrier, conjunctival barrier, and tear barrier) to improve drug absorption.

[0007] Cationic micelles are nano-delivery systems based on amphiphilic polymers that can simultaneously encapsulate hydrophobic (such as RES) and hydrophilic (such as CAT) drugs. Their positive surface charge enhances adhesion to negatively charged ocular tissues, such as the cornea, improving ocular retention and targeting. This allows drugs to more effectively cross the ocular delivery barrier, increasing ocular absorption and enhancing the therapeutic efficacy of drugs for AMD. Based on this, the present invention proposes encapsulating resveratrol and catalase in cationic micelles to achieve co-delivery of active ingredients of traditional Chinese medicines and protein drugs. This, combined with topical eye drops, enhances drug delivery efficiency and improves AMD treatment. Summary of the Invention

[0008] In view of the problems existing in the prior art, the present invention proposes a RES and CAT dual-drug-loaded cationic mixed micelle to improve the efficacy of drugs on AMD.

[0009] To achieve the above object, the technical solution of the present invention is as follows: A dual-drug-loaded cationic mixed micelle, consisting of the following components: the hydrophobic drug resveratrol; Hydrophilic drug catalase; A mixed micelle material composed of a cationic polymer polyethyleneimine-stearamide and an amphiphilic polymer DSPE-PEG2000, wherein the polyethyleneimine-stearamide is formed by amide bond condensation of polyethyleneimine with a molecular weight of 1800 g / mol and stearic acid; The mass ratio of the polyethyleneimine-stearamide to DSPE-PEG2000 is 0.5-3:1, the mass ratio of the polyethyleneimine-stearamide to catalase is 1:0.1-0.5, the resveratrol accounts for 1-5% of the mass of the mixed micelle material, and the Zeta potential of the micelle is positive, and the particle size is 100-300 nm.

[0010] Preferably, the mass ratio of the polyethyleneimine-stearamide to DSPE-PEG2000 is 1-2:1, and the optimal ratio is 1:1.

[0011] Preferably, the mass ratio of the polyethyleneimine-stearamide to catalase is 1:0.1-0.3, and the optimal mass ratio is 1:0.2.

[0012] Preferably, the resveratrol accounts for 3-5% by weight of the mixed micelle material, and optimally 3.5%.

[0013] The present invention further provides a method for preparing the dual-drug-loaded cationic mixed micelles, comprising the following steps: (1) Synthesis of polyethyleneimine-stearamide: Stearic acid, EDC, and NHS were dissolved in DMSO to activate the carboxyl groups, and polyethyleneimine with a molecular weight of 1800 g / mol was added for reaction. The mixture was purified by dialysis and freeze-dried. (2) Preparation of resveratrol-loaded cationic mixed micelles: dissolving polyethyleneimine-stearamide, resveratrol, and DSPE-PEG2000 in an organic solvent to form an organic phase, injecting it into the stirred aqueous phase, removing the organic solvent by rotary evaporation, and filtering; (3) Co-loading catalase: add catalase solution dropwise to the resveratrol-loaded cationic mixed micelle solution obtained in step (2), and vortex mix.

[0014] Preferably, the organic solvent is chloroform-methanol, with a volume ratio of 1-5:1, and the optimal volume ratio is 1:1.

[0015] Preferably, the volume ratio of the organic phase to the aqueous phase is 1:2-5, and optimally 1:4.

[0016] The micelles prepared by this invention penetrate the corneal barrier through topical eye drops, targeting the retinal pigment epithelium (RPE) and choroid. RES inhibits oxidative stress and inflammatory factors, while CAT catalyzes the decomposition of hydrogen peroxide. The synergistic effect of these two enhances the treatment of AMD. DSPE-PEG2000 neutralizes the cytotoxicity of polyethyleneimine-stearamide and enhances ocular tissue permeability. These micelles also offer advantages such as high drug loading, stable properties, high bioavailability, ease of use, and minimal complications.

[0017] For more detailed technical solutions, please refer to the specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Stability determination results of dual-drug loaded nanomicelles.

[0019] Figure 2 : Distribution of dual-drug loaded nanomicelles in the fundus.

[0020] Figure 3 : Results of catalase activity assay of dual-drug loaded nanomicelles, ***P<0.005.

[0021] Figure 4 : In vitro stability determination of catalase in dual-drug loaded nanomicelles.

[0022] Figure 5 : Toxicity of dual-drug loaded nanomicelles to HCE-T cells.

[0023] Figure 6 : Flow cytometry was used to detect the uptake of nanomicelles by HCE-T cells, *P<0.05, ****P<0.001.

[0024] Figure 7 : DHE staining of mouse eyeball sections, in which DAPI represents nucleus staining and blue represents nucleus; Merge represents the merged image of DAPI and DHE staining, red represents ROS (reactive oxygen species), and purple represents the overlapping area of ​​nucleus and ROS staining. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to specific embodiments.

[0026] Key material description: Polyethyleneimine: molecular weight 1800 g / mol, purchased from Aladdin.

[0027] DSPE-PEG2000: distearoylphosphatidylethanolamine-polyethylene glycol 2000, purchased from Ruixibio.

[0028] Resveratrol: purity 98%, purchased from Yuanye Company.

[0029] Catalase: enzyme activity 2000-5000 u / mg, purchased from Yuanye Company.

[0030] HCE-T cells: human corneal epithelial cells, purchased from Procell.

[0031] Other materials not described herein are conventional materials in this field and can be obtained through commercial channels.

[0032] Example 1 Step 1: Synthesis of PEI-stearamide cationic micelles First, 71.2 mg of stearic acid, 57.5 mg of EDC, and 34.5 mg of NHS were weighed and dissolved in DMSO. The mixture was stirred to fully activate the carboxyl groups of the stearic acid. Next, 752.1 mg of polyethyleneimine (molecular weight 1800 g / mol, PEI-1.8K) was dissolved in DMSO and added to the stirring mixture. Stirring was continued for 24 hours. Finally, the product was purified by dialysis using a MWCO 1K bag for 2 days. The supernatant was collected by centrifugation (13,000 rpm, 30 minutes), filtered through a 0.22 μm filter, and the filtrate was freeze-dried to obtain a white, fluffy PEI-stearamide (hereafter referred to as PSA).

[0033] The PEI skeleton itself is hydrophilic, and the long-chain stearoyl groups (C 17 Alkyl chains) are strongly hydrophobic groups. This structure gives the product the characteristics of a surfactant or amphiphilic polymer, which can aggregate at the water / oil interface to form micelles.

[0034] Step 2: Preparation of resveratrol-loaded cationic mixed micelles Drug-loaded micelles were prepared using the injection method: 0.21 mg of resveratrol, 3 mg of PSA, and 3 mg of DSPE-PEG2000 were dissolved in 0.5 mL of a mixed solvent of chloroform and methanol (1:1, v / v) as the organic phase; 2 mL of pure water was placed in a vial as the aqueous phase; under magnetic stirring at 600 rpm, the organic phase was injected into the aqueous phase using a syringe. After 20 minutes, the solution in the vial was transferred to a round-bottom flask, rotary evaporated at 50°C to remove the organic solvent, and filtered with a 0.22 µm filter to obtain resveratrol-loaded cationic mixed micelles (hereinafter referred to as RES / PSA@PEG).

[0035] The cationic mixed micelles are composed of DSPE-PEG and PSA. DSPE-PEG is a phospholipid-polyethylene glycol conjugate with biocompatibility and amphiphilic properties. It has strong membrane fluidity and can form micelles upon self-hydration. Through a specific preparation process, DSPE-PEG can combine with other drugs or active ingredients to form a stable micelle structure. The polyethyleneimine in PSA is a cationic material that generally exhibits good adhesion and can tightly adhere to negatively charged surfaces. The cornea is typically negatively charged due to the presence of negatively charged molecules such as glycoproteins and phospholipids on the surface of corneal epithelial cells, which allows polyethyleneimine to effectively bind to them. This binding ensures close contact between the drug or carrier and the cornea, thereby improving drug absorption efficiency. However, cationic materials have certain toxicities and may damage the integrity of cell membranes. Therefore, by introducing DSPE-PEG to neutralize the toxicity of PSA and enhance its fluidity, the mixed micelle system can promote the penetration of drugs or carriers into the eye, improving delivery efficiency.

[0036] Step 3: Preparation of resveratrol and catalase co-loaded cationic mixed micelles 100 μL of the prepared RES / PSA@PEG solution was taken and CAT solution was added dropwise to make the mass ratio of PSA / CAT 1:0.2. The mixture was vortexed to obtain the co-drug-loaded cationic mixed micelle solution (hereinafter referred to as RES / PSA@PEG / CAT).

[0037] Example 2 Step 1: Synthesis of PEI-stearamide cationic micelles The synthesis method of PSA is the same as that in Example 1.

[0038] Step 2: Preparation of resveratrol-loaded cationic mixed micelles Drug-loaded micelles were prepared using the injection method: 0.3 mg of resveratrol, 4.5 mg of PSA, and 1.5 mg of DSPE-PEG2000 were dissolved in 0.5 mL of a mixed solvent of chloroform and methanol (4:1, v / v) as the organic phase; 2.5 mL of pure water was placed in a vial as the aqueous phase; under magnetic stirring at 600 rpm, the organic phase was injected into the aqueous phase using a syringe. After 20 minutes, the solution in the vial was transferred to a round-bottom flask, rotary evaporated at 50°C to remove the organic solvent, and filtered with a 0.22 µm filter to obtain RES / PSA@PEG.

[0039] Step 3: Preparation of resveratrol and catalase co-loaded cationic mixed micelles 100 μL of the prepared RES / PSA@PEG solution was taken and CAT solution was added dropwise to make the mass ratio of PSA / CAT 1:0.1. The mixture was vortexed to obtain RES / PSA@PEG / CAT.

[0040] Example 3 Step 1: Synthesis of PEI-stearamide cationic micelles The synthesis method of PSA is the same as that in Example 1.

[0041] Step 2: Preparation of resveratrol-loaded cationic mixed micelles Drug-loaded micelles were prepared using the injection method: 0.15 mg of resveratrol, 4.2 mg of PSA, and 1.8 mg of DSPE-PEG2000 were dissolved in 0.5 mL of a mixed solvent of chloroform and methanol (3:1, v / v) as the organic phase; 1.5 mL of pure water was placed in a vial as the aqueous phase; under magnetic stirring at 600 rpm, the organic phase was injected into the aqueous phase using a syringe. After 20 minutes, the solution in the vial was transferred to a round-bottom flask, rotary evaporated at 50°C to remove the organic solvent, and filtered with a 0.22 µm filter to obtain RES / PSA@PEG.

[0042] Step 3: Preparation of resveratrol and catalase co-loaded cationic mixed micelles 100 μL of the prepared RES / PSA@PEG solution was taken and CAT solution was added dropwise to make the mass ratio of PSA / CAT 1:0.5. The mixture was vortexed to obtain RES / PSA@PEG / CAT.

[0043] Example 4 Step 1: Synthesis of PEI-stearamide cationic micelles The synthesis method of PSA is the same as that in Example 1.

[0044] Step 2: Preparation of resveratrol-loaded cationic mixed micelles Drug-loaded micelles were prepared using the injection method: 0.09 mg of resveratrol, 2 mg of PSA, and 4 mg of DSPE-PEG2000 were dissolved in 0.5 mL of a mixed solvent of chloroform and methanol (2:1, v / v) as the organic phase; 1 mL of pure water was placed in a vial as the aqueous phase; under magnetic stirring at 600 rpm, the organic phase was injected into the aqueous phase using a syringe. After 20 minutes, the solution in the vial was transferred to a round-bottom flask, rotary evaporated at 50°C to remove the organic solvent, and filtered with a 0.22 µm filter to obtain RES / PSA@PEG.

[0045] Step 3: Preparation of resveratrol and catalase co-loaded cationic mixed micelles 100 μL of the prepared RES / PSA@PEG solution was taken and CAT solution was added dropwise to make the mass ratio of PSA / CAT 1:0.3. The mixture was vortexed to obtain RES / PSA@PEG / CAT.

[0046] Comparative Example 0.21 mg of resveratrol and 6 mg of PSA were dissolved in 0.5 mL of a 1:1, v / v, chloroform-methanol mixture to form the organic phase. 2 mL of pure water was placed in a vial to form the aqueous phase. Under magnetic stirring at 600 rpm, the organic phase was syringed into the aqueous phase. After 20 minutes, the solution in the vial was transferred to a round-bottom flask, where the organic solvent was removed by rotary evaporation at 50°C. The solution was then filtered through a 0.22 µm filter to prepare resveratrol-loaded cationic micelles (RES / PSA). To 100 μL of the prepared RES / PSA solution, CAT solution was added dropwise to a PSA / CAT ratio of 1:0.2. The solution was vortexed to obtain the co-drug-loaded cationic micelle solution (RES / PSA / CAT).

[0047] Test example 1. Determination of physical and chemical properties 1) Determination of particle size, PDI, and Zeta potential The particle size and potential of the cationic mixed micelles were measured using a ZEN3600 series nanoparticle size analyzer. 0.1 mL of the cationic mixed micelles was diluted to 1 mL. The nanoparticle size analyzer temperature was set to 25°C, the equilibrium time was 2 minutes, and the measurement was repeated three times for each sample.

[0048] 2) The encapsulation efficiency and drug loading were determined as follows: The RES content was determined by HPLC. The chromatographic column was InertSustain AQ-C18 (250 mm × 4.6 mm, 5 μm); the mobile phase was methanol-water (42:58, v / v); the detection wavelength was 305 nm; the volume flow rate was 1.0 mL / min; the column temperature was 35°C; and the injection volume was 10 μL.

[0049] Place 1 ml of RES / PSA@PEG / CAT solution in an ultrafiltration tube (molecular weight cutoff 10 kDa) and centrifuge at 3500 rpm for 20 minutes. Collect the free drug in the lower layer, filter through a membrane, and determine the amount of free drug by HPLC, which is recorded as W1. Separately, add 0.5 ml of RES / PSA@PEG / CAT solution to a three-fold volume of methanol to break the emulsion. Filter through a 0.22 μm membrane and determine the total amount of drug in the sample, W2, by HPLC. Wm is the total mass of the carrier and drug. Calculate the concentration of RES encapsulated in the cationic micelles, and then calculate the encapsulation efficiency and drug loading of RES according to the following formula: Encapsulation efficiency (EE%) = (W2-W1) / W2×100% Drug loading (DL%) = (W2-W1) / W m ×100% Table 1: Results of physicochemical properties of mixed micelles

[0050] 2. Stability Determination The samples prepared in Example 1 and the comparative example were placed in a refrigerator at 4°C in the dark for 7 days, and their particle size and PDI changes were measured. Figure 1 As shown in the figure, the particle size and PDI of the prepared RES / PSA@PEG / CAT were more stable than those of RES / PSA / CAT, with no obvious changes within two weeks, no visible insoluble particles, and no obvious aggregation and precipitation.

[0051] 3. Observation of the distribution of mixed micelles in the fundus by frozen section In order to investigate the distribution of cationic mixed micelles in the mouse eye, healthy C57BL6J mice were administered eye drops. The administration volume for Example 1 and the comparative example was 7 μL. 4 hours after administration, the mice were killed, the eyeballs were removed, and fixed with eyeball fixative for 24 hours. Then, the eyeballs were transferred to a 15% sucrose aqueous solution, and after dehydration and sinking to the bottom, they were further dehydrated with a 30% sucrose solution for 24 hours. Then, the eyeballs were frozen and sectioned. Finally, they were stained with DAPI for 5 minutes and observed under a fluorescence microscope.

[0052] like Figure 2 As shown, it can be seen that the cationic mixed micelles can enter the RPE and choroid layers, which shows that the addition of DSPE-PEG can promote the delivery of drugs into the eye.

[0053] 4. Catalase Activity Assay The activity was determined using a catalase activity kit. For specific methods, refer to the kit instructions.

[0054] like Figure 3 As shown in the figure, the prepared RES / PSA@PEG / CAT not only maintains the original enzymatic activity of CAT, but also significantly improves the enzymatic activity compared with CAT.

[0055] 5. In vitro stability determination of CAT We tested the duration of activity of CAT and RES / PSA@PEG / CAT at 37°C. The samples were dissolved in PBS and incubated at 37°C for 72 h. RES / PSA@PEG / CAT still showed high activity ( Figure 4 ), which can ensure the therapeutic effect of CAT in the body.

[0056] 6. Cytotoxicity Evaluation HCE-T cells with good growth status were taken and seeded into 96-well plates (cell density was 1×10 4Cells were plated in a humidified atmosphere at 37°C and 5% CO₂ for 24 hours. RES / PSA@PEG / CAT micelles at varying concentrations (10-200 μg / mL) were then added to the plates and incubated for 24 hours (five replicate wells per group). Cell viability was then assessed using the CCK8 assay. First, the supernatant was removed from the wells. 100 μL of 0.1 M CCK8 culture medium was then added to each well. After an additional 30-minute incubation, the absorbance at 450 nm was recorded using a microplate reader. The control absorbance (OD) was set as 100%, and cell viability was calculated using the formula.

[0057] Cell viability (%) = [OD (drug) - OD (blank)] / [OD (control) - OD (blank)] * 100% The results showed that at high concentrations, the cell survival rate of RES / PSA@PEG / CAT was significantly higher than that of RES / PSA / CAT ( Figure 5 ).

[0058] 7. Flow cytometry to detect cellular micelle uptake Well-grown HCE-T cells were counted using a cell counter and seeded into 6-well plates at approximately 500,000 cells per well. The cells were cultured for 24 hours. The cells were grouped into three groups: Blank, Free CAT, and RES / PSA@PEG / CAT, with triplicate wells per group. 1 mL of the formulation solution (containing 2 μg of FITC) was added to each well. After administration, the plates were transferred to an incubator and incubated for 2 hours. After incubation, the cells were trypsinized and digested with complete culture medium. The cells were then pipetted and resuspended, centrifuged at 1800 rpm for 3 minutes, and the supernatant was discarded. The cells were washed twice with ice-cold PBS and resuspended in a flow cytometer. Intracellular fluorescence intensity was measured using a flow cytometer using 10,000 cells at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0059] Flow cytometry results showed that ( Figure 6 ), in the HCE-T cell line, RES / PSA@PEG / CAT can effectively promote the cellular uptake of fluorescent labeled substances compared with free CAT, which indicates that the carrier improves the cellular uptake efficiency of CAT.

[0060] 8. In vivo efficacy experiments Experimental methods: Healthy male C57 / BL6J mice, weighing approximately 20 g, were housed for one week before the experiment. Models were established by injecting sodium iodate solution (50 mg / kg) into the tail vein of the C57 / BL6J mice. The experimental groups were as follows: Normal group (Control): healthy C57 mice were housed normally; Model group (Model): model mice were administered ocular drops with PBS; Free RES group: a certain amount of resveratrol was dissolved in PBS, sonicated to prepare a resveratrol suspension, and administered ocular drops to the model mice; Free CAT group: a certain amount of catalase was dissolved in PBS, vortexed, and administered ocular drops to the model mice; RES / PSA@PEG group: model mice were administered ocular drops with a RES / PSA@PEG micelle solution; PSA@PEG / CAT group: model mice were administered ocular drops with a PSA@PEG / CAT micelle solution; and RES / PSA@PEG / CAT group: model mice were administered ocular drops with a RES / PSA@PEG / CAT micelle solution. There were 3 experimental subjects in each group, for a total of 7 groups.

[0061] Model mice were administered eye drops daily at 8 am and 8 pm for 14 days. A volume of 7 μL (the free drug concentration was the same as that in the micelles, containing 0.735 μg resveratrol and 2.1 μg catalase) was administered. After 14 days of administration, eyeballs from three mice were removed and quickly frozen on dry ice. The frozen eyeballs were then transferred to OCT embedding medium and oriented, ensuring that the corneal-retinal axis was perpendicular to the sectioning plane. Serial sections were cut using a cryostat pre-chilled to -20°C, obtaining 7 μm-thick sagittal sections. The sections were mounted on pre-chilled, anti-smear slides and stored at -80°C. After thawing, sections were stained with 10 μM dihydroethidium (DHE) solution in the dark. After incubation at 37°C for 60 minutes, the sections were washed four times for 5 minutes each in PBS on a decolorizing shaker. After briefly drying the sections, add a drop of DAPI stain to the tissue and incubate at room temperature in the dark for 5 minutes. Wash the slides four times in PBS on a destaining shaker for 5 minutes each. Immediately after mounting, observe under a fluorescence microscope (excitation wavelength 520 nm, emission wavelength 605 nm).

[0062] Experimental results: The DHE staining of the eyeball sections in the model group showed a strong red fluorescence signal, indicating a large accumulation of ROS; after RES / PSA@PEG / CAT treatment, the fluorescence intensity was significantly weakened compared with the model group, and was significantly better than that of the free drug group and the single drug-loaded mixed micelle group ( Figure 7 The results indicate that RES and CAT co-loaded cationic mixed micelles can synergistically enhance the ROS scavenging ability.

Claims

1. A dual-drug-loaded cationic mixed micelle, characterized in that: It is composed of the following components: the hydrophobic drug resveratrol; Hydrophilic drug catalase; A mixed micelle material composed of a cationic polymer polyethyleneimine-stearamide and an amphiphilic polymer DSPE-PEG2000, wherein the polyethyleneimine-stearamide is formed by amide bond condensation of polyethyleneimine with a molecular weight of 1800 g / mol and stearic acid; The mass ratio of the polyethyleneimine-stearamide to DSPE-PEG2000 is 0.5-3:1, the mass ratio of the polyethyleneimine-stearamide to catalase is 1:0.1-0.5, the resveratrol accounts for 1-5% of the mass of the mixed micelle material, and the Zeta potential of the micelle is positive, and the particle size is 100-300 nm.

2. The dual-drug-loaded cationic mixed micelle according to claim 1, characterized in that: The mass ratio of the polyethyleneimine-stearamide to DSPE-PEG2000 is 1-2:

1.

3. The dual-drug-loaded cationic mixed micelle according to claim 1, characterized in that: The mass ratio of the polyethyleneimine-stearamide to the catalase is 1:0.1-0.

3.

4. The dual-drug-loaded cationic mixed micelle according to claim 1, characterized in that: The resveratrol accounts for 3-5% of the mass of the mixed micelle material.

5. A method for preparing the dual-drug-loaded cationic mixed micelles according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Synthesis of polyethyleneimine-stearamide: Stearic acid, EDC, and NHS were dissolved in DMSO to activate the carboxyl groups, and polyethyleneimine with a molecular weight of 1800 g / mol was added for reaction. The mixture was purified by dialysis and freeze-dried. (2) Preparation of resveratrol-loaded cationic mixed micelles: dissolving polyethyleneimine-stearamide, resveratrol, and DSPE-PEG2000 in an organic solvent to form an organic phase, injecting it into the stirred aqueous phase, removing the organic solvent by rotary evaporation, and filtering; (3) Co-loading catalase: add catalase solution dropwise to the resveratrol-loaded cationic mixed micelle solution obtained in step (2), and vortex mix.

6. The preparation method according to claim 5, wherein: The organic solvent is chloroform-methanol, with a volume ratio of 1-5:

1.

7. The preparation method according to claim 5, wherein: The volume ratio of the organic phase to the aqueous phase is 1:2-5.

8. Use of the dual-drug-loaded cationic mixed micelles according to any one of claims 1 to 4 in the preparation of a drug for treating age-related macular degeneration.

9. The use according to claim 8, characterized in that: The medicine is eye drops.

10. A drug for treating age-related macular degeneration, characterized in that: The medicine contains the dual-drug-loaded cationic mixed micelles according to any one of claims 1 to 4, and the medicine dosage form is eye drops.

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