Nanoparticles loaded with aflibercept and curcumin as well as preparation method and application of nanoparticles loaded with aflibercept and curcumin

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a PLGA / TPGS loaded aflibercept-curcumin nanoparticle as well as a preparation method and application thereof. According to the present invention, the PLGA / TPGS loaded aflibercept-curcumin nanoparticles (AFL-Cur (at) PT NPs) are prepared through the water-in-oil-in-water (Water / Oil / Water, W / O / W) multiple emulsion method, the nanoparticles can effectively inhibit CNV in vitro and in vivo, and the effect is significantly superior to that of pure aflibercept or curcumin. The invention provides a more efficient and safer treatment strategy for CNV-related eye diseases, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a PLGA / TPGS-loaded aflibercept-curcumin nanoparticle, its preparation method, and its application. Background Technology

[0002] Age-related macular degeneration (AMD) is a leading cause of vision loss in the elderly. In developed countries, AMD has become the leading cause of irreversible central vision loss. AMD is classified into two types: dry and wet. Wet AMD, also known as neovascular AMD (nAMD), is characterized primarily by choroidal neovascularization (CNV). CNV refers to the abnormal proliferation of choroidal capillaries, which breach Bruch's membrane and invade the retinal pigment epithelium (RPE) layer or subretinal space, leading to a rapid decline in vision.

[0003] Anti-VEGF therapy is currently the gold standard treatment for nAMD. However, due to the choroid's location deep within the fundus, this unique physiological position presents numerous limitations and challenges to anti-VEGF therapy. Effective drug delivery to the CNV requires overcoming four key challenges: navigating multiple physiological barriers, targeting specific fundus cell types, delivering the appropriate drug, and achieving sustained drug release over an extended period. Furthermore, anti-VEGF drugs typically have short half-lives, and while they can inhibit angiogenesis in the short term, frequent intravitreal injections are necessary. Frequent injections not only increase the risks associated with surgery such as infection, hemorrhage, retinal detachment, and cataracts, but also impose an additional economic burden on patients and society, significantly reducing patient adherence. Therefore, the primary challenge currently lies in extending the interval between intravitreal injections and ensuring sufficient drug concentration in the vitreous humor to improve treatment effectiveness. Summary of the Invention

[0004] This invention utilizes two materials, polylactic-co-glycolic acid copolymer (PLGA) and vitamin E polyethylene glycol succinate (TPGS), to simultaneously load aflibercept (AFL) and curcumin (Cur) onto PLGA / TPGS (PT) via a double emulsion method, synthesizing AFL-Cur@PT. Intravitreal injection of AFL-Cur@PT provides sustained release of anti-angiogenic and anti-inflammatory drugs, enhancing the inhibitory effect on CNV. It is safe to use and possesses broad application prospects and market value.

[0005] This invention is specifically implemented through the following scheme:

[0006] In a first aspect, the present invention provides aflibercept and curcumin nanoparticles, characterized in that they comprise aflibercept, curcumin, polylactic acid-glycolic acid copolymer and vitamin E polyethylene glycol succinate.

[0007] Furthermore, the aflibercept and curcumin nanoparticles are characterized in that, based on the mass ratio of raw materials, the mass ratio of aflibercept, curcumin, polylactic acid-glycolic acid copolymer and vitamin E polyethylene glycol succinate is 8:4:140:3.

[0008] On the other hand, the present invention provides a method for preparing nanoparticles loaded with aflibercept and curcumin as described in claim 1, characterized in that it includes the following steps:

[0009] S1: Dissolve curcumin and polylactic acid glycolic acid copolymer in a mixture of dichloromethane and acetone to obtain an oil phase solution; dissolve aflibercept in water to obtain a first aqueous phase solution;

[0010] S2: Add the first aqueous solution to the oil phase solution and mix by ultrasonication to obtain oil-in-water solution particles;

[0011] S3: Dissolve vitamin E polyethylene glycol succinate and polyvinyl alcohol in water to obtain a second aqueous phase;

[0012] S4: Add the oil-in-water solution from S2 to the second aqueous phase solution and mix by ultrasonication to obtain an oil-in-water solution.

[0013] S5: Stir the water-in-oil-in-water solution, centrifuge, remove the supernatant, and wash to obtain loaded aflibercept-curcumin nanoparticles.

[0014] Furthermore, the method for preparing nanoparticles loaded with aflibercept and curcumin is characterized in that step S1, the polylactic acid-glycolic acid copolymer, includes lactic acid and glycolic acid, wherein the mass ratio of lactic acid to glycolic acid is 75:25.

[0015] Furthermore, the method for preparing nanoparticles loaded with aflibercept and curcumin is characterized in that, in step S1, the mass of curcumin is 4-5 mg and the mass of polylactic acid-glycolic acid copolymer is 140-150 mg.

[0016] Furthermore, the method for preparing nanoparticles loaded with aflibercept and curcumin is characterized in that, in step S2, the concentration of aflibercept is 40~50 mg / mL.

[0017] Furthermore, the method for preparing nanoparticles loaded with aflibercept and curcumin is characterized in that, in step S2, the ultrasonic conditions are as follows: the ultrasonic disruptor is set to a power of 150~160 W, an amplitude of 60~70%, and the ultrasonic cycle is 5 seconds of working time and 7 seconds of rest.

[0018] Furthermore, the method for preparing the nanoparticles loaded with aflibercept and curcumin is characterized in that, in step S3, the concentration of polyvinyl alcohol is 2-3%, and the concentration of vitamin E polyethylene glycol succinate is 0.3-0.4 mg / mL.

[0019] Furthermore, the method for preparing nanoparticles loaded with aflibercept and curcumin is characterized in that, in step S4, the ultrasonic conditions are: stirring at 800-900 rpm for 5-6 hours, followed by centrifugation at 12000-13000 rpm for 30-40 minutes.

[0020] On the other hand, the present invention provides the application of nanoparticles loaded with aflibercept and curcumin as described above, or nanoparticles loaded with aflibercept and curcumin prepared as described above, in the preparation of drugs that inhibit choroidal angiogenesis. Attached Figure Description

[0021] Figure 1 The diagram shows the structural characterization of nanoparticles loaded with aflibercept and / or curcumin. Figures A and C represent the particle size (B), polymer dispersity index (PDI), and zeta potential (n=3), respectively. In the diagrams, PT represents PLGA / TPGS, AFL@PT represents aflibercept@PLGA / TPGS, Cur@PT represents curcumin@PLGA / TPGS, and AFL-Cur@PT represents aflibercept-curcumin@PLGA / TPGS.

[0022] Figure 2 The images show the transmission electron microscope (TEM) images and nanoparticle size distribution of AFL-Cur@PT, where A is the TEM image of AFL-Cur@PT and B is the nanoparticle size distribution curve of AFL-Cur@PT.

[0023] Figure 3 The infrared spectrum of AFL-Cur@PT;

[0024] Figure 4 The UV absorption spectrum of AFL-Cur@PT;

[0025] Figure 5The figures show the in vitro and in vivo drug release curves of AFL-Cur@PT, where A is the in vitro cumulative release curve of AFL and Cur in AFL-Cur@PT, and B is the drug concentration of AFL (ng / g) and Cur (μg / g) in the retinal / choroidal complex at different time points after intravitreal injection of 2 μL of AFL-Cur@PT (Cur: 5 mg / mL, AFL: 60 mg / mL) in mice.

[0026] Figure 6 The figure shows the results of the in vitro CCK-8 cytotoxicity assay for PT.

[0027] Figure 7 Figure showing the results of in vitro apoptosis experiments on PT NPs;

[0028] Figure 8 The figure shows the results of the ocular safety study of PT NPs; where A is the H&E staining and statistical diagram of mouse retinal sections, B is the TUNEL staining diagram of frozen sections of mouse retina, and C is the electroretinogram (ERG) and statistical diagram of mice.

[0029] Figure 9 The graph shows the results of the in vivo safety ELISA assay for PT.

[0030] Figure 10 The image shows the hematological results of PT's in vivo safety, where BUN is blood urea nitrogen (mmol / L), TP is total protein (g / L), ALB is alkaline phosphatase (g / L), ALT is alanine aminotransferase (U / L), ALP is alkaline phosphatase (U / L), AST is aspartate aminotransferase (U / L), and WBC is white blood cell count (10⁻⁶). 9 / L) RBC count is red blood cells (10 12 / L);

[0031] Figure 11 H&E staining images of tissue sections from the heart, liver, spleen, lung, and kidney after PT injection;

[0032] Figure 12 The image shows the Western Blot results of the apoptosis proteins Bax, Bcl-2, and Caspase3 in tissues after PT injection.

[0033] Figure 13 The figure shows the experimental results of HRVECs uptake of PT NPs;

[0034] Figure 14 Figure showing the results of the RF / 6A cell uptake experiment of PT NPs;

[0035] Figure 15 Figure showing the results of PT uptake experiment in 661W cells;

[0036] Figure 16 Figure showing the results of the ARPE-19 cell uptake experiment for PT;

[0037] Figure 17 Figure showing the results of the PT uptake experiment in Müller cells;

[0038] Figure 18 The image shows the results of the retinal uptake experiment of PT NPs.

[0039] Figure 19 Figure 1 shows the results of in vivo imaging experiments of PT NPs;

[0040] Figure 20 Figure showing the effect of AFL-Cur@PT on HRVECs cell proliferation;

[0041] Figure 21 Figure showing the effect of AFL-Cur@PT on HRVECs cell migration;

[0042] Figure 22 Figure showing the effect of AFL-Cur@PT on tube formation in HRVECs cells;

[0043] Figure 23 Figure showing the effect of AFL-Cur@PT on the proliferation of RF / 6A cells;

[0044] Figure 24 Figure showing the effect of AFL-Cur@PT on RF / 6A cell migration;

[0045] Figure 25 Figure showing the effect of AFL-Cur@PT on tube formation in RF / 6A cells;

[0046] Figure 26 Figure showing the effect of AFL-Cur@PT on laser-induced CNV;

[0047] Figure 27 Figure showing the effect of AFL-Cur@PT on laser-induced macrophage infiltration in CNV lesions;

[0048] Figure 28 Figure showing the effect of AFL-Cur@PT on laser-induced neutrophil infiltration in CNV lesions;

[0049] Figure 29 A schematic diagram showing the changes in intraocular pressure in CNV model mice after intravitreal injection treatment;

[0050] Figure 30 Figure showing the effect of AFL-Cur@PT on the mRNA and protein expression levels of VEGF in laser-induced CNV;

[0051] Figure 31 Figure showing the results of Western blotting experiments detecting proteins in the VEGF-related signaling pathway;

[0052] Figure 32 Figure showing the effect of AFL-Cur@PT on the mRNA expression of inflammatory factors IL-6, TNF-α, MCP-1, IL-1β, ICAM-1 and NFκB-1 in laser-induced CNV;

[0053] Figure 33 Figure showing the effect of AFL-Cur@PT on the expression of inflammatory factors IL-6, TNF-α, IL-1β and MCP-1 proteins in laser-induced CNV;

[0054] Figure 34 Figure showing the results of protein level analysis of downstream signaling pathways of curcumin, including p-P65, p-ERK1 / 2, ERK1 / 2, p-P38, p38, p-JNK, JNK, HO-1, Nrf2, and ICAM-1.

[0055] Figure 35 A schematic diagram illustrating the preparation of AFL-Cur@PT and its therapeutic mechanism in CNV models. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0057] It should be noted that the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0058] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0059] Example 1: Preparation of PLGA / TPGS-loaded aflibercept-curcumin nanoparticles (AFL-Cur@PT NPs) and its control group

[0060] AFL-Cur@PT NPs were prepared using a water-in-oil-in-water (W / O / W) double emulsion method. The specific steps are as follows:

[0061] 1. Accurately weigh 4 mg curcumin (Cur) and 140 mg polylactic acid-glycolic acid copolymer (PLGA), and fully dissolve them in 2 mL of a mixture of dichloromethane and acetone (dichloromethane to acetone volume ratio of 4:1). The polylactic acid-glycolic acid copolymer is copolymerized by copolymerizing lactic acid and glycolic acid in a molar percentage of 75:25 to obtain an oil phase solution (Oil, O).

[0062] 2. Accurately pipette 200 µL of aflibercept (AFL) solution with a concentration of 40 mg / mL as the first aqueous phase (Water, W), and slowly add it dropwise to the oil phase solution. Vortex mix the solution, then place the mixture on ice and sonicate it for 4.8 min using an ultrasonic disruptor (power: 150 W, amplitude: 60%, ultrasonic cycle: 5 s working period and 7 s interval) to obtain a water-in-oil (O / W) solution.

[0063] 3. Prepare a 2% (w / w) polyvinyl alcohol (PVA) aqueous solution. Add 3 mg TPGS to every 10 mL of PVA aqueous solution to obtain a second aqueous phase solution with a TPGS concentration of 0.3 mg / mL. Using a 1 mL syringe, draw the above oil-in-water solution and evenly drop it into 10 mL of the second aqueous phase solution. Sonicate for 9.6 min (power: 150 W, amplitude: 60%, ultrasonic cycle: 5 s working period and 7 s interval) to obtain a water-in-oil-in-water (W / O / W) solution.

[0064] 4. The above solution was centrifuged and stirred at 800 rpm for 5 h, followed by centrifugation at 12000 rpm for 30 min. The supernatant was removed and the solution was washed with ultrapure water (12000 rpm, 15 min). This process was repeated 3 times to obtain nanoparticles loaded with aflibercept and curcumin, named AFL-Cur@PT NPs.

[0065] 5. The AFL-Cur@PT NPs obtained in step 4 were freeze-dried using 5% trehalose as a freeze-drying protectant. The freeze-dried product was stored at -20 °C.

[0066] The method for preparing AFL@PT NPs by loading aflibercept (AFL) onto PLGA / TPGS is the same as the method for preparing AFL-Cur@PT NPs, except that Cur is not added in the first step.

[0067] The method for preparing Cur@PT NPs using PLGA / TPGS-loaded curcumin (Cur) is the same as that for AFL-Cur@PT NPs, except that 200 µL of AFL solution is replaced with 200 µL of water.

[0068] The preparation method of simple carrier PT NPs is the same as that of AFL-Cur@PT NPs, except that Cur is not added in the first step and 200 µL of AFL solution is replaced with 200 µL of water.

[0069] The preparation method for C6@PT NPs is the same as that for AFL-Cur@PT NPs, except that in step 1, 4 mg Cur is replaced with 4 mg coumarin 6 (C6) and 200 µL of AFL solution is replaced with 200 µL of water.

[0070] PT (6.7 μg / mL), Cur @PT (Cur: 5 μg / mL), AFL@PT (Aflibercept: 60 μg / mL), and AFL-Cur@PT (Cur: 5 μg / mL, AFL: 60 μg / mL) were prepared and characterized. The final experimental concentrations were obtained at certain dilutions: PT (6.7 μg / mL), Cur @PT (Cur: 3.3 μg / mL), AFL@PT (Aflibercept: 40 μg / mL), and AFL-Cur@PT (Cur: 3.3 μg / mL, AFL: 40 μg / mL).

[0071] This preparation method can simultaneously encapsulate water-soluble AFL and hydrophobic Cur, thereby preparing NPs with both anti-angiogenic and anti-inflammatory effects. The dual emulsion interface (W1 / O / W2) forms a physical barrier, reducing the burst release of AFL and Cur during the preparation process.

[0072] Example 2 Characterization of AFL-Cur@PT NPs

[0073] 2.1 Detection of NPs particle size, polymer dispersion index (PDI), and zeta potential

[0074] AFL-Cur@PT NPs were prepared by diluting them to a semi-transparent state with ultrapure water. The ideal dilution was confirmed by the Tyndall effect. The NPs were placed in cuvettes. Particle size, polymer dispersion index (PDI), and zeta potential were measured using a Litesizer DLS particle size analyzer. Each sample was tested three times to ensure data reliability. PDI is defined by the ratio of the polymer's weight-average molecular weight (Mw) to its number-average molecular weight (Mn). When the PDI value is close to 0, it indicates a highly uniform particle size distribution, such as ideal monodisperse nanospheres or standard calibration particles. When the PDI value is greater than 0.1, it indicates a significant difference in particle size; the higher the PDI, the wider the particle size distribution. The zeta potential reflects the strength of the electrostatic interaction between droplets and the adsorption of emulsifiers at the oil-water interface, and is considered an indicator of the stability of the colloidal system.

[0075] like Figure 1 As shown in Figure A, the particle sizes of NPs PT, AFL@PT, Cur@PT, and AFL-Cur@PT are (165.3±3.84) nm, (214±7.57) nm, (188.7±6.69) nm, and (236.3±8.99) nm, respectively. The PDI of all four NPs is <0.1. Figure 1 (See Figure B). The Zeta potentials of PT, AFL@PT, Cur@PT, and AFL-Cur@PT are (-22.93±1.09) mV, (-17.1±0.59) mV, (-18.97±0.75) mV, and (-18.3±0.57) mV, respectively. Figure 1 (Figure C in the middle)

[0076] One of the challenges in intraocular drug delivery lies in the multiple barriers present in posterior segment drug delivery, while the challenge in intravitreal drug delivery lies in drug diffusion within the vitreous. Both are closely related to nanoparticle size; previous studies have shown that smaller nanoparticles facilitate delivery. However, excessively small nanoparticle sizes can lead to short drug retention times within the eye, contradicting the initial goal of this study: to design a long-acting drug release system. Previous research has indicated that a nanoparticle size of around 200 nm ensures both good posterior segment delivery and vitreous diffusion while achieving sustained release over a longer period within the eye. AFL-Cur@PT, with a nanoparticle size of (236.3±8.99) nm, possesses a suitable nanoparticle size, enabling effective diffusion within the vitreous and avoiding over-cleansing.

[0077] PDI and Zeta potential results ensured the reproducibility and biostability of the preparation. PDI describes the uniformity of particle size distribution; a smaller PDI indicates a more uniform particle size distribution. In this study, the PDI of AFL-Cur@PT was < 0.1, indicating a uniform particle size distribution. Surface charge also affects the movement, distribution, and half-life of nanomaterials within the vitreous cavity. Generally, anionic nanocarriers diffuse into the retina, while cationic nanocarriers tend to aggregate in the vitreous region rather than diffuse. Zeta potential is an important indicator of the surface charge of nanomedicines, significantly influencing the stability and biocompatibility of nanomedicines delivered intraocularly. Previous studies have shown that nanoparticles with Zeta potentials > ±30 mV are considered strong cations and strong anions, respectively. Generally, nanoparticles with higher Zeta potentials exhibit stronger electrostatic repulsion, which helps stabilize them and prevent aggregation. Nanomaterials with Zeta potentials ≤ ±10 mV tend to coagulate, flocculate, and / or agglomerate. The results of this study show that the Zeta potential of AFL-Cur@PT is (-18.3±0.57) mV, indicating that it has good dispersibility and is more likely to spread into the retina.

[0078] 2.2 Transmission Electron Microscopy (TEM) Detection

[0079] To visually observe the microstructure of the NPs, AFL-Cur@PT negatively stained NPs were used to observe their morphology using transmission electron microscopy (TEM). The AFL-Cur@PT NPs were diluted to an appropriate concentration using ultrapure water. The appropriate dilution was determined using the drop film method, with the standard being that after drying on a copper grid, sparsely distributed individual particles formed against a clean background free of contaminants. The morphology and structure of the NPs were observed using TEM and photographed for recording.

[0080] TEM observation of the microstructure of AFL-Cur@PT Figure 2As shown in Figure A, the AFL-Cur@PT NPs exhibit a uniform spherical structure. The AFL-Cur@PT NPs are evenly distributed, as shown in the distribution curve. Figure 2 In the middle B path, the average particle size exhibits a unimodal distribution with a diameter of (236.3±8.99) nm.

[0081] TEM clearly shows that it exhibits a uniformly sized, evenly distributed spherical structure. Previous studies have indicated that the uniform structure of NPs suggests that the drug is evenly distributed within the nanoparticles, which can lead to more consistent and predictable drug release behavior. The uniform microsphere structure also better protects the encapsulated drug activity, enhancing efficacy. Furthermore, the smooth surface morphology and spherical structure suggest that NPs have better biocompatibility. In this study, the nanoparticle size distribution plot showed a unimodal distribution, which also indirectly indicates that it is uniform in size and does not easily aggregate.

[0082] 2.3 Fourier Transform Infrared Spectroscopy Detection

[0083] Next, this example uses Fourier Transform Infrared Spectroscopy (FTIR) to confirm whether the drug was successfully encapsulated. After the sample to be tested was ground uniformly, FTIR detection was performed, and the obtained infrared spectral data were analyzed for characteristic functional groups.

[0084] See relevant results Figure 3 The graph shows the infrared spectrum of a PLGA at 3000 cm⁻¹, where the horizontal axis represents wavelength and the vertical axis represents characteristic absorption peaks. -1 and 2950 cm -1 The absorption peak at 1754 cm⁻¹ corresponds to an aliphatic CH bond. -1 The stretching vibration of the carbonyl group (C=O) is observed at 1085 cm⁻¹. -1 The corresponding tensile vibration of CO is observed at 1645 cm⁻¹. AFL and AFL-Cur@PT exhibit tensile vibrations at 1645 cm⁻¹. -1 With 1545 cm -1 Similar characteristic peaks exist at this location. Curve is at 1427 cm⁻¹. -1 The absorption peaks are due to CH bond bending vibrations, and AFL-Cur@PT also exhibits similar characteristic peaks. These characteristic peak clusters confirm that AFL and Cur are successfully encapsulated in AFL-Cur@PT NPs.

[0085] 2.4 Ultraviolet Absorption Detection

[0086] Ultraviolet absorption assays (UV-spectrophotometer, Thermo Scientific, USA) were used to further verify the successful drug encapsulation. Several test samples—Cur, PT, Cur@PT, AFL@PT, and AFL-Cur@PT—were dissolved and diluted in a dichloromethane / methanol mixture (1:3, v / v), and then subjected to sequential UV spectral scanning to obtain characteristic UV absorption peaks. The sample concentrations were maintained at 5–10 μg / mL, and all sample concentrations were kept consistent during testing to ensure that the measured absorbance values ​​(A) fell within the instrument's optimal linear range (0.2–1.5), guaranteeing the accuracy and reliability of the data.

[0087] Figure 4 The figure shows the UV absorption spectrum of the NPs, where the horizontal axis represents wavelength and the vertical axis represents characteristic UV absorption peaks. As shown, Cur's characteristic UV absorption peak is at 430 nm. PT and AFL@PT show no obvious UV absorption peaks in the wavelength range of 300 nm-600 nm. However, Cur@PT and AFL-Cur@PT exhibit Cur's maximum absorption peak at 425 nm, consistent with Cur's absorption peak near 430 nm. Notably, encapsulating Cur with PLGA causes a slight leftward shift of the NPs' maximum absorption peak. UV absorption experiments further confirm the successful encapsulation of Cur.

[0088] 2.5 Determination of encapsulation efficiency and drug loading

[0089] The encapsulation efficiencies of AFL and Cur were calculated using an indirect method. The supernatant from washing the NPs in step 4 of Example 1 was collected, and the contents of AFL and Cur were measured by liquid chromatography-mass spectrometry (LC / MS). The encapsulation efficiency (EE) was calculated according to the formula.

[0090] AFL EE (100%) = (Total AFL input mass - AFL mass in supernatant) ÷ Total AFL input mass × 100%.

[0091] Cur EE (100%) = (Total input mass of Cur - Mass of supernatant Cur) ÷ Total input mass of Cur × 100%.

[0092] The lyophilized NPs obtained in Example 1 were weighed, and the drug loading (DL) was calculated by LC / MS using the following formula:

[0093] AFL DL (100%) = (Total AFL input mass - AFL mass in supernatant) / Total NPs mass × 100%.

[0094] Cur DL (100%) = (total input mass of Cur - mass of supernatant Cur) / total mass of NPs × 100%.

[0095] Before the determination, the supernatant sample of NPs needs to be processed as follows: Add 200 μL of organic phase (including internal standard) to 50 μL of liquid sample to precipitate, vortex for 1 min, centrifuge at 13200 rpm for 4 min, and take 100 μL for testing.

[0096] Parameter settings: Column: Agela Venusil MP C18 (100 × 4.6 mm, 3 μm); Column temperature: 50 ºC; Flow rate: 1.0 mL / min; Mobile phase A: organic phase (methanol containing 2 mmol / L ammonium acetate + 0.1% formic acid); Mobile phase B: aqueous phase (water containing 2 mmol / L ammonium acetate + 0.1% formic acid); Injection volume: 10 μL. (High-performance liquid chromatography-mass spectrometry, Shimadzu Corporation, Japan)

[0097] The EE and DL of the drugs were determined by LC / MS. As shown in Table 1, the EE of AFL in AFL@PT and AFL-Cur@PT were (73.26±1.33)% and (78.39±3.73)%, respectively. The EE of Cur in Cur@PT and AFL-Cur@PT were (16.24±1.09)% and (13.1±0.28)%, respectively. The DL of AFL in AFL@PT and AFL-Cur@PT were (40.7±0.56)% and (38.59±3.13)%, respectively. The DL of Cur in Cur@PT and AFL-Cur@PT were (4.78±0.21)% and (3.22±0.06)%, respectively.

[0098] The results demonstrate that each NP has high safety and efficacy, avoids drug waste and intraocular safety risks, and has greater potential for clinical translation.

[0099] Table 1 Drug Encapsulation Efficiency (EE) and Drug Loading (DL)

[0100]

[0101] Example 3: In vitro and in vivo drug release assays of AFL-Cur@PT

[0102] The dialysis process was performed using a ready-to-use dialysis device (Repligen, USA). The membrane tubing of the miniature dialysis device (molecular weight cutoff: 300 kDa) was removed from the cannula and immersed in 10% ethanol solution, followed by thorough rinsing and immersion in deionized water to remove as much glycerol as possible and maximize membrane permeability. After pre-wetting, the membrane was re-wetted with dialysis buffer (PBS, pH 7.4). 1 mL of freshly prepared AFL-Cur@PT (containing 0.5 mg Cur and 6 mg AFL) dissolved in deionized water was placed in the pre-wetted dialysis device, which was then completely immersed in 50 mL of dialysis buffer and agitated at 37°C and 100 rpm. 0.2 mL of dialysis buffer was collected from outside the dialysis tubing at 6 h, 12 h, 1 d, 3 d, 7 d, 14 d, 28 d, 42 d, and 56 d. After each sampling, an equal volume of fresh dialysate was added. The concentrations of AFL and Cur in the samples were detected using LC / MS. Based on the AFL and Cur drug masses measured at different time points, a cumulative drug release curve of AFL-Cur@PT over time was plotted. Sample processing method for in vitro dialysate: 50 μL of liquid sample was added to 200 μL of organic phase (containing internal standard) for precipitation, vortexed for 1 min, centrifuged at 13200 rpm for 4 min, and 100 μL was transferred for analysis.

[0103] like Figure 5 Figure A shows the in vitro drug release results of AFL-Cur@PT. In the first 12 hours, AFL was rapidly released (23.65%), after which the release rate gradually slowed down. At 28 days, approximately 84.06% of AFL was released, and at 56 days, almost all of it was released (99.7%). This burst release phenomenon is significant in long-term effective anti-VEGF drug therapy, as it allows the drug to rapidly reach a high concentration in the initial release phase, quickly inhibiting angiogenesis, and then enters a more stable release phase, achieving a long-term therapeutic effect and effectively prolonging the duration of drug action. In contrast, the release curve of Cur from AFL-Cur@PT was relatively stable, without a significant burst release phenomenon. At 6 hours, 12 hours, and 24 hours, Cur was released at approximately 9.4%, 13.3%, and 18.04%, respectively. At 28 days, 42 days, and 56 days, Cur was released at approximately 69.12%, 75.85%, and 83.15%, respectively.

[0104] Given the actual differences between in vitro and in vivo release experiments, this embodiment further conducted an in vivo drug release experiment of AFL-Cur@PT. The method included: intraocular injection of 2 μL of AFL-Cur@PT (Cur: 5 mg / mL, AFL: 60 mg / mL) into the eyeballs of C57BL / 6J mice. Mice were sacrificed at 0, 1, 3, 7, 14, 28, 42, and 56 days post-injection. Retinal / choroidal tissue was then isolated from the mice, and the concentrations of AFL and Cur were detected by LC / MS. The retinal / choroidal tissue was a solid sample; the total sample volume was weighed, and 200 μL of methanol-water (1:1) was added. The mixture was vortexed for 15 min, centrifuged at 13200 rpm for 4 min, and 50 μL of the supernatant was collected. 200 μL of organic phase (containing internal standard) was added to precipitate the precipitate, vortexed for 1 min, centrifuged at 13200 rpm for 4 min, and 100 μL was collected for analysis. Then, an in vivo drug release curve of AFL-Cur@PT was plotted.

[0105] In vivo release experiment results showed ( Figure 5 (Figure B) The concentration of AFL in the retinal / choroidal tissue peaked at 502 ng / g at 7 days, remained high at 289 ng / g at 28 days, and decreased to 48.5 ng / g at 42 days, all significantly higher than the effective anti-VEGF drug concentration of AFL (8 ng / g). By 56 days, it was almost undetectable. The concentration of Curl peaked at 9.84 μg / g at 14 days, was 2.2 μg / g at 42 days, and was still 0.4 μg / g at 56 days.

[0106] The above indicates that AFL's release time in vitro and in vivo is >42 days, and Cur's release time in vitro and in vivo is >56 days. The detectable duration of AFL-Cur@PT in retinal / choroidal tissue is significantly longer than that following AFL injection alone (up to approximately 7 days). AFL-Cur@PT can significantly delay AFL release time, thereby prolonging the therapeutic duration of angiogenesis inhibition. Although there is no definitive EC50 for Cur... 50 While the numerical values ​​are not ideal, considering that Curl is difficult to dissolve and is rapidly metabolized in vivo when simply taken orally or injected, this study encapsulated Curl with PLGA / TPGS, which significantly improved its bioavailability and release time in vivo, thereby maximizing its biological effects.

[0107] Example 4: In vitro safety study of PT NPs

[0108] 8.1 PT NPs Cytotoxicity Assay

[0109] Different concentrations of PT were co-cultured with HRVECs, RF / 6A cells, 661W cells, ARPE-19 cells, and Müller cells for 24 h, 48 h, and 72 h. The cytotoxicity of PT was evaluated using a CCK-8 cytotoxicity assay.

[0110] 100 μL of cells at the same density were evenly seeded into 96-well plates. After cell attachment, culture medium containing different concentrations of PVT was added to each well. After incubation for 48 hours in a cell culture incubator, 100 μL of culture medium containing 10 μL of CCK-8 was added to each well, and the plates were incubated at 37°C in the dark for 2 hours. The absorbance (Optical Density, OD) was measured at 450 nm using a spectrophotometer. The calculation formula is as follows:

[0111] Cell viability (%) = [OD(PT) - OD(blank)] ÷ [OD(0 PT) - OD(blank)] × 100%; where OD(dosed): wells with added cells, CCK-8 and PT solution, OD(0 dosed): wells with cells and CCK-8 but without PT, and OD(blank): wells without cells, CCK-8 and PT. Cell viability is equivalent to cytotoxicity.

[0112] Experimental results showed that when the PT concentration was below 20 mg / mL, it had no significant toxicity to these cells within 24 h. Figure 6 (See Figure A); and when the PT concentration was below 10 mg / mL, it did not produce significant toxicity to the above cells during 72 h of co-culture (Figure A). Figure 6 (Figure B in the middle)

[0113] 8.2 PT NPs apoptosis assay

[0114] To further investigate whether PT NPs lead to apoptosis, Annexin V-FITC / PI flow cytometry apoptosis assay was performed using the Annexin V-FITC apoptosis detection kit (A211-01, Nanjing Novizan Biotechnology Co., Ltd.).

[0115] Different concentrations of PT were co-cultured with HRVECs, RF / 6A cells, 661W cells, ARPE-19 cells, and Müller cells for 24 h. The apoptosis rate was then quantitatively detected. After aspirating the liquid and washing with PBS, 1 mL of trypsin (without EDTA) was used to digest the cells in each well. The cells were gently pipetted to help them detach from the cell wall, and then complete culture medium was immediately added to stop the digestion. The cell suspension was transferred to centrifuge tubes, and after centrifugation, the supernatant was aspirated. PBS was added to the cell pellet. 1-5 × 10⁶ cells were collected. 5Centrifuge cells, discard the supernatant, add 100 μL Binding Buffer, mix well, then add 5 μL Annexin V-FITC and 5 μL PI Staining Solution and mix well. Incubate at room temperature in the dark for 10 min, then add 400 μL Binding Buffer and mix well. Place the above detection solution in a flow cytometer as soon as possible for detection (complete the detection within 1 h after preparation).

[0116] The test results were processed and analyzed using FlowJo 7.6.5 software. After correctly setting compensation and quadrants for unstained and single-positive control samples, Annexin V-FITC+ / PI- cells were identified as early apoptotic cells, and Annexin V-FITC+ / PI+ cells were identified as late apoptotic or necrotic cells. The total apoptosis rate was the sum of the percentages of early and late apoptotic cells.

[0117] The results showed that after co-culturing with the various cell types mentioned above for 24 h, there were no statistically significant differences among the groups, indicating that PT (≤10 mg / mL) did not induce significant cell apoptosis. Figure 7 ).

[0118] Example 5: In vivo safety study of PT NPs

[0119] 5.1 Ocular safety studies of PT NPs

[0120] To assess the in vivo biosafety of PT NPs, their effects on ocular tissues were first examined. PT was administered intravitreally to mice at concentrations of 1 mg / mL or 10 mg / mL. Retinal sections were collected at 1 month and 3 months post-injection for H&E staining and pathological analysis. The experimental procedures are as follows.

[0121] Frozen sectioning and TUNEL staining: Fresh tissue samples were obtained whenever possible. The tissue was thoroughly infiltrated with 4% PFA (P0099-100ml, Beyotime Biotechnology Co., Ltd., China) and fixed overnight at 4°C. The tissue was then dehydrated using a gradient of sucrose solutions prepared in PBS. The tissue was then placed in a specially designed embedding cassette for frozen sections and thoroughly embedded using OCT gel (4583, SAKURA Co., Ltd., Japan). Sections were prepared using a cryostat, attached to glass slides, and stored at -20°C.

[0122] If frozen sections are removed from a -20°C freezer, place them in a humidified chamber for approximately 20 minutes to rewarm. Wash the frozen sections three times with PBS, 5 minutes each time. After removing any residual OCT gel, fix the tissue with 4% PFA for 30 minutes, then wash three times with PBS. Prepare PBS containing 5% BSA (BS114-500g, Biosharp, USA) and 0.1% Triton X-100 (BL934B, Biosharp, USA), and completely infiltrate the tissue with this solution. Incubate at room temperature for 1 hour for blocking and permeabilization. Wash the tissue 2-3 times with PBS. Add 5-10 μL of TUNEL assay solution (TUNEL apoptosis assay kit, C1086, Beyotime Biotechnology, China) to the tissue and incubate at 37°C in the dark for 1-2 hours. Wash three times with PBS, stain cell nuclei with DAPI (1:1000) (C1002, Beyotime Biotechnology Co., Ltd., China), mount the slides and record images using an immunofluorescence microscope (TUNEL: λexc = 450-500 nm, λem = 515-565 nm).

[0123] Preparation of the DNase I positive control group (TUNEL detection positive control preparation kit, C1082, Beyotime Biotechnology Co., Ltd., China): Process tissue sections to the step before fluorescent labeling, following the steps described above. Dilute the Reaction Buffer to 1× with double-distilled water. Add 50 μL of 1× Reaction Buffer to the sample and incubate at room temperature for 5 min. Accurately add 1 μL of centrifuged DNase I precipitate to every 100 μL of 1× Reaction Buffer, vortex to mix, and then add to the sample. Incubate at room temperature for 10 min, wash three times with PBS, and then proceed with the subsequent TUNEL staining and detection procedures as before.

[0124] For frozen sections and staining of C6 and C6@PT, since C6 is inherently fluorescent, no immunofluorescence staining other than DAPI is required. All tissue preparation, preservation, and detection processes must be conducted in the dark. Gentle handling is essential, especially when washing samples; liquid should be added slowly from beside the tissue to avoid damaging the specimen.

[0125] Paraffin sectioning and hematoxylin-eosin staining (BP-DL001-100mL, hematoxylin-eosin (HE) staining kit, Nanjing Senbega Biotechnology Co., Ltd.): Mouse tissues were removed and fixed with 4% PFA fixative at 4°C for 24 h. After graded alcohol dehydration, the tissues were cleared with xylene and then embedded in paraffin. Subsequently, the tissues were sectioned, dewaxed, and stained sequentially with hematoxylin and eosin. Clearing with xylene was performed again, and finally, the sections were mounted with neutral resin (BL704A, Biosharp, USA). After air-drying overnight, the sections were photographed and recorded under a microscope.

[0126] The results showed that, after intravitreal injection of PT, compared with the control group injected with PBS, there were no significant changes in the retinal structure of mice, and there was also no significant difference in retinal thickness. Figure 8 (Figure A in the middle)

[0127] No significant apoptotic fluorescence signal was detected in TUNEL immunofluorescence staining of frozen sections of mouse retina at 1 m and 3 m after injection of PT NPs. Figure 8 (Figure B in the middle)

[0128] In addition, electroretinography (ERG) experiments were performed using a visual electrophysiological testing system (DIAGNOSYS, USA). Mice were kept in complete darkness overnight before ERG recording. After anesthesia, compound tropicamide eye drops (Shenyang Xingqi Eye Drops Co., Ltd.) were applied topically to dilate the mouse pupils. The mouse contact electrode was placed on the central cornea of ​​the test eye, ensuring the cornea remained moist. The reference electrode and ground electrode were inserted into the skin of both cheeks, respectively. Flash lamp ERG recording was performed with bandpass filtering at 0.3 and 500 Hz, and the light waves were captured using the electrophysiological testing system. The amplitudes of waves A and B were measured and recorded.

[0129] The results showed that one month and three months after PT injection, there were no significant differences in A and B waves in the ERG test between mice and the control group. Figure 8 (Figure C). These results indicate that PT NPs have good biocompatibility in ocular tissues in vivo, and did not cause significant histopathological changes, apoptosis, or retinal dysfunction.

[0130] 5.2 PT In vivo inflammatory response assay

[0131] 2 μL of PBS was injected intravitreally into the vitreous cavity of mice as the Ctrl group, and 10 mg / mL PT was injected as the PT group. On day 5, the protein concentrations of IL-1β, IL-6, MCP-1, TNF-α and VEGF in the retina / choroid were detected by mouse IL-1β ELISA, mouse IL-6 ELISA, mouse MCP-1 ELISA, mouse TNF-α ELISA and VEGF ELISA kits (MLB00C-1, M6000B-1, DCP00, MTA00B-1 and DVE00, R&D Systems, USA).

[0132] After euthanizing mice, retinal / choroidal complex tissue was isolated and homogenized using an ultrasonic homogenizer on ice. The homogenate was then dissolved in RIPA lysis buffer containing protease inhibitors. Subsequently, the protein levels of IL-6, TNF-α, MCP-1, IL-1β, and VEGF were quantitatively measured using ELISA according to the manufacturer's instructions. Specifically, the OD value at 450 nm was measured using a UV-Vis spectrophotometer. After plotting a standard curve, the protein concentration in each sample was calculated based on the measured OD values, and finally, statistical analysis was performed.

[0133] The results showed that, compared with the Ctrl group, there were no statistically significant differences in the expression of the above-mentioned inflammatory factors and VEGF. Figure 9 This experiment demonstrates that intravitreal injection of PT NPs in mice does not lead to inflammation of the retina / choroid tissue or changes in VEGF factor protein levels.

[0134] 5.3 Hematological parameters of PT NPs injected intravitreally

[0135] After injecting 2 μL of 10 mg / mL PT into the vitreous cavity of mice, the hematological parameters of the mice were measured.

[0136] The results showed that, compared with the control group, there were no significant differences in blood urea nitrogen (BUN), total protein (TP), albumin (ALB), alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), white blood cell (WBC), and red blood cell (RBC) levels. Figure 10 This indicates that intravitreal injection of 10 mg / mL PT NPs in mice did not cause changes in hematological parameters.

[0137] 5.4 H&E staining of tissue sections from the heart, liver, spleen, lung, and kidneys after intravitreal injection of PT NPs.

[0138] 2 μL of 10 mg / mL PT was injected intravitreally into the vitreous cavity of mice as the PT experimental group, and 2 μL of PBS was injected intravitreally into the vitreous cavity of mice as the Ctrl group. At 1 month and 3 months, heart, liver, spleen, lung, and kidney sections from mice in both the PT and Ctrl groups were collected for hematoxylin-eosin (H&E) staining. The staining procedure is described above. Figure 11 As shown, compared with the Ctrl group, no significant tissue damage was observed in the major organs of the mice after intravitreal injection of PT.

[0139] 5.5 Detection of apoptosis-related proteins in tissues after intravitreal injection of PT NPs

[0140] 2 μL of 10 mg / mL PT was injected intravitreally as the PT group, and 2 μL of PBS was injected intravitreally as the Ctrl group. Eye, heart, liver, spleen, lung and kidney tissues were collected from mice at 1 month and 3 months, respectively. Apoptosis-related proteins were detected by Western blotting. The Western blotting experimental procedure is as follows.

[0141] I. Extraction and Preparation of Tissue Protein Samples

[0142] The mice were dissected on ice using pre-cooled instruments to separate the required tissues. The samples were then boiled at 100 °C for 10 min before use.

[0143] II. Electrophoresis

[0144] Prepare a separating gel according to the molecular weight of the protein to be detected. After electrophoresis, separate the lower gel layer and soak it in transfer buffer for later use.

[0145] III. Transfer and Color Development

[0146] The primary antibody (Anti-Caspase3 antibody, 9662S, CST Biotechnology, USA / Anti-Bax antibody, Cat No. 50599-2-Ig, Wuhan Sanying Biotechnology Co., Ltd. / Anti-Bcl-2 antibody, Cat No. 26593-1-AP, Wuhan Sanying Biotechnology Co., Ltd.) was used for overnight incubation on a shaker at 4°C, followed by incubation and color development with secondary antibody (goat anti-rabbit IgG (H+L) secondary antibody, HRP, 31460).

[0147] The results showed that, compared with the control group injected with PBS, there were no statistically significant differences in the expression of Bax, Bcl-2, and Caspase3, three proteins related to apoptosis, in the above tissues. Figure 12The above indicates that intravitreal injection of PT did not induce apoptosis in the eyes or other major organs throughout the body.

[0148] Example 6: In vitro and in vivo intake studies of PT NPs

[0149] 6.1 In vitro intake study of PT NPs

[0150] Coumarin 6 (λexc = 450 nm, λem = 505 nm, HY-N7131, MCE Biosciences, USA) was used as a fluorescent probe in microparticle drug delivery systems to study the in vivo tracking, cellular uptake, and transport mechanisms of active molecule drug delivery systems. C6 was dissolved to 10 nM using DMSO as the basal medium. Free C6 and C6@PT were then diluted to 10 μM using serum-free medium. Cells of the same concentration were seeded in confocal cell culture dishes. After cell adhesion, the cells were cultured in serum-free medium containing PBS, PT, free C6, and C6@PT at 37°C for different time periods (0 h, 2 h, 6 h, and 24 h). After aspirating the culture medium and fixing the cells with paraformaldehyde, they were washed 2-3 times with PBS. The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) for 3 min, washed with PBS, and photographed using a fluorescence microscope. The wavelengths of DAPI were λexc = 364 nm and λem = 450 nm.

[0151] The ratio of average fluorescence intensity to cell number was measured using ImageJ software to plot the uptake dynamics curves of PTNPs by various cell types. The results showed that, with increasing time, the uptake rate of C6-loaded PTNPs by HRVECs, RF / 6A cells, 661W cells, ARPE-19 cells, and Müller cells significantly increased, with fluorescence intensities significantly higher than those in the free C6 group. These results indicate that PTNPs can be effectively taken up by various cell types, and the uptake rate increases with time. Figures 13-17 ).

[0152] 6.2 In vivo intake study of PT NPs

[0153] 6.2.1 Retinal uptake experiment of PT NPs

[0154] After anesthetizing mice, 2 µL of PBS, PT (6.7 mg / mL), C6 (3.3 mg / mL), and C6@PT (C6: 3.3 mg / mL) were injected intravitreally using a microinjector under a microscope. Mice were sacrificed at 0 h, 18 h, 36 h, and 7 d, and their eyeballs were harvested. Retinal frozen sections were prepared according to the method described in Example 9, and cell nuclei were stained with DAPI (1:1000). Immunofluorescence microscopy with the same parameters was used to capture and record images, and the fluorescence intensity was quantified and analyzed using ImageJ software.

[0155] The fluorescence intensity distribution of different layers of the mouse retina was measured using ImageJ software, and retinal uptake dynamics curves of PT were plotted. The results showed that in the first 36 hours, with increasing time, the uptake rate of C6-loaded PT NPs in different retinal layers, especially the ganglion cell layer (GCL), photoreceptor layer (PRL), and retinal pigment epithelium (RPE), significantly increased, with fluorescence intensity significantly higher than the free C6 group. After 36 hours, the uptake rate of C6@PT in the GCL, PRL, and RPE layers gradually decreased. Figure 18 Retinal uptake experiments in mice confirmed that PT significantly enhanced the signal intensity and duration of C6 in all layers of the retina, suggesting its ability to promote drug delivery from the inner to the outer layers of the retina.

[0156] 6.2.2 In Vivo Imaging Systems (IVIS) Experiments with PT NPs

[0157] After anesthetizing mice, 2 µL of C6 (3.3 mg / mL) and C6@PT (C6: 3.3 mg / mL) were injected intravitreally using a microinjector under a microscope. Fluorescence intensity in the mouse eyes was measured on days 0, 2, 7, and 10 under IVIS (isofluorane gas anesthesia), and the results were analyzed using the built-in Living Image software. Retention curves of C6@PT and C6 in the mouse eyes were plotted. The results are as follows: Figure 19 As shown, compared with free C6, significant C6 fluorescence was still detected in the C6@PT group at 14 days, indicating that PT significantly prolonged the retention time of loaded C6 in the mouse eye. The IVIS experiment demonstrates that PT plays an important role in prolonging drug delivery time. Furthermore, no obvious fluorescence signal was observed outside the eye, which indirectly reflects the safety of PT NPs in intraocular drug delivery.

[0158] Example 7: In vitro functional experiment of AFL-Cur@PT

[0159] VEGF, as a potent pro-angiogenic factor, can induce angiogenesis by promoting the proliferation, migration, and tubule formation of endothelial cells, all of which are key steps in angiogenesis. HRVECs and RF / 6A cells retain the angiogenic characteristics of endothelial cells and are classic in vitro experimental cell lines for studying the anti-angiogenic effects of drugs on fundus diseases. Therefore, in this embodiment, VEGF was used to stimulate HRVECs and RF / 6A cells, respectively, and then the angiogenic function of AFL-Cur@PT in vitro was evaluated using EdU assays, Transwell assays, and tubule formation assays.

[0160] 7.1 In vitro experiment on the regulation of angiogenesis-related functions of AFL-Cur@PT in HRVECs

[0161] EdU cell proliferation, chamber cell migration, and cell tube formation experiments were divided into the following groups based on the different drugs used to treat the cells: control group (Ctrl), PT group (6.7 μg / mL), Cur group (3.3 μg / mL), Cur@PT group (Cur: 3.3 μg / mL), AFL group (40 μg / mL), AFL@PT group (AFL: 40 μg / mL), and AFL-Cur@PT group (Cur: 3.3 μg / mL, AFL: 40 μg / mL). For all cell function experiments, cells were incubated with 10 ng / mL VEGF for 12 h before drug treatment.

[0162] 7.1.1 EdU experiment.

[0163] The same number of cells (HRVECs) to be tested were evenly seeded in 24-well plates. After incubation with 10 ng / mL VEGF for 12 h, the original culture medium was removed, and serum-free culture medium containing different drugs was added and incubated for 24 h. Cell proliferation was detected using the BeyoClick™ EdU-488 Cell Proliferation Detection Kit (C0071S, Beyotime Biotechnology Co., Ltd., China). Specifically, after discarding the culture medium, EdU working solution was diluted with serum-free cell culture medium and preheated to 37°C. 200 μL of diluted EdU (10 μM) working solution was added to each well, and incubation continued for 2 h. After EdU labeling, the culture medium was removed, and paraformaldehyde fixative was added and fixed at room temperature for 20 min. After removing the fixative, the cells were washed three times with PBS for 3-5 min each time. Permeabilization was performed for 15 min at room temperature with PBS containing 0.3% Triton X-100. After removing the permeabilization solution, the cells were washed twice more with PBS for 3-5 min each time. Prepare the Click reaction solution by adding 1.72 mL Click Reaction Buffer, 80 μL CuSO4, 4 μL Azide 488 (λexc = 495 nm, λem = 519 nm), and 200 μL Click Additive Solution to each well, ensuring complete infiltration of cells. Incubate at room temperature in the dark for 30 min, then discard the reaction solution and wash three times with PBS for 3-5 min each time. After staining the cell nuclei with DAPI, photograph and record the results using an immunofluorescence microscope.

[0164] EdU experiments showed that PT alone had no significant inhibitory effect on VEGF-induced HRVEC proliferation, while Cur, Cur@PT, AFL, AFL@PT, and AFL-Cur@PT all showed some inhibitory effects. Among them, AFL-Cur@PT had the most significant inhibitory effect, showing a statistically significant difference compared with other drugs (e.g., Figure 20 As shown, where DAPI: blue fluorescence, EdU: green fluorescence, n=4, scale bar: 20 μm; *P<0.050).

[0165] 7.1.2 Cell migration experiment

[0166] The same number of HRVECs were seeded in 12-well plates and treated with VEGF and appropriate drugs. After digestion, the cells were resuspended in serum-free DMEM. 600 μL of DMEM complete medium containing 10% FBS was added to 24-well plates. Appropriately sized hanging cell culture chambers (8 μm pore size) were carefully placed in the 24-well plates using microforceps, ensuring the polycarbonate membrane at the bottom of the chamber was completely submerged in the medium. 200 μL of FBS-free DMEM cell resuspension solution (containing 1×10⁻⁶ cells / well) was added. 4 Add the treated cells to the chamber and incubate in a cell culture incubator for 12 hours. Remove the chamber, carefully discard the culture medium, and wash the chamber three times with PBS for 5 minutes each time. Fix the cells with methanol for 20 minutes, invert the chamber to air dry completely, stain with 0.5% crystal violet for 30 minutes, wash the chamber again with PBS 2-3 times, and finally carefully wipe away any remaining cells that have not penetrated the membrane from the top of the chamber with a cotton swab. Take pictures under a microscope for recording.

[0167] The results showed that PT alone had no significant effect on the cell migration function of VEGF-induced HRVECs, while Cur, Cur@PT, AFL, AFL@PT, and AFL-Cur@PT all had a certain inhibitory effect on VEGF-induced HRVEC cell migration. Among them, AFL-Cur@PT had the strongest inhibitory effect on cell migration compared with the other groups, and the difference was statistically significant (e.g., ...). Figure 21 As shown, where n=4, scale bar: 20 μm; *P<0.050).

[0168] 7.1.3 Cell tube formation experiment:

[0169] The day before the experiment, the matrix gel was thawed at 4°C, PBS was pre-chilled at 4°C, and the 24-well plates and pipette tips were pre-chilled at -20°C. All cell culture plates, matrix gel, and pipette tips were handled on ice. After adding pre-chilled PBS to the 24-well plates to wet them, the PBS was aspirated. 40 μL of matrix gel was then added evenly to each well, avoiding the formation of air bubbles. The prepared plates were incubated for 30 min to allow the matrix gel to solidify. HRVECs were treated as before: the same number of cells were digested, resuspended in FBS-free DMEM medium, and evenly added to the 24-well plates containing matrix gel (5 × 10⁶ cells per well). 4 (Number of cells). The 24-well plate with the cells was placed back in the incubator, and the cell tube formation was observed after 6 hours. The cells were photographed and recorded under a microscope. ImageJ software was used to analyze the cell tube formation.

[0170] The results showed that PT alone had no significant effect on VEGF-induced HRVEC cell tuberculosis, while Cur, Cur@PT, AFL, AFL@PT, and AFL-Cur@PT all had some inhibitory effect on VEGF-induced HRVEC cell tuberculosis. Among them, AFL-Cur@PT showed the strongest inhibitory effect on HRVEC cell tuberculosis compared to other drugs, and the difference was statistically significant (e.g., ...). Figure 22 As shown, where n=4, scale bar: 20 μm; *P<0.050).

[0171] 7.2 Effects of AFL-Cur@PT on angiogenesis-related functions of RF / 6A cells in vitro

[0172] The results of in vitro functional studies of RF / 6A cells were similar to those of HRVECs cells, and the experimental procedures were the same. The results showed that PT alone had no significant effect on VEGF-induced proliferation, migration, and tube formation of RF / 6A cells. However, Cur, Cur@PT, AFL, AFL@PT, and AFL-Cur@PT all had some inhibitory effect on VEGF-induced proliferation, migration, and tube formation of RF / 6A cells. Among these, AFL-Cur@PT showed the strongest inhibitory effect on cell proliferation, migration, and tube formation of RF / 6A cells at the in vitro level compared to other drugs, and the difference was statistically significant (e.g., ...). Figures 23-25 As shown, where Figure 23 DAPI: blue fluorescence, EdU: green fluorescence; n=4, scale bar: 20 μm; *P<0.050).

[0173] Example 8: In vivo functional experiment of AFL-Cur@PT

[0174] 8.1 Study on the inhibitory function of AFL-Cur@PT on laser-induced CNV

[0175] To further investigate the effect of AFL-Cur@PT on pathological angiogenesis in vivo, a CNV model was constructed in mice using laser-induced in vivo. This is a classic nAMD animal model.

[0176] Specific operating steps: ① First, weigh the mouse and anesthetize it by intraperitoneal injection of ketamine (80 mg / kg) and toluenethiazide (10 mg / kg). ② After the mouse is fully anesthetized, administer compound tropicamide eye drops (containing 0.5% tropicamide and 0.5% norepinephrine hydrochloride) to the operated eye to dilate the pupil, and administer sodium hyaluronate eye drops to keep the ocular surface moist. Carefully trim eyelashes, whiskers, etc. ③ After the mouse pupil is fully dilated, apply ofloxacin eye ointment to the cornea and attach a coverslip to the ointment. ④ Adjust the mouse's position so that the mouse's cornea and fundus are perpendicular to the 532 nm laser. Perform laser photocoagulation on the mouse's fundus, setting the parameters as follows: wavelength 532 nm, duration 0.1 s, power 120 mW, and spot size 50 µm. Induce four laser spots evenly around the optic disc, approximately 1-2 optic disc diameters away from the optic disc. ⑤ After successful modeling, place the mice on a 37°C warming table for resuscitation until they regain consciousness. Note that after laser photocoagulation, the appearance of a white subretinal bubble indicates successful destruction of the Bruch's membrane, signifying successful modeling and allowing for subsequent experiments. If the laser site becomes severely white or experiences significant bleeding, the modeling has failed and the mice must be discarded.

[0177] Following laser modeling, intravitreal drug injection was performed the next day. On days 14 and 28, flat slides of the mouse RPE-choroid-sclera complex were taken for immunofluorescence staining with Isolectin GS-IB4 (IB4) (1:100, I21411, Thermo Fisher Scientific, USA) and incubated in the dark for 4 hours to mark the neovascularization area.

[0178] Quantitative analysis of IB4 fluorescence area was performed using ImageJ. First, a uniform analysis region was delineated across the entire retinal patch image to eliminate peripheral artifacts. Then, the IB4-stained region was converted into a binary image by setting a consistent grayscale threshold. Finally, the software automatically calculated the percentage of IB4-positive signal area (% Area) within the analysis region.

[0179] The results showed that at 14 and 28 days, compared with the control group (Ctrl group) or the PT group, the Cur group, Cur@PT group, AFL group, AFL@PT group, and AFL-Cur@PT group all effectively reduced CNV area. There were no statistically significant differences between the Cur group and the Cur@PT group, or between the AFL group and the AFL@PT group. Compared with the AFL-Cur@PT group, there were statistically significant differences in the Cur group, Cur@PT group, AFL group, and AFL@PT group (e.g., ...). Figure 26 As shown, n=6, scale: 50 μm).

[0180] The above results indicate that within 28 days, Cur, Cur@PT, AFL, AFL@PT, and AFL-Cur@PT all inhibited laser-induced CNV, with AFL-Cur@PT showing the most significant inhibitory effect, and the difference was statistically significant.

[0181] 8.2 Study on the inhibitory effect of AFL-Cur@PT on inflammatory cells in laser-induced CNV lesions

[0182] Immunofluorescence staining was performed on the RPE-choroid-sclera complex flat slides of mice after laser modeling and drug injection using anti-F4 / 80 (ab300421, 1:50, Abcam), anti-Neutrophil (ab131260, 1:250, Abcam), and IB4. Mice were euthanized following the steps described above, and their eyeballs were removed and fixed with 4% PFA at room temperature for 30 min. After fixation, the eyeballs were dissected along the limbus under a surgical microscope, and the cornea, lens, retina, and sclera were carefully removed. Subsequently, the choroid was carefully dissected into 4-8 petal-shaped pieces and laid flat on a slide containing PBS, with the inner choroid facing upwards, avoiding damage to the laser spot during the process. After a series of fixation (4% PFA), washing (PBS), blocking (5% BSA), and permeabilization (0.1% Triton X-100), the diluted primary antibody was infiltrated into the choroid flat slides and incubated overnight at 4 °C. The next day, the samples were incubated with Alexa Fluor 594 goat anti-rabbit IgG (ab150080, Abcam) and Alexa Fluor 488 Isolectin GS-IB4 (1:100) at room temperature in the dark for 4 h. DAPI staining was performed as needed, and after washing with PBS, the samples were covered with a coverslip and images were captured and recorded using an inverted fluorescence microscope.

[0183] Quantitative analysis of inflammatory cells in CNV was performed, and the results showed (e.g.) Figure 27 and Figure 28 (where n=4, scale bar: 50 μm): Compared with the control group (Ctrl group) or the PT group alone, the Cur group, Cur@PT group, AFL group, AFL@PT group, and AFL-Cur@PT group all effectively reduced the area of ​​F4 / 80 positive cells and Neutrophil positive cells in CNV. There were no significant differences between the control group and the PT group, between the Cur group and the Cur@PT group, and between the AFL group and the AFL@PT group. Compared with the AFL-Cur@PT group, there were statistically significant differences in the Cur group, Cur@PT group, AFL group, and AFL@PT group.

[0184] The above results indicate that within 28 days, Cur, Cur@PT, AFL, AFL@PT, and AFL-Cur@PT can all inhibit the infiltration of macrophages and neutrophils in laser-induced CNV lesions, with AFL-Cur@PT showing the most significant effect in inhibiting inflammatory cells.

[0185] 8.3 Analysis of IOP changes after intravitreal injection of AFL-Cur@PT

[0186] To monitor whether intravitreal injection of drug a would cause fluctuations in intravitreal opacity (IOP), IOP was measured in the aforementioned laser-induced CNV mice before and after drug injection.

[0187] Intraocular opacity (IOP) was measured in mice using a non-invasive tonometer. The probe tip was positioned 1–4 mm from the corneal surface, ensuring perpendicular contact with the corneal center. For each IOP reading, the tonometer probe made six consecutive contacts with the corneal center. The IOP of this device was determined using an algorithm that relied on the incident velocity and deceleration of the detector after six successful measurements. The results were then displayed on a monitor, with IOP measured in mmHg.

[0188] The results showed no significant differences in IOP among the drug treatment groups before injection, at 1 week, 2 weeks, and 4 weeks. Figure 29 This indicates that no significant abnormalities were observed in the intravitreal artery occlusion (IOP) of the mice throughout the treatment process. Furthermore, no serious complications associated with intravitreal injection, such as infection, vitreous hemorrhage, or lens damage, were observed in any of the mice.

[0189] Example 9: Study on the anti-angiogenic mechanism of AFL-Cur@PT in inhibiting CNV

[0190] The VEGF levels in retinal / choroidal tissue after drug injection were detected by PCR and ELISA experiments. The PCR experimental steps are as follows:

[0191] Total RNA was extracted from mouse eyeball tissue and reverse transcribed using the Novizumi reverse transcription kit (HiScript IV All-in-One Ultra RT SuperMix for qPCR, R433-01, Novizumi Nanjing Co., Ltd.) to obtain cDNA. qRT-PCR was then performed using the Novizumi qPCR kit (ChamQ Universal SYBR qPCR Master Mix, Q711-02 / 03, Novizumi Nanjing Co., Ltd.).

[0192] Using β-actin as an internal reference gene, the sequences of primers VEGF-AF / R and β-actin-F / R were analyzed as follows:

[0193] VEGF-AF: 5'-GCACATAGAGAGAATGAGCTTCC-3'

[0194] VEGF-AR: ​​5'-CTCCGCTCTGAACAAGGCT-3'

[0195] The ELISA experimental procedure is as follows: After euthanizing mice, the retinal / choroidal complex tissue was isolated and homogenized into a tissue homogenate using an ultrasonic homogenizer on ice. The homogenate was then dissolved in RIPA lysis buffer containing protease inhibitors. Subsequently, the protein levels of IL-6, TNF-α, MCP-1, IL-1β, and VEGF were quantitatively measured using ELISA according to the manufacturer's instructions. Specifically, the OD value at 450 nm was measured using a UV-Vis spectrophotometer. After plotting a standard curve, the protein concentration in each sample was calculated based on the detected OD values, and finally, statistical analysis was performed.

[0196] PCR and ELISA experimental results are as follows Figure 30 As shown, after laser-induced CNV modeling, compared with the control group (Ctrl group), VEGF was significantly highly expressed in both mRNA and protein levels in retinal / choroidal tissue. Compared with the CNV group and PT group, both the AFL@PT group and the AFL-Cur@PT group significantly reduced VEGF mRNA levels (e.g., ...). Figure 30 (as shown in Figure A) and proteins (such as...) Figure 30 (As shown in Figure B). Data analysis showed that AFL-Cur@PT had a significant inhibitory effect on VEGF in the laser-induced CNV model, effectively downregulating the expression level of VEGF in retinal and choroidal tissues, confirming its potential therapeutic value in regulating pathological angiogenesis.

[0197] The expression of proteins in the VEGF-related signaling pathways p-VEGFR2, p-ERK1 / 2, p-P38, and p-JNK was detected using Western blotting.

[0198] like Figure 31As shown, compared with the control group (Ctrl group), the expression level of p-VEGFR2 in the retinal / choroidal tissue of the CNV group and PT group was significantly increased, while the expression level of p-VEGFR2 in both the AFL@PT group and AFL-Cur@PT group was decreased. Similarly, the expression level of p-ERK1 / 2 in the CNV group and PT group was increased compared with the control group, while the expression level of p-ERK1 / 2 in both the AFL@PT group and AFL-Cur@PT group was decreased, and the expression level of p-ERK1 / 2 protein in the AFL-Cur@PT group was further decreased, which was statistically different from that in the AFL@PT group. In addition, the expression levels of p-P38 and p-JNK proteins in the CNV group, PT group and AFL@PT group were increased compared with the control group, while the expression levels of p-P38 and p-JNK in the AFL-Cur@PT group were significantly lower than those in the above groups, which was statistically significant.

[0199] PCR, ELISA, and Western Blot results showed that intravitreal injection of AFL and AFL@PT significantly reduced the mRNA and protein levels of VEGF and inhibited VEGFR2 phosphorylation, consistent with previous studies on AFL's inhibition of the VEGF / VEGFR2 signaling pathway.

[0200] The MAPK signaling pathway mainly includes three classic pathways: ERK, p38, and JNK. This study shows that AFL-Cur@PT can broadly inhibit the activation of the MAPK signaling pathway. Among them, the ERK1 / 2 pathway plays a crucial role in regulating cell proliferation, differentiation, survival, and migration, and is closely related to angiogenesis. Previous studies have shown that AFL can inhibit angiogenesis by inhibiting ERK1 / 2 phosphorylation, which is consistent with our findings. Notably, compared with AFL@PT alone, AFL-Cur@PT exhibits a stronger inhibitory effect on the ERK pathway. This synergistic enhancement may stem from the fact that Cur itself also has inhibitory functions on the ERK pathway, and the combination of the two achieves a more efficient signal blockade.

[0201] Example 10: Experiment on AFL-Cur@PT in reducing CNV-related inflammatory response

[0202] After laser-induced CNV modeling in mice, the mRNA expression of inflammatory factors IL-6, TNF-α, IL-1β, MCP-1, ICAM-1, and NFκB-1 in retinal / choroidal tissues was detected by PCR. The Ctrl group did not receive laser treatment, while the other groups used laser photocoagulation to create CNV models in C57BL / 6 mice. One day later, 2 μL of PBS (CNV group), PT (Ctrl and PT groups), Cur@PT (Cur: 3.3 mg / mL), or AFL-Cur@PT (Cur: 3.3 mg / mL, AFL: 40 mg / mL) were injected intravitreally. Retinal / choroidal tissues were harvested on day 5. Primer sequences for the relevant inflammatory factors were designed as follows:

[0203] Mouse IL-6 upstream primer: 5'-ATGCTTCCAATCTGGGTTCAATC-3'

[0204] Mouse-derived IL-6 downstream primer: 5'-CCAGGATCTTGGTACTCATGTGC-3'

[0205] Mouse TNF-α upstream primer: 5'-TTCTCCTTCCTCCTCGTCGCAG-3'

[0206] Mouse TNF-α downstream primer: 5'-CTACTGGCTTGTCACTTGGGGTTC-3'

[0207] Mouse-derived MCP-1 upstream primer: 5'-TAAAAACCTGGATCGGAACCAAA-3'

[0208] Mouse-derived MCP-1 downstream primer: 5'-GCATTAGCTTCAGATTTACGGGT-3'

[0209] upstream primer for mouse IL-1β: 5'-GCCTGTGTTTTCCTCCTTGC-3'

[0210] Mouse-derived IL-1β downstream primer: 5'-TGCTGCCTAATGTCCCCTTG-3'

[0211] Mouse-derived ICAM-1 upstream primer: 5'-TCCGCTACCATCACCGTGTAT-3'

[0212] Mouse-derived ICAM-1 downstream primer: 5'-TAGCCAGCACCGTGAATGTG-3'

[0213] upstream primer for mouse NfκB-1: 5'-ATGGCAGACGATGATCCCTAC-3'

[0214] Mouse-derived NfκB-1 downstream primer: 5'-CGGAATCGAAATCCCCTCTGTT-3'

[0215] upstream primer for mouse β-actin: 5'- GGGAAATCGTGCGTGAC-3'

[0216] Mouse β-actin downstream primer: 5'-AGGCTGGAAAAGAGCCT-3'

[0217] Compared with the control group, the mRNA expression of inflammatory factors IL-6, TNF-α, IL-1β, MCP-1, ICAM-1, and NFκB-1 in retinal / choroidal tissue was significantly upregulated. There was no significant difference between the PT group and the CNV group. Both the Cur@PT group and the AFL-Cur@PT group showed significant differences compared with the laser-induced modeling group, suggesting that both Cur@PT and AFL-Cur@PT can significantly reduce the mRNA expression of the above inflammatory factors in laser-induced CNV tissue. Figure 32 ).

[0218] Simultaneously, the levels of various inflammation-related factors such as IL-6, TNF-α, MCP-1, and IL-1β in the tissue were detected using an ELISA assay. The ELISA assay procedure is as described above.

[0219] ELISA results showed that after laser-induced CNV modeling in mice, the protein levels of IL-6, TNF-α, IL-1β, and MCP-1 in the retina / choroidal tissue were significantly increased compared to the control group. There was no significant difference between the PT group and the CNV group. Compared with the laser-induced modeling group, both the Cur@PT group and the AFL-Cur@PT group showed significant differences, indicating that both Cur@PT and AFL-Cur@PT can significantly reduce the protein levels of the above-mentioned inflammatory factors in laser-induced CNV tissue. Figure 33 ).

[0220] To explore the mechanism of AFL-Cur@PT in laser-induced CNV, this embodiment uses Western blotting to further detect the expression of proteins p-P65, p65, p-ERK1 / 2, ERK1 / 2, p-P38, p38, p-JNK, JNK, HO-1, Nrf2 and ICAM-1.

[0221] like Figure 34The results showed that, compared with the control group (Ctrl group), the expression level of p-P65 protein in the retinal / choroidal tissues of the CNV group and PT group was increased, while the expression of p-P65 protein in the Cur@PT group and AFL-Cur@PT group was significantly decreased. P65 is a key protein in the NF-κB signaling pathway, an important signaling pathway for regulating inflammation in vivo. Its phosphorylation can activate the NF-κB pathway. The results of this study indicate that both Cur@PT and AFL-Cur@PT can significantly inhibit the phosphorylation of p65, thereby inhibiting inflammation.

[0222] The expression levels of p-ERK1 / 2 protein in the CNV and PT groups were higher than those in the control group, while the expression of p-ERK1 / 2 was reduced in both the Cur@PT and AFL-Cur@PT groups. Compared with AFL@PT, AFL-Cur@PT showed a further decrease in ERK1 / 2 phosphorylation expression, while there was no significant difference in ERK1 / 2 phosphorylation expression between AFL-Cur@PT and Cur@PT. This confirms that Cur plays a major role in inhibiting the CNV ERK1 / 2 pathway.

[0223] The expression levels of p-P38 and p-JNK proteins in both the CNV and PT groups were significantly increased compared to the control group. Compared to the CNV group, there was no significant difference in JNK phosphorylation in the AFL@PT group, but compared to AFL@PT, JNK phosphorylation was decreased in the AFL-Cur@PT group, indicating that Cur has an inhibitory effect on the JNK signaling pathway. Compared to Cur@PT, AFL-Cur@PT further reduced p38 phosphorylation expression, demonstrating that Cur has an inhibitory effect on the p38 signaling pathway.

[0224] The expression levels of HO-1 and Nrf2 proteins in the CNV and PT groups were increased compared to the control group, while the levels of HO-1 and Nrf2 proteins in the Cur@PT and AFL-Cur@PT groups were further increased, showing significant differences compared to the laser-induced modeling group. The expression level of ICAM-1 protein in the CNV and PT groups was increased compared to the control group, while the expression level of ICAM-1 protein in the Cur@PT and AFL-Cur@PT groups was significantly decreased.

[0225] In summary, AFL-Cur@PT can inhibit the phosphorylation of proteins P65, ERK1 / 2, P38, and JNK, while upregulating Nrf2 / HO-1 protein expression and inhibiting ICAM-1 protein expression. AFL-Cur@PT exerts its anti-inflammatory effect by inhibiting the MAPK signaling pathway, while activating the Nrf2 / HO-1 signaling pathway for antioxidant activity.

[0226] The above study on the mechanism of action of AFL-Cur@PT on CNV shows that AFL-Cur@PT can significantly reduce the expression of factors such as VEGF, IL-6, TNF-α, IL-1β, MCP-1, ICAM-1, and NFκB-1. Its mechanism of action is to inhibit the VEGF / VEGFR2 and MAPK signaling pathways while activating the Nrf2 / HO-1 signaling pathway to achieve the inhibitory effect on CNV.

[0227] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A nanoparticle loaded with aflibercept and curcumin, characterized in that, It includes aflibercept, curcumin, polylactic acid-glycolic acid copolymer, and vitamin E polyethylene glycol succinate.

2. A nanoparticle loaded with aflibercept and curcumin, characterized in that, Based on the mass ratio of raw materials, the mass ratio of aflibercept, curcumin, polylactic acid-glycolic acid copolymer and vitamin E polyethylene glycol succinate is 8:4:140:

3.

3. A method for preparing nanoparticles loaded with aflibercept and curcumin as described in claim 1, characterized in that, Includes the following steps: S1: Dissolve curcumin and polylactic acid glycolic acid copolymer in a mixture of dichloromethane and acetone to obtain an oil phase solution; dissolve aflibercept in water to obtain a first aqueous phase solution; S2: Add the first aqueous phase solution to the oil phase solution and mix by ultrasonication to obtain an oil-in-water solution; S3: Dissolve vitamin E polyethylene glycol succinate and polyvinyl alcohol in water to obtain a second aqueous solution; S4: Add the oil-in-water solution from S2 to the second aqueous phase solution and mix by ultrasonication to obtain an oil-in-water solution. S5: Stir the water-in-oil-in-water solution, centrifuge, remove the supernatant, and wash to obtain nanoparticles loaded with aflibercept and curcumin.

4. The method for preparing nanoparticles loaded with aflibercept and curcumin according to claim 2, characterized in that, Step S1: The polylactic acid-glycolic acid copolymer includes lactic acid and glycolic acid, wherein the mass ratio of lactic acid to glycolic acid is 75:

25. The volume ratio of dichloromethane to acetone in the dichloromethane and acetone mixture is 4:

1.

5. The method for preparing nanoparticles loaded with aflibercept and curcumin according to claim 2, characterized in that, In step S1, the concentration of curcumin is 2-3 mg / mL, the concentration of polylactic acid glycolic acid copolymer is 70-80 mg / mL, and the concentration of aflibercept is 40-50 mg / mL.

6. The method for preparing nanoparticles loaded with aflibercept and curcumin according to claim 2, characterized in that, In step S1, the volume ratio of the oil phase solution to the first aqueous phase solution is 10:

1.

7. The method for preparing nanoparticles loaded with aflibercept and curcumin according to claim 2, characterized in that, In step S2, the conditions for the ultrasound are as follows: the power of the ultrasonic disruptor is set to 150~160 W, the amplitude is 60~70%, and the ultrasonic cycle is 5 seconds for the working period and 7 seconds for the rest period.

8. The method for preparing nanoparticles loaded with aflibercept and curcumin according to claim 2, characterized in that, In step S3, the polyvinyl alcohol concentration is 2-3% and the vitamin E polyethylene glycol succinate concentration is 0.3-0.4 mg / mL.

9. The method for preparing nanoparticles loaded with aflibercept and curcumin according to claim 3, characterized in that, In step S4, the volume ratio of the oil-in-water solution and the second aqueous solution is 1:4.6; The ultrasonic conditions were as follows: 9.6 minutes of ultrasound, power: 150-160 W, amplitude: 60-70%, and an ultrasonic cycle of 5-6 seconds for the working period and 7-8 seconds for the rest period. In step S5, the stirring conditions are: stirring at 800-900 rpm for 5-6 hours, followed by centrifugation at 12000-13000 rpm for 30-40 minutes.

10. The use of nanoparticles loaded with aflibercept and curcumin as described in any one of claims 1-2, or nanoparticles loaded with aflibercept and curcumin prepared by the preparation method as described in any one of claims 3-9, in the preparation of drugs that inhibit choroidal angiogenesis.

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