Nanoparticle, nanodrop eye drops, preparation method and application thereof

Abstract

The present application relates to a kind of nanoparticles, nano drop eye drops and its preparation method and application, belong to medical technology field.The present application provides a kind of nanoparticle, the nanoparticle is composed of tyrosine kinase inhibitor and carrier protein;The mass ratio of tyrosine kinase inhibitor and carrier protein is 2~4:1.The present application prepares a kind of nanoparticle loaded tyrosine kinase inhibitor, creatively combines it with light-sensitive agent loaded lipid vesicle, obtains a kind of synergistic photodynamic-VEGF inhibits bifunctional light-responsive nano drop eye drops, its particle size is uniform, preparation method is simple and easy to operate, and stability is good, realizes the efficient co-delivery of light-sensitive agent, tyrosine kinase inhibitor, with laser response release behavior, and can be long time in eye tissue Retention, not only greatly improve the bioavailability of hydrophobic drug, and realize the final curative effect of anti-ocular angiogenesis, with potential clinical application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a nanoparticle, a nano eye drop, its preparation method, and its application. Background Technology

[0002] With the aging population becoming increasingly severe, more and more elderly people are experiencing vision impairment due to chronic diseases such as diabetes. It is predicted that by 2040, the global prevalence of age-related macular degeneration (AMD) will exceed 300 million cases, making it a leading cause of permanent vision loss in middle-aged and elderly people. In blinding retinal diseases, including diabetic retinopathy (DR) and age-related macular degeneration (AMD), the formation of neovascularization in the retina or choroid is a key pathological marker. Previous studies have shown that abnormal activation of vascular endothelial growth factor (VEGF)-mediated signaling pathways is the core molecular mechanism driving the progression of neovascular eye diseases. Currently, although intravitreal injection of anti-VEGF drugs (such as ranibizumab, conbercept, aflibercept, bevacizumab, etc.) remains the first-line standard treatment, these drugs can only inhibit the progression of new blood vessels and cannot cause the degeneration of established pathological vascular structures. Furthermore, long-term repeated intravitreal injections increase the cumulative risk of complications, including endophthalmitis and iatrogenic retinal detachment; poor treatment adherence and increased patient burden greatly limit their clinical application.

[0003] In recent years, photodynamic therapy (PDT) has emerged as an important new treatment for neovascular eye diseases. Its core mechanism involves a specific wavelength of laser activating a photosensitizer, which generates short-lived singlet oxygen and free radicals through a photochemical reaction. These active substances induce local vascular endothelial cell damage, platelet activation, and thrombus formation, selectively blocking neovascularization in the eye. However, the clinical application of photosensitizers is limited by systemic intravenous administration. Drug accumulation in non-target tissues (such as the skin) can cause systemic phototoxicity, and only a small portion (3%–5%) of the drug reaches the retinal lesion site, resulting in limited efficacy and significantly restricting its widespread use in neovascular eye diseases.

[0004] Topical eye drops, as a non-invasive technique, have significant advantages in the treatment of eye diseases. However, due to anatomical limitations of the eye tissue (including corneal permeability limitations, rapid tear clearance, and pre-corneal metabolic degradation), the diffusion of drugs into the posterior segment of the eye is hindered, resulting in low bioavailability (<5%) of traditional eye drops.

[0005] Based on this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide nanoparticles, nano-eye drops, their preparation methods and applications, in order to solve the problem of low bioavailability of traditional eye drops in the prior art.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides nanoparticles loaded with a tyrosine kinase inhibitor, the nanoparticles being composed of a tyrosine kinase inhibitor and a carrier protein;

[0009] The mass ratio of the tyrosine kinase inhibitor to the carrier protein is 2-4:1.

[0010] Preferably, the tyrosine kinase inhibitor includes axitinib;

[0011] The carrier protein is lactoferrin or silk fibroin.

[0012] This invention provides a method for preparing the aforementioned nanoparticles, comprising the following steps:

[0013] (1) Mix the tyrosine kinase inhibitor with dimethylformamide to obtain solution 1;

[0014] (2) Mix the carrier protein with water to obtain solution 2;

[0015] (3) Mix solution 1 and solution 2 to obtain a mixed solution, stir for 10-20 min, homogenize under high pressure for 25-35 min, remove solvent to obtain nanoparticles loaded with tyrosine kinase inhibitor.

[0016] This invention provides the application of the nanoparticles loaded with tyrosine kinase inhibitors, or the nanoparticles loaded with tyrosine kinase inhibitors prepared by the aforementioned method, in the preparation of drugs for treating neovascular eye diseases.

[0017] This invention provides a nano eye drop, which consists of a core and a shell;

[0018] The core is a nanoparticle loaded with a tyrosine kinase inhibitor; the outer shell is a liposome vesicle loaded with a photosensitizer.

[0019] The mass ratio of the tyrosine kinase inhibitor to the photosensitizer is 1~2:1~2;

[0020] The nanoparticles loaded with tyrosine kinase inhibitors are either the nanoparticles loaded with tyrosine kinase inhibitors described above or the nanoparticles loaded with tyrosine kinase inhibitors prepared by the preparation method described above.

[0021] Preferably, the liposome vesicles loaded with photosensitizers are composed of photosensitizers, lecithin, cholesterol, and phospholipid-polyethylene glycol.

[0022] Preferably, the molar ratio of the photosensitizer, lecithin, cholesterol, and phospholipid-polyethylene glycol is 1~10:120~130:37~47:2.7~6.7.

[0023] Preferably, the photosensitizer includes vertiporfin.

[0024] This invention provides a method for preparing the aforementioned nano eye drops, comprising the following steps:

[0025] (1) Mix photosensitizer, lecithin, cholesterol, phospholipid-polyethylene glycol and chloroform to obtain a mixed solution;

[0026] (2) After evaporating chloroform in the mixed solution under reduced pressure, liposome vesicles loaded with photosensitizer are obtained. Nanoparticles loaded with tyrosine kinase inhibitor are added, and the mixture is vortexed and ultrasonically hydrated. High-pressure homogenization is then performed to obtain nano-eye drops.

[0027] This invention provides the application of the nano eye drops described above or the nano eye drops prepared by the method described above in the preparation of drugs for treating neovascular eye diseases.

[0028] The present invention has the following technical effects and advantages:

[0029] This invention prepares nanoparticles loaded with a tyrosine kinase inhibitor and creatively combines them with lipid vesicles loaded with a photosensitizer to obtain a synergistic photodynamic-VEGF inhibition bifunctional photoresponsive nano-eye drops. The nanoparticles have uniform particle size, are simple and easy to prepare, and have good stability. They achieve efficient co-delivery of photosensitizer and tyrosine kinase inhibitor, exhibit laser-responsive release behavior, and can remain in ocular tissue for a long time. This not only greatly improves the bioavailability of hydrophobic drugs but also achieves the ultimate therapeutic effect of anti-ocular angiogenesis, and has potential clinical application value. Attached Figure Description

[0030] Figure 1 Particle size distributions for Axi / SF NPs and Lips-NPs;

[0031] Figure 2 Release curves of axitinib in different formulations;

[0032] Figure 3 The ability to generate singlet oxygen for different formulations;

[0033] Figure 4 In vitro cytotoxicity of different formulations;

[0034] Figure 5 The in vitro anti-angiogenic ability of different concentration formulations;

[0035] Figure 6 The retention of different formulations in the mouse eye;

[0036] Figure 7 The effect of different formulations on the area of ​​neovascularization in rats. Detailed Implementation

[0037] This invention provides nanoparticles loaded with a tyrosine kinase inhibitor, the nanoparticles being composed of a tyrosine kinase inhibitor and a carrier protein;

[0038] The mass ratio of the tyrosine kinase inhibitor to the carrier protein is 2-4:1, preferably 3:1.

[0039] In this invention, the tyrosine kinase inhibitor includes axitinib;

[0040] The carrier protein is lactoferrin or silk fibroin.

[0041] This invention provides a method for preparing the aforementioned nanoparticles, comprising the following steps:

[0042] (1) Mix the tyrosine kinase inhibitor with dimethylformamide to obtain solution 1;

[0043] (2) Mix the carrier protein with water to obtain solution 2;

[0044] (3) Mix solution 1 and solution 2 to obtain a mixed solution, stir for 10-20 min, homogenize under high pressure for 25-35 min, remove solvent to obtain nanoparticles loaded with tyrosine kinase inhibitor;

[0045] The stirring time is preferably 15 minutes; the high-pressure homogenization time is preferably 30 minutes.

[0046] This invention provides the application of the nanoparticles loaded with tyrosine kinase inhibitors, or the nanoparticles loaded with tyrosine kinase inhibitors prepared by the aforementioned method, in the preparation of drugs for treating neovascular eye diseases.

[0047] This invention provides a nano eye drop, which consists of a core and a shell;

[0048] The core is a nanoparticle loaded with a tyrosine kinase inhibitor; the outer shell is a liposome vesicle loaded with a photosensitizer.

[0049] The mass ratio of the tyrosine kinase inhibitor to the photosensitizer is 1~2:1~2, preferably 1:1;

[0050] The nanoparticles loaded with tyrosine kinase inhibitors are either the nanoparticles loaded with tyrosine kinase inhibitors described above or the nanoparticles loaded with tyrosine kinase inhibitors prepared by the preparation method described above.

[0051] In this invention, the liposome vesicles loaded with photosensitizers are composed of photosensitizers, lecithin, cholesterol, and phospholipid-polyethylene glycol.

[0052] In this invention, the molar ratio of photosensitizer, lecithin, cholesterol and phospholipid-polyethylene glycol is 1~10:120~130:37~47:2.7~6.7, preferably 5:125:42:4.7.

[0053] In this invention, the photosensitizer includes vertiporfin.

[0054] This invention provides a method for preparing the aforementioned nano eye drops, comprising the following steps:

[0055] (1) Mix photosensitizer, lecithin, cholesterol, phospholipid-polyethylene glycol and chloroform to obtain a mixed solution;

[0056] (2) After evaporating chloroform in the mixed solution under reduced pressure, liposome vesicles loaded with photosensitizer are obtained. Nanoparticles loaded with tyrosine kinase inhibitor are added, and the mixture is vortexed and ultrasonically hydrated. High-pressure homogenization is then performed to obtain nano-eye drops.

[0057] Preferably, after high-pressure homogenization, the mixture needs to be modified with cell-penetrating peptides.

[0058] This invention provides the application of the nano eye drops described above or the nano eye drops prepared by the method described above in the preparation of drugs for treating neovascular eye diseases.

[0059] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] The drugs and reagents used in the examples are all commercially available products.

[0061] Example 1: Experimental Study on the Interaction between Tyrosine Kinase Inhibitors and Protein Carriers

[0062] Fluorescence spectra of lactoferrin (LF) and silk fibroin (SF) were obtained using a fluorescence spectrophotometer. The excitation wavelength for silk fibroin was set to 290 nm, the emission wavelength range to 280–500 nm, the excitation and emission slit widths to 10 nm each, and the scan rate to 1200 nm / min. The excitation wavelength for lactoferrin was set to 277 nm, the emission wavelength range to 200–500 nm, the excitation and emission slit widths to 5 nm each, and the scan rate to 1200 nm / min. Lactoferrin and silk fibroin (1.5 μM) were dissolved separately in 10 mM phosphate buffered aqueous solution (pH=7.4) to obtain protein solutions. Axitinib (Axi), dissolved in a mixture of methanol and phosphate buffered aqueous solution (pH=7.4), was added to the protein solutions at different molar ratios. Mixed solutions of silk fibroin and axitinib with molar ratios of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 were obtained, along with mixed solutions of lactoferrin and axitinib with molar ratios of 1:0.5, 1:0.75, 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, 1:2.25, and 1:2.5. All solutions were thoroughly mixed and equilibrated in a water bath at the appropriate temperature for 30 minutes. Fluorescence quenching spectra of silk fibroin in the mixed solutions of silk fibroin and axitinib with different molar ratios, and of lactoferrin in the mixed solutions of lactoferrin and axitinib with different molar ratios, were measured at different temperatures (298.15 K, 308.15 K, and 318.15 K).

[0063] The fluorescence data obtained above were fitted and calculated to determine the thermodynamic parameters of the interaction between lactoferrin, silk fibroin and axitinib at different temperatures. The results are shown in Table 1.

[0064] Table 1. Thermodynamic parameters of the interaction between lactoferrin, silk fibroin and axitinib at different temperatures.

[0065]

[0066] As shown in Table 1, the quenching mechanism of both silk fibroin / axitinib (SF / Axi) and lactoferrin / axitinib (LF / Axi) is static quenching. The binding constant of axitinib Axi with silk fibroin SF or lactoferrin LF is 1. Their interaction is carried out in a self-heating manner, driven by entropy. Hydrophobic interaction plays a key role in their binding process, and the interaction between silk fibroin SF and axitinib Axi is stronger.

[0067] Experimental Example 2: Preparation of Nanoparticles Loaded with Tyrosine Kinase Inhibitors and Nanoparticle Eye Drops

[0068] 1. Preparation of nanoparticles loaded with tyrosine kinase inhibitors

[0069] Solution 1 was prepared by mixing a tyrosine kinase inhibitor (Axi) with 1 mL of dimethylformamide (DMF); solution 2 was prepared by dissolving silk fibroin (SF) in 15 mL of pure water and stirring for 15 min; solution 1 was added dropwise to solution 2 under ice bath conditions and stirred for 15 min, and then homogenized under high pressure for 30 min to remove the solvent, thereby obtaining nanoparticles loaded with the tyrosine kinase inhibitor (Axi / SF nanoparticles).

[0070] Solution 1 was prepared by mixing a tyrosine kinase inhibitor (Axi) with 1 mL of dimethylformamide (DMF); solution 2 was prepared by dissolving lactoferrin (LF) in 15 mL of pure water and stirring for 15 min; solution 1 was added dropwise to solution 2 under ice bath conditions and stirred for 15 min, and then homogenized under high pressure for 30 min to remove the solvent, thereby obtaining nanoparticles loaded with the tyrosine kinase inhibitor (Axi / LF nanoparticles).

[0071] 2. Preparation of Nano-sized Eye Drops

[0072] Photosensitizer (verteporfen Vp), lecithin, cholesterol, and phospholipid-polyethylene glycol (DSPE-PEG2000) were accurately weighed and dissolved in 10 mL of chloroform at a molar ratio of 5:125:42:4.7 to obtain a mixed solution. After evaporating the chloroform from the mixed solution under reduced pressure, liposome vesicles loaded with photosensitizer (verteporfen Vp) were obtained. Nanoparticles loaded with tyrosine kinase inhibitor (Axi / Axi / SF NPs) were added at a mass ratio of photosensitizer (verteporfen Vp) to tyrosine kinase inhibitor (Axi) of 1:1. The mixture was vortexed and sonicated, homogenized under high pressure, and modified with cell-penetrating peptide (TAT) to obtain nano-eye drops (Lips-NPs).

[0073] The particle size and potential of Axi / SF NPs, Axi / LF NPs, and Lips-NPs were determined using a nanoparticle size analyzer. The results are shown in Table 2. The particle sizes of Axi / SF NPs (Axi NPs) and Lips-NPs are as follows: Figure 1 As shown, Figure 1 In this text, A represents the particle size of Lips-NPs, and B represents the particle size of Axi / SF NPs.

[0074] Table 2. Particle size and potential of Axi / SF NPs, Axi / LF NPs and Lips-NPs

[0075]

[0076] according to Figure 1As shown in Table 2, Axi / SF NPs (Axi NPs) have relatively small particle sizes, with an average particle size of 262.6 nm, while Lips-NPs have a particle size of 232.6 nm, and both have a single-peaked particle size distribution.

[0077] Example 3: In vitro release experiments of Axi / LF NPs and Lips-NPs under laser irradiation conditions

[0078] The experiment was divided into three groups: Lips-NPs+L group, Lips-NPs in dark group, and Axi NPs in dark group. Specifically, 100 mL of PBS (10 mM, pH=7.4) containing 1% (w / v) SDS was used as the release medium. A certain amount of sample was placed in a dialysis bag (MWCO=8000~14000), sealed at both ends, and incubated in the release medium at 37°C in a water bath with a constant temperature shaker (150 rpm). The Lips-NPs+L group tested Lips-NPs, which were then tested using a 650 nm laser (5 mV / cm²) during the experiment. 2 Continuous irradiation was used. The Lips-NPs in dark group consisted of Lips-NPs, incubated in darkness without laser irradiation during the experiment; the Axi NPs in dark group consisted of Axi / SF NPs, incubated in darkness without laser irradiation during the experiment. At the same specific time point in each group, 3 mL of sample was taken, and 3 mL of blank release medium at 37°C was added simultaneously. The axitinib content in the release medium at different time points was determined using liquid chromatography, and release curves of axitinib in different formulations were plotted against time with the cumulative percentage of drug release. The results are shown below. Figure 2 As shown in the figure, the horizontal axis represents time, and the vertical axis represents the cumulative percentage of axitinib release.

[0079] according to Figure 2 It was observed that Axi / LF NPs exhibited a sustained release trend, with a cumulative release rate of 50% within 24 hours and 65% within 72 hours. The cumulative release rate of Axi in Lips-NPs was significantly higher than that in Axi / LF NPs. This is attributed to the enhanced solubility of Axi due to the emulsifying effect of phospholipids, which prevented Axi crystallization. Notably, compared to Lips-NPs without laser irradiation, the release of Lips-NPs increased significantly after 4 hours of laser irradiation. This was primarily due to the reactive oxygen species (ROS) induced by the photosensitizer verteporfin (Vp) triggering lipid peroxidation in the liposomes of Lips-NPs. This confirms the photoresponsive drug release characteristics of Lips-NPs, suggesting their potential for targeted cytotoxic effects in angiogenesis regions while minimizing systemic toxicity.

[0080] Example 4: Determining the ability of different formulations to generate singlet oxygen

[0081] The singlet oxygen produced by different formulations was determined using the singlet oxygen green fluorescent probe Singlet Oxygen Sensor Green (SOSG, purchased from Dalian Meilun Biotechnology Co., Ltd.). 1 The ability of O2).

[0082] The experiment was divided into four groups: Free Drug group, Lips-NPs group, Axi NPs group, and control group. The Free Drug group contained the same Axi and Vp as the Lips-NPs group, the Lips-NPs group contained Lips-NPs, the Axi NPs group contained Axi / SFNPs, and the control group contained phosphate-buffered saline (PBS).

[0083] The experimental method was as follows: Each group was diluted with PBS buffer to a specific concentration (the concentrations of Axi and VP in the solution were both equivalent to 10 µg / mL) to obtain diluents for each group. Then, SOSG was added to each group's diluent and mixed to a final concentration of 2 µM to obtain a mixed solution. The mixed solutions of each group were then subjected to laser treatment (650 nm, 5 mW / cm²). 2 After irradiation for different times, the sample was placed in a fluorescence spectrophotometer with the experimental conditions set as follows: Ex = 498 nm, Em = 525.8 nm, and a slit width of 5 nm. The fluorescence intensity of SOSG-EP, the oxidation product of SOSG, was measured to compare the ability of different formulations to generate singlet oxygen. The results are as follows: Figure 3 As shown in the figure, the horizontal axis represents the laser irradiation time, and the vertical axis represents the fluorescence intensity of the oxidation product SOSG-EP.

[0084] according to Figure 3 It can be seen that the fluorescence intensity of SOSG-EP (oxidized SOSG) in the Lips-NPs group and the Free drug group was significantly enhanced compared with the control group (PBS) and the Axi NPs group, indicating that laser-induced SOSG-EP was generated. 1 O2 originates from Vp encapsulated within the liposome vesicle structure. Furthermore, the Lips-NPs group generates... 1 The rate of O2 production increased with increasing laser irradiation time, while the production of free drug group... 1 The faster rate of O2 generation is likely due to self-quenching caused by excessively high Vp concentrations in liposome vesicles. These results indicate that the Lips-NPs group can efficiently and continuously generate ROS under laser irradiation, thereby improving the efficiency of photodynamic therapy.

[0085] Example 5: In vitro cytotoxicity of different formulations

[0086] The MTT assay was used to evaluate the in vitro cytotoxicity of different formulations.

[0087] Experimental groups: Axi group, Vp group, VP+L group, Free Drug group, Free Drug+L group, Lips-NPs group, and Lips-NPs+L group. Among them, Axi group, Vp group, Lips-NPs group, and Free Drug group were normal groups, while VP+L group, Free Drug+L group, and Lips-NPs+L group were laser irradiation groups.

[0088] The experimental method involved using EA.hy.926 cells (5 × 10⁻⁶). 3 Cells (purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection) were seeded in 96-well plates. After 24 hours of culture, the medium was replaced with fresh medium containing different concentrations of Lips-NPs, FreeDrug (containing the same mass of Axi and Vp as Lips-NPs), or Axi or Vp. The normal group was incubated in the dark for 24 hours, the laser-treated group was incubated in the dark for 2 hours, and the laser-irradiated group was treated with 650 nm laser at 500 mW / cm². 2 Cells were treated with laser for 10 min, then incubated in the dark for 22 h. 20 μL of MTT (thiazolyl blue, purchased from Shanghai Beyotime Biotechnology Co., Ltd.) solution (5 mg / mL) was added to each well, and incubation was continued for 4 h. After removing the MTT reagent, 150 μL of DMSO was added to each well, and the absorbance at 570 nm was recorded using a microplate reader. Cell viability was calculated for each group using the following formula: Cell viability (%) = ODtreated / ODcontrol × 100%. The in vitro cytotoxicity of different formulations was compared, and the results are as follows: Figure 4 As shown, Figure 4 The x-axis represents the concentrations of Axi and Vp, and the y-axis represents cell viability. The half-maximal inhibitory concentration (IC50) for different treatment groups was calculated using GraphPad Prism 5.0 and CompuSyn 1.0. 50 ) and Co-existence Index (CI) 50 The results are shown in Table 3.

[0089] Table 3. Half-maximal inhibitory concentrations (IC50) of different treatment groups 50 ) and Co-existence Index (CI) 50 )

[0090]

[0091] according to Figure 4As shown in Table 3, the Lips-NPs+L group (IC50 value of 0.0819 μg / mL), Free drug+L group (IC50 value of 0.2299 μg / mL), Vp+L group (IC50 value of 0.4343 μg / mL), and Axi (IC50 value of 0.7487 μg / mL) all exhibited a significant decrease in EA.hy926 cell viability in a dose-dependent manner. In contrast, the Lips-NPs group and Vp group maintained higher cell viability (>60%). These results confirm the synergistic effect of the Lips-NPs+L group combined with anti-VEGF therapy and photodynamic therapy (CI50 value less than 0.2).

[0092] Example 6: In vitro anti-angiogenic ability of different concentrations of formulations

[0093] The in vitro anti-angiogenic ability of different concentrations of the formulation was evaluated using tube formation experiments.

[0094] Experimental groups: The experimental groups were divided into a blank control group, a Lips-NPs+L group, an Axi NPs group, and a Free drug+L group. The blank control group was a PBS buffer solution, the Lips-NPs+L group was a Lips-NPs group, the Axi NPs group was an Axi / SF NPs group, and the Free drug+L group was a group containing the same mass of Axi and Vp as the Lips-NPs group.

[0095] The experimental method involved preparing solutions of Lips-NPs+L, Axi NPs, and Free drug+L in PBS buffer at concentrations of 1 μg / mL and 10 μg / mL, respectively. Pre-chilled Matrigel (50 μL / well, purchased from Corning) was then added to 96-well plates and gelled at 37°C for 30 minutes. The cell density was adjusted to 4 × 10⁶ cells / well. 4 The concentration of cells / well was determined and mixed with treatment group solutions of different concentrations (1 μg / mL and 10 μg / mL). Both the Lips-NPs+L group and the Free drug+L group were irradiated with laser (650 nm, 500 mW / cm²) after 2 h. 2 The irradiation time was 10 min. The images were observed and photographed using an inverted fluorescence microscope, and the number of vascular branch points and branch length were quantified using ImageJ software. The in vitro anti-angiogenic ability of different concentrations of the formulation was compared, and the results are as follows: Figure 5 As shown in the figure, Figure A represents the effect of different concentrations of the formulation on the number of vascular branch points, with the horizontal axis representing the formulation concentration and the vertical axis representing the number of vascular branch points. Figure B represents the effect of different concentrations of the formulation on the length of vascular branches, with the horizontal axis representing the formulation concentration and the vertical axis representing the length of vascular branches.

[0096] according to Figure 5The results showed that the Lips-NPs+L group significantly reduced angiogenesis compared to the blank control and Axi NPs groups, decreasing the number of vascular branch points by 30.1% (1 μg / mL) and 41.6% (10 μg / mL), respectively, and the total branch length by 50.5% (1 μg / mL) and 40.0% (10 μg / mL), respectively (P<0.001). The anti-angiogenic ability of the Lips-NPs group was attributed to the synergistic effect of anti-VEGF therapy and photodynamic therapy.

[0097] Example 7: Determination of retention of different formulations in the mouse eye

[0098] The retention of different formulations in the eyes of normal male BALB / c mice (5-6 weeks old, weighing 18-20g, Jinan Pengyue Experimental Animal Breeding Co., Ltd.) was evaluated using a non-invasive fluorescence imaging system (PerkinElmer IVIS, Lumina XRMS Series III, Waltham, MA, USA).

[0099] Experimental groups: The experiment was divided into Lips-NPs, Free drug, and Axi NPs. The Lips-NPs group consisted of DiD-labeled Lips-NPs. The Free drug group consisted of Axi and Vp with the same mass as Lips-NPs, where both Axi and Vp were labeled with DiD. The Axi NPs group consisted of DiD-labeled Axi / SF NPs.

[0100] Experimental Methods: Lips-NPs, Free drug, and Axi NPs groups were prepared into 10 μg / mL solutions using PBS buffer. 5 μL of each treatment solution was instilled into the mouse eye, and imaging was performed 0.5, 1, 2, and 4 hours after instillation. Mice were completely anesthetized with isoflurane before imaging. Retention time of different formulations in the mouse eye was measured, and the results are as follows: Figure 6 As shown in the figure, the horizontal axis represents the droplet time, and the vertical axis represents the average radiation efficiency.

[0101] according to Figure 6It was observed that the Lips-NPs group showed a high fluorescence signal in the eye at 0.5 h, and the fluorescence intensity remained strong even after 4 h. In contrast, the Free drug group showed an average decrease of 14.5-fold in ocular fluorescence signal intensity compared to the Lips-NPs group (P<0.001); the Axi NPs group showed similar fluorescence signal intensity to the free drug group (P>0.05). The corneal epithelium, stroma, and blood-eye barrier, as well as frequent blinking and tear drainage, limit the retention of Axi NPs and the free drug group on the ocular surface. Lips-NPs possess a liposome bilayer structure similar to a cell membrane, and surface modification with TAT can improve their intraocular delivery efficiency, thereby prolonging their ocular surface retention time.

[0102] Example 8

[0103] A rat model of corneal neovascularization (CNV) induced by alkali burn was established: Rats (6-8 weeks old, weighing 160-180g, from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were completely anesthetized with isoflurane, and their pupils were dilated with compound tropicamide eye drops. Local anesthesia was achieved with 0.5% propacaine hydrochloride. A 4mm sterile filter paper disc saturated with 1M NaOH solution was placed on the central corneal surface for 30 seconds and then removed. The eyes were thoroughly rinsed with physiological saline to prevent further burns. To prevent infection, the eyes were rinsed with physiological saline daily and treated with levofloxacin eye drops (twice daily for 3 consecutive days). Corneal changes were monitored using a slit-lamp microscope. On the second day after the alkali burn, abnormal neovascularization appeared at the corneal margin, indicating successful establishment of the rat corneal neovascularization model.

[0104] Experimental groups: blank control, Lips-NPs+L group, Free drug+L group and Axi NPs group. The blank control group received no treatment. The Lips-NPs+L group consisted of Lips-NPs. The Free drug group consisted of Axi and Vp with the same mass as Lips-NPs. The Axi NPs group consisted of Axi / SF NPs.

[0105] Experimental Methods: The Lips-NPs+L group, Free drug+L group, and Axi NPs group were prepared into solutions with a concentration of 10 μg / mL using PBS buffer. After successful establishment of the rat corneal neovascularization model, 20 μL of each treatment group's preparation was instilled into the eyes of rats once daily for 14 days. The Lips-NPs+L group and Free drug+L group underwent laser irradiation (650 nm, 500 mW / cm²) 2 hours after administration. 2 (2 min). Ocular surface examination was performed daily using a slit-lamp microscope, and the neovascularization area on day 6 was quantified using ImageJ software. The effects of different formulations on the neovascularization area of ​​rats were compared, and the results are as follows: Figure 7 As shown in the figure, the horizontal axis represents different treatment groups, and the vertical axis represents the area of ​​neovascularization.

[0106] according to Figure 7 It was found that the blank control group had the largest area of ​​neovascularization, followed by the Axi NPs group. Both the Free drug+L group and the Lips-NPs+L group showed certain therapeutic effects, while the Lips-NPs+L group had the smallest area of ​​neovascularization (P<0.05), indicating that it had a significant anti-angiogenic effect, which provides a possibility for the application of Lips-NPs in the treatment of neovascular eye diseases.

[0107] As can be seen from the above embodiments, the present invention provides nanoparticles, nano-eye drops, their preparation methods, and applications. The present invention prepares nanoparticles loaded with a tyrosine kinase inhibitor and creatively combines them with lipid vesicles loaded with a photosensitizer to obtain a synergistic photodynamic-VEGF inhibition bifunctional photoresponsive nano-eye drop. This nano-eye drop has uniform particle size, a simple and easy preparation method, and good stability. It achieves efficient co-delivery of the photosensitizer and the tyrosine kinase inhibitor, exhibits laser-responsive release behavior, and can remain in ocular tissue for a long time. This not only greatly improves the bioavailability of hydrophobic drugs but also achieves the ultimate therapeutic effect of anti-ocular angiogenesis, possessing potential clinical application value.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nano eye drop, characterized in that, The nano eye drops consist of a core and a shell; The core is a nanoparticle loaded with a tyrosine kinase inhibitor; the outer shell is a liposome vesicle loaded with a photosensitizer. The mass ratio of the tyrosine kinase inhibitor to the photosensitizer is 1~2:1~2; The nanoparticles are composed of tyrosine kinase inhibitors and carrier proteins; The mass ratio of the tyrosine kinase inhibitor to the carrier protein is 2-4:1; The tyrosine kinase inhibitors include axitinib; The carrier protein is silk fibroin; The liposome vesicles loaded with photosensitizers are composed of photosensitizers, lecithin, cholesterol, and phospholipid-polyethylene glycol; The molar ratio of photosensitizer, lecithin, cholesterol, and phospholipid-polyethylene glycol is 1~10:120~130:37~47:2.7~6.7; The photosensitizer includes vertiporfin.

2. The nano eye drops according to claim 1, characterized in that, The method for preparing the nanoparticles includes the following steps: (1) Mix the tyrosine kinase inhibitor with dimethylformamide to obtain solution 1; (2) Mix the carrier protein with water to obtain solution 2; (3) Mix solution 1 and solution 2 to obtain a mixed solution, stir for 10-20 min, homogenize under high pressure for 25-35 min, remove solvent to obtain nanoparticles loaded with tyrosine kinase inhibitor.

3. The method for preparing the nano-eye drops according to claim 1 or 2, characterized in that, Includes the following steps: (1) Mix photosensitizer, lecithin, cholesterol, phospholipid-polyethylene glycol and chloroform to obtain a mixed solution; (2) After evaporating chloroform in the mixed solution under reduced pressure, liposome vesicles loaded with photosensitizer are obtained. Nanoparticles loaded with tyrosine kinase inhibitor are added, and the mixture is vortexed and ultrasonically hydrated. High-pressure homogenization is then performed to obtain nano-eye drops.

4. The use of the nano eye drops according to claim 1 or 2 or the nano eye drops prepared by the preparation method according to claim 3 in the preparation of drugs for treating neovascular eye diseases.

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