Nanoparticles, nano eye drops and preparation method and application of nano eye drops

Abstract

The invention relates to nanoparticles, nano eye drops as well as a preparation method and application thereof, and belongs to the technical field of medicines. The invention provides a nanoparticle. The nanoparticle is composed of a tyrosine kinase inhibitor and a carrier protein, the mass ratio of the tyrosine kinase inhibitor to the carrier protein is (2-4): 1. The tyrosine kinase inhibitor-loaded nanoparticles are prepared and creatively combined with the photosensitizer-loaded lipid vesicles to obtain the synergistic photodynamic-VEGF inhibition difunctional photoresponsive nano eye drops which are uniform in particle size, simple and feasible in preparation method and good in stability; the photosensitizer and tyrosine kinase inhibitor co-delivery hydrogel prepared by the method realizes efficient co-delivery of the photosensitizer and the tyrosine kinase inhibitor, has a laser response release behavior, can be detained in eye tissues for a long time, not only greatly improves the bioavailability of hydrophobic drugs, but also realizes the final curative effect of resisting ocular angiogenesis, and has potential clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a kind of nanoparticle, nano eye drop and its preparation method and application. BACKGROUND

[0002] With the phenomenon of population aging becoming more and more serious, more and more old people have visual impairment due to chronic diseases such as diabetes. According to the prediction, the global prevalence of age-related macular degeneration (AMD) will exceed 300 million cases by 2040, making it the leading cause of permanent visual impairment in the elderly population. Among the blinding retinal diseases, including diabetic retinopathy (DR) and age-related macular degeneration (AMD), the formation of retinal or choroidal neovascularization 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.) is still the first-line standard therapy. However, these drugs can only inhibit the progression of new blood vessels, but cannot make the established pathological vascular structure regress. And long-term repeated intraocular injection can increase the cumulative risk of complications, including endophthalmitis and iatrogenic retinal detachment; poor treatment compliance, increased patient burden, which greatly limits its clinical application.

[0003] In recent years, photodynamic therapy (PDT) has become one of the important emerging methods for the treatment of neovascular eye diseases. Its core mechanism involves the activation of photosensitizers by specific wavelength laser, which produces short-lived singlet oxygen and free radicals through photochemical reaction. These active substances induce local vascular endothelial cell damage, platelet activation and thrombosis, selectively blocking ocular neovascularization. However, the clinical application of photosensitizers is limited by systemic intravenous administration, and the accumulation of drugs in non-target tissues (such as skin) can cause systemic phototoxicity, and only a small part (3%~5%) of the drug reaches the retinal lesion site, resulting in limited efficacy, which greatly limits its widespread application 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 the anatomical limitations of the eye tissue (including corneal permeability restriction, rapid tear clearance and pre-corneal metabolic degradation, etc.), the diffusion of drugs to the posterior segment of the eye is hindered, resulting in low bioavailability (<5%) of traditional eye drops.

[0005] Based on this, the present application is proposed. SUMMARY

[0006] The purpose of the present application is to provide a kind of nanoparticle, nano eye drop and its preparation method and application, to solve the problem of low bioavailability of traditional eye drops in the prior art.

[0007] To achieve the above-mentioned object, the present application provides the following technical solutions. The present application provides a kind of nanoparticles loaded tyrosine kinase inhibitor, 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.

[0008] Preferably, the tyrosine kinase inhibitor includes axitinib; The carrier protein is lactoferrin or silk fibroin.

[0009] The present application provides the preparation method of the nanoparticle, comprising the following steps: (1) tyrosine kinase inhibitor is mixed with dimethylformamide to obtain solution 1; (2) carrier protein is mixed with water to obtain solution 2; (3) solution 1 is mixed with solution 2 to obtain a mixed solution, stir 10-20 min, high-pressure homogenization 25-35 min, remove solvent to obtain nanoparticles loaded tyrosine kinase inhibitor.

[0010] The present application provides the application of the nanoparticle loaded tyrosine kinase inhibitor or the nanoparticle loaded tyrosine kinase inhibitor prepared by the preparation method in preparing a drug for treating neovascular eye disease.

[0011] The present application provides a kind of nanodrop eye drops, the nanodrop eye drops are composed of core and shell; The core is nanoparticle loaded tyrosine kinase inhibitor;The shell is photosensitizer-loaded liposome vesicle; The mass ratio of tyrosine kinase inhibitor and photosensitizer is 1-2:1-2; The nanoparticle loaded tyrosine kinase inhibitor is the nanoparticle loaded tyrosine kinase inhibitor or the nanoparticle loaded tyrosine kinase inhibitor prepared by the preparation method.

[0012] Preferably, the photosensitizer-loaded liposome vesicle is composed of photosensitizer, lecithin, cholesterol and phospholipid-polyethylene glycol.

[0013] Preferably, the molar ratio of photosensitizer, lecithin, cholesterol and phospholipid-polyethylene glycol is 1-10:120-130:37-47:2.7-6.7.

[0014] Preferably, the photosensitizer includes verteporfin.

[0015] The present application provides the preparation method of the nanodrop eye drops, comprising the following steps: (1) mixing photosensitizer, lecithin, cholesterol, phospholipid-polyethylene glycol with chloroform to obtain a mixed solution; (2) after evaporating chloroform in the mixed solution under reduced pressure, obtaining liposome vesicles loaded with photosensitizer, adding nanoparticles loaded with tyrosine kinase inhibitor, vortexing and hydrating, and high-pressure homogenization to obtain nanodrop eye drops.

[0016] The application provides application of the nanodrop eye drops or the nanodrop eye drops prepared by the preparation method in preparation of a medicine for treating neovascular eye diseases.

[0017] The application has the following technical effects and advantages: The application prepares nanoparticles loaded with tyrosine kinase inhibitor, creatively combines the nanoparticles with liposome vesicles loaded with photosensitizer, and obtains a synergistic photodynamic-VEGF inhibiting bifunctional light-responsive nanodrop eye drops, which has uniform particle size, a simple and easy-to-operate preparation method, good stability, realizes efficient co-delivery of photosensitizer and tyrosine kinase inhibitor, has laser response release behavior, and can be long-stored in eye tissues, thereby greatly improving the bioavailability of hydrophobic drugs, realizing the final curative effect of anti-eye angiogenesis, and having potential clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Fig. 1 is a particle size diagram of Axi / SF NPs and Lips-NPs; Figure 2 Fig. 2 is a release curve diagram of axitinib in different preparations; Figure 3 Fig. 3 is the ability of different preparations to generate singlet oxygen; Figure 4 Fig. 4 is the in-vitro cytotoxicity of different preparations; Figure 5 Fig. 5 is the in-vitro anti-angiogenic ability of different concentration preparations; Figure 6 Fig. 6 is the retention of different preparations in the eyes of mice; Figure 7 Fig. 7 is the influence of different preparations on the area of new blood vessels of rats. DETAILED DESCRIPTION

[0019] The application provides a nanoparticle loaded with tyrosine kinase inhibitor, which is composed of tyrosine kinase inhibitor and carrier protein. The mass ratio of the tyrosine kinase inhibitor and the carrier protein is 2-4:1, preferably 3:1.

[0020] In the application, the tyrosine kinase inhibitor includes axitinib. The carrier protein is lactoferrin or silk fibroin.

[0021] The application provides a preparation method of the nanoparticles, which comprises the following steps: (1) mixing a tyrosine kinase inhibitor with dimethylformamide to obtain solution 1; (2) mixing a carrier protein with water to obtain solution 2; (3) mixing solution 1 with solution 2 to obtain a mixed solution, stirring for 10-20 min, high-pressure homogenization for 25-35 min, and removing the solvent to obtain the nanoparticles loaded with the tyrosine kinase inhibitor; Preferably, the stirring time is 15 min; and the high-pressure homogenization time is 30 min.

[0022] The application provides application of the nanoparticles loaded with the tyrosine kinase inhibitor or the nanoparticles loaded with the tyrosine kinase inhibitor prepared by the preparation method in preparation of a drug for treating neovascular eye diseases.

[0023] The application provides a nano eye drop, which is composed of an inner core and a shell. The inner core is the nanoparticles loaded with the tyrosine kinase inhibitor; and the 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, and preferably 1:1. The nanoparticles loaded with the tyrosine kinase inhibitor are the nanoparticles loaded with the tyrosine kinase inhibitor or the nanoparticles loaded with the tyrosine kinase inhibitor prepared by the preparation method.

[0024] In the application, the liposome vesicle loaded with the photosensitizer is composed of the photosensitizer, lecithin, cholesterol and phospholipid-polyethylene glycol.

[0025] In the application, the molar ratio of the photosensitizer, lecithin, cholesterol and phospholipid-polyethylene glycol is 1-10:120-130:37-47:2.7-6.7, and preferably 5:125:42:4.7.

[0026] In the application, the photosensitizer comprises verteporfin.

[0027] The application provides a preparation method of the nano eye drop, which comprises the following steps: (1) mixing a photosensitizer, lecithin, cholesterol, phospholipid-polyethylene glycol and chloroform to obtain a mixed solution; (2) after evaporating the chloroform in the mixed solution under reduced pressure, obtaining the liposome vesicle loaded with the photosensitizer, adding the nanoparticles loaded with the tyrosine kinase inhibitor, vortexing and ultrasonic hydrating, and high-pressure homogenization to obtain the nano eye drop; Preferably, the high-pressure homogenization is followed by modification with a cell-penetrating peptide.

[0028] The application provides application of the nanodrop eye drops or the nanodrop eye drops prepared by the preparation method in preparation of a medicine for treating neovascular eye diseases.

[0029] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0030] The corresponding drugs and reagents in the examples are all commercially available products.

[0031] Example 1: Study on interaction between tyrosine kinase inhibitors and protein carriers

[0032] The fluorescence spectrometer was used to obtain the fluorescence spectra of lactoferrin (LF) and silk fibroin (SF), the excitation wavelength of silk fibroin was set to 290 nm, the emission wavelength range was 280-500 nm, the excitation and emission slit width was both 10 nm, and the scanning rate was 1200 nm / min; the excitation wavelength of lactoferrin was set to 277 nm, the emission wavelength range was 200-500 nm, the excitation and emission slit width was both 5 nm, and the scanning rate was 1200 nm / min. Lactoferrin and silk fibroin (1.5 μM) were respectively dissolved in 10 mM phosphate buffer aqueous solution (pH=7.4) to obtain protein solutions. Axitinib (Axi) dissolved in a mixed solution of methanol and phosphate buffer aqueous solution (pH=7.4) was added to the protein solutions in different molar ratios. The molar ratios of silk fibroin to axitinib in the mixed solutions were 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10, and the molar ratios of lactoferrin to axitinib in the mixed solutions were 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 the solutions were mixed thoroughly and equilibrated in a water bath at the corresponding temperature for 30 minutes. The fluorescence quenching spectra of silk fibroin in the mixed solutions of silk fibroin and axitinib and lactoferrin in the mixed solutions of lactoferrin and axitinib at different temperatures (298.15 K, 308.15 K and 318.15 K) were determined.

[0033] 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, and the results are shown in Table 1.

[0034] Table 1 Thermodynamic parameters of the interaction between lactoferrin, silk fibroin and axitinib at different temperatures Table 1 Thermodynamic parameters of the interaction between lactoferrin, silk fibroin and axitinib at different temperatures

[0035] According to Table 1, the quenching mechanism of 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, the interaction is carried out in a spontaneous exothermic manner, is driven by entropy, and the hydrophobic interaction plays a key role in the binding process, and the interaction of silk fibroin SF with axitinib Axi is stronger.

[0036] Experimental Example 2: Preparation of tyrosine kinase inhibitor-loaded nanoparticles and nanodrops

[0037] 1. Preparation of tyrosine kinase inhibitor-loaded nanoparticles

[0038] The tyrosine kinase inhibitor (axitinib Axi) was mixed with 1 mL of dimethylformamide (DMF) to obtain solution 1; silk fibroin (SF) was dissolved in 15 mL of pure water and stirred for 15 min to obtain solution 2; solution 1 was added dropwise to solution 2 under ice bath conditions, stirred for 15 min, and high-pressure homogenization was performed using a homogenizer for 30 min, and the solvent was removed to obtain tyrosine kinase inhibitor-loaded nanoparticles (axitinib / silk fibroin nanoparticle Axi / SF NPs).

[0039] The tyrosine kinase inhibitor (axitinib Axi) was mixed with 1 mL of dimethylformamide (DMF) to obtain solution 1; lactoferrin (LF) was dissolved in 15 mL of pure water and stirred for 15 min to obtain solution 2; solution 1 was added dropwise to solution 2 under ice bath conditions, stirred for 15 min, and high-pressure homogenization was performed using a homogenizer for 30 min, and the solvent was removed to obtain tyrosine kinase inhibitor-loaded nanoparticles (axitinib / lactoferrin nanoparticle Axi / LF NPs).

[0040] 2. Preparation of nanodrops

[0041] The photosensitizer (verteporfin Vp), lecithin, cholesterol, and phospholipid-polyethylene glycol (DSPE-PEG2000) were accurately weighed according to the molar ratio of 5:125:42:4.7 and dissolved in 10 mL of chloroform to obtain a mixed solution; after removing the chloroform in the mixed solution by reduced pressure evaporation, the photosensitizer (verteporfin Vp)-loaded liposome vesicle was obtained, the tyrosine kinase inhibitor (axitinib Axi)-loaded nanoparticles (axitinib / silk fibroin nanoparticle Axi / SF NPs) were added according to the mass ratio of 1:1, vortexed and ultrasonically hydrated, high-pressure homogenized, and modified with cell penetrating peptide (TAT) to obtain nanodrops (Lips-NPs).

[0042] The particle size and potential of Axi / SF NPs, Axi / LF NPs and Lips-NPs were determined by using a nanoparticle size analyzer, and the results are shown in Table 2. The particle sizes of Axi / SF NPs (Axi NPs) and Lips-NPs are shown in Table 2. Figure 1 Figure 1 In Table 2, A represents the particle size of Lips-NPs, and B represents the particle size of Axi / SF NPs.

[0043] Table 2 Particle size and potential of Axi / SF NPs, Axi / LF NPs and Lips-NPs

[0044] According to Figure 1 and Table 2, the particle size of Axi / SF NPs (Axi NPs) is relatively small, with an average particle size of 262.6 nm, and the particle size of Lips-NPs is 232.6 nm, and the particle size distribution of both is unimodal.

[0045] Example 3: In vitro release experiment of Axi / LF NPs and Lips-NPs under the condition of laser irradiation or not

[0046] The experiment was divided into three groups, namely Lips-NPs+L group, Lips-NPs in dark group and Axi NPs in dark group. The specific method is as follows: 100 mL of PBS (10 mM, pH = 7.4) containing 1% (w / v) SDS was used as the release medium, a certain amount of test sample was loaded into the dialysis bag (MWCO = 8000-14000), and after sealing the two ends, it was placed in the release medium in a 37°C water bath constant temperature oscillator (150 rpm). The test sample of Lips-NPs+L group was Lips-NPs, which was continuously irradiated with a 650 nm laser (5 mV / cm 2 ) during the experiment. The test sample of Lips-NPs in dark group was Lips-NPs, which was incubated in the dark during the experiment without laser irradiation; the test sample of Axi NPs in dark group was Axi / SF NPs, which was incubated in the dark during the experiment without laser irradiation. 3 mL of sample was taken at the same specific time point in each group, and 3 mL of blank release medium with a temperature of 37°C was added at the same time. The content of axitinib in the release liquid at different time points was determined by liquid chromatography, and the release curve of axitinib in different preparations was plotted with the cumulative release percentage of axitinib against time, as shown in Figure 2 .

[0047] According to Figure 2 ​It can be seen that the Axi of Axi / LF NPs showed a sustained release trend, with a cumulative release rate of 50% within 24 h and a cumulative release rate of 65% within 72 h. The cumulative release rate of Axi of Lips-NPs was significantly higher than that of Axi / LF NPs, which was due to the emulsification of phospholipids, which enhanced the solubility of Axi and prevented the crystallization of Axi. It is worth noting that compared with Lips-NPs without laser irradiation, the release of Lips-NPs was significantly increased after laser irradiation for 4 h. This was mainly due to the active oxygen (ROS) triggered by the photosensitizer verteporfin (Vp) induced to produce the lipid peroxidation of the liposome vesicle of Lips-NPs. It was confirmed that the Lips-NPs had a light-responsive drug release property, which was expected to achieve a targeted cytotoxic effect in the new blood vessel area while minimizing systemic toxicity.

[0048] Example 4: Determination of the ability of different preparations to generate singlet oxygen

[0049] The ability of different preparations to generate singlet oxygen (O2) was determined by using the singlet oxygen green fluorescent probe Singlet Oxygen Sensor Green (SOSG, purchased from Dalian Melun Biotechnology Co., Ltd.). 1

[0050] The experiment was divided into four groups, namely Free Drug group, Lips-NPs group, Axi NPs group and control group, wherein the Free Drug group contained the same Axi and Vp as the Lips-NPs, the Lips-NPs group was Lips-NPs, the Axi NPs group was Axi / SF NPs, and the control group was phosphate buffered saline (PBS).

[0051] The experimental method was as follows: each group was diluted to a specific concentration (the concentration of Axi and VP in the solution was equivalent to 10 µg / mL) using PBS buffer to obtain the diluent of each group, then the diluent of each group was added to SOSG and mixed to obtain a mixed solution with a final concentration of 2 µM, and then each group of mixed solutions was irradiated with a laser (650 nm, 5 mW / cm 2 ) for different times, and then placed in a fluorescence spectrophotometer with the experimental conditions set as Ex=498 nm, Em=525.8 nm, and slit width of 5 nm. The fluorescence intensity of the oxidation product SOSG-EP of SOSG was determined, and the ability of different preparations to generate singlet oxygen was compared. The results are shown in Figure 3 , wherein the abscissa represents the laser irradiation time, and the ordinate represents the fluorescence intensity of the oxidation product SOSG-EP.

[0052] 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 the laser-induced generation of 1 O2in the Lips-NPs group was derived from Vp encapsulated in the liposome vesicular structure. In addition, the Lips-NPs group generated 1 O2at a rate that increased with the increase of laser irradiation time in a time-dependent manner, while the Free drug group generated 1 O2at a faster rate, which may be due to the self-quenching of Vp at a high concentration in the liposome vesicle. These results show that the Lips-NPs group can efficiently and continuously generate ROS under laser irradiation, thereby improving the efficiency of photodynamic therapy.

[0053] Example 5: In vitro cytotoxicity of different preparations

[0054] MTT method was used to evaluate the in vitro cytotoxicity of different preparations.

[0055] The experimental groups were Axi group, Vp group, VP+L group, Free Drug group, Free Drug+L group, Lips-NPs group, and Lips-NPs+L group, among which the Axi group, Vp group, Lips-NPs group, and Free Drug group were normal groups, and the VP+L group, Free Drug+L group, and Lips-NPs+L group were laser irradiation groups.

[0056] The experimental method was to inoculate EA.hy.926 cells (5×10 3 cells / well, purchased from the Chinese Academy of Sciences Typical Culture Collection Cell Library) in a 96-well plate. After 24 h of culture, the culture medium was replaced with fresh culture medium containing different concentrations of Lips-NPs, Free Drug (containing the same mass of Axi and Vp compared with Lips-NPs), Axi, or Vp. The normal groups were incubated in the dark for 24 h, the laser treatment groups were incubated in the dark for 2 h, and the laser irradiation groups were treated with a 650 nm, 500 mW / cm 2 2laser for 10 min, and then incubated in the dark for another 22 h. 20 μL of MTT (thiazolyl blue, purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) solution (5 mg / mL) was added to each well, and incubated for 4 hours. 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 detector. The cell viability of each group was calculated, and the in vitro cytotoxicity of different preparations was compared. The results are shown in Figure 4 Figure 4 ​The horizontal axis represents the concentration of Axi and Vp, and the vertical axis represents cell viability. The half maximal inhibitory concentration (IC 50 ) and the combination index (CI 50 ) of different treatment groups were calculated using GraphPad Prism 5.0 and CompuSyn 1.0, respectively, and the results are shown in Table 3.

[0057] Table 3. Half maximal inhibitory concentration (IC 50 ) and combination index (CI 50 ) of different treatment groups

[0058] According to Figure 4 and Table 3, the Lips-NPs+L group (IC50value of 0.0819 μg / mL), the Free drug+L group (IC50value of 0.2299 μg / mL), the Vp+L group (IC50value of 0.4343 μg / mL) and Axi (IC50value of 0.7487 μg / mL) all showed a significant decrease in EA.hy926 cell viability in a dose-dependent manner. In contrast, the Lips-NPs group and the Vp group maintained a higher cell viability (>60%). These results confirmed the synergistic effect of the Lips-NPs+L group in combination with anti-VEGF therapy and photodynamic therapy (CI50value less than 0.2).

[0059] Example 6: In vitro anti-angiogenic ability of different concentrations of preparations

[0060] The in vitro anti-angiogenic ability of different concentrations of preparations was evaluated using a tube formation experiment.

[0061] Experimental grouping: blank control group, Lips-NPs+L group, Axi NPs group and Free drug+L group, wherein the blank control group is a PBS buffer solution, the Lips-NPs+L group is Lips-NPs, the Axi NPs group is Axi / SF NPs, and the Free drug+L group contains the same mass of Axi and Vp as compared with the Lips-NPs.

[0062] The experimental method is that the Lips-NPs+L group, the Axi NPs group and the Free drug+L group are respectively prepared into solutions with concentrations of 1 μg / mL and 10 μg / mL using a PBS buffer solution, and then pre-cooled Matrigel matrix glue (50 μL / well, purchased from Corning) is added to a 96-well plate, which is gelled at 37°C for 30 minutes. The cells are adjusted to 4×10 4concentration of 1 pg / mL and 10 pg / mL. The Lips-NPs+L group and the Free drug+L group were irradiated with laser (650 nm, 500 mW / cm2) for 10 min after 2 h, and the vascular branch points and branch length were quantified using an inverted fluorescence microscope and Image J software. The anti-angiogenic ability of different concentrations of the preparation was compared, and the results are shown in 2 Figure 5

[0063] According to the results shown in Figure 5 , the inhibitory effect of the Lips-NPs+L group on angiogenesis was 30.1% (1 pg / mL) and 41.6% (10 pg / mL) lower than that of the blank control and the Axi NPs group, respectively, in terms of vascular branch points, and 50.5% (1 pg / mL) and 40.0% (10 pg / mL) lower in terms of total branch length (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.

[0064] Example 7: Determination of the retention of different preparations in the eyes of mice

[0065] The retention of different preparations in the eyes of normal male BALB / c mice (5-6 weeks old, weighing 18-20 g, 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).

[0066] Experimental groups: The Lips-NPs group, the Free drug group, and the Axi NPs group were divided, in which the Lips-NPs group was DiD-labeled Lips-NPs, the Free drug group contained the same mass of Axi and Vp as the Lips-NPs, both Axi and Vp were labeled with DiD, and the Axi NPs group was DiD-labeled Axi / SF NPs.

[0067] ​​The experimental method will be Lips-NPs group, Free drug group and Axi NPs group respectively using PBS buffer solution prepared into a concentration of 10 μg / mL solution, then respectively 5 μL of different treatment group solution into the mouse eye, and 0.5, 1, 2 and 4 h after imaging, before imaging, the mouse was completely anesthetized with isoflurane. The retention of different preparations in the mouse eye was determined, and the results are shown in Figure 6 The horizontal axis represents the drop time, and the vertical axis represents the average radiation efficiency.

[0068] According to Figure 6 It can be seen that the Lips-NPs group can observe a higher fluorescence signal in the eye at 0.5 h, and the fluorescence intensity is still strong after 4 h. Compared with Lips-NPs, the fluorescence signal intensity of the Free drug group is reduced by an average of 14.5 times (P<0.001); the fluorescence signal intensity of the Axi NPs group is similar to that of the free drug group (P>0.05). The corneal epithelium, interstitium and blood-ocular barrier of the eye tissue, as well as frequent blinking and tear drainage, result in limited retention of the Axi NPs group and the free drug group on the ocular surface. Lips-NPs has a liposome double-layer structure shell similar to the cell membrane, and the modification of its surface with TAT also helps to improve its intraocular delivery efficiency, thereby prolonging its ocular surface retention time.

[0069] Example 8

[0070] The rat corneal alkali burn-induced corneal neovascularization (CNV) model was established: the rats (6-8 weeks, body weight 160-180 g, Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were completely anesthetized with isoflurane, and compound tropicamide eye drops were used to dilate the pupils, and 0.5% propakaine hydrochloride was used for local anesthesia. Take a 4 mm sterile filter paper disc saturated with 1M NaOH solution, place it on the central surface of the cornea for 30 seconds, then take it out and rinse the eye thoroughly with normal saline to prevent further burns. In order to prevent infection, the eyes were rinsed with normal saline every day, and treated with levofloxacin eye drops (twice a day for 3 consecutive days), and the changes in the cornea were monitored by slit lamp microscopy. On the second day after alkali burn, abnormal neovascularization appeared on the edge of the cornea, indicating that the rat corneal neovascularization model was successfully established.

[0071] Experimental grouping: blank control, Lips-NPs+L group, Free drug+L group and Axi NPs group, wherein the blank control group is not treated, the Lips-NPs+L group is Lips-NPs, the Free drug group contains the same mass of Axi and Vp compared with Lips-NPs, and the Axi NPs group is Axi / SF NPs.

[0072] The experimental method is that the Lips-NPs+L group, the Free drug+L group and the Axi NPs group are respectively prepared into a solution with a concentration of 10 μg / mL by using a PBS buffer solution, after the rat corneal neovascularization model is successfully established, 20 μL of different treatment group preparations are respectively dropped into the eyes of rats, once a day, for 14 days, wherein the Lips-NPs+L group and the Free drug+L group are irradiated by laser (650 nm, 500 mW / cm 2 , 2 min) after 2 h of administration. The ocular surface is examined by using a slit lamp microscope every day, and the area of neovascularization on the 6th day is quantified by using Image J software. The effects of different preparations on the area of neovascularization of rats are compared, and the results are shown in Figure 7 , wherein the abscissa represents different treatment groups, and the ordinate represents the area of the neovascularization region.

[0073] According to Figure 7 , the area of neovascularization of the blank control group is the largest, followed by the Axi NPs group. The Free drug+L group and the Lips-NPs+L group both have a certain therapeutic effect, and the area of neovascularization of the Lips-NPs+L group is the smallest (P<0.05), indicating that the anti-angiogenic effect is significant, which provides a possibility for the application of Lips-NPs in the treatment of neovascular eye diseases.

[0074] It can be known from the above examples that the application provides a kind of nanoparticle, nano eye drop and its preparation method and application. The application prepares a kind of nanoparticle loaded with tyrosine kinase inhibitor, creatively combines it with a lipid vesicle loaded with photosensitizer, obtains a kind of synergistic photodynamic-VEGF inhibiting bifunctional light-responsive nano eye drop, which has uniform particle size, simple and easy-to-operate preparation method, good stability, realizes efficient co-delivery of photosensitizer and tyrosine kinase inhibitor, has laser response release behavior, and can be retained in eye tissue for a long time, not only greatly improves the bioavailability of hydrophobic drugs, but also realizes the final curative effect of anti-ocular neovascularization, and has potential clinical application value.

[0075] The above only describes the preferred embodiments of the application, and it should be noted that, for those skilled in the art, without departing from the principles of the application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the application.

Claims

1. A nanoparticle loaded with a tyrosine kinase inhibitor, characterized in that, The nanoparticles are composed of a tyrosine kinase inhibitor and a carrier protein; The mass ratio of the tyrosine kinase inhibitor and the carrier protein is 2-4:

1.

2. The nanoparticle of claim 1, wherein, The tyrosine kinase inhibitor includes axitinib; The carrier protein is lactoferrin or silk fibroin.

3. The method of producing nanoparticles as claimed in claim 1 or 2, characterized in that, The method comprises the following steps: (1) mixing the tyrosine kinase inhibitor with dimethylformamide to obtain solution 1; (2) mixing the carrier protein with water to obtain solution 2; (3) mixing solution 1 with solution 2 to obtain a mixed solution, stirring for 10-20 min, high-pressure homogenization for 25-35 min, and removing the solvent to obtain the nanoparticles loaded with the tyrosine kinase inhibitor.

4. The nanoparticles loaded with the tyrosine kinase inhibitor of claim 1 or 2 or prepared by the preparation method of claim 3 are used for preparing a drug for treating neovascular eye diseases.

5. A nanodrop eye drop solution, characterized in that, The nanodrop eye drops are composed of an inner core and an outer shell; The inner core is the nanoparticles loaded with the tyrosine kinase inhibitor; and the outer shell is a liposome vesicle loaded with a photosensitizer; The mass ratio of the tyrosine kinase inhibitor and the photosensitizer is 1-2:1-2; The nanoparticles loaded with the tyrosine kinase inhibitor are the nanoparticles loaded with the tyrosine kinase inhibitor of claim 1 or 2 or prepared by the preparation method of claim 3.

6. The nanodrop eye drop of claim 5, wherein, The liposome vesicle loaded with the photosensitizer is composed of a photosensitizer, lecithin, cholesterol, and phospholipid-polyethylene glycol.

7. The nanodrop eye drop of claim 6, wherein, The molar ratio of the photosensitizer, lecithin, cholesterol, and phospholipid-polyethylene glycol is 1-10:120-130:37-47:2.7-6.

7.

8. The nanodrop eye drop of claim 7, wherein, The photosensitizer includes verteporfin.

9. The method of preparing the nanodrop eye drop solution as claimed in any one of claims 5 to 8, wherein, The method comprises the following steps: (1) mixing the photosensitizer, lecithin, cholesterol, phospholipid-polyethylene glycol, and chloroform to obtain a mixed solution; (2) after evaporating the chloroform in the mixed solution under reduced pressure, obtaining the liposome vesicle loaded with the photosensitizer, adding the nanoparticles loaded with the tyrosine kinase inhibitor, vortexing and ultrasonic hydration, and high-pressure homogenization to obtain the nanodrop eye drops.

10. The nanodrop eye drops of any one of claims 5-8 or prepared by the preparation method of claim 9 are used for preparing a drug for treating neovascular eye diseases.

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