Application of amphiphilic cationic modified mPEG-PCL nanoparticles in the preparation of drugs for treating retinal diseases

Abstract

The present invention belongs to the technical field of polymer compounds, and specifically relates to the use of amphiphilic cationic modified mPEG-PCL nanoparticles in the preparation of drugs for treating retinal diseases. The technical problem to be solved by the present invention is the lack of nanomedicines for the treatment of retinal diseases. The technical solution of the present invention is the use of amphiphilic cationic modified mPEG-PCL nanoparticles in the preparation of drugs for treating eye diseases. The nanoparticles of the present invention have low cytotoxicity; can effectively inhibit the development of choroidal neovascularization; and can also be used as carriers for other eye disease-related drugs, enabling combined drug use and the treatment of various eye diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer compounds, and particularly relates to the use of mPEG-PCL nanoparticles modified with amphiphilic cationic substances in the preparation of drugs for treating retinal diseases. Background Art

[0002] Age-related macular degeneration (AMD) is a common retinal disease that occurs in middle-aged and elderly people. It is one of the main causes of vision impairment and even blindness worldwide, especially in economically developed regions. With the economic development of my country and the improvement of people's living standards, the number of people suffering from AMD is increasing, which also brings about an increasing demand for medical treatment. Clinically, AMD is divided into early (drusen formation, retinal pigment changes) and late (choroidal neovascularization, atrophy) stages. Among them, late AMD characterized by choroidal neovascularization (CNV) is also called wet AMD. Compared with late AMD characterized by atrophy (dry AMD), it progresses faster and vision may deteriorate severely within a few months or even weeks.

[0003] Treatment for wet AMD primarily targets CNV. Currently, the most effective treatment is intravitreal injection of VEGF inhibitors such as aflibercept and ranibizumab. No treatment has been approved for dry AMD. It's worth noting that anti-VEGF agents are extremely expensive, require repeated use, and lack clear criteria for discontinuation. This places a significant burden on both individual patients and national health insurance. Therefore, there remains a significant clinical need for alternative treatment options.

[0004] Nanotechnology is becoming increasingly important in the treatment of various diseases. Currently, nanoparticles are the primary form of nanoformulations used for treatment, and they are classified according to their charge: cationic, anionic, and neutral nanoparticles. Nanoparticles can be modified through covalent attachment, electrostatic adsorption, or encapsulation to provide targeted or non-targeted treatment for diseases. Nanoparticles are widely used as drug delivery vehicles, but the immunogenicity associated with their inherent charge properties limits the potential of nanoformulations to a certain extent. Utilizing the immunomodulatory effects of nanoparticles could be a new strategy for disease treatment.

[0005] Currently, most research on nanomedicines focuses on cancer treatment, however, the use of nanoparticles in the treatment of retinal diseases has rarely been reported. Therefore, nanomedicines have great potential in the treatment of retinal diseases such as CNV. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that there is still a lack of nanomedicines in the treatment of retinal diseases.

[0007] The technical solution of the present invention is the use of amphiphilic cationic substance-modified mPEG-PCL nanoparticles in the preparation of drugs for treating eye diseases.

[0008] Wherein, the amphiphilic cationic substance is DOTAP ((2,3-dioleyloxypropyl)trimethylammonium chloride).

[0009] Among them, the molecular weight ratio of mPEG and PCL in mPEG-PCL is 1:1, and the total molecular weight range of mPEG-PCL is 4000Da~8000Da.

[0010] Specifically, the mass ratio of the amphiphilic cationic substance to mPEG-PCL is 1-30:70-99.

[0011] Preferably, the mass ratio of the amphiphilic cationic substance to mPEG-PCL is 5:45.

[0012] Wherein, the solvent is at least one of dichloromethane, chloroform, acetone, tetrachloromethane, ethanol, methanol, ether, pentane, ethyl acetate, and cyclohexane.

[0013] Specifically, the hydration solution is double distilled water, deionized water, pure water or physiological saline.

[0014] Specifically, the preparation method of the nanoparticles includes the following steps: dissolving an amphiphilic cationic substance and mPEG-PCL in a solvent, then evaporating the solvent, adding a hydration solution to hydrate until completely dissolved, the resulting solution is a nanoparticle solution, and drying to obtain nanoparticles; the mass ratio of the amphiphilic cationic substance to mPEG-PCL (methoxy polyethylene glycol-polylactic acid) is 1-30:70-99.

[0015] Among them, the eye diseases in the above applications are eyelid and lacrimal diseases, conjunctival and scleral diseases, corneal diseases, lens diseases, glaucoma, uveal diseases, vitreous diseases or retinal diseases.

[0016] Furthermore, the retinal disease is age-related macular degeneration.

[0017] Particularly, the age-related macular degeneration is age-related macular degeneration caused by drusen formation, retinal pigment changes, choroidal neovascularization or choroidal neovascularization atrophy.

[0018] Furthermore, the dosage form of the drug in the above application is injection, tablet or drop.

[0019] In particular, the main component of the drug in the above application is the nanoparticles of mPEG-PCL modified with an amphiphilic cationic substance.

[0020] Furthermore, the amphiphilic cationic substance-modified mPEG-PCL nanoparticles are also loaded with other drugs for treating eye diseases and / or other pharmaceutically acceptable auxiliary ingredients.

[0021] Beneficial effects of the present invention: The mPEG-PCL nanoparticles modified with amphiphilic cationic substances have an average particle size of 86.48 nm and an average potential of +42.57 mV. In vitro, they exhibited lower cytotoxicity than the gold-labeled transfection material PEI 25K. In vivo, a choroidal neovascularization model was established, and treatment was performed via intravitreal injection. The choroidal neovascularization and vascular leakage in each group were compared; it was found that the nanoparticles of the present invention significantly reduced choroidal neovascularization and inflammation in mice. Therefore, the nanoparticles of the present invention can be used to treat eye diseases (particularly CNV) in vitro and in vivo. These nanoparticles can effectively inhibit the development of choroidal neovascularization and have promising application prospects in ophthalmic maculopathy research and clinical applications, providing a new approach and potential option for the treatment of choroidal neovascularization. Furthermore, the nanoparticles of the present invention can be used alone to treat CNV; they can also be used as carriers to carry other CNV-treating drugs to further enhance the therapeutic effect; and they can also be loaded with other eye-related drugs for combination therapy to treat a variety of eye diseases. In addition, the nanoparticles of the present invention are simple to prepare (self-assembly is sufficient), are degradable, and are safe and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 、DOTAP structural formula.

[0023] Figure 2 , mPEG-PCL structural formula, x and y are positive integers.

[0024] Figure 3 , particle size distribution diagram of nanoparticles.

[0025] Figure 4 , potential distribution of nanoparticles.

[0026] Figure 5 , the morphology of the prepared DMP nanoparticles was observed under a scanning transmission electron microscope.

[0027] Figure 6 , comparison of the cytotoxicity of DMP nanoparticles and the gold standard transfection material PEI 25K.

[0028] Figure 7 , FFA was performed on rats to evaluate neovascularization.

[0029] Figure 8 , Observation and statistics of the area of ​​new blood vessels in rats. DETAILED DESCRIPTION

[0030] To address the technical problems of the present invention, the amphiphilic cationic substance and mPEG-PCL employed in the present invention were used to prepare nanoparticles using a self-assembly method. In one embodiment of the present invention, DOTAP was selected as the amphiphilic cationic substance. The mPEG-PCL (methoxypolyethylene glycol-polycaprolactone) employed is amphiphilic and biocompatible, increases the drug's circulation time and bioavailability in the blood, and provides sustained release and targeted delivery, thereby increasing drug efficacy and reducing side effects. The prepared nanoparticles have a uniform particle size, stable positive charge, and low cytotoxicity.

[0031] In in vitro experiments, the inventors found that the nanoparticles have low cytotoxicity and can be used as a carrier for drug delivery. Moreover, the inventors found during the experiment that the nanoparticles can also treat vascular leakage caused by CNV without loading drugs. The inventors analyzed that the unique charge characteristics of cationic nanomaterials can adsorb substances with negative charge characteristics (protein molecules) such as inflammatory factors related to the inflammatory site, and have the effect of inhibiting the occurrence of inflammation. Furthermore, the inventors verified through rat model experiments that the nanoparticles of the present invention can significantly reduce the formation of choroidal neovascularization and the occurrence of inflammation in mice. The above experimental results also lay the foundation for the application of the nanoparticles of the present invention. They can be used to treat CNV alone, or as a carrier to load other drugs for treating CNV, further improving the treatment effect; they can also load other eye disease-related drugs to achieve combined medication and treat a variety of eye diseases.

[0032] The present invention is further described in detail below with reference to examples.

[0033] Main reagents and their sources:

[0034] DOTAP, Sigma-Aldrich, product number D6182;

[0035] mPEG-PCL, manufacturer Xi'an Ruixi Biotechnology Co., Ltd., molecular weight 4000Da, R-PL1103-4K;

[0036] PEI25K, Sigma-Aldrich, 900743.

[0037] Example 1 Preparation method of DMP nanoparticles

[0038] DMP nanoparticles were prepared by self-assembly: 5 mg of DOTAP (structural formula see Figure 1) and 45 mg mPEG-PCL (structural formula see Figure 2 ) copolymers were dissolved in dichloromethane solution and transferred to a flask for thorough mixing. Connect a rotary evaporator and place the mixed solution in a 60°C waterbath under vacuum for 45 minutes. Wait until the dichloromethane is completely evaporated, forming a transparent film on the bottom of the round-bottom flask. Add an appropriate amount of pure water to the desired concentration and gently shake in a 60°C waterbath until completely dissolved. The resulting solution is the DMP nanoparticle aqueous solution and is stored in a refrigerator at 4°C until needed.

[0039] Example 2 Study on the particle size, potential and morphology characteristics of DMP nanoparticles

[0040] Particle size and potential of DMP nanoparticles: The particle size and potential of DMP nanoparticles were measured using a Zetasizer Nano ZS Malvern Instruments (Malvern Instruments, Worcestershire, UK) at 25°C and equilibrated for 2 minutes before measurement. The results were averaged over three measurements. The average particle size of the DMP nanoparticles was 86.48 nm, and the average potential was +42.57 mV. The particle size distribution of the nanoparticles ( Figure 3 ) and potential distribution ( Figure 4 ).

[0041] The morphology of DMP nanoparticles was observed using a scanning transmission electron microscope (STEM). A portion of DMP was drawn up using a capillary tube and drained onto a copper mesh, which was then completely covered. After two minutes, the membrane was used to absorb the water. A drop of phosphotungstic acid was added to stain for 30 seconds, the staining solution was then absorbed, and the copper membrane was placed under a scanning transmission electron microscope to observe the morphology and take photos. Figure 5 As shown, under a scanning transmission electron microscope, the DMP nanoparticles are spherical particles with relatively uniform size and a diameter of about 86.48 nm.

[0042] Example 3 Cytotoxicity Detection of DMP Nanoparticles

[0043] The cytotoxicity of DMP nanoparticles was compared with that of the gold standard transfection material PEI 25K and tested by cell viability assay as follows:

[0044] (1) One day before drug addition, Muller cells in logarithmic phase were obtained and digested with trypsin to prepare cell suspension. Cell count was performed to obtain 4×10 3 The cells were seeded into 96-well plates at a density of 100 cells / well, 100 μL of cell suspension was added to each well, and the plates were placed in a 37°C, 5% CO2 constant temperature incubator for 24 hours.

[0045] (2) Using DMEM medium as the solvent, 100 μL of a series of DMP nanoparticle solutions and PEI25K solutions at different concentrations (0 μg / mL, 150 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, and 1200 μg / mL) were prepared and added to a 96-well plate, with six replicate wells for each concentration. After drug addition, the cells were incubated in a 37°C, 5% CO2 incubator for 48 hours.

[0046] (3) After incubation, add 20 μL of MTT reagent (5 mg / mL) to each well and incubate the cells in a 37°C, 5% CO2 constant temperature incubator in the dark for 4 hours. Remove the cells and observe the purple-blue crystal violet in the cells under an inverted microscope. The darker the color, the greater the cell viability.

[0047] (4) After discarding the culture medium, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, and the 96-well plate was placed on a shaker for 15 minutes. After the shaking was completed, the 96-well plate was read on a microplate reader at a wavelength of 570 nm (OD = 570).

[0048] See the results Figure 6 In Muller cells, PEI 25K is highly toxic to cells, with IC50 < 150 mg / mL; while DMP nanocarriers are much less toxic to these cells, with IC50 > 300 mg / mL.

[0049] Example 4 In vivo experiment

[0050] (1) Establishment of rat CNV model: Ten male Brown-Norway (BN) rats aged 6 to 8 weeks were purchased and housed at the Animal Experiment Center of West China Hospital, Sichuan University. The rats were intraperitoneally injected with sodium pentobarbital saline solution (2%) (30 mg / kg body weight). After general anesthesia, the rats were fully dilated using compound tropicamide eye drops (Medo-Li). The right eye of each rat was used as the model eye: a cover glass was coated with artificial tears (Xiaolaiwei, sodium carboxymethylcellulose eye drops) and then covered on the surface of the rat cornea. The optic disc and retinal blood vessels were visible under the slit lamp. A 532nm fundus laser machine (Mida, Tianjin Maida Medical Technology Co., Ltd.) was used to evenly perform laser treatment at 5 to 6 points around the optic disc, about 3 optic disc diameters away from the center of the optic disc, avoiding blood vessels. The spot size was 50 μm, the energy was 250 mW, and the treatment time was 100 ms. Brief blistering was observed at the laser site under the slit lamp, indicating that the rat Bruch's membrane was damaged, which could stimulate the formation of CNV. Only laser sites with successful modeling were included in subsequent analyses. The left eye remained untreated. After laser surgery, levofloxacin hydrochloride ophthalmic gel (Jeqi) was applied to the corneal surface of both rats to prevent infection and maintain corneal moisture. After waking up on a warming blanket, the rats were returned to the animal laboratory for continued maintenance.

[0051] (2) Random grouping: Rats were randomly divided into two groups, 5 in each group, namely the normal saline group and the DMP treatment group.

[0052] (3) Intravitreal injection of DMP in rats: One day after the CNV model was established, the rats were anesthetized with the same anesthesia method and the pupils were fully dilated with Midori. The rat ocular surface was anesthetized with oxybuprocaine hydrochloride eye drops (Benoxi). Under a stereomicroscope, a 32G microneedle was used to puncture the sclera on the nasal side of the rat's right palpebral fissure, about 1 mm away from the corneoscleral limbus, to make a pre-incision. A Hamilton microsyringe was used to draw 2 μL of saline or DMP aqueous solution (10 mg / mL), and the needle was inserted from the pre-incision toward the optic disc. The syringe needle was seen in the vitreous cavity through the pupil. The contents of the syringe were slowly pushed in, and after standing for 20 seconds, the needle was pulled out. At this time, there should be no or only a small amount of exudate in the incision.

[0053] (4) Fundus infrared photography (IR) and fluorescein angiography (FFA) image acquisition: Heidelberg high-resolution fundus optical coherence tomography (HRA-OCT) was used for image acquisition. One week after CNV modeling, rats were anesthetized and dilated as before. Both eyes were placed with Xiaolaiwei to keep the cornea moist. The rats were placed in a prone position on the operating table with the right eye facing the equipment lens. IR images were first acquired at a distance where the light spot was clear. A 10% concentration of fluorescein sodium contrast agent (Lishede) was prepared and injected intraperitoneally into the rats at a dose of 1 mL per kg body weight. The timing was started immediately after the injection. Early and late FFA images were acquired within 2 minutes and 5 to 10 minutes, respectively. The FFA image scoring criteria are as follows: 0, "no leakage", very weak fluorescence enhancement or mottled fluorescence enhancement; 1, "suspicious leakage", there is a fluorescence enhancement lesion, but the size and intensity do not increase progressively; 2, "leakage", strong fluorescence with progressively increasing intensity but unchanged size; 3, "obvious pathological leakage", strong fluorescence with progressively increasing size and intensity. After the image acquisition was completed, the rat's eyes were repaired and it was returned to the animal experimental center for maintenance after it woke up on a warm blanket.

[0054] (5) Choroidal neovascularization staining and imaging: 24 hours after the injection, the rats were killed by overdose anesthesia (intraperitoneal injection of sodium pentobarbital 200 mg / kg body weight), and the rat eyeballs were dissected. The extraocular muscles, fat and other excess connective tissues outside the sclera were stripped under a stereomicroscope. A 24G needle was used to make an incision at the limbus of the cornea and sclera. A circular incision was made along the limbus of the cornea and sclera using ophthalmic scissors. The cornea, iris and vitreous were removed, and the neural retina was carefully stripped with the help of ophthalmic forceps, leaving only the retinal pigment epithelium-choroid-sclera complex. The complex was immersed in 4% paraformaldehyde solution and fixed at room temperature for 30 minutes. After fixation, it was washed with PBS three times, each time for 5 minutes. The endothelial cell dye Isolectin B4 coupled to FITC was diluted 1:200 in PBS solution containing 0.5% Triton-X100, and the retinal pigment epithelium-choroid-sclera complex was incubated at 4°C in the dark overnight. The next day, it was washed with PBS three times, each time for 5 minutes. The tissue was transferred to a glass slide. Under a stereomicroscope, four to six radial incisions were made around the perimeter of the tissue, avoiding the spot of light. The tissue was spread flat on the slide in a petal-like pattern. Anti-fluorescence fading mounting medium was applied to the tissue and the slide was sealed with a coverslip. After it dried slightly, nail polish was applied around the coverslip to further seal the slide. Images were acquired using a Leica fully motorized fluorescence microscope, and neovascularization area was quantified using LAS X.

[0055] After laser fundus modeling in rats, DMP was injected into the vitreous cavity for treatment. During the process of CNV formation, FFA examination was performed on the rats to evaluate the neovascularization and IR examination was performed to determine the location of the laser spot. Figure 7At the location of the laser spot indicated by IR, the late FFA images of the saline control group showed significantly more fluorescein leakage than the early FFA images, while the late fluorescein leakage of the DMP treatment group was weaker than that of the saline control group. The fluorescence leakage scores of the saline control group and the DMP treatment group were 2.043 and 1.125, respectively. The DMP group had a significantly lower score than the saline group (P < 0.0001). Two weeks later, the rats were sacrificed and choroidal endothelial staining was performed on the choroidal flat mounts. The new blood vessel area of ​​the saline control group and the DMP treatment group was 169537μm, respectively. 2 、79700μm 2 The DMP treatment group was significantly lower than the normal saline group (P < 0.05). Figure 8 .

Claims

1. Use of amphiphilic cationic substance-modified mPEG-PCL nanoparticles in the preparation of drugs for treating eye diseases; the amphiphilic cationic substance is DOTAP ((2,3-dioleyloxypropyl)trimethylammonium chloride); the method for preparing the nanoparticles comprises the following steps: dissolving the amphiphilic cationic substance and mPEG-PCL in a solvent, then evaporating the solvent, adding a hydration solution to hydrate until completely dissolved, the resulting solution being a nanoparticle solution, which is dried to obtain nanoparticles; the eye disease is age-related macular degeneration characterized by choroidal neovascularization.

2. The use according to claim 1, characterized in that: The mass ratio of the amphiphilic cationic substance to mPEG-PCL is 1-30:70-99.

3. The use according to claim 2, characterized in that: The mass ratio of the amphiphilic cationic substance to mPEG-PCL is 5:

45.

4. The application according to claim 1, characterized in that: The molecular weight of the mPEG-PCL is 4000Da to 8000Da, and the molecular weight ratio of the mPEG block to the PCL block in the mPEG-PCL is 1:

1.

5. The application according to claim 1, characterized in that: The solvent is at least one of dichloromethane, chloroform, acetone, tetrachloromethane, ethanol, methanol, ether, pentane, ethyl acetate or cyclohexane.

6. The application according to claim 1, characterized in that: The hydration solution is pure water or physiological saline.

7. The use according to claim 1, characterized in that: The hydration solution is double distilled water or deionized water.

8. The application according to claim 1, characterized in that: The dosage form of the medicine is injection or drops.

9. The use according to any one of claims 1 to 8, characterized in that: The nanoparticles are also loaded with other drugs for treating eye diseases.

10. The use according to any one of claims 1 to 8, characterized in that: The nanoparticles are also loaded with other pharmaceutically acceptable auxiliary components.

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