Olantinib nano eye medicine as well as preparation method and application thereof

Abstract

The invention provides a preparation method of olantinib nano eye medicine. The preparation method comprises the following steps: S1, preparing olantinib pure medicine nano particles; s2, preparing a cell-penetrating peptide modified mesenchymal stem cell membrane; and S3, fully mixing the olantinib pure drug nanoparticles and the cell membrane of the mesenchymal stem cell modified by the cell-penetrating peptide, and carrying out ultrasonic treatment to obtain the olantinib nano eye medicine. The nano olantinib eye drop coated with the cell-penetrating peptide cell membrane, prepared by the preparation method disclosed by the invention, has relatively high drug concentration and has long-time drug stability.

Description

Technical Field

[0001] The invention relates to an olantinib nano eye drop, a preparation method and application thereof, and belongs to the technical field of biomedicine. Background Art

[0002] Corneal neovascularization (CNV) is an eye disease. Under normal physiological conditions, the cornea is transparent and avascular and lymphatic. This avascular state is maintained by low levels of pro-angiogenic factors and high levels of anti-angiogenic factors in the corneal microenvironment. However, when the eyes are affected by diseases such as dry eye disease, inflammation, diabetes, age-related macular degeneration, bacterial and fungal infections, or trauma, the balance between these cytokines is broken, leading to the occurrence of corneal neovascularization (CNV). The new blood vessels will affect the transparency of the cornea and thus affect vision. At present, CNV has become one of the most dangerous causes of blindness. However, the current treatment of CNV includes surgical and non-surgical methods. In the process of surgical removal of CNV, new pro-angiogenic factors may be introduced, leading to the recurrence of CNV; conventional laser photocoagulation uses high-energy lasers as light sources during surgery, which may cause other complications. Non-surgical methods include non-steroidal anti-inflammatory drugs and anti-vascular endothelial growth factor drugs such as bevacizumab, ranibizumab and aflibercept, which have shown the effect of inhibiting CNV. However, the traditional drug treatment of CNV has disadvantages such as repeated injections, low corneal penetration, high drug clearance, short duration of action, and drug resistance, resulting in poor efficacy. In view of the problems currently encountered in the treatment of CNV, precise treatment through cross-disciplinary research can bring into play a variety of advantages and provide a safe and effective means for the treatment of the disease.

[0003] Among anti-angiogenic drugs, olantinib (SU6668) is a small molecule inhibitor of the tyrosine kinase activity of angiogenic receptors VEGFR2, PDGFRβ and FGFR1, and is considered to be an effective substance that antagonizes the central pathway of angiogenic signal transduction. However, its poor water solubility inhibits its further application as eye drops. Summary of the invention

[0004] The present invention provides an olantinib nano eye drop and a preparation method and application thereof, which can effectively solve the above problems.

[0005] The present invention is achieved in that:

[0006] A method for preparing olantinib nano eye drops comprises the following steps:

[0007] S1, preparation of pure olantinib nanoparticles;

[0008] S2, preparation of mesenchymal stem cell membrane modified with cell-penetrating peptide;

[0009] S3, the olantinib pure drug nanoparticles and the cell membrane of the mesenchymal stem cells modified by the cell-penetrating peptide are fully mixed and then sonicated to obtain the olantinib nano eye drops.

[0010] In step S1, the steps of preparing pure olantinib drug nanoparticles include:

[0011] S11, dissolving olantinib in a first organic solvent, and then adding dropwise a second organic solvent to obtain an olantinib mixed solution;

[0012] S12, spraying the olantinib mixed solution into supercritical carbon dioxide to nanosize it;

[0013] S13, then continue to rinse with supercritical carbon dioxide to obtain pure olantinib drug nanoparticles;

[0014] The supercritical carbon dioxide has a pressure of 10-30 MPa and a temperature of 37-55° C.

[0015] The injection speed of the olantinib mixed solution into the supercritical carbon dioxide is 1-5 mL / min; the supercritical carbon dioxide continues to flush for 1-4 hours.

[0016] In some embodiments, the concentration of olantinib in the olantinib mixed solution is 1-20 mg / mL.

[0017] In some embodiments, the first solvent is one of methanol, ethanol, and isopropanol; the second solvent is one of dichloromethane, chloroform, dimethyl sulfoxide, and acetone.

[0018] In some embodiments, the pressure of the supercritical carbon dioxide is 20 MPa and the temperature is 37°C.

[0019] In some embodiments, the injection rate of the olantinib mixed solution into the supercritical carbon dioxide is 2.5 mL / min; the supercritical carbon dioxide continues to flush for 2 hours.

[0020] In some embodiments, the step of preparing the cell membrane of mesenchymal stem cells modified with a cell-penetrating peptide is: incubating the cell membrane of the mesenchymal stem cells with a cell-penetrating peptide having an active ester at the C-terminus at 3-5° C. for 10-14 hours to obtain the cell membrane.

[0021] In some embodiments, the mass ratio of the pure olantinib nanoparticles to the cell-penetrating peptide-modified mesenchymal stem cell membrane is 2-1:1-3.

[0022] In some embodiments, the sequence of the cell-penetrating peptide is one of GRKKRRQRRRPPQQ, GKKALKLAAKLLKK, and RRRRRRRRR.

[0023] An olantinib nano eye drop prepared by the above method.

[0024] A use of the above-mentioned olantinib nano eye drops in the preparation of drugs for treating corneal neovascularization, dry eye disease and keratitis.

[0025] The beneficial effects of the present invention are:

[0026] The present invention uses a supercritical carbon dioxide method to prepare olantinib nanoparticles, aiming to significantly improve the water solubility of the drug olantinib. Through this method, the water solubility of olantinib is greatly improved from the original 31 μg / ml to 10 mg / ml. This significant improvement provides a solid foundation for the efficacy of olantinib as an eye drop, ensuring that the drug can achieve the expected therapeutic effect when used in the eye.

[0027] In the present invention, mesenchymal stem cell membranes are used to wrap nanoscale olantinib drug particles. The mesenchymal stem cell membrane itself has natural anti-inflammatory properties, while nanosized olantinib can effectively inhibit the signaling pathway of vascular endothelial growth factor (VEGF). This combination allows the advantages of the two materials to be brought into play at the same time, thereby achieving a synergistic effect during the treatment process and enhancing the therapeutic effect of the drug.

[0028] The present invention also utilizes the cell membrane-penetrating peptide molecules, which can help drugs pass through the barrier structure of the corneal epithelium. In this way, the drugs can more easily enter the eye tissue, promote the local enrichment of the drugs, and thus improve the efficacy and duration of action of the drugs.

[0029] The present invention adopts a supercritical pure nano drug preparation method to prepare pure drug nanoparticles of olantinib. This method uses supercritical carbon dioxide as a solvent, which can not only ensure the high purity of the drug, but also provide additional protection for the safety of eye drops because supercritical carbon dioxide does not leave any residue during the operation process. This preparation method not only improves the quality of the drug, but also ensures that patients will not be affected by potentially harmful residues during use. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1The pictures of the olantinib nano eye drops prepared in Example 1 of the present invention include: (a) pictures of olantinib and olantinib nanoparticles after dissolution; (b) transmission electron microscopy pictures of olantinib nanoparticles; and (c) transmission electron microscopy pictures of nano olantinib wrapped by cell membrane-penetrating peptides.

[0032] Figure 2 This is a graph showing the cellular uptake results of nano-olantinib encapsulated by the cell membrane of the cell-penetrating peptide of Example 2 of the present invention.

[0033] Figure 3 This is a fluorescence quantitative graph of cell uptake in Example 2 of the present invention.

[0034] Figure 4 This is an evaluation of the therapeutic effect of the cell membrane-penetrating peptide nano-olantinib eye drops in Example 3 of the present invention in the rat corneal neovascularization model.

[0035] Figure 5 These are the neovascularization scores of each group before and after treatment with normal saline, MSCm, NanoSU6668, MNS, and T-MNS in Example 3 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] A method for preparing olantinib nano eye drops comprises the following steps:

[0038] S1, preparation of pure olantinib nanoparticles;

[0039] S2, preparation of mesenchymal stem cell membrane modified with cell-penetrating peptide;

[0040] S3, the olantinib pure drug nanoparticles and the cell membrane of the mesenchymal stem cells modified by the cell-penetrating peptide are fully mixed and then sonicated to obtain the olantinib nano eye drops.

[0041] In some specific embodiments, step S1 involves a process for preparing pure olantinib drug nanoparticles, and the specific steps are as follows:

[0042] First, in step S11, we took measures to dissolve olantinib in a first organic solvent. After completing this step, we then slowly dripped this solution into a second organic solvent, and in this way, we successfully obtained a mixed solution containing olantinib. This process ensures the uniform dispersion of olantinib in the organic solvent, laying a good foundation for the subsequent nano-processing.

[0043] Next, in step S12, we took measures to spray the prepared olantinib mixed solution into a supercritical carbon dioxide environment. Through this spraying technology, we achieved the nano-processing of olantinib. Supercritical carbon dioxide, as a unique medium, can not only effectively promote the refinement of drug particles, but also maintain the original chemical properties of the drug without introducing any chemical changes.

[0044] Finally, in step S13, we continue to use supercritical carbon dioxide to rinse the nanosized olantinib particles. This rinsing step helps to remove any organic solvent that may remain on the surface of the particles, ensuring that the final olantinib pure drug nanoparticles have extremely high purity. Through this series of treatments, we finally obtained the desired olantinib pure drug nanoparticles.

[0045] The core advantage of the present invention is that chemical drugs are nano-processed by physical means, and this process does not cause any changes to the chemical properties of the drugs, nor does it introduce any new impurities. At the same time, this nano-processing significantly improves the solubility and stability of the drug, which is of great significance for the absorption and efficacy of the drug. The pure drug nanoparticles of olantinib prepared by this method can not only improve the bioavailability of the drug, but also provide patients with more accurate and efficient treatment options.

[0046] In some embodiments, the concentration of olantinib in the olantinib mixed solution is 1-20 mg / mL.

[0047] In some specific embodiments, the first solvent may be any one of methanol, ethanol or isopropanol; and the second solvent may be any one of dichloromethane, chloroform, dimethyl sulfoxide or acetone.

[0048] In these embodiments, the volume ratio of the first solvent to the second solvent can be any value between 1:9 and 9:1, which means that the volume of the first solvent can account for 10% to 90% of the total volume, and the volume of the second solvent can account for 90% to 10% of the total volume accordingly. This flexible adjustment of the ratio can optimize the performance of the solvent according to the requirements of a specific experiment or application to achieve the best dissolution effect or reaction conditions.

[0049] In certain embodiments, the pressure of supercritical carbon dioxide is set in the range of 10 to 30 megapascals (MPa), while the temperature is maintained at 37 to 55 degrees Celsius (°C). These specific conditions are crucial for the formation of nanoparticles, because only under such pressure and temperature conditions can the desired nanoparticles be successfully prepared. If the pressure or temperature exceeds this range, the formation process of the nanoparticles will not proceed smoothly. In certain embodiments, the pressure of supercritical carbon dioxide is precisely set to 20 MPa, while the temperature is maintained at 37°C, which helps to ensure that the formation process of the nanoparticles is both efficient and stable.

[0050] Similarly, in certain embodiments, the mixed solution of olantinib is sprayed into supercritical carbon dioxide at a spray rate of 1 to 5 milliliters per minute (mL / min), and the supercritical carbon dioxide is then continued to be flushed for 1 to 4 hours. These operating conditions are also crucial for the formation of nanoparticles, because the spray rate and flushing time directly affect the size, morphology and distribution of the nanoparticles. If the spray rate or flushing time does not meet these specific conditions, the formation process of the nanoparticles may be disturbed, thereby affecting the quality of the final product. In a specific embodiment, the spray rate is set to 2.5 milliliters per minute, and the supercritical carbon dioxide flushing time is 2 hours. Such operating conditions help to ensure that the formation process of the nanoparticles is both efficient and controllable, thereby obtaining an ideal nanoparticle product.

[0051] In some specific embodiments, the process of preparing a mesenchymal stem cell membrane modified with a cell-penetrating peptide comprises the following steps: first, the mesenchymal stem cell membrane is co-incubated with a cell-penetrating peptide having an active ester group at the C-terminus at a temperature of 3-5°C for about 10-14 hours. In this way, a cell-penetrating peptide-modified mesenchymal stem cell membrane can be successfully obtained. In these embodiments, the mesenchymal stem cell can be one of a bone marrow-derived mesenchymal stem cell, an adipose-derived mesenchymal stem cell, or an umbilical cord-derived mesenchymal stem cell. In order to obtain the cell membrane of these stem cells, differential centrifugation can be used. As for the cell-penetrating peptide, it can be one of the following three: GRKKRRQRRRPPQQ (TAT, SEQ ID NO: 1), GKKALKLAAKLLKK (SEQ ID NO: 2), RRRRRRRRR (SEQ ID NO: 3). In these embodiments, the pure drug nanoparticles of olantinib and the solvent used to encapsulate the cell membrane are sterile saline. By encapsulating these nano-drugs in cell membranes modified with cell-penetrating peptides, the drugs can effectively penetrate the corneal barrier and reach the lesion area. Such modification not only helps to effectively enrich the drugs, but also enables the drugs to stay in the target area for a long time, thereby improving the therapeutic effect.

[0052] In certain specific embodiments, the mass ratio between the pure olantinib nanoparticles and the cell membrane of the mesenchymal stem cells modified with the cell-penetrating peptide can be set within the range of 2-1:1-3. For example, this mass ratio can be 2:1, 1:1, 1:2, or 1:3. This ratio setting is intended to ensure that the pure olantinib nanoparticles and the cell membrane-modified mesenchymal stem cells can be effectively combined, thereby achieving the best therapeutic effect in the drug delivery system. By carefully adjusting this ratio, the drug release rate and cell targeting ability can be optimized, thereby improving the accuracy and efficiency of the treatment.

[0053] An olantinib nano eye drop prepared by a specific method, which is in the form of a uniformly dispersed yellow solution and is very suitable for use as eye drops for the treatment of various eye diseases. During the preparation process of this olantinib nano eye drop, the nanoscale size of the drug particles is ensured through fine process control, thereby improving the bioavailability and efficacy of the drug. It can effectively penetrate into the ocular tissue and has a significant therapeutic effect on a variety of eye diseases such as corneal neovascularization, dry eye disease, and keratitis. Due to its unique nanoscale structure, this eye drop can stay in the eye for a longer time, ensuring that the drug ingredients can be continuously released, thereby reducing the frequency of medication and improving patient compliance. In addition, the preparation method of this olantinib nano eye drop also has the advantages of simple operation and low cost, which makes it have broad application prospects in clinical applications.

[0054] Example 1

[0055] Preparation of pure olantinib nanoparticles

[0056] Weigh 200 mg of olantinib and stir to dissolve in 14 mL of anhydrous ethanol, add 6 mL of dimethyl sulfoxide to the solution, mix well, and pump the mixed solution into the autoclave at a flow rate of 1 mL / min using a suction pump. The conditions in the autoclave are pressure 20 MPa, temperature 37 ° C, and carbon dioxide flow rate of 2.5 ml / min. After the mixed solution is pumped in, continue to pump supercritical carbon dioxide at the same carbon dioxide flow rate for 2 hours to fully rinse and dry the nanoparticles, and obtain olantinib pure drug nanoparticles NanoSU6668.

[0057] Preparation of Mesenchymal Stem Cell Membranes

[0058] Mesenchymal stem cells were collected and 5×10 6 The cells were resuspended in 1 mL of saline (0.9% sodium chloride solution), ultrasonicated at 40W for 1 min under ice bath conditions, and centrifuged at 5000 rpm for 10 min in a 4 ° C centrifuge to remove organelles. The cell membrane was harvested by centrifugation at 15000 rpm for 60 min in a 4 ° C centrifuge. The cell membrane was labeled with cell-penetrating peptide active ester (TAT-NHS). 1 mg of cell membrane (in terms of protein) was resuspended in 1 ml of saline with pH = 7.4, and then 0.15 mg of TAT-NHS was added, mixed and kept overnight at 4 ° C. Centrifuged at 15000 rpm for 60 min in a 4 ° C centrifuge, the cell membrane was collected, and resuspended with saline. Repeat three times to remove excess TAT-NHS. Thus, a mesenchymal cell membrane labeled with a cell-penetrating peptide was obtained. The sequence of TAT is GRKKRRQRRRPPQQ (SEQ ID NO: 1).

[0059] Preparation of mesenchymal stem cell membrane-encapsulated nano-olantinib eye drops

[0060] Take an appropriate amount of mesenchymal stem cell membrane and resuspend it with physiological saline, then add pure olantinib nanoparticles at a mass ratio of 1:1, ultrasonicate at 40W for 5 minutes, and centrifuge at 12000rpm for 5 minutes to obtain nano-olantinib eye drops wrapped with the membrane-penetrating peptide mesenchymal stem cell membrane.

[0061] Figure 1 a shows the appearance of a 1 mg / mL olantinib solution (SU6668) and a pure olantinib nanoparticle (NanoSU6668) at the same concentration. From the figure, two forms of olantinib can be clearly observed, one is the drug form dissolved in the solution, and the other is the drug form in the form of nanoparticles. Figure 1 b shows a projection electron microscope image of the pure olantinib nanoparticles (NanoSU6668) after preparation in Example 1. From this image, it can be seen in detail that the particle size of the nanoparticles is about 100 nanometers, and the size distribution of the particles is relatively uniform, without obvious aggregation. Figure 1 Figure c shows a projection electron microscope image of the nano-olantinib nanoparticles (T-MNS) coated with the membrane-penetrating peptide mesenchymal stem cell prepared in Example 1 after phosphotungstic acid counterstaining. In this image, we can observe the specially treated nanoparticles, which are wrapped by a layer of membrane-penetrating peptide mesenchymal stem cell membrane. This wrapping structure helps to improve the stability and targeting of the drug, thereby possibly enhancing the efficacy of the drug. Through these images, we can intuitively understand the morphological characteristics of olantinib nanoparticles and their specific performance at different preparation stages.

[0062] According to the solubility test, in water at 25°C, the solubility of olantinib (SU6668) was 31 μg / ml, and the solubility of olantinib pure drug nanoparticles (NanoSU6668) was 10 mg / ml.

[0063] Comparative Example 1

[0064] Olantinib pure drug nanoparticles were prepared according to the method of Example 1, except that the pressure in the autoclave was 5 MPa, and other operations were the same as in Example 1. The experimental results showed that nanoparticles that could be trapped were not formed because the pressure in the autoclave was too low.

[0065] Comparative Example 2

[0066] Olantinib pure drug nanoparticles were prepared according to the method of Example 1, except that the pressure in the autoclave was 40 MPa, and other operations were the same as in Example 1. The experimental results showed that due to the high pressure in the autoclave, nanoparticles that could be trapped could not be formed.

[0067] Comparative Example 3

[0068] Olantinib pure drug nanoparticles were prepared according to the method of Example 1, except that the injection speed into supercritical carbon dioxide was 0.5 mL / min, and other operations were the same as Example 1. The experimental results showed that nanoparticles that could be trapped were not formed because the injection speed was too low.

[0069] Comparative Example 4

[0070] Olantinib pure drug nanoparticles were prepared according to the method of Example 1, except that the injection speed into the supercritical carbon dioxide was 10 ml / min, and other operations were the same as in Example 1. The experimental results showed that due to the too high injection speed, nanoparticles that could be trapped could not be formed.

[0071] Comparative Example 5

[0072] Olantinib pure drug nanoparticles were prepared according to the method of Example 1, except that the temperature of the supercritical carbon dioxide was 20° C., and other operations were the same as in Example 1. The experimental results showed that due to the low temperature, nanoparticles that could be trapped were not formed.

[0073] Comparative Example 6

[0074] Olantinib pure drug nanoparticles were prepared according to the method of Example 1, except that the temperature of the supercritical carbon dioxide was 60° C., and other operations were the same as those of Example 1. The experimental results showed that due to the high temperature, nanoparticles that could be trapped could not be formed.

[0075] Comparative Example 7

[0076] The olantinib pure drug nanoparticles were prepared according to the method of Example 1, except that the concentration of olantinib in the olantinib mixed solution was 30 mg / mL, and other operations were the same as Example 1. The experimental results showed that due to the high concentration of olantinib, nanoparticles that could be trapped could not be formed.

[0077] Comparative Example 8

[0078] The olantinib pure drug nanoparticles were prepared according to the method of Example 1, except that the olantinib concentration in the olantinib mixed solution was 0.5 mg / mL, and other operations were the same as Example 1. Since the olantinib concentration was too low, nanoparticles that could be trapped were not formed.

[0079] The comparison of the results of Example 1 and Comparative Examples 1-6 illustrates that the preparation process of the pure drug nanoparticles prepared in this embodiment, such as the pressure of the reactor, the injection speed into the supercritical carbon dioxide, the temperature and other operating steps are very critical. If certain conditions are not met, it is difficult to form a qualified nanostructure. The comparison of the results of Example 1 and Comparative Examples 7-8 illustrates that the drug concentration when preparing the pure drug nanoparticles in this embodiment is also very critical. Only under the appropriate concentration conditions can the nanostructure be formed, otherwise it is difficult to form a nanostructure.

[0080] Example 2

[0081] The cell uptake experiment was performed on the cell-penetrating peptide mesenchymal stem cell membrane-encapsulated nano-olantinib (T-MNS) prepared in Example 1 of the present invention, and the olantinib nanoparticles (NanoSU6668) prepared in Example 1 and the mesenchymal stem cell membrane-encapsulated nano-olantinib (MNS) were used as controls. Indocyanine green (ICG) was used for fluorescent labeling of the nanoparticles, and the specific steps were as follows:

[0082] (1) HUVEC cells were inoculated into three groups of 24-well cell culture plates, with 5×10 cells per well. 4 After adding 500 μL of ECM medium to each well of the culture medium for overnight culture, the culture medium was removed and 500 μL of different drugs containing 20 μg / ml olantinib nanoparticles were added to the experimental groups respectively, and the culture was continued for 6 h;

[0083] (2) The supernatant in the well plate was aspirated, the cells were washed with PBS (phosphate buffered saline), the cell nuclei were stained with DAPI dye, and the cells were observed and photographed under a laser confocal microscope.

[0084] Figure 2 The specific results of the cell uptake experiment are shown, where the red part represents the ICG fluorescence signal and the blue part represents the DAPI fluorescence signal. In the figure, we can see three different columns of images: the first column shows the DAPI fluorescence signal, the second column shows the ICG fluorescence signal, and the third column is an image that merges these two signals. In addition, the figure also contains three rows of different experimental results: the first row shows the uptake of olantinib nanoparticles (NanoSU6668) by cells, the second row shows the uptake of nano-olantinib (MNS) wrapped in mesenchymal stem cell membrane by cells, and the third row shows the uptake of nano-olantinib (T-MNS) wrapped in transmembrane peptide mesenchymal stem cell membrane by cells. Figure 3 Yes Figure 2 From the statistical results, we can observe that the ICG fluorescence signal in the third row is significantly stronger than that in the control group. By quantitatively analyzing these fluorescence signals, we have made an important discovery: the fluorescence intensity of the nano-olantinib (T-MNS) coated with the membrane of transmembrane peptide mesenchymal stem cells is 6.3 times that of the simple olantinib nanoparticles (NanoSU6668), and it is also 3 times that of the nano-olantinib (MNS) coated with the membrane of mesenchymal stem cells. This result clearly shows that the labeling of transmembrane peptides significantly increases the intracellular uptake rate of nanoparticles, thereby enhancing the distribution and effect of nanoparticles in cells.

[0085] Example 3

[0086] The cell-penetrating peptide mesenchymal stem cell membrane-encapsulated nano-olantinib (T-MNS) prepared in Example 1 of the present invention was used to treat rat CNV, and normal saline (0.9% NaCl) was used as a blank control (control), and a mesenchymal stem cell membrane group (MSCm), an olantinib nanoparticle group (NanoSU6668), and a mesenchymal stem cell membrane-encapsulated nano-olantinib group (MNS) were set as parallel controls. The specific steps are as follows:

[0087] Prepare 310μg / mL of cell-penetrating peptide mesenchymal stem cell membrane-coated nano-olantinib eye drops (1mM in terms of olantinib) and the same concentration of mesenchymal stem cell membrane (MSCm), olantinib nanoparticles (NanoSU6668), and mesenchymal stem cell membrane-coated nano-olantinib (MNS). 10μL / eye was administered each time, once every 12 hours, for 4 consecutive days, and the therapeutic effect of corneal neovascularization was tracked.

[0088] Figure 4 and Figure 5 A detailed comparison of the effects of choroidal neovascularization (CNV) treatment in rats is shown. In these figures, we can clearly see the effects of different treatment regimens on neovascularization. Specifically, each row in the figure represents a different experimental group. The first row presents the experimental results of the control group, which did not receive any treatment and served as a baseline for comparison. The second row shows the treatment effects of the mesenchymal stem cell membrane group (MSCm), which was treated with mesenchymal stem cell membranes. The third row shows the treatment results of the olantinib nanoparticle group (NanoSU6668), which was treated with olantinib nanoparticles. The last row, the fourth row, shows the treatment effects of the mesenchymal stem cell membrane-wrapped nano-olantinib group (MNS), which combines the treatment regimen of mesenchymal stem cell membranes and olantinib nanoparticles.

[0089] By comparing the results of these groups, we can clearly observe significant differences between the experimental group and the control group. After treatment, the growth of new blood vessels in the experimental group was effectively inhibited and improved. In particular, the nano-olantinib group (MNS) wrapped with mesenchymal stem cell membranes had a particularly outstanding therapeutic effect. In order to further quantify these observations, the researchers accurately measured and calculated the density of new blood vessels. Through quantitative analysis, we found that compared with the control group, the number of new blood vessels decreased by 80% after T-MNS treatment. In contrast, the MSCm group had basically no change, the NanoSU6668 group decreased by 30%, and the MNS group decreased by 60%. This data not only intuitively demonstrates the high efficiency of the T-MNS treatment regimen, but also has good efficacy at extremely low concentrations (1mM in terms of olantinib), reducing the concentration of olantinib and reducing the side effects of olantinib. It also provides strong evidence for its potential in clinical applications.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing olantinib nano eye drops, characterized in that: The following steps are involved: S1, preparation of pure olantinib nanoparticles; S2, preparation of mesenchymal stem cell membrane modified with cell-penetrating peptide; S3, fully mixing the olantinib pure drug nanoparticles and the cell membrane of the mesenchymal stem cells modified by the cell-penetrating peptide, and then sonicating to obtain the olantinib nano eye drops; In step S1, the steps of preparing pure olantinib drug nanoparticles include: S11, dissolving olantinib in a first organic solvent, and then adding dropwise a second organic solvent to obtain an olantinib mixed solution; S12, spraying the olantinib mixed solution into supercritical carbon dioxide to nanosize it; S13, then continue to wash with supercritical carbon dioxide to obtain pure olantinib drug nanoparticles; The supercritical carbon dioxide has a pressure of 10-30 MPa and a temperature of 37-55° C. The injection speed of the olantinib mixed solution into the supercritical carbon dioxide is 1-5 mL / min; the supercritical carbon dioxide continues to flush for 1-4 hours.

2. The method for preparing the olantinib nano eye drops according to claim 1, characterized in that: The concentration of olantinib in the olantinib mixed solution is 1-20 mg / mL.

3. The method for preparing the olantinib nano eye drops according to claim 1, characterized in that: The first solvent is one of methanol, ethanol and isopropanol; the second solvent is one of dichloromethane, chloroform, dimethyl sulfoxide and acetone.

4. The method for preparing the olantinib nano eye drops according to claim 1, characterized in that: The pressure of the supercritical carbon dioxide is 20 MPa and the temperature is 37°C.

5. The method for preparing the olantinib nano eye drops according to claim 2, characterized in that: The injection speed of the olantinib mixed solution into the supercritical carbon dioxide is 2.5 mL / min; the supercritical carbon dioxide continues to flush for 2 hours.

6. The method for preparing the olantinib nano eye drops according to claim 1, characterized in that: In step S2, the step of preparing the cell membrane of mesenchymal stem cells modified with the cell-penetrating peptide is as follows: the cell membrane of the mesenchymal stem cells is co-incubated with the cell-penetrating peptide having an active ester at the C-terminus at 3-5° C. for 10-14 hours.

7. The method for preparing the olantinib nano eye drops according to claim 1, characterized in that: The mass ratio of the pure olantinib nanoparticles to the mesenchymal stem cell membrane modified by the cell-penetrating peptide is 2-1:1-3.

8. The method for preparing olantinib nano eye drops according to claim 1, characterized in that: The sequence of the cell-penetrating peptide is one of GRKKRRQRRRPPQQ, GKKALKLAAKLLKK, and RRRRRRRRR.

9. An olantinib nano eye drop prepared by the method according to any one of claims 1 to 8.

10. Use of the olantinib nano eye drops according to claim 9 in the preparation of drugs for treating corneal neovascularization, dry eye disease and keratitis.

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