Thalidomide and magnesium acetyltaurate nano eye medicine as well as preparation method and application thereof

Abstract

The invention provides thalidomide and magnesium acetyltaurate nano eye medicine as well as a preparation method and application thereof. The preparation method comprises the following steps: S1, preparing thalidomide and magnesium acetyltaurate co-assembled nano particles; s2, preparing BHK21 cell membrane vesicles with high expression of transferrin; s3, the thalidomide and magnesium acetyltaurate co-assembled nanoparticles and the BHK21 cell membrane vesicles with high expression of transferrin are fully mixed and then subjected to ultrasonic treatment, and the thalidomide and magnesium acetyltaurate nano eye medicine is obtained. The thalidomide and magnesium acetyltaurate nano eye medicine has relatively high medicine concentration and has long-time medicine stability.

Description

Technical Field

[0001] The present invention relates to a thalidomide and magnesium acetyltaurinate nano-eye drop and its preparation method and application, belonging to the field of biomedical technology. Background Art

[0002] Abnormal angiogenesis, including retinal and choroidal neovascularization, such as retinopathy of prematurity, diabetic retinopathy, wet age-related macular degeneration, not only causes retinal and vitreous hemorrhage, retinal detachment, but in severe cases, it will damage the macula and cause blindness. Among them, retinopathy of prematurity mainly occurs in extremely low gestational age neonates. Low concentrations of oxygen provided in the uterus, which are important factors for development, will prevent the formation of normal blood vessels in the very immature retinas of extremely premature infants, leading to the formation of choroidal neovascularization. Research statistics show that about 10% of births worldwide occur in premature infants, and among them, the probability of retinopathy of prematurity is 30 - 70%. As the main drug treatment strategy, anti-VEGF still has the problems that the effect cannot last long and the traumatic risk caused by multiple intravitreal injections. Therefore, there is an urgent need for more effective alternative treatment approaches.

[0003] In view of the problems in the current treatment of retinal neovascularization, precise treatment through interdisciplinary research can bring multiple advantages and provide a safe and effective means for the treatment of diseases. Among anti-angiogenic drugs, thalidomide (Tha) has significant advantages. In addition, it can also be used as an anti-inflammatory and immunomodulatory drug, which can inhibit the activation of NF-kB and reduce the production of pro-inflammatory factors. Magnesium acetyltaurinate combines the treatment advantages of taurine and Mg 2+ It can not only break through the blood-brain barrier, effectively reach the treatment site, and effectively improve the survival rate of ganglion cells in the NMDA-induced glaucoma mouse model, showing significant neuroprotective effects. However, thalidomide has very poor water solubility, and magnesium acetyltaurinate has a short half-life, which greatly limits its further application as an eye drop. Summary of the Invention

[0004] The present invention provides a thalidomide and magnesium acetyltaurinate nano-eye drop and its preparation method and application, which can effectively solve the above problems.

[0005] The present invention is implemented as follows:

[0006] A preparation method of a thalidomide and magnesium acetyltaurinate nano-eye drop, comprising the following steps:

[0007] S1, preparing thalidomide and magnesium acetyltaurinate co-assembled nanoparticles;

[0008] S2, preparing cell membrane vesicles with high expression of transferrin;

[0009] S3. After thoroughly mixing the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate and the cell membrane vesicles highly expressing transferrin, sonicate them to obtain the thalidomide and magnesium acetyltaurinate nano-eye drops.

[0010] In some embodiments, in step S1, the steps of preparing the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate include:

[0011] S11. Dissolve thalidomide and magnesium acetyltaurinate in a first organic solvent, and then dropwise add a second organic solvent to obtain a mixed solution of thalidomide and magnesium acetyltaurinate.

[0012] S12. Spray the mixed solution of thalidomide and magnesium acetyltaurinate into supercritical carbon dioxide to nanoscale it.

[0013] S13. Then continue to rinse with supercritical carbon dioxide to obtain the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate.

[0014] The pressure of the supercritical carbon dioxide is 12 - 20 Mpa; the temperature is 37 - 50 °C.

[0015] The spraying rate of the mixed solution of thalidomide and magnesium acetyltaurinate into supercritical carbon dioxide is 1 - 5 mL / min; the time for continuous rinsing with supercritical carbon dioxide is 1.5 - 6 h.

[0016] In some embodiments, the concentration of thalidomide in the mixed solution of thalidomide and magnesium acetyltaurinate is 240 - 260 mg / mL, and the concentration of magnesium acetyltaurinate is 240 - 260 mg / mL.

[0017] In some embodiments, the first solvent is dimethyl sulfoxide; the second solvent is ethanol.

[0018] In some embodiments, the pressure of the supercritical carbon dioxide is 16 Mpa; the temperature is 45 °C.

[0019] In some embodiments, the spraying rate of the mixed solution of thalidomide and magnesium acetyltaurinate into supercritical carbon dioxide is 2 mL / min; the time for continuous rinsing with supercritical carbon dioxide is 2 h.

[0020] In some embodiments, the steps for the cell membrane vesicles highly expressing transferrin are as follows: First, introduce the plasmid highly expressing transferrin into lentivirus, and then transfect this lentivirus into BHK21 cells. High-expressing transferrin BHK21 cells can be obtained by monoclonal technology screening.

[0021] In some embodiments, the mass ratio of the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate to the cell membrane vesicles highly expressing transferrin is 1:0.8 - 1.2.

[0022] A thalidomide and magnesium acetyltaurinate nano-eye drop prepared by the above method.

[0023] Use of the above-mentioned thalidomide and magnesium acetyltaurinate nano-eye drop in the preparation of a drug for treating retinal neovascularization and choroidal neovascularization diseases.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention adopts a supercritical carbon dioxide method to prepare co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate, aiming to significantly improve the water solubility of thalidomide. By this method, the water solubility of thalidomide has been greatly improved, from the original 51.2 μg / L to 10 mg / mL. This significant improvement provides a solid foundation for the efficacy of thalidomide as an eye drop, ensuring that the drug can achieve the expected therapeutic effect when applied to the eye.

[0026] In the present invention, cell membrane vesicles with high expression of transferrin are used to load the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate. Transferrin is widely expressed in retinal tissues, including retinal vascular endothelial cells, retinal pigment epithelial cells, photoreceptor cells and ganglion cells. Therefore, the encapsulation of cell membrane vesicles is beneficial for the drug to cross the retinal barrier and enter the retinal tissue more easily, promoting the local enrichment of the drug, thereby improving the efficacy and action time of the drug. In addition, transferrin itself can regulate iron circulation and has properties such as antioxidant stress and protection of photoreceptor cells. The co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate can not only effectively inhibit the signal pathway of vascular endothelial growth factor (VEGF), but also protect photoreceptor cells and ganglion cells. This combination enables the advantages of the three components to be exerted simultaneously, thus achieving a synergistic effect during the treatment process and enhancing the therapeutic effect of the drug.

[0027] The present invention adopts a supercritical pure nano-drug preparation method to prepare co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate. This method uses supercritical carbon dioxide as a solvent, which can not only ensure the high purity of the drug, but also provide additional guarantee for the safety of the eye drop because supercritical carbon dioxide will not leave any residues 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. Description of the Drawings

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the accompanying drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0029] Figure 1 This is a picture of the thalidomide and magnesium acetyltaurinate nano-eye drops prepared in Example 1 of the present invention. Among them, (a) is a picture of the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate; (b) is a transmission electron microscope picture of the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate; (c) is a transmission electron microscope picture of the cell membrane vesicles highly expressing transferrin loaded with nano-scale thalidomide and magnesium acetyltaurinate nanoparticles.

[0030] Figure 2 This is a graph of the cell uptake results of the thalidomide and magnesium acetyltaurinate nano-eye drops in Example 3 of the present invention.

[0031] Figure 3 This is a small animal in vivo imaging picture of the thalidomide and magnesium acetyltaurinate nano-eye drops in Example 3 of the present invention and the nano-eye drops with ordinary BHK21 cell membrane as the carrier in the mouse vitreous cavity retention.

[0032] Figure 4 This is a statistical graph of the fluorescence intensity of the thalidomide and magnesium acetyltaurinate nano-eye drops in Example 3 of the present invention and the nano-eye drops with ordinary BHK21 cell membrane as the carrier in the mouse vitreous cavity retention.

[0033] Figure 5 This is the evaluation of the therapeutic effect of the thalidomide and magnesium acetyltaurinate nano-eye drops in Example 3 of the present invention in the mouse retinal neovascularization model.

[0034] Figure 6 This is a statistical graph of the evaluation of the therapeutic effect of the thalidomide and magnesium acetyltaurinate nano-eye drops in Example 3 of the present invention in the mouse retinal neovascularization model.

[0035] Figure 7 This is the evaluation of the protective effect of the thalidomide and magnesium acetyltaurinate nano-eye drops in Example 3 of the present invention on retinal ganglion cells (RGC).

[0036] Figure 8 This is a statistical graph of the evaluation of the protective effect of the thalidomide and magnesium acetyltaurinate nano-eye drops in Example 3 of the present invention on retinal ganglion cells (RGC). Detailed implementation manners

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying 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 them. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to 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 present invention to be protected, but merely represents the selected embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0038] An embodiment of the present invention provides a method for preparing thalidomide and magnesium acetyltaurinate nano-eye drops, comprising the following steps:

[0039] S1. Prepare co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate;

[0040] S2. Prepare cell membrane vesicles with high expression of transferrin;

[0041] S3. After fully mixing the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate and the cell membrane vesicles with high expression of transferrin, perform ultrasonic treatment to obtain thalidomide and magnesium acetyltaurinate nano-eye drops.

[0042] In some specific embodiments, step S1 involves the process of preparing co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate, and the specific steps are as follows:

[0043] First, in step S11, we take the measure of dissolving thalidomide and magnesium acetyltaurinate in a first organic solvent. After completing this step, we then slowly drop this solution into a second organic solvent. In this way, we successfully obtain a mixed solution containing thalidomide and magnesium acetyltaurinate. This process ensures the uniform dispersion of thalidomide and magnesium acetyltaurinate in the organic solvent, laying a good foundation for subsequent nanometerization treatment.

[0044] Immediately afterwards, in step S12, we take the measure of spraying the above-prepared mixed solution of thalidomide and magnesium acetyltaurinate into a supercritical carbon dioxide environment. Through this spraying technique, we achieve the nanometerization treatment of thalidomide and magnesium acetyltaurinate. As a unique medium, supercritical carbon dioxide 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.

[0045] Finally, in step S13, we continue to rinse the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate that have been nanoparticleized with supercritical carbon dioxide. This rinsing step helps to remove any organic solvents that may remain on the particle surface, ensuring that the finally obtained co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate have extremely high purity. Through this series of treatments, we finally obtain the desired co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate.

[0046] The core advantage of the present invention is that the chemical drug is nanoparticleized by physical means, and this process will not cause any change to the chemical properties of the drug, nor will it introduce any new impurities. At the same time, this nanoparticleization treatment significantly improves the solubility and stability of the drug, which is of extremely important significance for the absorption and efficacy of the drug. The co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate prepared by this method can not only improve the bioavailability of the drug, but also provide a more precise and efficient treatment plan for patients.

[0047] In some embodiments, the concentration of thalidomide in the thalidomide and magnesium acetyltaurinate mixed solution is 240 - 260 mg / mL, and the concentration of magnesium acetyltaurinate is 240 - 260 mg / mL.

[0048] In certain specific embodiments, the first solvent mentioned is dimethyl sulfoxide; and the second solvent is ethanol.

[0049] In these embodiments, the volume ratio of the first solvent to the second solvent can be any value between 1:50 and 1:9, which means that the volume of the first solvent can account for 2% to 10% of the total volume, while the volume of the second solvent correspondingly accounts for 90% to 98% of the total volume. This flexible adjustment of the ratio can optimize the performance of the solvent according to the specific experimental or application requirements to achieve the best dissolution effect or reaction conditions.

[0050] In some embodiments, the pressure of supercritical carbon dioxide is set in the range of 12 to 20 megapascals (MPa), and the temperature is maintained at 37 to 50 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 a specific embodiment, the pressure of supercritical carbon dioxide is precisely set to 16 megapascals, and the temperature is maintained at 45 °C. Such conditions help to ensure that the formation process of the nanoparticles is both efficient and stable.

[0051] Similarly, in some embodiments, a mixed solution of thalidomide and magnesium acetyltaurinate is sprayed into supercritical carbon dioxide at a spraying rate of 1 to 5 milliliters per minute (mL / min), and then the supercritical carbon dioxide continues to be flushed for 1.5 to 6 hours. These operating conditions are also crucial for the formation of nanoparticles because the spraying rate and flushing time directly affect the size, morphology, and distribution of the nanoparticles. If the spraying rate or flushing time does not meet these specific conditions, the nanoparticle formation process may be disrupted, thereby affecting the quality of the final product. In a specific embodiment, the spraying rate is set to 2 mL / min, and the flushing time of the supercritical carbon dioxide is 2 hours. Such operating conditions help ensure that the nanoparticle formation process is both efficient and controllable, thereby obtaining an ideal nanoparticle product.

[0052] In some specific embodiments, the process of preparing cell membrane vesicles highly expressing transferrin includes the following steps: First, plasmids containing transferrin and GFP target genes are introduced into lentiviruses, and then the lentiviruses are extracted and transfected into BHK21 cells, and BHK21 cells highly expressing transferrin are screened by monoclonal technology. Finally, cell membrane vesicles highly expressing transferrin are extracted. In these embodiments, lentiviruses can transfect BHK21 cells or 293T cells. To obtain the cell membranes of these cells, differential centrifugation can be used. In these embodiments, both the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate and the solvent used to dissolve the cell membranes are sterile PBS. By loading these nano-drugs into cell membrane vesicles highly expressing transferrin, the drugs can effectively target the endothelial cells of retinal neovascularization. Such modification not only helps the effective enrichment of the drugs but also enables the long-term retention of the drugs in the target area, thereby improving the therapeutic effect.

[0053] In certain specific embodiments, the mass ratio between the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate and the cell membrane vesicles highly expressing transferrin is 1:0.8 - 1.2. Such a ratio setting aims to ensure that the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate and the cell membrane vesicles highly expressing transferrin can effectively bind, thereby exerting the best therapeutic effect in the drug delivery system. By carefully adjusting this ratio, the drug release rate and the cell targeting ability can be optimized, thereby improving the accuracy and efficiency of treatment.

[0054] A thalidomide and magnesium acetyltaurinate nano-eye drop prepared by a specific method, which presents a colorless and transparent solution state with uniform dispersion. It is very suitable for posterior segment ocular drug delivery and does not affect the optical imaging of the eye. During the preparation of this thalidomide and magnesium acetyltaurinate nano-eye drop, through fine process control, the nano-scale size of drug particles is ensured, thereby improving the bioavailability and efficacy of the drug. It can effectively target neovascular endothelial cells and has significant therapeutic effects on various ocular diseases such as retinal neovascularization and choroidal neovascularization. Due to its unique nano-scale structure, this eye drop can stay in the posterior segment of the eye for a longer time, ensuring the continuous release of drug components, thereby reducing the frequency of drug use and improving patient compliance. In addition, the preparation method of this thalidomide and magnesium acetyltaurinate nano-eye drop also has the advantages of simple operation and low cost, making it have broad application prospects in clinical applications.

[0055] Example 1

[0056] Preparation of co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate

[0057] Weigh 250 mg of thalidomide and 250 mg of magnesium acetyltaurinate, dissolve them in 5 mL of dimethyl sulfoxide by stirring. Then add 45 mL of ethanol to the solution, mix evenly, and pump the mixed solution into the autoclave at a flow rate of 2 mL / min with a pipette pump. The conditions in the autoclave are a pressure of 16 MPa, a temperature of 45 °C, and a carbon dioxide flow rate of 2.5 mL / min. After the pumping of the mixed solution is completed, continue to pump supercritical carbon dioxide at the same carbon dioxide flow rate for 2 hours to fully rinse and dry the nanoparticles, thus obtaining co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate.

[0058] Preparation of cell membrane vesicles with high expression of transferrin

[0059] Introduce the plasmid containing transferrin and GFP target genes into lentivirus, then extract the lentivirus transfected and expressed BHK21 cells, and screen the expression cells with high expression of transferrin by monoclonal technology. Finally, extract the cell membrane vesicles with high expression of transferrin. Collect BHK21 cells with high expression of transferrin (the amino acid sequence of transferrin is shown in Appendix I), resuspend 4×10 6 cells in 1 mL of physiological saline (0.9% sodium chloride solution), under the condition of ice bath, sonicate at 40 W for 1 min, and centrifuge at 5000 rpm for 10 min in a 4 °C centrifuge to remove cell organelles. Centrifuge at 14000 rpm for 60 min in a 4 °C centrifuge to harvest the cell membrane. Resuspend the cell membrane (counted by protein) in 1 mL of physiological saline with pH = 7.4. Thus, cell membrane vesicles with high expression of transferrin are obtained.

[0060] Preparation of Thalidomide and Magnesium N-acetyltaurinate Nano-eye Drops

[0061] After an appropriate amount of cell membrane vesicles with high expression of transferrin was resuspended with physiological saline, thalidomide and magnesium N-acetyltaurinate co-assembled nanoparticles were added in a mass ratio of 1:1. After ultrasonic treatment at 30W for 5 min, centrifugation was carried out at 14,000 rpm for 10 min to obtain thalidomide and magnesium N-acetyltaurinate nano-eye drops.

[0062] Appendix I: Amino Acid Sequence of Transferrin

[0063] MRLAVGALLV CAVLGLCLAV PDKTVRWCAV SEHEATKCQS FRDHMKSVIP SDGPSVACVKKASYLDCIRAIAANEADAVT LDAGLVYDAY LAPNNLKPVV AEFYGSKEDP QTFYYAVAVV KKDSGFQMNQLRGKKSCHTG LGRSAGWNIP IGLLYCDLPE PRKPLEKAVA NFFSGSCAPC ADGTDFPQLC QLCPGCGCSTLNQYFGYSGA FKCLKDGAGD VAFVKHSTIF ENLANKADRD QYELLCLDNT RKPVDEYKDC HLAQVPSHTVVARSMGGKED LIWELLNQAQ EHFGKDKSKE FQLFSSPHGK DLLFKDSAHG FLKVPPRMDA KMYLGYEYVTAIRNLREGTC PEAPTDECKP VKWCALSHHE RLKCDEWSVN SVGKIECVSA ETTEDCIAKIMNGEADAMSLDGGFVYIAGK CGLVPVLAEN YNKSDNCEDTPEAGYFAIAV VKKSASDLTW DNLKGKKSCH TAVGRTAGWNIPMGLLYNKINHCRFDEFFS EGCAPGSKKD SSLCKLCMGS GLNLCEPNNKEGYYGYTGAF RCLVEKGDVAFVKHQTVPQN TGGKNPDPWA KNLNEKDYEL LCLDGTRKPV EEYANCHLAR APNHAVVTRKDKEACVHKILRQQQHLFGSN VTDCSGNFCL FRSETKDLLF RDDTVCLAKLHDRNTYEKYL GEEYVKAVGN LRKCSTSSLLEACTFRRP

[0064] Figure 1a shows the appearance photo of the co-assembled nanoparticles of dry thalidomide and magnesium acetyltaurinate. Figure 1 b presents the transmission electron microscopy image of the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate (MT NPs) after the preparation of Example 1. From this image, it can be clearly seen that the particle size of the nanoparticles is approximately 100 - 200 nm, and the size distribution of the particles is relatively uniform without obvious aggregation. Figure 1 c shows the transmission electron microscopy image of the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate loaded in the BHK21 cell membrane vesicles with high expression of transferrin (Tf@MT NPs) prepared in Example 1 after negative staining with phosphotungstic acid. In this image, we can observe the specially treated nanoparticles, which are wrapped by a cell membrane vesicle with high expression of transferrin. This wrapping structure helps to improve the stability and targeting of the drug, thereby potentially enhancing the efficacy of the drug. Through these images, we can intuitively understand the morphological characteristics of the thalidomide and magnesium acetyltaurinate nano-drugs and their specific performances at different preparation stages.

[0065] Comparative Example 1

[0066] The co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate were prepared according to the method of Example 1. The difference from Example 1 is that the condition in the autoclave is a pressure of 10 MPa, and other operations are the same as those in Example 1. The experimental results show that due to the too small pressure in the autoclave, nanoparticles that can be retained were not formed.

[0067] Comparative Example 2

[0068] The co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate were prepared according to the method of Example 1. The difference from Example 1 is that the condition in the autoclave is a pressure of 30 MPa, and other operations are the same as those in Example 1. The experimental results show that due to the too large pressure in the autoclave, nanoparticles that can be retained were not formed.

[0069] Comparative Example 3

[0070] The co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate were prepared according to the method of Example 1. The difference from Example 1 is that the spraying speed into supercritical carbon dioxide is 0.5 mL / min, and other operations are the same as those in Example 1. The experimental results show that due to the too small spraying speed, nanoparticles that can be retained were not formed.

[0071] Comparative Example 4

[0072] The co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate were prepared according to the method of Example 1. The difference from Example 1 was that the spraying speed into supercritical carbon dioxide was 7 mL / min, and other operations were the same as those in Example 1. The experimental results showed that due to the too high spraying speed, nanoparticles that could be retained were not formed.

[0073] Comparative Example 5

[0074] The co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate were prepared according to the method of Example 1. The difference from Example 1 was that the temperature of the supercritical carbon dioxide was 15 °C, and other operations were the same as those in Example 1. The experimental results showed that due to the too low temperature, nanoparticles that could be retained were not formed.

[0075] Comparative Example 6

[0076] The co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate were prepared according to the method of Example 1. The difference from Example 1 was that the temperature of the supercritical carbon dioxide was 55 °C, and other operations were the same as those in Example 1. The experimental results showed that due to the too high temperature, nanoparticles that could be retained were not formed.

[0077] Comparative Example 7

[0078] The co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate were prepared according to the method of Example 1. The difference from Example 1 was that the concentration of thalidomide in the mixed solution of thalidomide and magnesium acetyltaurinate was 100 mg / mL, and other operations were the same as those in Example 1. The experimental results showed that due to the too low concentration of thalidomide, nanoparticles that could be retained were not formed.

[0079] Comparative Example 8

[0080] The co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate were prepared according to the method of Example 1. The difference from Example 1 was that the concentration of thalidomide in the mixed solution of thalidomide and magnesium acetyltaurinate was 500 mg / mL, and other operations were the same as those in Example 1. Due to the too high concentration of thalidomide, nanoparticles that could be retained were not formed.

[0081] Comparative Example 9

[0082] The co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate were prepared according to the method of Example 1. The difference from Example 1 was that the concentration of magnesium acetyltaurinate in the mixed solution of thalidomide and magnesium acetyltaurinate was 100 mg / mL, and other operations were the same as those in Example 1. The experimental results showed that due to the too low concentration of magnesium acetyltaurinate, nanoparticles that could be retained were not formed.

[0083] Comparative Example 10

[0084] Prepare thalidomide and magnesium acetyltaurinate co-assembled nanoparticles according to the method of Example 1. The difference from Example 1 is that the concentration of magnesium acetyltaurinate in the thalidomide and magnesium acetyltaurinate mixed solution is 500 mg / mL, and other operations are the same as those in Example 1. The experimental results show that due to the too high concentration of magnesium acetyltaurinate, nanoparticles that can be retained were not formed.

[0085] The result comparison between Example 1 and Comparative Examples 1-6 illustrates the preparation process of the nanoparticles prepared in this example. Operation steps such as the pressure in the reaction kettle, the injection speed into supercritical carbon dioxide, and the temperature are very crucial. It is difficult to form qualified nanostructures if certain conditions are not met. The result comparison between Example 1 and Comparative Examples 7-10 illustrates that the drug concentration when preparing the nanoparticles in this example is also very crucial. Nanostructures can be formed only under suitable concentration conditions, otherwise it is difficult to form nanostructures.

[0086] Example 2

[0087] Perform a cell uptake experiment on the thalidomide and magnesium acetyltaurinate nano-eye drops (Tf@MT NPs) prepared in Example 1 of the present invention, using the thalidomide and magnesium acetyltaurinate co-assembled nanoparticles loaded on the ordinary BHK21 cell membrane (BHK21m@MT NPs) as a control. Fluorescently label these two cell membrane vesicles using a cell membrane dye. The specific steps are as follows:

[0088] (1) Seed HRMEC cells in a confocal dish, with 3×10 4 cells per well. After overnight culture with 1 mL of DMEM complete medium in each well, remove the medium and add 1 mL of the solution containing 10 μg / mL thalidomide and magnesium acetyltaurinate nano-eye drops, and continue to culture for 6 h;

[0089] (2) Aspirate the supernatant in the well plate, rinse the cells with PBS (phosphate buffer solution), stain the cell nuclei with DAPI dye, and observe and take pictures under a laser confocal microscope.

[0090] Figure 2Shows the specific results of the cell uptake experiment, where the green part represents the fluorescence signal of the cell membrane dye, and the blue part represents the DAPI fluorescence signal. In the figure, we can see four different columns of images: the first column shows the DAPI fluorescence signal, the second column shows the fluorescence signal of the cell membrane vesicles, the third column shows the fluorescence signal of the live cell membrane, and the fourth column is the image of the fusion of these three signals. In addition, the figure also contains two different rows of experimental results: the first row shows the cell uptake of thalidomide and magnesium acetyltaurinate co-assembled nanoparticles (BHK21m@MT NPs) loaded in normal BHK21 cell membrane vesicles, and the second row shows the cell uptake of thalidomide and magnesium acetyltaurinate co-assembled nanoparticles (Tf@MT NPs) loaded in cell membrane vesicles with high expression of transferrin. We can observe that there are thalidomide and magnesium acetyltaurinate fluorescence signals both inside and outside the HRMEC cells in the second row, while there are fewer in the first row. This result clearly shows that the cell membrane vesicles with high expression of transferrin significantly improve the intracellular uptake rate of the nanoparticles, thus enhancing the distribution and action of the nanoparticles inside the cells.

[0091] Figure 3 Shows the small animal in vivo imaging of the retention of BHK21m@MT NPs and Tf@MT NPs in the vitreous cavity of mice. Both cell membranes were labeled with DIR and loaded with thalidomide and magnesium acetyltaurinate co-assembled nanoparticles. Intravitreal injection was performed on the 12th day after birth (P12) in mice, and small animal in vivo imaging was performed at P12, P13, P15, P17, P19, and P21, with red-yellow fluorescence signals presented at the eyeballs. Figure 4 Is the fluorescence intensity statistical chart of the retention of two kinds of nano-eye drops, BHK21m@MT NPs / DIR and Tf@MT NPs / DIR, in the vitreous cavity at different time periods. The results show that the nano-eye drops loaded with normal BHK21m have a short retention time in the eye, and the fluorescence intensity shows a rapid decay state, with a half-life of only 1 day. While the nano-eye drops loaded with cell membrane vesicles with high expression of transferrin show a slow decay trend in the eye, and still show a relatively high fluorescence intensity at P19, and the half-life is about 6 times that of BHK21m@MT NPs / DIR. This result clearly shows that the cell membrane vesicles with high expression of transferrin significantly improve the retention time of the nanoparticles in the eye and extend the half-life of thalidomide and magnesium acetyltaurinate co-assembled nanoparticles.

[0092] Example 3

[0093] The thalidomide and magnesium acetyltaurinate (Tf@MT NPs) prepared in Example 1 of the present invention were used for the treatment of RNV, with PBS as the blank control (Ctr). At the same time, a thalidomide group (Tha), a magnesium acetyltaurinate group (MgAT), a cell membrane vesicle group with high expression of transferrin (Tf), and a co-assembled nanoparticle group of thalidomide and magnesium acetyltaurinate (MT NPs) were set as parallel controls. The specific steps are as follows:

[0094] Prepare 10 μg / mL of thalidomide and magnesium acetyltaurinate nano-eye drops, as well as cell membrane vesicles with high expression of transferrin (Tf) and co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate (MT NPs) at the same concentration. At the same time, prepare 9 μg / mL of thalidomide (Tha) and 1 μg / mL of magnesium acetyltaurinate (MgAT). Administer 2 μL per eye cavity each time. On the fifth day after administration, a follow-up study was conducted on the therapeutic effect of retinal neovascularization.

[0095] Figure 5 and Figure 6 Shows a detailed comparison of the therapeutic effects of retinal neovascularization (RNV) in an OIR mouse model (that is, neonatal mice on the seventh day after birth were respectively placed in a high-oxygen atmosphere box with an oxygen content of 75% (i.e., the high-oxygen period), and then on the twelfth day, they were placed in an environment with an oxygen content of 21%, simulating the state of hypoxia in the fetal period and high oxygen in the neonatal period after premature birth of newborns to generate an OIR model). In these figures, we can clearly see the inhibitory effects of different treatment regimens on neovascularization. Specifically, each column in the figure represents a different experimental group. The first column presents the experimental results of the control group, which did not receive any treatment and serves as the benchmark for comparison. The second column shows the treatment results of the magnesium acetyltaurinate group (MgAT), which used magnesium acetyltaurinate for treatment. The third column is the treatment effect of the thalidomide group (Tha), which received thalidomide treatment. The fourth column shows the treatment results of the cell membrane vesicle group with high expression of transferrin (Tf), which used cell membrane vesicles with high expression of transferrin for treatment. The fifth column shows the treatment results of the co-assembled nanoparticle group of thalidomide and magnesium acetyltaurinate (MT NPs), which used co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate for treatment. The sixth column shows the treatment results of the thalidomide and magnesium acetyltaurinate group (Tf@MT NPs), which used thalidomide and magnesium acetyltaurinate for treatment. Each row represents different observation indicators. The first row represents the area of retinal neovascularization after treatment in different groups, the second row represents the local map of the retinal neovascularization area, the third row represents the area of the avascular region of the retina after treatment in different groups, and the fourth row represents the local map of the avascular region of the retina.

[0096] 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, and the area of the avascular region also decreased significantly. In particular, the thalidomide and magnesium acetyltaurinate group (Tf@MT NPs) showed particularly prominent treatment effects. To further quantify these observations, the researchers precisely measured and calculated the areas of the new blood vessel region and the avascular region. Through quantitative analysis, we found that compared with the control group, after treatment with Tf@MT NPs, the area of new blood vessels decreased by 84%, and the area of the avascular region decreased by 80%; in contrast, in the MgAT group, the area of new blood vessels decreased by 48%, and the area of the avascular region decreased by 37%; in the Tha group, the area of new blood vessels decreased by 64%, and the area of the avascular region decreased by 51%; in the Tf group, the area of new blood vessels decreased by 46%, and the area of the avascular region decreased by 46%; in the MT NPs group, the area of new blood vessels decreased by 72%, and the area of the avascular region decreased by 67%.

[0097] Figure 7 and Figure 8 and

[0098] show a detailed comparison of the protective effects of different treatment regimens on retinal ganglion cells (RGCs) in the OIR mouse model. In these figures, we can clearly see the protective effects of different treatment regimens on retinal ganglion cells. Specifically, each column in the figure represents a different experimental group. The first column presents the experimental results of the control group, which did not receive any treatment and serves as a comparison benchmark. The second column shows the treatment results of the magnesium acetyltaurinate group (MgAT), which used magnesium acetyltaurinate for treatment. The third column shows the treatment effect of the thalidomide group (Tha), which received thalidomide treatment. The fourth column shows the treatment results of the cell membrane vesicles group with high expression of transferrin (Tf), which used cell membrane vesicles with high expression of transferrin for treatment. The fifth column shows the treatment results of the thalidomide and magnesium acetyltaurinate co-assembled nanoparticles group (MT NPs), which used thalidomide and magnesium acetyltaurinate co-assembled nanoparticles for treatment. The sixth column shows the treatment results of the thalidomide and magnesium acetyltaurinate group (Tf@MT NPs), which used thalidomide and magnesium acetyltaurinate for treatment.

[0098] By comparing the results of these groups, we can clearly observe the significant differences between the experimental group and the control group. The number of RGCs in the experimental group increased significantly after treatment. Especially in the thalidomide and magnesium acetyltaurinate group (Tf@MTNPs), the treatment effect was particularly prominent. To further quantify these observations, the researchers precisely counted the number of RGCs. Through quantitative analysis, we found that compared with the control group, after treatment with Tf@MT NPs, the number of RGCs was 1.8 times higher than that of the control group. In contrast, the number of RGCs in the MgAT group was 1.47 times higher than that of the control group, the number of RGCs in the Tha group was 1.39 times higher than that of the control group, the number of RGCs in the Tf group was 1.07 times higher than that of the control group, and the number of RGCs in the MTNPs group was 1.66 times higher than that of the control group.

[0099] This data not only intuitively demonstrates the protective effect of the Tf@MT NPs treatment regimen on ganglion cells, but also has good efficacy at a lower concentration (10 μg / mL), reducing the side effects of thalidomide. It also provides strong evidence for its potential in clinical applications.

[0100] The above description is only the preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of thalidomide and magnesium acetyltaurinate nano-eye drops, characterized in that, It includes the following steps: S1. Prepare the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate; S2. Prepare the cell membrane vesicles highly expressing transferrin; S3. After fully mixing the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate and the cell membrane vesicles highly expressing transferrin, perform ultrasonic treatment to obtain the thalidomide and magnesium acetyltaurinate nano-eye drops.

2. The preparation method of thalidomide and magnesium acetyltaurinate nano eye drops according to claim 1, characterized in that, In step S1, the steps for preparing the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate include: S11. Dissolve thalidomide and magnesium acetyltaurinate in the first organic solvent, and then drop into the second organic solvent to obtain a mixed solution of thalidomide and magnesium acetyltaurinate; S12. Spray the mixed solution of thalidomide and magnesium acetyltaurinate into supercritical carbon dioxide to nano-scale it; S13. Then continue to rinse with supercritical carbon dioxide to obtain the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate; The pressure of the supercritical carbon dioxide is 12 - 20 Mpa; the temperature is 37 - 50 °C; The spraying speed of spraying the mixed solution of thalidomide and magnesium acetyltaurinate into supercritical carbon dioxide is 1 - 5 mL / min; the time for continuous rinsing with supercritical carbon dioxide is 1.5 - 6 h.

3. The preparation method of the thalidomide and magnesium acetyltaurinate nano-eye drops according to claim 2, characterized in that, In the mixed solution of thalidomide and magnesium acetyltaurinate, the concentration of thalidomide is 240 - 260 mg / mL, and the concentration of magnesium acetyltaurinate is 240 - 260 mg / mL.

4. The preparation method of thalidomide and magnesium acetyltaurinate nano-eye drops according to claim 2, characterized in that, The first solvent is dimethyl sulfoxide; the second solvent is ethanol.

5. The preparation method of thalidomide and magnesium acetyltaurinate nano-eye drops according to claim 2, characterized in that, The pressure of the supercritical carbon dioxide is 16 Mpa; the temperature is 45 °C.

6. The preparation method of the thalidomide and magnesium acetyltaurinate nano-eye drops according to claim 2, characterized in that, The spraying speed of spraying the mixed solution of thalidomide and magnesium acetyltaurinate into supercritical carbon dioxide is 2 mL / min; the time for continuous rinsing with supercritical carbon dioxide is 2 h.

7. The preparation method of the thalidomide and magnesium acetyltaurinate nano-eye drops according to claim 1, characterized in that, In step S2, the preparation steps of the cell membrane vesicles highly expressing transferrin are as follows: First, introduce the plasmid containing the transferrin highly expressing gene into lentivirus, and transfect this lentivirus into BHK21 cells, and then screen the BHK21 cells highly expressing transferrin through monoclonal technology to obtain them.

8. The preparation method of thalidomide and magnesium acetyltaurinate nano-eye drops according to claim 1, characterized in that The mass ratio of the co-assembled nanoparticles of thalidomide and magnesium acetyltaurinate to the cell membrane vesicles highly expressing transferrin is 1:0.8 - 1.

2.

9. The thalidomide and magnesium acetyltaurinate nano-eye drops prepared by the method according to any one of claims 1 to 8.

10. Use of the thalidomide and magnesium acetyltaurinate nano-eye drops according to claim 9 in the preparation of a drug for treating retinal neovascularization or choroidal neovascularization diseases.

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