Exosome-like nanovesicle from chrysanthemum morifolium, preparation method and application in treatment of optic nerve retina degenerative disease

Abstract

This invention discloses an exosome-like nanovesicle derived from Hangzhou white chrysanthemum, its preparation method, and its application in the treatment of optic nerve and retinal degenerative diseases. In vitro experiments show that the exosome-like nanovesicles derived from Hangzhou white chrysanthemum provided by this invention can effectively inhibit glutamate-induced oxidative stress and mitochondrial damage in retinal cells, and enhance cell viability. In vivo experiments show that intravitreal injection of these exosome-like nanovesicles derived from Hangzhou white chrysanthemum can significantly improve the survival rate of ganglion cells in the retina of mice with NMDA damage and improve their visual electrophysiological function. This invention provides a new approach and method for the treatment of optic nerve and retinal degenerative diseases, and has broad application prospects. Furthermore, the preparation method of the exosome-like nanovesicles derived from Hangzhou white chrysanthemum provided by this invention is simple and has advantages such as low cost, wide applicability, good safety, and absence of animal-derived components.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an exosome-like nanovesicle derived from Hangzhou white chrysanthemum, its preparation method, and its application in the treatment of optic nerve and retinal degenerative diseases. Background Technology

[0002] Glaucoma is a neurodegenerative disease characterized primarily by damage to retinal ganglion cells (RGCs). In China, the prevalence of glaucoma is rising annually, reaching 3.6% in people over 40 years of age, and it is the second leading cause of blindness. Glaucoma treatment focuses on two main aspects: lowering intraocular pressure to relieve mechanical compression of the optic nerve, and protecting RGC function to delay or prevent irreversible damage. Currently, a wide variety of drugs are available for lowering intraocular pressure, providing diverse options for disease intervention. However, controlling intraocular pressure alone is insufficient to prevent the progressive death of RGCs and the loss of visual function; therefore, protecting RGCs and preventing their death is of paramount importance.

[0003] Apoptosis of retinoid globulins (RGCs) is a core pathological challenge faced by various ophthalmic diseases, including glaucoma, diabetic retinopathy, and optic neuritis. Their survival status directly affects the integrity of optic nerve function and the patient's visual prognosis. Among the many mechanisms inducing RGC apoptosis, inflammation plays a key driving role: inflammation at the lesion site leads to abnormal accumulation of reactive oxygen species (ROS); ROS then synergistically interact with pro-inflammatory cytokines, amplifying local inflammatory damage to the retina and directly exacerbating the structural destruction and functional loss of RGCs, ultimately resulting in optic nerve damage. Therefore, developing innovative intervention strategies that can simultaneously inhibit inflammatory responses, clear excess ROS, and thus efficiently promote RGC survival has become a core focus in the research of ophthalmic disease pathological mechanisms and the development of therapeutic drugs, and is also a key technical challenge that urgently needs to be overcome.

[0004] Plant-derived exosome-like nanovesicles (PELNVs) are natural nanocarriers containing lipids, proteins, DNA, and miRNAs. Hangzhou white chrysanthemum (Chrysanthemummorifolium Ramat.cv. Hangbaiju) is an important cultivated variety of chrysanthemum used for both medicinal and edible purposes; research has primarily focused on the chemical composition and bioactivity of its extracts.

[0005] There are currently no reports on the use of exosome-like nanovesicles (CRELNVs) derived from Hangzhou white chrysanthemum in the treatment of optic nerve and retinal degenerative diseases. Based on the applicant's findings, this invention is proposed. Summary of the Invention

[0006] The first objective of this invention is to provide an exosome-like nanovesicle derived from chrysanthemum, the second objective is to provide a method for preparing the nanovesicle, and the third objective is to provide the medicinal uses of the nanovesicle.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] A plant-derived exosome-like nanovesicle, said exosome-like nanovesicle is derived from Hangzhou white chrysanthemum.

[0009] A method for preparing the above-mentioned exosome-like nanovesicles includes the following steps:

[0010] Step S1: Immerse the Hangzhou white chrysanthemum tissue in pre-cooled buffer solution;

[0011] Step S2: Centrifuge the soaking solution three times at differential speed and then filter;

[0012] Step S3: Centrifuge the filtrate obtained in step S2 twice at ultraspeed, collect the precipitate, and obtain the final product.

[0013] Preferably, in step S1, the Hangzhou white chrysanthemum tissue is the capitulum of Hangzhou white chrysanthemum.

[0014] Preferably, in step S2, the three differential centrifugation parameters are 3000g, 10min, 4℃; 5000g, 20min, 4℃; and 12000g, 20min, 4℃.

[0015] Preferably, in step S3, the centrifugal force of the ultracentrifugation is 130,000 g, and the time for each centrifugation is 70 min.

[0016] The above-mentioned exosome-like nanovesicles are used in the preparation of drugs for the prevention and / or treatment of optic nerve and retinal degenerative diseases.

[0017] Preferably, the optic nerve-retinal degenerative disease is a disease characterized by damage or apoptosis of retinal ganglion cells.

[0018] More preferably, the disease characterized by retinal ganglion cell damage or apoptosis is glaucoma, diabetic retinopathy, optic neuritis, or ischemic optic neuropathy.

[0019] More preferably, the above-mentioned exosome-like nanovesicles are used as the active ingredient and formulated into a pharmaceutically acceptable dosage form using a pharmaceutically acceptable carrier or excipient.

[0020] More preferably, the carrier or excipient is a solid, liquid, or semi-solid carrier or excipient; the dosage form is selected from one of injections, drops, sprays, and liposomes.

[0021] Beneficial effects:

[0022] In vitro experiments showed that the exosome-like nanovesicles (CRELNVs) derived from *Chrysanthemum indicum* prepared in this invention can effectively improve optic nerve damage in a glutamate excitotoxicity model, protect retinal ganglion cells, target and improve mitochondrial dysfunction, repair cellular energy metabolism imbalance, and effectively inhibit glutamate-induced retinal cell oxidative stress. In vivo experiments showed that intravitreal injection of CRELNVs significantly improved the survival rate of ganglion cells in the retina of mice with NMDA injury and improved their visual electrophysiological function. This invention provides a new approach and method for the treatment of optic nerve and retinal degenerative diseases and has broad application prospects. Furthermore, the CRELNVs preparation method provided by this invention is simple and has advantages such as low cost, wide applicability, good safety, and absence of animal-derived components. Attached Figure Description

[0023] Figure 1 A schematic diagram of the extraction and separation of CRELNVs;

[0024] Figure 2 The images show the identification of CRELNVs; where A is a transmission electron microscope image, B is a ZETA potential image, C is an SDS-PAGE (10%) and Coomassie brilliant blue staining image, DE is an NTA immunofluorescence image, and FH is a nanoflow cytometry image.

[0025] Figure 3 A diagram of the hemolysis test for CRELNVs;

[0026] Figure 4 The statistical results of CCK8 cells are shown in the figure; where A represents the toxicity of different concentrations of CRELNVs to R28 cells, and B and C represent the effects of different concentrations of CRELNVs on the viability of GLU-induced R28 cells.

[0027] Figure 5 Statistical results of Calcein AM / PI staining for each group of cells;

[0028] Figure 6 A graph showing the statistical results of LDH in each group of cells;

[0029] Figure 7 In the figure, A and B are statistical graphs of ROS detection in each group of cells, and C is a cell flow cytometry graph;

[0030] Figure 8 The graph shows the ATP levels in each group of cells.

[0031] Figure 9 In the diagram, A represents the morphology of mitochondria in each group of cells, B represents the number of mitochondria in each group of cells, and C represents the length of mitochondria in each group of cells.

[0032] Figure 10The image shows the uptake of CRELNVs by retinal precursor cells R28 and mouse retinal tissue; where A and B are the particle size distribution and fluorescence signal intensity of DiD-labeled CRELNVs as identified by nanoflow cytometry, and C is the uptake of CRELNVs by R28.

[0033] Figure 11 The figure shows the effect of CRELNVs on the survival rate of retinal RGCs in a mouse NMDA injury model; where A is an example of immunofluorescence staining of retinal patches in different treatment groups (Ctr, Ctr+CRELNVs, NMDA, NMDA+CRELNVs); and B is a statistical graph of the number of ganglion cells in retinal patches in different treatment groups.

[0034] Figure 12 Figure 1 shows HE staining of retinal RGCs by CRELNVs in a mouse NMDA injury model. Figure 2 shows an example of immunohistochemical staining of paraffin sections of the retina in different treatment groups (Ctr, Ctr+CRELNVs, NMDA, NMDA+CRELNVs); Figure 3 shows a statistical graph of ganglion cell survival in paraffin sections of the retina in different treatment groups of mice.

[0035] Figure 13 Figure A shows the effect of CRELNVs on the repair of visual function in a mouse NMDA injury model; Figure A is an example of the amplitude curve of the mouse FVEP electrophysiological test; Figure B is a statistical graph of the amplitude of the mouse FVEP electrophysiological test. Detailed Implementation

[0036] The following detailed description of the invention, with reference to specific embodiments, illustrates the essential content of the present invention. However, those skilled in the art should understand that the scope of protection of the invention should not be limited to these specific embodiments. Unless otherwise specified, all raw materials and reagents described herein are commercially available. Unless otherwise specified, all methods described are conventional operating procedures.

[0037] Example 1: Preparation, separation and purification of CRELNVs

[0038] The preparation process diagram is as follows: Figure 1 As shown, the specific steps are as follows:

[0039] Step 1: Weigh 50g of the flower heads of Hangzhou white chrysanthemum and soak them in 500mL of pre-cooled PBS overnight at 4℃.

[0040] Step 2: Take the soaking solution and centrifuge it using the differential centrifugation method: 3000g, 10min, 4℃ to remove large precipitates and collect the supernatant.

[0041] Step 3: Centrifuge at 5000g for 20 minutes at 4℃ to remove cells, cell debris, protoplasts and other large particulate impurities, and collect the supernatant.

[0042] Step 4: Centrifuge at 12000g for 20 minutes at 4℃ to remove plant fibers, cell debris, large vesicles and other impurities, and collect the supernatant.

[0043] Step 5: Use a 0.45μm filter to remove large vesicles larger than 0.45μm.

[0044] Step 6: Centrifuge the supernatant in an ultra-high-speed refrigerated centrifuge at 130,000 × g for 70 min at 4 °C, twice. Resuspend the precipitate in 2 mL of PBS, filter sterilize using a 0.22 μm filter membrane, store at -80 °C, and aliquot to avoid repeated freeze-thaw cycles.

[0045] The protein concentration of CRELNVs samples was determined using the BCA protein assay kit for quantification.

[0046] Example 2: Characterization Analysis of CRELNVs

[0047] Morphological identification of CRELNVs was performed using transmission electron microscopy, and the results are shown in the figure. Figure 2 As shown in Figure A, CRELNVs are shaped like round vesicles.

[0048] The surface charge distribution of CRELNVs was measured by scanning at 25℃ using a nanoparticle zeta potential analyzer, and the results are as follows: Figure 2 As shown in B.

[0049] CRELNVs stained with SDS-PAGE (10%) and Coomassie Brilliant Blue showed the following results: Figure 2 As shown in C.

[0050] The particle size distribution of CRELNVs was determined using a NanoSight NS300 nanoparticle tracking analyzer (NTA) with a laser wavelength of 405 nm and an sCMOS camera. The results are as follows: Figure 2 D and E in the middle.

[0051] The particle size distribution and concentration of CRELNVs were further analyzed using a NanoFCM nanoflow cytometer, and the results are as follows: Figure 2 F, G, H.

[0052] The results showed that the CRELNVs prepared by this invention were uniform in size, with a particle size of 50–150 nm, a Zeta potential of -10.37 ± 0.54 mV, and a particle concentration of 1.5 × 10⁻⁶. 10 The particles / mL exhibit the typical bilayer lipid membrane structure of exosomes.

[0053] Example 3: Safety Assessment of CRELNVs

[0054] To assess the biocompatibility of CRELNVs, we performed a hemolysis assay. This assay involved incubating different concentrations of CRELNVs with diluted fresh anticoagulated blood for 30 minutes, followed by centrifugation and observation of the supernatant color. Hemolytic activity was quantitatively determined by measuring the amount of hemoglobin released at 540 nm using a spectrophotometer. Results are as follows: Figure 3 As shown, the results indicate that no obvious hemolytic reaction was observed at different concentrations of CRELNVs, demonstrating its reliable blood safety.

[0055] Example 4:

[0056] 1. Cell CCK8 assay (cytotoxicity)

[0057] (1) R28 (rat retinal progenitor cells) in the logarithmic growth phase were subjected to a growth rate of 7.5 × 10⁻⁶. 3 - 1.5×10 4 Inoculate the cells at a density of cells / well into 96-well plates, add 100 μL of complete culture medium to each well, and incubate at 37°C and 5% CO2 for 24 hours to allow them to adhere to the plate.

[0058] (2) Discard the old culture medium, add culture medium containing different concentrations of CRELNVs to the experimental group, and add an equal amount of blank culture medium without CRELNVs to the control group. Set up 6 replicates for each group and continue incubation for 24 hours.

[0059] (3) After incubation, discard the old culture medium and add 100 μL of serum-free culture medium containing 10% CCK8 detection reagent to each well. Gently shake the 96-well plate to mix the reagent and culture medium thoroughly.

[0060] (4) Place the 96-well plate back into the incubator and incubate in the dark for 2 hours to allow the CCK8 reagent to fully react with the intracellular dehydrogenase.

[0061] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm and record the experimental data.

[0062] (6) Calculate the relative survival rate of cells in the experimental group based on the OD value of the control group.

[0063] The results are as follows Figure 4 As shown in Figure A, the results indicate that CRELNVs did not exhibit cytotoxicity at concentrations ranging from 0.1 to 25 ng / μL.

[0064] 2. Cell CCK8 assay (cell viability)

[0065] Experimental groups: control group (Ctr), drug control group (Ctr+CRELNVs), model group (GLU), experimental group (GLU+CRELNVs with different concentration gradients).

[0066] The steps are the same as "1. Cell CCK8 assay (cytotoxicity)", and the results are as follows: Figure 4 As shown in B and C.

[0067] The results showed that 1 ng / μL of CRELNVs exhibited the best therapeutic effect among the tested concentrations, which is the concentration of CRELNVs used in subsequent cell experiments.

[0068] Example 5: Cell PI staining

[0069] Experimental groups: control group (Ctr), drug control group (Ctr+CRELNVs), model group (GLU), and treatment group (GLU+1ng / μL CRELNVs).

[0070] (1) Take R28 cells in the logarithmic growth phase and use 5×10 5 -2.5×10 5 Inoculate each well with 1 cell per well into a 24-well plate, add 500 μL of complete culture medium, and incubate at 37°C and 5% CO2 for 24 hours to allow the cells to adhere.

[0071] (2) Discard the culture medium. Add complete culture medium containing 1 ng / μL CRELNVs to the Ctr+CRELNVs group, add complete culture medium containing 10 mmol / L GLU to the GLU+CRELNVs group, add complete culture medium containing both GLU and 1 ng / μL CRELNVs to the GLU+CRELNVs group, and add an equal amount of complete culture medium to the Ctr group. Set up 6 replicates for each group and continue incubation for 12-24 hours.

[0072] (3) After the treatment is completed, discard the culture medium and wash with PBS 3 times.

[0073] (4) Discard the PBS, add AM / PI staining reagent, and incubate at 37°C for 30 minutes.

[0074] (5) After staining, the cells were washed twice with PBS and images were taken using a fluorescence microscope to analyze the survival and death of each group of cells.

[0075] The results are as follows Figure 5 As shown in the figure. Quantitative analysis showed that the cell survival rate in the GLU+CRELNVs treatment group was significantly higher than that in the GLU model group (P* < 0.05), confirming that CRELNVs have a clear protective effect against glutamate-induced cytotoxicity.

[0076] Example 6: Cellular LDH Detection

[0077] Experimental groups: control group (Ctr), drug control group (Ctr+CRELNVs), model group (GLU), and treatment group (GLU+1ng / μL CRELNVs).

[0078] (1) R28 cells were subjected to a concentration of 7.5 × 10⁻⁶ cells. 3 - 1.5×10 4 Inoculate each well with 100 μL of culture medium and incubate at 37°C and 5% CO2 for 24 hours to allow the cells to adhere.

[0079] (2) The Ctr+CRELNVs group was given complete medium containing 1 ng / μL CRELNVs, the GLU group was given complete medium containing 10 mmol / L GLU, the GLU+CRELNVs group was given complete medium containing both GLU and 1 ng / μL CRELNVs, and the Ctr group was given an equal volume of drug-free complete medium. Each group was divided into 6 replicates, and blank control wells (only medium was added, no cells) and maximum enzyme activity control wells (the same number of cells as the Ctr group, used for lysis to release all LDH) were also set up. The cells were incubated for 12-24 hours.

[0080] (3) One hour before the end of incubation, remove the reserved "maximum enzyme activity control well" from the incubator, discard the culture medium, add 100 μL of complete culture medium containing 10% LDH release agent (prepared according to the kit instructions) as the maximum enzyme activity control group, and put it back into the incubator to continue incubation for 1 hour.

[0081] (4) After incubation, carefully aspirate the supernatant from each well and centrifuge at 400g for 5 minutes.

[0082] (5) Remove the precipitate, take the supernatant, and transfer 120 μL of the supernatant to each well of the new 96-well plate.

[0083] (6) Add 60 μL of reaction solution to each well according to the LDH detection kit instructions, gently shake to mix, and incubate in a shaker at room temperature in the dark for 30 minutes.

[0084] (7) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value of each well at a wavelength of 490 nm, and subtract the OD value of the blank control wells of the culture medium to correct the background.

[0085] The results are as follows Figure 6The results showed that, based on the LDH release assay, the LDH release in the GLU model group was significantly increased compared to the Ctr group; while the LDH release in the GLU+CRELNVs treatment group was significantly decreased compared to the GLU model group.

[0086] Example 7: Cellular ROS Detection

[0087] Experimental groups: control group (Ctr), drug control group (Ctr+CRELNVs), model group (GLU), and treatment group (GLU+1ng / μL CRELNVs).

[0088] 1. Immunofluorescence:

[0089] (1) R28 cells were fed at a rate of 4 × 10⁻⁶ 5 -3×10 5 Cells were seeded per well onto cell spreaders in 6-well plates, 2 mL of complete culture medium was added, and the cells were incubated at 37°C and 5% CO2 for 24 hours to allow them to adhere.

[0090] (2) Discard the old culture medium, add complete culture medium containing 1 ng / μL CRELNVs to the Ctr+CRELNVs group, add complete culture medium containing 10 mmol / L GLU to the GLU+CRELNVs group, add complete culture medium containing both GLU and 1 ng / μL CRELNVs to the GLU+CRELNVs group, and add an equal amount of drug-free complete culture medium to the Ctr group, and continue incubation for 12-24 hours.

[0091] (3) Discard the culture medium, add DCFH-DA fluorescent probe solution (1:2000), and incubate at 37°C in the dark for 20-30 minutes.

[0092] (4) After incubation, wash the cells three times with pre-cooled PBS buffer to remove free probes that have not entered the cells.

[0093] (5) Use a fluorescence microscope to take images and observe the green fluorescence signal.

[0094] (6) Fluorescence intensity is positively correlated with intracellular ROS level; the stronger the green fluorescence, the higher the ROS level.

[0095] ROS detection results are as follows: Figure 7 Figures A and B show that the reactive oxygen species (ROS) level in the GLU model group was significantly higher than that in the Ctr group, while the ROS level in the GLU+CRELNVs treatment group was significantly reversed, indicating that CRELNVs can effectively alleviate glutamate-induced oxidative stress.

[0096] 2. Cell flow cytometry

[0097] (1) R28 cells were fed at a rate of 4 × 10⁻⁶ 5 -3×10 5 Cells were seeded per well onto cell spreaders in 6-well plates, 2 mL of complete culture medium was added, and the cells were incubated at 37°C and 5% CO2 for 24 hours to allow them to adhere.

[0098] (2) Discard the old culture medium, add complete culture medium containing 1 ng / μL CRELNVs to the Ctr+CRELNVs group, add complete culture medium containing 10 mmol / L GLU to the GLU+CRELNVs group, add complete culture medium containing both GLU and 1 ng / μL CRELNVs to the GLU+CRELNVs group, and add an equal amount of drug-free complete culture medium to the Ctr group, and continue incubation for 12-24 hours.

[0099] (3) Discard the culture medium, gently wash the cells once with pre-cooled PBS buffer, add an appropriate amount of trypsin to digest the cells, and after the cells detach from the cell wall, add complete culture medium to stop the digestion. Gently pipette to prepare a single-cell suspension, collect it into a flow cytometry centrifuge tube, centrifuge at 1000 rpm at room temperature for 5 min, discard the supernatant, resuspend the cells in pre-cooled PBS, and adjust the cell concentration to 1×10⁻⁶. 6 per mL.

[0100] (4) Add the DCFH-DA fluorescent probe solution (1:2000) diluted with serum-free culture medium and incubate at 37°C and 5% CO2 in the dark for 20-30 minutes.

[0101] (5) After incubation, discard the probe solution and wash the cells three times with serum-free culture medium to fully remove free probes that have not entered the cells and avoid background fluorescence interference.

[0102] (6) The prepared single-cell suspension was tested by flow cytometer. The excitation wavelength was 488 nm and the emission wavelength was 525 nm. The green fluorescence signal of each group of cells was collected. Three replicates were set up for each group. The average fluorescence intensity value of each group of cells was detected and recorded.

[0103] Fluorescence intensity was positively correlated with intracellular ROS levels; stronger green fluorescence indicated higher ROS levels, as shown in Figure 7C.

[0104] Example 8: Detection of cellular ATP

[0105] Experimental groups: control group (Ctr), drug control group (Ctr+CRELNVs), model group (GLU), and treatment group (GLU+1ng / μL CRELNVs).

[0106] (1) Take 4 × 10 cells in the logarithmic growth phase. 5 -3×10 5 Cells were seeded per well in 6-well plates, 2 ml of complete culture medium was added, and the plates were incubated at 37°C and 5% CO2 for 24 hours to allow the cells to adhere.

[0107] (2) Discard the original culture medium, add complete culture medium containing 1 ng / μL CRELNVs to the Ctr+CRELNVs group, add complete culture medium containing 10 mmol / L GLU to the GLU group, add complete culture medium containing both GLU and 1 ng / μL CRELNVs to the GLU+CRELNVs group, and add an equal amount of drug-free complete culture medium to the Ctr group. Continue to incubate in a 37℃, 5% CO2 incubator for 12-24 hours.

[0108] (3) After the treatment is completed, carefully discard the culture medium in each well, and gently wash the cells three times with pre-cooled PBS buffer. Discard any residual PBS after each wash to avoid affecting subsequent detection.

[0109] (4) Add an appropriate amount of pre-prepared cell lysis buffer to each well, gently shake the 6-well plate to ensure that the lysis buffer evenly covers all cells at the bottom of the well, lyse on ice for 10-15 minutes to allow the cells to fully lyse and release ATP, scrape the cells off with a cell scraper and put them into a grinding tube to grind the cells.

[0110] (5) Centrifuge the ground cells: 12000g, 10min, 4℃.

[0111] (6) Carefully transfer the lysed cell suspension into a 96-well fluorescent microplate, with 4 replicates for each sample to ensure data reliability.

[0112] (7) Quickly mix the luciferase reagent and substrate evenly, and immediately add an equal amount of the mixed reagent to each well of the 96-well plate. Gently shake to mix, and then place it in the luciferase reader.

[0113] (8) Set the detection parameters of the microplate reader (excitation wavelength and emission wavelength refer to the kit instructions), and detect the fluorescence intensity value of each well. The results are as follows: Figure 8 .

[0114] ATP assay results showed that the relative ATP content of cells in the GLU+CRELNVs treatment group was significantly higher than that in the GLU model group, further confirming that CRELNVs have a protective effect against glutamate-induced cytotoxicity and can maintain cellular energy metabolism homeostasis.

[0115] Example 9: Cellular Mitochondrial Morphology

[0116] Experimental groups: control group (Ctr), drug control group (Ctr+CRELNVs), model group (GLU), and treatment group (GLU+1ng / μL CRELNVs).

[0117] (1) Take R28 cells in the logarithmic growth phase and use 5×10 5 -2.5×10 5 Cells were seeded per well in 24-well plates, and 500 μL of complete culture medium was added. The plates were then incubated at 37°C and 5% CO2 for 24 hours to allow the cells to adhere.

[0118] (2) Discard the original culture medium, add complete culture medium containing 1 ng / μL CRELNVs to the Ctr+CRELNVs group, add complete culture medium containing 10 mmol / L GLU to the GLU group, add complete culture medium containing both GLU and 1 ng / μL CRELNVs to the GLU+CRELNVs group, and add an equal amount of drug-free complete culture medium to the Ctr group. Continue to incubate in a 37℃, 5% CO2 incubator for 12-24 hours.

[0119] (3) After the treatment, carefully discard the culture medium in each well and gently wash the cells twice with PBS buffer preheated at 37°C to avoid damage to the mitochondrial structure due to low temperature.

[0120] (4) Dilute the mitotracker dye at a ratio of 1:2000 with serum-free medium.

[0121] (5) Take out the slide, drop the diluted Mitotracker working solution onto the slide, and incubate in a 37°C, 5% CO2 incubator for 20 minutes in the dark to ensure that the dye fully penetrates the mitochondria and binds specifically.

[0122] (6) After incubation, discard the dye working solution and wash the cells three times with preheated complete culture medium at 37°C for 5 minutes each time to completely remove unbound free dye.

[0123] (7) Cell localization was performed by staining cell nuclei with Hoechst (1:200), incubating at room temperature in the dark for 5 minutes, and then washing three times with preheated complete culture medium.

[0124] (8) The mitochondrial morphology can be recorded by taking out the smear and placing it on a glass slide, and using a laser confocal microscope to image it.

[0125] (9) The number and length of mitochondria were quantitatively analyzed using Fiji software. The relative number and length of each group were calculated based on the Ctr group to reflect the integrity and functional status of mitochondria.

[0126] The results are as follows Figure 9 Quantitative analysis showed that the mitochondria in the GLU+CRELNVs treatment group were more intact (with reduced fragmentation), confirming that CRELNVs can reduce glutamate-induced cytotoxicity by protecting mitochondrial structure and function.

[0127] Example 10: Identification of CRELNVs uptake by cells in vivo and in vitro

[0128] (1) Label CRELNVs with the lipid-soluble fluorescent dye DiD. The staining conditions are to incubate at room temperature in the dark for 15 minutes.

[0129] (2) After staining, centrifuge at 130000×g for 30 min at 4℃ using an ultracentrifuge, and resuspend and wash once with PBS buffer to remove excess unbound dye.

[0130] (3) The fluorescence signal and particle size distribution of DiD-labeled CRELNVs were analyzed and detected using a NanoFCM nanoflow cytometer. The results are as follows: Figure 10 A.

[0131] (4) The fluorescently labeled CRELNVs were co-incubated with R28 retinal precursor cells at a concentration of 10 μg / mL for 6-12 hours.

[0132] (5) After incubation, wash the cells three times with PBS to remove untaken vesicles, fix with PFA for 20-30 min, permeate with 0.5% Triton X-100 for 15 min, block in 5% bovine serum albumin (BSA) for 30 min, and then add antibody Actin-tracker green-488 (1:200) and incubate at room temperature for 30 min.

[0133] (6) After washing three times with PBS, add DAPI and incubate at room temperature for 5 minutes.

[0134] (7) After washing three times with PBS, observation under a fluorescence microscope showed that the red fluorescent signal of the vesicles was located in the cytoplasm, indicating effective uptake. The results are as follows: Figure 10 B.

[0135] (8) Inject fluorescently labeled CRELNVs into the vitreous of C57BL / 6 mice, 2 μL per eye, at a concentration of 1 μg / μL.

[0136] (9) 6-24 hours after injection, the eyeball is taken out and the retina is laid flat. After DAPI staining of the nucleus, fluorescence imaging is performed.

[0137] (10) Fluorescence images show that CRELNVs can be taken up by neuronal cells. See Figure 10 C.

[0138] Cellular and animal uptake experiments confirmed that CRELNVs have good cell permeability and tissue targeting, providing a foundation for their subsequent efficacy.

[0139] Example 11: Protective effect of CRELNVs on the survival rate of mouse retinal RGCs (retinal ganglion cells) in an NMDA injury model (N-methyl-D-aspartate injury model).

[0140] Experimental groups: Ctr, Ctr+CRELNVs, NMDA, NMDA+CRELNVs.

[0141] Animals and models: C57BL / 6J mice, 6-8 weeks old, bilateral NMDA model; all doses are "per eye", administration route is intravitreal injection (IVT).

[0142] Administration:

[0143] (1) Ctr: No damage control; IVT injection of 1.5 μL of PBS.

[0144] (2) Ctr+CRELNVs: IVT injection of CRELNVs (CRELNVs resuspended in PBS, CRELNVs concentration of 200μg / μL) total volume 1.5μL, single injection.

[0145] (3) NMDA: IVT injection of 1.5 μL of NMDA (resuspended in PBS, concentration of 20 mM), once.

[0146] (4) NMDA+CRELNVs: IVT injection of NMDA and CRELNVs (NMDA and CRELNVs resuspended in PBS, NMDA concentration of 20mM, CRELNVs concentration of 200μg / μL), total volume 1.5μL, single injection.

[0147] Three days after modeling, the mice were sacrificed and their eyeballs were harvested to observe the number of RGCs in the mouse retina.

[0148] Detection:

[0149] 1. To assess the viability of retinal ganglion cells, immunofluorescence staining of retinal smears was used to detect the number of Brn3a-positive cells.

[0150] (1) Immediately after the mice were euthanized, their eyeballs were removed and fixed in 4% paraformaldehyde (PFA) at room temperature for 2 hours.

[0151] (2) After fixation, the cornea and lens of the eyeball are removed, and then the complete retinal tissue is separated, laid flat on a glass slide, and cut into four quadrants for later use.

[0152] (3) After the retina was permeated with 0.5% Triton X-100 for 15 minutes, it was blocked in 5% bovine serum albumin (BSA) for 30 minutes, and then anti-Brn3a antibody (1:500) was added and incubated at 4°C overnight.

[0153] (4) The next day, after washing three times with PBS, add Alexa Fluor 488 labeled secondary antibody and incubate at room temperature for 1 hour, then mount the slide.

[0154] (5) To perform cell counting analysis, we used a fluorescence microscope to photograph the four quadrants of each retina. Then, we randomly selected one quadrant from each retina and selected equal-sized fields of view within the inner, middle, and outer regions of that quadrant for image acquisition and cell counting. The results are as follows: Figure 11 .

[0155] The results of the film placement test showed that ( Figure 11 In the NMDA+CRELNVs group (group A), the overall retinal structure was well preserved, the ganglion cell layer (GCL) was tightly arranged, and the cell number was significantly increased compared to the NMDA group. Immunofluorescence staining was used to detect the expression of the RGCs-specific marker Brn3a. Figure 11 (B) The results showed that the number of RGCs-positive cells in the NMDA+CRELNVs group was significantly higher than that in the NMDA group, suggesting that CRELNVs treatment can promote RGC survival.

[0156] 2. To assess changes in retinal structure, mouse eyeballs were harvested for HE staining.

[0157] (1) The eyeballs of the mice were removed immediately after the mice were euthanized and fixed in FAS fixative (FAS eye fixative) at 4°C for 24 hours.

[0158] (2) After the fixed eyeball was embedded in paraffin, it was sliced ​​along the longitudinal axis of the optic nerve with a thickness of 4 μm and stained with hematoxylin and eosin according to the standard procedure.

[0159] (3) Select sections containing the optic nerve stumps for microscopic imaging, and use standardized analysis methods to ensure consistent results.

[0160] (4) At least three non-contiguous slices were selected from each animal for image acquisition. CaseViewer software was used to count the number of cells in the GCL (ganglionic cell layer) and analyze its tissue morphology. Results are shown in […]. Figure 12 .

[0161] HE staining results further showed that the retinal layer structure of the NMDA+CRELNVs treatment group was clearer, especially the cells in the GCL region were neatly arranged and had a higher density. Compared with the NMDA model group, it had a significant tissue protection effect and could effectively reduce retinal tissue damage caused by ischemia-reperfusion injury.

[0162] Example 12: The protective effect of CRELNVs on visual function in mice in an NMDA injury model.

[0163] Experimental groups: Ctr, Ctr+CRELNVs, NMDA, NMDA+CRELNVs.

[0164] Animals and models: C57BL / 6J mice, 6-8 weeks old, bilateral NMDA model; all doses are "per eye", administration route is intravitreal injection (IVT).

[0165] Dosing and grouping (same as in Example 11):

[0166] (1) Ctr: No damage control; IVT injection of 1.5 μL of PBS.

[0167] (2) Ctr+CRELNVs: IVT injection of CRELNVs (CRELNVs resuspended in PBS, CRELNVs concentration of 200μg / μL) total volume 1.5μL, single injection.

[0168] (3) NMDA: IVT injection of 1.5 μL of NMDA (resuspended in PBS, concentration of 20 mM), once.

[0169] (4) NMDA+CRELNVs: IVT injection of NMDA and CRELNVs (NMDA and CRELNVs resuspended in PBS, NMDA concentration of 20mM, CRELNVs concentration of 200μg / μL), total volume 1.5μL, single injection.

[0170] To evaluate the impact of CRELNVs on visual function, flash visual evoked potential (FVEP) testing was performed. After anesthetizing mice, a recording electrode was inserted in the midline behind the ear (occipital region), a reference electrode was inserted subcutaneously in the nose, and a ground electrode was inserted in the tail.

[0171] In the experiment, one eye was covered, and the FVEP waveform of the other eye was recorded to obtain the amplitude change of N1-P1. Then the recording of the other eye was repeated.

[0172] All visual function-related electrophysiological data were statistically analyzed using GraphPad Prism 9.0, and comparisons between groups were performed using analysis of variance. Results are shown below. Figure 13 .

[0173] The test results showed that the VEP amplitude in the NMDA+CRELNVs treatment group was significantly higher than that in the NMDA model group, and the difference was statistically significant. These results suggest that CRELNVs treatment significantly improves the excitability of RGCs and the function of nerve signal transduction.

[0174] In summary:

[0175] In vitro experiments showed that exosome-like nanovesicles (CRELNVs) derived from Chrysanthemum morifolium could effectively improve optic nerve damage in a glutamate excitotoxicity model, protect retinal ganglion cells, improve mitochondrial dysfunction, repair cellular energy metabolism imbalance, and effectively inhibit glutamate-induced retinal cell oxidative stress.

[0176] In vivo experiments showed that intravitreal injection of CRELNVs significantly improved the survival rate of ganglion cells in the retina of mice with NMDA injury and improved their visual electrophysiological function.

[0177] This invention provides new ideas and methods for the treatment of optic nerve and retinal degenerative diseases, and has broad application prospects. Furthermore, the CRELNVs preparation method provided by this invention is simple and has advantages such as low cost, wide applicability, good safety, and absence of animal-derived components.

[0178] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. The application of plant-derived exosome-like nanovesicles in the preparation of drugs for treating glaucoma, characterized in that, The exosome-like nanovesicles were derived from the capitulum of Chrysanthemum morifolium.

2. The application according to claim 1, characterized in that, The method for preparing the exosome-like nanovesicles includes the following steps: Step S1: Immerse the Hangzhou white chrysanthemum tissue in pre-cooled buffer solution; Step S2: Centrifuge the soaking solution three times at differential speed and then filter; Step S3: Centrifuge the filtrate obtained in step S2 twice at ultraspeed, collect the precipitate, and obtain the final product.

3. The application according to claim 2, characterized in that: In step S2, the parameters for the three differential centrifugations are 3000g, 10min, 4℃; 5000g, 20min, 4℃; and 12000g, 20min, 4℃.

4. The application according to claim 2, characterized in that: In step S3, the centrifugal force of the ultracentrifugation is 130,000 g, and the time for each centrifugation is 70 min.

5. The application according to claim 1, characterized in that: The drug is formulated as a pharmaceutically acceptable dosage form using exosome-like nanovesicles as its active ingredient and pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that: The excipients are solid, liquid, or semi-solid excipients; the dosage form is selected from one of injections, drops, sprays, and liposomes.

Images