Targeted retina drug-loading extracellular vesicle as well as preparation method and application thereof

Abstract

The invention discloses a targeted retina drug-loaded extracellular vesicle as well as a preparation method and application thereof. The targeted retina drug-loaded extracellular vesicle comprises a lactobacillus-derived extracellular vesicle and a retina ischemia-reperfusion injury related disease treatment drug loaded in the extracellular vesicle, the medicine for treating the diseases related to the retinal ischemia reperfusion injury is indolepropionic acid; the loading capacity of the targeted retina drug-loading extracellular vesicle to indolepropionic acid is (6-8) * 10 <-8 > microgram / particle. The drug-loaded extracellular vesicle has the advantages of high drug loading rate, good biocompatibility, good gastrointestinal tract stability, high bioavailability and good safety. Besides, the extracellular vesicles loaded with indolepropionic acid can carry related marker proteins of retinal neurons through a membrane fusion technology, and can reach an eye retina area more efficiently through peripheral vein administration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a targeted retinal drug-loaded extracellular vesicle, its preparation method, and its application. Background Technology

[0002] Retinal ischemia-reperfusion injury (RIRI) is a major cause of blindness, with typical examples including diabetic retinopathy, retinal vein occlusion, and especially acute ocular hypertension in glaucoma. A self-reinforcing cascade of destructive events is a key characteristic of RIRI, including mitochondrial dysfunction, energy metabolism failure, calcium overload, oxidative stress, inflammatory responses, and ultimately, neuronal death. However, current treatments primarily focus on symptom control; for example, glaucoma treatment mainly involves lowering intraocular pressure through medication or surgery, but disease progression remains common. Treating RIRI remains a significant challenge, particularly due to the lack of effective neuroprotective agents. Protecting or reducing damage to or loss of retinal ganglion cells is crucial in the treatment of RIRI, as it is a core pathological mechanism leading to blindness in RIRI-related diseases.

[0003] Extracellular vesicles (LrEVs) are a class of nanoscale lipid bilayer vesicles that can be released by almost all types of cells, carrying important biomolecules such as nucleic acids, proteins, and lipids. Compared with extracellular vesicles derived from animals or plants, bacterial-derived extracellular vesicles have advantages such as low production cost and suitability for large-scale production. Their low toxicity, high drug loading capacity, ease of modification, and industrialization also make bacterial extracellular vesicles ideal lipid-layer nanocarriers for treating various diseases. Furthermore, compared with ordinary bacterial-derived extracellular vesicles, probiotic-derived extracellular vesicles exhibit higher biocompatibility, stronger immunomodulatory and antioxidant capabilities, better intestinal barrier protection, and higher targeting and biocompatibility. Existing technologies have reported some applications of probiotic-derived extracellular vesicles. For example, Chinese patent document CN118956679A discloses a method for preparing a probiotic extracellular vesicle mimic and its application in the preparation of anti-inflammatory drugs. For example, Chinese patent document CN118853471A discloses that compound probiotic vesicles loaded with luteolin improve the stability and bioavailability of luteolin, and have strong antioxidant, anti-inflammatory, intestinal flora regulating and intestinal barrier repair effects, and have a synergistic effect in the prevention and treatment of ulcerative colitis.

[0004] Although the therapeutic potential of probiotics and their derivatives, including extracellular vesicles and metabolites, in various diseases has been widely reported, their application in the treatment of RIRI-related diseases remains in its early stages and has not been fully explored. In particular, the application of Lactobacillus-derived extracellular vesicles and indolepropionic acid in RIRI-related diseases has not been reported, and the construction of retinal-targeted drug delivery systems remains challenging. Currently, treatments for RIRI-related diseases have significant limitations in terms of optic nerve protection, disease progression control, and patient compliance. Therefore, developing novel therapeutic strategies based on probiotics and their derivatives, especially interventions targeting optic nerve protection, anti-inflammation, and anti-oxidative stress, is of great significance in addressing the shortcomings of existing treatments. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a targeted retinal drug-loaded extracellular vesicle, its preparation method and application.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: A targeted retinal drug-loaded extracellular vesicle comprises extracellular vesicles derived from lactobacillus and a therapeutic drug for retinal ischemia-reperfusion injury-related diseases loaded within the extracellular vesicles; the therapeutic drug for retinal ischemia-reperfusion injury-related diseases is indolepropionic acid; the loading capacity of the targeted retinal drug-loaded extracellular vesicles for indolepropionic acid is 6~8×10⁻⁶. -8 Micrograms per particle.

[0007] As a further improvement, the targeted retinal drug-loaded extracellular vesicles are bilayer phospholipid vesicles with a particle size mainly distributed in the range of 100-300 nm and a zeta potential of -8 to -12 mV.

[0008] As a further improvement, the encapsulation rate of indolepropionic acid in the targeted retinal drug-loaded extracellular vesicles is 50-60%.

[0009] The present invention provides a method for preparing targeted retinal drug-loaded extracellular vesicles, comprising the following steps: (1) Preparation of extracellular vesicles derived from lactobacillus; (2) Indolepropionic acid was loaded onto extracellular vesicles derived from Lactobacillus to obtain the targeted retinal drug-loaded extracellular vesicles.

[0010] As a further improvement, step (1) includes: centrifuging the cultured lactobacillus to remove the bacterial cells and obtaining the supernatant, and then filtering, concentrating and ultracentrifuging the supernatant to obtain a crude extract of extracellular vesicles derived from lactobacillus.

[0011] As a further improvement, step (2) includes: The crude extract of Lactobacillus-derived extracellular vesicles was mixed with PBS solution and centrifuged at ultraspeed to obtain Lactobacillus-derived extracellular vesicles. The Lactobacillus-derived extracellular vesicles and indolepropionic acid solution were mixed at a volume ratio of (1~5):1 and drug-loaded extracellular vesicles were obtained by co-incubation. Alternatively, the crude extract of extracellular vesicles derived from Lactobacillus and the indolepropionic acid solution can be mixed at a volume ratio of (1~5):1, and drug-loaded extracellular vesicles can be obtained by co-incubation.

[0012] As a further improvement, the concentration of the indolepropionic acid solution in step (2) is 0.8~1.2 mg / mL, and the concentration of the Lactobacillus-derived extracellular vesicles or crude extract of Lactobacillus-derived extracellular vesicles is 1~3 mg / mL.

[0013] The present invention provides a fusion vesicle targeting the retina, comprising the aforementioned drug-loaded extracellular vesicle for targeting the retina, and a cell membrane containing retinal neuron-associated marker proteins; wherein the cell membrane containing retinal neuron-associated marker proteins is the cell membrane of a 661W cell.

[0014] The present invention provides a method for preparing the fusion vesicles targeting the retina, comprising the following steps: mixing the drug-loaded extracellular vesicles with the cell membrane of 661W cells, fusing them by ultrasonic treatment to obtain the fusion vesicles targeting the retina.

[0015] The present invention also provides the use of the aforementioned targeted retinal drug-loaded extracellular vesicles or the fused retinal targeted vesicles in the preparation of drugs for treating retinal ischemia-reperfusion injury-related diseases.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a lactobacillus-derived extracellular vesicle loaded with drug molecules, its preparation method, and its application in the treatment of retinal ischemia-reperfusion injury-related diseases (RIRI). An engineering strategy enhances the retinal tissue targeting of the extracellular vesicles. In this invention, the extracellular vesicles are loaded with indolepropionic acid to form a drug delivery system. The drug-loaded extracellular vesicles exhibit high drug loading rates, good biocompatibility, and good gastrointestinal stability, offering advantages such as high bioavailability and excellent safety. Furthermore, the indolepropionic acid-loaded extracellular vesicles carry retinal neuron-related marker proteins via membrane fusion technology, allowing for more efficient delivery to the retinal region via peripheral intravenous administration. This invention demonstrates that the drug-loaded lactobacillus-derived extracellular vesicles synergistically treat RIRI-related diseases and improve the effectiveness and safety of drug delivery, providing a novel drug delivery strategy for the treatment of RIRI-related diseases and neuroprotection. It has potential value in both local ocular and systemic drug administration in the treatment of RIRI-related diseases. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The target lactobacillus in Example 1 of this invention exhibits its culture performance on MRS agar plates and MRS liquid culture medium.

[0019] Figure 2 The results of NTA and transmission electron microscopy identification of Lactobacillus-derived extracellular vesicles and Lactobacillus-derived extracellular vesicles loaded with indolepropionic acid in Example 3 of the present invention are shown.

[0020] Figure 3 This is a mass spectrum of the indolepropionic acid standard and the release of indolepropionic acid from extracellular vesicles of Lactobacillus-derived cells loaded with indolepropionic acid in Example 3 of the present invention.

[0021] Figure 4 The results of the toxicity test on R28 cells of indolepropionic acid, lactobacillus-derived extracellular vesicles, and lactobacillus-derived extracellular vesicles loaded with indolepropionic acid in Example 4 of the present invention are as follows.

[0022] Figure 5 This invention relates to the protective effects of indolepropionic acid, lactobacillus-derived extracellular vesicles, and lactobacillus-derived extracellular vesicles loaded with indolepropionic acid on R28 cell damage induced by the OGD / R model, as described in Example 4 of this invention.

[0023] Figure 6 This describes the uptake of extracellular vesicles derived from lactobacillus by R28 cells in Example 5 of this invention.

[0024] Figure 7 This invention relates to the protective effect of Lactobacillus-derived extracellular vesicles and Lactobacillus-derived extracellular vesicles loaded with indolepropionic acid on retinal ganglion cells in mice with retinal ischemia-reperfusion injury, as described in Example 6 of this invention.

[0025] Figure 8 This invention demonstrates the protective effect of Lactobacillus-derived extracellular vesicles and Lactobacillus-derived extracellular vesicles loaded with indolepropionic acid on visual function in mice with retinal ischemia-reperfusion injury, as described in Example 7 of this invention.

[0026] Figure 9 This relates to the construction of engineered neurons and retinal-targeted fusion vesicles in Embodiment 8 of the present invention.

[0027] Figure 10 This refers to the application of engineered neurons and retinal-targeted fusion vesicles in Embodiment 8 of the present invention. Detailed Implementation

[0028] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0029] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0030] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0031] In a first aspect, the targeted retinal drug-loaded extracellular vesicles provided by the present invention are drug-loaded extracellular vesicles derived from lactobacillus, which include extracellular vesicles derived from lactobacillus and therapeutic drugs for definite or potential retinal ischemia-reperfusion injury-related diseases loaded in the extracellular vesicles.

[0032] Lactobacillus-derived extracellular vesicles, when used as drug delivery carriers for RIRI-related diseases, not only demonstrate advantages in stability, biocompatibility, and cell internalization, but also possess intrinsic therapeutic activity, enabling them to synergistically interact with drugs and exert better anti-inflammatory, antioxidant, and neuroprotective effects.

[0033] In some embodiments, the drug-loaded lactobacillus-derived extracellular vesicles have a bilayer phospholipid vesicle structure, which can encapsulate hydrophilic drugs and contain hydrophobic drugs, thereby improving the insolubility and stability of the drugs and enhancing their efficacy.

[0034] In some embodiments, the particle size of the drug-loaded lactobacillus-derived extracellular vesicles is mainly distributed in the range of 100-300 nm.

[0035] In some embodiments, the zeta potential of the drug-loaded lactobacillus-derived extracellular vesicles is approximately -8 to -12 mV, preferably -10 mV, which ensures that the drug-loaded lactobacillus-derived extracellular vesicle system is in a relatively stable state.

[0036] In some embodiments, the loaded drug used in this invention comprises at least one of indole compounds such as indole-3-propionic acid (IPA), short-chain fatty acids such as butyric acid, or other probiotic metabolites, or drugs that may be associated with other retinal ischemia-reperfusion injury-related diseases. The chemical structure of IPA is as follows: .

[0037] In some embodiments, the encapsulation efficiency of indolepropionic acid in the drug-loaded Lactobacillus-derived extracellular vesicles (the percentage of indolepropionic acid loaded in the vesicles relative to the total amount of added indolepropionic acid) is 50-60%, preferably about 54.97%, and the loading capacity (mass of indolepropionic acid loaded in the vesicles / number of extracellular vesicle particles) is 6-8 × 10⁻⁶. -8 Micrograms per particle, preferably about 6.25 × 10⁻⁶. -8 Micrograms per particle.

[0038] Secondly, the method for preparing targeted retinal drug-loaded extracellular vesicles provided by the present invention includes the following steps: (1) Preparation of extracellular vesicles derived from lactobacilli; In some embodiments, after culturing Lactobacillus, centrifugation is used to remove the bacterial cells to obtain a supernatant. The supernatant is then filtered, concentrated, and subjected to ultra-high-speed centrifugation to obtain extracellular vesicles derived from Lactobacillus.

[0039] In some embodiments, the following sub-steps are included: 1) Lactobacillus culture method: Inoculate Lactobacillus into MRS broth medium (de Man, Rogosa and Sharpe Broth) and culture it on a constant temperature shaker at 37℃ with a rotation speed of 220 rpm until the logarithmic growth phase is reached.

[0040] 2) Extraction of extracellular vesicles from Lactobacillus: The fermentation culture was centrifuged at low speed (4000 rpm, 45-60 min) to remove the lactic acid bacteria cells, and the supernatant was collected. The supernatant was filtered through a sterile filter (0.22 μm); then concentrated using an ultrafiltration membrane (50-100 kDa), and the concentrate was collected. The concentrate was ultracentrifuged at 100,000-150,000 g (standard gravity acceleration) at 4℃ for 2 h, and the precipitate was resuspended in 2 mL-5 mL PBS solution to obtain the crude extract of extracellular vesicles from Lactobacillus.

[0041] (2) Drugs were loaded onto extracellular vesicles of lactobacillus to obtain drug-loaded extracellular vesicles of lactobacillus.

[0042] In some embodiments, the loading of the drug onto Lactobacillus-derived extracellular vesicles comprises any of the following methods: Method 1: The crude extract of Lactobacillus-derived extracellular vesicles was mixed with an appropriate amount of PBS solution and then ultracentrifuged at 100,000-150,000g for 2 hours at 4°C. The precipitate was then resuspended with 1-2 mL of PBS solution to obtain Lactobacillus-derived extracellular vesicles. The Lactobacillus-derived extracellular vesicles and the drug solution were then mixed at a volume ratio of (1-5):1 and co-incubated at 37°C for 4-8 hours. The drug-loaded Lactobacillus-derived extracellular vesicles were then obtained by purification column purification or by ultracentrifugation again.

[0043] Method 2: Mix the crude extract of Lactobacillus-derived extracellular vesicles with the drug solution at a volume ratio of (1~5):1, and incubate at 37 °C for 4h~8h. Then, obtain drug-loaded Lactobacillus-derived extracellular vesicles by ultra-high speed centrifugation.

[0044] Co-incubation is the simplest and most commonly used method for loading drugs into extracellular vesicles. Drugs are encapsulated in extracellular vesicles through hydrophobic interactions, diffusion, or electrostatic interactions, or they can be loaded into extracellular vesicles along a concentration gradient. The drug loading efficiency depends on the hydrophobicity of the drug molecules. Hydrophobic drugs can interact with the lipid layer of the vesicle membrane and improve the drug loading efficiency.

[0045] In some embodiments, the drug solution is an indolepropionic acid (IPA) solution prepared with PBS solution, and the IPA concentration is 0.8-1.2 mg / mL, preferably 1 mg / mL. The concentration of the Lactobacillus-derived extracellular vesicles or the crude extract of Lactobacillus-derived extracellular vesicles is 1-3 mg / mL, preferably 1.5 mg / mL. The two are mixed at a volume ratio of (1-5):1.

[0046] In some embodiments, because the drug is insoluble in water or has low solubility, the drug needs to be pre-dissolved in ethanol before being loaded onto extracellular vesicles derived from lactobacillus. After loading, the solution is stirred at 55-65°C to allow the ethanol in the solution to evaporate, and finally filtered through a 0.22 μm filter membrane.

[0047] In some embodiments, because the drug is insoluble in water or has low solubility, a cosolvent such as polyethylene glycol may be added to the solution to improve the drug solubility.

[0048] Thirdly, the fusion vesicles targeting the retina provided by the present invention include extracellular vesicles derived from the above-mentioned drug-loaded lactobacillus, and cell membranes containing retinal neuron-related marker proteins (e.g., cell membranes of 661W cells).

[0049] In some embodiments, retinal neuron-like cells are cultured and their cell membranes are extracted. Extracellular vesicles derived from drug-loaded lactobacilli are then fused with the cell membranes to obtain fused vesicles targeting the retina.

[0050] In some embodiments, the fusion vesicles are prepared by fusing extracellular vesicles derived from drug-loaded Lactobacillus with the cell membrane of 661W cells. This fusion strategy aims to utilize specific proteins or recognition molecules on the 661W cell membrane to endow the engineered vesicles with the ability to actively target retinal tissue, thereby significantly improving their targeting efficiency and enrichment concentration during intraocular delivery.

[0051] In some embodiments, the preparation of fusion vesicles targeting the retina includes: mixing extracellular vesicles derived from drug-loaded lactobacilli with the cell membranes of 661W cells at a weight ratio of 1:1 and sonicating for 8-15 minutes.

[0052] Fourthly, the application of the above-mentioned drug-loaded lactobacillus-derived extracellular vesicles or retinal-targeting fused vesicles of the present invention in the preparation of drugs for treating retinal ischemia-reperfusion injury-related diseases.

[0053] The drug-loaded Lactobacillus-derived extracellular vesicles provided by this invention can effectively reduce the inflammatory response and oxidative stress levels in the retina in a mouse model of retinal ischemia-reperfusion injury. This invention can address the problems of retinal ganglion cell loss and visual function impairment in retinal ischemia-reperfusion injury-related diseases. Compared with single Lactobacillus-derived extracellular vesicles or drugs, this invention exhibits better neuroprotective effects and therapeutic efficacy, providing a novel drug delivery strategy for the treatment of retinal ischemia-reperfusion injury-related diseases. It is expected to become an effective strategy for the clinical treatment of retinal ischemia-reperfusion injury-related diseases.

[0054] In some embodiments, the medicament for treating retinal ischemia-reperfusion injury-related diseases may be in dosage forms including, but not limited to, the following: topical preparations for local ocular administration, such as eye drops, ophthalmic gels, or ointments; intravitreal injection preparations, which deliver the drug directly into the eye via injection and are suitable for situations requiring rapid or locally high concentrations of drug treatment; oral preparations, such as tablets, capsules, or oral solutions, which exert their therapeutic effect through systemic absorption; and nasal inhalation preparations, which are absorbed through the nasal mucosa to achieve rapid drug delivery and systemic effects.

[0055] Example 1 Example 1 of this invention provides a method for preparing extracellular vesicles derived from Lactobacillus loaded with indolepropionic acid, comprising the following steps: S1.1, Lactobacillus rhamnosus ( L.rhamnosus The fermentation broth was inoculated into MRS medium and cultured at 37°C with shaking at 220 rpm for 12 h. An appropriate amount of fermentation broth was then inoculated into fresh MRS medium and cultured under the same conditions for another 8 h. Next, the fermentation broth and MRS medium were mixed at a volume ratio of 1:50 and cultured at 37°C with shaking at 220 rpm for 24 h for extracellular vesicle isolation. Figure 1 As shown, A. The results of the selected lactobacilli inoculated on MRS agar plates for identification, ensuring that the subsequent cultured strains are lactobacilli. B. The fermentation broth obtained by inoculating the selected lactobacilli on MRS medium and culturing for 12 hours.

[0056] S1.2. Centrifuge the fermentation broth obtained above at low speed (10000g) for one hour to remove bacterial cells. Filter the supernatant through a sterile filter (0.22 μm); then concentrate using an ultrafiltration membrane (50-100 kDa) and collect the concentrate.

[0057] S1.3. The concentrated solution obtained above was centrifuged at 120,000g at 4°C for 2 hours, and the precipitate was resuspended with 2-5 mL of PBS solution to obtain crude extract of extracellular vesicles from lactobacillus.

[0058] S1.4. The crude extract of Lactobacillus-derived extracellular vesicles was mixed with 1-2 mL of PBS solution and then centrifuged at 120,000 g for 2 h at 4 °C. The precipitate was then resuspended with 1-2 mL of PBS solution to obtain Lactobacillus-derived extracellular vesicles (LrEV).

[0059] S2. Dissolve 1 mg of indolepropionic acid (IPA) in 1 mL of PBS solution to prepare a 1 mg / mL IPA solution. Mix the IPA solution with the Lactobacillus-derived LrEV (concentration 1.5 mg / mL) obtained in S1.4 at a 1:1 volume ratio and incubate at room temperature and 100 rpm for 6 h on a shaker. Centrifuge the mixture at 120,000 g at 4 °C for 2 h. Resuspend the precipitate in 1 mL of PBS solution and filter through a 0.22 μm filter membrane to obtain Lactobacillus-derived extracellular vesicles loaded with indolepropionic acid (IPA-LrEV).

[0060] Example 2 Example 2 of this invention provides a method for preparing Lactobacillus-derived extracellular vesicles loaded with indolepropionic acid. The difference from Example 1 is that step S1.4 is omitted. In step S2: the crude extract of Lactobacillus-derived extracellular vesicles is mixed with IPA solution at a volume ratio of 1:1 and incubated on a shaker at room temperature and 100 rpm for 6 h; the mixture is ultracentrifuged at 120,000 g at 4 °C for 2 h, the precipitate is resuspended with 1 mL PBS solution and filtered through a 0.22 μm filter membrane to obtain Lactobacillus-derived extracellular vesicles loaded with indolepropionic acid (IPA-LrEV).

[0061] Example 3 The performance of LrEV and IPA-LrEV prepared in Example 1 was tested, including... 1) The average particle size and zeta potential of LrEV and IPA-LrEV were measured by nanoparticle tracking analysis (NTA) and dynamic light scattering. The measurements were performed in three parallel measurements, and the results are shown in Table 1.

[0062] Table 1: Average particle size and zeta potential of LrEV and IPA-LrEV

[0063] Figure 2 A represents the NTA result of LrEV; Figure 2 B represents the NTA results of IPA-LrEV. The results show that there is no significant difference in particle size between LrEV and IPA-LrEV, and both conform to the distribution range of Lactobacillus-derived extracellular vesicles, exhibiting a normal distribution. Their zeta potentials are similar and consistent with those of Lactobacillus-derived extracellular vesicles.

[0064] 2) Transmission electron microscopy determination After diluting LrEV and IPA-LrEV with PBS solution, 10 μL of the diluted LrEV and IPA-LrEV were pipetted onto a microporous copper grid. After air drying, the grid was stained with 2% phosphotungstic acid for 3 min. The residual phosphotungstic acid was washed with distilled water and then blotted dry with filter paper. The copper grid was then placed under a transmission electron microscope to observe its morphology and structure and photographed for preservation.

[0065] The transmission electron microscopy results of LrEV are as follows: Figure 2 As shown in Figure C, LrEVs were found to possess a phospholipid bilayer structure, with approximately spherical morphology and varying particle sizes, but all distributed within the defined range of extracellular vesicles derived from Lactobacillus. Transmission electron microscopy results of IPA-LrEVs are shown below. Figure 2 As shown in Figure D, IPA-LrEV also exhibits a phospholipid bilayer structure with an approximately circular morphology, almost identical in size and shape to LrEV.

[0066] 3) Encapsulation efficiency and load capacity determination The concentration of IPA in IPA-LrEV was quantitatively determined using liquid chromatography-mass spectrometry (LC-MS). Specifically, an appropriate amount of IPA standard was accurately weighed and a stock solution of 2.00 mg / mL was prepared with methanol. The stock solution was then diluted with methanol to prepare standard curve working solutions at concentrations of 10000, 8000, 5000, 2000, 500, 200, 50, and 10 ng / mL. These solutions were then detected using chromatographs and mass spectrometers. A standard curve was obtained by regressing the peak area of ​​IPA (the target analyte) on the ordinate and the concentration on the abscissa, using the weighting coefficient Equal. The final IPA content in IPA-LrEV was determined by calculating the IPA sample data based on the standard curve. Figure 3 As shown, A. the peak elution time of IPA standard was approximately 2.41 min; B. the peak elution time of IPA in IPA-LrEV samples detected by chromatograph and mass spectrometer was approximately 2.42 min. Our results indicate that co-incubation of IPA with LrEV successfully loaded IPA into LrEV.

[0067] Encapsulation efficiency (EE) is the percentage of drug successfully encapsulated in micelles or nanoparticles, calculated using the following formula:

[0068] The formula for calculating load capacity (LC) is as follows:

[0069] The calculated encapsulation efficiency is 54.97%, and the load capacity is 6.25 × 10⁻⁶. -8 Micrograms per particle.

[0070] Example 4 Cytological evaluation of IPA, LrEV and IPA-LrEV 1) Safe concentrations of IPA, LrEV, and IPA-LrEV for R28 cells The safe concentrations of IPA, LrEV, and IPA-LrEV for R28 cells were screened using the CCK-8 assay. R28 cells are a retinal progenitor cell line with differentiation potential, commonly used in vitro to study the neuroprotective and pathological mechanisms of retinopathy of prematurity (RGC). R28 cells were cultured in DMEM low-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Specific steps included: R28 cells were cultured at 3 × 10⁻⁶ cells / cells. 4 After seeding cells at a density of 1 cell / well into 96-well plates, the plates were placed in a cell culture incubator. Once the cells adhered, the old culture medium was removed, and IPA solutions of 1 μM, 10 μM, 50 μM, 100 μM, 200 μM, and 500 μM, LrEV solutions of 1 μg / mL, 3 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 30 μg / mL, and IPA-LrEV solutions (LrEV and IPA-LrEV obtained in Example 1 were diluted with PBS) were added. Six replicates were set for different concentrations of different samples. After incubation for 24 h, the supernatant was discarded, and the plates were washed twice with PBS. 10 μL of CCK-8 solution was added to each well, and the plates were placed in an incubator in the dark for 2 h. After incubation in the dark, the 96-well plates were removed, and the absorbance at 450 nm was measured.

[0071] like Figure 4 As shown, R28 cells showed no significant cytotoxicity in the concentration range of 1-500 μM after 24 h of IPA treatment, and no significant toxicity was observed in the range of 1-30 μg / mL after 24 h of LrEV and IPA-LrEV treatment.

[0072] 2) Protective effects of IPA, LrEV and IPA-LrEV on R28 cell damage induced by the OGD / R model. R28 cells were cultured as described above, with R28 cells cultured at 3 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured in a cell culture incubator. The oxygen-glucose deprivation (OGD / R) model was established as follows: First, the cell culture medium was replaced with serum-free and glucose-free medium, and the cells were cultured in an anoxic incubator (2% O2, 5% CO2, 93% N2) for 6 hours. Then, the medium was replaced with normal medium (serum-free), and the cells were cultured under standard conditions until the experiment was completed. IPA solution, LrEV solution, and IPA-LrEV solution were added before each of the two medium replacements.

[0073] Cell viability assay: Before each culture medium change, 1 μM, 10 μM, 50 μM, 100 μM, 200 μM, 500 μM, and 1000 μM IPA solutions, as well as 1 μg / mL, 3 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL LrEV solutions and IPA-LrEV solutions were added. Before the experiment was completed, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the dark for 2 h. After incubation, the 96-well plate was removed from the dark, and the absorbance at 450 nm was measured.

[0074] Live / dead staining: R28 cells were seeded into 24-well plates. Before each culture medium change, 5 μg / mL LrEV solution and IPA-LrEV solution were added, respectively. The culture medium for each group was removed, washed once with PBS, and Calcein-AM / PI staining reagent and low-glucose basal medium were prepared at a ratio of 1:2000. After incubation at 37°C in the dark for 30 minutes, the staining solution was removed and placed in PBS. The staining status of each group was observed and photographed under a fluorescence microscope.

[0075] like Figure 5As shown, in the oxygen-glucose deprivation model, A. Cell viability was highest at an IPA concentration of 100 μM, approximately 1.2 times that of the OGD / R group. B. A LrEV concentration of 5 μg / mL could rescue R28 cell viability under the oxygen-glucose deprivation model, approximately 1.2 times that of the OGD / R group; LrEV concentrations of 1 or 3 μg / mL did not show a rescue effect, while concentrations of 10 or 20 μg / mL had similar effects to 5 μg / mL. C. An IPA-LrEV concentration of 5 μg / mL significantly rescued R28 cell viability under the oxygen-glucose deprivation model, approximately 1.5 times that of the OGD / R group, with a better effect than concentrations of 1 or 3 μg / mL. D. Under the same oxygen-glucose deprivation modeling injury conditions, the rescue effect of LrEV (5 μg / mL) or IPA (100 μM) on R28 cell viability under the oxygen-glucose deprivation model was not as good as that of IPA-LrEV (5 μg / mL). E. Cell death rate was detected using the Calcein AM / PI double staining method. Calcein AM is a lipophilic, non-fluorescent precursor dye that can penetrate the intact cell membrane of living cells and enter the cell. It is hydrolyzed by intracellular esterases into a green fluorescent substance, thus living cells exhibit green fluorescence. PI (propidium iodide) is a nucleic acid dye that cannot penetrate the intact cell membrane of living cells, but can enter dead cells with damaged cell membranes. After binding to DNA, it emits red fluorescence, thus dead cells exhibit red fluorescence. Cell death rate was assessed by merging the green and red fluorescence channels in the same field of view and counting the corresponding cells. The number of PI-positive cells was significantly increased in the OGD / R group. After treatment with LrEV, IPA-LrEV, or IPA, the number of PI-positive cells was significantly reduced, indicating that LrEV, IPA-LrEV, and IPA can all reduce OGD / R-induced R28 cell death, with IPA-LrEV showing a more significant effect. F. Percentage of PI-negative R28 cells in each treatment group under the oxygen-glucose deprivation model.

[0076] Example 5 R28 cell uptake of LrEV assessment In this embodiment, PKH26-labeled LrEV was used to assess whether R28 cells took up LrEV. Specifically, 20 μL of PKH26 dye working solution was added to 20 μg of LrEV protein (obtained in Example 1), followed by vortexing and incubation for 10 min; 10 mL of PBS was added to the incubated LrEV-dye complex and mixed; ultracentrifugation was performed to remove excess dye; an appropriate amount of PBS was taken to resuspend the precipitate, which was the stained exosome.

[0077] R28 cell culture was performed as described above. R28 cells were seeded into 24-well plates. After cell adhesion, PKH26-labeled LrEV was added, and the cells were cultured for another 6 hours. The culture medium was aspirated, and the cells were washed five times with PBS to remove untaken LrEV. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 minutes and washed three times with PBS. The membranes were then perforated with 0.1% PBST and washed three times with PBS, followed by blocking with 5% BSA. Actin-specific fluorescent staining and nuclear staining were performed sequentially using a microfilament green fluorescent probe and DAPI. The staining was observed and photographed under a fluorescence microscope. Figure 6 As shown, PKH26-labeled extracellular vesicles (red) are located around most cell nuclei (DAPI staining, blue) and distributed in the cytoplasm (Actin staining, green), indicating that R28 cells can effectively take up LrEV.

[0078] Example 6 Protective effects of LrEV and IPA-LrEV on retinal ganglion cells damaged in mice with retinal ischemia-reperfusion injury-related disease. Animal experiments: An acute ocular hypertension (AOH) mouse model was established using wild-type C57BL / 6J mice (8 weeks old, 18-20 g) by increasing intraocular pressure (120 mmHg, sustained for 60 minutes). In short, mice with dilated pupils and corneal anesthesia were placed on a heated worktable. A 30-gauge needle connected to a saline infusion device was carefully inserted into the anterior chamber of the mouse, and the anterior chamber pressure was gradually increased to 120 mmHg; this pressure was maintained for 60 minutes. The control group underwent sham surgery without increasing intraocular pressure.

[0079] Twenty-four hours after establishing the acute ocular hypertension model, 0.2 μL of LrEV or IPA-LrEV from Example 1 was administered intravitreally. Mice were weighed and anesthetized with an intraperitoneal injection of 1% sodium pentobarbital (prepared with physiological saline) at a dose of 10 ml / kg. Specifically, after anesthesia, mice were locally anesthetized with oxybuprofen hydrochloride eye drops (Santen Pharmaceutical Co., Ltd., Japan), mydriasis was achieved with compound tropicamide eye drops (Santen Pharmaceutical Co., Ltd., Japan), and the ocular surface was disinfected with levofloxacin eye drops (Bausch & Lomb Freda Pharmaceutical Co., Ltd., Shandong, China) and povidone-iodine (Yintao Pharmaceutical Co., Ltd., Jiangxi, China). After adequate mydriasis, the mice were fixed under a microscope, and 0.2 μL of each solution was injected into the vitreous cavity of both eyes through a 10 μL microsyringe with a 33-gauge needle (Hamilton Pharmaceuticals, Switzerland). Control mice received the same volume of solvent. After injection, the needle was slowly withdrawn, and tobramycin-dexamethasone eye ointment (ALCON, Spain) was applied to the mouse's eye surface before the mouse was placed back in its cage to recover from anesthesia. On day 7 post-modeling, relevant phenotypic tests were performed. All animal experiments were reviewed and approved by the Animal Care and Use Committee of the Experimental Animal Research Center, Xiangya Medical College, Central South University.

[0080] Specific groupings: Ctr: normal control group; IR: ischemia-reperfusion model group; IR+LrEV: ischemia-reperfusion + LrEV treatment group; IR+IPA-LrEV: ischemia-reperfusion + IPA-LrEV treatment group; IR+IPA: ischemia-reperfusion + IPA treatment group.

[0081] Retinal mounting: Mouse eyeballs were enucleated and fixed with 4% paraformaldehyde (m / v) for 2 hours at room temperature. The cornea and lens were then removed. After further fixation in 4% PFA (m / v) for 1 hour at room temperature, the retina was dissected, transferred to a glass slide, and cut into four equal quadrants. The retina was permeated in PBS containing 0.3% Triton X-100 (v / v) for 10 minutes, then blocked in blocking solution (5% BSA) (m / v) for 1 hour, followed by staining with anti-Brn3a (1:500) and incubation overnight at 4°C. After rinsing three times with PBS, the retina was incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at room temperature for 2 hours, and then mounted. Images were acquired using a fluorescence microscope (Leica, Wetzlar, Germany). Each retina was divided into four quadrants (sharing a central quadrant), and Brn3a-positive cells were counted using Image J to determine the RGC density of that retina. The mean density of all retinas in each group was calculated. Using the control sample as a reference, the percentage of relative RGC density in each group relative to the mean was calculated. Statistical analysis of the relative Brn3a+ cells in each group was performed using GraphPad Prism software.

[0082] like Figure 7 As shown, the number of Brn3a+ cells in the central, middle, and peripheral retina were counted. Seven days after intravitreal injection of LrEV, IPA-LrEV, or IPA, retinal smear results showed that the number of Brn3a+ cells recovered significantly compared with the simple IR group; and the protective effect of IPA-LrEV was better than that of LrEV or IPA.

[0083] Example 7 Protective effects of LrEV and IPA-LrEV on visual function in mice with retinal ischemia-reperfusion injury-related diseases Flash VEP (FVEP) detection After completing the intervention in Example 6, mice were anesthetized with 1% sodium pentobarbital to fully dilate their pupils. The mice were then fixed prone on the operating table, and a heating pad was maintained at 37°C. Carboxymethyl cellulose eye drops were instilled into both eyes to keep the corneas moist. Three needle electrodes were inserted subcutaneously into the mice, specifically as follows: the recording electrode was inserted at the midpoint between the bases of the ears, contacting the surface of the occipital bone; the reference electrode was inserted subcutaneously into the nose; and the grounding electrode was inserted subcutaneously into the tail. One eye was completely covered, and testing was performed according to international standard procedures. After testing, the above steps were repeated for the other eye. The N1-P1 amplitude values ​​for each group were analyzed.

[0084] like Figure 8 As shown, FVEP detection revealed that the N1-P1 amplitude value of AOH model mice was significantly lower than that of the normal group. After treatment with LrEV, IPA-LrEV or IPA, the N1-P1 amplitude value recovered significantly compared with that of AOH mice alone, and the IPA-LrEV treatment had the best effect.

[0085] Example 8 Tissue-targeted fusion vesicle construction and application based on LrEV To enhance the tissue targeting of LrEVs and expand their application scope, taking retinal targeting as an example, a fusion vesicle of retinal neuron cell membrane and LrEV, NeuroRetina-LrEV, was constructed. Specifically, LrEVs were fused with 661W cell membranes.

[0086] The 661W cell membrane was obtained as follows: The 661W cell line was cultured in cell culture dishes for 48 hours. Cells were scraped off using a cell scraper and pipetteed off. Cells were collected by centrifugation, and the supernatant was aspirated, leaving the cell pellet for later use. The cell pellet was gently resuspended in a suitable amount of ice-cold PBS. The collected cells were centrifuged at 600g for 5 minutes at 4°C to precipitate the cells. The supernatant was discarded, and the cells were then centrifuged at 600g for 1 minute at 4°C to remove residual liquid from the centrifuge tube wall and further precipitate the cells. 1 ml of membrane protein extraction reagent A (pre-added with PMSF) was added to an appropriate amount of cells, and the cells were gently and thoroughly resuspended. The cells were incubated on ice for 10-15 minutes. Lysis was then performed using a freeze-thaw cycle, followed by centrifugation at 700g for 15 minutes at 4°C. The supernatant was collected and centrifuged at 14000g for 30 minutes at 4°C, and the supernatant was discarded. Then, an appropriate amount of membrane protein extraction reagent B was added. The precipitate was resuspended by high-speed, vigorous vortexing for 5 seconds, followed by an ice bath for 5-10 minutes, repeated twice. Subsequently, it was centrifuged at 14000g for 5 minutes at 4°C, and the supernatant was collected as the cell membrane protein solution.

[0087] Construction of fusion vesicles: The LrEV solution (1.5 mg / mL) obtained in Example 1 was sonicated for 10 minutes, and then mixed with 661W membrane solution (1.5 mg / mL) at a 1:1 weight ratio. To obtain the fusion membrane, the mixture was sonicated at 37°C for 10 minutes. Figure 9 As shown, to demonstrate the successful fusion of LrEV and 661W membranes, PKH-26 and DiO were used to stain the LrEV and 661W membranes, respectively. A. PKH-26-labeled LrEV solution and DiO-labeled 661W membrane were mixed but not sonicated; B. PKH-26-labeled LrEV solution and DiO-labeled 661W membrane were mixed and sonicated. The results showed that sonication effectively promoted the fusion of LrEV and 661W membranes.

[0088] To verify the retinal and neuronal targeting of NeuroRetina-LrEV, equal volumes of DiR-labeled LrEV and NeuroRetina-LrEV were injected via the tail vein, and fluorescence distribution at different time points was observed using the IVIS small animal in vivo imaging system. Figure 10As shown, compared with LrEV, NeuroRetina-LrEV exhibited stronger fluorescence signals in the brain and eyes 5 minutes after tail vein injection, suggesting potentially stronger retinal and neuronal targeting. At 8, 24, and 32 hours post-injection, the ocular fluorescence signals in the NeuroRetina-LrEV group were significantly stronger than those in the LrEV group. After 32 hours, mice were harvested (① liver, ② eye, ③ heart, ④ kidney) for in vitro re-examination. The results showed that the liver of mice injected with both LrEV and NeuroRetina-LrEV via tail vein had the strongest signal, suggesting that the vesicle membrane may be degraded and primarily processed by the liver after 32 hours. However, the ocular signal of mice injected with NeuroRetina-LrEV via tail vein was significantly stronger than that of mice injected with LrEV via tail vein, suggesting that the retinal targeting of NeuroRetina-LrEV may be significantly superior to that of LrEV.

[0089] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.

Claims

1. A targeted retinal drug-loaded extracellular vesicle, characterized in that, It includes extracellular vesicles derived from lactobacilli and a therapeutic drug for retinal ischemia-reperfusion injury-related diseases loaded within the extracellular vesicles; the therapeutic drug for retinal ischemia-reperfusion injury-related diseases is indolepropionic acid; the loading capacity of the targeted retinal drug-loaded extracellular vesicles for indolepropionic acid is 6~8×10⁻⁶. -8 Micrograms per particle.

2. The targeted retinal drug-loaded extracellular vesicles according to claim 1, characterized in that, The targeted retinal drug-loaded extracellular vesicles have a bilayer phospholipid vesicle structure with a particle size mainly distributed in the range of 100-300 nm and a zeta potential of -8 to -12 mV.

3. The targeted retinal drug-loaded extracellular vesicles according to claim 1, characterized in that, The encapsulation rate of indolepropionic acid in the targeted retinal drug-loaded extracellular vesicles is 50-60%.

4. A method for preparing targeted retinal drug-loaded extracellular vesicles according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Preparation of extracellular vesicles derived from lactobacillus; (2) Indolepropionic acid was loaded onto extracellular vesicles derived from Lactobacillus to obtain the targeted retinal drug-loaded extracellular vesicles.

5. The preparation method according to claim 4, characterized in that, Step (1) includes: after culturing Lactobacillus, centrifuging to remove the bacterial cells to obtain supernatant, the supernatant is successively filtered, concentrated and ultracentrifuged to obtain crude extract of Lactobacillus-derived extracellular vesicles.

6. The preparation method according to claim 5, characterized in that, Step (2) includes: The crude extract of Lactobacillus-derived extracellular vesicles was mixed with PBS solution and centrifuged at ultraspeed to obtain Lactobacillus-derived extracellular vesicles. The Lactobacillus-derived extracellular vesicles and indolepropionic acid solution were mixed at a volume ratio of (1~5):1 and drug-loaded extracellular vesicles were obtained by co-incubation. Alternatively, the crude extract of extracellular vesicles derived from Lactobacillus and the indolepropionic acid solution can be mixed at a volume ratio of (1~5):1, and drug-loaded extracellular vesicles can be obtained by co-incubation.

7. The preparation method according to claim 6, characterized in that, The concentration of the indolepropionic acid solution in step (2) is 0.8~1.2 mg / mL, and the concentration of the Lactobacillus-derived extracellular vesicles or crude extract of Lactobacillus-derived extracellular vesicles is 1~3 mg / mL.

8. A fusion vesicle targeting the retina, characterized in that, It includes the targeted retinal drug-loaded extracellular vesicles as described in any one of claims 1 to 3, and a cell membrane containing retinal neuron-associated marker proteins; wherein the cell membrane containing retinal neuron-associated marker proteins is the cell membrane of 661W cells.

9. A method for preparing the retinal-targeting fusion vesicle as described in claim 8, characterized in that, The process includes the following steps: mixing the drug-loaded extracellular vesicles with the cell membrane of 661W cells and fusing them by ultrasound to obtain the fused vesicles targeting the retina.

10. The use of a targeted retinal drug-loaded extracellular vesicle according to any one of claims 1 to 3 or a targeted retinal fusion vesicle according to any one of claims 8 to 9 in the preparation of a medicament for treating retinal ischemia-reperfusion injury-related diseases.

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