Lipid bilayer delivery system loaded with quercetin and application of lipid bilayer delivery system

Abstract

The invention provides a quercetin-loaded lipid bilayer delivery system and application thereof, and relates to the technical field of myopia prevention and control. The invention adopts exosome to load quercetin, prepares a lipid bilayer delivery system loaded with quercetin, and provides a specific preparation method of the lipid bilayer delivery system and application of the lipid bilayer delivery system in preparation of drugs for treating and / or preventing myopia. The preparation method of the quercetin-loaded lipid bilayer delivery system is simple and convenient, short in preparation time and suitable for popularization and production. Moreover, the water solubility of the natural product quercetin can be improved through the method, the obtained product has good biocompatibility and tissue penetrating ability, bioavailability and high biological safety, the myopia progress can be effectively controlled, a new strategy is provided for prevention and control of myopia, and a new choice is expected to be provided for drug prevention and control of myopia.

Description

Technical Field

[0001] The present application relates to the technical field of myopia prevention and control, and particularly relates to a lipid bilayer delivery system loaded with quercetin and its application. Background Art

[0002] The global myopia prevalence rate is rising at an alarming rate. Especially in some Asian countries, myopia has approached crisis levels. The "myopia pandemic" has become a serious global public health problem, and myopia prevention and control is imminent. Although the current myopia prevention and control methods have achieved initial effects, they cannot completely inhibit myopia progression, and these methods also have their own challenges in practical applications. Currently, the control effect of orthokeratology lenses is relatively ideal, and the control rate can reach 43% - 63%. However, it has potential complications, and the professional fitting technology requirements and high economic costs limit its large-scale clinical application. The long-term prevention and control effect of red light therapy is still unknown, and there is a lack of safety assessment criteria and specific monitoring mechanisms for evaluating adverse events such as retinal photochemical damage. Atropine eye drops can slow down myopia progression in a concentration-dependent manner, but high concentrations are prone to safety problems such as photophobia, myopia rebound after drug withdrawal, dry eye, and allergic reactions, which limit its long-term clinical application. Therefore, there is an urgent need to develop new and safe alternative drugs to obtain better myopia prevention and control effects.

[0003] As potential drug leads, natural products are widely distributed, have low toxicity, and rich biological activities. They are an important source of innovative drug treatment preparations. As an important natural flavonoid compound, quercetin has been proven to have strong antioxidant, anti-inflammatory, immunomodulatory, antifibrotic, anticancer, neuroprotective, and vasoprotective biological activities, and has extensive benefits in the treatment of various ophthalmic diseases. Although quercetin has good biological activities and medicinal values, its poor water solubility leads to low bioavailability, which restricts its practical clinical application. Therefore, the preparation of novel quercetin nano-drugs has important clinical significance.

[0004] As a nano-scale extracellular vesicle, exosome can freely cross cell membranes and is an attractive drug carrier. The unique lipid bilayer structure of exosome can effectively encapsulate and control the release of various natural active ingredients, enhance the solubility of natural products, freely cross biological barriers, significantly improve drug bioavailability, and have little impact on cytotoxicity and immunogenicity. Drug-loaded exosomes provide a new perspective for the drug prevention and treatment of diseases. Summary of the Invention

[0005] The object of the present invention is to provide a lipid bilayer delivery system loaded with quercetin, its preparation method and application. The lipid bilayer delivery system loaded with quercetin has strong tissue penetration ability, low immunogenicity, good water solubility, excellent biocompatibility and long-term stability, and can also prolong the residence time of the drug on the anterior corneal surface, effectively control the elongation of the myopic eye axis, and has a good effect on treating / preventing myopia.

[0006] On the one hand, the present application provides a preparation method of a lipid bilayer delivery system loaded with quercetin for preventing and treating myopia, and the method includes: mixing quercetin with exosomes or liposomes and incubating, and the exosomes are exosomes derived from human umbilical cord mesenchymal stem cells.

[0007] In a preferred embodiment, the quercetin is a quercetin solution, and the solvent can be DMSO solvent.

[0008] Those skilled in the art can understand that the solvent here is only for facilitating the dissolution of quercetin, and any non-toxic and harmless common organic solvent can be arbitrarily selected as the carrier, and no excessive limitation is imposed here.

[0009] Further, the concentration of the quercetin is 0.1 - 5 mg / mL; preferably, 1 mg / mL.

[0010] Among them, the concentration of the quercetin can be any value of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL.

[0011] Further, the number of exosomes or liposomes is 1000 - 100000 billion; preferably, 10000 billion.

[0012] Among them, the number of exosomes or liposomes is any value of 1000 billion, 2000 billion, 3000 billion, 4000 billion, 5000 billion, 6000 billion, 7000 billion, 8000 billion, 9000 billion, 10000 billion, 20000 billion, 30000 billion, 40000 billion, 50000 billion, 60000 billion, 70000 billion, 80000 billion, 90000 billion, 100000 billion.

[0013] Further, the volume ratio of the quercetin to the exosomes or liposomes is 1:(1 - 20); preferably, 1:9.

[0014] Among them, the volume ratio of the quercetin to the exosomes or liposomes is any value among 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20.

[0015] Furthermore, the conditions for the incubation include: incubating at 35°C to 40°C for 1 to 5 h; preferably, incubating at 37°C for 2 h.

[0016] Among them, the conditions for the incubation include: incubating at 35°C, 36°C, 37°C, 38°C, 39°C, 40°C for 1, 2, 3, 4, 5 h.

[0017] Those skilled in the art can understand that exosomes or liposomes can be prepared by conventional methods or obtained by purchase.

[0018] In a preferred embodiment, the method for preparing the mesenchymal stem cell exosomes includes: inoculating mesenchymal stem cells in a culture medium for culture, when the cells reach 80% to 95% confluence, collecting the cells at 4°C, centrifuging at 2000 g for 20 to 30 min to remove dead cells and debris, taking the supernatant at 4°C, centrifuging at 10000 to 12000 g for 20 to 30 min to remove microbubbles or protein aggregates, and taking the supernatant. Filtering the sample with a 0.22 μm filter to remove apoptotic bodies and microbubbles, and taking the filtered supernatant to isolate exosomes from the cell supernatant using an ultra-rapid separation system (EXODUS) device.

[0019] Preferably, the preparation method further includes a step of separation and purification.

[0020] The separation and purification can be carried out by conventional methods in the art. In a preferred embodiment, the purification method can be to use an ultra-rapid separation system (EXODUS) to separate unbound drugs and solvents.

[0021] In a preferred embodiment, the method includes: mixing quercetin with a concentration of 0.1 to 5 mg / mL and human umbilical cord-derived mesenchymal stem cell exosomes with a particle number of 1000 to 100000 billion at a volume ratio of 1:(1 to 20) for incubation, and the conditions for the incubation include: incubating at 35°C to 40°C for 1 to 5 h.

[0022] On the other hand, the present application also provides a lipid bilayer delivery system loaded with quercetin prepared by the method.

[0023] Furthermore, the particle size of the lipid bilayer delivery system loaded with quercetin is between 100 nm and 200 nm.

[0024] Among them, the particle size of the quercetin-loaded lipid bilayer delivery system can be any value among 100 nm, 110 nm, 120 nm, 125 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, and 200 nm.

[0025] Preferably, it is 125 nm to 150 nm.

[0026] Preferably, the potential is between -70 mV and -30 mV; more preferably, it is between -50 mV and -30 mV.

[0027] The quercetin-loaded lipid bilayer delivery system prepared by the method of the present application has no obvious change in particle size before and after drug loading, enabling the drug-loaded exosomes to maintain their unique immunogenicity, good penetrability, targeting properties, etc., which is beneficial for efficient drug delivery.

[0028] Moreover, the appearance shape, particle size, potential, and marker protein of the quercetin-loaded lipid bilayer delivery system have not changed significantly before and after drug loading, and it has long-term stability after drug loading, and can still maintain good stability after being stored at -80 °C for 30 days.

[0029] And the quercetin-loaded lipid bilayer delivery system has good biocompatibility, strong tissue penetration ability, high bioavailability, good biosafety, and has a good control effect on myopia progression, and is expected to provide a new option for the drug prevention and control of myopia.

[0030] On the other hand, the present application also provides a composition comprising the quercetin-loaded lipid bilayer delivery system.

[0031] The composition of the present application can also be added with excipients, and the excipients can be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.

[0032] Preferably, the solvent can be water.

[0033] The quercetin-loaded exosomes can be prepared into single-agent preparations, compound preparations, drug-loaded contact lenses, etc. for myopia prevention and control. The dosage forms of the single-agent preparation or compound preparation include powders, pills, granules, tablets, capsules, suspensions, gels, suppositories, creams, emulsions, sprays, injections, injections, etc.

[0034] The composition described in the present application can be prepared by a general method, and one or more diluents or carriers can be added. Preferably, the composition can be prepared into a sterile ophthalmic preparation such as an ophthalmic suspension.

[0035] In a preferred embodiment, the number of particles of the lipid bilayer delivery system loaded with quercetin in the composition is 2.0×10 10 per mL.

[0036] On the other hand, the present application also provides the use of the method or the lipid bilayer delivery system loaded with quercetin or the composition in the preparation of a drug for treating and / or preventing myopia. The lipid bilayer delivery system loaded with quercetin includes exosomes or liposomes loaded with quercetin.

[0037] Preferably, the exosomes are mesenchymal stem cell exosomes derived from human umbilical cord.

[0038] Furthermore, the use includes delaying the growth of the eye axis; preferably, the use includes inhibiting ferroptosis and endoplasmic reticulum stress to improve the remodeling of the scleral stroma caused by form deprivation and delay the progression of myopia; more preferably, the use includes an increase in the content of TGF-β1, a decrease in the content of MMP2 and / or a decrease in the content of α-SMA.

[0039] Preferably, the increase in the content of TGF-β1 and the decrease in the content of MMP2 are achieved by inhibiting ferroptosis and endoplasmic reticulum stress. The decrease in the content of α-SMA inhibits the transformation of fibroblasts into myofibroblasts and thus delays the progression of myopia.

[0040] Among them, the lipid bilayer delivery system loaded with quercetin described in the present application can improve the thinning and sparseness of scleral fibers caused by form deprivation, inhibit scleral stroma remodeling, delay eye axis growth and / or delay the progression of myopia by increasing the content of TGF-β1, decreasing MMP2 and / or decreasing the content of α-SMA, thereby playing a role in treating and / or preventing myopia, and providing a new drug for the field of myopia treatment.

[0041] Among them, the preparation process of the exosomes or liposomes loaded with quercetin includes conventional loading methods such as co-incubation method, electroporation method, saponin treatment method, freeze-thaw cycle method, ultrasonic method and extrusion method.

[0042] On the other hand, the present application also provides the use of the method or the lipid bilayer delivery system loaded with quercetin or the composition in prolonging the residence time of the drug on the anterior corneal surface, improving the drug penetration efficiency, improving the drug utilization rate, promoting cell proliferation, promoting cell migration and / or promoting cell injury repair.

[0043] Furthermore, the cells include corneal epithelial cells and / or scleral fibroblasts.

[0044] Preferably, the cell uptake process can be completed in a relatively short time, preferably within 15 - 30 minutes.

[0045] Preferably, it can remain on the anterior corneal surface for a long time, preferably within 40 minutes, more preferably within 10 minutes.

[0046] Preferably, the improvement of drug penetration efficiency is to increase the permeability of the drug in corneal epithelial cells and / or various cells in the drug delivery pathway, and the improvement of drug utilization rate is achieved by increasing the residence time of the drug in front of the cornea and its uptake rate in cells.

[0047] Preferably, the promotion of cell proliferation is to promote the proliferation of corneal epithelial cells.

[0048] Preferably, the promotion of cell migration is to promote the migration of corneal epithelial cells.

[0049] Preferably, the promotion of cell damage repair is to promote the damage repair of corneal epithelial cells.

[0050] Those skilled in the art can understand that appropriate concentrations and dosages can be selected according to the actual situation, and the specific use concentration of the quercetin-loaded lipid bilayer delivery system is not specifically limited here.

[0051] In a preferred embodiment, the number of particles of the quercetin-loaded lipid bilayer delivery system (Exo-Que) is 2.0×10 10 per mL, and the dosage is 10 μL each time, 6 times a day.

[0052] The present invention has the following beneficial effects: In the present invention, exosomes or liposomes are used to load quercetin to prepare a quercetin-loaded lipid bilayer delivery system, and its specific preparation method and application are provided. The preparation method of the quercetin-loaded lipid bilayer delivery system is simple and the preparation time is short, which is suitable for popularization and production.

[0053] Since quercetin itself is insoluble in water and soluble in the organic solvent DMSO, and the organic solvent DMSO has potential cytotoxicity and cannot be directly used for eye drops. Moreover, conventional eye drops have a short residence time on the ocular surface and low bioavailability, which greatly limits the use and therapeutic effect of quercetin. The lipid bilayer delivery system loaded with quercetin prepared by the method in this application can improve the water solubility of the natural product quercetin, making the obtained product have good solubility, biocompatibility and tissue penetration ability. The obtained product also has the characteristics of long-term stability, high bioavailability and high biosafety. In addition, the product can also prolong the residence time of the drug on the anterior surface of the cornea, so that the drug has more time to penetrate into the cornea; its strong tissue barrier penetration ability enables them to effectively deliver the drug to specific cells or tissues, while avoiding immune responses and lysosomal degradation, solving the problems of short residence time on the ocular surface and low bioavailability of traditional eye drops. The product can effectively control the progression of myopia, providing a new strategy for the prevention and control of myopia, and is expected to provide a new option for the drug prevention and control of myopia. Brief Description of the Drawings

[0054] The drawings described herein are used to provide a further understanding of the present application and form a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation of the present application. In the drawings: Figure 1 It is a diagram for the preparation, characterization and stability evaluation of Exo-Que. Among them, Figure 1 A is a schematic diagram of the synthesis process of Exo-Que, Figure 1 B is a transmission electron microscope image of exosomes in different states, Figure 1 C is a diagram of the particle size and potential of exosomes in different states, Figure 1 D is an identification result diagram of exosome marker proteins; Figure 2 It is a diagram for the in vitro safety study of Exo-Que, Figure 2 A is the result of cytotoxicity detection, Figure 2 B is a cell scratch experiment, Figure 2 C is a migration quantitative analysis result diagram; Figure 3 It is a diagram for the evaluation of cell uptake and ocular surface residence of Exo-Que, Figure 3 A is the uptake of Exo-Que by corneal epithelial cells and scleral fibroblasts, Figure 3 B is a fluorescence quantitative analysis diagram of the pre-corneal retention of Exo-Que labeled with a fluorescent probe, Figure 3 C is a small animal in vivo imaging diagram of the retention of Exo-Que labeled with a fluorescent probe on the ocular surface; Figure 4 It is a diagram for the myopia prevention and control effect study of Exo-Que. Among them, Figure 4Figure A is the experimental flow chart of treating the guinea pig form deprivation myopia model with Exo-Que eye drops. Figure 4 Figure B shows the changes in refractive status of guinea pigs in different groups. Figure 4 Figure C shows the changes in axial length of guinea pigs in different groups. Figure 5 Figure is the in vivo biocompatibility evaluation diagram of Exo-Que. Among them, Figure 5 Figure A is the slit lamp evaluation of the ocular surface health status of the guinea pig form deprivation myopia model treated with Exo-Que eye drops. Figure 5 Figure B is the tissue sections of important organs of guinea pigs in different groups. Figure 5 Figure C is the quantitative analysis results of blood biochemical detection indexes of guinea pigs in different groups. Figure 6 Figure is the research diagram of the myopia prevention and control mechanism of Exo-Que. Among them, Figure 6 Figure A and Figure 6 Figure B show the changes in collagen fibers of scleral tissue in different intervention groups. Figure 6 Figure C is the WB detection diagram of the changes in related signaling molecules of scleral matrix remodeling. From left to right are the NC group, FDM group, and FDM + Exo-Que group. Figure 6 Figure D is the quantitative analysis result diagram of scleral tissue. Specific implementation manners

[0055] In order to more clearly illustrate the overall concept of the present application, the following will be described in detail by way of examples in conjunction with the accompanying drawings of the specification. In the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some well-known technical features in the art are not described.

[0056] Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation manners described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific implementation manners, rather than limiting the protection scope of the present invention. The test methods without specific conditions noted in the following examples are usually carried out under conventional conditions or according to the conditions recommended by each manufacturer.

[0057] It should be noted that the terms used herein are only for the purpose of describing specific implementation manners and are not intended to limit the exemplary implementation manners according to the present application. As used herein, unless otherwise clearly specified in the context, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. When numerical ranges are given in the examples, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value therebetween can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, equipment, and materials used in the examples, any methods, equipment, and materials of the prior art similar or equivalent to those described in the examples of the present invention can also be used to implement the present invention according to the knowledge of those skilled in the art of this technology and the description of the present invention.

[0058] Unless otherwise specified, in the following embodiments, for the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained by commercial purchase.

[0059] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, medicine, and related fields in this technology.

[0060] In addition, the "water" described in the present invention includes any feasible water that can be used in this field, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, purified water, etc.

[0061] In the following examples, unless otherwise specifically stated, % represents wt%, that is, weight percentage.

[0062] Example 1 Preparation of Exo-Que (1) Human umbilical cord-derived mesenchymal stem cells were inoculated in a serum-free exosome medium for culture. When the cells reached 90% confluence, the cells were collected at 4°C and centrifuged at 2000 g for 20 min to remove dead cells and debris. The supernatant was taken and centrifuged again at 4°C and 12000 g for 30 min to remove microbubbles or protein aggregates. The supernatant was taken. The sample was filtered through a 0.22 μm filter to remove apoptotic bodies and microbubbles, and the filtered supernatant was used to isolate exosomes from the cell supernatant using an ultra-rapid separation system (EXODUS) device.

[0063] (2) Quercetin was dissolved in DMSO solvent to make the drug concentration 1 mg / mL. 1 mL of the quercetin solution was added to the exosome solution (the number of exosome particles was 1000 billion) at a volume ratio of 1:9. After mixing, it was incubated at 37°C for 2 h. Then, the unbound drug and solvent were separated using an ultra-rapid separation system (EXODUS) to obtain Exo-Que (exosomes loaded with quercetin) by purification.

[0064] The prepared Exo-Que can be resuspended in pure water to prepare eye drops. The number of Exo-Que particles in the eye drops of this example is 2.0×10 10 per mL, and the following experiments are carried out using this Exo-Que.

[0065] Example 2 Characterization and Stability Evaluation of Exo-Que In this example, Exo-Que was characterized and its stability was evaluated. The specific methods include: Place 10 μL of Exo-Que on a carbon-coated copper grid, stain it with 2% uranyl acetate for 2 min, then air-dry the exosomes and Exo-Que at 100 kV and observe their morphology under a transmission electron microscope, and observe their morphology after storing at -80 °C for 15 days and 30 days to judge their long-term stability. Nano particle tracking analysis was used to detect the concentration, zeta potential and particle size distribution of Exo-Que; QubitTM Protein Assay Kit was used to detect the protein concentration of Exo-Que and the expression of exosome surface markers CD9, CD81, TSG101, and Alix was detected by western blot.

[0066] The results of the characterization and stability evaluation of Exo-Que are as Figure 1 shown, where Figure 1 A shows the synthesis process of Exo-Que: Quercetin was successfully prepared with exosomes by co-incubation.

[0067] Figure 1 B is the transmission electron microscope of exosomes. The appearance of exosomes before and after drug loading did not change, and it still showed a saucer-like appearance. Moreover, Exo-Que had good stability after storing at -80 °C for 15 days and 30 days, proving that Exo-Que has long-term stability.

[0068] Figure 1 C shows the particle size and potential of exosomes in different states. The results show that the particle size of exosomes is between 125 nm and 150 nm, and the particle size before and after drug loading did not change significantly, which enables the drug-loaded exosomes to maintain their unique immunogenicity, good penetrability and targeting properties, etc., which is beneficial to efficient drug delivery. In addition, the particle size of Exo-Que did not change significantly after storing at -80 °C for 15 days and 30 days. There was no difference in the potential of exosomes before and after drug loading, which was about -40 mV. The potential of Exo-Que became significantly negative after storing at -80 °C for 15 days and 30 days, reaching -70 mV.

[0069] Figure 1D is the identification of exosome marker proteins. The results showed that the exosome marker proteins Alix, TSG101, CD9, and CD81 did not change before and after drug loading.

[0070] Example 3 In vitro safety study of Exo-Que In this example, relevant studies on the in vitro safety of Exo-Que were carried out. The specific methods include: The CCK-8 method was used to detect the effects of Exo-Que on the activity and cytotoxicity of HCECs. The scratch assay was used to evaluate the cell migration ability of HCECs, and the in vitro safety of Exo-Que was evaluated.

[0071] In the cell proliferation experiment, HCECs were seeded into 96-well plates at a density of 10,000 cells per well and cultured in a standard medium (DMEM / F12, 10% FBS, and 1% penicillin-streptomycin) at 37 °C and 5% CO2 for 24 hours. Then, the medium was replaced with 100 μL of DMEM / F12 containing different concentrations of Exo-Que (0, 1.0 × 10¹ 0 particles / mL, 2.0 × 10¹ 0 particles / mL). After culturing for 12, 24, 36, and 48 h, the absorbance of the cells was measured using a CCK-8 assay kit to evaluate the cell proliferation ability.

[0072] In the scratch assay, HCECs were seeded into 12-well plates at a density of 2×10 5 cells / well. When the cells reached 90% confluence, the cell layer was scratched with a 200 μL pipette tip, washed with PBS, and the damaged cells were removed. Fresh medium containing different concentrations of Exo-Que (0, 1.0×10 10 particles / mL, 2.0×10 10 particles / mL) was added, and the cells were co-incubated for 48 h. Wound surface photos were taken at 0, 12, 24, 36, and 48 h, and analyzed using ImageJ software.

[0073] The results of the in vitro safety study of Exo-Que are shown in Figure 2 and Table 1. Specifically, the effects of Exo-Que on cell proliferation and migration ability were evaluated.

[0074] Figure 2 As shown in A, different concentrations of Exo-Que had no effect on the activity of human corneal epithelial cells. The cell survival rate of each group was greater than 95%, and no obvious cytotoxicity of Exo-Que was found compared with the control group.

[0075] Figure 2 As shown in Figure B, the scratch assay showed that Exo-Que promoted the proliferation of human corneal epithelial cells.

[0076] Figure 2 Figures C and Table 1 are for migration quantitative analysis. The scratch area was analyzed using Image J. The results showed that when the particle number of Exo-Que was 2.0 × 10 10 particles / mL, it could significantly promote cell migration and wound repair at about 24 h.

[0077] Table 1 Relative migration rate of cells in each group (%)

[0078] Example 4 Evaluation of cellular uptake and ocular surface residence time of Exo-Que In this example, the cellular uptake and ocular surface residence time of Exo-Que were evaluated. The specific methods included: (1) Uptake of Exo-Que by HCECs and HSCFs HCECs and HSCFs were seeded in observation dishes at a density of 1.5×10 4 cells / dish. After attachment, the cells were seeded in media containing different concentrations of DiD-labeled Exo-Que (0, 1.0×10 10 particles / mL, 2.0×10 10 particles / mL), and the cells were fixed at different time points (0 min, 15 min, 30 min, 60 min) to observe the uptake of Exo-Que by the cells. For confocal analysis, the culture medium on the cell slides was removed, and the cells were washed 3 times with PBS; fixed with 4% paraformaldehyde for 30 min, washed 3 times with PBS for 5 min each time, 0.1% Triton X-100 solution was added to permeabilize the membrane for 20 min; then washed 3 times and stained with DAPI for 10 min. Finally, the cellular uptake was observed using a laser scanning confocal microscope.

[0079] (2) Evaluation of pre-corneal retention of Exo-Que 10 μL of free DiR (5 μM) and DiR-labeled Exo-Que eye drops were respectively dropped into the eyes of anesthetized guinea pigs. The heads of the guinea pigs were imaged at different time points using an IVIS Lumina imaging system (PerkinElmer) to evaluate the ocular surface retention of Exo-Que eye drops. Data analysis was performed using live Image software (version 4.5.5). The experiment was repeated 3 times.

[0080] The test results of cell uptake and residence time on the ocular surface of Exo-Que are as Figure 3 shown in and Table 2, where Figure 3 A. The uptake of Exo-Que in corneal epithelial cells and human scleral fibroblasts was evaluated. The results showed that with the prolongation of the incubation time, Exo-Que labeled with DiD and showing red fluorescence accumulated extensively around the perinuclear region (as shown by blue fluorescence). Cell uptake was basically completed within a short period of time (15 - 30 min).

[0081] Figure 3 B, Table 2 and Figure 3 C. The retention of Exo-Que in front of the cornea was studied using in vivo imaging technology. The results showed that the fluorescence intensity of the free DiR group decreased rapidly, only 54% of the fluorescence was retained within 10 min, and it decreased by more than 30% within 40 min, verifying the short and time-dependent nature of the retention in front of the cornea. The retention performance of DiR-labeled Exo-Que on the ocular surface was enhanced. Over time, the fluorescence signal of DiR-labeled Exo-Que was significantly stronger than that of free DiR fluorescence signal. 90% of the fluorescence could be retained within 10 min, and about 74% of the fluorescence could still be retained after 40 min. These results indicate that Exo-Que can prolong the residence time of drugs on the anterior corneal surface, enabling the drugs to have more time to penetrate into the cornea, and solving the problems of short residence time on the ocular surface and low bioavailability of traditional eye drops.

[0082] Table 2 Fluorescence quantitative analysis of Exo-Que retention on the ocular surface in small animals by in vivo imaging (%)

[0083] Example 5 Research on the myopia prevention and control effect of Exo-Que In this example, an evaluation test was conducted on the myopia prevention and control effect of Exo-Que. The specific methods include: Using the FDM (form deprivation myopia) model as the material, it was divided into an NC group (normal control group, without treatment), an FDM group, an FDM+Exo-Que group, and an FDM+Que group. The FDM+Exo-Que group and the FDM+Que group were respectively instilled with Exo-Que and Que eye drops (with the same Que concentration) into the eyes, 6 times a day, 10 μL each time. The FDM group only removed and re-wore the 3D headgear when the other groups instilled eye drops. To reduce the influence of light on the construction of the FDM model, eye drops were instilled under dim conditions, and all operations were completed within 10 s. After instilling the eye drops, the 3D headgear was immediately worn. Before treatment, 2 weeks after treatment, and 4 weeks after treatment, all animals were measured for changes in ocular biological parameters using an A-mode ultrasound instrument, and the refractive power after cycloplegia was measured using an infrared refractometer. The eye axis of each eye was measured 10 times repeatedly, and the refractive power was measured 3 times, and the average value was taken as the final result.

[0084] The test results of the myopia prevention and control effect of Exo-Que are as Figure 4 , shown in Table 3 and Table 4, where Figure 4 A is the experimental flowchart of treating the guinea pig form deprivation myopia model with Exo-Que eye drops.

[0085] Figure 4 B and Table 3 show the changes in the eye axis length of guinea pigs in different groups. The results show that after instilling Exo-Que into the eyes, at 2 weeks and 4 weeks, the eye axis length of guinea pigs did not increase significantly compared with the normal control group, and was significantly lower than that of the simple quercetin treatment group (FDM+Que) and the myopia group (FDM), indicating that Exo-Que can effectively delay the growth of the eye axis, and the curative effect is better than that of the simple quercetin treatment group.

[0086] Table 3 Changes in refractive status of each group

[0087] Figure 4 C and Table 4 show the changes in the refractive status of guinea pigs in different groups. The myopia group (FDM) and the simple quercetin treatment group (FDM+Que) showed different degrees of myopia at 2 weeks and 4 weeks of intervention, while the Exo-Que instillation group and the normal group did not show myopia, indicating that Exo-Que has a good myopia prevention and control effect.

[0088] Table 4 Changes in eye axis length of each group

[0089] Example 6 In vivo biocompatibility evaluation of Exo-Que In this example, the in vivo biocompatibility of Exo-Que was evaluated and tested. The specific methods include: After 4 weeks of intervention using the method of Example 5, 6 guinea pigs were randomly selected from each group (NC group, FDM group, and FDM+Exo-Que group) to observe the cornea for any inflammatory reactions, edema, neovascularization, etc. under a slit lamp microscope, and the eye health status was photographed and recorded. Subsequently, the guinea pigs were euthanized by intraperitoneal injection of 4% pentobarbital, and the right eye globes and major internal organs (including the liver, kidney, lung, and heart) were removed and fixed for hematoxylin-eosin (HE) staining to observe the histological changes. At the same time, orbital blood of the guinea pigs was collected for biochemical examination to evaluate the biosafety of Exo-Que.

[0090] The in vivo biocompatibility test results of Exo-Que are as Figure 5 shown, among which, Figure 5 A The results showed that after instilling Exo-Que into the eyes, no side effects such as inflammatory reactions were caused.

[0091] Figure 5 B, Figure 5 C are the tissue sections of the important organs of the guinea pigs and the detection indexes of liver and kidney functions. The results showed that after instilling Exo-Que into the eyes, the microstructure of each internal organ was intact, no obvious histological changes were found, and there were no obvious inflammatory cells. The liver and kidney functions remained within the normal parameter range, and there was no significant difference between groups. It shows that Exo-Que has no obvious toxic effect and has good biocompatibility.

[0092] Example 7 Research on the mechanism of myopia prevention and control by Exo-Que In this example, the mechanism of myopia prevention and control by Exo-Que was studied. The specific methods include: (1) After 2 weeks and 4 weeks of treatment using the method of Example 5, 3 guinea pigs were randomly selected from each group, the scleral tissue was isolated, and about 1 mm 3 sized scleral tissue at the same posterior pole part was intercepted, fixed at 4°C with TEM for 48 h, and then fixed with 1% osmium at room temperature for 2 h in the dark. After dehydration, the tissue was embedded in an epoxy resin mixture, treated at 60°C for 48 hours, and cut into 50-60 nm sections for transmission electron microscopy examination. Before observing with the transmission electron microscope, all sections were first stained with 2% uranyl acetate saturated ethanol solution in the dark for 10 min, and then stained with 2.6% lead citrate to avoid CO2 for 10 min.

[0093] (2) The relative expression levels of TGF-β1, MMP2, a-SMA, and β-actin proteins were detected using a Protein Simple Abby capillary protein blot analyzer.

[0094] The research results on the mechanism of myopia prevention and control by Exo-Que are as Figure 6 shown, among which, Figure 6 The results of A and B showed that Exo-Que could significantly improve the thinning and sparseness of scleral fibers caused by form deprivation, inhibit scleral matrix remodeling, and delay the progression of myopia.

[0095] Figure 6 C and D were the WB detection and quantitative analysis results of scleral tissue. As shown in the figure, TGF-β1 in the scleral tissue of form deprivation myopia was significantly reduced, the expression of MMP2 increased, the expression of α-SMA increased, the transformation of scleral fibroblasts into myofibroblasts occurred, and the continuous elongation of the eye axis and the continuous increase of myopia were caused by scleral matrix remodeling; after instilling Exo-Que eye drops, the expression levels of the above indicators were reversed, scleral matrix remodeling was alleviated, and the progression of myopia was effectively controlled.

[0096] Conclusion: Through the above experimental studies, it was found that Exo-Que had good biocompatibility, strong tissue penetration ability, high bioavailability, good biosafety, and had a good control effect on the progression of myopia, and was expected to provide a new option for the drug prevention and control of myopia.

[0097] Comparative Example 1 The only difference between this comparative example and Example 1 was that anthocyanin was used instead of quercetin.

[0098] Comparative Example 2 The only difference between this comparative example and Example 1 was that nanoliposomes were used instead of mesenchymal stem cell exosomes.

[0099] Comparative Example 3 The only difference between this comparative example and Example 1 was that quercetin solution was added to the exosome solution at a volume ratio of 1:5.

[0100] Comparative Example 4 In this comparative example, commercially available 0.1% atropine was used as the sample.

[0101] In this factual example, Example 1 and Comparative Examples 1-4 were used as samples, and the myopia prevention and control effect was studied by the method of Example 5. The results are shown in Tables 5 and 6.

[0102] Table 5 Changes in refractive status of each group

[0103] Table 6 Changes in axial length of each group

[0104] As can be seen from Table 5 and Table 6, when exosomes are used to load quercetin in the treatment of myopia, the physicochemical properties of quercetin are significantly improved, its drug utilization rate is increased, and its therapeutic effect is comparable to that of atropine, providing a new drug component for myopia prevention and treatment.

[0105] The above are only examples of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A preparation method of a quercetin-loaded lipid bilayer delivery system for preventing and treating myopia, characterized in that, The method includes: mixing quercetin with exosomes for incubation, wherein the exosomes are exosomes derived from human umbilical cord mesenchymal stem cells, the concentration of quercetin is 0.1 - 5 mg / mL, the number of exosome particles is 1000 - 100000 billion, and the volume ratio of quercetin to exosomes is 1:(1 - 20).

2. The preparation method according to claim 1, wherein The conditions of the incubation include: incubating at 35°C - 40°C for 1 - 5 h.

3. The lipid bilayer delivery system loaded with quercetin prepared by the method according to claim 1 or 2, characterized in that, The particle size of the lipid bilayer delivery system loaded with quercetin is 100 nm - 200 nm.

4. A composition comprising the lipid bilayer delivery system loaded with quercetin as claimed in claim 3.

5. Use of the method according to claim 1 or 2, or the quercetin-loaded lipid bilayer delivery system according to claim 3, or the composition according to claim 4 in the preparation of a medicament for treating and / or preventing myopia, characterized in that, The lipid bilayer delivery system loaded with quercetin includes exosomes loaded with quercetin.

6. The application according to claim 5, wherein The application includes delaying the growth of the eye axis.

7. Use of the method as claimed in claim 1 or 2 or the lipid bilayer delivery system loaded with quercetin as claimed in claim 3 or the composition as claimed in claim 4 in the preparation of a drug for prolonging the residence time of a drug on the anterior corneal surface, a drug for improving the penetration efficiency of a drug, a drug for improving the utilization rate of a drug, a drug for promoting cell proliferation, a drug for promoting cell migration, and / or a drug for promoting cell injury repair.

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