A liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor, its preparation method and application

Abstract

This invention relates to the field of biomedicine, specifically to a liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF), its preparation method, and its application. This multifunctional hybrid vesicle comprises human umbilical cord mesenchymal stem cell exosomes and liposomes, with a particle number ratio of 1-4:1-4 for the human umbilical cord mesenchymal stem cell exosomes to liposomes. It is an innovative delivery system with dual functions, retaining the inherent natural targeting and biocompatibility of exosomes while integrating the efficient encapsulation and protection capabilities of liposomes for CNTF. In application, this multifunctional hybrid vesicle is used in a treatment model of traumatic optic nerve injury. This system can deliver bioactive CNTF to the injury site, effectively promoting the survival of retinal ganglion cells and axonal regeneration, demonstrating excellent therapeutic effects and providing a highly promising strategy for the treatment of central nervous system diseases such as optic nerve injury.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor, its preparation method, and its application. Background Technology

[0002] Traumatic optic nerve injury (TON) is a severe ocular trauma, often caused by direct or indirect force to the head or orbital region. This injury leads to mechanical damage and secondary degeneration of the optic nerve axons, resulting in a rapid decline in vision or even permanent blindness. The pathophysiological mechanisms of TON are complex, involving multiple aspects such as inflammatory response, oxidative stress, apoptosis, and a deficiency of neurotrophic factors. Because the regenerative capacity of the optic nerve is extremely limited once damaged, the treatment of TON remains a significant challenge, and currently, there are no effective treatments in clinical practice to promote optic nerve regeneration.

[0003] Ciliary neurotrophic factor (CNTF) is a widely proven protein that promotes neuronal survival, axonal regeneration, and myelination, showing great potential in the treatment of various neurological diseases. However, CNTF itself has extremely poor stability in vivo and is highly susceptible to degradation by proteases; for example, its half-life in rat plasma is only 2.9 minutes, which severely limits its further clinical application in the field of nerve repair.

[0004] To overcome the stability issues of CNTFs and improve their effective delivery to target sites, the development of drug delivery systems has become an important research direction. Among them, liposomes and exosomes, as two promising nanocarriers, have attracted widespread attention.

[0005] Liposomes, as biocompatible and biodegradable lipid bilayer extracellular vesicles, possess many excellent properties due to their unique amphiphilic structure. This structure allows liposomes to simultaneously encapsulate both hydrophilic and lipophilic drugs, achieving effective drug protection and targeted delivery. Furthermore, liposomes exhibit good biocompatibility and low immunogenicity, demonstrating great potential in drug delivery. However, liposomes also have some drawbacks, mainly including poor stability, susceptibility to clearance by the reticuloendothelial system, insufficient targeting, and high costs associated with large-scale production. These limitations restrict the further development of liposomes in clinical applications.

[0006] Exosomes are membrane vesicles naturally secreted by various cells, widely distributed in all body fluids, and involved in intercellular communication. As an emerging drug delivery vehicle, exosomes have attracted considerable attention due to their natural biocompatibility, low immunogenicity, ability to cross biological barriers (such as the blood-brain barrier), and potential targeting capabilities. The diverse bioactive molecules (proteins, lipids, and nucleic acids) they naturally carry also endow them with additional therapeutic potential. However, exosomes also face challenges in drug delivery, mainly manifested in low drug encapsulation efficiency, limited yield, and complex purification and separation processes. How to efficiently encapsulate therapeutic drugs into exosomes and achieve large-scale production is a current research focus and challenge.

[0007] To overcome the limitations of liposomes and exosomes, researchers have proposed a strategy to construct a hybrid exosome-liposome delivery system. This hybrid system aims to combine the advantages of both, achieving complementary benefits and thus superior drug delivery performance. For example, by fusing or co-assembling exosomes with liposomes, the ease of large-scale preparation and drug encapsulation capabilities of liposomes can be utilized, while leveraging the natural targeting and biological barrier penetration capabilities of exosomes. This hybrid delivery system holds promise for improving drug encapsulation efficiency and stability, enhancing specific delivery to target cells or tissues, reducing systemic toxicity, and ultimately improving therapeutic efficacy. Future research will focus on optimizing the construction strategy of the hybrid system and evaluating its in vitro and in vivo delivery efficiency and safety, with the aim of providing a more efficient and safer new drug delivery platform for disease treatment. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF), its preparation method, and its application.

[0009] To achieve this objective, the present invention employs the following technical solution: a liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF), wherein the liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor comprises human umbilical cord mesenchymal stem cell exosomes and liposomes; the particle number ratio of the human umbilical cord mesenchymal stem cell exosomes to liposomes is 1-4:1-4.

[0010] In some embodiments of the present invention, the ratio of the number of exosomes to liposomes of the human umbilical cord mesenchymal stem cells is 1:2.

[0011] In some embodiments of the present invention, the human umbilical cord mesenchymal stem cell exosomes are prepared by using a culture medium of human umbilical cord mesenchymal stem cells and successfully separating human umbilical cord mesenchymal stem cell exosomes by ultra-high speed centrifugation.

[0012] More preferably, the liposomes are prepared by mixing and dissolving 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), cholesterol, and phospholipid-polyethylene glycol (DSPE-PEG-2000) in ethanol, then rotary evaporating to form a membrane, hydrating the membrane with ciliary neurotrophic factor, and subjecting it to intermittent sonication; then performing three liquid nitrogen freeze-thaw cycles, allowing it to stand overnight, and filtering the liposome solution with a filter membrane the next day to obtain the liposomes.

[0013] In some embodiments of the present invention, the mass ratio of the 1,2-dioleoyl-sn-glycerol-3-phosphocholine to the cholesterol and the phospholipid-polyethylene glycol mixture is 19:1:21.

[0014] On the other hand, the present invention provides a method for preparing the liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factors, the preparation method comprising the following steps: (1) The exosomes of human umbilical cord mesenchymal stem cells and the liposomes loaded with ciliary neurotrophic factor were counted using a nanoparticle tracking analyzer; (2) Install two syringes on the microfluidic chip to fuse human umbilical cord mesenchymal stem cell exosomes and liposomes at a certain ratio and a certain flow rate; (3) Collect droplets at the end of the microfluidic chip, which are liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factors.

[0015] Preferably, in step (1), the method for preparing the liposomes loaded with ciliary neurotrophic factor includes the following steps: (1) 1,2-dioleoyl-sn-glycerol-3-phosphocholine, cholesterol and phospholipid-polyethylene glycol were dissolved in ethanol at a certain mass ratio and formed into a film by rotary evaporation to obtain a lipid film; (2) The lipid membrane was then hydrated and subjected to intermittent sonication on ice; (3) After three liquid nitrogen freeze-thaw cycles, the solution was left to stand overnight at 4°C. The next day, the liposome solution was filtered twice using a 0.22 µm filter membrane.

[0016] More preferably, in step (1), the mass ratio of the mixture of 1,2-dioleoyl-sn-glycerol-3-phosphocholine, cholesterol, and phospholipid-polyethylene glycol is 19:1:21; in step (2), a liposome film suspension is obtained after hydration; the intermittent ultrasonic treatment is performed at 30% power, with a 5-second pause every 5 seconds for a total of 10 minutes.

[0017] In some embodiments of the present invention, the microfluidic chip has a structure comprising a main channel and several periodically arranged "V"-shaped micron-scale barrier structures; the ratio of the number of particles fusing human umbilical cord mesenchymal stem cell exosomes and liposomes in the microfluidic chip is 1-4:1-4, and the flow rate is 0.5-2.5 μl / s; the syringe has a volume of 1 ml.

[0018] Preferably, the ratio of human umbilical cord mesenchymal stem cell exosomes to liposomes in the microfluidic chip is 4:1, 2:1, 1:1, 1:2 or 1:4, and the flow rate is 0.5 μl / s, 1 μl / s, 1.5 μl / s, 2 μl / s or 2.5 μl / s.

[0019] In another aspect, the present invention provides the application of the liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor or the liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor prepared by the above preparation method in the preparation of drugs for treating optic nerve injury.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF) provided by the present invention solves the problem of poor in vivo stability and easy degradation of CNTF; it makes full use of the biocompatibility and targeting of exosomes, combined with the encapsulation efficiency and protective effect of liposomes, to provide a stable carrier for CNTF, prolong its half-life in vivo, and achieve effective delivery to diseased nerve tissue.

[0021] (2) The liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF) provided by the present invention is an innovative delivery system with dual functions: it retains the inherent natural targeting and biocompatibility of exosomes, and integrates the efficient encapsulation and protection capabilities of liposomes for CNTF, thereby significantly prolonging the half-life of CNTF in vivo and improving its stability.

[0022] (3) The present invention provides a method for preparing liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor (CNTF). Through synergistic innovation of microfluidic chip structure and fusion mechanism, and by inducing fusion with unique fluid dynamics, a specific microchannel geometry is designed to generate unique fluid shear forces and diffusion modes, thereby effectively triggering membrane fusion between exosomes and liposomes under mild conditions. The membrane fusion mediated by microfluidic chip technology leverages its advantages of high efficiency, mildness, precise control, and high throughput. By precisely manipulating fluids within microscale channels, microfluidic technology can… Achieving precise control over the mixing, interaction time, and fusion conditions of nanomembrane particles, thereby improving fusion efficiency and product uniformity; a microfluidic fusion strategy for efficient CNTF encapsulation and protection: microfluidic chip technology promotes the fusion of CNTF-loaded lipid nanoparticles and exosomes. During the fusion process, CNTF remains inactive and does not degrade, maximizing the protection of the inherent properties of exosomes and CNTF; balancing high throughput and high uniformity: high fusion efficiency and high uniformity of fusion products ensure batch-to-batch stability. Fixed chip flow rate and particle concentration minimize batch-to-batch variability.

[0023] (4) The present invention provides a liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF) in an application level. The multifunctional hybrid vesicle is applied to a treatment model of traumatic optic nerve injury. The experimental results confirm that the system can deliver bioactive CNTF to the injury site, effectively promote the survival of retinal ganglion cells and axon regeneration, and show excellent therapeutic effect. It provides a novel strategy with great potential for the treatment of central nervous system diseases such as optic nerve injury. Attached Figure Description

[0024] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 The results show the Umsc-ev electron microscopy characterization and NTA. Figure 1 In the figure, A represents the morphology of exosome particles observed by TEM. Figure 1 In this context, B represents the exosome particle size analyzed by NTA. Figure 1 In the figure, C represents the marker protein of exosomes detected by Western blotting at protein concentrations of 5 μg and 10 μg.

[0025] Figure 2 The results show the electron microscopy characterization and NTA of liposomes; among them, Figure 2 In the figure, A represents the morphology of blank liposome particles observed by TEM. Figure 2 In the figure, B represents the liposome particle size analyzed by NTA.

[0026] Figure 3 To observe the morphology of CNTF protein-loaded liposome particles using TEM.

[0027] Figure 4 To test the 18-day stability (size, PDI, zeta) of liposomes using a DLS instrument; among which, Figure 4 In the figure, A represents the changes in particle size and PDI of liposomes observed over 18 days. Figure 4 B in the figure represents the zeta potential change detection of liposomes observed over 18 days.

[0028] Figure 5 Electron microscopy characterization and NTA results of hybrid fusion vesicles; among them, Figure 5 In the figure, A represents the morphology of the fused vesicles after the microfluidic chip was fused with exosomes and liposomes, as observed by TEM. Figure 5 In section B, NTA analysis was used to compare the particle size of fusion vesicles, exosomes, and liposomes. Figure 5 In the middle, C represents the changes in particle size and zeta potential of fusion vesicles, exosomes, and liposomes analyzed by DLS.

[0029] Figure 6 This image shows the results of Western blotting (WB) analysis to identify marker proteins before and after exosome fusion.

[0030] Figure 7 The prepared blank liposome suspension (concentration of 1) 10 12 (each / ml).

[0031] Figure 8 Estimate the encapsulation efficiency after liposomes are encapsulated with 100 μg / ml CNTF; Figure 9 This diagram illustrates the principle of the FRET experiment and the FRET effect after hybridization (including direct incubation for 4 hours and chip hybridization); among which, Figure 9 In the diagram, A represents the Fret experiment. Figure 9 In Figure B, the Fret effect variation curves after 4 hours of incubation and fusion with the microfluidic chip are shown. Figure 9 C represents the degree of change in the Fret effect after 4 hours of incubation and fusion with the microfluidic chip.

[0032] Figure 10 A statistical plot of Fret results was used to screen for the optimal flow rate and lipid exosome ratio; among which, Figure 10 Figure A shows a comparison of the degree of change in the Fret effect when the microfluidic chip is fused at different speeds. Figure 10 B shows a comparison of the degree of change in the Fret effect when using a fusion ratio of liposomes and exosomes in a microfluidic chip (labeled). (Represents the conditions for the final selection).

[0033] Figure 11 To directly observe the localization effect and PCC results of microglia phagocytosis of fused and unfused particles under confocal microscopy.

[0034] Figure 12 This is a schematic diagram of a microfluidic chip.

[0035] Figure 13 The results of voltage drop and nanoparticle collision tests on the chip were performed using COMSOL software; among them, Figure 13 Image A shows the pressure drop difference of the liquid flowing through the microfluidic chip without the "V" shape, as observed using COMSOL software, compared to the microfluidic chip with multiple "V" shapes in this study. Figure 13 In the figure, B represents the particle distribution of 100 nm particles flowing through a microfluidic chip without a "V" shape, as simulated by COMSOL software, and the multi-V "V" shaped microfluidic chip used in this study.

[0036] Figure 14 This diagram illustrates the fabrication and assembly of a microfluidic chip and provides a visual representation of fluid fusion using two dyes.

[0037] Figure 15 Images of optic nerve axons after injury in mice that have undergone specified treatment, including fluorescent staining and quantitative analysis of the number of optic nerve axons; among them, Figure 15 Image A shows fluorescently stained optic nerve axons on day 14 after injury in mice treated with the specified method. The axons were traced and labeled with cholera toxin B subunit-488 (CTB-488) (green), and the optic nerve pinch site was marked with a red asterisk. Figure 15 B represents the quantitative analysis results of the number of optic nerve axons within a range of 100-1300 μm from the injury site.

[0038] Figure 16 The images show a complete retinal patch specimen and the results of quantitative analysis of retinal ganglion cells in mice after injury following specified treatment. Figure 16 In the image, A represents the central, intermediate, and peripheral areas of a complete retinal specimen (C) taken 14 days after intravitreal injection (IVT). Figure 16 Image B shows the quantitative analysis results of RBPMS-positive retinal ganglion cells (RGCs) in different regions of the retinal patch on day 14 after optic nerve clipping (ONC). Figure 16 C is a complete retinal smear specimen taken 14 days after intravitreal injection (IVT).

[0039] Figure 17 This image shows optical coherence tomography (OCT) images of the thickness of each retinal layer and quantitative analysis results of the thickness of the retinal ganglion cell complex in mice after injury following specified treatment. Figure 17Image A shows representative images from different processing groups, displaying corresponding optical coherence tomography (OCT) images of the thickness of each retinal layer; Figure 17 Image B represents the quantitative analysis results of the thickness of the retinal ganglion cell complex (GCC) on day 14 after specified treatment following optic nerve clipping (ONC). Detailed Implementation

[0040] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0041] Example 1: This example is a liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF). A liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF) comprises human umbilical cord mesenchymal stem cell exosomes and liposomes; the particle ratio of the human umbilical cord mesenchymal stem cell exosomes to liposomes is 1:2. Preparation of the human umbilical cord mesenchymal stem cell exosomes: Human umbilical cord mesenchymal stem cell exosomes were successfully separated by ultracentrifugation using a culture medium containing human umbilical cord mesenchymal stem cells. Preparation of the liposomes: 1,2-dioleoyl-sn-glycerol-3-phosphocholine, cholesterol, phospholipids, and polyethylene glycol were mixed in a mass ratio of 19:1:21, dissolved in ethanol, and then rotary evaporated to form a membrane. The membrane was hydrated with PBS or a drug solution and subjected to intermittent sonication. After three liquid nitrogen freeze-thaw cycles, the membrane was incubated overnight at 4°C, and the liposome solution was filtered through a filter membrane the following day.

[0042] The above-mentioned method for preparing liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor (CNTF) includes the following steps: (1) The exosomes of human umbilical cord mesenchymal stem cells and liposomes carrying ciliary neurotrophic factor were counted using an analyzer; the preparation method of the liposomes carrying ciliary neurotrophic factor included the following steps: 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), cholesterol and phospholipid-polyethylene glycol (DSPE-PEG-2000) were dissolved in ethanol at a mass ratio of 19:1:21 and formed into a membrane by rotary evaporation; then the membrane was hydrated and subjected to intermittent ultrasonic treatment on ice (30% power, 5s on, 5s off, 10 minutes); after three liquid nitrogen freeze-thaw cycles, it was left to stand overnight at 4°C, and the liposome solution was filtered twice the next day using a 0.22 µm filter membrane.

[0043] (2) Install two syringes on the microfluidic chip to fuse human umbilical cord mesenchymal stem cell exosomes and liposomes at a certain ratio and a certain flow rate; the ratio of the number of particles of human umbilical cord mesenchymal stem cell exosomes and liposomes fused in the microfluidic chip is 1:2, and the flow rate is 1 µl / s; the volume of the syringe is 1 ml.

[0044] (3) Collect droplets at the end of the chip, which are ciliary neurotrophic factor exosome-liposome hybrid vesicles.

[0045] The microfluidic chip features a main channel (160 μm high, 180 μm wide) and a series of periodically arranged micron-sized herringbone-shaped obstruction structures (80 μm high, 80 μm wide), with a minimum dimension of 180×80×80 μm. To verify the hydrodynamic performance of this design, the inventors conducted numerical simulations using COMSOL software. Compared to a conventional channel without obstruction structures, the pressure drop of the microfluidic chip increased to three times, indicating that it can induce more vigorous fluid collisions. Particle tracking simulations also showed that this design significantly enhances the contact frequency between nanoparticles and the channel walls. Furthermore, to evaluate its actual mixing and flow performance, red and blue dyes were introduced at a 1:1 flow rate ratio. Figure 12-14 As shown, the dye solution rapidly mixes into purple at the intersection of the two channels and flows stably throughout the chip, demonstrating its excellent mixing efficiency and flow stability. Figure 12 This is a schematic diagram of a microfluidic chip. Figure 13 The results of voltage drop and nanoparticle collision tests on the chip were performed using COMSOL software; among them, Figure 13 Image A shows the pressure drop difference of the liquid flowing through the microfluidic chip without the "V" shape, as observed using COMSOL software, compared to the microfluidic chip with multiple "V" shapes in this study. Figure 13 In the figure, B represents the particle distribution of 100nm particles flowing through a microfluidic chip without a "V" shape, as simulated by COMSOL software, and the multi-V "V" shaped microfluidic chip used in this study. Figure 14 This diagram illustrates the fabrication and assembly of a microfluidic chip and provides a visual representation of fluid fusion using two dyes.

[0046] Example 2: This example is a liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF). The liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor (CNTF) described in this embodiment are identical to those in Example 1, except that the ratio of human umbilical cord mesenchymal stem cell exosomes to liposomes is 1:4, and the preparation method is the same as in Example 1, except that the ratio of human umbilical cord mesenchymal stem cell exosomes to liposomes fused in the microfluidic chip is 1:4 and the flow rate is 1 µl / s.

[0047] Example 3: This example is a liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF). The liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor (CNTF) described in this embodiment are identical to those in Example 1, except that the ratio of human umbilical cord mesenchymal stem cell exosomes to liposomes is 1:1, and the preparation method is the same as in Example 1, except that the ratio of human umbilical cord mesenchymal stem cell exosomes to liposomes fused in the microfluidic chip is 1:1 and the flow rate is 0.5 µl / s.

[0048] Example 4: This example is a liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF). The liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor (CNTF) described in this embodiment are identical to those in Example 1, except that the ratio of human umbilical cord mesenchymal stem cell exosomes to liposomes is 4:1, and the preparation method is the same as in Example 1, except that the ratio of human umbilical cord mesenchymal stem cell exosomes to liposomes fused in the microfluidic chip is 4:1 and the flow rate is 2.5 µl / s.

[0049] Example 5: This example is a liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF). The liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor (CNTF) described in this embodiment are identical to those in Example 1, except that the ratio of human umbilical cord mesenchymal stem cell exosomes to liposomes is 2:1, and the preparation method is the same as in Example 1, except that the ratio of human umbilical cord mesenchymal stem cell exosomes to liposomes fused in the microfluidic chip is 2:1 and the flow rate is 2 µl / s.

[0050] The following sample and instrument parameter settings and data acquisition, as well as a liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor (CNTF) from Example 1, were used in Experiments 1-6.

[0051] Sample preparation: The samples were stored in an icebox to maintain their stability. Before particle size analysis, the samples were diluted to the appropriate measurement concentration using 0.1 µm filtered PBS. Before loading, the samples were vortexed for 30 seconds to ensure homogeneity. The samples were UMSC-EVs (human umbilical cord mesenchymal stem cell exosomes), liposomes, and ciliary neurotrophic factor exosome-liposome hybrid vesicles from Example 1.

[0052] Instrument parameter settings and data acquisition: In the nanoparticle tracking analyzer software, each sample was measured three times repeatedly, with each recording time set to 30 seconds, the detection threshold set to 30, and the autosampler flow rate set to 30. Ideally, the measurement field of view should contain 20 to 80 particles per frame. To ensure data comparability, the measurement parameters for all samples must be consistent. After measurement, the particle size distribution map and corresponding concentration data for each UMSC-EV (human umbilical cord mesenchymal stem cell exosome) sample should be saved for subsequent analysis.

[0053] Experimental Example 1: Observation of the morphology of exosomes and liposomes using transmission electron microscopy (TEM) and analysis of their particle size distribution using an NTA analyzer. Experimental methods: 1. Transmission electron microscopy (TEM) observation of exosome morphology: According to the sample preparation requirements for EV (umbilical vein mesenchymal stem cell exosomes), all reagents used in the experiment must be filtered through a 0.1-micron filter membrane. The sample preparation steps are as follows: (1) Sample fixation: Take 20 μL of well-mixed sample and mix it with 20 μL of 4% paraformaldehyde in equal volume, and gently blow and mix.

[0054] (2) Sample adsorption: Drop the mixed sample onto the sealing film, cover it with a carbon mesh using clean tweezers, and let it stand for 30 minutes.

[0055] (3) Sample cleaning: Clean the carbon mesh with PBS for 2 minutes each time, repeat twice.

[0056] (4) Secondary fixation: Fix with 1% glutaraldehyde PBS solution for 5 min.

[0057] (5) Sample cleaning: Clean with ddH2O for 30s, repeat 3-5 times.

[0058] (6) Background negative staining: Negative stain with 2% uranium acetate for 30s, remove excess liquid, and dry overnight at room temperature.

[0059] (7) Morphological observation: Observed under a 200kV transmission electron microscope.

[0060] 2. Observation of liposome particle morphology using transmission electron microscopy (TEM): Liposome concentration diluted to 1×10⁻⁶. 12 Samples were collected at a density of 1 sample / ml, covered with a carbon mesh, and allowed to stand for 30 minutes. Moisture was absorbed with filter paper, and the sample was then placed in uranium acetate for 30 seconds. Residue was absorbed with filter paper, and the sample was dried overnight in a desiccator. Morphological observation: Images were taken using a Talos F200S microscope at 200 kV.

[0061] Experimental results: Figure 1To collect culture medium for human umbilical cord mesenchymal stem cells, UMSC-EV (human umbilical cord mesenchymal stem cell exosomes) were successfully isolated and obtained by ultracentrifugation. The inventors of this application conducted a comprehensive identification and analysis of the collected MSC-EV suspension. Figure 1 In the figure, A represents the morphology of exosome particles observed by TEM. Figure 1 In this context, B represents the exosome particle size analyzed by NTA. Figure 1 In the figure, C represents the marker protein of exosomes detected by Western blotting at protein concentrations of 5 μg and 10 μg.

[0062] First, the particle size of the vesicles was determined using nanoparticle tracking analysis (NTA), showing that the diameter of MSC-EVs ranged from 30 to 200 nanometers, consistent with the typical size characteristics of extracellular vesicles. Second, Western blot analysis detected specific molecular markers of extracellular vesicles, including CD63, CD9, and Flot1. The positive expression of these markers further confirmed the EV characteristics of the isolated product. Furthermore, the inventors used transmission electron microscopy (TEM) to observe the ultrastructure of the vesicles, clearly capturing the unique cup-shaped or disc-shaped morphology of extracellular vesicles. Based on the above results and in accordance with the isolation and identification standards published by the International Society for Extracellular Vesicle Research (ISEV), it was confirmed that purified extracellular vesicles were successfully isolated and extracted from human umbilical cord mesenchymal stem cells.

[0063] Figure 2 and Figure 3 Analysis: The inventors used transmission electron microscopy (TEM) to observe the ultrastructure of liposomes, clearly capturing the lipid bilayer morphology of liposomes. Using TEM to observe liposomes encapsulating CNTF protein, it was observed that the hollow structure disappeared, indicating that the liposomes had encapsulated the protein. Figure 2 The results show the electron microscopy characterization and NTA of liposomes; among them, Figure 2 In the figure, A represents the morphology of blank liposome particles observed by TEM. Figure 2 In the figure, B represents the liposome particle size analyzed by NTA. Figure 3 To observe the morphology of CNTF protein-loaded liposome particles using TEM.

[0064] Experimental Example 2: The size, zeta potential, and PDI of the liposomes from Example 1 were determined by dynamic light scattering (DLS, Zetasizer Nano-ZS90, Malvern Instruments Limited, UK). Figure 4To test the 18-day stability (size, PDI, zeta) of liposomes using a DLS instrument, the liposomes were prepared and left to stand overnight before testing began the following day. The particle size of the vesicles was determined using NTA technology, showing a diameter range of 100-150 nm. The size, PDI, and zeta were measured using a DLS instrument (Zetasizer Nano (Malvern, UK)). The results showed a liposome size of approximately 120 nm, consistent with the NTA results. The PDI was between 0.1 and 0.3, indicating good uniformity of the prepared liposomes. The zeta potential was around -30 mV, consistent with the potential set for the lipid components in this application. To determine the stability of the liposomes, the inventors also monitored their colloidal stability for up to 18 days. The liposomes exhibited good stability in terms of PDI, size, and zeta potential. Figure 4 To test the 18-day stability (size, PDI, zeta) of liposomes using a DLS instrument; among which, Figure 4 In the figure, A represents the changes in particle size and PDI of liposomes observed over 18 days. Figure 4 B in the figure represents the zeta potential change detection of liposomes observed over 18 days.

[0065] Experimental Example 3: Characterization of liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor (CNTF) under electron microscopy and analysis of nanoparticle size distribution using an NTA analyzer; Western blotting to verify biological characteristics before and after exosome fusion. Figure 5 Electron microscopy characterization and NTA results of hybrid fusion vesicles; among them, Figure 5 In the figure, A represents the morphology of the fused vesicles after the microfluidic chip was fused with exosomes and liposomes, as observed by TEM. Figure 5 In section B, NTA analysis was used to compare the particle size of fusion vesicles, exosomes, and liposomes. Figure 5 C represents the changes in zeta potential of fused vesicles, exosomes, and liposomes analyzed by DLS. The inventors used transmission electron microscopy (TEM) to observe the ultrastructure of the fused particles on the chip. The image clearly shows the fusion of the two types of particles, indicating their fusion morphology. NTA results show that after the chip fused with exosomes and liposomes, the particle size increased slightly, and the zeta potential decreased slightly compared to liposomes.

[0066] Figure 6To verify that the biological characteristics of exosomes did not change significantly before and after fusion by Western blotting, and to verify that the fused exosomes retained their original biological characteristics, Western blotting experiments were performed to verify the characteristic proteins of EVs, Flottin-1, Alix, and CD9. The Western blotting results showed that there was no significant difference in the specific molecular markers of extracellular vesicles between simple exosomes and fused exosomes. The lack of significant difference in these markers before and after fusion indicates that the characteristics of the fused exosomes did not change significantly.

[0067] Experiment Example 4: Specific steps or methods for microfluidic fusion of exosomes and liposomes To verify the formation of exosome-liposome hybrids, the inventors used the FRET experiment for evaluation. The FRET experiment steps are as follows: (1) Labeling exosomes: Add a certain amount of Dil and CY5-NHS fluorescent dye to the exosomes, place them on a vertical shaker, and incubate at room temperature for 30 minutes. Use a 3KD ultrafiltration tube, wash three times with PBS to remove unbound dye.

[0068] (2) Incubation group: Exosomes and liposomes were added to EP tubes in a certain ratio and incubated in a constant temperature shaker with a final volume of 200 μl. Chip set: Exosomes and liposomes were quantified by NTA counting. The final particle concentration was the same as that of the incubation group. Exosomes were added to well 1 and liposomes were added to well 2. The injection rates and volumes of the two wells were equal.

[0069] (3) Fluorescence spectroscopy analysis: The sample was excited at 550 nm and the emission spectrum between 550 nm and 700 nm was measured to analyze the FRET effect.

[0070] Chip flow rate: The ratio of exosomes to liposomes was set to 1:1, and the concentration was 1. 10 10 Samples were collected at concentrations of 1 sample per ml and microfluidic injection rates of 0.5 μl / s, 1 μl / s, 1.5 μl / s, 2 μl / s, and 2.5 μl / s, respectively, for fluorescence spectroscopy analysis.

[0071] Exosome to liposome ratio: Exosomes and liposomes were added to the two injection ports at particle number ratios of 4:1, 2:1, 1:1, 1:2, and 1:4, respectively, with the total volume and liposome number being consistent, while varying the number of exosomes. Microfluidic injection was performed at a rate of 1 μl / s, and samples were collected for fluorescence spectroscopy analysis.

[0072] Experimental results: Figure 7 The prepared blank liposome suspension (concentration of 1) 10 12 (pcs / ml) Figure 8 Estimate the encapsulation efficiency after encapsulating 100 μg / ml CNTF[LL1.1] with liposomes; Figure 9 This diagram illustrates the principle of the FRET experiment and the FRET effect after hybridization (including direct incubation for 4 hours and chip hybridization); among which, Figure 9 In the diagram, A represents the Fret experiment. Figure 9 In Figure B, the Fret effect variation curves after 4 hours of incubation and fusion with the microfluidic chip are shown. Figure 9 C represents the degree of change in the Fret effect after 4 hours of incubation and fusion with the microfluidic chip. Figure 10 A statistical plot of Fret results was used to screen for the optimal flow rate and lipid exosome ratio; among which, Figure 10 Figure A shows a comparison of the degree of change in the Fret effect when the microfluidic chip is fused at different speeds. Figure 10 B shows a comparison of the degree of change in the Fret effect when using a fusion ratio of liposomes and exosomes in a microfluidic chip (labeled). (Representing the final selected conditions). The liposome spectrum represents before hybridization, and the HE spectrum and 4-hour incubation represent after hybridization. The FRET effect weakens after hybridization, which can only occur when the distance between FRET pairs increases. This indicates that EVs were inserted into the lipid bilayer of the liposomes, confirming the success of hybridization. Furthermore, comparing the results after 4 hours of incubation with those after chip hybridization, it is evident that chip hybridization shows greater recovery of energy transfer, indicating that our chip hybridization achieves a higher degree and efficiency of fusion than direct incubation. To optimize hybridization conditions, the optimal flow rate and liposome ratio were screened by quantifying the FRET intensity (calculated as: FRET intensity = (F670 / (F567 + F670)) × 100), where F567 = fluorescence emission at 567 nm and F670 = fluorescence emission at 670 nm. Flow rate optimization results show that flow rates of 1 µL / s and below can achieve efficient FRET recovery, with 1 µL / s ensuring both fusion effect and highest flow efficiency, thus it was selected as the condition for subsequent experiments. Optimization of the liposome to exosome ratio showed that the fusion efficiency reached its peak when the mass ratio of liposomes to exosomes was 2:1. Therefore, subsequent experiments were conducted using a flow rate of 1 µL / s and a liposome to exosome ratio of 2:1.

[0073] Experiment Example 5: Observation of the fusion effect of liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor (CNTF) under confocal microscopy Experimental Methods: Microglia were grown to 50% using 24-well plate slides. ① Dio-lipo and DiI-EV were fused at a 2:1 ratio using microfluidic technology at a flow rate of 1 μl / s, and the concentration was measured according to NTA. ② Equal numbers of DiI-EV and Dio-lipo were directly added. Cells were incubated for 6 hours after addition. All subsequent operations were performed under dark conditions.

[0074] Experimental results: Colocalization analysis was performed by observing the uptake of EV+Liposome and Hybrid EV by microglia using confocal microscopy, and the degree of colocalization between fluorescence signals of different EV-Dil and Liposome-Dio was quantified by Pearson correlation coefficient (PCC). Figure 11 The fusion effect of exosome-liposome hybrid vesicles was observed under a confocal microscope. The figures show that the hybrid group exhibited high colocalization, with a PCC of approximately 0.91; while the group directly adding EVs and liposomes showed low colocalization, with a PCC of approximately 0.57. This further confirms the fusion between exosomes and liposomes.

[0075] Experimental Example 6: Application of the liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor (CNTF) of Example 1 of the present invention in the treatment of optic nerve injury. Experimental Methods: C57BL / 6 mice (4-6 weeks old) were deeply anesthetized with aphthylamine. After topical anesthesia with propofol in both eyes, the eyeballs were removed from their sockets, and surrounding tissues were separated and trimmed to expose the optic nerve. Using cross-locking forceps, the optic nerve was clamped 2 mm posterior to the eyeball for 3 seconds. The eyeballs were repositioned, eye ointment was applied, and analgesia was administered. Postoperatively, the mice were placed on a heating pad for recovery, and the ocular infection was checked every 12-24 hours.

[0076] Two days prior to euthanasia, cholera toxin B subunit (CTB) labeled with Alexa Fluor 488 (ThermoFisher, USA) was injected intravitreally to anterogradely track regenerating axons in the optic nerve (ON). The optic nerve was fixed, dehydrated, embedded in a compound for optimal cutting temperature (SAKURA, Japan), and frozen in liquid nitrogen. Longitudinal sections (14 μm) were cut, and the CTB-labeled regenerating axons were imaged using a fluorescence microscope (DM4B, Leica, Germany). The number of individual axons was manually counted according to previous studies. The total number of regenerating axons (Σad) was quantified using the following formula: Σad = πr² x (average number of axons) / (2r xt), where the radius of the nerve (r) was measured at the point (d) where the total number of axons was calculated, and the section thickness was t = 14 μm.

[0077] Mice were euthanized and their hearts were perfused with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Eyes were removed and fixed with 4% PFA for 2 hours at room temperature. After washing three times in PBS, the retina was dissected into a clover shape and blocked at 37°C with 0.5% Triton X-100 and 10% donkey serum for 2 hours. Next, the retina was incubated overnight on a shaker at 4°C with anti-RBPMS antibody (1:500; ProteinTech, China). After washing again, the retina was incubated with secondary antibody at 37°C for 2 hours. Finally, the retina was transferred to a glass slide and mounted using anti-quenching mounting medium (Sigma, USA). The fat-mounted retina was observed under a 4B fluorescence microscope (Leica, Germany) with a 20x objective lens. Images were collected in each of the four quadrants of the retina using ImageJ software (multi-point function), starting from the optic nerve head, at 1.5, 2.5, and 3.5 mm, respectively, and the overall RGC survival rate was estimated by calculating the number of RGCs in 12 different regions.

[0078] The ganglion cell complex (GCC), composed of the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), and inner plexiform layer (IPL), is a reliable biomarker for assessing the integrity of the retinal ganglion cell / optic nerve (RGC / ON). Optical coherence tomography (OCT) was used to measure the thickness of the GCC. After anesthesia, mice were instilled with tropicamide eye drops to dilate the pupils. Subsequently, OCT images were acquired using a MicronIV retinal imaging microscope (Phoenix, USA) in peripapillary circular scanning mode. Built-in software was used to segment the GCC and quantify its thickness.

[0079] Experimental results: Figure 15 Images of optic nerve axons after injury in mice that have undergone specified treatment, including fluorescent staining and quantitative analysis of the number of optic nerve axons; among them, Figure 15 Image A shows a fluorescently stained optic nerve axon on day 14 after injury in mice that have undergone the specified treatment. The axons are traced and labeled with cholera toxin B subunit-488 (CTB-488) (green), and the red asterisks indicate the optic nerve pinch site. Figure 15 B represents the quantitative analysis results of the number of optic nerve axons within a range of 100-1300 μm from the injury site.

[0080] Figure 16 The images show a complete retinal patch specimen and the results of quantitative analysis of retinal ganglion cells in mice after injury following specified treatment. Figure 16 In the image, A represents the central, intermediate, and peripheral areas of a complete retinal specimen (C) taken 14 days after intravitreal injection (IVT). Figure 16Image B shows the quantitative analysis results of RBPMS-positive retinal ganglion cells (RGCs) in different regions of the retinal patch on day 14 after optic nerve clipping (ONC). Figure 16 C is a complete retinal smear specimen taken 14 days after intravitreal injection (IVT).

[0081] Figure 17 This image shows optical coherence tomography (OCT) images of the thickness of each retinal layer and quantitative analysis results of the thickness of the retinal ganglion cell complex in mice after injury following specified treatment. Figure 17 Image A shows representative images from different processing groups, displaying corresponding optical coherence tomography (OCT) images of the thickness of each retinal layer; Figure 17 Image B represents the quantitative analysis results of the thickness of the retinal ganglion cell complex (GCC) on day 14 after specified treatment following optic nerve clipping (ONC).

[0082] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor, characterized in that, The liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor include human umbilical cord mesenchymal stem cell exosomes and liposomes; the particle number ratio of the human umbilical cord mesenchymal stem cell exosomes to liposomes is 1-4:1-4.

2. The liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor as described in claim 1, characterized in that, The ratio of exosomes to liposomes in the human umbilical cord mesenchymal stem cells is 1:

2.

3. The liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor as described in claim 2, characterized in that, Preparation of human umbilical cord mesenchymal stem cell exosomes: Human umbilical cord mesenchymal stem cell exosomes were successfully obtained by using culture medium of human umbilical cord mesenchymal stem cells and ultra-high speed centrifugation. The liposomes were prepared by dissolving a mixture of 1,2-dioleoyl-sn-glycerol-3-phosphocholine, cholesterol, phospholipids, and polyethylene glycol in ethanol, followed by rotary evaporation to form a membrane. The membrane was then hydrated with ciliary neurotrophic factor and subjected to intermittent sonication. After three liquid nitrogen freeze-thaw cycles, the solution was allowed to stand overnight, and the liposome solution was filtered through a filter membrane the next day to obtain the liposomes.

4. The liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor as described in claim 3, characterized in that, The mass ratio of the mixture of 1,2-dioleoyl-sn-glycerol-3-phosphocholine, cholesterol, and phospholipid-polyethylene glycol is 19:1:

21.

5. A method for preparing liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factor according to any one of claims 1-4, the method comprising the following steps: (1) The exosomes of human umbilical cord mesenchymal stem cells and the liposomes loaded with ciliary neurotrophic factor were counted using a nanoparticle tracking analyzer; (2) Install two syringes on the microfluidic chip to fuse human umbilical cord mesenchymal stem cell exosomes and liposomes at a certain ratio and a certain flow rate; (3) Collect droplets at the end of the microfluidic chip, which are liposome-exosome hybrid vesicles loaded with ciliary neurotrophic factors.

6. The preparation method according to claim 5, characterized in that, In step (1), the method for preparing the liposomes loaded with ciliary neurotrophic factor includes the following steps: (1) 1,2-dioleoyl-sn-glycerol-3-phosphocholine, cholesterol and phospholipid-polyethylene glycol were dissolved in ethanol at a certain mass ratio and formed into a film by rotary evaporation to obtain a lipid film; (2) The lipid membrane was then hydrated and subjected to intermittent sonication on ice; (3) After three liquid nitrogen freeze-thaw cycles, the solution was left to stand overnight at 4°C. The next day, the liposome solution was filtered twice using a 0.22 µm filter membrane.

7. The preparation method according to claim 6, characterized in that, In step (1), the mass ratio of the mixture of 1,2-dioleoyl-sn-glycerol-3-phosphocholine, cholesterol, and phospholipid-polyethylene glycol is 19:1:21; In step (2), a liposome membrane suspension is obtained after hydration; the intermittent ultrasonic treatment is performed at 30% power, with a 5-second interval between treatments and a 5-second pause, for a total of 10 minutes.

8. The preparation method according to claim 5, characterized in that, The structure of the microfluidic chip includes a main channel and several periodically arranged "herringbone" micron-sized barrier structures. In the microfluidic chip, the ratio of exosomes of human umbilical cord mesenchymal stem cells to liposomes is 1-4:1-4, and the flow rate is 0.5-2.5 μl / s; the volume of the syringe is 1 ml.

9. The preparation method according to claim 8, characterized in that, In the microfluidic chip, the particle ratio of human umbilical cord mesenchymal stem cell exosomes fused with liposomes is 4:1, 2:1, 1:1, 1:2 or 1:4, and the flow rate is 0.5 μl / s, 1 μl / s, 1.5 μl / s, 2 μl / s or 2.5 μl / s.

10. The use of the liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor according to any one of claims 1-4, or the liposome-exosome hybrid vesicle loaded with ciliary neurotrophic factor prepared by the preparation method according to any one of claims 5-9, in the preparation of a medicament for treating optic nerve injury.

Images