CN116763945A - Exosome diagnosis and treatment agent based on M2 microglial cells, preparation method and application thereof in preparation of medical imaging probes - Google Patents

Abstract

The present invention provides an exosome diagnostic and therapeutic agent based on M2 microglia, a preparation method and its application in preparing medical imaging probes. Exosomes derived from M2 microglia are prepared and labeled and modified to be able to target. Small molecule ligands to brain neurons can actively target and migrate specifically to the stroke area at high doses, improving the retention ability of exosome therapeutic agents in the stroke area and improving their repair efficacy in the stroke area. For the first time, chelator-free direct co-labeling of fluorescent dyes, SPECT signal radionuclide ¹²⁵ I and magnetic resonance _T2- weighted contrast agent superparamagnetic iron oxide has been achieved, which satisfies the requirements of multi-modality equipment for fluorescence imaging, nuclear medicine and magnetic resonance imaging. The demand for imaging probes enables non-invasive, accurate, real-time and dynamic in vivo monitoring of the migration, homing and distribution of exosome therapeutic agents in the target area under different administration routes, and at the same time evaluates the treatment effect in order to adjust the administration time and achieve Effective repair of stroke further improves the therapeutic effect.

Description

Exosome therapeutic agents based on M2 microglia, preparation methods and their preparation Applications in Medical Imaging Probes

Technical field

The invention belongs to the field of biomedicine, and in particular relates to an exosome therapeutic agent based on M2 microglia, a preparation method and its application in preparing medical imaging probes.

Background technique

Stroke is the main cause of disability and death worldwide. Due to the limitations of treatment time window and treatment methods, the diagnosis and treatment situation is grim. In recent years, extracellular vesicles (EVs), including exosomes, have shown great potential in treating stroke. Our previous studies have shown that M2 microglia-derived exosomes can reduce neuroinflammation, protect mouse brains from ischemia-reperfusion injury, and promote white matter repair and remodeling in stroke mice. However, the lack of effective strategies for in vivo visualization based on EVs treatment to clarify the target site migration efficiency during EVs treatment has seriously hindered its clinical translation. In addition, in the face of the promising treatment of EVs, there is currently controversy over which method of administration is optimal for the best therapeutic effect in stroke. Therefore, a comprehensive visualization and understanding of the directional migration and distribution of EVs during therapy is crucial for their further clinical translation.

In order to address the above unresolved needs, there is an urgent need for advanced imaging technologies that can perform non-invasive, reproducible and quantitative monitoring of exogenously introduced EVs. In terms of imaging diagnosis, conventional computed tomography (CT) imaging and magnetic resonance imaging (MRI) are the main imaging methods used for the diagnosis of stroke patients. Nuclear medicine imaging represented by PET and SPECT, as well as imaging technologies such as SPECT/CT, PET/CT, PET/MRI and fluorescence imaging that combine nuclear medicine with conventional imaging technologies, have been widely used for non-invasive EVs tracing in the brain. Basic research on stroke plays an increasingly important role. Although each imaging modality has made certain progress, they all have limitations in accurately monitoring the migration and efficacy of EVs at target sites in vivo from the whole body to the single cell level. For example, fluorescence imaging can study cellular and subcellular processes, but is always affected by non-specific "background" signals and cannot achieve non-invasive imaging under most conditions. MRI provides ultra-high spatial resolution and soft tissue contrast, while its sensitivity is insufficient to achieve whole-body imaging and quantitative tracking of EVs. Due to the high sensitivity of nuclear medicine imaging, it allows whole-body visualization and dynamic monitoring of the in vivo biodistribution of exogenous EVs, but it is unable to provide sufficient spatial anatomical information, especially in ischemic encephalopathy. Therefore, developing an appropriate labeling strategy for EVs allows for multi-modal imaging and comprehensive monitoring of the directional migration and distribution of EVs at target sites in the body after different administration routes, which is important for dissecting the mechanism of EVs-based treatment. is important and conducive to its further clinical transformation.

Contents of the invention

An object of the present invention is to provide an exosome therapeutic agent based on M2 microglia, a preparation method and its application in preparing medical imaging probes, and to provide at least the advantages that will be explained below.

Another object of the present invention is to provide an exosome therapeutic agent based on M2 microglia, a preparation method and its application in preparing medical imaging probes. Exosomes derived from M2 microglia are prepared. , labeled and modified small molecule ligands that can target brain neurons can specifically and actively target migration at high doses in the stroke area, improve the retention ability of exosome therapeutic agents in the stroke area, and improve their response to the stroke area. Restorative efficacy in stroke areas. For the first time, direct co-labeling of fluorescent dyes, SPECT signal radionuclide iodine-125, and magnetic resonance T2-weighted contrast agent superparamagnetic iron oxide (SPIO) without chelating was achieved, which meets the requirements of fluorescence imaging, nuclear medicine and magnetic resonance imaging equipment. The demand for multi-modal imaging probes enables non-invasive, accurate, real-time and dynamic in vivo monitoring of exosome therapeutic agents in the target area under different administration routes (tail vein i.a., carotid artery i.v and/or nasal cavity i.n. administration) migration, homing and distribution, and at the same time, the therapeutic effect was evaluated in order to adjust the administration time to achieve effective repair of stroke and further improve its therapeutic effect.

The technical solution of the present invention is as follows:

M2-type microglia-based exosome therapeutic agents, which include M2-type microglia-based exosomes, exosome surface-modified fluorescent dyes, dopamine or dopamine derivatives, radionuclides, and magnetic resonance imaging ions, neuron-targeting ligands.

Preferably, among the exosome therapeutic agents based on M2 microglia,

The source of exosomes is M2 type microglia;

The fluorescent dye is selected from one of DiR, Cy5.5, and Cy7;

The dopamine derivative is selected from dopamine hydrochloride;

The radioactive nuclide is selected from one of iodine-125, iodine-131, gallium-68, and copper-64;

The magnetic resonance imaging ions are selected from one or more of Gd ³⁺ , Mn ²⁺ , Fe ³⁺ , Cu ²⁺ , and Ni ³⁺ ;

The neuron-targeting ligand is selected from RVG.

A method for preparing exosome therapeutic agents based on M2 microglia, which includes the following steps:

1) Cultivate undifferentiated M0 cells in vitro, promote their differentiation into M2 cells by adding polarizing factors, and secrete exosomes;

2) Separate the product of step 1) by centrifugation, collect and purify exosomes derived from M2 cells;

3) All or part of the exosomes obtained in step 2) are stained and labeled with fluorescent dyes;

4) Coat all or part of the surface of the fluorescent dye-labeled exosomes obtained in step 3) with a dopamine or dopamine derivative layer;

5) Mix radionuclides, magnetic resonance imaging ions and the product of step 4);

6) Graft the surface of the product of step 5) with a neuron-targeting ligand to obtain the exosomes.

Preferably, in the preparation method of the exosome therapeutic agent based on M2 type microglia, in step 1), the M0 type cells can be microglia, and the polarizing factor can be IL4. , based on the total volume of the total culture system, the culture volume is controlled to 15 mL, the concentration of polarization factors added is 20 ng/mL, and the polarization culture time is 36-48 hours. After the culture is completed, collect the stimulated M2 small cells The supernatant of glial cells was stored at -20°C until exosomes were extracted.

Preferably, in the method for preparing the exosome therapeutic agent based on M2 microglia, the step 2) is specifically:

Collect the conditioned medium of stimulated M2 microglia and perform ultracentrifugation at 4°C at 300g for 10 minutes, 2000g for 15 minutes to remove dead cells, and 10000g for 30 minutes to remove cell debris. And the exosomes were precipitated by continuous ultracentrifugation at 100,000 g for 70 minutes, and the EVs were washed once with 100,000 g of PBS for 70 minutes and suspended for further characterization;

The structure of the collected exosomes was identified using 120 kV transmission electron microscopy, nanoparticle tracking analysis was used to measure the diameter and particles of exosomes, BCA method was used to determine the content of exosomes, and Western blotting was used to analyze exosome markers CD63 and TSG101.

Preferably, in the method for preparing the exosome therapeutic agent based on M2 microglia, step 3) specifically uses the near-infrared fluorescent dye DiR and the red fluorescent dye PKH26 to modify the exosomes;

For the DiR labeling process, exosomes and 1 μL of diluted DiR solution were incubated at 37°C for 5 mins;

For in vivo exosome uptake experiments, PKH26 diluted 1:500 was incubated with exosomes at room temperature for 5 min;

EVs-free serum is used throughout the process;

The labeled exosomes were washed in 100,000 g of PBS for 1 hour to wash away non-specific labeling, and the exosomes were resuspended in PBS for later use.

Preferably, in the method for preparing the exosome therapeutic agent based on M2 microglia, step 4) specifically includes:

Mix 200 μL EVs and PBS at a ratio of 1:1, add 1 mg PDA dissolved in 8 mL Tris buffer (pH=8.5) into the solution and react for 10 mins to make the concentration of dopamine 0.1 mg/mL;

Afterwards, the PDA-coated EVs were suspended in a dialysis tube, separated by 10000 g ultrafiltration for 8 minutes, and then washed three times with PBS. The concentration of dopamine-coated exosomes in the final product was approximately 1.2 × 10 ¹¹ particles/mL.

Preferably, in the preparation method of the exosome therapeutic agent based on M2 microglia,

Radioactive iodine-125 labeling in step 5) is achieved by the classic Iodogen method. PDA@EVs suspension (0.5 mL, ≈1.2×10 ¹¹ particles/mL) is added to a glass tube coated with 20 μg Iodogen at the bottom. middle. Add the newly prepared Na ¹²⁵ I solution (500 μCi, 18.5 MBq) into the tube, oscillate intermittently to avoid PDA@EVs deposition, and after incubating at room temperature for 30 mins, purify the final product by ultracentrifugation and wash three times with PBS;

PDA@EVs were labeled with amino-functionalized superparamagnetic iron oxide (SPIO) at a concentration of 20 μg/mL for 1 h, followed by washing and ultrafiltration to discard excess SPIO particles.

Preferably, in the preparation method of the exosome therapeutic agent based on M2 microglia, the step 6) specifically includes: adding 50 μg RVG to 300 μL of EVs suspension, and reacting at room temperature. 2-4 hours. RVG-modified EVs were then purified by ultrafiltration and washed with PBS.

Application of exosome therapeutic agents based on M2 microglia in the preparation of medical imaging probes, where the medical imaging is selected from any one or more of nuclear medicine imaging, magnetic resonance imaging, and CT imaging.

The present invention is useful both in repairing stroke and in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and brain trauma diseases.

Other advantages, objects, and features of the present invention will be apparent in part from the description below, and in part will be understood by those skilled in the art through study and practice of the present invention.

Description of drawings

Figure 1 is a schematic diagram of the exosome therapeutic agent RVG- ^125I /SPIO-PDA@DiR-EVs of the present invention.

Figure 2 is a schematic diagram of labeling of RVG- ^125I /SPIO-PDA@DiR-EVs, the exosome therapeutic agent of the present invention.

Figure 3 shows the characterization of 125I/SPIO-PDA@EVs of the present invention; A: TEM images of EVs and PDA@EVs. B: Nanoparticle identification of EVs (left) and PDA@EVs (right); C: Identification of EVs markers CD63 and TSG101; D: Coomassie brilliant blue identification of cell lysates, EVs and PDA@EVs; E: TEM showing PDA and changes in EVs morphology after SPIO labeling; F&D: Zeta potential and particle size show the stability of PDA and SPIO labeling; H&I: Stability of radioactive element iodine-125 labeling; J: The distribution of elements after EDS mapping shows successful labeling .

Figure 4 shows the different distribution patterns of SPIO-labeled EVs of the present invention after ischemic stroke through different administration routes in MRI imaging; comparing the cerebral blood flow of ischemic stroke after injection in three ways: i.a., i.v. and i.n. The change. A: Schematic diagram of in vivo experiments. B&C: Laser speckle shows changes and quantification of cerebral blood flow after tMACO; D, E&F: T2-weighted magnetic resonance imaging of SPIO-PDA@EVs injected i.a. (D), i.v. (E) and i.n. 24 hours after tMCAO in stroke mice Resonance imaging.

Figure 5 is in vivo SPECT/CT imaging showing the dynamic distribution of 125I-labeled EVs of the present invention in stroke mice through different administration routes; A-F: In vivo biodistribution and various organs of SPIO-PDA@EVs after different administration routes , Quantitative signal analysis of tissue; G: Radioactivity intensity of EVs in the brains of stroke mice by different administration routes at different time points.

Figure 6 shows fluorescence imaging of DiR-labeled EVs through different administration routes; A: In vivo distribution of DiR-labeled EVs in stroke mice; B: Brain and major organs (liver, spleen, lung, heart and kidney) Ex vivo fluorescence signal of DiR-labeled EVs; C: Quantification of brain and major organ homogenates of stroke mice treated with DiR-labeled EVs; D: HE staining showing the brain, heart, lungs, and liver of stroke mice , spleen and kidney cellular structure (above). Confocal images of brain sections showing ex vivo distribution of DiR-labeled EVs (red) in stroke mice 48 hours after i.a., i.v., or i.n. injection; E: brain, heart, lungs, liver, spleen, and kidneys Quantification of DiR fluorescence intensity; F: Confocal imaging of brain slices shows the distribution of DiR-labeled EVs within various types of cells; G: Bar chart shows quantification of phagocytosis index in different cell types.

Figure 7 shows that the RVG modification of the present invention increases the neuronal uptake of EVs in vitro; A: STEM images of PDA@EVs and RVG-PDA@EVs; B: Dynamic light scattering (DLS) measurement of PDA@EVs and RVG-PDA@EVs ; C: Zeta potential of PDA@EVs and RVG-PDA@EVs; D: Confocal imaging shows that PKH26-labeled EVs (red) co-labeled with primary neurons and astrocytes; E: RVG modification increases EVs For neuron targeting; F: Quantification of the difference in phagocytosis between PKH26-labeled EVs and neurons and astrocytes before and after modification; G: Quantitative display of neuron phagocytosis index in different groups; H: Using EVs and PDA@EVs, Cell viability of neurons treated with RVG-PDA@EVs and RVG+RVG-PDA@EVs.

Figure 8 shows that the RVG modification of the present invention promotes the targeted delivery of EVs in ischemic brain.

A: Magnetic resonance imaging of the brain of stroke mice at different time points before and after injection of SPIO-PDA@EVs and RVG-SPIO-PDA@EVs; B&C: Prussian blue staining of brain sections 48 hours after injection shows that SPIO-PDA@EVs are phagocytosed by cells. and quantification; D&E: Imaging and quantitative data after arterial injection of ¹²⁵ I-PDA@EVs and RVG- ¹²⁵ I-PDA@EVs into stroke mice; F: ¹²⁵ I-PDA@EVs and RVG- ¹²⁵ I-PDA@EVs Representative fluorescence images after arterial injection; G: Quantification of fluorescence intensity of EVs in ipsilateral brain homogenates; H&I: Confocal imaging showing colocalization and quantification of EVs and neurons.

Figure 9 shows that RVG-modified M2 EVs of the present invention further reduces neuronal apoptosis and promotes neurological function recovery in mice after tMCAO; A-D: TUNEL and Fluoro-Jeded B staining shows PBS, M2-EVs or RVG-M2- Neuronal death and quantification in stroke mice treated with EVs; E&F: M2-EVs and RVG-M2-EVs inhibited the expression and quantification of Cleaved caspase-3; G&H: M2-EVs and RVG-M2-EVs improved stroke mNSS score and rotarod test in mice.

Figure 10 shows that M2-EVs transcriptomic sequencing of the present invention up-regulates apoptosis-related miRNAs; A: Principal component (PC) analysis of miRNA array data of M0-EVs, M2-EVs and RVG-M2-EVs; B: The bar graph shows the differentially expressed miRNAs in M2 EVs and M0 EVs; C&D: The heat map and volcano plot show that the differentially expressed miRNAs between the M2 and M0 groups are the highest; E: The dot plot shows that compared with the M0 group, the M2 group The top 20 enriched pathways that were upregulated.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it with reference to the text of the description.

It will be understood that terms such as "having," "comprising," and "including" as used herein do not connote the presence or addition of one or more other elements or combinations thereof.

Direct and indirect labeling strategies for EVs have been developed to achieve multimodal imaging tracing. However, the indirect labeling method requires genetic modification of the parental cells in advance, which is time-consuming and inefficient. Therefore, direct labeling is more convenient and effective for labeling isolated EVs, but it often causes irreversible damage to exosomes and further affects their biological activity. According to reports, EVs can directly label radioactive iodine-125 through physical adsorption. However, physically adsorbed radioactive iodine-125 is extremely unstable under physiological conditions and is easily delabeled. Non-specific uptake of radioactive iodine-125 is achieved in the body, thereby affecting imaging. Effect. Therefore, it is necessary to establish a simple but effective multimodal EVs visualization method without losing the biological activity of EVs. In recent years, polydopamine (PDA) has attracted widespread interest due to its self-polymerizing properties and multifunctional chemical reactivity. Compared with other complex labeling methods, dopamine can be wrapped on the surface of EVs through the self-aggregation of catechol under weakly alkaline conditions, and the catechol and anthraquinone bonds on the PDA coating can easily bind to many metals. Coordination reactions occur with ions, such as Fe ³⁺ , Gd ³⁺ , and Pt ⁴⁺ . In addition, PDA can also react with halogens, sulfhydryl groups and amino groups through the Michael addition reaction, thereby helping to modify EVs with radioactive isotopes, polyethylene glycol (PEG) and targeted small molecule complexes for visual imaging of EVs. , to further improve the stability of EVs under physiological conditions and the targeting ability during treatment.

Based on the above premise, we collected and purified the exosomes secreted by M2 microglia and stained them with fluorescent dyes. Then, we wrapped the above products with dopamine self-aggregation, and used magnetic resonance imaging contrast agents and radionuclides to Directly coupled to the PDA wrapping layer, we successfully constructed a diagnostic and therapeutic agent that can be used for stroke repair and nuclear medicine/magnetic resonance imaging/fluorescence three-functional imaging to non-invasively and quantitatively track the impact of EVs on the brain under different administration routes. Targeted migration, repair, and efficacy assessment of stroke areas. Real-time visual dynamic monitoring of diagnostic and therapeutic agents in vivo at the individual, organ/tissue, cellular and molecular levels is achieved. By conjugating exosome therapeutic agents with specific ligands targeting brain neurons, the amount of targeted migration of Evs to neurons in cerebral ischemic areas is significantly increased, thereby improving the therapeutic effect on stroke. Through miRNA sequencing of EVs, miRNAs related to neuronal apoptosis were identified, elucidating the potential therapeutic mechanism of M2 microglia-derived therapeutic agents in ischemic brain. The present invention provides a powerful labeling method and functional modification approach for the future clinical application and promotion of EVs in cerebrovascular diseases and even neurodegenerative related diseases.

The present invention shows great potential for treating extracellular vesicles (EVs) including exosomes, and provides a solution for the comprehensive visualization and understanding of the directional migration and distribution of EVs during stroke treatment.

The present invention provides a nanometer-sized exosome diagnostic and therapeutic agent probe. The exosome therapeutic agent at least includes exosomes, fluorescent dyes that mark exosomes, dopamine or dopamine derivatives on the surface of exosomes, radioactive Nuclides, magnetic resonance imaging ions, ligands targeting neurons.

The source of exosomes is selected from microglia.

The source cells of the exosomes are subjected to the following pretreatment: Interleukin 4 induces M0 type microglia for at least 48 hours and then differentiates into M2 type microglia.

In certain embodiments of the present invention, the obtained exosomes are stained with a fluorescent dye selected from one or more of DiR, Cy5.5, and Cy7.

The dopamine derivative is selected from dopamine hydrochloride.

The radioactive nuclide is selected from one of iodine-125, iodine-131, gallium-68, and copper-64.

The magnetic resonance imaging ion is a T1 or T2 contrast agent, selected from one or more of Gd ³⁺ , Mn ²⁺ , Fe ³⁺ , Cu ²⁺ , and Ni ³⁺ ; the T1 contrast agent or T2 contrast agent The agent is provided by paramagnetic or superparamagnetic contrast agent particles, and the paramagnetic metal salt includes one or more of gadolinium chloride, gadolinium nitrate, gadolinium acetate, manganese chloride, manganese nitrate, manganese acetate, and ferric oxide. kind.

The neuron-targeting ligand is one or more of a protein, a monoclonal antibody, or a small molecule inhibitor. In certain embodiments of the invention, the neuron-targeting ligand is selected from one or more RVGs.

The exosome therapeutic agent is a probe that can be used for medical imaging. The medical imaging is selected from any one or more of nuclear medicine imaging (such as PET, SPECT), magnetic resonance imaging (MRI), and CT imaging. For example, the probe can be used as a PET/MRI imaging probe, a PET/CT imaging probe, a SPECT/MRI imaging probe, or a SPECT/CT imaging probe.

In some embodiments of the present invention, a schematic structural diagram of the probe is shown in Figure 1.

The invention also provides a method for preparing the exosomes, which includes the following steps:

Undifferentiated M0 type cells are cultured in vitro, and polarizing factors are added to promote their differentiation into M2 type cells and secrete exosomes; the microglia cells in step 1) are separated by centrifugation, and exosomes derived from M2 type cells are collected and purified. Exosomes; all or part of the exosomes obtained in step 2) are stained and labeled with fluorescent dyes;

Coat all or part of the surface of the fluorescent dye-labeled exosomes obtained in step 3) with a dopamine or dopamine derivative layer; mix radionuclides and magnetic resonance imaging ions with the product of step 4); 6) 5) The surface of the product is grafted with ligands targeting neurons to obtain the exosome therapeutic agent.

In certain preferred embodiments of the present invention, step 1) directed differentiation of M0 type cells in vitro includes: adding polarizing factors to M0 type cells to promote their differentiation into M2 type cells and secrete exosomes. In one embodiment, M0 type cells can be selected as microglia, and the polarizing factor can be selected as IL4. Based on the total volume of the total culture system, the culture volume is controlled to be 15 mL, and the concentration of the polarizing factor added is 20 ng/mL, and the polarization culture time is 36-48 hours. After the culture, collect the supernatant of the stimulated M2 microglia and store it at -20°C until exosomes are extracted.

In some preferred embodiments of the present invention, the conditioned medium of the stimulated M2 microglia cells collected in step 2) is ultracentrifuged under the conditions of continuous centrifugation at 300 g for 10 minutes at 4°C and continuous centrifugation at 2000 g for 15 minutes. minutes to remove dead cells, continuous centrifugation at 10,000 g for 30 minutes to remove cell debris, and continuous ultracentrifugation at 100,000 g for 70 minutes to precipitate exosomes. EVs were washed once with 100,000 g of PBS for 70 minutes and suspended for further characterization. The collected exosomes were characterized by transmission electron microscopy (TEM) at 120 kV to identify the structure, nanoparticle tracking analysis (NTA) to measure the diameter and particles of exosomes, BCA method to determine the content of exosomes, and protein immunoblotting to analyze exosomes. Markers CD63, TSG101.

In some preferred embodiments of the present invention, step 3) specifically includes: according to the instructions, using the near-infrared fluorescent dye DiR and the red fluorescent dye PKH26 to modify the exosomes. For the DiR labeling process, exosomes were incubated with 1 μL of diluted DiR solution at 37°C for 5 mins. For in vivo exosome uptake experiments, PKH26 diluted 1:500 was incubated with exosomes for 5 min at room temperature. In order to prevent over-labeling during the above process, EVs-free serum was used throughout the process. The labeled exosomes were washed in 100,000 g of PBS for 1 hour to wash away non-specific labeling, and the exosomes were resuspended in PBS for later use.

In some preferred embodiments of the present invention, step 4) specifically includes: mixing 200 μL EVs and PBS (1:1), and adding 1 mg PDA dissolved in 8 mL Tris buffer (pH=8.5) to the solution React for 10 mins to make the concentration of dopamine 0.1 mg/mL. After that, the PDA-coated EVs were suspended in a dialysis tube (100 kDa-MWCO), separated by 10000 g ultrafiltration for 8 minutes, and then washed three times with PBS. The concentration of dopamine-encapsulated exosomes in the final product was approximately 1.2 × 10 ¹¹ particles/mL. PDA was conjugated with amino-functionalized indocyanine green (ICG) to evaluate the labeling efficiency, and the fluorescence intensity of ICG-PDA@EVs was measured with a microplate reader before and after ultrafiltration to evaluate the PDA labeling efficiency.

In certain preferred embodiments of the present invention, step 5) specifically includes: radioactive iodine-125 labeling is achieved by the classic Iodogen method. PDA@EVs suspension (0.5 mL, ≈1.2 × 10 ¹¹ particles/mL) was added to a glass tube coated with 20 μg Iodogen at the bottom. Add freshly prepared Na ¹²⁵ I solution (500 μCi, 18.5 MBq) into the tube and oscillate intermittently to avoid PDA@EVs deposition. After incubation at room temperature for 30 mins, the final product was purified by ultracentrifugation and washed three times with PBS. Radioactive thin-layer chromatography was performed using a gamma detector to assess labeling efficiency. The stability of the radioactive probe was evaluated by incubating 5 μL samples in 200 μL 10% FBS for different times (1, 3, 6, 12, and 24 h) in DMEM high glucose solution at 37 °C. After incubation, the probes were collected by centrifugation and the radioactivity retained on the particles was counted. Silica gel chromatography paper and 0.9% sodium chloride were used as stationary and mobile phases. ^{The 125} I-labeled SPIO-PDA@EVs remains at the origin, while the free ¹²⁵ I remains at the front with the mobile phase. The radiochemical purity of the ¹²⁵ I-labeled SPIO-PDA@EVs is expressed as the SPIO-PDA@EVs probe retained at the origin. The radioactive dose to the needle as a percentage of the total radioactive dose.

In certain preferred embodiments of the present invention, step 5) specifically includes: PDA@EVs are labeled with amino-functionalized superparamagnetic iron oxide (SPIO) at a concentration of 20 μg/mL for 1 hour, followed by washing and ultrafiltration to Discard excess SPIO pellets. To check the labeling stability of SPIO, SPIO-PDA@EVs were suspended in DMEM high glucose culture medium containing 10% FBS at 37 °C and inducted with inductor at 0, 1, 3, 6, 12, 24 and 48 h. Coupled plasma optical emission spectrometry (ICP-OES) measures isolated SPIO particles. The T2 relaxation time of labeled EVs (1 × ¹⁰ particles/mL) was measured using a 1.41 T minispec mq 60 NMR analyzer at 37 °C. By co-incubating 1 × ¹⁰ probes in 5 mL DMEM high-glucose culture medium containing 10% FBS at 37 °C for different times (0, 1, 3, 6, 12, 24, and 48 hours), all The possible release of SPIO from the probe was studied in triplicate. After incubation, the probe was collected by ultrafiltration and free SPIO in serum was measured by ICP-OES. The stability of SPIO is expressed as the total amount of SPIO retained on PDA@EVs relative to the probe. Use DLS to measure the zeta potential of labeled EVs at each step during probe preparation. To evaluate the stability of SPIO labeling, hydrodynamic diameter and surface charge of EVs, PDA@EVs and SPIO-PDA@EVs in 10% FBS (v/v) solution at 0, 1, 2, 3 and 4 days Check via DLS.

In certain preferred embodiments of the present invention, step 6) specifically includes: adding 50 μg RVG to the EVs suspension (300 μL), and reacting at room temperature for 2-4 hours. RVG-modified EVs were then purified by ultrafiltration and washed with PBS. The fluorescence intensity of FITC-RVG-PDA@EVs before and after purification was quantified using a microplate system to evaluate the grafting rate.

In the embodiment of the present invention, the characterization of exosomes specifically includes:

ICP-OES measurement. Add 100 μL of SPIO to the exosomes that have been treated with PDA for 45 min and let it react for 5 minutes. The resulting SPIO-PDA@EVs were suspended in DMEM high glucose containing 10% FBS and incubated at 37°C for 1, 3, 6, 12, 24, and 48 h. The labeling stability was detected using an inductively coupled plasma optical emission spectrometer by measuring SPIO-PDA@EVs after adding KI, ultracentrifugation and dilution with deionized water. In addition, SPIO-PDA@EVs (≈3.8 × 10 ¹² particles/mL) and an equal amount of pure SPIO were heated at 120 °C, and 50 μL nitric acid was added to each ml of the product for ablation for 1 hour before comparison. The labeling rate is calculated as (Fe ion concentration in SPIO-PDA@EVs/ion concentration of SPIO) * 100%.

STEM imaging. 5 μL SPIO-PDA@EVs were placed on the hydrophilic carbon membrane and allowed to incubate for 2 min. The incubated copper grid and sample were washed three times with deionized water and dried at room temperature for 10 s each time to reduce salt deposition. Next, 4 μL of phosphotungstic acid was added to the sample and dried for 1 min. In order to prevent SPIO-PDA@EVs from entering the pole shoe during imaging, a carbon film was covered on the top of the sample. Perform brightfield and high-angle annular darkfield (HAADF) imaging for 5-10 minutes using weight percent (wt%) or atomic percent (at%) scanning. Energy dispersive spectrometer-detector (EDS) mapping analysis SPIO-PDA@EVs and STEM have the same elemental distribution of iron, iodine, nitrogen, carbon and oxygen elements

In the embodiment of exosome imaging of the present invention, it specifically includes:

Magnetic resonance imaging. A T2-weighted spin echo sequence was used with the following parameters: repetition time (TR) = 2500 ms, echo time (TE) = 33 ms, field of view (FOV) = 20 × 20 mm, matrix = 256 × 256, slice thickness = 700 μm. MR images were analyzed using ParaVision 6 software. The average signal intensity within a region of interest (ROI) drawn around the tumor was calculated for each image. Relative signal intensity enhancement (rSIE) is defined as the ratio of the average intensity in the brain after injection to the average intensity before injection. After final in vivo imaging, mice were immediately sacrificed, and brains were collected and fixed in 4% paraformaldehyde solution for histological analysis.

SPECT/CT imaging. Mice were injected with ^125I -labeled EVs via different administration methods (tail vein, carotid artery and/or nasal administration) and imaged at 0.5, 6, 24 and 48 hours after injection. The radiation dose per mouse was 300 μCi. The amount of ¹²⁵ I-PDA@EVs was measured by the injected dose density per volume of organ from the region of interest (ROI). The CT images provide an anatomical reference for the mouse's position. SPECT images were acquired for 32 mappings at 360°C (radius of rotation = 7.6 cm, 30 sec/mapping). Use InVivoScope to visualize and co-register reconstructed data from SPECT and CT. ROIs were drawn in major organs exhibiting significant radioactivity.

Fluorescence imaging. For in vivo fluorescence imaging, mice were injected with DiR-labeled EVs via different administration methods (tail vein, carotid artery, and/or nasal administration), and the IVIS system was used at 0.5, 6, 24, and 48 hours after injection at 748 and imaged at excitation and emission wavelengths of 780nm. ROIs for ipsilateral and contralateral brain, heart, lung, stomach, liver, spleen, kidney, and background were selected from equally sized regions containing the same number of pixels. Quantification of uptake was determined by plotting the ROI for each organ. The removed organs were placed on ice, weighed, and stored at minus 80°C to verify homogenate fluorescence. Briefly, 80-150 mg of each organ was added to 1 mL of lysate buffer containing 1× RIPA buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% Ige-Pal (NP40), 0.5 % sodium deoxycholate, 0.1% SDS, and 1% protease inhibitor cocktail. The sample was then homogenized and centrifuged to collect the supernatant, 100 µL of the homogenate was added to a black 96-well opaque microplate, and the fluorescence intensity in each well was immediately measured to calculate the fluorescence intensity per gram of tissue.

In the embodiment of the exosome treatment of the present invention, it specifically includes: adult ICR mice are subjected to a transient middle cerebral artery ischemia model, and one day after the modeling, 100 ug of exosomes are injected through the vein, artery and nasal cavity respectively. (1.0×10 ¹¹ particles). Neurobehavioral testing and brain slices were collected for immunofluorescence staining at 0, 1, and 3 days after surgery to identify the types of nerve cells that take up exosomes, and to further clarify the role of labeled exosomes in ischemic stroke. Postneuronal cell targeting and therapy.

The present invention detected the morphological structure and particle size of normal and PDA-coated exosomes through transmission electron microscopy and NTA respectively (as shown in Figure 3A-B), and found that PDA coating did not affect the expression of exosomes in exosomes. Expression changes of sub-markers CD63 and TSG101 (Figure 3C-D). STEM imaging showed that there was a particle coverage of about 10 nm superparamagnetic iron tetroxide on the exosome membrane (Figure 3E), so that magnetic resonance imaging could show the spatial distribution of exosomes. By detecting the zeta potential and particle size of labeled exosomes over time (Figure 3F-G), we can know that our iron particle labeling is successful and stable. To enable us to perform SPET/CT imaging, we labeled exosomes with the classic iodine-125, and radionuclide imaging showed that the labeling rate was as high as 90% (Figure 3H). Radioactive thin-layer chromatography showed that the labeling rate remained about 85% after 24 h, indicating that the radioactive labeling was stable (Figure 3I). In addition, energy spectrometer mapping showed that iodine and Fe elements were accumulated on the surface of individual exosomes, indicating successful labeling (Figure 3J).

Using a multimodal imaging system, the present invention verified the tracking effect of labeled exosomes in mice with transient ischemic stroke. First, laser speckle cerebral blood flow quantification confirmed the success of occlusion and reperfusion in mice during tMCAO surgery (Figure 4B-C). To determine the optimal route of administration, stroke mice were injected with 100 μg (1 × 10 ¹¹ particles) of exosomes via intraarterial (ia), intravenous (iv), and intranasal (in) respectively. Using 7T magnetic resonance, T2-weighted imaging was performed on mice before and after injection one day after modeling (Figure 4E-F). It can be seen that strong contrast was detected in the infarct area 30 minutes after intra-arterial injection, but almost no contrast was detected after 24 hours. disappeared, indicating rapid uptake and degradation of EVs in the ischemic brain. In contrast, brain uptake after intravenous and intranasal injections was much lower than intraarterial injections.

In order to observe and track the migration of EVs in major organs and tissues, the present invention treated stroke mice with radiolabeled exosomes and scanned them with SPECT/CT. As shown in Figure 5, it can be seen that EVs were detected to have significant organ targeting and biodistribution characteristics in different administration routes. SPECT/CT imaging results showed that 30 minutes after arterial administration, labeled exosomes mainly appeared in the liver/spleen and bladder, with a small amount accumulated in the lungs and heart, and a large amount accumulated in the ipsilateral brain. After 24 hours, after being cleared by the renal system, a large number of EVs still accumulated in the liver/spleen. At the same time, the signal in the brain was significantly reduced by nearly 50%; after 48 hours, almost no EVs were found in the brain. The results showed that after arterial injection EVs, uptake and cellular internalization are efficient. Compared with arterial injection, EVs accumulated strongly in the liver, kidney and thyroid 30 min after intravenous injection, but the signal in the brain was much lower. The signal disappeared after 24 h, indicating lower initial uptake of EVs in the brain and faster pharmacokinetic acceleration after intravenous injection. Compared with the first two injection methods, EVs administered intranasally did not migrate to other organs or tissues except the thyroid gland. EVs tended to migrate to the gastrointestinal tract after 6 hours and were excreted 48 hours after injection. SPET/CT results showed that compared with intravenous and nasal injection, 6 h after intraarterial injection of EVs, nuclear signals in the brain increased 2.4 times and 2.0 times respectively.

The dynamics of EVs distribution in different organs was verified by using fluorescence imaging. The near-infrared dye DiR is inserted into the membrane of EVs, and small animal in vivo imaging systems are used for in vitro and in vivo detection. In vivo imaging results of small animals show (Figure 6A-B) that in the first 6 hours of arterial and intravenous injection of exosomes, DIR signals appear in the abdomen and pelvis, and the signals decrease significantly after 24 hours, while nasal injection first occurs at the injection site. The signal is high and then continues to decrease. Ex vivo imaging showed different distribution characteristics, with arterial and intravenous injections mainly concentrated in the liver, lungs, kidneys and damaged brain hemispheres, while nasal injections showed strong signals in the stomach, lungs and spleen, with minimal signals in the brain. The results were consistent with MRI and SPECT/CT imaging, indicating that compared with the three injection methods, intra-arterial injection produced stronger signals in the ipsilateral brain, which means that intra-arterial injection has significant brain targeting advantages. At the same time, frozen sections of different organs were imaged under a confocal microscope (Figure 6D) to verify the distribution of EVs. HE staining showed the structure of the major organs, and confocal micrographs showed that the fluorescence intensity levels in the liver and spleen of mice injected intra-arterially and intravenously were similar, but the accumulated EVs in the injured striatum of mice injected intra-arterially were obvious. increased, which further supports the aforementioned imaging results. Statistics of DiR fluorescence signals in different organs and tissues showed (Figure 6E) that compared with intravenous or intranasal injection, the fluorescence intensity of EVs in the mouse brain was significantly higher after intra-arterial injection. The present invention uses immunostaining to determine the cellular uptake characteristics of intra-arterially injected EVs in the brain. 3D reconstructed images showed that most EVs were taken up by microglia (Iba-1 ⁺ ) and neurons (MAP2 ⁺ ) rather than endothelial cells (CD31 ⁺ ) or astrocytes (GFAP ⁺ ).

The present invention monitors the pharmacokinetics of a multifunctional EVs probe through multiple imaging modalities, demonstrating that intra-arterial injection is the best way to achieve maximum visualization of EVs delivery to the stroke brain at the systemic and organ/tissue levels. In order to improve the targeting of EVs uptake by neurons, RVG was connected to PDA to facilitate specific binding to acetylcholine receptors expressed by neurons. Electron microscopy and DLS testing proved that RVG modification did not affect the morphological characteristics of exosomes. Confocal 3D fluorescence images showed (Figure 6F) that RVG modification significantly increased the MAP2 ⁺ neuronal uptake rate, indicating that the RVG peptide successfully competed for neuronal binding but failed to reverse neuronal phagocytosis. To examine whether RVG binding would affect the viability of neurons, a CCK-8 assay was performed, and it was found that incubation with 10 μg/mL EVs for 6 h did not affect the cell viability of neurons.

To investigate whether RVG modification can improve the uptake of EVs in the brain, we examined stroke mice treated with RVG-modified EVs and untreated EVs by MR/SPECT/FL imaging. MRI showed (Figure 8A) that more RVG-modified EVs accumulated in the infarct area of the ischemic brain at 4.5 h after injection, and even after 48 h after injection, there were still many scattered low-intensity spots localized in the diseased brain. Prussian blue staining also showed (Figure 8B-C) that SPIO-labeled EVs were clearly located in the brain infarct core and peri-infarct areas, and RVG modification significantly increased the accumulation of EVs in the ipsilateral cerebral hemisphere. From the SPECT/CT images (Figure 8D-E), we noticed that RVG modification significantly improved brain targeting 0.5 h after injection. At 6 h after injection, the initially accumulated EVs tended to migrate and spread within the diseased brain area. Ex vivo imaging showed that RVG modification indeed improved the targeting of EVs in the brain. RVG modification not only improved the uptake of EVs in the brain, but further staining showed that RVG significantly increased the uptake of EVs by neurons in the infarcted area of the striatum. After ischemic stroke, a large number of neurons in the ischemic penumbra undergo apoptosis within hours and persist for days. Given the time-limited therapeutic window for stroke, it is critical to evaluate the appropriate time point for EVs administration. We injected exosomes 2 h after stroke (Figure 9A-D). Compared with the PBS group and M2-EVs, we found that the number of FJ-B ⁺ and TUNEL ⁺ cells was significantly reduced after 3 d, and was further reduced after RVG-M2-EVs treatment. neuronal degeneration and apoptosis. WB results showed (Figure 9E) that RVG-modified exosome treatment reduced the expression of cleaved-caspase3 (cleaved-caspase3), and lower neurological severity scores and better rotarod test were detected Performance. The results indicate that RVG modification enhances the neuroprotective effect of EVs on ischemic stroke.

In order to study the potential mechanism of M2 EVs-mediated neuroprotection, the present invention performed EVs-miRNA sequencing (Figure 10). Principal component analysis showed that except for one sample of RVG-M2-EVs, which was excluded, the other samples were well dispersed. M0-EVs and M2-EVs exhibit different gene expression profiles, while RVG-M2-EVs and M2 EVs have similar genetic composition, indicating that the RVG-PDA labeling strategy does not change EV miRNA content. Compared with M0 EVs, 35 differentially expressed miRNAs were identified in M2 EVs, of which 21 were up-regulated and 14 were down-regulated. Further KEGG analysis showed that some metabolic signaling, synapse, vascular function and apoptosis-related pathways were enriched in M2 EVs. Apoptosis-related miRNAs, including miR-423-3p, miR-7688-5p, miR-106b-3p, miR-532-5p, miR-151-3p and miR-146a-5p, are particularly evident. These results indicate that anti-apoptosis-related miRNAs enriched in M2 EVs have neuroprotective effects in mice with acute ischemic stroke.

Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and embodiments. They can be applied to various fields suitable for the present invention. For those familiar with the art, they can easily Additional modifications may be made, and the invention is therefore not limited to the specific details and illustrations shown and described herein without departing from the general concept defined by the claims and equivalent scope.

Claims (10)

1. Exosome therapeutic agents based on M2 microglia, characterized by including exosomes based on M2 microglia, fluorescent dyes modified on the exosome surface, dopamine or dopamine derivatives, radioactive nuclei ligands, magnetic resonance imaging ions, and neuron-targeting ligands. 2. The exosome therapeutic agent based on M2 type microglia according to claim 1, characterized in that, The source of exosomes is M2 type microglia; The fluorescent dye is selected from one of DiR, Cy5.5, and Cy7; The dopamine derivative is selected from dopamine hydrochloride; The radioactive nuclide is selected from one of iodine-125, iodine-131, gallium-68, and copper-64; The magnetic resonance imaging ions are selected from one or more of Gd ³⁺ , Mn ²⁺ , Fe ³⁺ , Cu ²⁺ , and Ni ³⁺ ; The neuron-targeting ligand is selected from RVG. 3. The preparation method of exosome therapeutic agent based on M2 type microglia according to claim 1 or 2, characterized in that it includes the following steps: 1) Cultivate undifferentiated M0 cells in vitro, promote their differentiation into M2 cells by adding polarizing factors, and secrete exosomes; 2) Separate the product of step 1) by centrifugation, collect and purify exosomes derived from M2 cells; 3) All or part of the exosomes obtained in step 2) are stained and labeled with fluorescent dyes; 4) Coat all or part of the surface of the fluorescent dye-labeled exosomes obtained in step 3) with a dopamine or dopamine derivative layer; 5) Mix radionuclides, magnetic resonance imaging ions and the product of step 4); 6) Graft the surface of the product of step 5) with a neuron-targeting ligand to obtain the exosomes. 4. The preparation method of exosome therapeutic agent based on M2 type microglia as claimed in claim 1, characterized in that in step 1), M0 type cells can be selected as microglia, polarized The factor can be selected as IL4, based on the total volume of the total culture system. The control culture volume is 15 mL. The concentration of the polarization factor added is 20 ng/mL. The polarization culture time is 36-48 hours. After the culture is completed, collect The supernatant of M2 type microglia after stimulation was stored at -20°C until exosomes were extracted. 5. The preparation method of exosome therapeutic agent based on M2 type microglia according to claim 4, characterized in that the step 2) is specifically: Collect the conditioned medium of stimulated M2 microglia and perform ultracentrifugation at 4°C at 300g for 10 minutes, 2000g for 15 minutes to remove dead cells, and 10000g for 30 minutes to remove cell debris. And the exosomes were precipitated by continuous ultracentrifugation at 100,000 g for 70 minutes, and the EVs were washed once with 100,000 g of PBS for 70 minutes and suspended for further characterization; The structure of the collected exosomes was identified using 120 kV transmission electron microscopy, nanoparticle tracking analysis was used to measure the diameter and particles of exosomes, BCA method was used to determine the content of exosomes, and Western blotting was used to analyze exosome markers CD63 and TSG101. 6. The preparation method of exosome therapeutic agent based on M2 type microglia as claimed in claim 5, characterized in that step 3) specifically involves using near-infrared fluorescent dye DiR and red fluorescent dye PKH26 to modify the exosomes. secretion body; For the DiR labeling process, exosomes and 1 μL of diluted DiR solution were incubated at 37°C for 5 mins; For in vivo exosome uptake experiments, PKH26 diluted 1:500 was incubated with exosomes at room temperature for 5 min; EVs-free serum is used throughout the process; The labeled exosomes were washed in 100,000 g of PBS for 1 hour to wash away non-specific labeling, and the exosomes were resuspended in PBS for later use. 7. The method for preparing exosome therapeutic agents based on M2 microglia according to claim 6, characterized in that said step 4) specifically includes: Mix 200 μL EVs and PBS at a ratio of 1:1, add 1 mg PDA dissolved in 8 mL Tris buffer (pH=8.5) into the solution and react for 10 mins to make the concentration of dopamine 0.1 mg/mL; Afterwards, the PDA-coated EVs were suspended in a dialysis tube, separated by 10000 g ultrafiltration for 8 minutes, and then washed three times with PBS. The concentration of dopamine-coated exosomes in the final product was approximately 1.2 × 10 ¹¹ particles/mL. 8. The preparation method of exosome therapeutic agent based on M2 type microglia according to claim 7, characterized in that, Radioactive iodine-125 labeling in step 5) is achieved by the classic Iodogen method. PDA@EVs suspension (0.5 mL, ≈1.2×10 ¹¹ particles/mL) is added to a glass tube coated with 20 μg Iodogen at the bottom. middle. Add the newly prepared Na ¹²⁵ I solution (500 μCi, 18.5 MBq) into the tube, oscillate intermittently to avoid PDA@EVs deposition, and after incubating at room temperature for 30 mins, purify the final product by ultracentrifugation and wash three times with PBS; PDA@EVs were labeled with amino-functionalized superparamagnetic iron oxide (SPIO) at a concentration of 20 μg/mL for 1 h, followed by washing and ultrafiltration to discard excess SPIO particles. 9. The preparation method of exosome therapeutic agent based on M2 type microglia as claimed in claim 8, characterized in that the step 6) specifically includes: adding 50 μg RVG to 300 μL of EVs suspension, And react at room temperature for 2-4 hours. RVG-modified EVs were then purified by ultrafiltration and washed with PBS. 10. Application of exosome therapeutic agents based on M2 microglia in the preparation of medical imaging probes, characterized in that the medical imaging is selected from any one of nuclear medicine imaging, magnetic resonance imaging, and CT imaging. or more.