CN116763945A - Exosome diagnosis and treatment agent based on M2 microglial cells, preparation method and application thereof in preparation of medical imaging probes - Google Patents
Exosome diagnosis and treatment agent based on M2 microglial cells, preparation method and application thereof in preparation of medical imaging probes Download PDFInfo
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Abstract
The invention provides an exosome diagnosis and treatment agent based on M2 microglial cells, a preparation method and application thereof in preparing a medical imaging probe, and the exosome from the M2 microglial cells is prepared, and small molecule ligands capable of targeting cerebral neurons are marked and modified, so that the exosome diagnosis and treatment agent can be specifically used for high-dose initiative in cerebral stroke areasTargeting migration improves the detention capability of an exosome diagnosis and treatment agent in a cerebral apoplexy region and improves the healing efficacy of the exosome diagnosis and treatment agent on the cerebral apoplexy region. For the first time realize fluorescent dye and SPECT signal radionuclide 125 I and magnetic resonance T 2 The direct co-labeling of the weighted contrast agent superparamagnetic iron oxide without chelating agent meets the requirements of fluorescence imaging, nuclear medicine and magnetic resonance imaging equipment on the multi-mode imaging probe, realizes noninvasive, accurate, real-time and dynamic in-vivo monitoring of migration, homing and distribution conditions of exosome diagnosis and treatment agents in a target area under different administration routes, and simultaneously evaluates treatment effects so as to adjust administration time, realize effective repair of cerebral apoplexy and further improve treatment effects.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to an exosome diagnosis and treatment agent based on M2 type microglial cells, a preparation method and application thereof in preparation of medical imaging probes.
Background
Stroke is a major cause of disability and mortality worldwide, and diagnosis and treatment are severe due to limitations in the time window of treatment and the means of treatment. In recent years, extracellular Vesicles (EVs) including exosomes have shown great potential in the treatment of stroke. Our previous studies have shown that M2 microglial-derived exosomes can reduce neuroinflammation, protect the mouse brain from ischemia reperfusion injury, and promote white matter repair and remodeling in stroke mice. However, due to the lack of an effective strategy for in vivo visualization based on EVs treatment, the efficiency of target site migration during EVs treatment is clear, severely hampering its clinical transformation. In addition, in the face of promising treatments for EVs, it is currently also controversial which is the optimal mode of administration for the best therapeutic effect of stroke. Thus, a comprehensive visualization and understanding of directional migration and distribution during EVs treatment is critical to its further clinical transformation.
In order to address the above-unsolved need, there is a pressing need for advanced imaging techniques that enable noninvasive, 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 primary imaging means for stroke patient diagnosis. Nuclear medicine imaging represented by PET and SPECT, and SPECT/CT, PET/MRI, and fluorescence imaging technologies combining nuclear medicine with conventional imaging technologies have been widely used for noninvasive EVs tracing, and play an increasingly important role in basic research of cerebral stroke. Although each imaging modality has achieved some progress, they have limitations in accurately monitoring EVs from systemic to single cell levels for migration and therapeutic efficacy at target sites in vivo. For example, fluorescence imaging is capable of studying cellular and subcellular processes, but is always affected by nonspecific "background" signals, and non-invasive imaging is not possible under most conditions. MRI provides ultra-high spatial resolution and soft tissue contrast, while its sensitivity is insufficient to enable whole-body imaging and quantitative tracking of EVs. Due to the high sensitivity of nuclear medicine imaging, systemic visualization and dynamic monitoring of the in vivo biodistribution of exogenous EVs is allowed, but not capable of providing sufficient spatial anatomical information, particularly in ischemic encephalopathy. Thus, developing an appropriate labeling strategy for EVs allows for multimodal imaging thereof, comprehensively monitors the directional migration and distribution of EVs at target sites in vivo after different routes of administration, is critical for profiling EVs-based therapeutic mechanisms, and facilitates further clinical transformations thereof.
Disclosure of Invention
It is an object of the present invention to provide an M2 microglial cell-based exosome diagnostic agent, a method of preparation and use thereof in the preparation of medical imaging probes, and to provide at least the advantages as will be described later.
The invention further aims to provide an exosome diagnosis and treatment agent based on M2 microglial cells, a preparation method and application thereof in preparing a medical imaging probe, exosomes derived from the M2 microglial cells are prepared, small molecule ligands capable of targeting cerebral neurons are marked and modified, and can specifically and actively migrate in a cerebral stroke area in a high-dose and targeted manner, so that the detention capacity of the exosome diagnosis and treatment agent in the cerebral stroke area is improved, and the healing curative effect of the exosomes diagnosis and treatment agent on the cerebral stroke area is improved. The method realizes direct co-labeling of fluorescent dye, SPECT signal radionuclide iodine-125 and magnetic resonance T2 weighted contrast agent superparamagnetic iron oxide (SPIO) without chelating agent for the first time, meets the requirements of fluorescence imaging, nuclear medicine and magnetic resonance imaging equipment on a multi-mode imaging probe, realizes noninvasive, accurate, real-time and dynamic in-vivo monitoring of migration, homing and distribution conditions of exosome diagnosis and treatment agents (tail vein i.a., carotid artery i.v and/or nasal cavity i.n. administration) in a target area under different administration routes, and simultaneously evaluates treatment effects so as to adjust administration time, realize effective repair of cerebral apoplexy and further improve the treatment effects of cerebral apoplexy.
The technical scheme of the invention is as follows:
an exosome diagnosis and treatment agent based on M2 type microglial cells comprises exosomes based on M2 type microglial cells, fluorescent dye with surface modification of the exosomes, dopamine or dopamine derivatives, radionuclides, magnetic resonance imaging ions and ligands for targeting neurons.
Preferably, in the M2 microglial cell-based exosome diagnosis and treatment agent,
the exosome source is M2 type microglial cells;
the fluorescent dye is one of DiR, cy5.5 and Cy 7;
the dopamine derivative is selected from dopamine hydrochloride;
the radionuclide is selected from one of iodine-125, iodine-131, gallium-68 and copper-64;
the magnetic resonance imaging ion is selected from Gd 3+ 、Mn 2+ 、Fe 3+ 、Cu 2+ 、Ni 3+ One or more of the following;
the ligand targeting neurons is selected from RVG.
The preparation method of the M2 microglial cell-based exosome diagnosis and treatment agent comprises the following steps:
1) Culturing undifferentiated M0 type cells in vitro, promoting differentiation to M2 type cells by adding polarization factors, and secreting exosomes;
2) Centrifugally separating the product of the step 1), and collecting and purifying to obtain an exosome of M2 type cell source;
3) All or part of the exosomes obtained in the step 2) are marked by fluorescent dye;
4) Coating all or part of the surface of the exosome subjected to fluorescent dye staining marking obtained in the step 3) with a dopamine or dopamine derivative layer;
5) Mixing a radionuclide, magnetic resonance imaging ions, and the product of step 4);
6) Grafting the ligand of the targeted neuron on the surface of the product obtained in the step 5) to obtain the exosome.
Preferably, in the preparation method of the M2 microglial cell-based exosome diagnosis and treatment agent, in the step 1), the M0 type cells are selected as microglial cells, the polarization factor is selected as IL4, the total volume of the total culture system is taken as a reference, the culture volume is controlled to be 15 mL, the concentration of the polarization factor is added to be 20 ng/mL, the polarization culture time is 36-48 hours, and after the culture is finished, the supernatant of the M2 type microglial cells after stimulation is collected and stored at the temperature of minus 20 ℃ to extract exosomes.
Preferably, in the preparation method of the M2 microglial cell-based exosome diagnosis and treatment agent, the step 2) specifically includes:
conditioned medium ultracentrifugation of stimulated M2 microglial cells was collected, conditioned at 4 ℃ by continuous centrifugation at 300 g for 10 min, continuous centrifugation at 2000 g for 15 min to remove dead cells, continuous centrifugation at 10000 g for 30 min to remove cell debris, and continuous ultracentrifugation at 100000g for 70 min to pellet the exosomes, EV washed once with 100000g PBS for 70 min and suspended for further characterization;
collecting the obtained exosomes, identifying the structure by adopting a transmission electron microscope of 120 kV, measuring the diameter and particles of the exosomes by nanoparticle tracking analysis, measuring the content of the exosomes by a BCA method, and analyzing exosome markers CD63 and TSG101 by western immunoblotting.
Preferably, in the preparation method of the M2 microglial cell-based exosome diagnosis and treatment agent, the step 3) specifically comprises using a near infrared fluorescent dye DiR and a red fluorescent dye PKH26 to modify exosomes;
for the labeling procedure of DiR, the exosomes and 1 μl of diluted DiR solution were incubated for 5 mins at 37 ℃;
for in vitro and in vivo urinary uptake experiments, 1: the 500 diluted PKH26 and exosomes were incubated for 5 mins at room temperature;
serum without EVs is adopted in the whole process;
the labeled exosomes were washed in 100000g PBS for 1 hour to wash off non-specific labels, and the exosomes were resuspended in PBS for use.
Preferably, in the preparation method of the M2 microglial cell-based exosome diagnosis and treatment agent, the step 4) specifically includes:
200. Mu.L of EVs were mixed with PBS at a ratio of 1:1, and 1 mg of PDA dissolved in 8 mL of Tris buffer (pH=8.5) was added to the solution to react for 10 minutes to give a dopamine concentration of 0.1 mg/mL;
thereafter, the PDA-coated EVs were suspended in dialysis tubing, isolated by ultrafiltration at 10000 g for 8 minutes, then washed three times with PBS, and the final product had a dopamine-coated exosome concentration of about 1.2X10 11 particles/mL.
Preferably, in the preparation method of the M2 microglial cell-based exosome diagnosis and treatment agent,
the radioiodine-125 labelling in step 5) was achieved by classical Iodogen methods, by suspending PDA@EVs (0.5 mL, # 1.2X10) 11 Individual particles/mL) was added to a glass tube with a bottom coated with 20 μg Iodogen. To freshly prepared Na 125 Solution I (500 μci,18.5 MBq) was added to the tube, shaken intermittently to avoid pda@evs deposition, after incubation for 30 mins at room temperature, the final product was purified by ultracentrifugation and washed three times with PBS;
pda@evs were labeled with amino-functionalized superparamagnetic iron oxide (SPIO) at a concentration of 20 μg/mL for 1 hour, then washed and ultrafiltered to discard excess SPIO particles.
Preferably, in the preparation method of the M2 microglial cell-based exosome diagnosis and treatment agent, the step 6) specifically includes: mu.g RVG was added to 300. Mu.L of EVs suspension and reacted at room temperature for 2-4 hours. RVG modified EVs were then purified by ultrafiltration and washed with PBS.
Use of an M2 microglial cell-based exosome diagnostic agent in the preparation of a medical imaging probe, the medical imaging being selected from any one or more of nuclear medicine imaging, magnetic resonance imaging, CT imaging.
The invention is useful in both stroke repair and neurodegenerative diseases, including senile dementia, parkinson's disease and brain trauma.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 shows an exosome diagnostic agent RVG- 125 I/SPIO-PDA@DiR-EVs schematic diagram.
FIG. 2 shows the exosome diagnostic agent RVG- 125 I/SPIO-PDA@DiR-EVs marking schematic diagram.
FIG. 3 is a representation 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 TSG 101; d: coomassie brilliant blue identified cell lysates, EVs and pda@evs; e: TEM shows changes in morphology of EVs after PDA and SPIO labeling; f & D: zeta potential and particle size showed stability of PDA and SPIO markers; h & I: stability of the radioactive elemental iodine-125 label; j: the distribution of display elements after EDS mapping shows that the marking is successful.
FIG. 4 is a graph showing the different distribution patterns of SPIO labeled EVs of the present invention after ischemic stroke by different routes of administration in an MRI imaging display; comparison of the changes in ischemic stroke cerebral blood flow following three modes of injection i.a., i.v., and i.n. A: schematic of in vivo experiments. B & C, laser speckle after tMACO shows the change and quantification of cerebral blood flow; D. SPIO-pda@evs T2 weighted magnetic resonance imaging of mice by i.a. (D), i.v. (E) and i.n. injection stroke 24 hours after tMCAO.
FIG. 5 is an in vivo SPECT/CT imaging showing the dynamic distribution of 125I-labeled EVs of the invention in stroke mice by different routes of administration; A-F, in vivo biodistribution and quantitative signal analysis of each organ and tissue after different administration routes of SPIO-PDA@EVs; different administration routes the radioactivity intensity of EVs in brain of mice with cerebral apoplexy at different time points.
FIG. 6 is a fluorescence imaging of DiR-labeled EV by different routes of administration; a: in vivo distribution of DiR-tagged EVs in stroke mice; b: ex vivo fluorescence signals of DiR-labeled EVs in brain and major organs (liver, spleen, lung, heart and kidney); c: quantification of brain and major organ homogenates in stroke mice treated with DiR-labeled EVs; d: HE staining shows the cellular structure of brain, heart, lung, liver, spleen and kidney in stroke mice (upper panel). Confocal images of brain sections showed in vitro distribution of DiR-labeled EV (red) in stroke mice 48 hours after i.a., i.v., or i.n. injection; e: quantification of DiR fluorescence intensity in brain, heart, lung, liver, spleen and kidney; f: confocal imaging of brain sections showed the distribution of DiR-labeled EVs in various cells; g: the bar graph shows quantification of phagocytic index for different cell types.
Figure 7 is a graph of RVG modification of the present invention that increases neuronal uptake of EVs in vitro; a: STEM images of PDA@EVs and RVG-PDA@EVs; b: dynamic Light Scattering (DLS) measurements PDA@EVs and RVG-PDA@EVs; c: zeta potentials of PDA@EVs and RVG-PDA@EVs; d: confocal imaging showed that PKH 26-labeled EVs (red) were co-labeled with primary neurons and astrocytes; e: RVG modification increases targeting of EVs to neurons; f: quantitative differences in phagocytosis of PKH 26-labeled EVs with neurons and astrocytes before and after modification; g: quantitatively displaying different sets of neuronal phagocytosis indices; h: cell viability of neurons treated with EVs, PDA@EVs, RVG-PDA@EVs and RVG+RVG-PDA@EVs.
Figure 8 is a graph of RVG modification of the present invention that promotes targeted delivery of EVs in ischemic brain.
A: cerebral magnetic resonance imaging of mice with cerebral apoplexy at different time points before and after injection of SPIO-PDA@EVs and RVG-SPIO-PDA@EVs; b (B)&C: prussian blue staining of brain sections 48 hours post injection showed SPIO-pda@evs phagocytized and quantified by cells; d (D)&E: 125 I-PDA@EVs and RVG- 125 Imaging and quantifying data of the mice after the cerebral apoplexy is arterial injected by the I-PDA@EVs; f: 125 I-PDA@EVs and RVG- 125 Representative fluorescence image after I-pda@evs arterial injection; g: quantification of EVs fluorescence intensity in ipsilateral brain homogenates; h&I: confocal imaging showed that EVs co-localized and quantified with neurons.
Figure 9 is a graph of RVG modified M2 EVs of the invention further reducing neuronal apoptosis and promoting restoration of neurological function in mice following tMCAO; TUNEL and Fluoro-selected B staining showed neuronal death and quantification in PBS, M2-EVs or RVG-M2-EVs treated stroke mice; e & F: M2-EVs and RVG-M2-EVs inhibit the expression and quantification of clear caspase-3; g & H M2-EVs and RVG-M2-EVs improved mNSS scoring and stick rotation testing in stroke mice.
FIG. 10 is a schematic representation of an M2-EVs of the invention that up-regulates apoptosis-related miRNAs by transcriptomic sequencing; a: principal Component (PC) analysis of miRNA array data for M0-EVs, M2-EVs, and RVG-M2-EVs; b: bar graphs show differentially expressed mirnas in M2 EVs and M0 EVs; c & D: the heat and volcanic plots show that the differentially expressed mirnas between the M2 and M0 groups are highest; e: the dot plot shows the first 20 enrichment pathways upregulated in the M2 group compared to the M0 group.
Detailed Description
The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.
It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Marking strategies for EVs, both direct and indirect, have been developed to enable multi-modality imaging tracking. However, the indirect marking method needs to be performed in advanceThe parent cell is genetically modified, and the process is time consuming and inefficient. Thus, the isolated EVs are marked more simply and effectively by a direct marking mode, but irreversible damage is often caused to exosomes, and the biological activity of the EVs is further influenced. It has been reported that EVs can directly label radioiodine-125 by physical adsorption, but the physically adsorbed radioiodine-125 is extremely unstable under physiological conditions, is very easy to label off, realizes nonspecific uptake of the radioiodine-125 in vivo, and further affects imaging effects. Thus, it is necessary to establish a simple but effective method of multi-modal EVs visualization without loss of EVs bioactivity. In recent years, polydopamine (PDA) has attracted a great deal of interest due to its self-polymerizing properties and multifunctional chemical reactivity. In contrast to other complex labelling methods, dopamine can be entrapped on the surface of EVs by catechol self-aggregation under weakly alkaline conditions, and catechol and anthraquinone bonds on PDA coatings can readily coordinate with many metal ions, such as Fe 3+ 、Gd 3+ 、Pt 4+ . In addition, PDA can also react with halogen, sulfhydryl and amino through Michael addition reaction, thereby being beneficial to modifying EVs with radioisotope, polyethylene glycol (PEG), targeting small molecule complex and the like, being used for visual imaging of EVs, and further improving stability of EVs under physiological condition and targeting ability in treatment process.
Based on the premise, we successfully construct a three-functional imaging diagnosis and treatment agent for cerebral apoplexy repair and nuclear medicine/magnetic resonance imaging/fluorescence by collecting and purifying exosomes secreted by M2 type microglia and staining the exosomes by using fluorescent dye, wrapping the products by using dopamine self-aggregation, and directly coupling a magnetic resonance imaging contrast agent and a radionuclide to a PDA wrapping layer, so as to noninvasively and quantitatively track the directional migration, repair and curative effect evaluation of EVs to cerebral apoplexy areas under different administration routes. The in vivo real-time visual dynamic monitoring of the diagnosis and treatment agent on the level of biological individuals, organs/tissues, cells and molecules is realized. The targeted migration quantity of Evs to neurons at cerebral ischemia sites is remarkably improved through coupling specific ligands of targeted cerebral neurons to exosome diagnosis and treatment agents, so that the treatment effect on cerebral apoplexy is improved. miRNA related to neuronal apoptosis is identified through miRNA sequencing of EVs, and the potential therapeutic mechanism of M2 microglial-derived diagnosis and treatment agents in ischemic brain is clarified. The invention provides a powerful marking method and a functional modification approach for clinical application and popularization of EVs in cerebrovascular diseases and even neurodegenerative related diseases in future.
The invention shows great potential in treating stroke aiming at Extracellular Vesicles (EVs) of exosomes, and provides a solution for the comprehensive visualization and understanding of directional migration and distribution of EVs in the stroke treatment process.
The invention provides a nanometer-sized exosome diagnosis and treatment agent probe, which at least comprises exosome, fluorescent dye for marking the exosome, dopamine or dopamine derivative on the surface of the exosome, radionuclide, magnetic resonance imaging ion and ligand for targeting neurons.
The exosome is derived from microglial cells
The source cells of the exosomes are pretreated as follows: interleukin 4 induced M0 microglial cells to differentiate directionally into M2 microglial cells at least 48 hours later.
In certain embodiments of the invention, the obtained exosomes are stained by a fluorescent dye selected from one or more of DiR, cy5.5, cy 7.
The dopamine derivative is selected from dopamine hydrochloride.
The radionuclide is selected from one of iodine-125, iodine-131, gallium-68 and copper-64.
The magnetic resonance imaging ion is T1 or T2 contrast agent, and is selected from Gd 3+ 、Mn 2+ 、Fe 3+ 、Cu 2+ 、Ni 3+ One or more of the following; the T1 contrast agent or the T2 contrast agent is provided by paramagnetic or superparamagnetic contrast agent particles, and the paramagnetic metal salt comprises one or more of gadolinium chloride, gadolinium nitrate, gadolinium acetate, manganese chloride, manganese nitrate, manganese acetate and ferroferric oxide.
The ligand of the targeted neuron is one or more of protein, monoclonal antibody or small molecule inhibitor. In certain embodiments of the invention, the neuron-targeting ligand is selected from one or more of RVG.
The exosome diagnostic agent is a probe that can be used for medical imaging. The medical imaging is selected from any one or more of nuclear medical imaging (e.g. PET, SPECT), magnetic Resonance Imaging (MRI), CT imaging. For example, the probe may 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 certain embodiments of the invention, the probe is schematically depicted in FIG. 1.
The invention also provides a preparation method of the exosome, which comprises the following steps:
culturing undifferentiated M0 type cells in vitro, promoting differentiation to M2 type cells by adding polarization factors, and secreting exosomes; collecting and purifying microglial cells obtained in the step 1) through centrifugal separation to obtain an exosome of M2 type cell sources; all or part of the exosomes obtained in the step 2) are marked by fluorescent dye;
coating all or part of the surface of the exosome subjected to fluorescent dye staining marking obtained in the step 3) with a dopamine or dopamine derivative layer; mixing a radionuclide, magnetic resonance imaging ions, and the product of step 4); 6) Grafting the ligand of the targeted neuron on the surface of the product obtained in the step 5) to obtain the exosome diagnosis and treatment agent.
In certain preferred embodiments of the present invention, the step 1) of in vitro directed differentiation of M0 type cells comprises: the polarization factor is added to the M0 type cell, promotes its differentiation into the M2 type cell, and secretes exosomes. In one embodiment, the M0 type cells are selected from microglial cells, the polarization factor is selected from IL4, the total volume of the total culture system is taken as the reference, the culture volume is controlled to be 15 mL, the concentration of the polarization factor is added to be 20 ng/mL, the polarization culture time is 36-48 hours, after the culture is finished, the supernatant of the M2 type microglial cells after stimulation is collected, and the exosomes to be extracted are stored at the temperature of minus 20 ℃.
In certain preferred embodiments of the invention, the conditioned medium after collection of stimulated M2 microglial cells in step 2) is ultracentrifuged under conditions of 300 g continuous centrifugation for 10 minutes, 2000 g continuous centrifugation for 15 minutes, 10000 g continuous centrifugation for 30 minutes to remove cell debris, and 100000g continuous ultracentrifugation for 70 minutes to pellet the exosomes, and the EV is washed once with 100000g of PBS for 70 minutes and suspended for further characterization. The obtained exosomes are collected and used for identifying the structure by adopting a Transmission Electron Microscope (TEM) of 120 kV, the diameter and the particles of the exosomes are measured by Nanoparticle Tracking Analysis (NTA), the content of the exosomes is measured by a BCA method, and the exosome markers CD63 and TSG101 are analyzed by western immunoblotting.
In certain preferred embodiments of the present invention, step 3) specifically comprises: according to the instructions, the exosomes were modified using the near infrared fluorescent dye DiR and the red fluorescent dye PKH 26. For the labeling procedure of DiR, the exosomes and 1 μl of diluted DiR solution were incubated for 5 mins at 37 ℃. For in vitro and in vivo urinary uptake experiments, 1: PKH26 after 500 dilutions was incubated with exosomes for 5 mins at room temperature. In the above process, serum free of EVs is used throughout the course to prevent excessive labeling. The labeled exosomes were washed in 100000g PBS for 1 hour to wash off non-specific labels, and the exosomes were resuspended in PBS for use.
In certain preferred embodiments of the present invention, step 4) specifically comprises: 200. Mu.L of EVs were mixed with PBS (1:1), and 1 mg of PDA dissolved in 8 mL Tris buffer (pH=8.5) was added to the solution and reacted for 10 minutes to give a dopamine concentration of 0.1 mg/mL. Thereafter, PDA-coated EVs were suspended in dialysis tubing (100 kDa-MWCO), isolated by ultrafiltration at 10000 g for 8 minutes, and washed three times with PBS, resulting in a final product with a dopamine-coated exosome concentration of about 1.2X10 11 particles/mL. PDA was attached to amino-functionalized indocyanine green (ICG) to assess labeling efficiency, and fluorescence intensity ICG-PDA@EVs was measured with a microplate reader before and after ultrafiltration to assess PDA labeling rate.
In certain preferred embodiments of the present invention, step 5) specifically comprises: radioiodine-125 labeling was achieved by classical Iodogen methods. PDA@EVs suspension0.5 mL，≈1.2×10 11 Individual particles/mL) was added to a glass tube with a bottom coated with 20 μg Iodogen. To freshly prepared Na 125 The I solution (500. Mu. Ci,18.5 MBq) was added to the tube and intermittently shaken to avoid PDA@EVs deposition. After incubation for 30 mins at room temperature, the final product was purified by ultracentrifugation and washed three times with PBS. The labeling efficiency was evaluated by performing radioactive thin layer chromatography using a gamma detector. The stability of the radioactive probe was evaluated by co-incubating 5 μl samples in 200 μl of 10% FBS for different times (1, 3, 6, 12 and 24 hours) in DMEM high sugar solution at 37 ℃. 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. 125 I-labeled SPIO-PDA@EVs remain at the origin, while free 125 I remains with the mobile phase at the leading edge, 125 the radiochemical purity of the I-labeled SPIO-pda@evs is expressed as a percentage of the total radioactive dose to the SPIO-pda@evs probe remaining at the origin.
In certain preferred embodiments of the present invention, step 5) specifically comprises: pda@evs were labeled with amino-functionalized superparamagnetic iron oxide (SPIO) at a concentration of 20 μg/mL for 1 hour, then washed and ultrafiltered to discard excess SPIO particles. To check the labeling stability of SPIO-pda@evs were suspended in DMEM high-sugar culture broth containing 10% FBS at 37 ℃ and isolated SPIO particles were measured with inductively coupled plasma-emission spectrometry (ICP-OES) at 0,1, 3, 6, 12, 24 and 48 hours. Labeled EVs were measured at 37 ℃ using a 1.41T minispec mq 60 NMR analyzer (1×10 9 Individual particles/mL) T2 relaxation time. By mixing 1X 10 9 The individual probes were incubated at 37℃for different times (0, 1, 3, 6, 12, 24 and 48 hours) in 5 mL of DMEM high-sugar culture medium containing 10% FBS, and the possible release of SPIO from the probes was studied in triplicate. After incubation, the probes were 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 at pda@evs relative to the probe. The Zeta potential of the labeled EV was measured using DLS for each step in the probe preparation process. To evaluate stability of SPIO markers, EV, PDA @Hydrodynamic diameters and surface charges of EVs and SPIO-PDA@EVs were examined by DLS in 10% FBS (v/v) solutions for 0,1, 2, 3, 4 days.
In certain preferred embodiments of the present invention, step 6) specifically comprises: 50. Mu.g RVG was added to the EVs suspension (300. Mu.L) and reacted at room temperature for 2-4 hours. RVG modified EVs were then purified by ultrafiltration and washed with PBS. Fluorescence intensity FITC-RVG-pda@evs were quantified using a microplate system before and after purification to assess the grafting rate.
In an embodiment of the invention for characterizing exosomes, specifically comprising:
ICP-OES measurement. 100. Mu.L of SPIO was added to the exosomes treated with 45 mins PDA and allowed to react for 5 minutes. The SPIO-pda@evs thus produced were suspended in DMEM high glucose containing 10% FBS and incubated at 37 ℃ for 1, 3, 6, 12, 24 and 48 hours. The stability of the label was measured by measuring SPIO-pda@evs after KI addition, ultracentrifugation and deionized water dilution using an inductively coupled plasma emission spectrometer. Furthermore, SPIO-PDA@EVs (. Apprxeq.3.8X10) 12 particle/mL) and an equal amount of pure SPIO were heated at 120 ℃, and compared after ablation for 1 hour with 50 μl of nitric acid added per mL of product. The mark rate was calculated as (Fe ion concentration/SPIO ion concentration in SPIO-pda@evs) ×100%.
STEM imaging. 5. mu.L of SPIO-PDA@EVs were placed on a hydrophilic carbon film and incubated for 2 minutes. The copper mesh and samples after incubation were washed three times with deionized water, each time dried 10 a s a at room temperature to reduce salt deposition. Next, 4. Mu.L of phosphotungstic acid was added to the sample and dried for 1 minute. In order to prevent SPIO-pda@evs from entering the pole piece during imaging, the top of the sample was covered with a carbon film. Bright field and high angle annular dark field imaging (HAADF) imaging was performed using weight percent (wt%) or atomic percent (at%) scans for 5-10 minutes. Analysis of energy dispersive spectrometer-detector (EDS) mapping SPIO-pda@evs and STEM have elemental distributions of the same iron, iodine, nitrogen, carbon and oxygen elements
In an embodiment of the exosome imaging of the present invention, specifically comprising:
magnetic resonance imaging. Using a T2 weighted spin echo sequence, the parameters are as follows: 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 the ParaVision 6 software. An average signal intensity within a region of interest (ROI) drawn around the tumor is calculated for each image. The 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. Immediately after final in vivo imaging, mice were sacrificed and brains were collected and fixed in 4% paraformaldehyde solution for histological analysis.
SPECT/CT imaging. Injection into mice by different modes of administration (tail vein, carotid artery and/or nasal administration) 125 I-labeled EVs, and imaged at 0.5, 6, 24, and 48 hours post-injection, each mouse was irradiated at a dose of 300 μci. 125 The amount of I-pda@evs was measured by the injection dose density per volume of organ from the region of interest (ROI). The CT image provides an anatomical reference to the location of the mouse. SPECT images were obtained at 32 maps at 360 ℃ (radius of rotation=7.6 cm,30 seconds/map). Reconstruction data from SPECT and CT were visualized and co-registered using an insivoscope. The ROI was drawn in the major organ exhibiting significant radioactivity.
Fluorescence imaging. For in vivo fluorescence imaging, mice were injected with DiR-labeled EVs by different modes of administration (tail vein, carotid artery, and/or nasal administration) and imaged at excitation and emission wavelengths of 748 and 780nm, respectively, using an IVIS system 0.5, 6, 24, and 48 hours after injection. The ipsilateral and contralateral brain, heart, lung, stomach, liver, spleen, kidney and background ROIs are selected from equally sized regions containing the same number of pixels. Quantification of uptake was determined by mapping the ROI of each organ. The removed organs were placed on ice and weighed and stored to minus 80 ℃ to verify the fluorescence of the homogenate. Briefly, 80-150 mg of each organ was added to 1 mL lysate buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% Ige-pal (NP 40), 0.5% sodium deoxycholate, 0.1% SDS, and 1% protease inhibitor cocktail. The samples were 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 measured immediately, thereby calculating the fluorescence intensity per gram of tissue.
In an embodiment of the exosome treatment of the present invention, specifically comprising: adult ICR mice were subjected to a transient middle cerebral artery ischemia model, and 100 ug exosomes (1.0X10) were injected intravenously, arterially, and nasally, respectively, one day after molding 11 Individual particles). Neurobehavioral detection and brain slice collection were performed at 0,1 and 3 days post-surgery for immunofluorescent staining, identifying the type of neuronal cells that took up the exosomes, and further defining the targeting and treatment of the labeled exosome-treated body to neuronal cells after ischemic stroke.
The invention detects the morphology and the particle size of the exosomes after normal and PDA coating respectively through a transmission electron microscope and NTA (shown in figures 3A-B), and discovers that the expression changes of exon markers CD63 and TSG101 in the exosomes are not influenced after PDA coating (figures 3C-D). STEM imaging showed a particle coverage of superparamagnetic ferroferric oxide of around 10 nm on the exosome membrane (fig. 3E) in order to show the spatial distribution of the exosome by magnetic resonance imaging. By detecting the Zeta potential and particle size of the labeled exosomes over time (fig. 3F-G), it was found that our iron particle labeling was successful and stable. To enable us to perform SPET/CT imaging, we label exosomes with classical iodine-125, with nuclide imaging showing a labeling rate as high as 90% (FIG. 3H). The radiolabeled layer showed that the labeling rate remained around 85% after 24 h, indicating that the radiolabeling was stable (fig. 3I). In addition, the spectrometer mapping showed that iodine and Fe were accumulated on the surface of the single exosomes, indicating successful labeling (fig. 3J).
By utilizing a multi-modality imaging system, the present invention demonstrates the exosome tracing effect of markers in mice with transient ischemic strokes. First, laser speckle cerebral blood flow quantification confirmed the success of mice occlusion and reperfusion during tMCAO surgery (fig. 4B-C). To determine the optimal route of administration, stroke mice were injected with 100 μg (1×10) by intraarterial (i.a.), intravenous (i.v.), and intranasal (i.n.), respectively 11 Individual particles) exosomes. Using 7T magnetic resonance, T2 weighted imaging (fig. 4E-F) was performed before and after injection of mice one day after molding, and it was seen that after intra-arterial injection for 30 min, a strong contrast was detected in the infarct zone, but almost disappeared after 24 h, indicating rapid uptake and degradation of EVs in the ischemic brain. In contrast, brain uptake was much lower after intravenous and intranasal injection than after intra-arterial injection.
To observe and track migration of EVs in major organs and tissues, the present invention treats stroke mice with radiolabeled exosomes and scans with SPECT/CT. As shown in fig. 5, it can be seen that EVs were detected with significant organ targeting and biodistribution characteristics in different routes of administration. SPECT/CT imaging results indicated: after 30 min of arterial administration, labeled exosomes appear mainly in the liver/spleen and bladder, accumulating in small amounts in the lungs and heart, and also in large amounts in the ipsilateral brain. 24 After h, there is still a significant accumulation of EVs in the liver/spleen through clearance of the renal system, while the signal in the brain is significantly reduced by nearly 50%;48 After h, little or no EVs were found in the brain, and the results indicate that EVs after arterial injection, uptake and cellular internalization were efficient. After 30 mins intravenous injection, EVs accumulate strongly in the liver, kidneys and thyroid, but the signal in the brain is much lower than in arterial injection. 24 After h the signal disappeared, indicating a lower initial uptake of EVs in the brain after intravenous injection and a faster pharmacokinetic acceleration. In contrast to the first two injection modes, nasal delivery of EVs, with the exception of thyroid, did not migrate to other organs or tissues, with EVs tending to migrate to the gastrointestinal tract after 6 h and exit the body at 48 h post injection. SPET/CT results show a 2.4-fold and 2.0-fold increase in nuclear signal in the brain, respectively, compared to 6 h after arterial injection of EVs, both intravenous and nasal injection.
The kinetics of the distribution of EVs in different organs was verified by using fluorescence imaging. Near infrared dye DiR was inserted into the membranes of EVs for ex vivo and in vivo detection using a small animal in vivo imaging system. The results of in vivo imaging of small animals showed (FIGS. 6A-B) that the DIR signal appeared at the abdominal and pelvic sites for the first 6 h of arterial and intravenous exosomes, the signal was significantly reduced after 24 h, whereas nasal injection was first performed at the site of injectionThe site signal is higher and then continuously falls. Ex vivo imaging shows different distribution profiles, with arterial and intravenous injection focused mainly on the liver, lung, kidney and damaged hemispheres of the brain, whereas nasal injection gives strong signals in the stomach, lung and spleen with very little brain signal. The results are consistent with MRI and SPECT/CT imaging, indicating that intra-arterial injection produces a stronger signal in the ipsilateral brain than the three injection modes, 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 (fig. 6D) to verify the distribution of EVs. HE staining showed the structure of the major organs, confocal micrographs showed comparable levels of fluorescence intensity in the liver and spleen of intra-arterial and intravenous mice, but significantly increased EVs accumulated in the injured striatum of intra-arterial mice, further supporting the aforementioned imaging results. Statistics of DiR fluorescence signals in different organs and tissues showed (fig. 6E) that the fluorescence intensity of EVs in the brain of mice after intra-arterial injection was significantly higher compared to intravenous injection or intranasal injection. The invention determines the cell uptake characteristics of EVs in the intra-arterial injection brain by immunostaining. The 3D reconstructed images indicate that most EVs are surrounded by microglia (Iba-1 + ) And neurons (MAP 2) + ) Absorbing, but not endothelial cells (CD 31 + ) Or astrocytes (GFAP) + ) Absorbing.
The invention monitors the pharmacokinetics of the multifunctional EVs probe through various imaging modes, and proves that intra-arterial injection is the best mode for realizing the maximum visualization of EVs delivery to the brain of stroke on the whole body and organ/tissue level. To improve targeting of EVs for uptake by neurons, RVG is linked to PDA to facilitate specific binding to neuronal expressed acetylcholine receptors. Detection by electron microscopy and DLS demonstrated that modification of RVG did not affect morphological features of exosomes. Confocal 3D fluorescence plot showed (fig. 6F), significant increase in MAP2 after RVG modification + Neuronal uptake rates indicate that RVG peptides successfully compete for neuronal binding, but fail to reverse neuronal phagocytosis. To examine whether RVG binding would affect neuronal viability, CCK-8 assays were performed, which found that incubation of 10. Mu.g/mL EVs 6 hDoes not affect the cell viability of the neurons.
To investigate whether RVG modification can improve uptake of EVs in brain, the present invention examined stroke mice treated with RVG modified EVs and untreated EVs by MR/SPECT/FL imaging. MRI showed (figure 8A) that 4.5 h post injection, more RVG modified EVs accumulated in the infarct zone of the ischemic brain, even after 48 h post injection, with many scattered low intensity points located in the diseased brain. Prussian blue staining also showed (FIGS. 8B-C), SPIO-labeled EVs clearly determined to be localized to the core and peri-infarct area, with RVG modifications significantly increasing the accumulation of EVs in the ipsilateral hemispheres. From SPECT/CT images (figures 8D-E), we note that RVG modification significantly improved brain targeting at 0.5 h after injection. After injection, 6 h, the initially aggregated EVs tend to migrate and spread within the diseased brain area. Ex vivo imaging shows that RVG modification does improve targeting of EVs in the brain. RVG modification not only improved uptake of EVs in the brain, but further staining indicated that RVG significantly increased uptake of EV by neurons in the striatal infarct zone. After ischemic stroke, a large number of neurons in the ischemic penumbra undergo apoptosis within hours and last for days. Considering the time-limited therapeutic window of stroke, it is important to assess the appropriate time point for EVs administration. We injected exosomes at post-stroke 2 h (FIGS. 9A-D), compared to PBS group and M2-EVs, found FJ-B after 3D + And TUNEL + The number of cells is significantly reduced and RVG-M2-EVs treatment further reduces neuronal degeneration and apoptosis. WB results showed (fig. 9E) that RVG modified exosome treatment reduced expression of cleaved caspase-3 (clear-caspase 3) and lower nerve severity scores and better rotarod test performance were detected. The results indicate that RVG modification enhances neuroprotection of EVs against ischemic stroke.
To investigate the underlying mechanism of M2 EVs-mediated neuroprotection, EVs-miRNA sequencing was performed (fig. 10). Principal component analysis showed that samples were well dispersed except for one of the RVG-M2-EVs samples. M0-EVs and M2-EVs exhibited different gene expression profiles, while RVG-M2-EVs and M2 EV had similar gene compositions, indicating that RVG-PDA labeling strategies did not alter EV miRNA content. 35 differentially expressed mirnas were identified in M2 EVs compared to M0 EVs, 21 of which were up-regulated and 14 were down-regulated. Further KEGG analysis indicated that some metabolic signaling, synapses, vascular function, and apoptosis-related pathways are enriched in M2 EVs. miRNAs related to apoptosis, including miR-423-3p, miR-7688-5p, miR-106b-3p, miR-532-5p, miR-151-3p, miR-146a-5p and the like are particularly obvious. These results indicate that M2 EVs-enriched anti-apoptosis-related mirnas have neuroprotective effects on acute ischemic stroke mice.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (10)
1. An M2 microglial cell-based exosome diagnosis and treatment agent, which is characterized by comprising an exosome based on M2 microglial cells, a fluorescent dye with surface modification of the exosome, dopamine or a dopamine derivative, a radionuclide, a magnetic resonance imaging ion and a ligand of a targeted neuron.
2. The M2 microglial cell-based exosome therapeutic agent according to claim 1,
the exosome source is M2 type microglial cells;
the fluorescent dye is one of DiR, cy5.5 and Cy 7;
the dopamine derivative is selected from dopamine hydrochloride;
the radionuclide is selected from one of iodine-125, iodine-131, gallium-68 and copper-64;
the magnetic resonance imaging ion is selected from Gd 3+ 、Mn 2+ 、Fe 3+ 、Cu 2+ 、Ni 3+ One or more of the following;
the ligand targeting neurons is selected from RVG.
3. A method for preparing an M2 microglial cell-based exosome therapeutic agent according to claim 1 or 2, comprising the steps of:
1) Culturing undifferentiated M0 type cells in vitro, promoting differentiation to M2 type cells by adding polarization factors, and secreting exosomes;
2) Centrifugally separating the product of the step 1), and collecting and purifying to obtain an exosome of M2 type cell source;
3) All or part of the exosomes obtained in the step 2) are marked by fluorescent dye;
4) Coating all or part of the surface of the exosome subjected to fluorescent dye staining marking obtained in the step 3) with a dopamine or dopamine derivative layer;
5) Mixing a radionuclide, magnetic resonance imaging ions, and the product of step 4);
6) Grafting the ligand of the targeted neuron on the surface of the product obtained in the step 5) to obtain the exosome.
4. The method for preparing an M2 microglial cell-based exosome diagnosis and treatment agent according to claim 1, wherein in the step 1), M0 type cells are selected as microglial cells, polarization factors are selected as IL4, the total volume of the total culture system is taken as a reference, the culture volume is controlled to be 15 mL, the concentration of the added polarization factors is 20 ng/mL, the polarization culture time is 36-48 hours, and after the culture is finished, the supernatant of the M2 type microglial cells after stimulation is collected and stored at-20 ℃ to extract exosomes.
5. The method for preparing an M2 microglial cell-based exosome therapeutic agent according to claim 4, wherein the step 2) specifically comprises:
conditioned medium ultracentrifugation of stimulated M2 microglial cells was collected, conditioned at 4 ℃ by continuous centrifugation at 300 g for 10 min, continuous centrifugation at 2000 g for 15 min to remove dead cells, continuous centrifugation at 10000 g for 30 min to remove cell debris, and continuous ultracentrifugation at 100000g for 70 min to pellet the exosomes, EV washed once with 100000g PBS for 70 min and suspended for further characterization;
collecting the obtained exosomes, identifying the structure by adopting a transmission electron microscope of 120 kV, measuring the diameter and particles of the exosomes by nanoparticle tracking analysis, measuring the content of the exosomes by a BCA method, and analyzing exosome markers CD63 and TSG101 by western immunoblotting.
6. The method for preparing an M2 microglial cell-based exosome therapeutic agent according to claim 5, wherein the step 3) is specifically to modify exosomes using a near infrared fluorescent dye DiR and a red fluorescent dye PKH 26;
for the labeling procedure of DiR, the exosomes and 1 μl of diluted DiR solution were incubated for 5 mins at 37 ℃;
for in vitro and in vivo urinary uptake experiments, 1: the 500 diluted PKH26 and exosomes were incubated for 5 mins at room temperature;
serum without EVs is adopted in the whole process;
the labeled exosomes were washed in 100000g of PBS for 1 hour to wash off non-specific labels, and the exosomes were resuspended in PBS for use.
7. The method for preparing an M2 microglial cell-based exosome therapeutic agent according to claim 6, wherein the step 4) specifically comprises:
200. Mu.L of EVs were mixed with PBS at a ratio of 1:1, and 1 mg of PDA dissolved in 8 mL of Tris buffer (pH=8.5) was added to the solution to react for 10 minutes to give a dopamine concentration of 0.1 mg/mL;
thereafter, the PDA-coated EVs were suspended in dialysis tubing, isolated by ultrafiltration at 10000 g for 8 minutes, then washed three times with PBS, and the final product had a dopamine-coated exosome concentration of about 1.2X10 11 particles/mL.
8. The method for preparing an M2 microglial cell-based exosome therapeutic agent according to claim 7,
the radioiodine-125 labelling in step 5) was achieved by classical Iodogen methods, by suspending PDA@EVs (0.5 mL, # 1.2X10) 11 Individual particles/mL) was added to a glass tube with a bottom coated with 20 μg Iodogen. To freshly prepared Na 125 Solution I (500 μci,18.5 MBq) was added to the tube, shaken intermittently to avoid pda@evs deposition, after incubation for 30 mins at room temperature, the final product was purified by ultracentrifugation and washed three times with PBS;
pda@evs were labeled with amino-functionalized superparamagnetic iron oxide (SPIO) at a concentration of 20 μg/mL for 1 hour, then washed and ultrafiltered to discard excess SPIO particles.
9. The method for preparing an M2 microglial cell-based exosome therapeutic agent according to claim 8, wherein the step 6) specifically comprises: mu.g RVG was added to 300. Mu.L of EVs suspension and reacted at room temperature for 2-4 hours. RVG modified EVs were then purified by ultrafiltration and washed with PBS.
10. Use of an M2 microglial cell-based exosome diagnostic agent in the preparation of a medical imaging probe, characterized in that the medical imaging is selected from any one or more of nuclear medical imaging, magnetic resonance imaging, CT imaging.
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