A 99m Tc-labeled extracellular vesicles, methods of making and using the same

Abstract

This invention discloses a 99m Tc-labeled extracellular vesicles, their preparation method, and applications. The preparation method includes: S1, mixing equal volumes of stannous chloride solution and extracellular vesicle solution and incubating for 5-10 minutes to obtain pre-stannous extracellular vesicles; S2, adding... 99m Tc-NaTcO4 solution, co-incubated for 25 min to 40 min, to obtain 99m Tc-labeled extracellular vesicles. Applications include... 99m The application of Tc-labeled extracellular vesicles in the preparation of drugs for myocarditis, drugs for liver injury, and kits for the identification of liver injury. This invention... 99m Tc-labeled extracellular vesicles can effectively reduce inflammatory markers in myocarditis and improve myocardial enzymes, providing a new direction for the research of anti-myocarditis drugs.

Description

Technical Field

[0001] This invention belongs to the field of radiolabeled biological applications technology, specifically relating to a... 99m Tc-labeled extracellular vesicles, their preparation methods, and applications. Background Technology

[0002] Myocarditis, especially acute myocarditis, is an inflammatory disease of the myocardium caused by infection (such as viruses or bacteria) or autoimmune diseases. Its clinical manifestations are diverse, ranging from asymptomatic to fulminant myocarditis, leading to acute heart failure, cardiogenic shock, and even sudden death. It is one of the leading causes of sudden cardiac death in adolescents.

[0003] Clinical challenges in myocarditis include: 1. Diagnostic difficulties: Endocardial biopsy is the "gold standard," but its invasiveness limits its widespread application. Non-invasive biomarkers such as troponin and cardiac magnetic resonance imaging still have insufficient sensitivity and specificity. 2. Limited treatment options: Currently, there is a lack of specific targeted therapies. Mainstream treatments include supportive care (such as rest, diuretics, and angiotensin-converting enzyme inhibitors) and immunosuppressive therapy for severe cases. However, immunosuppressive therapy may carry the risk of infection and is ineffective in some patients.

[0004] Extracellular vesicles of mesenchymal stromal cells (MSCs) are nanoscale vesicles secreted by MSCs, carrying bioactive substances such as mRNA, miRNA, proteins, and lipids from the mother cells. They are considered key "paracrine" mediators for the therapeutic effects of MSCs. Their main functions include: regulating the function of T cells, B cells, and macrophages (promoting M2 anti-inflammatory phenotype polarization) and inhibiting excessive immune responses by delivering substances such as TGF-β, HLA-G, miR-17-5p, and let-7b-5p; inhibiting excessive activation of cardiomyocytes and apoptosis signaling pathways by delivering miR-19a and miR-45-2; activating vascular endothelial cells and promoting angiogenesis by being rich in miR-126 and miR-210; and exhibiting a homing effect by specifically migrating to sites of inflammation and injury.

[0005] In recent years, the potential of MSCs-Exo in the treatment of myocarditis has begun to attract attention. A study published in *Circulation Research* by Zhou et al. (2023) showed that intravenous infusion of MSCs-Exo significantly reduced myocardial inflammatory infiltration, decreased cardiomyocyte apoptosis, and improved cardiac function in an experimental autoimmune myocarditis mouse model. The mechanism was confirmed to be closely related to the exosome-carried miR-21 targeting and inhibiting the PDCD4 pathway, thereby promoting macrophage polarization towards the M2 type. However, these encouraging results are almost entirely limited to small animal models; the translation into large preclinical animal models is an essential step towards clinical application.

[0006] In preclinical research, finding animal models that can highly mimic human diseases is crucial. As non-human primates, cynomolgus monkeys are highly similar to humans in terms of genetics, physiology, and the immune system. Spontaneous myocarditis has been reported in cynomolgus monkeys, and this model, compared to chemically or virally induced models, more realistically reflects the complex, multifactorial disease process of human myocarditis, particularly the central role played by the immune system.

[0007] Real-time, in vivo, and quantitative tracking of exosome distribution is crucial for optimizing dosing regimens and understanding their mechanisms of action. Current tracking technologies primarily include: optical imaging, such as near-infrared fluorescence (NIRF) and bioluminescence imaging (BLI). Optical imaging requires prior labeling of exosomes with fluorescent dyes (e.g., DIR, DiR) or luciferase. Radionuclide imaging, such as positron emission tomography (PET), uses zirconium-89 (… 89 Zr), Copper-64 ( 64 Radiolabeled exosomes with radioactive isotopes such as Cu can achieve highly sensitive quantitative tracking of deep tissues. Multimodal imaging, such as combining optical imaging and radionuclide imaging or combining multiple other techniques, can simultaneously obtain high-sensitivity and high-resolution information.

[0008] However, these methods also have significant drawbacks. For example, labeling efficiency and tracer accuracy are problematic: fluorescent dye labeling may leak or produce false positive signals (such as being re-released after being taken up by the reticuloendothelial system); radioactive labeling may alter the surface properties of exosome membranes, affecting their natural tropism. Quantification is also challenging: optical imaging signals are easily affected by tissue penetration depth, making precise quantification difficult. While PET can quantify, it cannot distinguish between "active" and "inactive" exosomes: current technologies can only show the physical location of exosomes, but cannot differentiate whether they maintain their complete biological function and are effectively taken up by target cells.

[0009] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0010] To address the aforementioned problems in the prior art, the present invention provides a... 99m Tc-labeled extracellular vesicles, their preparation methods, and applications. The technical problem to be solved by this invention is achieved through the following technical solution: In a first aspect, the present invention provides a 99m The method for preparing Tc-labeled extracellular vesicles includes the following steps: S1. Mix equal volumes of stannous chloride solution and extracellular vesicle solution and incubate for 5-10 minutes to obtain a pre-stannous extracellular vesicle solution; wherein the mass concentration of stannous chloride is 0.005%-0.01%, and the concentration of the extracellular vesicle solution is (3-3.75)×10⁻⁶. 11 cells / mL (concentration of 3~3.75×10⁻⁶) 11 / mL of extracellular vesicles contains approximately 0.75 mg / mL of total protein). S2, Add to the pre-tinned extracellular vesicle solution 99m Tc-NaTcO4 solution, co-incubated for 25 min to 40 min, to obtain 99m Tc-labeled extracellular vesicles.

[0011] In one embodiment of the present invention, the extracellular vesicle solution is mesenchymal matrix extracellular vesicles, and the concentration per 1 mL is (3-3.75) × 10⁻⁶. 11 Add the above to the extracellular vesicles of the mesenchymal matrix per mL. 99m The dose of technetium-99m in Tc-NaTcO4 solution is 7.5 mCi to 30 mCi.

[0012] In one embodiment of the present invention, the 99m The Tc-NaTcO4 solution was obtained by rinsing, and the 99m The Tc-NaTcO4 solution should be stored for less than 3 hours.

[0013] Secondly, the present invention provides 99m Tc-labeled extracellular vesicles were obtained using the preparation method described above.

[0014] Thirdly, the present invention provides the above-mentioned 99m Application of Tc-labeled extracellular vesicles in the preparation of drugs for myocarditis.

[0015] In one embodiment of the invention, the drug is prepared as a unit-dose formulation, each unit of which contains 99m The protein mass of Tc-labeled extracellular vesicles is no greater than 2.1 mg.

[0016] According to the embodiments, the protein content of extracellular vesicles in a unit dose of the formulation was calculated using the safe dose conversion from the cynomolgus monkeys in Example 4. Specifically, the conversion was based on body surface area (BSA), and the adult dose was calculated to be 21.14 mg using the average parameters of 175 cm² / 75 kg. A safety factor of 10 was introduced, meaning that the protein mass of extracellular vesicles in the initial dose was no more than 2.1 mg.

[0017] In one embodiment of the present invention, the drug is an injectable preparation.

[0018] Fourthly, the present invention provides the above-mentioned 99m The application of Tc-labeled extracellular vesicles in the preparation of drugs for liver injury, wherein the liver injury includes type 2 diabetes mellitus combined with non-alcoholic fatty liver disease and food-induced fatty liver disease.

[0019] Fifthly, the present invention provides the above-mentioned 99m Application of Tc-labeled extracellular vesicles in the preparation of kits for identifying liver injury.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention provides 99m Preparation method of Tc-labeled extracellular vesicles 99m Tc exhibits a high percentage of radioactivity when it binds to extracellular vesicles, and this high percentage of radioactivity is maintained even 24 hours after labeling.

[0021] 2. The present invention provides 99m Tc-labeled extracellular vesicles can effectively reduce inflammatory markers in myocarditis and improve myocardial enzymes, providing a new direction for myocarditis drug research.

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 This is provided by the embodiments of the present invention. 99m A schematic diagram showing the percentage of radioactivity that Tc-labeled MSC-EVs bind to external vesicles during 24 hours of in vitro incubation. Figure 2 This is a schematic diagram comparing the morphology and particle size distribution of MSC-EVs before and after labeling, provided in an embodiment of the present invention. Figure 3 This is a schematic diagram comparing Western Blot results before and after MSC-EVs labeling, provided in an embodiment of the present invention. Figure 4This is provided by the embodiments of the present invention. 99m Schematic diagram of the biological effects of co-incubation of Tc-labeled MSC-EVs with LO2 cells, BMDM cells, and BV2 cells; Figure 5 These are different dosages provided in the embodiments of the present invention. 99m A diagram showing the percentage of Tc radioactivity bound to external vesicles when labeling MSC-EVs; Figure 6 This is a schematic diagram of the whole-body SPECT / CT tomographic fusion imaging results of db / db mice provided in an embodiment of the present invention; Figure 7 This is a schematic diagram of the whole-body SPECT / CT tomographic fusion imaging results of HFD mice provided in an embodiment of the present invention; Figure 8 This is provided by the embodiments of the present invention. 99m Electrophoretic patterns and thin-layer chromatography (ITLC) characterization of Tc-labeled MSC-EVs after membrane perforation; Figure 9 The membrane rupture provided in the embodiments of the present invention 99m SPECT / CT images of Tc-labeled MSC-EVs after intravenous injection and intestinal perfusion into mice; Figure 10 This is an example of intravenous injection of a normal cynomolgus monkey provided in this invention. 99m Schematic diagram of whole-body SPECT / CT tomographic fusion imaging results at different time points after Tc-labeled MSC-EVs; Figure 11 This is an example of intravenous injection of a normal cynomolgus monkey provided in this invention. 99m A schematic diagram of organ distribution percentage (%ID) kinetic analysis after Tc-labeled MSC-EVs; Figure 12 This is an example of intravenous injection of a normal cynomolgus monkey provided in this invention. 99m Schematic diagram of organ distribution concentration (%ID / mL) kinetics analysis after Tc-labeled MSC-EVs; Figure 13 This is an example of intravenous injection of a normal cynomolgus monkey provided in this invention. 99m Schematic diagram of the blood half-life of Tc-labeled MSC-EVs; Figure 14 This is an example of intravenous injection of a normal cynomolgus monkey provided in this invention. 99m Schematic diagram of blood AUC after Tc-labeled MSC-EVs; Figure 15 This is an example of intravenous injection of a normal cynomolgus monkey provided in this invention. 99m Schematic diagram of whole-body retention dynamics analysis after Tc-labeled MSC-EVs; Figure 16 This invention provides an embodiment of intravenous injection of different doses into normal cynomolgus monkeys. 99m Comparison of blood routine, liver and kidney function, and myocardial enzyme spectrum results after Tc-labeled MSC-EVs; Figure 17 The embodiment of this invention provides a spontaneous myocarditis cynomolgus monkey injected with 0.75 mg of extravesicular vesicle protein. 99m Comparison of blood routine, liver and kidney function, and myocardial enzyme profiles of Tc-labeled MSC-EVs with those of cynomolgus monkeys with spontaneous myocarditis injected with saline. Detailed Implementation

[0024] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following description, in conjunction with the accompanying drawings and specific embodiments, explains the proposed invention. 99m This paper provides a detailed explanation of Tc-labeled extracellular vesicles, their preparation methods, and applications.

[0025] The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of specific embodiments in conjunction with the accompanying drawings. Through the description of the specific embodiments, a more in-depth and concrete understanding can be gained of the technical means and effects adopted by the present invention to achieve its intended purpose. However, the accompanying drawings are for reference and illustration only and are not intended to limit the technical solutions of the present invention.

[0026] Example 1 This embodiment provides a 99m The method for preparing Tc-labeled extracellular vesicles specifically includes the following steps: Step 1: Obtain mesenchymal extracellular vesicle (MSC-EVs) solution In this embodiment, umbilical cord blood was used. After screening and culture, mesenchymal stromal cells were obtained. After qualitative and activity tests on the mesenchymal stromal cells, the supernatant was collected. The supernatant was centrifuged using a gradient centrifugation method, and then subjected to ultracentrifugation and particle size screening to select particles with a diameter <200nm. Qualitative and quantitative tests were performed using Western blotting, electron microscopy, and omics to obtain mesenchymal stromal cell extracellular vesicles with good activity and quality.

[0027] Step 2: Pre-tinning of extracellular vesicles in mesenchymal matrix The concentration of extracellular vesicles in the mesenchymal matrix was adjusted to 4 × 10⁻⁶. 11 / mL; take 0.1mL (4×10 10 Mesenchymal extracellular vesicles (MSC-EVs with a protein content of 100 μg) were added to 0.1 mL of 0.005% stannous chloride solution and co-incubated for 5 min to obtain a pre-stannous extracellular vesicle solution.

[0028] In this pre-tinning process, Sn 2+ Ions can be adsorbed on the cell membrane surface, and some can even penetrate into the phospholipid bilayer membrane and enter the vesicles.

[0029] Step 3, Add 99m Tc-NaTcO4 solution was used 99m Tc mark Add to pre-tinned extracellular vesicle solution 99m The solution is a Tc-NaTcO4 solution, in which the dose of technetium-99m is 3mCi. 99m Tc-NaTcO4 solution 99m TcO4 - Sn adsorbed on or inside the membrane 2+ Rapid reduction produces a reduced state of technetium (typically +4 or +5 valence). 99m Tc is highly reactive and rapidly forms stable covalent or coordination bonds (complexes) with proteins, glutathione, or other sulfur-containing (-SH) or amino (-NH2) biomolecules within the outer vesicles, thereby obtaining... 99m Tc-labeled extracellular vesicles of mesenchymal matrix ( 99m Tc-labeled MSC-EVs).

[0030] In this step, the following steps are used 99m The Tc-NaTcO4 solution is used to rinse fresh technetium-99m, with a storage time of less than 3 hours. The shorter the storage time after rinsing technetium-99m, the higher the radioactive activity of technetium-99m for labeling cells or extravesicles of equal protein content. Technetium-99m rinsed from the molybdenum-technetium generator for too long (≥2 hours) will have more radionuclides decay into long-lived technetium-99, resulting in lower labelable radioactivity.

[0031] It should be noted that the present invention provides 99m The Tc-labeled extravesicle method is applicable to various extravesicles, such as milk extravesicles, bovine umbilical cord extravesicles, and tool cell extravesicles; it can also be used to... 99m Tc-labeled cells can be used to label somatic cells such as mesenchymal stem cells (MSCs), human hepatocyte line LO2, bone marrow macrophages (iBMDM), and neuronal microglia (BV2), as well as tumor cells such as mouse triple-negative breast cancer cells (4TI), human malignant melanoma cells (B16-F10), and human cervical cancer cells (HeLa cells).

[0032] The initial material prepared in step 3 99mTc-labeled MSC-EVs were analyzed using thin-layer chromatography with silica gel plates (ITLC-SG) and a radiometric scanner, achieving a labeling efficiency greater than 95%. To further improve purification, Sephadex G-25 column chromatography was used for purification, yielding purified Tc-labeled MSC-EVs. 99m Tc-labeled MSC-EVs. The following describes the purified... 99m Tc-labeled MSC-EVs were used for performance testing.

[0033] Performance testing: 1. In vitro stability test: The purified product... 99m Tc-labeled MSC-EVs were placed in fetal bovine serum (FBS) at 37°C to simulate a physiological environment and incubated in vitro for 24 hours. The percentage of radioactivity still bound to the outer vesicles in the samples was determined by thin-layer chromatography at 1 hour, 6 hours, 12 hours, and 24 hours. Results are as follows: Figure 1 As shown, Figure 1 Figure (A) illustrates the instantaneous thin-layer chromatography (ITLC) radiochromatographic spectra at 1 h, 6 h, 12 h, and 24 h after labeling. At each time point, the radioactive peaks are concentrated at the origin (Region 1), indicating... 99m Tc was tightly bound to MSC-EVs, with no obvious free Tc observed. 99m TcO4 – Impurity peaks. Six hours after labeling, 99m The percentage of radioactivity bound to the outer vesicles by Tc is as high as 99%; 24 hours after labeling, 99m The percentage of radioactivity of Tc bound to extracellular vesicles remains at 95%. Figure 1 Figure (B) illustrates the curve of labeling efficiency over time, quantitatively demonstrating... 99m The stability trend of Tc-labeled MSC-EVs. After incubation at 37°C for 24 hours, the labeling efficiency remained above 95%, indicating that the Tc-labeled MSC-EVs prepared according to the embodiments of the present invention... 99m Tc-labeled MSC-EVs exhibit good in vitro and in vivo stability, meeting the needs of subsequent long-term imaging studies. Figure 1 (C) indicates free 99m TcO4 – The control figure shows that incubation in fetal bovine serum (FBS) at 37°C maintained extremely high radiochemical purity with prolonged incubation time (compared to 100% chromatographic behavior after 1 hour). The free pertechnetate peak clearly migrated to the solvent front (top of the chromatographic plate), forming a sharp contrast with the marked EVs peak position in Figure A, verifying the effectiveness of the chromatographic identification system.

[0034] 2. Morphological Detection: Recording was performed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). 99m Tc-labeled pre-MSC-EVs and99m Changes in morphology and particle size of Tc-labeled MSC-EVs. For example... Figure 2 The image in (A) shows a TEM image of unlabeled MSC-EVs, which exhibit typical cup-shaped or near-circular vesicle morphology with complete membrane structures. Figure 2 As shown in (B) 99m TEM images of Tc-labeled MSC-EVs show that the labeled MSC-EVs maintained their intact vesicle structure, with no obvious particle aggregation or membrane fragmentation, indicating that... 99m The Tc direct labeling method does not significantly damage the basic morphology of EVs. Figure 2 (C) shows the particle size distribution comparison curves, with the unlabeled group (green curve) and the particle size distribution measured using NTA technology. 99m The particle size distribution of the Tc-labeled group (red curve) is shown. The peak particle size of both groups of vesicles is distributed in the range of 100-200 nm. Statistical results show that... 99m The particle size distribution characteristics of EVs before and after Tc labeling highly overlap, indicating that 99m The Tc labeling process did not cause significant changes in vesicle size.

[0035] 3. Western blotting was used to analyze the radioisotopes of MSC-EVs before and after labeling. 99m The effect of Tc labeling on antigen expression. Simultaneously, on... 99m The proteins in MSCs before and after Tc labeling were analyzed. For example... Figure 3 The results show the detection of transmembrane protein families (CD81, CD9, CD63) and intracellular protein Tsg101. In MSC-EVs (before labeling) and 99m In the detection results of Tc-MSC-EVs (post-labeling), these marker proteins all showed strong positive expression, and there was no significant difference in band abundance before and after labeling, proving that the labeling process did not change the protein composition of the exome. Figure 3 As shown, cellular component markers: Compared with the MSC-EVs group, the MSCs cell group exhibited a characteristic protein distribution. Negative marker (Calnexin): The endoplasmic reticulum protein Calnexin, used as a negative control, was mainly present in cellular components, while it was almost not expressed in the extracted EVs group (including before and after labeling), confirming the high purity of the exome sample and the absence of cell debris contamination. Molecular weight annotation: The bands corresponding to each protein were all located near their standard molecular weights (CD81 / CD9 / CD63 approximately 26 kDa, Tsg101 approximately 45 kDa, Calnexin approximately 70 kDa). Experimental data indicate that... 99mTc labeling does not affect the protein expression profile of MSCs themselves, nor does it lead to the loss of surface characteristic proteins of their derived MSC-EVs, thus ensuring the authenticity of biological behavior in subsequent in vivo tracking experiments.

[0036] 4. 99m Toxicity detection of Tc-labeled MSC-EVs: 99m Tc-labeled MSC-EVs were co-cultured with various somatic cell lines to verify the effect of labeled MSC-EVs on the biological activity of normal somatic cells.

[0037] Experimental group 1 ( 99m Tc-MSC-EVs Group 1): 99m Tc-labeled MSC-EVs+ human normal hepatocytes (LO2 cells); Normal control group 1 (MSC-EVs group 1): Unlabeled MSC-EVs + normal human hepatocytes (LO2 cells); Blank control 1 (NS group 1): physiological saline + normal human hepatocytes (LO2 cells).

[0038] Experimental group 2 ( 99m Tc-MSC-EVs Group 2): 99m Tc-labeled MSC-EVs+ mouse bone marrow-derived macrophages (BMDM cells); Normal control group 2 (MSC-EVs group 2): Unlabeled MSC-EVs + mouse bone marrow-derived macrophages (BMDM cells); Blank control 2 (NS group 2): physiological saline + mouse bone marrow-derived macrophages (BMDM cells).

[0039] Experimental group 3 ( 99m Tc-MSC-EVs Group 3): 99m Tc-labeled MSC-EVs+ mouse microglia (BV2 cells). Normal control group 3 (MSC-EVs group 3): Unlabeled MSC-EVs + mouse microglia (BV2 cells); Blank control group 3 (NS group 3): physiological saline + mouse microglia (BV2 cells).

[0040] The LO2 cell slurry volumes and concentrations were identical across all groups to assess potential toxicity to metabolic organ cells. The BMDM cell slurry volumes and concentrations were identical to assess effects on immune system cells. The BV2 cell slurry volumes and concentrations were identical to assess effects on the activity of cells related to the central nervous system. 99mThe number of Tc-labeled MSC-EVs and the volume of added MSC-EVs were the same as those of unlabeled MSC-EVs; the volume of physiological saline was the same as the volume of MSC-EVs.

[0041] During the co-culture process of the above groups, cell viability was assessed using the CCK-8 assay at 1h, 6h, 24h, 48h, and 96h, and OD values ​​were measured at a wavelength of 450nm. Results are shown below. Figure 4 .like Figure 4 As shown, during a continuous monitoring period of 96 hours, the three cell groups (NS group, MSC-EVs group, ...) 99m The absorbance (OD 450nm) growth curves of the Tc-MSC-EVs group highly overlapped. This indicates that... 99m Neither the Tc labeling process nor the trace radiation irradiation interfered with the basic cellular biological functions of MSC-EVs, that is... 99m Tc-labeled MSC-EVs showed no significant toxicity to somatic cells, meeting the requirements for in vivo application.

[0042] Example 2 Using the method in Example 1 99m In the preparation method of Tc-labeled extracellular vesicles, this embodiment uses different dosages of technetium-99m in step 3 to study the relationship between the dosage of technetium-99m and the labeling rate.

[0043] Specifically, technetium-99m was used at doses of 0 mCi, 1 mCi, 2 mCi, 3 mCi, 3.5 mCi, 4 mCi, 4.5 mCi, 5 mCi, 6 mCi, 7 mCi, 8 mCi, and 9 mCi, respectively, to treat 4 × 10 10 MSC-EVs (100 μg protein content) were labeled, purified, and then detected in the corresponding samples. 99m The percentage of Tc radioactivity bound to MSC-EVs. See results below. Figure 5 When the dose of technetium-99m was between 1 mCi and 4 mCi, the percentage of bound radioactivity was greater than or equal to 99%. Beyond 4 mCi, the percentage of bound radioactivity began to gradually decrease, indicating that within this dose range, there were sufficient binding sites on the surface of extracellular vesicles to completely bind reduced technetium ions in solution. However, a dose saturation effect was observed; when the injected dose exceeded 4 mCi, the labeling efficiency showed a significant decreasing trend with further increases in radioactive dose. This indicates that the effective binding sites on the surface of extracellular vesicles were approaching saturation, and excessive free... 99m Tc can no longer bind to the vesicle surface.

[0044] It should be noted that for 4×10 10Each MSC-EV was labeled with technetium-99m at a dose of 1 mCi to 4 mCi; this translates to a concentration of (3 to 3.75) × 10⁻⁶. 11 Labeling was performed using MSC-EVs per mL (volume 1 mL), with technetium-99m doses ranging from 7.5 mCi to 30 mCi. The labeling rate decreased when the dose exceeded 30 mCi.

[0045] Example 3 This embodiment uses the preparation method from Example 1. 99m Tc-labeled MSC-EVs were used in mouse models to verify... 99m Application of Tc-labeled MSC-EVs.

[0046] (1) db / db mice (non-alcoholic fatty liver combined with type 2 diabetes mouse model) Negative control group (free) 99m Tc group): The average weight of db / db mice was 50 g, and 1 mCi was released. 99m Tc(Na) 99m TcO4 was injected into db / db mice via the tail vein, and the results were as follows: Figure 6 As shown in AD.

[0047] experimental group ( 99m Tc-EVs group): The average weight of db / db mice was 50 g. 99m 0.05 mL of Tc-labeled MSC-EVs (EV concentration was 4 × 10⁻⁵) 11 cells / mL 99m (Tc concentration was 20 mCi / mL); at this time, each mouse was injected with 2×10 mCi / mL via the tail vein. 10 50 μg of MSC-EVs and 1 mCi 99m Tc, results as follows Figure 6 As shown in EH.

[0048] Results analysis: In the negative control group, such as Figure 6 As shown in Figures A and B, due to the natural affinity of free pertechnetate ions for organs expressing sodium / iodine isotransporters (NIS), imaging 24 hours after injection revealed that radioactivity was primarily concentrated in the thyroid, stomach, and bladder. Figure 6 As shown in C and D, quantitative analysis confirmed that the uptake rates in the stomach and thyroid were extremely high and lasted for a long time, while intestinal signals rose slowly over time, and liver uptake was extremely low.

[0049] In the experimental group, such as Figure 6 As shown in Figures E and F, imaging 24 hours post-injection revealed distinctly different distribution patterns. The radioactive signal primarily accumulated in the liver, followed by the bladder, with almost no accumulation in the thyroid and stomach. This indicates... 99mThe binding of Tc to extracellular vesicles was very stable, and no significant radionuclide shedding occurred. For example... Figure 6 As shown in G and H, the signal in the liver (red) initially increases and then slowly decreases, making it the core accumulation organ for MSC-EVs in db / db mice. The signal in the bladder (blue) shows an extremely high excretion peak in the initial period after injection (1 hour), followed by a rapid decline and a slow excretion phase. These experimental results indicate... 99m Tc-MSC-EVs exhibit high in vivo stability in an obesity / diabetes pathology model. The "liver enrichment-early kidney excretion" pattern in this model differs significantly from that of free radionuclides, providing precise tracking data for subsequent studies on the targeting and therapeutic effects of extracellular vesicles in metabolic diseases.

[0050] (2) HFD mice (a high-fat fed mouse model of fatty liver disease) Negative control group: (free) 99m Tc group): The average weight of HFD mice was 35 g, and 1 mCi of free... 99m Tc(Na) 99m TcO4 was injected into HFD mice via the tail vein, and the results were as follows: Figure 7 As shown in AD.

[0051] experimental group ( 99m Tc-EVs group): The average weight of HFD mice was 35 g. 99m 0.05 mL of Tc-labeled MSC-EVs (EV concentration was 4 × 10⁻⁵) 11 cells / mL 99m (Tc concentration was 20 mCi / mL); at this time, each mouse was injected with 2×10 mCi / mL via the tail vein. 10 50 μg of MSC-EVs and 1 mCi 99m Tc, results as follows Figure 7 As shown in EH.

[0052] Results Analysis: SPECT / CT imaging and in vitro quantitative analysis of tissue were used to compare and study the effects of intravenously injected free tissue. 99m TcO4-( Figure 7 (AD) and 99m Tc-MSC-EVs ( Figure 7 Differences in the biological behavior of EH in HFD-STZ-induced diabetic mice. In the negative control group, such as Figure 7 As shown in Figures A and B, because free pertechnetate ions are readily taken up by organs expressing sodium / iodine cotransporters (NIS), planar and tomographic fusion imaging 24 hours after injection shows that the radioactive signal is specifically and highly enriched in the thyroid and stomach. Figure 7As shown in C and D, tissue distribution data confirm that the stomach and thyroid gland are the main organs of radioactive accumulation, maintaining high levels over 48 hours, while liver uptake is extremely low.

[0053] In the experimental group, such as Figure 7 As shown in Figures E and F, imaging 24 hours post-injection revealed a pattern completely different from the negative control group. The radioactive signal was primarily concentrated in the liver region, with almost no signal concentration observed in the thyroid and stomach. These results indicate... 99m Tc binds tightly to extracellular vesicles, and labeled EVs exhibit stable in vivo biodistribution characteristics. For example... Figure 7 As shown in G and H, the liver (red curve) is the primary organ for EV accumulation, with uptake gradually increasing over time and peaking around 24 hours. Excretion pathway: The bladder (blue curve) shows a high concentration signal in the initial stage of injection (1 hour), followed by a rapid decline, indicating that some EVs or their metabolites are rapidly cleared early through the renal system.

[0054] The results of HFD mouse experiments showed that, under the HFD-STZ pathological model, 99m Tc-MSC-EVs exhibited excellent labeling stability and in vivo targeting patterns (primarily enriched in the liver). Their distribution characteristics, significantly different from those of free radionuclides, excluded and validated the interference of label shedding under pathological conditions, providing a reliable foundation for subsequent research on the tracing and treatment of extracellular vesicles in diabetes-related complications.

[0055] Furthermore, radiolabeling can delabel in vivo, making it impossible to determine whether extracellular vesicles retain their complete biological function and are effectively taken up by target cells. Additionally, delabeled free technetium-99m naturally tends towards the thyroid gland and stomach. To address this issue, this embodiment physically perforates technetium-99m-labeled extracellular vesicles in vitro (labeling rate > 99%), and performs radiochromatographic and protein gel electrophoresis analyses on the perforated technetium-99m-labeled extracellular vesicles. See [link to relevant documentation]. Figure 8 In the middle (A) and (B), technetium-99m-labeled extracellular vesicles after membrane rupture and completely free vesicles are shown. 99m A schematic diagram comparing Tc protein gel electrophoresis, (A) showing the electrophoretic migration characteristics and peak distribution of technetium-99m-labeled extracellular vesicles in different molecular weight (KD) ranges after membrane perforation; (B) showing the free pertechnetium salt ( 99m Tc O4 – Electrophoretic migration behavior and molecular weight distribution characteristics of ). Figure 8 Image C shows the radiochromatographic characterization of technetium-99m-labeled extracellular vesicles after membrane rupture (ITLC band analysis results), displaying the free vesicles after membrane rupture. 99m Tc and binding state 99mThe curve showing the change in radioactivity counts (counts) of Tc-MSC-EVs with migration distance (mm). Figure 8 The detection results showed that the ruptured vesicles contained both technetium-labeled vesicle fragments and free technetium-99m.

[0056] Technetium-99m-labeled extracellular vesicles, after rupture, were injected into normal mice via intravenous injection and intestinal perfusion, respectively, followed by SPECT / CT imaging. Figure 9 As shown, (A) is a planar and tomographic image of the mouse body 3 hours after intravenous injection, with radioactive signals mainly concentrated in the thyroid, liver, stomach, and bladder; (B) is a planar and tomographic image of the mouse body 3 hours after intestinal perfusion, with radioactive signals significantly concentrated in the thyroid, large intestine, and feces. This indicates that after the outer vesicles rupture in vivo, free technetium-99m rapidly enters the thyroid and stomach, and this image is a signal of outer vesicle rupture. However, this was not observed in the mice in this embodiment of the invention or in the subsequent cynomolgus monkey experiments. This indicates that the outer vesicles did not rupture during the observation period of the experiment and maintained their intact vesicle structure.

[0057] Example 4 This embodiment uses the preparation method provided in Example 1. 99m Tc-labeled MSC-EVs were applied to cynomolgus monkeys, and further validation was performed using primates as experimental subjects. 99m Application of Tc-labeled MSC-EVs.

[0058] (1) Normal cynomolgus monkeys were used as the research subjects. The average weight of normal cynomolgus monkeys was 3.0±0.15 Kg, and there were 3 cynomolgus monkeys in each group.

[0059] Experimental group 1: Each cynomolgus monkey was intravenously injected with... 99m 20 mL of Tc-labeled MSC-EVs solution, containing 0.75 mg of MSC-EVs protein (3.0 × 10⁻⁶ extracellular vesicles). 11 (each), the dose of technetium-99m is 10mCi.

[0060] Experimental group 2: Each cynomolgus monkey was intravenously injected with... 99m 20 mL of Tc-labeled MSC-EVs solution, containing 1.5 mg of MSC-EVs protein (6.0 × 10⁻⁶ extracellular vesicle particles). 11 (each), the dose of technetium-99m is 10mCi.

[0061] Experimental group 3: Each cynomolgus monkey was intravenously injected with... 99m 20 mL of Tc-labeled MSC-EVs solution, containing 2 mg of MSC-EVs protein (8.0 × 10⁻⁶ extracellular vesicle particles). 11 (each), the dose of technetium-99m is 10mCi.

[0062] Experimental group 4: Each cynomolgus monkey was intravenously injected with... 99m 20 mL of Tc-labeled MSC-EVs solution, containing 3 mg of MSC-EVs protein (12.0 × 10⁻⁶ extracellular vesicle particles). 11 (each), the dose of technetium-99m is 10mCi.

[0063] Control group: Each cynomolgus monkey was intravenously injected with 20 mL of physiological saline containing 10 mCi technetium-99 m.

[0064] Result detection: 1. Analysis of drug metabolism and distribution: The cynomolgus monkeys in each experimental group (including experimental groups 1-4) were injected with... 99m After Tc-labeling of MSC-EVs, whole-body SPECT / CT tomographic fusion imaging was performed at 5 min, 0.5 h, 1 h, 3 h, 6 h, 24 h, and 48 h. The scanning mode was a scanning table movement speed of 12 cm / min, and the scanning range was 80 cm from head to toe. Figure 10 As shown in Figure AF, the experimental group of cynomolgus monkeys were intravenously injected with... 99m Following Tc labeling of extracellular vesicles in mesenchymal stem cells (MSCs), early distribution (0.5h-1h): MSCs rapidly enter the bloodstream after injection, with early imaging showing blood pooling and significant radioactivity concentration in the heart (cyan arrow). Subsequently, MSC-EVs rapidly accumulate in the liver (blue arrow), spleen (yellow arrow), and both kidneys (green arrow). Increased radioactivity in the bladder (pink arrow) suggests that some metabolites are excreted through the urinary system. Mid-term distribution (3h-6h): Over time, the radioactivity in the blood pool gradually decreases. Significant imaging begins in the biliary system (purple arrow), followed by a gradual shift of radioactivity to the intestines (red arrow). Long-term distribution (24h-48h): Imaging shows a clear enterohepatic circulation characteristic of the tracer. At 24h and 48h, radioactivity remains in the liver, while further concentration and displacement of radioactivity in the intestines (red arrow) indicate the excretion and possible reabsorption of EV metabolites in the digestive tract. 99m The metabolic distribution of Tc-MSC-EVs in healthy cynomolgus monkeys is shown in Tables 1 and 2.

[0065] Table 1. 99mSystemic biodistribution of Tc-MSC-EVs in healthy cynomolgus monkeys (percentage of injected dose %ID)

[0066] See Table 1 for a summary of the data. Figure 11 , Figure 11 (A) shows the time to recovery at 0.5h, 1h, 6h, 24h, and 48h after injection. 99m Percentage of Tc-MSC-EVs distribution in major organs and tissues throughout the body (including brain, thyroid, heart, lungs, liver, stomach, kidneys, intestines, bones, muscles, etc.) (% ID). 99m Tc-MSC-EVs rapidly enter the bloodstream in the early post-injection period and are mainly enriched in the liver and bladder. Over time, liver uptake gradually decreases, while intestinal radioactivity significantly increases after 6 hours, indicating the presence of a clear hepatobiliary excretion pathway. Figure 11 (B) shows the distribution curves of major organs over time, quantitatively displaying the distribution in the bladder, liver, gallbladder, spleen, kidneys, and intestines. 99m Metabolic kinetics of Tc-MSC-EVs: Liver: Shows an initial increase followed by a slow decrease, indicating it is the primary processing organ in the early and mid-stages. Intestine: The percentage of radioactivity peaks at 6 hours and remains at a high level, confirming enterohepatic circulation and subsequent gastrointestinal excretion. Bladder: Extremely high transient enrichment occurs 0.5-1 hours after injection, followed by a rapid decrease, indicating that some MSC-EVs or their degradation fragments are rapidly cleared through the urinary system.

[0067] Table 2. Tc 99m -Spanish-temporal radioactivity concentration of MSC-EVs (%ID / mL)

[0068] like Figure 12 Figure (A) shows the radioactive distribution concentration (%ID / mL) in various tissues and organs at 0.5h, 1h, 6h, 24h, and 48h after injection. The results indicate that MSC-EVs rapidly distribute throughout the body in the early post-injection period, with the gallbladder, liver, and bladder exhibiting higher initial distribution concentrations. Over time, the distribution concentrations in each organ exhibited differentiated metabolic clearance kinetics. Figure 12 Figure (B) illustrates the concentration distribution curves over time in major organs. Gallbladder: maintained a high concentration for a relatively long period, suggesting that the hepatobiliary system is the main metabolic pathway for MSC-EVs. Bladder: peaked at the initial injection stage (0.5 h) and then rapidly declined, reflecting the early and rapid clearance of some EVs or their degradation products via the urinary system. Liver and kidneys: showed stable distribution and a plateau in metabolism. All data in Tables 1 and 2 are expressed as mean ± standard error (Mean ± SE). Figure 11 The data points in section 10 correspond to the data in Table 1, and the data points in section 10 correspond to the data in Table 2. It is particularly important to emphasize that... Figure 11 and Figure 12 All % ID and % ID / mL results shown have been corrected for physical attenuation and subtracted. 99m The effect of Tc physical half-life on the decay of radioactivity counts. Therefore, the concentration distribution and evolution shown in the figure truly and accurately reflect the biological distribution and metabolic kinetics of MSC-EVs external vesicles in cynomolgus monkeys.

[0069] Combining Table 1, Table 2 and Figure 11 and Figure 12 Early stage (0-1 hour): rapid systemic circulation and initial isolation pattern are similar to rodent models.

[0070] Within the first hour after injection 99m Tc-labeled MSC-EVs rapidly entered the systemic circulation, with blood radioactivity decreasing sharply from 72.34 ± 1.29% ID at 5 minutes to 15.39 ± 0.83% ID at 30 minutes. This rapid systemic clearance was accompanied by a dual clearance pathway: early urinary excretion and significant liver isolation. SPECT / CT imaging showed significant radioactive accumulation in both kidneys and bladder, indicating that the urinary system is the primary pathway for early MSC-EV elimination in NHPs (non-human primates). Simultaneously, rapid blood flushing was accompanied by significant liver isolation, with uptake peaking at 1 hour (30.84 ± 1.27% ID). This early distribution closely correlates with the liver-dominant isolation pattern established in previous rodent studies, confirming that liver tissue serves as a conserved primary filter for systemic MSC-EVs across species.

[0071] Unlike rodent models, in NHPs (healthy cynomolgus monkeys), early radioactivity was observed in the gallbladder within the first hour, and this high radioactivity persisted for up to 48 hours. Figure 10 F). Quantitative pharmacokinetic analysis confirmed intensive processing within the biliary system, with the gallbladder showing a sustained increase in MSC-EV concentrations, peaking at 0.5966 ± 0.0139% ID / mL at 1 hour. Notably, the synchronization between the liver isolation plateau (%ID) and the rise in bile concentration (%ID / mL) provides clear evidence of the active transition of MSC-EVs from the liver parenchyma to the bile, marking the initiation of the hepatobiliary-gut axis.

[0072] Late stage (3-48 hours): Primate-specific "hepatobiliary-gut" clearance axis of MSC-EVs. Compared to the persistent liver-spleen isolation typically observed in rodents, MSC-EVs in NHPs begin a progressive spatiotemporal shift towards the hepatobiliary-gut clearance pathway 3 hours post-injection. Although high-intensity radioactivity persists in the liver, significant accumulation gradually occurs in the gallbladder and intestines. Figure 10 C, D). Longitudinal SPECT / CT monitoring showed that MSC-EVs bypassed rapid fecal clearance, instead exhibiting 24-48 hours of intestinal retention, primarily confined to the colon. Figure 10 E, F). Table 2 shows the “delayed intestinal surge” observed between 3 and 6 hours, indicating the pathway of intestinal entry; at 30 minutes, although systemic blood levels peaked, intestinal uptake remained negligible (0.0081 ± 0.0005 %ID / mL), ruling out direct systemic perfusion as the primary source of intestinal MSC-EVs. Subsequently, as shown in Table 2, a significant influx of MSC-EVs into the intestine was detected at 3 hours. By 6 hours, the intestine became the primary anatomical reservoir, with the injected dose percentage at 37.70 ± 0.53% ID, representing a nearly 25-fold increase in local concentration (0.0085 → 0.2170% ID / mL). This intestinal surge reflects the simultaneous decline in hepatobiliary signaling, and since the surge occurred long after systemic blood clearance, these data confirm a “relay” transport mechanism: the sequential migration of MSC-EVs from liver parenchyma → bile → gallbladder → intestinal lumen.

[0073] During the terminal phase (24–48 h), although the local concentration (%ID / mL) gradually decreased, the gut remained the largest reservoir of MSC-EVs (27.94% ID at 24 h and 21.19% ID at 48 h). Figure 10 E, F). This persistently high percentage indicates slow luminal transport and potential circulatory circulation within the intestine. Meanwhile, after 3 hours, the contributions from the urinary tract and cardiac blood pools decrease to baseline levels. Figure 10 DF marks a clear transition from early renal clearance to sustained hepatobiliary-enteric isolation.

[0074] like Figure 13 As shown, through 99m Tc radioactive tracer technology was used to quantitatively analyze the clearance kinetics of MSC-EVs in the blood of normal cynomolgus monkeys, and the results are expressed as a curve of the logarithm of the injected dose percentage [%ID(Log10)] versus time. The results showed that... 99mFollowing injection, Tc-MSC-EVs exhibited a distinct biphasic clearance characteristic. The initial phase was a rapid distribution phase, with a rapid decrease in extracellular vesicle concentration in the blood during the first few hours after injection, reflecting the rapid distribution of MSC-EVs from the bloodstream to various tissues and organs throughout the body (such as the liver, spleen, and kidneys). This was followed by a slow elimination phase, after which the blood concentration entered a period of steady decline, demonstrating the metabolic and clearance rate of MSC-EVs in vivo.

[0075] like Figure 14 As shown, through 99m The cumulative exposure (AUC) of MSC-EVs in the blood circulation of normal cynomolgus monkeys, monitored by Tc radioactive tracer technology, is expressed as the change in the area under the curve [AUC (%ID / mL)] per unit volume accumulation rate over time. The results indicate that... 99m Following Tc-MSC-EVs injection, the AUC in the blood exhibits a very early, explosive increase, reflecting the initial high exposure level after the extracellular vesicles enter the bloodstream. The curve then rapidly flattens out, indicating the metabolic equilibrium process as the extracellular vesicles distribute widely throughout the body's tissues and organs from the bloodstream. The dynamic evolution of blood AUC is a core indicator for assessing the bioavailability and in vivo retention time of extracellular vesicles, reflecting the overall exposure level of the body to MSC-EVs.

[0076] like Figure 15 As shown, the systemic elimination and retention patterns of MSC-EVs in cynomolgus monkeys were quantitatively analyzed through dynamic monitoring of radioactive counts. Based on the fitted experimental data, the retention rate of MSC-EVs in vivo exhibited a clear double-exponential decay characteristic: the first stage (0~6h) was a rapid elimination phase, with an elimination rate constant k1 = 0.0322 h. -1 This phase primarily reflects the early excretion of the external vesicles through the urinary system (bladder). The second phase (t ≥ 6 h) is a slow clearance phase, with an elimination rate constant k2 = 0.0177 h. -1 As the extravesicles distribute to deeper tissues or enter the enterohepatic circulation, the elimination rate slows significantly. Limit of Detection (LOD) and Duration of Detection: The lower limit of detection (LOD) was set at a point where the systemic retention rate decreased to 5% of the initial dose. LOD = 5%. Based on model calculations, the detectable retention time (t) of the external vesicles in the cynomolgus monkey is... LOD It takes approximately 158 hours.

[0077] To ensure statistical reliability, quantitative organ-level analysis was limited to a 48-hour time window. A weak signal was observed at 72 hours, corresponding to a physical half-life of 12 Tc. 99m Period (T) 1 / 2 = 6.007 h; 1 / 2 12(Approximately 0.024% initial activity), below the sensitivity threshold for robust volumetric quantification. In summary, these findings underscore the necessity of NHP models for capturing the complex species-specific physiological processes of therapeutic EVs and identify a functional hepatobiliary-enterogulary clearance axis. Quantitative pharmacokinetic analyses (expressed as organ-specific uptake percentage ID / mL) revealed that MSC-EVs exhibited high selectivity and sustained accumulation in the liver, biliary system, and intestine. These three organs collectively constitute a functional hepatobiliary-enterogulary clearance axis, suggesting that their distribution is co-regulated by active transport mechanisms and organ tropism, rather than passive retention.

[0078] 2. Peripheral blood samples were collected from experimental groups 1-4 and the control group of cynomolgus monkeys. Routine peripheral blood tests, liver and kidney function tests, and myocardial enzyme profiles were performed within 240 hours. Results are shown below. Figure 16 In the figure, WBC represents white blood cells, RBC represents red blood cells, NEUT% represents the percentage of neutrophils, MONO% represents the percentage of monocytes, ALT represents alanine aminotransferase, AST represents aspartate aminotransferase, TBIL represents total bilirubin, LDH represents lactate dehydrogenase, CK represents creatine kinase, and HBDH represents α-hydroxybutyrate dehydrogenase.

[0079] from Figure 16 As can be seen from the figure (the dashed lines in the figure represent the maximum and minimum values ​​of the normal reference range, respectively), in Example 1, when the used 99m The amount of extracellular vesicle protein in Tc-labeled mesenchymal matrix extracellular vesicles was 0.75 mg. There was only a transient increase in white blood cells and neutrophils around 48 hours, but this did not exceed the maximum threshold. Liver function tests, including transaminases and kidney function, showed no significant abnormalities. (When used...) 99m Tc-labeled extracellular vesicle protein in mesenchymal matrix cells was 1.5 mg (Example 2). This resulted in a transient increase in white blood cell (WBC) and lactate dehydrogenase (LDH) levels, while AST levels rose to the upper limit of normal within a short period around 70 hours; indicating that at this dose, there may be a transient burden on the liver and myocardium in cynomolgus monkeys. When used... 99m Tc-labeled extracellular vesicle protein in mesenchymal matrix cells at a concentration of 2 mg (Example 3) resulted in a transient and significant increase in leukocytes and neutrophils, as well as mild hepatotoxicity (elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and cardiotoxicity (elevated lactate dehydrogenase (LDH)). When used... 99mThe amount of extracellular vesicle protein in Tc-labeled mesenchymal matrix extracellular vesicles was 3 mg (Example 4). The number of white blood cells and neutrophils was higher than the upper limit of normal, and significant liver toxicity (alanine aminotransferase ALT and aspartate aminotransferase AST were significantly elevated) and cardiotoxicity (lactate dehydrogenase LDH, α-hydroxybutyrate dehydrogenase HDBH and creatine kinase CK were all elevated).

[0080] Experimental results showed that at low doses (0.75 mg), most indicators remained within the normal reference range, demonstrating good biocompatibility; however, high doses (≥2.0 mg) led to reversible abnormal fluctuations in multiple physiological indicators. Based on the above experiments in normal cynomolgus monkeys, the 0.75 mg dose of extracellular vesicle protein in the experiment only caused slight, transient, and completely reversible fluctuations in leukocyte and neutrophil parameters within the physiological range, and no adverse clinical events were observed. This dose was determined as the No Observed Adverse Effect Level (NOAEL) and recommended as a safe starting dose for non-human primate studies. This embodiment, through rigorous characterization of the multi-organ distribution dynamics of extracellular vesicles in a non-human primate model and determination of its safe window for therapeutic feasibility, provides crucial mechanism-based preclinical data for subsequent clinical trial design.

[0081] (2) The study subjects were cynomolgus monkeys with spontaneous myocarditis.

[0082] Experimental group (IM): Three cynomolgus monkeys with spontaneous myocarditis had an average weight of 3.15 kg. 99m A single intravenous injection of 0.75 mg of Tc-labeled extracellular vesicle protein from mesenchymal matrix cells was administered.

[0083] Control group (NC): Three normal cynomolgus monkeys with an average weight of 3.15 kg were used. 99m A single intravenous injection of 0.75 mg of Tc-labeled extracellular vesicle protein from mesenchymal matrix cells was administered.

[0084] Peripheral blood was collected from the experimental group and the control group for testing. Results are shown below. Figure 17 For injection 99m Trend of changes in extracellular vesicles of mesenchymal matrix within 240 hours after Tc labeling. Figure 17 The red and green dashed lines represent the upper limit (Max) and lower limit (Min) of the normal reference range, respectively. Figure 17 (A) and (B) are inflammation-related markers, which are observed during injection. 99mIn the early stages of Tc-MSC-EVs, the WBC level in the experimental group was significantly higher than the upper limit of the normal reference range, indicating a significant systemic inflammatory response in the myocarditis model. Over time, the WBC level gradually decreased and approached normal levels. In contrast, the control group maintained levels within the normal range throughout. The NEUT level in the experimental group was high at the initial stage (0h), gradually decreasing over time after injection and approaching normal levels after 72h. Figure 17 (C)-(E) represent biochemical indicators related to myocardial injury. In the experimental group, the initial LDH value was significantly higher than the normal range, reflecting tissue damage caused by myocarditis. After injection of MSC-EVs, this indicator showed a steady downward trend. As a key indicator for evaluating myocardial injury, the experimental group showed extremely high CK levels at the beginning of administration, followed by a significant decrease, indicating that MSC-EVs have a potential ameliorative or regulatory effect on damaged myocardium. Consistent with the trend of CK, the HBDH level in the experimental group gradually decreased from an elevated state, while the control group remained stable throughout the experimental period. These results indicate that administration of MSC-EVs did not increase the biochemical burden on the body, and that extracellular vesicles of mesenchymal matrix cells played a significant therapeutic role in spontaneous myocarditis. Inflammatory and myocardial injury indicators largely recovered or approached the normal reference range within 240 hours.

[0085] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A kind 99m The method for preparing Tc-labeled extracellular vesicles is characterized by, Includes the following steps: S1. Mix equal volumes of stannous chloride solution and extracellular vesicle solution and incubate for 5-10 minutes to obtain a pre-stannous extracellular vesicle solution; wherein the mass concentration of stannous chloride is 0.005%-0.01%, and the concentration of the extracellular vesicle solution is (3-3.75)×10⁻⁶. 11 Cells / mL; S2, Add to the pre-tinned extracellular vesicle solution 99m Tc-NaTcO4 solution, co-incubated for 25 min to 40 min, to obtain 99m Tc-labeled extracellular vesicles.

2. As described in claim 1 99m The method for preparing Tc-labeled extracellular vesicles is characterized by, The extracellular vesicle solution is mesenchymal matrix extracellular vesicles, and the concentration per 1 mL is (3–3.75) × 10⁻⁶. 11 The extracellular vesicles of the mesenchymal matrix per mL were supplemented with the Na 99m The dose of technetium-99m in TcO4 solution is 7.5 mCi to 30 mCi.

3. As described in claim 1 99m The method for preparing Tc-labeled extracellular vesicles is characterized by, The 99m The Tc-NaTcO4 solution was obtained by rinsing, and the 99m The Tc-NaTcO4 solution should be stored for less than 3 hours.

4. A kind 99m Tc-labeled extracellular vesicles, characterized in that... It is obtained by the preparation method described in claim 1.

5. The claim 4 99m Application of Tc-labeled extracellular vesicles in the preparation of drugs for myocarditis.

6. The application according to claim 5, characterized in that, The drug is prepared into a unit-dose formulation, each unit of which contains 99m The protein content of Tc-labeled extracellular vesicles is no more than 2.1 mg.

7. The application according to claim 6, characterized in that, The drug is an injectable form.

8. The claim 4 99m The application of Tc-labeled extracellular vesicles in the preparation of drugs for liver injury, characterized in that... The liver injury includes type 2 diabetes mellitus combined with non-alcoholic fatty liver disease and food-induced fatty liver disease.

9. The claim 4 99m Application of Tc-labeled extracellular vesicles in the preparation of kits for identifying liver injury.

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