Use of reagents for detecting chemokine ligand 9 in the manufacture of a product for the early detection of myocarditis

Abstract

The application discloses application of a reagent for detecting chemokine ligand 9 in preparation of a myocarditis early detection product. The application identifies immune cell characteristics in a mouse myocarditis early stage by using single cell RNA sequencing technology, and finds that compared with healthy mice, myocarditis mice have more macrophages infiltrating myocardial tissue and expressing chemokine ligand 9, which reveals that the chemokine ligand 9 can be used as a specific target for detecting acute myocarditis. Further, the application uses BPBBT, DSPE-mPEG 2000 and DSPE-PEG 2000 -MAL to prepare nanoparticles, designs a targeting peptide capable of specifically combining with the chemokine ligand 9 and connects the targeting peptide to a surface of the nano material, and thus myocarditis targeted nano probes are prepared. The myocarditis targeted nano probes can specifically gather in an inflammation area, and thus myocarditis can be accurately detected.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of a reagent for detecting chemokine ligand 9 in the preparation of an early detection product for myocarditis. Background Art

[0002] Myocarditis is a common inflammatory heart disease, accounting for 1% to 7% of all cases. Initial symptoms are subtle, but as inflammation worsens, it can lead to sudden, fatal cardiovascular events, including heart failure, cardiogenic shock, and even sudden death. Therefore, early and accurate diagnosis of myocarditis is crucial for early and proactive treatment to reduce the incidence of fatal cardiovascular events.

[0003] Currently, the primary methods for detecting myocarditis rely on either endomyocardial biopsy or cardiovascular magnetic resonance imaging. However, endomyocardial biopsy, the gold standard for detecting myocarditis, has certain limitations. This invasive procedure not only has a limited sampling range but also carries the risk of sampling errors, making it difficult to widely implement in clinical practice.

[0004] On cardiovascular magnetic resonance imaging, myocarditis presents similarly to myocardial infarction, cardiomyopathy, and cardiac amyloidosis, requiring the combination of other tests for more reliable detection. It is noteworthy that myocarditis can be clinically divided into acute, subacute, and chronic phases. Early therapeutic intervention in the acute phase can often restore cardiac function, but this is not the case in the chronic phase. Therefore, the development of advanced detection technologies to improve the accuracy of myocarditis detection is urgently needed.

[0005] In recent years, fluorescence imaging technology has emerged as a promising technology in bioimaging applications, offering real-time, on-site, and noninvasive imaging with exceptionally high spatial and temporal resolution. Meanwhile, nanotechnology is also transforming our understanding of detection and therapeutic agent delivery. Nanoparticles possess numerous advantageous properties, such as nanoscale size effects, excellent dispersibility and water solubility, superior blood circulation, and targeted modifiability, enabling precise delivery of diagnostic or therapeutic agents to specific locations.

[0006] By combining the advantages of nanotechnology and fluorescence imaging, we can develop nanoprobes that are highly efficient and specifically targeted to lesions, allowing for accurate identification of myocarditis at its early stages. This new detection method has the potential to not only improve detection accuracy but also reduce misdiagnosis rates, providing strong support for the early diagnosis of acute myocarditis. Summary of the Invention

[0007] In response to the deficiencies of existing technologies and actual needs, the present invention provides a reagent for detecting chemokine ligand 9 protein for use in early detection of myocarditis. The present invention explores new targets for early detection of myocarditis and further develops detection schemes in order to achieve rapid and accurate early detection of myocarditis.

[0008] Compared with the prior art, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides use of a reagent for detecting chemokine ligand 9 in preparing a product for early detection of myocarditis.

[0010] In the present invention, we successfully constructed an experimental immune myocarditis (EAM) mouse model. By using single-cell RNA sequencing technology, we deeply analyzed the characteristics of immune cells in the early stages of mouse myocarditis and found significant differential expression of chemokine ligand 9. The differential expression of chemokine ligand 9 was further verified in vitro using independent mouse and human macrophage models. Compared with healthy mice, there were more macrophage infiltrations expressing chemokine ligand 9 in the myocardial tissue of myocarditis mice. We also collected myocardial samples from clinical myocarditis patients and verified them using database comparison and histochemical experiments. The results showed that the expression of chemokine ligand 9 was significantly higher in myocarditis patients than in the myocardium of healthy people. These data fully demonstrate that chemokine ligand 9 has the potential to be a specific target for detecting acute myocarditis.

[0011] Preferably, the reagent for detecting chemokine ligand 9 includes a reagent for detecting cells expressing chemokine ligand 9.

[0012] In the second aspect, the present invention provides a myocarditis targeting nanoprobe, wherein the nanoprobe targets and binds to the chemokine ligand 9, and the myocarditis targeting nanoprobe comprises 1,4-di[4-(octyloxy)-triphenylamine)]-benzo[1,2-c:4,5-c'] and [1,2,5] thiadiazole (BPBBT), phosphoethanolamine phospholipid-polyvinyl alcohol 2000 (DSPE-mPEG 2000 ), phosphoethanolamine phospholipid-polyvinyl alcohol 2000 -Maleimide (DSPE-PEG 2000 -MAL) and chemokine ligand 9 peptide, the BPBBT, DSPE-mPEG 2000 and DSPE-PEG 2000 -MAL self-assembles into nanoparticles, and the chemokine ligand 9 peptide is modified on the surface of the nanoparticles; the amino acid sequence of the chemokine ligand 9 peptide is shown in SEQ ID NO.1.

[0013] SEQ ID NO.1: LKVRKSQRSRQKKTTC.

[0014] In the present invention, BPBBT is a high-brightness near-infrared region II (NIR-II) emitting aggregation-induced luminescence agent (AIEgens), using BPBBT, DSPE-mPEG2000 and DSPE-PEG 2000 By using a chemokine-specific ligand 9 (CNS)-MAL nanoparticle preparation method, and designing a targeting peptide that specifically binds to CNS chemokine ligand 9, the researchers modified the nanoparticle surface with a targeting peptide that specifically binds to CNS chemokine ligand 9. This resulted in myocarditis-targeted nanoprobes (TNPs). These targeted nanoprobes can specifically accumulate in areas of inflammation, enabling accurate detection of acute myocarditis. Furthermore, TNP-based fluorescence imaging technology can be used to monitor the efficacy of drugs used to treat myocarditis.

[0015] Preferably, the nanoparticles are further modified with fluorescent markers.

[0016] Preferably, the fluorescent marker is modified on DSPE-PEG 2000 -MAL.

[0017] Preferably, the fluorescent label includes cyanine dye Cy7 and / or cyanine dye Cy5.5.

[0018] Preferably, the terminal end of the chemokine ligand 9 peptide is modified with a thiol group.

[0019] Preferably, BPBBT, DSPE-mPEG in the myocarditis targeting nanoprobe 2000 , DSPE-PEG 2000 The mass ratio of -MAL to chemokine ligand 9 peptide is 1:(2-4):(0.5-1):(2-3), preferably 1:3.2:0.8:2.4.

[0020] In a third aspect, the present invention provides a method for preparing the myocarditis-targeted nanoprobe according to the first aspect, the preparation method comprising:

[0021] BPBBT and DSPE-mPEG 2000 The mixture is mixed, and ultrasonic treatment and dialysis treatment are performed in sequence. The dialysis product is mixed with the chemokine ligand 9-peptide and dialyzed to obtain the product.

[0022] In a fourth aspect, the present invention provides a kit for early detection of myocarditis, comprising the myocarditis-targeted nanoprobe described in the second aspect.

[0023] The present invention designs a myocarditis-targeted nanoprobe (named TNPs) and uses a near-infrared second-zone TNPs system to perform optical imaging detection of myocarditis. It has high specificity, safety and reliability. TNPs are sensitive and accurate in detecting inflammation. TNPs can accurately detect the reduction of inflammation after PX478 treatment. TNPs can be effectively used to detect myocardial inflammation and evaluate the effectiveness of drug treatment.

[0024] In a fifth aspect, the present invention provides use of the myocarditis-targeted nanoprobe described in the second aspect in detecting cells expressing chemokine ligand 9.

[0025] The myocarditis-targeted nanoprobe designed by the present invention can also be applied to fields other than disease detection, such as in vitro detection and analysis of cells expressing chemokine ligand 9, and basic cell-related research.

[0026] Preferably, the cells expressing chemokine ligand 9 include chemokine ligand 9-expressing inflammatory macrophages.

[0027] In a sixth aspect, the present invention provides a method for detecting cells expressing chemokine ligand 9, the method comprising: mixing a sample to be detected with the myocarditis-targeted nanoprobe described in the second aspect, and performing fluorescence detection.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention uses single-cell RNA sequencing technology to identify the characteristics of immune cells in the acute phase of mouse myocarditis and finds that myocarditis mice have more macrophages expressing chemokine ligand 9 infiltrating myocardial tissue compared with healthy mice, indicating that chemokine ligand 9 can be used as a specific target for detecting acute myocarditis. 2000 and DSPE-PEG 2000 -MAL nanoparticles were prepared, and a targeting peptide that specifically binds to chemokine ligand 9 was designed to create a myocarditis-targeted nanoprobe. This myocarditis-targeted nanoprobe can specifically accumulate in areas of inflammation, thereby accurately detecting myocarditis. Furthermore, fluorescence imaging technology based on the myocarditis-targeted nanoprobe can be used to monitor the efficacy of drugs used to treat myocarditis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a cross-section of the t-distributed random neighborhood embedding of 34,665 immune cells, where the elliptical box indicates the changing trend of the number of macrophage cluster-2 during EAM progression;

[0031] Figure 2 Violin plots of chemokine ligand 9 marker gene expression in each macrophage cluster;

[0032] Figure 3 The percentage of chemokine ligand 9-positive cells in early myocarditis at different time points;

[0033] Figure 4 This is a graph showing the correlation between the percentage of chemokine ligand 9-positive cells and the degree of inflammation at different stages;

[0034] Figure 5The expression of chemokine ligand 9 in RAW264.7 and U937 cells was detected by Western blotting;

[0035] Figure 6 The relative values ​​of chemokine ligand 9 / GAPDH are shown in gray stripes (n=3);

[0036] Figure 7 is the fluorescence emission spectrum of BPBBT (10 μM) in water / tetrahydrofuran mixture, with the proportion of water ranging from 0 to 99% when excited at a wavelength of 660 nm;

[0037] Figure 8 is the fluorescence intensity of BPBBT (10 μM) in a tetrahydrofuran / water (v / v) mixed solvent when excited at a wavelength of 660 nm with increasing water content;

[0038] Figure 9 Representative TEM images and size distribution diagrams of TNPs;

[0039] Figure 10 are the absorption and fluorescence spectra of TNPs in water;

[0040] Figure 11 Fluorescence images of TNPs, NPs, and PBS samples;

[0041] Figure 12 In vivo near-infrared-II imaging of the EAM model under different treatments in the acute phase (day 14);

[0042] Figure 13 for Figure 12 Semi-quantitative fluorescence bar graph;

[0043] Figure 14 、 Figure 15 and Figure 16 These are the in vivo near-infrared-II imaging of the EAM model under different treatments, and the in vitro near-infrared-II imaging of the harvested hearts of the corresponding groups;

[0044] Figure 17 、 Figure 18 and Figure 19 They are Figure 14 、 Figure 15 and Figure 16 Semi-quantitative fluorescence bar graphs of in vivo and in vitro;

[0045] Figure 20 Representative fluorescence images of normal and myocarditis specimens obtained from freshly excised human normal and myocarditis specimens after incubation with saline, NPs, or TNPs for 2 h.

[0046] Figure 21 for Figure 20 Semi-quantitative fluorescence statistics. DETAILED DESCRIPTION

[0047] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0048] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0049] Histological examination

[0050] An EAM mouse model was established, and cardiac tissue was collected from EAM mice. Clinical myocardial tissue samples from human myocarditis were collected. The samples were fixed overnight in 4% (v / v) formalin solution, embedded in paraffin, and cut into 5 μm thick sections. The sections were then subjected to H&E, immunohistochemistry, and immunohistochemical fluorescence staining for histopathological evaluation. H&E and immunohistochemical images were examined by digital microscopy. Fluorescent images were observed under a stereomicroscope (Leica DMi8, Leica Microsystems, Germany) and quantified using ImageJ.

[0051] Ethical approval and consent to participate

[0052] This study complied with the ethical guidelines of the 2013 revised version of the Declaration of Helsinki. All patients provided written informed consent for the use of clinical specimens for medical research. Experiments using human research specimens and mice were approved by the Ethics Committee (Fuwai Hospital, Chinese Academy of Medical Sciences, Shenzhen Hospital), and experiments were conducted according to standard guidelines approved by the Animal Welfare Committee (Fuwai Hospital, Chinese Academy of Medical Sciences, Shenzhen Hospital, China).

[0053] Statistical analysis

[0054] Unless otherwise indicated, all data are presented as mean ± standard deviation. Statistical differences between treatment and control groups were analyzed by one-way or two-way analysis of variance with Tukey's post hoc test (for multiple comparisons) using GraphPad Prism version 8.0 software. Survival data were analyzed using the log-rank test. * indicates statistically significant differences, p < 0.05; **, p < 0.01; and ***, p < 0.001.

[0055] Example 1

[0056] In this example, an experimental autoimmune myocarditis mouse model was established.

[0057] Six-week-old male Balb / c mice (purchased from Guangzhou Jicui Pharmaceutical Co., Ltd.) were acclimated for at least seven days before the experiment and had free access to fresh food and water. On days 0 and 7, mice were subcutaneously injected with 250 μg of α-MyHC peptide (Ac-RSLKLMATLFSTYASADR-OH; AnaSpec, AS-62554) in complete Freund's adjuvant (Sigma, F5881; 1:1 weight ratio) to induce experimental autoimmune myocarditis (EAM).

[0058] Example 2

[0059] This example performs single cell analysis and target screening.

[0060] Preparation of single cell suspension: Prepare collagenase 2 (CLS2, Sigma) at a concentration of 400 U / mL; take the myocardial tissue of the myocarditis mouse model and put it in PBS and cut it into 1 mm 3 small pieces; then rinse with PBS until the tissue fragments turn white, digest with 400U / mL CLS210 mL, and digest in a 37℃ water bath for 15 minutes without stopping shaking; aspirate the supernatant through a 40μm sieve and collect it in a 50mL tube; differential centrifugation: 4℃, 100g centrifugation for 1 minute, take the supernatant, and then centrifuge at 400×g for 5 minutes, discard the supernatant; resuspend with 1mL 2% FBS / RPMI; repeat the steps for a total of 4 digestion cycles until the tissue is completely digested; collect all the cells and perform differential centrifugation; resuspend the cells with 4 times the volume of red blood cell lysis buffer, lyse on ice for 2 minutes, dilute to 10 times the volume, centrifuge at 400×g for 5 minutes, and discard the supernatant.

[0061] Sorting CD45 + Cells: Preparation: Flow cytometry tube blocking, incubate with 20% FBS / PBS at 25°C for 3 hours; Staining control design: Control tube: 1: isotype ctrl, 2: 7-AAD single positive, 3: Cd45 single positive; Use the cell with the most cells in the sample tube as the control, only need to take 10 5 cells / tube; resuspend the cell pellets of the four experimental groups (normal control) in 1 mL PBS; use Countstar to count, and the Fc block should be 1 μg block (2 μL) / 10 6Add 5 μL of CD45 antibody to the cell solution at a ratio of 1:200 (not including 7-AAD single-positive tube) and stain on ice for 30 minutes; centrifuge at 400 rpm for 5 minutes; wash twice with PBS; prepare 7-AAD staining solution: dilute 7-AAD 1:20 in PBS, and the 7-AAD should be 5 μL 7-AAD / 10 6 Stain cells / 100μL PBS; resuspend isotype ctrl and CD45 single positive with PBS; resuspend 7-AAD single positive with 300μL 7-AAD staining solution; resuspend the remaining sample tubes with 1mL 7-AAD staining solution; pass all cells through a 40μm sieve, transfer to a flow tube, and sort on the machine; first adjust the voltage and compensation of tubes 1 / 2 / 3; the voltage is FSC: 210; SSC: 300; FITC: 430; PerCP-Cy5-5: 520; analyze 10,000 cells of each sample first, adjust the gate, and save the image; pour out the blocking buffer in the flow tube, add 500μL 2% FBS / RPMI; sort the cells in each sample tube, sort the cells into the culture medium, and collect 400,000 cells; aspirate the cells in the culture medium into a 1.5mL centrifuge tube, and centrifuge at 3000rpm. After 5 minutes of centrifugation, a clear precipitate should be visible. Aspirate the supernatant with a pipette and resuspend in 50 μL PBS. Take 10 μL of cells and mix with 10 μL of AOPI. Use Countstar to count the cells and determine the cell concentration, viability, nucleation rate, and clumping rate. The cell suspension should have a viable cell concentration of 800 cells / μL, a viability greater than 90%, a clumping rate less than 5%, and a nucleation rate greater than 70%. Cell suspensions that meet the above criteria can be used for single-cell library construction.

[0062] The present invention uses the EAM model to analyze myocarditis immune cells in order to identify specific targets. According to the H&E staining results of cardiac tissue, the infiltration of inflammatory cells in the myocardial tissue of the EAM group was more obvious. In addition, the degree of inflammation in myocarditis has a certain correlation with the duration of illness. It is generally believed that myocarditis can be divided into acute, subacute and chronic stages. In order to find the signature targets of immune cells in the acute phase of myocarditis, we conducted single-cell RNA sequencing studies on myocardial tissue in the acute phase (14 days), subacute phase (21 days) and chronic phase (60 days) of the EAM model to evaluate stage-specific cell clusters.

[0063] The results showed that myocarditis induces a new cell cluster, macrophage cluster 2. This cell cluster is most abundant during the acute phase of myocarditis and gradually decreases as the onset of the disease progresses. Figure 1The Violin graph shows the expression of the chemokine ligand 9 (CXCL9) marker gene in each macrophage cluster. It can be seen that the expression of chemokine ligand 9 is the highest in macrophage cluster 2. Further analysis shows that the proportion of chemokine ligand 9 positive cells is different in different stages of EAM. On day 14 of the acute phase ( Figure 2 ) The proportion of chemokine ligand 9-positive cells was the highest and was positively correlated with the degree of inflammation in EAM (R2=0.9, p=0.04; Figure 3 Since chemokine ligand 9 is highly expressed in inflammatory macrophages, this suggests that EAM mice have more chemokine ligand 9-expressing macrophages infiltrating myocardial tissue compared to healthy mice, which is a significant difference.

[0064] In summary, it can be speculated that chemokine ligand 9 can be used as a specific target for detecting acute myocarditis. This discovery is of great significance for deepening the understanding of the pathogenesis of myocarditis and developing new treatment strategies.

[0065] Example 3

[0066] This example performs cell culture and in vitro verification of chemokine ligand 9 expression.

[0067] Mouse mononuclear leukemia cell line (RAW264.7) and human lymphoma cell line (U937) were obtained from the Cell Bank for Type Culture Collection, Chinese Academy of Sciences. RAW264.7 and U937 cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO2.

[0068] In the LPS-induced macrophage inflammation assay, macrophages were stimulated with 100 ng / mL LPS at 37°C for 4 hours. Western blotting and immunohistochemical fluorescence detection were performed to verify the expression level of chemokine ligand 9. Based on the results of single-cell RNA sequencing and histological analysis, we further verified the level of chemokine ligand 9 in vitro. Western results showed that the level of chemokine ligand 9 was significantly increased in LPS-induced mouse and human (RAW264.7 and U937) macrophages ( Figure 5 and Figure 6 ). The above results showed that chemokine ligand 9 was differentially expressed in inflammatory macrophages as determined by histological examination.

[0069] Example 4

[0070] In this example, myocarditis-targeted nanoprobes (TNPs) were prepared and characterized.

[0071] According to the effective sequence of the chemokine ligand 9 antibody protein, a chemokine ligand 9 targeting binding peptide was prepared, the sequence of which is LKVRKSQRSRQKKTTC (terminal thiol group), and the peptide was synthesized by Shanghai Qiangyao Company. BPBBT was prepared according to the method previously published by the applicant team (Shuai Gao et.al., Nat. Commun. 2019, 10: 2206). When preparing TNPs, 1 mg of BPBBT, 3.2 mg of DSPE-mPEG 2000 (Xi'an Ruixi) and 0.8mg DSPE-PEG 2000 A 1 mL THF solution of -MAL (Xi'an Ruixi) was poured into 9 mL ultrapure water and then sonicated for 2 min using a microtip probe sonicator (VCX150, SONICS & MATERIALSINC, USA). The mixture was transferred to a dialysis bag (molecular weight cutoff: 1000 Da) and dialyzed with deionized water for 24 h. The dialysate was filtered with a 0.45 μm syringe filter. Anti-chemokine ligand 9 polypeptide (2.4 mg) with terminal modified thiol groups was added and stirred at 25°C overnight. The reaction mixture was dialyzed again as described above. The dialysate was concentrated by ultrafiltration and centrifugation, and the obtained TNPs could be stored at 4°C until further use.

[0072] For non-targeted nanoparticles (NPs), 1 mg BPBBT and 4 mg DSPE-mPEG 2000 1 mL of THF solution was poured into 9 mL of ultrapure water and then sonicated for 2 min using a microtip probe sonicator (VCX150, SONICS & MATERIALS INC, USA). The mixture was transferred to a dialysis bag (molecular weight cutoff: 1000 Da) and dialyzed against deionized water for 24 h. The dialysate was filtered with a 0.45 μm syringe filter. The dialysate was concentrated by ultrafiltration and centrifugation, and the obtained NPs could be stored at 4 °C until further use. The size and zeta potential of the prepared TNPs and NPs in aqueous solution were monitored using dynamic light scattering (DLS), and their morphology was observed using transmission electron microscopy (TEM).

[0073] TNPs in vitro experiments

[0074] In order to achieve deep tissue penetration and high-precision detection, the NIR-II emission wavelength of the AIE molecule BPBBT was selected when preparing the nanoprobe. In order to obtain ideal water solubility, good biocompatibility and outstanding macrophage targeting ability, the hydrophobic BPBBT was combined with the amphiphilic copolymer DSPE-mPEG by nanoprecipitation. 2000 and DSPE-PEG 2000-MAL was encapsulated into nanoparticles at a ratio of 1:4. Subsequently, the anti-chemokine ligand 9 peptide was modified on the surface of the nanoparticles to form TNPs (Scheme 1). Therefore, the anti-chemokine ligand 9 peptide was used to provide TNPs with targeting ability to inflammatory macrophages that highly express chemokine ligand 9. Only BPBBT and DSPE-mPEG were used to prepare NPs. 2000 As a control, the AIE characteristics of BPBBT molecules were first studied. When water was added to tetrahydrofuran, the polarity of the solvent was increased and the solubility of BPBBT was reduced. As the proportion of water gradually increased, the fluorescence intensity of BPBBT also increased. When the proportion of water reached 80%, the fluorescence intensity of BPBBT reached the highest ( Figure 7 and Figure 8 ). This result shows that when the water ratio is lower than 40%, the fluorescence intensity of BPBBT is dominated by the distorted intramolecular charge transfer state, while when the water ratio increases further, the balance of fluorescence intensity shifts to the AIE state. The average diameter and morphology of the prepared NPs and TNPs were then characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Figure 9 As shown in the figure, the average sizes of the prepared TNPs and NPs are approximately 141 nm and 138 nm, respectively. Their sizes are actually between 100-200 nm, which is the optimal range for effective accumulation at the site of inflammation through the enhanced permeability and retention (EPR) effect. TEM images show that these nanoparticles are uniformly spherical with a diameter of approximately 100 nm ( Figure 9 ), which is smaller than the hydrodynamic diameter measured by DLS, which may be due to the lack of a hydration layer on the surface of the nanoparticles. The good aggregation-induced emission characteristics and regular morphology prompted us to study the FL properties of the nanoprobes. The prepared TNPs and NPs have a broad and strong absorption in almost the entire NIR-I band (650-900nm) ( Figure 10 ). The longer absorption wavelength in the near-infrared region is one of the key prerequisites for the application of light detection in vivo, because the photoactivity of TNPs and NPs is triggered by near-infrared light, which can penetrate tissues deeper and cause less damage to tissues than ultraviolet or visible light. Due to the typical AIE characteristics and stronger DA interactions in the structure, both TNPs and NPs exhibit larger Stokes shifts (about 293nm) and stronger near-infrared-II fluorescence. Their emission spectra are mainly located in the NIR-II region (700-1400nm), which indicates that both TNPs and NPs can achieve deeper tissue penetration, high resolution, high imaging fidelity and higher signal-to-noise ratio in vivo. As Figure 11 As shown, the FL signal was found to be significantly different from that of the control group.

[0075] In vitro NIR-II imaging experiments

[0076] Mice were divided into four groups (n = 5 / group) according to the different treatments: 1) healthy group + saline; 2) healthy group + TNPs; 3) EAM + NPs; and 4) EAM + TNPs. Each group received a tail vein injection of TNPs or NPs in saline, along with 200 μg of BPBBT per mouse. In vivo NIR-II imaging was performed 12 hours after injection, and on days 7 and 14, respectively. Images were acquired using a commercial Series II 900 / 1700 imaging system with an 1100 nm long-pass (LP) filter. Fluorescence images were quantitatively analyzed using ImageJ.

[0077] Based on the confirmation that chemokine ligand 9 can be targeted and that macrophages expressing chemokine ligand 9 are abundantly accumulated in the EAM model, we used EAM mice to perform in vivo imaging of acute myocarditis in the near-infrared-II region. We designed a protocol to investigate the efficacy of in vivo imaging in the acute phase of EAM (day 14). We found that the EAM+TNPs group emitted a clear optical signal, and the cardiac outline was clearly visible ( Figure 12 ). The healthy + saline group and the healthy + NPs group served as control groups, and almost no optical signals were detected in these two groups. The healthy + TNPs group and the EAM + NPs group emitted a slight sternum-like signal in the center of the chest cavity, which was due to the accumulation of some nanoparticles in the bone marrow through blood circulation. This effect did not affect the detection of myocarditis. In particular, obvious fluorescence signals were observed in the chest cavity of mice in the EAM + TNPs group. Semi-quantitative fluorescence statistics showed that the fluorescence signals in the heart area of ​​mice in the EAM + TNPs group were significantly different from those in other groups ( Figure 13 These results suggest that TNPs can provide a significant optical signal in EAM mice, which is useful for detecting acute-stage myocarditis.

[0078] Experimental study on drug efficacy evaluation using NIR-II

[0079] According to different treatments, mice were divided into six groups (n=5 / group): 1) healthy + NPs; 2) healthy + TNPs; 3) EAM + NPs; 4) EAM + TNPs; 5) EAM + PX478 + NPs; 6) EAM + PX478 + TNPs. In the PX478 treatment group, EAM mice were intraperitoneally injected with PX478 (50 mg / kg) in saline once a day before sacrifice. -1The mice were injected with TNPs or NPs (200 pg BPBBT per mouse) in saline via tail vein for one week. After the in vivo imaging, the mice were sacrificed and the hearts were collected for ex vivo NIR-II imaging. The images were acquired using Series II 900 / 1700 imaging system with a long-pass (LP) filter at 1100 nm. The fluorescence images were quantitatively analyzed using ImageJ.

[0080] The mortality of acute myocarditis will be greatly reduced if the myocarditis is found in the early stage (acute phase) and combined with appropriate treatment (including medication). Given the excellent imaging ability and biocompatibility of TNPs, we tried to evaluate the efficacy of anti-inflammatory drugs using TNPs. It has been reported that the inhibitor of Hifl a, PX478, can inhibit the inflammatory response of EAM. In the healthy + NPs group and the healthy + TNPs group, no obvious fluorescence signal was observed in the heart region in vivo and ex vivo images( Figure 14 ). This indicates that NPs and TNPs do not accumulate in the healthy heart. No obvious fluorescence signal was detected in the EAM + NPs group, which indicates that NPs cannot target the inflammatory area. In contrast, in the EAM + TNPs group, in vivo images showed that there was a significant signal in the heart region. In the ex vivo images, the heart tissue also showed strong fluorescence signal( Figure 15 ), which indicates that TNPs can target and accumulate in the myocarditis site, consistent with the results of the above experiment. The signal of the EAM + PX478 + TNPs group was weaker than that of the EAM + TNPs group, indicating that the degree of inflammation was reduced after PX478 treatment( Figure 16 ). The semi-quantitative fluorescence column chart shows that the signal contrast of the EAM + TNPs group and the EAM + PX478 + TNPs group has statistical difference with other groups( Figure 17 、 Figure 18 and Figure 19 ). This result indicates that TNPs can not only accurately detect acute myocarditis, but also provide a method for rapid detection and evaluation of drug efficacy. Therefore, this method can be used for drug screening in experimental animals.

[0081] Human clinical sample chemokine ligand 9 targeted localization experiment

[0082] The Human Ethics Committee of Shenzhen Fuwai Hospital, Chinese Academy of Medical Sciences, approved the use of human tissue in this study. Each patient provided written informed consent. A series of cardiac samples were collected from patients undergoing heart transplantation. The human cardiac tissue samples were divided into two groups: a myocarditis group (n=5) and a healthy heart group (n=5). Healthy heart samples were collected from brain-dead donors who were unsuitable for transplantation due to technical or non-cardiac reasons (such as weight mismatch), according to the recommendations of the China Organ Transplant Service Center. The donors had a normal circulatory system. The samples were placed in 24-well plates. After rinsing with saline (3 × 1 min), the two groups of samples were incubated with 1 mL of NPs and TNPs (both labeled with DSPE-PEG-Cy7) for 2 h. The samples were then rinsed with saline (3 × 5 min) to remove unbound nanoparticles. Fluorescence imaging was then performed using an IVIS system, and the mean fluorescence intensity of the samples was quantified using the measurement panel of the IVIS software.

[0083] We further explored the ability of TNPs to detect human myocarditis samples. To verify its effectiveness, we collected normal human myocardial tissue and myocarditis tissue samples from the clinic and conducted experiments. During the experiment, human myocardial samples were incubated with different nanoprobes, with the normal saline group serving as a fluorescence background control. The experimental results showed that compared with NPs, TNPs targeting chemokine ligand 9 can more accurately distinguish between myocarditis tissue and normal myocardial tissue. In addition, in myocarditis samples, the fluorescence intensity of the TNPs group was about five times that of the NPs group ( Figure 20 and Figure 21 These data fully demonstrate that the TNPs we invented have extremely high selectivity and precise targeting to human myocarditis tissue.

[0084] To image myocarditis using nanoprobes, two key challenges must be addressed: 1) how to specifically localize myocardial lesions; and 2) how to improve the probe's sensitivity. To identify specific targets within myocardial lesions, we employed single-cell RNA sequencing to conduct in-depth analysis of myocardial inflammation samples from EAM mice. The results revealed that macrophage population 2 is a unique immune cell population in acute myocarditis. Further screening revealed that chemokine ligand 9 (CL9) is significantly associated with CL2 during acute myocarditis, particularly on day 14 of EAM. At this stage, cardiac fibroblasts exhibit immune activation and overexpress chemokine ligand 9 and chemokine ligand 10. These chemokines are commonly involved in immune regulation and inflammation and can further contribute to T cell recruitment. Single-cell RNA sequencing also revealed a high proportion of CL9-positive cells in acute myocarditis, with distinct patterns of changes. Therefore, we hypothesized that targeting CL9-positive cells could be a promising approach for detecting acute myocarditis. Staining of sections of mouse and human myocarditis samples showed that mouse myocarditis tissues expressed varying degrees of chemokine ligand 9 receptor. Therefore, we selected chemokine ligand 9 as the target for subsequent nanoprobe detection and imaging.

[0085] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A myocarditis-targeted nanoprobe, characterized in that: The nanoprobe targets and binds to the chemokine ligand 9, and the myocarditis-targeted nanoprobe includes 1,4-di[4-(octyloxy)-triphenylamine)]-benzo[1,2-c:4,5-c'] and [1,2,5] thiadiazole, phosphoethanolamine phospholipid-polyvinyl alcohol 2000 , phosphoethanolamine phospholipid-polyvinyl alcohol 2000 -maleimide and chemokine ligand 9 peptide, the 1,4-bis[4-(octyloxy)-triphenylamine)]-benzo[1,2-c:4,5-c'] and [1,2,5] thiadiazole, phosphoethanolamine phospholipid-polyvinyl alcohol 2000 and phosphoethanolamine phospholipid-polyvinyl alcohol 2000 - Maleimide self-assembles into nanoparticles, and the chemokine ligand 9 peptide is modified on the surface of the nanoparticles; The chemokine ligand 9 peptide is a targeting peptide that can specifically bind to chemokine ligand 9; the amino acid sequence of the targeting peptide that specifically binds to chemokine ligand 9 is shown in SEQ ID NO.1; The myocarditis targeting nanoprobe contains 1,4-di[4-(octyloxy)-triphenylamine)]-benzo[1,2-c:4,5-c']-[1,2,5]thiadiazole, phosphoethanolamine phospholipid-polyvinyl alcohol 2000 , phosphoethanolamine phospholipid-polyvinyl alcohol 2000 -The mass ratio of maleimide to chemokine ligand 9 peptide is 1:3.2:0.8:2.4; The size of the nanoparticles is 100-200 nm; The myocarditis targeting nanoprobe is prepared by the following steps: 1,4-bis[4-(octyloxy)-triphenylamine)]-benzo[1,2-c:4,5-c'] and[1,2,5]thiadiazole and phosphoethanolamine phospholipid-polyvinyl alcohol 2000 , phosphoethanolamine phospholipid-polyvinyl alcohol 2000 -maleimide, and sequentially subjected to ultrasonic treatment and dialysis treatment, and the dialysis product is mixed with the chemokine ligand 9 peptide and dialyzed to obtain.

2. The myocarditis-targeted nanoprobe according to claim 1, characterized in that: The surface of the nanoparticles is also modified with fluorescent markers.

3. The myocarditis-targeted nanoprobe according to claim 2, characterized in that: The fluorescent label includes cyanine dye Cy7 and / or cyanine dye Cy5.

5.

4. The myocarditis-targeted nanoprobe according to claim 1, characterized in that: The terminal of the chemokine ligand 9 peptide is modified with a thiol group.

5. A method for preparing the myocarditis-targeting nanoprobe according to any one of claims 1 to 4, characterized in that: The preparation method comprises: 1,4-bis[4-(octyloxy)-triphenylamine)]-benzo[1,2-c:4,5-c'] and[1,2,5]thiadiazole and phosphoethanolamine phospholipid-polyvinyl alcohol 2000 , phosphoethanolamine phospholipid-polyvinyl alcohol 2000 -maleimide, and sequentially subjected to ultrasonic treatment and dialysis treatment, and the dialysis product is mixed with the chemokine ligand 9 peptide and dialyzed to obtain.

6. A kit for early detection of myocarditis, characterized in that: The kit comprises the myocarditis-targeting nanoprobe according to any one of claims 1 to 4.

7. Use of the myocarditis-targeted nanoprobe according to any one of claims 1 to 4 in detecting cells expressing chemokine ligand 9.

8. The use according to claim 7, characterized in that The cells expressing chemokine ligand 9 include chemokine ligand 9-expressing inflammatory macrophages.

9. A method for detecting cells expressing chemokine ligand 9, characterized in that: The method comprises: mixing a sample to be detected with the myocarditis-targeting nanoprobe according to any one of claims 1 to 4, and performing fluorescence detection.

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