CN117538537A - Application of reagent for detecting chemokine ligand 9 in preparation of myocarditis early detection product - Google Patents
Application of reagent for detecting chemokine ligand 9 in preparation of myocarditis early detection product Download PDFInfo
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Abstract
本发明公开了检测趋化因子配体9的试剂在制备心肌炎早期检测产品中的应用。本发明利用单细胞RNA测序技术鉴定了小鼠心肌炎早期阶段的免疫细胞特征，并发现心肌炎小鼠与健康小鼠相比，有更多表达趋化因子配体9的巨噬细胞浸润心肌组织，这一发现揭示了趋化因子配体9可作为检测急性心肌炎的特异性靶点。进一步地，我们利用BPBBT，DSPE‑mPEG2000和DSPE‑PEG2000‑MAL制备了纳米颗粒，并设计出能与趋化因子配体9特异性结合的靶向肽并将其连接到纳米材料表面，制备出了心肌炎靶向纳米探针。所述心肌炎靶向纳米探针能特异性地聚集在炎症区域，从而准确检测心肌炎。
The invention discloses the application of a reagent for detecting chemokine ligand 9 in the preparation of early detection products for myocarditis. The present invention uses single-cell RNA sequencing technology to identify the characteristics of immune cells in the early stages of mouse myocarditis, and finds that compared with healthy mice, mice with myocarditis have more macrophages expressing chemokine ligand 9 infiltrating the myocardial tissue. This finding reveals that chemokine ligand 9 can serve as a specific target for the detection of acute myocarditis. Furthermore, we prepared nanoparticles using BPBBT, DSPE‑mPEG 2000 and DSPE‑PEG 2000 ‑MAL, and designed a targeting peptide that can specifically bind to chemokine ligand 9 and attached it to the surface of the nanomaterial. Myocarditis-targeted nanoprobes were prepared. The myocarditis-targeted nanoprobe can specifically gather in the inflammatory area, thereby accurately detecting myocarditis.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to application of a reagent for detecting chemokine ligand 9 in preparation of an early detection product for myocarditis.
Background
Myocarditis is a common inflammatory heart disease, wherein 1% -7% of myocarditis is the myocarditis. Symptoms are not obvious at the beginning, and if inflammation is aggravated, sudden fatal cardiovascular events can be caused, including heart failure, cardiogenic shock and even sudden death. Thus, early accurate diagnosis of myocarditis is of great importance for early positive treatment to reduce the incidence of fatal cardiovascular events.
Currently, detection of myocarditis is mainly dependent on two modes of endocardial myocardial biopsy or cardiovascular magnetic resonance imaging. However, endocardial myocardial biopsies have certain limitations as gold standard for detecting myocarditis. Such invasive tests are not only limited in sampling range, but also have a probability of sampling errors, and thus are difficult to be widely conducted in clinical practice.
In cardiovascular magnetic resonance imaging detection, myocarditis behaves similarly to myocardial infarction, cardiomyopathy and myocardial amyloidosis, and other examinations are required to make more reliable detection. Notably, myocarditis can be clinically divided into acute, subacute and chronic phases. Early therapeutic interventions in patients during the acute phase generally restore cardiac function, but not during the chronic phase. Therefore, development of advanced detection techniques to improve the detection accuracy of myocarditis is needed.
In recent years, fluorescence imaging detection techniques have been used in biological imaging applications at a very wide angle, which can provide real-time, on-site, and noninvasive imaging modes, and have extremely high spatial and temporal resolution. Nanotechnology, on the other hand, is changing our detection and therapeutic agent delivery concepts. The nanoparticle has many excellent properties such as nanoscale size effect, good dispersibility and water solubility, excellent blood circulation capacity, targeting modification property and the like, and can help us to accurately deliver diagnostic or therapeutic reagents to specific sites.
By combining the advantages of nanotechnology and fluorescence imaging, we can develop efficient, specific focal-targeting nanoprobes to accurately determine myocarditis at an early stage. The novel detection method is not only expected to improve the detection accuracy, but also can reduce the misdiagnosis rate, and provides powerful support for early diagnosis of acute myocarditis.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides a reagent for detecting chemokine ligand 9 protein for an early detection product of myocarditis, and the invention digs a novel early detection target spot of myocarditis and further develops a detection scheme so as to realize rapid and accurate early detection of myocarditis.
Compared with the prior art, the invention adopts the following technical scheme:
in a first aspect, the invention provides the use of a reagent for detecting chemokine ligand 9 in the preparation of an early myocarditis detection product.
In the present invention, we successfully constructed an experimental immune myocarditis (EAM) mouse model. By applying single cell RNA sequencing technology, we have further analyzed immune cell characteristics at early stages of mouse myocarditis and found significant differential expression of chemokine ligand 9. Differential expression of chemokine ligand 9 was further validated in vitro by independent mouse and human macrophage models. There was more macrophage infiltration of chemokine ligand 9-expressing myocardium tissue in myocarditis mice than in healthy mice. We also collected myocardial samples from patients with clinical myocarditis and validated using database comparison and histochemical experiments. The results show significantly higher expression of chemokine ligand 9 in myocarditis patients compared to healthy human myocardium. These data fully demonstrate the potential of chemokine ligand 9 as a specific target for detection of acute myocarditis.
Preferably, the reagent for detecting chemokine ligand 9 comprises a reagent for detecting cells expressing chemokine ligand 9.
In a second aspect, the present invention provides a myocarditis-targeted nanoprobe, which is targeted for bindingChemokine ligand 9, said myocarditis targeting nanoprobe comprising 1, 4-bis [4- (octyloxy) -triphenylamine]1, 2-c-4, 5-c 'radical-benzo']And [1,2,5 ]]Thiadiazole (BPBBT), phosphoethanolamine phospholipid-polyvinyl alcohol 2000 (DSPE-mPEG 2000 ) Phosphorylethanolamine phospholipid-polyvinyl alcohol 2000 Maleimide (DSPE-PEG) 2000 -MAL) and chemokine ligand 9 peptide, said BPBBT, DSPE-mPEG 2000 And DSPE-PEG 2000 -MAL self-assembly into nanoparticles, the chemokine ligand 9 peptide being modified at the nanoparticle surface; the amino acid sequence of the chemokine ligand 9 peptide is shown as SEQ ID NO. 1.
SEQ ID NO.1：LKVRKSQRSRQKKTTC。
In the invention, BPBBT is a high-brightness near infrared two-region (NIR-II) emission aggregation-induced emission agent (AIEgens) and is utilized to prepare the fluorescent dye by using BPBBT and DSPE-mPEG 2000 And DSPE-PEG 2000 -MAL preparation of nanoparticles, designing targeting peptides capable of specifically binding to chemokine ligand 9, and modifying the targeting peptides specifically binding to chemokine ligand 9 on the surface of nanoparticles to obtain myocarditis Targeting Nanoprobes (TNPs) capable of specifically aggregating in inflammatory regions, thereby accurately detecting the acute phase of myocarditis. In addition, TNPs-based fluorescence imaging techniques can also be used to monitor the efficacy of drugs for the treatment of myocarditis.
Preferably, the nanoparticle is further modified with a fluorescent label.
Preferably, the fluorescent label is modified at DSPE-PEG 2000 -on MAL.
Preferably, the fluorescent label comprises the cyanine dye Cy7 and/or the cyanine dye Cy5.5.
Preferably, the chemokine ligand 9 peptide is terminally modified with a sulfhydryl group.
Preferably, BPBBT and DSPE-mPEG in the myocarditis targeting nano probe 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.
In a third aspect, the present invention provides a method for preparing the myocarditis-targeted nanoprobe according to the first aspect, the method comprising:
BPBBT and DSPE-mPEG 2000 Mixing, sequentially performing ultrasonic treatment and dialysis treatment, mixing the dialyzed product with chemokine ligand 9 peptide, and performing dialysis treatment.
In a fourth aspect, the invention provides an early detection kit for myocarditis, which comprises the myocarditis targeting nanoprobe in the second aspect.
The invention designs the myocarditis targeting nano probe (named TNPs), utilizes a near infrared two-region TNPs system to carry out optical imaging detection of the myocarditis, has high specificity, safety and reliability, is sensitive and accurate in detection of the inflammation by the TNPs, can accurately detect the reduction of the inflammation after PX478 treatment, and can be effectively applied and used for detecting myocardial inflammation, evaluating the treatment effect of medicaments and the like.
In a fifth aspect, the invention provides the use of a myocarditis-targeted nanoprobe according to the second aspect for detecting cells expressing chemokine ligand 9.
The myocarditis targeting nano-probe designed by the invention can also be applied to the field of non-disease detection, such as in-vitro detection and analysis of cells of chemokine ligand 9, cell related basic research and the like.
Preferably, the chemokine ligand 9 expressing cell comprises an inflammatory macrophage expressing chemokine ligand 9.
In a sixth aspect, the invention provides a method of detecting a cell expressing chemokine ligand 9, said method comprising: mixing a sample to be detected with the myocarditis targeting nano probe in the second aspect, and performing fluorescence detection.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, immune cell characteristics of the acute phase of the myocarditis of the mice are identified by utilizing a single-cell RNA sequencing technology, and compared with healthy mice, the myocarditis mice are found to have more macrophages for expressing the chemokine ligand 9 to infiltrate myocardial tissues, so that the chemokine ligand 9 can be used as a specific target for detecting the acute myocarditis. Further utilize BPBBT, DSPE-mPEG 2000 And DSPE-PEG 2000 -MAL preparation of nanoparticles, designing targeting peptides capable of binding specifically to chemokine ligand 9, preparing myocarditis targeting nanoprobes capable of specifically aggregating in inflammatory areas, thereby accurately detecting myocarditis. In addition, the fluorescent imaging technology based on the myocarditis targeting nano probe can also be used for monitoring the curative effect of the medicament for treating myocarditis.
Drawings
FIG. 1 is a cross section of a t-distribution random neighborhood embedding diagram of 34665 immune cells, wherein the trend of the number of macrophage cluster-2 during EAM progression is shown in an oval box;
FIG. 2 is a view of the Violin expression of chemokine ligand 9 marker genes in each macrophage cluster;
FIG. 3 is a graph showing the percentage of chemokine ligand 9 positive cells in early myocarditis at various time points;
FIG. 4 is a graph showing the correlation of the percentage of chemokine ligand 9 positive cells at various stages with the degree of inflammation;
FIG. 5 is a graph showing the detection of chemokine ligand 9 expression on RAW264.7 and U937 cells by Western blotting;
FIG. 6 is a graph of relative values (n=3) of chemokine ligand 9/GAPDH stripe gray;
FIG. 7 is a fluorescence emission spectrum of BPBBT (10. Mu.M) in a water/tetrahydrofuran mixture, the ratio of water at excitation at 660nm being from 0 to 99%;
FIG. 8 is the fluorescence intensity of BPBBT (10. Mu.M) at 660nm wavelength excitation in tetrahydrofuran/water (v/v) mixed solvent with increasing water content;
FIG. 9 is a representative TEM image and size distribution diagram of TNPs;
FIG. 10 shows the absorption spectrum and fluorescence spectrum of TNPs in water;
FIG. 11 is a fluorescence image of TNPs, NPs and PBS samples;
FIG. 12 is an in vivo near infrared-II imaging of the EAM model for various treatments during the acute phase (day 14);
FIG. 13 is a semi-quantitative fluorescence bar graph of FIG. 12;
FIGS. 14, 15 and 16 are in vivo near infrared-II imaging of the EAM model under different treatments, and in vitro near infrared-II imaging of the corresponding set of harvested hearts, respectively;
FIGS. 17, 18 and 19 are in vivo and ex vivo semi-quantitative fluorescent bar graphs of FIGS. 14, 15 and 16, respectively;
FIG. 20 is a fluorescence imaging of representative normal myocardial and myocarditis specimens obtained from freshly excised human normal myocardial and myocarditis specimens after 2h incubation with saline, NPs or TNPs;
FIG. 21 is a semi-quantitative statistical plot of the fluorescence of FIG. 20.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
Histological examination
An EAM mouse model is constructed, and heart tissues of the EAM mouse are collected. Collecting clinical myocardial tissue samples of human myocarditis. Samples were fixed in 4% (v/v) formalin solution overnight, embedded in paraffin, and sectioned into 5 μm thick sections. The sections were then subjected to H & E, immunohistochemical and immunohistochemical fluorescent staining for histopathological evaluation. H & E and immunohistochemical images were examined by digital microscopy. The fluorescence images were observed under a stereoscopic microscope (Leica DMi8, leica microsystems, germany) and quantified using ImageJ.
Ethical approval and consent participation
The study was in accordance with the ethical guidelines of the revision 2013 of the declaration of helsinki. All patients provided written informed consent for medical studies using clinical specimens. The laboratory animals (Shenzhen Hospital Fuforeign Hospital, china) were subjected to ethical committee approval (Shenzhen Hospital, fungial, china) according to the standard guidelines approved by the animal welfare Committee, using human study specimens and mouse experiments.
Statistical analysis
All data are expressed as mean ± standard deviation, unless otherwise indicated. Statistical differences between treatment and control groups were analyzed by single or two-factor anova and Tukey post-hoc test (multiple comparisons) using graphpad prism version 8.0 software. The survival data were tested using log-rank. * Indicating that the difference is statistically significant, p <0.05; * P <0.01; and p <0.001.
Example 1
The present example performs the establishment of a mouse model of experimental autoimmune myocarditis.
Six week old male Balb/c mice (purchased from Guangzhou Jiujiakang corporation) were taken. Mice were acclimatized for at least seven days prior to the experiment, and were free to eat fresh food and water. On days 0 and 7, mice were subcutaneously injected with 250 μg of alpha-MyHC peptide (Ac-RSLKLMATLFSTYASADR-OH; anaSpec, AS-62554) and complete Freund's adjuvant (Sigma, F5881;1:1, weight ratio) to induce autoimmune myocarditis (Experimental Autoimmune Myocarditis, EAM).
Example 2
In this example, single cell analysis and target screening were performed.
Single cell suspension preparation: collagenase 2 (CLS 2, sigma), concentration 400U/mL; cutting myocardial tissue of mouse model with myocarditis in PBS to 1mm 3 A small block; subsequently rinsed with PBS until the tissue fragments whiten, 400U/mL CLS210 mL, digested in a 37℃water bath for 15min, where shaking cannot be stopped; the supernatant was aspirated through a 40 μm sieve and collected in a 50mL tube; and (3) differential centrifugation: centrifuging at 4deg.C for 1min at 100g, collecting supernatant, centrifuging at 400×g for 5min, and discarding supernatant; resuspension with 1mL 2% FBS/RPMI; repeating the steps for 4 total cycles until the tissue is completely digested; collecting all cells, and centrifuging at a differential speed; the cells were resuspended in 4 volumes of erythrocyte lysate, lysed on ice for 2min, diluted 10 volumes, centrifuged at 400 Xg for 5min and discardedClearing.
Sorting CD45 + And (3) cells: preparation: flow tube blocking, incubation with 20% FBS/PBS25℃for 3h; dyeing control design: control tube: 1: isotype ctrl,2:7-AAD monocyang, 3: cd45 monocyang; the most cells in the sample tube are used as a control, and only 10 cells are needed to be taken 5 Cells/tubes; the cell pellet from experiment 4 above (normal control) was resuspended in 1mL PBS, respectively; counting was performed using a Countstar, and Fc block was calculated to be 1. Mu.g block (2. Mu.L)/10 6 The cells/100. Mu.L PBS ratio was added to the cell fluid and blocked on ice for 5min (approximately 20. Mu.L Fc Block); according to the following steps of 1:200 adding 5 mu L of Cd45 antibody (No. 2 tube: 7-AAD single positive tube is not added), and dyeing for 30min on ice in dark; centrifuging at 400rpm for 5 min; washing with PBS for 2 times; preparing 7-AAD dye liquor: 7-AAD 1:20 in PBS, 7-AAD should be diluted in 5. Mu.L of 7-AAD/10 6 Cells/100 μl PBS staining; iso type ctrl and CD45 single cations were resuspended with PBS; the 7-AAD single cation was resuspended using 300. Mu.L of 7-AAD dye liquor; the remaining sample tubes were resuspended with 1mL of 7-AAD dye; all cells were sieved through a 40 μm sieve, transferred to a flow tube, and sorted on an on-machine; firstly, adjusting voltage and adjusting compensation of a No. 1/2/3 tube; the voltage is FSC 210; SSC 300; FITC 430; perCP-Cy5-5:520; analyzing 1 ten thousand cells of each sample, performing gate control adjustment, and storing a picture; pouring out the blocking buffer in the flow tube, and adding 500 mu L of 2% FBS/RPMI; sorting cells in each sample tube, sorting the cells into a culture medium, and collecting 40 ten thousand cells; cells in the medium were aspirated into 1.5mL centrifuge tubes, and centrifuged at 3000rpm for 5min in a conventional centrifuge to see a significant pellet; the supernatant was blotted with a gun and resuspended in 50 μl PBS; taking 10 mu L of cells and 10 mu L of AOPI, uniformly mixing, using a countstar count, and determining the cell concentration, the activity rate, the nucleated rate and the agglomeration rate, so that the viable cell concentration of the cell suspension is 800 cells/mu L, the activity rate is more than 90%, the agglomeration rate is less than 5%, the nucleated rate is more than 70%, and the cell suspension meeting the standard can be subjected to single cell library construction.
The invention utilizes EAM model to analyze myocarditis immune cells, and aims at determining specific targets. According to the H & E staining result of heart tissue, inflammatory cell infiltration phenomenon in EAM myocardial tissue is obvious. In addition, the degree of inflammation of myocarditis has a certain correlation with the time of onset. Myocarditis is generally considered to be divided into acute, subacute and chronic phases. To find the marker targets for myocarditis acute phase immune cells, we performed single cell RNA sequencing studies on acute phase (14 days), subacute phase (21 days) and chronic phase (60 days) myocardial tissue of the EAM model to evaluate phase-specific cell clusters.
The research result shows that after myocarditis is happened, a new cell cluster, namely macrophage cluster 2 is induced. The number of cells is greatest in the acute phase of myocarditis and gradually decreases with the increase of the onset time. FIG. 1 shows a Violin map of chemokine ligand 9 (CXCL 9) marker gene expression in each macrophage colony, and shows that chemokine ligand 9 is expressed to the greatest extent in macrophage colony 2. Further analysis showed that the proportion of chemokine ligand 9 positive cells was different at different stages of EAM. The highest proportion of chemokine ligand 9 positive cells was at day 14 of the acute phase (fig. 2) and correlated positively with the degree of inflammation of EAM (r2=0.9, p=0.04; fig. 3). Since chemokine ligand 9 was highly expressed in inflammatory macrophages, this suggests that EAM mice have more macrophages expressing chemokine ligand 9 infiltrating myocardial tissue than healthy mice, which is a significant difference.
From the above, it is assumed that chemokine ligand 9 can be used as a specific target for the detection of acute myocarditis. This finding is of great importance for the in-depth understanding of the pathogenesis of myocarditis and for the development of new therapeutic strategies.
Example 3
Cell culture and in vitro validation of chemokine ligand 9 expression were performed in this example.
The mouse mononuclear macrophage leukemia cell line (RAW 264.7) and the human lymphoma cell line (U937) were from the national academy of sciences cell bank (Cell Bank forType Culture Collection). RAW264.7 and U937 cells were cultured in 10% FBS-added high sugar Dulbecco's Modified Eagle Medium (DMEM), 37℃and 5% CO 2 Incubation under conditions.
In the LPS-induced macrophage inflammatory assay, macrophages were stimulated with 100ng/mL LPS for 4h at 37 ℃. Western blot and immunohistochemical fluorescence detection were performed to verify expression levels of chemokine ligand 9. Based on single cell RNA sequencing and histological analysis, we further validated chemokine ligand 9 levels in vitro. Western results showed that the levels of chemokine ligand 9 were significantly elevated in both LPS-induced mouse and human (RAW 264.7 and U937) macrophages (FIGS. 5 and 6). The above results indicate that chemokine ligand 9 is differentially expressed in inflammatory macrophages as measured by histology.
Example 4
myocarditis-Targeted Nanoprobes (TNPs) and characterization were prepared in this example.
According to the effective sequence of the chemokine ligand 9 antibody protein, a chemokine ligand 9 targeting binding polypeptide is prepared, the sequence is LKVRKSQRSRQKKTTC (end-linked sulfhydryl), and the polypeptide is synthesized by Shanghai blaze company. BPBBT is prepared according to the method of applicant's team prepit (Shuai Gao et al, nat. Commun.2019, 10:2206). In the preparation of TNPs, the TNPs will contain 1mg of BPBBT, 3.2mg of DSPE-mPEG 2000 (Simaroubrious) and 0.8mg DSPE-PEG 2000 1mL of THF solution of-MAL (Simarouble) was poured into 9mL of ultrapure water, and then a microtip probe sonicator (VCX 150, SONES) was used&MATERIALS INC, usa) for 2min. The mixture was transferred to a dialysis bag (molecular weight cut-off: 1000 Da) and dialyzed against deionized water for 24h. The dialysate was filtered with a 0.45 μm syringe filter. An end-group modified sulfhydryl anti-chemokine ligand 9 polypeptide (2.4 mg) was added and stirred overnight at 25 ℃. The reaction mixture was dialyzed again as described above. The dialysate is concentrated by ultrafiltration and centrifugation, and the obtained TNPs can be stored at 4deg.C until further use.
For non-targeted Nanoparticles (NPs), will contain 1mg BPBBT and 4mg DSPE-mPEG 2000 Is poured into 9mL of ultrapure water, and then a microtip probe sonicator (VCX 150, SONES)&MATERIALS INC, usa) for 2min. The mixture was transferred to a dialysis bag (molecular weight cut-off: 1000 Da) and dialyzed against deionized water for 24h. The dialysate was filtered through a 0.45 μm syringe filter. The dialysate is concentrated by ultrafiltration and centrifugation and the resulting NPs can be stored at 4 ℃ until further use. Monitoring of prepared TNPs and N using Dynamic Light Scattering (DLS)The size and zeta potential of Ps in aqueous solution and its morphology was observed using Transmission Electron Microscopy (TEM).
TNPs in vitro experiments
In order to realize deep tissue penetration and high-precision detection, the NIR-II emission wavelength of AIE molecule BPBBT is selected when the nano probe is prepared. In order to obtain ideal water solubility, good biocompatibility and outstanding macrophage targeting ability, hydrophobic BPBBT and amphiphilic copolymer DSPE-mPEG are prepared by a nano precipitation method 2000 And DSPE-PEG 2000 MAL is packed into nanoparticles in a 1:4 ratio. Subsequently, anti-chemokine ligand 9 peptides were modified on the nanoparticle surface to form TNPs (scheme 1). Thus, anti-chemokine ligand 9 peptides are used to provide TNPs with targeting ability to inflammatory macrophages that highly express chemokine ligand 9. Preparation of NPs Using BPBBT and DSPE-mPEG only 2000 As a control. The AIE properties of BPBBT molecules were first studied. The polarity of the solvent is enlarged and the solubility of BPBBT is reduced when water is added into tetrahydrofuran. As the proportion of water increases, the fluorescence intensity of BPBBT increases. When the proportion of water reached 80%, the fluorescence intensity of BPBBT reached the highest (fig. 7 and 8). This result shows that when the proportion of water is less than 40%, the fluorescence intensity of BPBBT is dominated by a distorted intramolecular charge transfer state, and when the proportion of water is further increased, the balance of fluorescence intensity shifts to the AIE state. The average diameter and morphology of the NPs and TNPs produced were then characterized by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). As shown in FIG. 9, the average sizes of the TNPs and NPs prepared were about 141nm and 138nm, respectively. Their size is practically between 100-200nm, which is the optimal range for effective accumulation at the site of inflammation by enhancing the permeability and retention (EPR) effect. TEM images show that these nanoparticles are uniformly spherical, with a diameter of about 100nm (FIG. 9), less than the hydrodynamic diameter measured by DLS, probably due to the absence of a hydrated layer on the nanoparticle surface. Good aggregation-induced emission properties and regular morphology prompted us to investigate the FL properties of nanoprobes. TNPs and NPs prepared have broad and strong absorption almost throughout the NIR-I band (650-900 nm) (FIG. 10). Longer absorption wavelength in the near infrared regionOne of the key prerequisites for in vivo application of light detection is that TNPs and NPs are photo-active due to near infrared light, which penetrates tissue deeper than ultraviolet or visible light, and cause less damage to tissue. TNPs and NPs both exhibit greater Stokes shift (about 293 nm) and stronger near infrared-II fluorescence due to typical AIE properties and stronger D-A interactions in structure. The emission spectrum is mainly in the NIR-II region (700-1400 nm), which shows that TNPs and NPs can realize deeper tissue penetration, high resolution, high imaging fidelity and higher signal-to-noise ratio in vivo. As shown in fig. 11, a significant difference in FL signal from the control group was found.
In vitro NIR-II imaging experiments
Mice were divided into four groups (n=5/group) according to different treatment methods: 1) Healthy group + saline; 2) Healthy group + TNPs; 3) Eam+nps; 4) EAM+TNPs. Each group of mice was injected with TNPs or NPs in physiological saline by tail vein, 200. Mu.g of BPBBT per mouse. Mice were examined for in vivo NIR-II imaging at 12h post injection at 7d and 14d, respectively. Images were detected using a commercial Series II 900/1700 imaging system with a Long Pass (LP) filter of 1100nm. The fluorescent images were quantitatively analyzed using ImageJ.
Based on the confirmation that chemokine ligand 9 can be targeted and that macrophages expressing chemokine ligand 9 in the EAM model aggregate in large amounts, we used EAM mice to perform in vivo imaging detection of acute phase myocarditis in the near infrared-II region. We designed a protocol to study the efficacy of EAM acute phase (day 14) in vivo imaging. We found that the eam+tnps group emitted clear optical signals and that the heart contours were clearly visible (fig. 12). The healthy + saline group and the healthy + NPs group served as control groups, and these two groups hardly detected any optical signal. The healthy + TNPs group and EAM + NPs group emitted a slight sternal-like signal in the center of the chest, due to the accumulation of some nanoparticles in the bone marrow through blood circulation. This effect does not affect the detection of myocarditis. In particular, a clear fluorescent signal was observed in the chest of mice in the eam+tnps group. Semi-quantitative fluorescence statistics showed that the fluorescence signal of heart regions of mice in the eam+tnps group was significantly different from that of other groups (fig. 13). These results indicate that TNPs can provide significant optical signals to EAM mice, helping to detect acute phase myocarditis.
Evaluation experiment of drug efficacy Using NIR-II
According to the different treatments, the mice were divided into six groups (n=5/group): 1) Health + NPs; 2) Health + TNPs; 3) Eam+nps; 4) EAM+TNPs; 5) Eam+px478+nps; 6) EAM+PX478+TNPs. In the PX 478-treated group, EAM mice were intraperitoneally injected with PX478 (50 mg/kg) in normal saline once daily prior to sacrifice -1 Body weight) for one week. Each group of mice was injected with TNPs or NPs in physiological saline via tail vein, 200. Mu.g of BPBBT per mouse. After in vivo imaging, mice were sacrificed, hearts of each group of mice were collected and subjected to NIR-II in vitro imaging monitoring. Images were acquired using a Series II 900/1700 imaging system with a Long Pass (LP) filter at a wavelength of 1100nm. The fluorescent images were quantitatively analyzed using ImageJ.
Myocarditis is found in early (acute) phase, and the mortality rate of acute myocarditis is greatly reduced in combination with proper treatment (including medication). In view of the excellent imaging ability and biocompatibility of TNPs, we have attempted to evaluate the efficacy of anti-inflammatory drugs using TNPs. Inhibitors of hif1α, PX478, have been reported to inhibit the inflammatory response of EAM. In both the healthy + NPs group and the healthy + TNPs group, no significant fluorescence signal was observed in the heart region in vivo and in vitro images (fig. 14). This suggests that NPs and TNPs do not accumulate in healthy hearts. No significant fluorescent signal was detected in the eam+nps group, indicating that NPs were unable to target the inflammatory region. In contrast, in the eam+tnps group, in vivo images showed significant signs in the heart region. In vitro images, cardiac tissue also showed strong fluorescent signals (fig. 15), indicating that TNPs could target the accumulation at the myocarditis sites, consistent with the experimental results described above. The eam+px478+tnps group had weaker signal than the eam+tnps group, indicating a reduction in the degree of inflammation after PX478 treatment (fig. 16). Semi-quantitative fluorescence histograms showed that the signal comparisons for the eam+tnps group and the eam+px478+tnps group were statistically different from the other groups (fig. 17, 18, and 19). The result shows that TNPs can not only accurately detect acute myocarditis, but also provide a method for rapidly detecting and evaluating the curative effect of medicaments. Thus, this method can be used for drug screening of experimental animals.
Human clinical sample chemokine ligand 9 targeting experiment
The human ethical committee of Shenzhen Fuectohospital, national academy of medical science, approved the use of human tissue in this study. Each patient provided written informed consent. A series of heart samples were collected by researchers from patients undergoing heart transplant surgery. Human heart tissue samples are divided into two groups: myocarditis group (5 persons) and healthy heart group (5 persons). Healthy heart samples were taken from brain dead donors unsuitable for transplantation due to technical or non-cardiac reasons (e.g. weight mismatch) according to the recommendations of the chinese organ transplant service center, with normal circulatory system. Samples were placed in 24-well plates. After washing with physiological saline (3X 1 min), the two groups of samples were incubated with 1mL of NPs and TNPs (NPs and TNPs were labeled with DSPE-PEG-Cy 7), respectively, for 2h. Then, the sample was rinsed with physiological saline (3×5 min) to remove unbound nanoparticles. Fluorescence imaging was then performed with the IVIS system and the average fluorescence intensity of the samples was quantified using the measurement panel of the IVIS software.
We further examined the ability of TNPs to detect samples of human myocarditis. To verify its effectiveness, we collected human normal myocardial tissue and myocarditis tissue samples from the clinic and performed experiments. In the experimental process, human myocardial samples are respectively incubated with different nano probes, wherein a physiological saline group is used as a fluorescent background control. Experimental results show that TNPs targeting chemokine ligand 9 are able to more accurately distinguish myocarditis tissue from normal myocardial tissue than NPs. Furthermore, in the myocarditis samples, the fluorescent intensity of the TNPs group was approximately five times that of the NPs group (fig. 20 and 21). These data fully demonstrate that our invented TNPs have extremely high selectivity and accurate targeting to human myocarditis tissue.
In order to perform nanoprobe imaging of myocarditis, two main problems must be solved: 1) How to target the myocarditis focus; 2) How to improve the detection sensitivity of the probe. In order to determine specific targets of myocarditis lesions, single-cell RNA sequencing technology is adopted, and myocarditis samples of EAM mice are deeply analyzed. The results show that macrophage population 2 is a unique immune cell population in acute phase myocarditis. After further screening, chemokine ligand 9 was found to have a significant correlation with macrophage population 2 in acute phase myocarditis (especially day 14 of EAM). At this time, the myocardial fibroblasts exhibit activation of immune processes, and overexpress chemokine ligand 9 and chemokine ligand 10. These chemokines are generally involved in immunomodulation and inflammatory processes, and may further be involved in recruitment of T cells. The single cell RNA sequencing results also show that the proportion of chemokine ligand 9 positive cells in acute phase myocarditis is high, and the change characteristics are obvious. Thus, we propose a hypothesis to detect acute phase myocarditis by targeting chemokine ligand 9 positive cells. Staining of the sections of the mouse and human myocarditis samples showed varying degrees of chemokine ligand 9 receptor expression in the mouse myocarditis tissue. Thus, we selected chemokine ligand 9 as the target for subsequent nanoprobe detection imaging.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011155949A (en) * | 2010-02-03 | 2011-08-18 | Kitasato Institute | Establishment of fluorescence-labeled myocarditis inducing cell strain and application thereof to clinical state elucidation of autoimmune myocarditis |
| WO2012094512A2 (en) * | 2011-01-05 | 2012-07-12 | The Cleveland Clinic Foundation | Myocardial tissue targeting peptides |
| US20190381195A1 (en) * | 2017-03-29 | 2019-12-19 | Shanghai Jiao Tong University | Conjugated polymer-based nanoprobe and preparation method and application thereof |
| CN111693495A (en) * | 2019-03-15 | 2020-09-22 | 复旦大学 | Near-infrared double-channel fluorescence living body microscopic imaging method |
| CN113214822A (en) * | 2021-04-12 | 2021-08-06 | 华南理工大学 | AIE nanofiber probe for targeted staining of cell membrane and preparation method thereof |
| US20210338842A1 (en) * | 2020-05-03 | 2021-11-04 | West China Hospital Of Sichuan University | Dual-modality nanoprobe targeting glioblastoma and preparation method thereof |
| CN116531344A (en) * | 2023-06-07 | 2023-08-04 | 重庆医科大学附属儿童医院 | Macrophage membrane modified nano-particle and preparation method and application thereof |
-
2023
- 2023-11-10 CN CN202311492674.3A patent/CN117538537B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011155949A (en) * | 2010-02-03 | 2011-08-18 | Kitasato Institute | Establishment of fluorescence-labeled myocarditis inducing cell strain and application thereof to clinical state elucidation of autoimmune myocarditis |
| WO2012094512A2 (en) * | 2011-01-05 | 2012-07-12 | The Cleveland Clinic Foundation | Myocardial tissue targeting peptides |
| US20190381195A1 (en) * | 2017-03-29 | 2019-12-19 | Shanghai Jiao Tong University | Conjugated polymer-based nanoprobe and preparation method and application thereof |
| CN111693495A (en) * | 2019-03-15 | 2020-09-22 | 复旦大学 | Near-infrared double-channel fluorescence living body microscopic imaging method |
| US20210338842A1 (en) * | 2020-05-03 | 2021-11-04 | West China Hospital Of Sichuan University | Dual-modality nanoprobe targeting glioblastoma and preparation method thereof |
| CN113214822A (en) * | 2021-04-12 | 2021-08-06 | 华南理工大学 | AIE nanofiber probe for targeted staining of cell membrane and preparation method thereof |
| CN116531344A (en) * | 2023-06-07 | 2023-08-04 | 重庆医科大学附属儿童医院 | Macrophage membrane modified nano-particle and preparation method and application thereof |
Non-Patent Citations (8)
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119074926A (en) * | 2024-08-29 | 2024-12-06 | 中国医学科学院阜外医院深圳医院(深圳市孙逸仙心血管医院) | Application of CAR-T cells targeting FAP in the preparation of drugs for treating cardiac fibrosis in chronic myocarditis |
| CN119074926B (en) * | 2024-08-29 | 2025-11-25 | 中国医学科学院阜外医院深圳医院(深圳市孙逸仙心血管医院) | Application of FAP-targeting CAR-T cells in the preparation of drugs for treating chronic myocarditis and cardiac fibrosis |
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