Nanoscale mitochondrial DNA scavenger and application thereof

Abstract

The invention relates to the technical field of polypeptide drugs, in particular to a nanoscale mitochondrial DNA scavenger and application thereof. The polypeptide targeting myocardial cells in the nanoscale mitochondrial DNA scavenger provided by the invention can target the myocardial cells, so that the STING inhibitor is enriched in the myocardial cells; the mtDNA probe can be combined with mtDNA in a targeted manner to form an NS-mtDNA compound; the self-assembly peptide is a convertible self-assembly unit, forms a nano structure, can transport an NS-mtDNA compound to lysosome to promote degradation, can prolong the retention time and stability of the nano-scale mitochondrial DNA scavenger and continuously release an STING inhibitor, the released STING inhibitor can inhibit an STING pathway, and the self-assembly peptide can be used for inhibiting the activity of the nano-scale mitochondrial DNA scavenger, so that the activity of the nano-scale mitochondrial DNA scavenger can be improved, and the activity of the nano-scale mitochondrial DNA scavenger is improved. Therefore, myocarditis or inflammations related to myocarditis are improved, and a new technical support is provided for treatment of myocarditis.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs, and in particular to a nanometer-level mitochondrial DNA scavenger and application thereof. Background Art

[0002] It is generally believed that mtDNA-induced inflammation is involved in a variety of inflammatory diseases. Existing literature shows that the clearance of mtDNA can regulate the inflammatory manifestations of autoimmune diseases, such as autoimmune encephalomyelitis and psoriasis. However, it has not been reported in chronic myocarditis, and existing drugs for the treatment of chronic myocarditis have problems such as large side effects and poor targeting. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a nano-scale mitochondrial DNA scavenger and its application. The nano-scale mitochondrial DNA scavenger provided by the present invention can not only be enriched in myocardial cells, with strong targeting, but also can target and bind to mtDNA, while releasing STING inhibitors to promote the targeted degradation of mtDNA, thereby improving myocarditis or inflammation related to myocarditis, and providing new technical support for the treatment of myocarditis.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a nano-scale mitochondrial DNA scavenger, comprising a nano-mtDNA target and a STING inhibitor; the nano-mtDNA target comprises a mtDNA probe, a self-assembling peptide and a polypeptide targeting myocardial cells coupled in sequence; the amino acid sequence of the self-assembling peptide is shown in SEQ ID NO.3.

[0006] Preferably, the amino acid sequence of the polypeptide targeting cardiomyocytes is as shown in SEQ ID NO.2.

[0007] Preferably, the STING inhibitor comprises C-176.

[0008] Preferably, the STING inhibitor is encapsulated in the nano-mtDNA targeter.

[0009] Preferably, the molar ratio of the STING inhibitor to the nano-mtDNA target is 1:2.

[0010] Preferably, the mtDNA probe comprises: one or more of SYBR Green I, superoxide dehydrogenase 2 antibody, any one of the amino acids shown in SEQ ID NO.4 to SEQ ID NO.24, or tail anchor protein antibody.

[0011] Preferably, the molar ratio of the mtDNA probe, the self-assembling peptide and the polypeptide targeting cardiomyocytes is 1:1:1.

[0012] The present invention provides a method for preparing the nano-scale mitochondrial DNA scavenger described in the above technical solution, comprising the following steps:

[0013] The nanometer mtDNA target and the STING inhibitor are mixed to obtain the nanometer-scale mitochondrial DNA remover.

[0014] The present invention provides the use of the nanoscale mitochondrial DNA scavenger described in the above technical solution or the nanoscale mitochondrial DNA scavenger prepared by the preparation method described in the above technical solution in the preparation of drugs for treating diseases, wherein the diseases are mtDNA-induced diseases.

[0015] Preferably, the disease comprises myocarditis or inflammation associated with myocarditis.

[0016] Beneficial effects:

[0017] The present invention provides a nano-scale mitochondrial DNA scavenger, including a nano-mtDNA target (NS) and a STING inhibitor; the nano-mtDNA target includes a sequentially coupled mtDNA probe, a self-assembling peptide and a polypeptide targeting myocardial cells; the amino acid sequence of the self-assembling peptide is shown in SEQ ID NO.3. The polypeptide targeting myocardial cells in the nano-scale mitochondrial DNA scavenger provided by the present invention can target myocardial cells to enrich the STING inhibitor in myocardial cells; the mtDNA probe can target and bind to mtDNA to form a NS-mtDNA complex; the self-assembling peptide is a convertible self-assembling unit, forming a nanostructure, which can transport the NS-mtDNA complex to the lysosome to promote degradation, and the self-assembling peptide can also prolong the retention time and stability of the nano-scale mitochondrial DNA scavenger, continuously release the STING inhibitor, and the released STING inhibitor can inhibit the STING pathway, thereby improving myocarditis or inflammation associated with myocarditis, and providing new technical support for the treatment of myocarditis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 The time-of-flight mass spectrometry results of NS and NP in Example 1;

[0020] Figure 2 is the HPLC chromatogram of NS and NP in Example 1;

[0021] Figure 3This is a molecular synthesis diagram of mtDNA nano-scavenger;

[0022] Figure 4 This is the fitting curve result of mtDNA nano-scavenger in plasma;

[0023] Figure 5 This is a diagram of the interaction between mtDNA nanoscavengers and cells;

[0024] Figure 6 The results of cytotoxicity experiments of NS and NP with AC16 cells and HUVEC cells, respectively;

[0025] Figure 7 This is the in vivo retention time diagram of the mtDNA nano-scavenger;

[0026] Figure 8 Results of hematoxylin and eosin staining of major organs after administration of NS or NP;

[0027] Fig. 9 Blood biochemical analysis results of PBS group, NS group and NP group;

[0028] Fig.10 This is a diagram showing the therapeutic effect of mtDNA nano-scavenger in vivo. DETAILED DESCRIPTION

[0029] The present invention provides a nano-scale mitochondrial DNA scavenger, comprising a nano-mtDNA target (NS) and a STING inhibitor; the nano-mtDNA target comprises a mtDNA probe, a self-assembling peptide and a polypeptide targeting myocardial cells coupled in sequence; the amino acid sequence of the self-assembling peptide is shown in SEQ ID NO.3, specifically as follows: FFVDF.

[0030] In the present invention, the amino acid sequence of the polypeptide targeting cardiomyocytes is preferably as shown in SEQ ID NO.2, specifically as follows: APWHLSSQYSRT; the STING inhibitor preferably includes C-176; the STING inhibitor is preferably encapsulated in the nano mtDNA target; the molar ratio of the STING inhibitor to the nano mtDNA target is preferably 1:2; the mtDNA probe preferably includes: SYBR Green I, superoxide dehydrogenase 2 antibody, any one of the amino acids shown in SEQ ID NO.4 to SEQ ID NO.24 or one or more of the tail anchor protein antibodies, more preferably SYBR Green I; the molar ratio of the mtDNA probe, the self-assembling peptide and the polypeptide targeting cardiomyocytes is preferably 1:1:1.

[0031] SEQ ID NO.4:GPZLSRLLSYAGC;

[0032] SEQ ID NO.5:GPOLSRLLSYAGC;

[0033] SEQ ID NO.6:GPBLSRLLSYAGC;

[0034] SEQ ID NO.7:GPRZSRLLSYAGC;

[0035] SEQ ID NO.8:GPROSRLLSYAGC;

[0036] SEQ ID NO.9:GPRBSRLLSYAGC;

[0037] SEQ ID NO.10:GPRLZRLLSYAGC;

[0038] SEQ ID NO.11:GPRLORLLSYAGC;

[0039] SEQ ID NO.12:GPRLBRLLSYAGC;

[0040] SEQ ID NO.13:GPRLSZLLSYAGC;

[0041] SEQ ID NO.14:GPRLSOLLSYAGC;

[0042] SEQ ID NO.15:GPRLSBLLSYAGC;

[0043] SEQ ID NO.16:GPRLSRZLSYAGC;

[0044] SEQ ID NO.17:GPRLSROLSYAGC;

[0045] SEQ ID NO.18:GPRLSRBLSYAGC;

[0046] SEQ ID NO.19:GPRLSRLZSYAGC;

[0047] SEQ ID NO.20:GPRLSRLOSYAGC;

[0048] SEQ ID NO.21:GPRLSRLBSYAGC;

[0049] SEQ ID NO.22:GPRLSRLLZYAGC;

[0050] SEQ ID NO.23:GPRLSRLLOYAGC;

[0051] SEQ ID NO.24: GPRLSRLLBYAGC;

[0052] Among them, the N-termini of the amino acid sequences shown in SEQ ID NO.4 to SEQ ID NO.24 are preferably modified with an X group, and the X group preferably includes CH3, HCO, CH3CO or C2H5CO; Z is A, V, I, L, M, F, Y or W; O is G, A, S, T, D, E, N or Q; and B is A, P, K, H or R.

[0053] The NS in the nanoscale mitochondrial DNA scavenger of the present invention creates a favorable biological environment for the sustained release of the STING inhibitor, and this process enhances the targeted accumulation of the STING inhibitor in the pathological site; at the same time, it promotes the targeted degradation of mtDNA, thereby improving autoimmune myocarditis (EAM) or inflammation associated with myocarditis, and provides a new solution for the treatment of myocarditis.

[0054] The present invention also provides a method for preparing the nano-scale mitochondrial DNA scavenger described in the above technical solution, comprising the following steps:

[0055] The nanometer mtDNA target and the STING inhibitor are mixed to obtain the nanometer-scale mitochondrial DNA remover.

[0056] The present invention also provides the use of the nano-scale mitochondrial DNA scavenger described in the above technical solution or the nano-scale mitochondrial DNA scavenger prepared by the preparation method described in the above technical solution in the preparation of a drug for treating a disease, wherein the disease is a mtDNA-induced disease. In the present invention, the disease preferably includes myocarditis or inflammation related to myocarditis; the myocarditis preferably includes autoimmune myocarditis.

[0057] In order to further illustrate the present invention, a nanometer-scale mitochondrial DNA remover and its application provided by the present invention are described in detail below in combination with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0058] Example 1

[0059] The NP peptide shown in SEQ ID NO.1 (FFVDFAPWHLSSQYSRT) was synthesized by standard Fmoc chemistry solid phase peptide synthesis, and wang resin was used as a solid phase carrier.

[0060] The NP peptide was coupled with SYBR Green I (denoted as SG, CAS No.: 163795-75-3) to obtain the SG-FFVDF-APWHLSSQYSRT peptide (denoted as NS, molecular structure as shown in Figure 3As shown in A), the method is as follows:

[0061] The NP peptide and SYBR Green I were mixed in a molar ratio of 1:1, dissolved in 3 mL of N,N-dimethylformamide (DMF) under vacuum conditions, and reacted at 80°C for 5 hours; the reaction was monitored by liquid chromatography-mass spectrometry (LC-MS), the heating was turned off, and the mixture was cooled to room temperature; then the solvent was evaporated, and the NS was obtained after purification by reverse phase preparative liquid chromatography.

[0062] NS (50 μM) and NP (50 μM) were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (ESI-MS, Brook Dalton, USA). Figure 1 , where A is the time-of-flight mass spectrometry result of NS, and B is the time-of-flight mass spectrometry result of NP.

[0063] NS (50 μM) and NP (50 μM) were characterized by high performance liquid chromatography (HPLC). Figure 2 , wherein A is the HPLC chromatogram of NS, and B is the HPLC chromatogram of NP.

[0064] Depend on Figure 1 and Figure 2 It can be seen that both NS and NP peptides were successfully synthesized.

[0065] Test Example 1

[0066] Fluorescence analysis of mtDNA

[0067] mtDNA was extracted by mitochondrial DNA isolation kit (abcam, ab65321). 20 μM NS (prepared in Example 1) was incubated in PBS (10.0 mM, pH = 7.5) containing different concentrations of mtDNA for 0.5 h. The concentrations of mtDNA were 0, 5 μg / mL, 10 μg / mL, 30 μg / mL, and 60 μg / mL, respectively. The fluorescence response of NS to mtDNA was analyzed at an excitation wavelength of 490 nm. The results are shown in Table 1. Figure 3 B. The results show that in the absence of mtDNA, the green fluorescence of NS is zero, but after incubation with mtDNA, the green fluorescence of NS reaches a peak at 525nm, indicating that NS has a strong mtDNA targeting ability.

[0068] Stability of NS in human plasma

[0069] The enzymatic stability of the NS prepared in Example 1 was evaluated using the human plasma degradation method. NS was dissolved in 1 mL of human plasma at 37°C to a concentration of 1 mg / mL; the plasma came from family members of patients at the Fuwai Hospital of the Chinese Academy of Medical Sciences. At different time points (0, 6h, 12h, 24h, 48h), 40 μL of stop solution was added, which consisted of equal volumes of trifluoroacetic acid (TFA) and H2O. Take 20 μL of the supernatant and centrifuge (13000 rpm, 10 min) for HPLC and MS analysis. According to the fitting curve (see Figure 4 ) to determine the half-life of NP in human plasma. Figure 3 C in. From the results, we can see that the half-life of NS is about 10.3h.

[0070] Analysis of NS self-assembled nanostructures

[0071] Schematic diagram of NS self-assembly and fibril conversion of deformable peptide monomers is shown in Figure 3 D in.

[0072] A schematic diagram of the interaction region indicator (IRI) of the NS dimer is shown in Figure 3 E in; different colors are used to represent different interactions, red: steric effect, green: van der Waals interaction, cyan: π-π stacking and hydrogen bonding; IRI data were calculated by Multiwfn 3.8 (dev) program based on the calculation results of ORCA5.0.3 program (B3LYP / 6-31g), and the picture was drawn by VMD 1.9.3 program based on the data.

[0073] NS characterization

[0074] The NS in Example 1 was resuspended to a concentration of 50 μM using PBS solution to prepare a NS solution. The NS solution was placed for 0, 4, 12 and 24 hours, respectively, and then measured. The measurement method was as follows: the DLS hydrodynamic particle size measurement method of the American ZetasizerNano ZS company was used to test the NS solution placed for different times, and the morphological changes were observed under a transmission electron microscope (TEM): the NS solution was drop-coated on a carbon-coated copper grid; after the droplet was in contact with the copper grid for 5 minutes, the excess droplet was removed with filter paper; the transmission electron microscope sample was then stained with uranyl acetate for 1 minute; before observation under a transmission electron microscope, all samples were dried under vacuum. The TEM images of the initial NS converted into nanofibers at different placement time points (0, 4h, 12h and 24h) were observed, and the results are shown in FIG. Figure 3 G in Figure 2, the scale bar is 200 nm. The changes in the initial NS size distribution at different time points are shown in Figure 3 F in.

[0075] The results show that at 0 h, the diameter of NS was about 10 nm, and then gradually increased to 20 nm. A nanofiber structure with a diameter of about 9 nm was observed, the number of nanofibers increased within 4 h, and the nanofiber network was clearly visible after 24 h with a wide size distribution. DLS measurements showed the distribution size of NS aggregates, confirming the structural transition from nanoparticles to nanofibers. These results indicate that NS has a stable nanostructure.

[0076] Test Example 2

[0077] Confocal laser scanning microscopy (CLSM) was used to observe the interaction between NS and cells.

[0078] To prepare Cy-5 labeled NS, the steps are as follows:

[0079] 1) Dissolve 1.0 mg of Cy5-NHS ester in 400 μL of DMSO; 2) Add the dissolved Cy5-NHS ester to the solution containing the NS to be labeled at a volume ratio of 1:1; In order to promote the reaction, add a small amount of triethylamine to the mixture to adjust the pH value and promote the hydrolysis of NHS ester and subsequent amine reaction; 3) Stir the mixture overnight at room temperature in the dark to allow the Cy5-NHS ester to react with the amino group of the polypeptide to form a stable covalent bond.

[0080] HUVEC cells and AC16 cells were cultured in complete DMEM medium at 37°C overnight in a humidified atmosphere containing 5% CO2; 50 μL of Cy-5 labeled NS (200 μM) was added. After different culture times, the cells were washed three times with PBS. The culture medium was then replaced with PBS, and the cells were imaged using a Zeiss LSM710 confocal laser scanning microscope (CLSM) in Jena, Germany with a 60x objective. All microscopy parameters were kept consistent across different samples to compare the binding capacity of NS. The fluorescence distribution images of NS interacting with AC16 cells and HUVECs within 30 minutes are shown in Figure 5 In A, the white curve represents the cell outline; Figure 5 The B in the figure is quantized Figure 5 Fluorescence intensity on the cell membrane of A, ***P<0.001; Figure 5 C in Figure 5 The fluorescence intensity curve results at the white curve position in A.

[0081] Confocal laser scanning microscopy (CLSM) observed that the surface of cardiomyocytes treated with NS had a strong red fluorescence signal. Compared with endothelial cells, most of the fluorescence signals stayed in endothelial cells. It can be seen that the NS provided by the present invention can specifically bind to cardiomyocytes.

[0082] Observation of the external morphology of cardiomyocytes using scanning electron microscopy (SEM)

[0083] AC16 cells with good morphological integrity were selected and incubated with 1×10 6 AC16 cells for 1 hour, targeting the cell surface. The cells were then removed from the wells, washed with PBS, and fixed in a 4% paraformaldehyde solution for 4 hours at room temperature. The cells were then dehydrated stepwise in an ethanol series from 30%, 50%, 70%, 90%, and 100%, and then critical point dried with isoamyl acetate. Similarly, after perfusion, the cells were excised, cut along the longest axis, fixed for 24 hours, and dehydrated with gradient ethanol. Finally, the cells and NS were observed using a scanning electron microscope (Hitachi S4800+EDS), and the results are shown in Figure 5 D in the figure, where untreated refers to AC16 cells that were not incubated with NS. The results show that nanoparticles exist on the surface of AC16 cells treated with NS; in contrast, no nanoparticle structure was detected on the surface of untreated cells. These experimental results indicate that NS can specifically bind to cardiomyocytes.

[0084] Cytotoxicity test

[0085] AC16 cells were plated at 1×10 4 The cells were seeded in 96-well plates at a density of 10 cells and allowed to adhere overnight. The cells were then exposed to different concentrations (0, 2, 5 and 10 μg / mL) of NS for 24 h. Cells that did not receive any treatment were used as controls. After the treatment, the culture medium was replaced with fresh complete culture medium containing CCK-8 reagent and incubated at 37 ° C for 2 h. The absorbance at 490 nm was then measured using a Tecan plate reader (Switzerland). Figure 5 E in the figure shows the interaction between different concentrations of NS and AC16 cells; the scale bar represents 10 μm, and the experiment was repeated three times in three parallel experiments; Figure 5 F in the figure is the quantitative fluorescence intensity of AC16 cells treated with different concentrations of NS for 24 hours. The results show that NS can target mtDNA.

[0086] The effects of different concentrations of NS and NP on the survival rate of cardiomyocytes (AC16) and endothelial cells (HUVEC) were determined as follows:

[0087] 1. Preparation of cell suspension: For suspension cells, directly collect cells in the logarithmic growth phase by centrifugation; for adherent cells, digest them with trypsin and collect them by centrifugation, resuspend them in serum-containing culture medium, and dilute them to a single cell suspension of appropriate concentration.

[0088] 2. Cell inoculation: Inoculate the cell suspension in a 96-well plate, 100 μl per well, set up different concentration groups, and design 3-6 replicate wells for each group, and set up a blank group and a control group.

[0089] 3. Pre-culture: Place the culture plate in an incubator for pre-culture for about 24 hours (37°C, 5% CO2).

[0090] 4. Drug treatment: Add different concentrations (12.5, 25, 50, 100, 200 μM) of NS or NP to each well of the culture plate and continue culturing for 6-96 hours.

[0091] 5. Add CCK-8 reagent: Add 10 μl CCK-8 solution to each well and gently shake the culture plate to help mix and avoid bubbles.

[0092] 6. Incubation: Place the culture plate back into the incubator and incubate for 1 to 4 hours.

[0093] 7. Absorbance determination: Use an ELISA reader to measure the absorbance (OD value) of each well at 450 nm.

[0094] 8. Data analysis: Calculate the cell inhibition rate or cell proliferation rate based on the measured OD value.

[0095] Results Figure 6 The results showed that the viability of cardiomyocytes (AC16) and endothelial cells (HUVEC) at 200 μM concentration was over 85%, as determined by CCK-8.

[0096] Test Example 3

[0097] The cardiac targeting of NS and NP was investigated using a mouse model (n=3), which provides a realistic simulation of the heart under a dynamic fluid environment. The experimental schematic is shown in Figure 7 A in the method is as follows:

[0098] NS and NP labeled with Cy-7 (labeling method refers to test example 2) were intravenously injected into BALB / c mice, and PBS was injected as the control group. The injection concentration of the NS and NP was 200 μM, and the injection volume was 150 μL / mouse; after 2 hours, the high fluorescence signal in the heart area was monitored, and the fluorescence signal was captured using an in vivo imaging system (IVIS, PerkinElmer). Then, the biological distribution of NS and NP in mice was observed, and the heart and various organs, including liver, spleen, lungs and kidneys, were removed and in vitro fluorescence analysis was performed using the same imaging system. The results are shown in Figure 7 , where B is a representative in vivo fluorescence image of mice 2 hours after tail vein injection of Cy-7-labeled NS and NP, and the black circle represents the position of the heart; C is a representative in vitro fluorescence image at different time points after injection; D is the average fluorescence intensity of different organs in C; E is a representative in vitro fluorescence image of the heart at different time points after injection.

[0099] The results showed that NS was mainly distributed in the heart, liver, and kidney, followed by the lung. This is mainly because the liver and kidney are the main metabolic organs, while the distribution in the lung may be caused by the cardiopulmonary circulation. The high fluorescence signal of the heart in the NS-treated group began to weaken at a slower rate from 2 hours after intravenous injection, showing better cardiac clearance resistance. Then a clear fluorescence signal could still be observed within 12 hours, while a weak fluorescence signal was visible in mice injected with NS intravenously after 24 hours. There was no visible fluorescence signal in mice injected with NS intravenously after 72 hours, while there was no fluorescence signal in the PBS group. The fluorescence quantitative intensity of the main organs was consistent with the experimental results of the NP group, indicating that the introduction of the mtDNA targeting unit SG had no significant effect on the cardiac targeting ability of NS.

[0100] Serum and major organs were obtained from mice in the PBS, NS, and NP groups to evaluate potential in vivo side effects. Hematoxylin and eosin staining of major organs after administration of NS or NP is shown in Figure 8 , scale bar is 20μm. The results showed that no obvious changes were found through pathological examination.

[0101] The blood biochemical analysis of PBS group, NS group and NP group was performed using biochemical tester. Fig. 9 . The results showed that there were no abnormal fluctuations in liver function markers including ALT, AST, ALB and GLB, indicating that NS neither caused liver cell damage nor interfered with protein synthesis. Compared with PBS-treated mice, there was no significant deviation in the kidney function marker (BUN) of NS-treated mice, indicating that NS and NP were not nephrotoxic. The results of blood routine examinations of WBC, Neu, Lym, Mon, RBC, HGB and PLT showed no significant differences among the three groups, thus verifying the biocompatibility and biosafety of NS and NP.

[0102] Test Example 4

[0103] This invention was approved by the Animal Ethics Committee of Fuwai Hospital (No. FW-2022-0015). All procedures complied with the guidelines of Directive 2010 / 63 / EU of the European Parliament on the protection of animals used for scientific purposes.

[0104] Construction and processing of EAM models

[0105] C-176 was dissolved in DMSO to obtain a C-176 solution; the NS (SG-FFVDF-CTP) prepared in Example 1 was mixed with the C-176 solution and kept at room temperature overnight to obtain a mtDNA nanoscavenger encapsulating C-176 (denoted as C-176@SG-FFVDF-CTP); the molar ratio of C-176 to NS was 1:2. The preparation method of C-176@FFVDF-CTP was the same as above, except that NS was replaced with NP (FFVDF-CTP) prepared in Example 1.

[0106] Six-week-old male BALB / c mice were obtained from Vital River Laboratories, Beijing, China, and were housed in a specific pathogen-free facility and given normal diet and water. α-MyHC peptide (AnaSpec, AS-62554) was mixed with complete Freund's adjuvant (Sigma, F5881) at a mass ratio of 1:1 to obtain the sensitizer. After 1 week of adaptive feeding, the sensitizer was subcutaneously injected on days 0 and 7 (the injection amount of α-MyHC peptide was 250 μg / mouse). After the mice were sensitized, EAM model mice were obtained.

[0107] The EAM model mice were randomly divided into 5 groups, 5 mice in each group, namely EAM group (no treatment), NS group (injection of C-176@SG-FFVDF-CTP), C-176 group (injection of C-176), NP1 group (injection of C-176@FFVDF-CTP), NP2 group (injection of SG-FFVDF-CTP) and PBS group (injection of PBS). The 5 groups of mice were injected with the corresponding reagents through the tail vein every 2 days. The injection concentration was 200μM and the injection volume was 150μL / mouse for 1 week. The hearts of mice in different groups were photographed, H&E stained and histologically analyzed, and the inflammation scores were summarized and the percentage of mtDNA+ cells was calculated. The standards for inflammation scores are shown in Table 1. Fig.10 , where A is a digital photo and a representative image of a mouse heart stained with H&E, scale bar: 500 μm, the scale bar of the enlarged image is 20 μm, and the immunohistochemical staining shows mtDNA+ cells, scale bar: 50 μm; B is the summary inflammation score of A, and C is the percentage of mtDNA+ cells. ***P<0.001.

[0108] Table 1 Standards for inflammation scoring

[0109] score Evaluate 0 No inflammation 1 1-5 areas of prominent mononuclear inflammation involving 5% or less of the cross-sectional area of ​​the heart 2 More than 5 areas of marked mononuclear inflammation or involving more than 5% but not more than 20% of the cardiac cross-sectional area 3 Prominent mononuclear infiltration involving more than 20% of the area, without necrosis 4 Diffuse inflammation of the heart with necrosis

[0110] After one week of treatment with NS, NP1, NP2, and C-176, histological analysis showed that mtDNA clearance and C-176 reduced the accumulation of leukocytes and alleviated inflammation in the hearts of EAM mice. The NS group showed good therapeutic effects, even the same as the results of the PBS group. Next, the NP1 group, then the C-176 group, and finally the NP2 group had the worst effect. This is attributed to the targeted delivery of NS to the heart, where it penetrates the cells and binds to mtDNA. The nanostructure of NS facilitates the transport of mtDNA-NS complexes to lysosomes for degradation, effectively clearing mtDNA. In addition, the deformation of ligand-receptor interactions within NS leads to the effective release of C-176, which increases the concentration and residence time of the drug in the heart. Therefore, a synergistic therapeutic approach was successfully achieved in the mouse myocarditis model.

[0111] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nanoscale mitochondrial DNA remover, characterized in that: It comprises a nanometer mtDNA target and a STING inhibitor; the nanometer mtDNA target comprises a mtDNA probe, a self-assembling peptide and a polypeptide targeting myocardial cells coupled in sequence; the amino acid sequence of the self-assembling peptide is shown in SEQ ID NO.

3.

2. The nanoscale mitochondrial DNA remover according to claim 1, characterized in that: The amino acid sequence of the polypeptide targeting myocardial cells is shown in SEQ ID NO.

2.

3. The nanoscale mitochondrial DNA remover according to claim 1, characterized in that: The STING inhibitors include C-176.

4. The nanoscale mitochondrial DNA remover according to claim 1 or 3, characterized in that: The STING inhibitor is encapsulated in the nano-mtDNA targeter.

5. The nanoscale mitochondrial DNA remover according to claim 1 or 3, characterized in that: The molar ratio of the STING inhibitor to the nano mtDNA target is 1:

2.

6. The nanoscale mitochondrial DNA remover according to claim 1, characterized in that: The mtDNA probe comprises: SYBR Green I, superoxide dehydrogenase 2 antibody, any one of amino acids shown in SEQ ID NO.4 to SEQ ID NO.24 or one or more of tail anchor protein antibodies.

7. The nanoscale mitochondrial DNA remover according to claim 1, characterized in that: The molar ratio of the mtDNA probe, the self-assembling peptide and the polypeptide targeting myocardial cells is 1:1:

1.

8. The method for preparing the nano-scale mitochondrial DNA remover according to any one of claims 1 to 7, characterized in that: The following steps are involved: The nanometer mtDNA target and the STING inhibitor are mixed to obtain the nanometer-scale mitochondrial DNA remover.

9. Use of the nanoscale mitochondrial DNA scavenger according to any one of claims 1 to 7 or the nanoscale mitochondrial DNA scavenger prepared by the preparation method according to claim 8 in the preparation of drugs for treating diseases, wherein the diseases are mtDNA-induced diseases.

10. The use according to claim 9, characterized in that: The disease includes myocarditis or inflammation associated with myocarditis.

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