Carbon nanotube complex loaded with gs alpha inhibitory cyclic peptide, and preparation method and application thereof

Abstract

The present application relates to a kind of carbon nanotube complex of Gs alpha inhibitory cyclic peptide and preparation method and application, belong to the field of biological medicine.The carbon nanotube complex of Gs alpha inhibitory cyclic peptide is with polyethylene glycol single-walled carbon nanotube as nano skeleton, Gs alpha inhibitory cyclic peptide cpGN13 is loaded on nano skeleton.The present application also provides the imaging type carbon nanotube complex of Gs alpha inhibitory cyclic peptide that near infrared fluorescent marker Cy5.5 is connected on carbon nanotube complex of Gs alpha inhibitory cyclic peptide, and the preparation method of the complex.The carbon nanotube complex of Gs alpha inhibitory cyclic peptide of the present application can preferentially recognize GTP binding active state Gs alpha, and inhibit downstream cAMP signal transduction by interfering the interaction of active Gs alpha and adenylate cyclase, to improve the state selectivity of Gs alpha intervention, realize the active state selective inhibition of Gs alpha.

Description

Technical Field

[0001] This invention relates to a carbon nanotube complex loaded with a Gsα-inhibiting cyclic peptide, its preparation method, and its application. More specifically, it relates to a single-walled carbon nanotube complex loaded with an active, selective Gsα-inhibiting cyclic peptide, its preparation method, and its application in the preparation of drugs for treating atherosclerosis and imaging agents for atherosclerotic lesions, belonging to the field of biomedical technology. Background Technology

[0002] Atherosclerosis is a crucial pathological basis for myocardial infarction, ischemic stroke, and peripheral artery disease. Macrophage uptake of oxidized low-density lipoprotein (oxLDL) and the formation of foam cells are hallmark events in plaque formation and progression. Current lipid-lowering, antithrombotic, and anti-inflammatory therapies can reduce overall cardiovascular risk, but they typically do not directly correct the abnormal oxLDL uptake process within plaque macrophages.

[0003] Previous studies have shown that oxLDL can activate macrophage Gsα and upregulate scavenger receptors CD36 and SR-A1 through the cAMP-PKA-CREB pathway, promoting oxLDL uptake, intracellular lipid accumulation, and plaque development. Therefore, active Gsα is an upstream target for inhibiting foam cell formation. However, Gsα is widely expressed and participates in the physiological functions of multiple systems, including the endocrine, renal, skeletal, vascular, and smooth muscle systems. Smooth muscle-specific Gsα deficiency can cause adverse phenotypes such as severe gastrointestinal motility disorders, suggesting a potential safety risk associated with systemic, widespread, and tissue-insensitive inhibition.

[0004] Dai's article discloses the Gsα-inhibiting cyclic peptide GN13, which preferentially recognizes GTP and binds to active Gsα, as well as the cell-entrant analog cyclic peptide cpGN13. The cyclic peptide cpGN13 primarily targets the Gsα switch II / α3 pocket, inhibiting the interaction between Gsα and adenylate cyclase, thereby reducing cAMP signaling. However, the preparation and application of cyclic peptide molecules may still be limited by factors such as cyclization efficiency, conformational heterogeneity, and formulation stability. Furthermore, existing cpGN13 is mainly used in the form of a free cyclic peptide, lacking spatial selectivity for atherosclerotic plaques and plaque macrophages, making it difficult to achieve effective enrichment and precise delivery within lesions, and it lacks lesion tracing or imaging capabilities. Therefore, its further application in the diagnosis and treatment of atherosclerosis remains limited.

[0005] Previous research on nanomedicines has shown that circulating nanomaterials can accumulate in plaques due to plaque vascular permeability, local retention, and phagocytic uptake. For example, nanoparticles that promote cell burial can be taken up by macrophages in lesions and alleviate atherosclerosis. Single-walled carbon nanotubes (SWCNTs) have also been used for photoacoustic imaging of inflammatory plaques or pH-dependent drug release. SWCNTs have characteristics such as high aspect ratio, tunable surface chemistry, near-infrared absorption, and endogenous photoacoustic contrast, but their biodispersibility, reticuloendothelial system enrichment, and long-term safety need to be optimized through surface modification and dosage control.

[0006] Therefore, there is an urgent need in the field to develop a Gsα-inhibiting nanocomposite with stable loading, high delivery efficiency, improved stability of cyclic peptide formulations, and both active state selectivity and plaque macrophage spatial selectivity, so as to leverage the function of cpGN13 in the treatment of atherosclerosis and lesion imaging. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a carbon nanotube composite loaded with Gsα-inhibitory cyclic peptides, its preparation method, and its application.

[0008] The technical solution of this invention is as follows:

[0009] A carbon nanotube complex loaded with a Gsα-inhibiting cyclic peptide is described, which uses polyethylene glycolated single-walled carbon nanotubes as a nanoframework and loads the Gsα-inhibiting cyclic peptide cpGN13 onto the nanoframework.

[0010] According to a preferred embodiment of the present invention, the polyethylene glycol-modified single-walled carbon nanotubes are prepared by the following method:

[0011] Single-walled carbon nanotubes were added to an aqueous medium and sonicated for 5 to 15 minutes under a 100-150W probe. Then, DSPE-PEG5000-amine solution was added and sonicated for another 1 to 2 hours. After centrifugation, the supernatant was collected to obtain polyethylene glycol-modified single-walled carbon nanotubes.

[0012] The single-walled carbon nanotubes were added at a concentration of 0.8–1.2 mg; the aqueous medium was sterile RPMI-1640 medium, and the volume was 1–2 mL; the concentration of the DSPE-PEG5000-amine solution was 3.5–4.5 mg / mL, and the volume added was 1.5–2.5 mL; the centrifugation conditions were 18,000–20,000 rpm for 4–5 h.

[0013] More preferably, the amount of single-walled carbon nanotubes added is 1.0 mg; the amount of sterile RPMI-1640 culture medium used is 2 mL; the power of the ultrasonic treatment under the probe is 120 W, and the pre-dispersion time is 10 min; the concentration of the DSPE-PEG5000-amine solution is 4.0 mg / mL, and the amount added is 2.0 mL; the duration of the continued ultrasonic treatment is 1.5 h, and the centrifugation conditions are 20000 rpm for 4 h.

[0014] According to a preferred embodiment of the present invention, the amino acid sequence of the Gsα-inhibiting cyclic peptide cpGN13 is: (D-Tyr)-FQSVYAIWGTLC-GSGSGS-RRRRRRRR, as shown in SEQ ID NO.1.

[0015] The preparation method of the above-mentioned carbon nanotube complex loaded with Gsα-inhibitory cyclic peptides includes the following steps:

[0016] The cpGN13 linear peptide precursor was added to the polyethylene glycolated single-walled carbon nanotube dispersion and mixed thoroughly. Acetonitrile and water were then added to form a reaction system. NH4HCO3 was added to the reaction system while stirring to adjust the pH to 7.8–8.2, allowing the cpGN13 linear peptide precursor to undergo in-situ cyclization in the presence of polyethylene glycolated single-walled carbon nanotubes for 12–16 hours. After purification by centrifugation and ultrafiltration, SWCNT-cpGN13 was obtained.

[0017] According to a preferred embodiment of the present invention, in the reaction system, the acetonitrile volume fraction is 15-25% (v / v), and the concentration of the cpGN13 linear peptide precursor is 1.5-2.5 mg / mL.

[0018] More preferably, the acetonitrile volume fraction in the reaction system is 20% (v / v), and the concentration of the cpGN13 linear peptide precursor is 2 mg / mL.

[0019] The above-mentioned carbon nanotube complex loaded with Gsα-inhibiting cyclic peptides is used in the preparation of drugs for treating atherosclerosis.

[0020] An imaging-enabled carbon nanotube complex loaded with Gsα-inhibitory cyclic peptides consists of a carbon nanotube complex loaded with Gsα-inhibitory cyclic peptides and a near-infrared fluorescent label Cy5.5 attached to it.

[0021] The preparation method of the above-mentioned imaging-type carbon nanotube complex loaded with Gsα-inhibitory cyclic peptides includes the following steps:

[0022] Prepare a carbon nanotube complex solution loaded with Gsα-inhibiting cyclic peptides;

[0023] Cy5.5-NHS ester was added to a carbon nanotube complex solution loaded with Gsα-inhibiting cyclic peptide at pH 7.0-7.5, and the reaction was carried out at 25-30℃ for 1.5-2.5 h. After centrifugation and filtration, an imaging-type carbon nanotube complex loaded with Gsα-inhibiting cyclic peptide (SWCNT-cpGN13-Cy5.5) was obtained.

[0024] According to a preferred embodiment of the present invention, the molar ratio of the Cy5.5-NHS ester to the carbon nanotube complex loaded with Gsα-inhibiting cyclic peptide is (8~12):1.

[0025] The above-mentioned imaging-type carbon nanotube complex loaded with Gsα-inhibitory cyclic peptides is used in the preparation of drugs for treating atherosclerosis or imaging formulations for atherosclerotic lesions.

[0026] Technical features and beneficial effects of the present invention:

[0027] 1. This invention involves in-situ cyclization and loading of the cpGN13 linear peptide precursor into a polyethylene glycol-modified single-walled carbon nanotube system to obtain a carbon nanotube complex loaded with a Gsα-inhibiting cyclic peptide. The resulting carbon nanotube complex loaded with the Gsα-inhibiting cyclic peptide preferentially recognizes GTP-bound active Gsα and inhibits downstream cAMP signaling by interfering with the interaction between active Gsα and adenylate cyclase, rather than indiscriminately inhibiting Gsα in different conformational states. This improves the state selectivity of Gsα intervention, achieving state-selective inhibition of Gsα.

[0028] 2. The carbon nanotube complex loaded with Gsα inhibitory cyclic peptide provided in this invention uses polyethylene glycolated single-walled carbon nanotubes as a carrier. After intravenous administration, polyethylene glycolated single-walled carbon nanotubes can preferentially accumulate in atherosclerotic lesions and be taken up by macrophages and foam cells within the plaque. This further extends the molecular state selectivity of free cpGN13 to spatial selectivity, improving the relative accumulation and effective local exposure of cpGN13 in atherosclerotic lesions and macrophages in the lesions, thus exhibiting good spatial selectivity for atherosclerotic lesions.

[0029] 3. Unlike simply mixing pre-prepared cyclic peptides with an inert carrier, this invention utilizes polyethylene glycol-modified single-walled carbon nanotubes (SWCNTs) as a nanoframework, providing a reaction and assembly interface for the in-situ cyclization, stable loading, and surface presentation of the cpGN13 linear precursor. Simultaneously, it functions as a carrier for cyclic peptide construction, loading, presentation, and imaging. Furthermore, the imaging-type carbon nanotube complex with Gsα-inhibiting cyclic peptides provided by this invention also utilizes the near-infrared absorption and photoacoustic properties of SWCNTs, combined with Cy5.5 near-infrared fluorescence labeling, to achieve lesion enrichment tracking and photoacoustic / fluorescence imaging of the nanocomplex, thereby integrating drug delivery and lesion imaging onto the same nanoplatform.

[0030] 4. The carbon nanotube complex loaded with Gsα-inhibiting cyclic peptide provided in this invention inhibits macrophage active Gsα and its downstream cAMP-related signals, reduces oxLDL uptake and intracellular lipid accumulation, and directly intervenes in the upstream signaling pathway of macrophage foaming, thereby inhibiting foam cell formation and the progression of atherosclerotic plaques. In animal experiments, the therapeutic effect occurred without significant changes in plasma total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), indicating that its anti-atherosclerotic effect is independent of systemic lipid-lowering.

[0031] 5. In vitro experiments showed that the carbon nanotube complex loaded with Gsα-inhibiting cyclic peptides provided in this invention can reduce oxLDL-induced Gsα activation and foam cell formation; in Ldlr - / - In a mouse model of atherosclerosis, it can reduce plaque burden and lipid accumulation in lesions. Single-cell RNA sequencing further showed that the proportion of lipid-related macrophages decreased after treatment, and lipid metabolism and foaming-related transcriptional programs were inhibited. The therapeutic effect was verified from different levels such as cellular function, overall pathology and molecular transcriptional status.

[0032] 6. Under the dosage and administration conditions adopted in this invention, no obvious hematological abnormalities were found during the 28-day observation period after using the carbon nanotube complex loaded with Gsα-inhibiting cyclic peptide. No obvious abnormalities were found in liver and kidney function indicators such as ALT, AST, blood urea nitrogen and creatinine. No obvious histological damage was found in major tissues such as heart, blood vessels, lungs, liver, spleen, pancreas and kidneys. This indicates that the nanocomposite has good short-term in vivo tolerance under the tested conditions. Attached Figure Description

[0033] Figure 1 The preparation and characterization results of SWCNT-cpGN13-Cy5.5;

[0034] In the figure, A is a schematic diagram of the preparation process of SWCNT-cpGN13-Cy5.5; B is a transmission electron microscope (TEM) image of polyethylene glycol-modified single-walled carbon nanotubes; C is the Raman signal imaging result and local magnification result of SWCNT; D is the Raman spectrum result of SWCNT; E is the Raman signal reconstruction result after macrophages take up SWCNT; F ​​is the atomic force microscope (AFM) image of SWCNT-cpGN13-Cy5.5; G is the AFM height contour analysis result of SWCNT-cpGN13-Cy5.5; H is the AFM three-dimensional morphology reconstruction result of SWCNT-cpGN13-Cy5.5.

[0035] Figure 2It exhibits preferential uptake by macrophages of SWCNT-cpGN13-Cy5.5 and inhibits foam cell formation;

[0036] In the figure, A shows the flow cytometry analysis results of SWCNT-Cy5.5 uptake by macrophages, endothelial cells, and smooth muscle cells; B shows the quantitative results of the proportion of Cy5.5-positive cells in different cell types; C shows the flow cytometry analysis results of SWCNT-Cy5.5 and SWCNT-cpGN13-Cy5.5 uptake by foam cells; D shows the quantitative results of the proportion of Cy5.5-positive cells in foam cells; E shows the immunofluorescence staining results of GTP-bound active Gsα (Gsα-GTP); F shows the quantitative results of Gsα-GTP signal; G shows the results of Oil Red O staining in foam cells; H shows the quantitative results of Oil Red O-positive lipid accumulation; I shows the Western blot detection results of CD36 and SR-A1 protein expression; J shows the quantitative results of CD36 and SR-A1 protein expression.

[0037] Figure 3 In vivo distribution and photoacoustic imaging of SWCNT-cpGN13-Cy5.5 in atherosclerotic mice;

[0038] In the figure, A shows the in vitro fluorescence imaging results of the aorta at different time points; B shows the in vitro fluorescence imaging results of the liver at different time points; C shows the in vitro fluorescence imaging results of the spleen and pancreas at different time points; D shows the in vitro fluorescence imaging results of the heart at different time points; E shows the in vitro fluorescence imaging results of the lungs at different time points; F shows the ultrasound and photoacoustic imaging results of the carotid artery region at different time points; and G shows the quantitative results of photoacoustic signals of atherosclerotic lesions.

[0039] Figure 4 The therapeutic effect of SWCNT-cpGN13-Cy5.5 on atherosclerosis;

[0040] In the diagram, A represents Ldlr. - / - A schematic diagram of the drug administration process in a mouse atherosclerosis model; B shows the results of mouse body weight, systolic blood pressure, and diastolic blood pressure measurements in each group; C shows the results of plasma total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol measurements in each group; D shows the staining results of whole aortic plaques in each group; E shows the quantitative results of whole aortic plaque area in each group; F shows the H&E staining and Oil Red O staining results of the aortic root in each group; G shows the quantitative results of aortic root plaque area and lipid accumulation area in each group.

[0041] Figure 5To reveal changes in plaque macrophage status after SWCNT-cpGN13-Cy5.5 treatment for atherosclerosis using single-cell transcriptomics;

[0042] In the figure, A is a schematic diagram of the single-cell RNA sequencing experiment and analysis process of aortic tissue; B is the result of the overall single-cell cluster analysis of aortic cells; C is the result of the analysis of the composition ratio of different cell types in aorta; D is the result of the cluster analysis of macrophage subsets; E is the result of the analysis of the composition ratio of different macrophage subsets; F is the result of the GO functional enrichment analysis of differentially expressed genes in lipid-related macrophages; and G is the result of the pseudo-temporal trajectory analysis of macrophage subsets.

[0043] Figure 6 This study aims to evaluate the short-term safety of SWCNT-cpGN13-Cy5.5 28 days after a single intravenous administration.

[0044] In the figure, A represents the results of hematological and biochemical index analysis; B represents the H&E staining results of major organs; and C represents the immunofluorescence results of tissue structure and cell markers. Detailed Implementation

[0045] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art.

[0046] Single-walled carbon nanotubes (SWCNTs) with diameters of 0.8–1.2 nm are available from Unidym.

[0047] DSPE-PEG5000-amine is available from GlpBio under product number GC69020.

[0048] The cpGN13 linear peptide precursor was custom-synthesized by DgPeptides Co., Ltd. (Hangzhou, China).

[0049] Male Ldlr with C57BL / 6 background - / - Mice are available from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0050] Unless otherwise specified, all raw materials and equipment are standard materials and can be purchased commercially.

[0051] The methods for obtaining and culturing mouse bone marrow-derived macrophages (BMDMs), mouse vascular smooth muscle cells (mVSMCs), and human umbilical vein endothelial cells (HUVECs) in this embodiment are as follows:

[0052] Mouse bone marrow cells were seeded in DMEM medium containing 10% fetal bovine serum and 15% L929 cell conditioned medium and cultured for 7 days in a 37°C, 5% CO2 saturated humidity incubator to differentiate them into mouse bone marrow-derived macrophages (BMDMs).

[0053] Mouse vascular smooth muscle cells (mVSMCs) were isolated from the mouse aorta and cultured in DMEM medium containing 10% fetal bovine serum.

[0054] Human umbilical vein endothelial cells (HUVECs) were cultured in Endothelial Cell Growth Medium-2 medium supplemented with 1% penicillin / streptomycin. All cells were cultured in a 37°C, 5% CO2 saturated humidity incubator. Before the experiment, BMDMs, mVSMCs, and HUVECs were seeded into 24-well plates and cultured to approximately 70% confluence.

[0055] Example 1

[0056] like Figure 1 As shown in Figure A, a method for preparing a carbon nanotube composite loaded with a Gsα-inhibiting cyclic peptide includes the following steps:

[0057] (1) 1.0 mg of SWCNT with a diameter of 1.0 nm was added to 2 mL of sterile RPMI-1640 medium and sonicated for 10 min with a 120 W probe for pre-dispersion; then 2.0 mL of DSPE-PEG5000-amine solution with a concentration of 4.0 mg / mL was added and sonicated for another 1.5 h to fully disperse the SWCNT and form a PEGylated coating layer; then centrifuged at 20,000 rpm for 4 h and collected the supernatant to obtain a polyethylene glycol-modified single-walled carbon nanotube dispersion.

[0058] (2) Add cpGN13 linear peptide precursor to polyethylene glycol-modified single-walled carbon nanotube dispersion, mix well, and continue to add acetonitrile and water to form a reaction system, so that the volume fraction of acetonitrile in the reaction system is 20% (v / v) and the concentration of cpGN13 linear peptide precursor is 2 mg / mL;

[0059] (3) While stirring, add NH4HCO3 to the reaction system obtained in step (2) and adjust the pH of the reaction system to 8.0. The cpGN13 linear peptide precursor is subjected to in-situ cyclization reaction in the presence of polyethylene glycol-modified single-walled carbon nanotubes for 16 h. After the reaction, the unreacted peptide and small molecule components are removed by centrifugal ultrafiltration device with a molecular weight cutoff of 100 kDa to obtain carbon nanotube complex loaded with Gsα-inhibited cyclic peptide (SWCNT-cpGN13).

[0060] Example 2

[0061] like Figure 1 As shown in Figure A, a method for preparing an imaging-enabled carbon nanotube composite loaded with Gsα-inhibitory cyclic peptides includes the following steps:

[0062] (1) 1.0 mg of SWCNT with a diameter of 1.0 nm was added to 2 mL of sterile RPMI-1640 medium and sonicated for 10 min with a 120 W probe for pre-dispersion; then 2.0 mL of DSPE-PEG5000-amine solution with a concentration of 4.0 mg / mL was added and sonicated for another 1.5 h to fully disperse the SWCNT and form a PEGylated coating layer; then centrifuged at 20,000 rpm for 4 h and collected the supernatant to obtain a polyethylene glycol-modified single-walled carbon nanotube dispersion.

[0063] (2) Add cpGN13 linear peptide precursor to polyethylene glycol-modified single-walled carbon nanotube dispersion, mix well, and continue to add acetonitrile and water to form a reaction system, so that the volume fraction of acetonitrile in the reaction system is 20% (v / v) and the concentration of cpGN13 linear peptide precursor is 2 mg / mL;

[0064] (3) While stirring, add NH4HCO3 to the reaction system obtained in step (2) to adjust the pH of the reaction system to 8.0, so that the cpGN13 linear peptide precursor can be cyclized in situ for 16 h in the presence of polyethylene glycol-modified single-walled carbon nanotubes. After the reaction, use a centrifugal ultrafiltration device with a molecular weight cutoff of 100 kDa to purify the unreacted peptides and small molecule components to obtain the carbon nanotube complex loaded with Gsα-inhibited cyclic peptide (SWCNT-cpGN13).

[0065] (4) Prepare a carbon nanotube complex solution loaded with Gsα-inhibiting cyclic peptide; add Cy5.5-NHS ester to the carbon nanotube complex solution loaded with Gsα-inhibiting cyclic peptide at pH=7.4 and react at 25℃ for 2 h; after the reaction, purify the solution using a centrifugal ultrafiltration device with a molecular weight cutoff of 100 kDa until the filtrate is colorless to obtain the imaging-type carbon nanotube complex loaded with Gsα-inhibiting cyclic peptide (SWCNT-cpGN13-Cy5.5).

[0066] The molar ratio of Cy5.5-NHS ester to carbon nanotube complex loaded with Gsα-inhibitory cyclic peptide is 10:1.

[0067] Comparative Example

[0068] A method for preparing an imaging-type carbon nanotube composite, comprising the following steps:

[0069] (1) 1.0 mg SWCNT was added to 2 mL of sterile RPMI-1640 medium and sonicated for 10 min with a 120 W probe for pre-dispersion; then 2.0 mL of DSPE-PEG5000-amine solution with a concentration of 4.0 mg / mL was added and sonicated for another 1.5 h to fully disperse SWCNT and form a PEGylated coating layer; then centrifuged at 20,000 rpm for 4 h and collected the supernatant to obtain a polyethylene glycol-modified single-walled carbon nanotube dispersion.

[0070] (2) At pH=7.4, Cy5.5-NHS ester was added to the polyethylene glycol-modified single-walled carbon nanotube dispersion and reacted at 25°C for 2 h. After the reaction, the mixture was purified by centrifugal ultrafiltration device with a molecular weight cutoff of 100 kDa until the filtrate was colorless, and the imaging carbon nanotube composite (SWCNT-Cy5.5) was obtained.

[0071] The molar ratio of Cy5.5-NHS ester to polyethylene glycolated single-walled carbon nanotubes is 10:1.

[0072] Example 3

[0073] 1. Observation using transmission electron microscopy (TEM)

[0074] 10 μL of the polyethylene glycol-modified single-walled carbon nanotube dispersion prepared in Example 1 was dropped onto a 400-mesh copper grid coated with an ultrathin carbon film. After standing for 10 min, it was stained with 1% uranium acetate and observed under a transmission electron microscope at 80 kV. The results are as follows. Figure 1 As shown in B in the diagram.

[0075] Depend on Figure 1 As can be seen from B, the functionalized polyethylene glycol-modified single-walled carbon nanotubes have a complete and continuous tubular structure.

[0076] 2. Raman spectroscopy imaging

[0077] The polyethylene glycol-modified single-walled carbon nanotube dispersion prepared in Example 1 was centrifuged and washed three times, and 2 μL of the sample was added to a clean substrate and dried overnight. Raman microscopy was used for detection, with an excitation wavelength of 532 nm, a laser power set to 10% of the instrument's maximum output power, and an integration time of 4 s. Raman spectra and Raman imaging data were acquired, and the results are as follows: Figure 1 As shown in C~D.

[0078] Depend on Figure 1 As can be seen from C~D, the polyethylene glycolated single-walled carbon nanotube dispersion has obvious characteristic vibrational peaks such as D band, G band and G′ band, indicating that the functionalized polyethylene glycolated single-walled carbon nanotubes still retain their characteristic carbon nanostructure.

[0079] BMDMs were added to the polyethylene glycolated single-walled carbon nanotube dispersion prepared in Example 1 to achieve a final concentration of 4 nM. After uniform dispersion, the dispersion was incubated at 37°C for 3 h. After washing with PBS buffer, Raman imaging and signal reconstruction were performed. The results are as follows: Figure 1 As shown in E in the figure.

[0080] Depend on Figure 1 As can be seen from E, after macrophages take up polyethylene glycol-modified single-walled carbon nanotubes, intracellular SWCNT-related Raman signals can be observed, indicating that the nanomaterial can be taken up by macrophages.

[0081] 3. Observation using atomic force microscopy (AFM)

[0082] 10 μL of the SWCNT-cpGN13-Cy5.5 dispersion prepared in Example 2 was dropped onto the surface of freshly peeled mica sheets. After drying at room temperature for 24 h, the morphology and height were measured using a BioScope Resolve atomic force microscope, with SWCNT as a control. The results are as follows: Figure 1 As shown in F~H.

[0083] Depend on Figure 1 As indicated by F~H in the figure, there are regions with increased local height on the surface of SWCNT-cpGN13-Cy5.5. Compared with SWCNT, the local site height of the functionalized polyethylene glycol-modified single-walled carbon nanotubes increases by approximately 0.5 nm, which is consistent with the changes in the surface structure of nanotubes after cpGN13 loading.

[0084] Example 4

[0085] 1. Intake test

[0086] BMDMs, mVSMCs, and HUVECs cultured to approximately 70% confluence were replaced with serum-free medium, and PBS blank group, SWCNT group, and SWCNT-Cy5.5 group were set up respectively.

[0087] Then, cells in the SWCNT-Cy5.5 group were added with SWCNT-Cy5.5 prepared in Comparative Example 1 at a final concentration of 4 nM, cells in the SWCNT group were added with an equal concentration of SWCNT, and cells in the PBS blank group were added with an equal volume of PBS buffer. All groups of cells were incubated at 37°C for 3 h. After incubation, the cells were washed with PBS, and fluorescence signals were collected using flow cytometry. The proportion of Cy5.5-positive cells in each group was analyzed using FlowJo 10.1.r5 software. The results are as follows: Figure 2 As shown in A~B in the diagram.

[0088] Depend on Figure 2As shown in A~B, BMDMs showed an uptake rate of 90% for SWCNT-Cy5.5, significantly higher than that of mVSMCs and HUVECs. The Cy5.5 positivity rate of mVSMCs and HUVECs was approximately 5-15%, indicating that SWCNT-Cy5.5 exhibited a clear macrophage-preferred uptake characteristic in the tested vascular-associated cells.

[0089] 2. Foam cell uptake experiment

[0090] BMDMs were separately treated with 50 μg / mL oxLDL for 24 h to induce foam cell formation. The resulting foam cells were divided into SWCNT-Cy5.5 group and SWCNT-cpGN13-Cy5.5 group. In this example, foam cells refer to BMDMs that showed significant intracellular lipid accumulation after oxLDL induction.

[0091] Then, the SWCNT-Cy5.5 group cells were added with SWCNT-Cy5.5 prepared in Comparative Example 1 at a final concentration of 4 nM, and the SWCNT-cpGN13-Cy5.5 group cells were added with SWCNT-cpGN13-Cy5.5 prepared in Example 2 at a final concentration of 4 nM. The proportion of Cy5.5 positive cells was then detected by flow cytometry, and the results are as follows: Figure 2 As shown in C~D.

[0092] Depend on Figure 2 As shown in C~D, both SWCNT-Cy5.5 and SWCNT-cpGN13-Cy5.5 can be efficiently taken up by foam cells, and the addition of cpGN13 did not significantly reduce the uptake capacity of foam cells for SWCNT-cpGN13-Cy5.5. This uptake level is similar to that of unlipid-loaded BMDMs.

[0093] 3. Gsα activation and foam cell formation experiment

[0094] BMDMs were treated with 50 μg / mL oxLDL for 24 h. At the 12th h of oxLDL treatment, the cells were set up as SWCNT-Cy5.5 group and SWCNT-cpGN13-Cy5.5 group, respectively. SWCNT-Cy5.5 prepared in Comparative Example 1 and SWCNT-cpGN13-Cy5.5 prepared in Example 2 were added to make the final concentration 4 nM, and the cells were incubated with oxLDL for another 12 h.

[0095] Gsα-GTP Immunofluorescence Detection: After treatment, cells were fixed with 4% paraformaldehyde and blocked with 5% BSA; active Gsα-specific antibody Gsα-GTP (NewEast Biosciences, 26906, 1:200) was added and incubated overnight at 4°C. Then, Alexa Fluor 594-labeled goat anti-mouse IgG secondary antibody was added for fluorescence development, and imaging was performed using a fluorescence microscope. The results are shown below. Figure 2 As shown in E~F.

[0096] Depend on Figure 2 As shown in E~F, compared with the SWCNT-Cy5.5 group, the Gsα-GTP fluorescence signal of the SWCNT-cpGN13-Cy5.5 group was significantly reduced, indicating that SWCNT-cpGN13-Cy5.5 can inhibit oxLDL-induced Gsα activation.

[0097] Oil Red O staining: After treatment, fix with 4% paraformaldehyde at room temperature for 10 min, wash with PBS buffer, and then stain with Oil Red O for 30 min, followed by washing with PBS buffer. Observe and calculate the accumulation of Oil Red O-positive lipids under a microscope. Analyze at least 6 fields of view for each sample. Results are as follows: Figure 2 As shown in G~H.

[0098] Depend on Figure 2 As indicated by G~H, compared with the SWCNT-Cy5.5 group, the Oil RedO positive lipids were significantly reduced after SWCNT-cpGN13-Cy5.5 treatment, indicating that SWCNT-cpGN13-Cy5.5 can inhibit oxLDL-induced macrophage foaming.

[0099] 4. Detection of CD36 and SR-A1 proteins

[0100] Simultaneously using the BMDMs from experiments 2 and 3, total cellular protein was extracted. The expression levels of scavenger receptors CD36 and SR-A1 were detected by Western blot, with β-actin used as an internal control. Gray-scale analysis of the target protein bands was performed, and normalization was performed using the corresponding β-actin signal. The results are as follows: Figure 2 As shown in I~J.

[0101] Depend on Figure 2 As shown in I~J, compared with the SWCNT-Cy5.5 group, the protein expression levels of CD36 and SR-A1 in the SWCNT-cpGN13-Cy5.5 group were significantly reduced, which is consistent with the results of decreased Gsα activation and reduced Oil Red O positive lipids.

[0102] Example 5

[0103] 1. The method for constructing a mouse model of atherosclerosis is as follows: Male Ldlr mice with a C57BL / 6 background were selected. - / - Mice, purchased at 7 weeks of age, were acclimatized for one week before being given purified Paigen feed (TP28640, Nantong Trofi Feed Technology Co., Ltd.) for free access to induce atherosclerosis. Mice were housed under standard conditions with free access to food and water. Mice used for in vivo distribution and imaging experiments were continuously fed Paigen feed before probe administration, and subsequent experiments were conducted after atherosclerosis was induced.

[0104] 2. Fluorescence distribution

[0105] Atherosclerosis mouse models were divided into a blank group (PBS), a SWCNT group (SWCNT), a SWCNT-Cy5.5 group (SWCNT-Cy5.5), and a SWCNT-cpGN13-Cy5.5 group (SWCNT-cpGN13-Cy5.5). Mice in the SWCNT, SWCNT-Cy5.5, and SWCNT-cpGN13-Cy5.5 groups were then administered 200 μL of 0.068 mg / mL SWCNT, the comparative SWCNT-Cy5.5, and the SWCNT-cpGN13-Cy5.5 prepared in Example 2 via a single dose via the tail vein. The control group was given an equal volume of PBS buffer. The aorta and major organs were perfused and harvested on days 0, 1, 3, and 7 after administration. In vitro fluorescence imaging was performed using the IVIS Spectrum 2 small animal imaging system. The excitation wavelength was set to 675 nm, and the emission wavelength was set to 700–720 nm. The same imaging parameters were maintained for each group and at each time point. Quantitative analysis of the fluorescence intensity of the aorta and major organs was performed. The results are as follows: Figure 3 As shown in A~E.

[0106] Depend on Figure 3 As shown in Figures A through E, on day 1 after administration, significant fluorescence signals appeared in the aortic region of both the SWCNT-Cy5.5 and SWCNT-cpGN13-Cy5.5 groups, and these signals persisted until day 7 after administration. Strong fluorescence signals were observed in the liver and spleen, while the heart and lungs mainly showed transient low-level signals. The overall distribution trends of SWCNT-Cy5.5 and SWCNT-cpGN13-Cy5.5 were similar, indicating that cpGN13 loading did not significantly alter the overall in vivo distribution characteristics of SWCNT-cpGN13-Cy5.5.

[0107] 3. Photoacoustic imaging

[0108] Atherosclerotic mice were administered a single dose of 200 μL of SWCNT-cpGN13 prepared in Example 1 at a concentration of 0.068 mg / mL via the tail vein. The absorption spectrum of SWCNT-cpGN13 was measured before imaging, and 715 nm was selected as the single-wavelength photoacoustic imaging wavelength based on its maximum absorption peak. The carotid artery region of the mice was imaged using a Vevo F2 LAZR-X ultrasound / photoacoustic imaging system at 0, 1, 3, and 7 days after administration, with the same imaging parameters maintained at each time point. Quantitative analysis of the photoacoustic signal intensity in the carotid plaque region was performed, and the results are as follows: Figure 3 As shown in F~G.

[0109] Depend on Figure 3 As shown in F~G, a significant and continuous photoacoustic signal can be detected in the atherosclerotic lesions after drug administration. The temporal variation trend is basically consistent with the Cy5.5 fluorescence distribution results, indicating that SWCNT-cpGN13 can be enriched in the atherosclerotic lesions and used for photoacoustic imaging.

[0110] Example 6

[0111] 1. The atherosclerotic mouse model was divided into three groups: a blank control group (PBS), a SWCNT-Cy5.5 group (SWCNT-Cy5.5), and a SWCNT-cpGN13-Cy5.5 group (SWCNT-cpGN13-Cy5.5), with n=6 in each group. The experimental design and drug administration procedure are as follows: Figure 4 As shown in A, all groups of mice continued to be fed Paigen diet.

[0112] Then, during the intervention phase, mice in the SWCNT-Cy5.5 group and the SWCNT-cpGN13-Cy5.5 group were administered 200 μL and 0.068 mg / mL of the comparative SWCNT-Cy5.5 and the SWCNT-cpGN13-Cy5.5 prepared in Example 2, respectively, via tail vein. The PBS group was administered an equal volume of PBS buffer. Administration was once weekly for 4 weeks. During the 4-week intervention period, the body weight, systolic blood pressure, and diastolic blood pressure of mice in each group were monitored. The results are as follows: Figure 4 As shown in B in the diagram.

[0113] Depend on Figure 4 As shown in B, no significant differences were observed in the body weight and blood pressure of the mice in each group during the observation period, indicating that SWCNT-cpGN13-Cy5.5 did not cause significant changes in systemic body weight or hemodynamics under the administration conditions used in this embodiment.

[0114] 2. After the intervention, blood was collected from mice in each group, plasma was separated, and the levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured. The results are as follows: Figure 4 As shown in C.

[0115] Depend on Figure 4 As shown in C, there were no significant differences in the levels of TC, TG, HDL-C, and LDL-C among the groups, suggesting that the anti-atherosclerotic effect of SWCNT-cpGN13-Cy5.5 does not primarily depend on reducing circulating blood lipids.

[0116] 3. After the intervention, mice in each group were sacrificed, and the intact aorta of each group was isolated. After longitudinal dissection, the aorta was stained with 0.5% Oil Red O for 30 min, and the percentage of Oil Red O positive plaque area to the total surface area of ​​the aorta was calculated using ImageJ software. The results are as follows: Figure 4 As shown in D~E.

[0117] Depend on Figure 4 As shown in D~E, compared with the PBS group and the SWCNT-Cy5.5 group, the total aortic plaque area of ​​the SWCNT-cpGN13-Cy5.5 group was significantly reduced, indicating that SWCNT-cpGN13-Cy5.5 can inhibit the formation and progression of atherosclerotic plaques.

[0118] 4. After the intervention, mice in each group were sacrificed, and aortic root tissue was collected from each group. After OCT embedding, frozen sections approximately 5 μm thick were continuously prepared starting at the aortic valve leaflet level. H&E staining and Oil Red O staining were performed, and the results are as follows: Figure 4 As shown in F~G.

[0119] Depend on Figure 4 As shown in F~G, compared with the PBS group and the SWCNT-Cy5.5 group, the aortic root plaque area and Oil Red O positive lipid area of ​​the SWCNT-cpGN13-Cy5.5 group were significantly reduced, further indicating that SWCNT-cpGN13-Cy5.5 can alleviate atherosclerotic lesions and lipid accumulation in plaques.

[0120] Example 7

[0121] The aortas of mice from the SWCNT-Cy5.5 group (SWCNT-Cy5.5) and the SWCNT-cpGN13-Cy5.5 group (SWCNT-cpGN13-Cy5.5) in Example 6 were cut into pieces approximately 1.5 mm in size. 3 The tissue blocks were digested at 37°C for 45 minutes and then filtered through a 40 μm filter to obtain a single-cell suspension.

[0122] Single-cell suspensions were used as samples. Cells were captured and whole transcriptome libraries were constructed using the BDRhapsody system, and PE150 sequencing was performed using Illumina NovaSeq 6000.

[0123] Quality control was performed by removing cells with a mitochondrial UMI exceeding 25%, a UMI less than 500, or fewer than 200 detected genes. Normalization, clustering, and annotation were performed using Seurat 4.0.4, and DDRTree pseudo-temporal analysis was conducted using Monocle 2.22.0. The results are as follows: Figure 5 As shown.

[0124] Depend on Figure 5 It was found that, compared with the SWCNT-Cy5.5 group, the overall macrophage ratio in the SWCNT-cpGN13-Cy5.5 group was slightly decreased (28.8% vs. 38.0%). The proportion of lipid-related macrophages decreased from 29.9% in the control group to 21.7%, indicating downregulation of lipid metabolism-related pathways and a reduction in macrophages that develop along lipid-related pathways.

[0125] Example 8

[0126] Atherosclerotic mice were given a single tail vein injection of 200 μL of 0.068 mg / mL SWCNT-cpGN13-Cy5.5 prepared in Example 2. Blood samples were collected from the mice at 0, 1, 3, 7, and 28 days after administration, and hematological and biochemical parameters were measured. The results are as follows: Figure 6 As shown in A in the diagram.

[0127] The hematological indicators include white blood cells, monocytes, granulocytes, lymphocytes, erythrocytes, hemoglobin, hematocrit, and platelets; the biochemical indicators include alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen, and creatinine. Both the hematological and biochemical indicators described herein can be detected using existing technologies.

[0128] Mice were sacrificed at 0, 1, 3, 7, and 28 days after drug administration. Heart, aorta, lung, liver, spleen, pancreas / islets, and kidneys were collected for H&E staining. Results are as follows: Figure 6 As shown in B in the diagram.

[0129] Simultaneously, immunofluorescence was performed on corresponding cell / structural markers: WGA and CD31 immunofluorescence staining were used for the heart to evaluate cardiomyocyte structure and microvascular endothelium; α-SMA and CD31 immunofluorescence staining were used for the aorta to evaluate vascular smooth muscle and endothelial structure; laminin and CD31 immunofluorescence staining were used for lung tissue to evaluate the basement membrane and vascular endothelial structure; LYVE1 and F4 / 80 immunofluorescence staining were used for the liver to evaluate sinusoidal structures and hepatic macrophage distribution; CD4 immunofluorescence staining was used for the spleen; insulin and glucose immunofluorescence staining were used for the pancreatic islets to evaluate the structure of pancreatic β-cells and α-cells; and AQP1 and nephrin immunofluorescence staining were used for the kidneys to evaluate renal tubular and glomerular structures. Fluorescence images were acquired using a DS-Ri2 fluorescence microscope (Nikon, Japan), and the results are as follows: Figure 6 As shown in C.

[0130] Depend on Figure 6 It was found that after administration of SWCNT-cpGN13-Cy5.5, no obvious hematological, hepatic and renal biochemical, or major organ histological abnormalities were observed in atherosclerotic mice, and no significant abnormal changes were observed in the related tissue structures and cell-specific marker distribution of each major organ. This indicates that the SWCNT-cpGN13-Cy5.5 prepared in this invention has good short-term in vivo tolerability under the single-dose administration and 28-day observation conditions used in this embodiment.

[0131] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A carbon nanotube complex loaded with a Gsα-inhibiting cyclic peptide, characterized in that, It uses polyethylene glycolated single-walled carbon nanotubes as the nanoframework, and loads the Gsα-inhibiting cyclic peptide cpGN13 on the nanoframework. The polyethylene glycol-modified single-walled carbon nanotubes were prepared according to the following method: Single-walled carbon nanotubes were added to an aqueous medium and sonicated for 5 to 15 minutes under a 100-150W probe. Then, DSPE-PEG5000-amine solution was added and sonicated for another 1 to 2 hours. After centrifugation, the supernatant was collected to obtain polyethylene glycol-modified single-walled carbon nanotubes. The single-walled carbon nanotubes were added at a concentration of 0.8–1.2 mg; the aqueous medium was sterile RPMI-1640 medium, and the volume was 1–2 mL; the concentration of the DSPE-PEG5000-amine solution was 3.5–4.5 mg / mL, and the volume added was 1.5–2.5 mL; the centrifugation conditions were 18,000–20,000 rpm for 4–5 h.

2. The carbon nanotube composite loaded with Gsα-inhibiting cyclic peptide as described in claim 1, characterized in that, The amount of single-walled carbon nanotubes added was 1.0 mg; the amount of sterile RPMI-1640 culture medium used was 2 mL; the power of the ultrasonic treatment under the probe was 120 W, and the pre-dispersion time was 10 min; the concentration of the DSPE-PEG5000-amine solution was 4.0 mg / mL, and the amount added was 2.0 mL; the ultrasonic treatment was continued for 1.5 h, and the centrifugation conditions were 20000 rpm for 4 h.

3. The carbon nanotube composite loaded with Gsα-inhibiting cyclic peptide as described in claim 1, characterized in that, The amino acid sequence of the Gsα-inhibitory cyclic peptide cpGN13 is shown in SEQ ID NO.

1.

4. The method for preparing the carbon nanotube composite loaded with Gsα-inhibiting cyclic peptide as described in claim 1, characterized in that, The steps are as follows: The cpGN13 linear peptide precursor was added to the polyethylene glycolated single-walled carbon nanotube dispersion and mixed thoroughly. Acetonitrile and water were then added to form a reaction system. NH4HCO3 was added to the reaction system while stirring to adjust the pH to 7.8–8.2, allowing the cpGN13 linear peptide precursor to undergo in-situ cyclization in the presence of polyethylene glycolated single-walled carbon nanotubes for 12–16 h. After purification by centrifugation and ultrafiltration, SWCNT-cpGN13 was obtained. In the reaction system, the volume fraction of acetonitrile is 15-25%, and the concentration of the cpGN13 linear peptide precursor is 1.5-2.5 mg / mL.

5. The method for preparing the carbon nanotube composite loaded with Gsα-inhibiting cyclic peptide as described in claim 4, characterized in that, The acetonitrile volume fraction in the reaction system was 20%, and the concentration of the cpGN13 linear peptide precursor was 2 mg / mL.

6. The use of the carbon nanotube complex loaded with Gsα-inhibiting cyclic peptide as described in claim 1 in the preparation of drugs for treating atherosclerosis.

7. An imaging-enabled carbon nanotube composite loaded with a Gsα-inhibiting cyclic peptide, characterized in that, The near-infrared fluorescent marker Cy5.5 is attached to the carbon nanotube complex loaded with the Gsα-inhibitory cyclic peptide as described in claim 1.

8. The method for preparing the imaging-type carbon nanotube composite loaded with Gsα-inhibitory cyclic peptide as described in claim 7, comprising the following steps: Prepare a carbon nanotube complex solution loaded with Gsα-inhibiting cyclic peptides; Cy5.5-NHS ester was added to a carbon nanotube composite solution loaded with Gsα-inhibiting cyclic peptides at pH 7.0–7.5, and the reaction was carried out at 25–30 °C for 1.5–2.5 h. After centrifugation and filtration, an imaging-type carbon nanotube composite loaded with Gsα-inhibiting cyclic peptides was obtained. The molar ratio of the Cy5.5-NHS ester to the carbon nanotube complex loaded with Gsα-inhibitory cyclic peptide is (8~12):

1.

9. The application of the imaging-type carbon nanotube complex loaded with Gsα-inhibitory cyclic peptide as described in claim 7 in the preparation of drugs for treating atherosclerosis or imaging formulations for atherosclerotic lesions.

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