Use of brd4 inhibitors in the manufacture of a product for the prevention or treatment of aortic dissection

Abstract

The application discloses application of a BRD4 inhibitor in preparation of a product for preventing and treating aortic dissection; BRD4 is significantly highly expressed in human and mouse AD pathological tissue, and a nano delivery system constructed can realize efficient enrichment of the BRD4 inhibitor at an aortic pathological site and intracellular nuclear internalization of a target cell, and effectively inhibit an inflammatory cascade reaction, protect a vascular smooth muscle cell function, maintain structural integrity of a blood vessel wall and reduce inflammatory cell infiltration, so as to significantly delay AD progression.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of BRD4 inhibitors in the preparation of products for the prevention and treatment of aortic dissection. Background Technology

[0002] Aortic dissection (AD) is a catastrophic cardiovascular disease with a rapid onset, rapid progression, and extremely high mortality rate. Its core pathological change is the rupture of the aortic intima, allowing circulating blood to flood into the aortic wall media, forming a false lumen that separates and expands along the aortic wall, leading to aortic wall dissection, rupture and hemorrhage, or occlusion of important branch vessels. Despite significant advancements in surgical procedures and endovascular repair techniques in recent years, patients with acute Stanford type B dissection or postoperative residual dissection still primarily rely on conservative medical treatments such as intensive blood pressure control. Furthermore, the potential side effects of surgery urgently need improvement and mitigation, all stemming from the lack of specific drugs targeting the core pathophysiological aspects of AD. Therefore, in-depth exploration of the molecular mechanisms of AD pathogenesis, identification and validation of new drug intervention targets, and development of novel and effective treatment strategies are major scientific challenges urgently needing to be addressed in the cardiovascular field.

[0003] The pathogenesis of aortic atrophy (AD) is a complex process involving multiple intertwined factors, including genetics, hemodynamics, inflammatory response, extracellular matrix degradation, vascular smooth muscle cell (VSMC) apoptosis, and phenotypic transformation. Uncontrolled inflammatory responses and massive VSMC apoptosis are considered key drivers leading to structural fragility and loss of integrity in the aortic wall. Abnormal regulation of gene expression plays a central role in this process. Epigenetics, as a bridge connecting environmental factors and gene expression, has received considerable attention in cardiovascular disease research in recent years. The bromodomain and extra-terminal (BET) protein family, particularly BRD4, acts as a crucial epigenetic "reader," recognizing and binding to acetylated lysine residues of histones. This recruits the transcriptional elongation complex, activates the transcription of target genes (such as c-Myc), enhances the transcriptional activity of inflammatory signaling pathways such as NF-κB, and mediates chromatin remodeling. Initially, it gained significant attention due to its extensive research in tumors. Current research has confirmed that BRD4 plays a crucial role in triggering inflammatory responses and tissue remodeling in various vascular remodeling diseases, including atherosclerosis, myocardial hypertrophy, and pulmonary hypertension. Furthermore, BRD4 can regulate the proliferation and migration of vascular smooth muscle cells. However, the specific role of BRD4 in the particular pathological process of aortic dissection and whether it can serve as an effective therapeutic target remain unclear and require further systematic and in-depth investigation.

[0004] Traditional drug development strategies, including small molecule inhibitors and nucleic acid drugs, rely on the former to occupy the active site of the target protein. This "occupation-driven" approach may require higher drug doses to maintain efficacy in some cases, increasing the risk of off-target effects or drug resistance. The latter, however, suffers from significant instability and low tissue penetration. To address these issues, protein degradation-targeting chimeras (PROTACs) technology has emerged. In recent years, PROTAC technology has received widespread attention, providing a revolutionary new approach to drug development. A PROTAC molecule is a heterobifunctional molecule; one end binds to the target protein, while the other end recruits an E3 ubiquitin ligase. By bringing the two ends closer, it induces ubiquitination of the target protein, which is then specifically degraded using the cell's own 26S proteasome system. This "event-driven" catalytic mode offers unique advantages such as low dosage, long-lasting action, and the ability to degrade "undruggable" targets. ARV825, as a highly efficient BRD4-PROTAC degrader, has shown promising application prospects in improving various tumor models and emphysema. However, PROTAC molecules typically have large molecular weights and poor pharmacokinetic properties, such as poor water solubility, low bioavailability, and rapid metabolism in vivo. These problems greatly limit their direct application in vivo, especially when targeting specific tissues such as blood vessel walls. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a BRD4 inhibitor in the preparation of products for the prevention and treatment of aortic dissection. BRD4 is significantly highly expressed in human and mouse AD lesion tissues. The nanodelivery system constructed in this invention can achieve efficient enrichment and internalization of BRD4 inhibitor in aortic lesion sites and target cell nuclei. By specifically degrading BRD4 protein, it can effectively inhibit the inflammatory cascade, protect the function of vascular smooth muscle cells, maintain the integrity of vascular wall structure, and reduce inflammatory cell infiltration, thereby significantly delaying the progression of AD.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: The first aspect of this invention provides the application of a BRD4 inhibitor in the preparation of products for the prevention and treatment of aortic dissection.

[0007] Preferably, the BRD4 inhibitor is ARV825; the product includes a drug.

[0008] A second aspect of the present invention provides a product for preventing and treating aortic dissection, the product comprising a therapeutically effective amount of a BRD4 inhibitor.

[0009] A third aspect of the present invention provides a method for constructing a multi-level nanodelivery system for preventing aortic dissection, the method comprising the following steps: (a) A solution containing BRD4 inhibitor was added dropwise to a PBS solution containing histones to obtain a nanocomposite. (b) The TAT membrane-penetrating peptide was coupled to the surface of the nanocomposite to obtain a nanocomposite coupled with the TAT membrane-penetrating peptide. (c) The aldehyde-modified polyethylene glycol and the nanocomposite coupled with TAT membrane-penetrating peptide are subjected to a Schiff base reaction to obtain the multi-level nanodelivery system with both nuclear targeting and long circulation functions.

[0010] Preferably, in step (a), the BRD4 inhibitor is ARV825; the molar ratio of the histone to the BRD4 inhibitor is 1:(2~4).

[0011] Preferably, in step (b), the coupling of the TAT transmembrane peptide to the surface of the nanocomposite specifically includes: (1) Histones were added to a cross-linking agent DMSO solution and reacted in the dark, followed by dialysis to obtain activated histones; (2) DTT was added to the PBS solution of TAT membrane-penetrating peptide for reduction treatment, and then permeabilized to obtain reduced TAT membrane-penetrating peptide. (3) The activated histone and reduced TAT membrane-penetrating peptide were subjected to a light-protected reaction, followed by dialysis to obtain a nanocomposite of TAT membrane-penetrating peptide.

[0012] Preferably, in step (1), the crosslinking agent is succinimide 3-(2-pyridyldithio)-propionate; the molar ratio of crosslinking agent to histone is (2~4):1; the temperature of the reaction in the dark is room temperature, and the time is 50~70 min.

[0013] Preferably, in step (2), the molar ratio of TAT membrane-penetrating peptide to DTT is 1:(0.8~1.2); the reduction treatment temperature is room temperature, and the time is 20~40 min; In step (3), the molar ratio of activated histone to reduced TAT transmembrane peptide is 1:(2~4); the reaction temperature is room temperature and the time is 6~10h.

[0014] Preferably, in step (c), the molar ratio of aldehyde-modified polyethylene glycol to the nanocomposite of coupled TAT membrane-penetrating peptide is (8~10):1; the Schiff base reaction is carried out at room temperature and the pH value is 7.5~8.5.

[0015] The fourth aspect of the present invention provides a multi-level nanodelivery system for preventing aortic dissection constructed by the above-described construction method.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least the following: This invention confirms that BRD4 is significantly highly expressed in human and mouse Alzheimer's disease (AD) lesions. Furthermore, a PT@hARV825 (multi-level nanodelivery system) with relatively uniform particle size (approximately 24 nm), positive surface potential, and good stability under physiological conditions was successfully constructed. In vitro experiments showed that PT@hARV825 can be efficiently taken up by HASMCs and RAW264.7 cells and achieve nuclear targeting, significantly reducing the expression levels of BRD4 protein and mRNA. Compared with the AD group, PT@hARV825 treatment effectively inhibited the mRNA expression of cellular inflammatory factors IL-6, IL-1β, and TNF-α, and downregulated the expression of MMP2 and MMP9. In addition, it can reduce the protein levels of the inflammation-related proteins P-STING / P-IκBα in HASMCs and inhibit their phenotypic transformation, proliferation, and migration abilities. In vivo experiments showed that PT@hARV825 exhibited good stability and long-lasting effect in blood circulation and could effectively accumulate in AD sites. Compared with the AD model group, the survival rate of mice in the PT@hARV825 treatment group was significantly improved, and the incidence, severity, and degree of vasodilation of AD were significantly reduced. Histopathological analysis showed that the aortic structure of mice in the treatment group maintained good integrity, collagen deposition and elastic fiber breakage were reduced, and the levels of macrophage infiltration and HASMC apoptosis were significantly lower than those in the control group. Furthermore, the expression of BRD4 protein in the aortic tissue was effectively inhibited. Safety evaluation results showed that PT@hARV825 has good biocompatibility, and no obvious toxic reactions or hemolysis were observed in vitro or in vivo. Serum biochemical indicators also showed no significant abnormalities. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0018] Figure 1 This describes the expression of BRD4 in human and mouse aortic dissection tissues in Example 1 of the present invention. Figure 2 The results of BRD4 expression and localization in key cells of AD lesions in Example 2 of this invention; Figure 3 The expression of BRD4 in HASMCs and RAW264.7 cells induced by AD-related pathological stimulation in Example 3 of this invention; Figure 4The BRD4 degradation efficiency and cytotoxicity of ARV825 in Example 4 of this invention; Figure 5 Preparation and characterization of PT@hARV825 nanoparticles in Example 5 of this invention; Figure 6 For the in vitro safety assessment of PT@hARV825 in Example 6 of this invention; Figure 7 This refers to the cellular uptake efficiency of PT@hARV825 in Example 7 of the present invention; Figure 8 This refers to the nuclear translocation state of BRD4 and the degradation ability of PT@hARV825 in Example 8 of the present invention; Figure 9 This relates to the regulation of apoptosis, phenotypic transition, and inflammatory response of HASMCs by PT@hARV825 in Example 8 of the present invention; Figure 10 This illustrates the effect of PT@hARV825 on the proliferation, migration, and cellular senescence of HASMCs in Example 8 of this invention. Figure 11 This describes the impact of PT@hARV825 on RAW264.7 related functions in Embodiment 9 of the present invention. Figure 12 This demonstrates the metabolic and targeting capabilities of PT@hARV825 in AD model mice in Example 10 of this invention. Figure 13 The in vivo biodistribution of the formulations in different treatment groups in Example 10 of the present invention; Figure 14 In Example 11 of this invention, PT@hARV825 effectively improves AD lesions; Figure 15 In Example 12 of this invention, PT@hARV825 inhibits pathological remodeling of the vascular wall; Figure 16 This describes the molecular mechanism of PT@hARV825 in preventing and treating AD in Example 13 of this invention. Figure 17 This is an in vivo safety evaluation of PT@hARV825 at therapeutic doses in Example 14 of the present invention. Detailed Implementation

[0019] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0020] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by those skilled in the art to which this invention pertains.

[0021] This invention provides an application of a BRD4 inhibitor in the preparation of products for the prevention and treatment of aortic dissection.

[0022] In one embodiment, the BRD4 inhibitor is ARV825.

[0023] Another embodiment of the present invention provides a product for preventing and treating aortic dissection, the product comprising a therapeutically effective amount of a BRD4 inhibitor.

[0024] Another embodiment of the present invention provides a method for constructing a multi-level nanodelivery system for preventing aortic dissection, the method comprising the following steps: (a) A solution containing BRD4 inhibitor was added dropwise to a PBS solution containing histones to obtain a nanocomposite. (b) The TAT membrane-penetrating peptide was coupled to the surface of the nanocomposite to obtain a nanocomposite coupled with the TAT membrane-penetrating peptide. (c) The aldehyde-modified polyethylene glycol and the nanocomposite coupled with TAT membrane-penetrating peptide are subjected to a Schiff base reaction to obtain the multi-level nanodelivery system with both nuclear targeting and long circulation functions.

[0025] In one embodiment, in step (a), the BRD4 inhibitor is ARV825; the molar ratio of the histone to the BRD4 inhibitor is 1:(2~4).

[0026] In one embodiment, step (b), specifically, involves coupling the TAT transmembrane peptide to the surface of the nanocomposite: (1) Histones were added to a cross-linking agent DMSO solution and reacted in the dark, followed by dialysis to obtain activated histones; (2) DTT was added to the PBS solution of TAT membrane-penetrating peptide for reduction treatment, and then permeabilized to obtain reduced TAT membrane-penetrating peptide. (3) The activated histone and reduced TAT membrane-penetrating peptide were subjected to a light-protected reaction, followed by dialysis to obtain a nanocomposite of TAT membrane-penetrating peptide.

[0027] In one embodiment, in step (1), the crosslinking agent is succinimide 3-(2-pyridyldithio)-propionate; the molar ratio of the crosslinking agent to histone is (2~4):1; the temperature of the reaction in the dark is room temperature, and the time is 50~70 min.

[0028] In one embodiment, in step (2), the molar ratio of TAT membrane-penetrating peptide to DTT is 1:(0.8~1.2); the reduction treatment is performed at room temperature for 20~40 min. In step (3), the molar ratio of activated histone to reduced TAT transmembrane peptide is 1:(2~4); the reaction temperature is room temperature and the time is 6~10h.

[0029] In one embodiment, in step (c), the molar ratio of aldehyde-modified polyethylene glycol to the nanocomposite of coupled TAT membrane-penetrating peptide is (8~10):1; the Schiff base reaction is carried out at room temperature and the pH value is 7.5~8.5.

[0030] Another embodiment of the present invention provides a multi-level nanodelivery system for preventing aortic dissection constructed by the above-described construction method.

[0031] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0032] Example 1 This example illustrates the difference in BRD4 levels between healthy and aortic dissection samples: To investigate whether BRD4 is involved in the pathogenesis of aortic dissection, we first analyzed RNA sequencing databases of human aortic samples (GSE98770, GSE47472, GSE52093, and GSE57691), finding a significantly elevated BRD4 expression level (Figure 1A). Next, we experimentally examined the expression level of BRD4 in clinical tissues of human aortic dissection. Western blot analysis revealed a significant upregulation of BRD4 protein expression in the aortic tissues of AD patients compared to healthy aortic tissues (Figure 1B, C). Consistent with this, real-time quantitative PCR results showed that the mRNA level of BRD4 in AD tissues was also significantly higher than that in the healthy control group (Figure 1D), suggesting that BRD4 overexpression may occur at the transcriptional level. To further verify the generalizability of this finding in vivo, a mouse aortic dissection model induced by BAPN combined with Ang II was constructed. In mouse aortic dissection (AD) tissues, changes consistent with those observed in human samples were observed: the protein expression level (E, F in Figure 1) and mRNA level (G in Figure 1) of BRD4 were significantly higher than those in the healthy control group. These results indicate that BRD4 exhibits specific high expression in both human and mouse aortic dissection lesions, suggesting that it may serve as a key regulatory factor in the pathogenesis of AD, providing an important theoretical basis for subsequent targeted interventions.

[0033] Example 2 This example illustrates the expression and co-localization of BRD4 in different cells: The pathological process of aortic dissection involves dysfunction of multiple cell types in the vessel wall, and clarifying the expression and distribution of BRD4 in various cells is crucial for precise intervention. First, the basal expression levels of BRD4 in human aortic endothelial cells, human aortic smooth muscle cells, and the mouse macrophage line RAW264.7 were examined. Western blot results showed that the expression level of BRD4 in HASMCs and RAW264.7 cells was significantly higher than that in HUVECs (Figure 2, A, B), which is consistent with the fact that BRD4 is mainly found in HASMCs and lays the foundation for focusing the research on smooth muscle cells and macrophages. To further clarify the cellular localization of BRD4 in AD tissue, immunofluorescence co-staining was performed on human AD tissue sections. The results showed that the fluorescence signal of BRD4 significantly co-localized with the macrophage marker CD68 and the smooth muscle cell marker α-SMA, and the fluorescence signal of BRD4 was firmly localized within the cell nucleus (Figure 2, CF). In mouse AD tissues, immunofluorescence staining also confirmed the intranuclear co-localization of BRD4 with CD68-positive macrophages (G, H in Figure 2) and α-SMA-positive smooth muscle cells (I, J in Figure 2). This result not only reaffirms the high expression of BRD4 in key pathological cells of AD, but more importantly, it clarifies its intranuclear localization characteristics, providing direct spatial localization basis for subsequent design of nuclear-targeted delivery strategies.

[0034] Example 3 This example studies the response of BRD4 in HASMC and RAW264.7 to pathological stimuli: To investigate which factors might induce BRD4 upregulation in the aortic dissection microenvironment, the effects of AD-related pathological stimuli on BRD4 expression were examined at the cellular level. Angiotensin II (Ang II) was used to treat high-intensity vascular smooth muscle cells (HASMCs) to simulate the hemodynamic stress experienced by vascular smooth muscle cells in AD. Western blot results showed that Ang II induced BRD4 protein expression in HASMCs in a time-dependent manner, reaching a peak at 24 hours of treatment (Figure 3A, B). Simultaneously, lipopolysaccharide (LPS) was used to treat RAW264.7 macrophages to simulate the inflammatory microenvironment in AD. Results showed that LPS also induced BRD4 expression in RAW264.7 cells in a time-dependent manner, also reaching a peak at 24 hours (Figure 3C, D). Furthermore, it was found that TNF-α also stimulated BRD4 expression in both cell types (EH in Figure 3), while H2O2 stimulation failed to induce BRD4 expression in HASMCs, and Ang II also failed to induce BRD4 expression in RAW264.7 cells (IL in Figure 3), indicating that BRD4 has a selective response to specific types of pathological stimuli. These results not only reveal the potential mechanism by which BRD4 is induced to be upregulated under AD pathological conditions, but also lay the foundation for subsequent in vitro experiments to select 24 hours as the stimulation time point and conduct functional studies.

[0035] Example 4 This example studies the cytotoxicity of ARV825 and its degradation efficiency against BRD4: After identifying BRD4 as a potential intervention target, the biological effects of the PROTAC degrader ARV825, which targets BRD4, were further evaluated in core target cells (Figure 4A) to determine the optimal experimental conditions for subsequent studies. Cells were treated with different concentrations (0, 0.04, 0.2, 1, 5, and 10 μM) of ARV825 for 24 hours. Western blot analysis showed that ARV825 significantly reduced BRD4 protein levels in both cell types in a concentration-dependent manner, exhibiting highly efficient degradation at a concentration of 1 μM (Figure 4BE). However, high concentrations of PROTAC molecules may pose a potential risk of cytotoxicity. MTT assay results showed that when the concentration of ARV825 reached 10 μM, the survival rate of HASMCs decreased significantly; while RAW264.7 cells were more sensitive, showing significant inhibition of viability (survival rate below 80%) at a concentration of 5 μM (Figure 4F, G), suggesting that macrophages have low tolerance to this compound. Taking into account the balance between degradation efficiency and cytotoxicity, 1 μMARV825 and 24 hours of treatment were determined as the standard working concentration for all subsequent in vitro functional experiments, laying a solid dosage basis for the subsequent evaluation of the synergistic effect of the nanodelivery system.

[0036] Example 5 This embodiment presents a method for constructing and characterizing a multi-stage nanodelivery system that combines nuclear targeting and long-cycle functionality. The construction strategy is as follows: Figure 5 As shown in Figure A, the construction method includes the following steps: (a) The BRD4 inhibitor was loaded onto histones to obtain a nanocomplex (denoted as hARV825). (b) The TAT membrane-penetrating peptide was coupled to the surface of the nanocomposite to obtain the nanocomposite coupled with the TAT membrane-penetrating peptide (denoted as T@hARV825). (c) The aldehyde-modified polyethylene glycol and the nanocomposite coupled with TAT membrane-penetrating peptide were subjected to a Schiff base reaction to obtain the multi-level nanodelivery system with both nuclear targeting and long circulation functions (denoted as PT@hARV825).

[0037] The construction process and final product were systematically characterized physicochemically. In optimizing drug loading conditions, it was found that the solution was clearest and showed no precipitation when the molar ratio of histone to ARV825 was 3:1, indicating the formation of a relatively stable complex (Figure 5, B). Transmission electron microscopy (TEM) observation showed that the native histone morphology was heterogeneous, while PT@hARV825 exhibited a more uniform spherical structure with a particle size of approximately 24 nm (Figure 5, C). Dynamic light scattering analysis determined its hydrated particle size to be approximately 30 nm, which is consistent with the TEM results (Figure 5, D). Zeta potential analysis showed that the native histone carries a strong positive charge, and the potential of PT@hARV825 decreased with the adsorption of ARV825 and after PEG modification (Figure 5, E). This is beneficial for reducing non-specific adsorption in vivo and for interacting with negatively charged cell membranes, thereby promoting endocytosis. Coomassie Brilliant Blue staining showed that the band position of hARV825 was consistent with that of pure histone, indicating that drug loading did not change the protein's charge properties; while the modification with TAT and PEG caused the band to diffuse or migrate, confirming the success of chemical coupling (F in Figure 5). UV-Vis spectroscopy analysis showed that PT@hARV825 retained the characteristic absorption peaks of histone at 220 nm and 280 nm, proving that its basic protein structure was maintained during the modification process (G in Figure 5). Fourier transform infrared spectroscopy analysis further confirmed the successful PEG linkage, especially at 1690 cm⁻¹. -1 Characteristic absorption peaks of Schiff base bonds appeared nearby (H in Figure 5). Particle size stability tests showed that hARV825 and PT@hARV825 exhibited no significant changes in particle size after 7 days of storage at 4℃ and 37℃, demonstrating excellent colloidal stability (I, J in Figure 5). In summary, a good nuclear-targeted nanodelivery system was successfully constructed.

[0038] Example 6 This example is a study on the in vitro safety of PT@hARV825: To assess the potential safety risks of the constructed PT@hARV825 in biomedical applications, a systematic in vitro cytotoxicity and blood compatibility evaluation was conducted. First, the effects of different formulations on the viability of two cell types were detected using the MTT assay. The results showed that when using the working concentration (ARV825 equivalent concentration 1 μM, corresponding to a histone concentration of approximately 50 μg / mL), histone alone, hARV825, and PT@hARV825 did not exhibit any cytotoxicity to either cell type (AD in Figure 6), preliminarily confirming the superior biocompatibility of histone as an endogenous carrier material. More importantly, when the ARV825 concentration was increased to 5 μM, the free ARV825 treatment group showed significant cytotoxicity, while the cell viability of the hARV825 and PT@hARV825 treatment groups, although slightly decreased, was still higher than that of the free drug group. This phenomenon indicates that loading ARV825 with histone can, to some extent, slow the drug release, reduce the direct contact between high concentrations of PROTAC molecules and cells, thereby mitigating its potential off-target toxicity. Besides cytotoxicity, blood compatibility is a crucial prerequisite for intravenous injection of nanomedicines. In vitro hemolysis experiments were used to evaluate the interactions between different formulations and erythrocytes. The results showed that at equivalent ARV825 concentrations of 1 μM and 5 μM, the hemoglobin release from the supernatant of ARV825, hARV825, and PT@hARV825 after co-incubation with mouse erythrocytes was extremely low, and the hemolysis rate was far below the 5% safety threshold (E, F in Figure 6). In conclusion, the PT@hARV825 nanosystem exhibits excellent in vitro biocompatibility, ensuring its subsequent in vivo intravenous injection application.

[0039] Example 7 This example illustrates a study on the cellular uptake of PT@hARV825: The key to efficient delivery of nanomedicines lies in their effective internalization by target cells. To verify the intracellular delivery efficiency of PT@hARV825, its distribution within cells was tracked using fluorescent labeling technology. FITC-labeled hARV825 and PT@hARV825 were prepared and co-incubated with HASMCs and RAW264.7 cells for different times, respectively. Intracellular fluorescence signals were observed using fluorescence microscopy. The results showed that with the extension of incubation time (2 h, 8 h, 24 h), the intracellular fluorescence signals of the hARV825 and PT@hARV825 groups continuously increased (Figure 7, A, B, D, E). Moreover, at the same time point, the fluorescence intensity of the PT@hARV825 treatment group was stronger than that of the hARV825 group (Figure 7, C, F). In HASMCs, nanoparticles showed a tendency to aggregate around the nucleus and disperse within the nucleus (Figure 7, A, B), while in RAW264.7, most of the nanomedicine was internalized within the nucleus (Figure 7, D, E). These results demonstrate that modification with the TAT transmembrane peptide significantly enhances the transmembrane capacity of nanoparticles, promoting endocytosis within cells. PEG modification, however, did not hinder this process; rather, it may have improved the efficient delivery of nanoparticles by stabilizing them. These findings provide direct evidence that nanoparticles can successfully deliver ARV825 to the vicinity of the nuclear target BRD4, thereby efficiently performing protein degradation. In conclusion, PT@hARV825, mediated by the TAT peptide and NLS, significantly enhances the uptake efficiency of target cells, laying a crucial delivery foundation for its efficient degradation of nuclear BRD4 at the cellular level.

[0040] Example 8 This embodiment is a study on the protection of HASMC physiological function by PT@hARV825: Dysfunction of vascular smooth muscle cells is a core cellular event in the development of aortic dissection. To investigate whether targeted degradation of BRD4 can reverse this pathological process, the therapeutic effect of PT@hARV825 was systematically evaluated using an Ang II-induced HASMCs pathological model. First, confocal microscopy results showed that Ang II treatment for 24 hours significantly upregulated the protein level of BRD4 in HASMCs, and its fluorescence signal was localized within the cell nucleus, indicating no translocation (Figure 8A). Furthermore, Western blotting showed that PT@hARV825 treatment could effectively reverse this high expression (Figure 8C, D), demonstrating its effective drug delivery.

[0041] Regarding apoptosis, TUNEL staining showed that PT@hARV825 pretreatment significantly reduced the number of TUNEL-positive cells, with better results than free ARV825 (Figure 9, A, B). Regarding phenotypic transformation, studies have shown that BRD4 can regulate smooth muscle cell differentiation. Results indicated that PT@hARV825 treatment effectively reversed the downregulation of smooth muscle cell contractile markers α-SMA and CNN1 induced by Ang II stimulation, as well as the upregulation of synthetic markers OPN and matrix metalloproteinases MMP2 and MMP9 (Figure 9, CH). Regarding inflammatory signaling pathways, similarly, studies have shown that the acetylated reader BRD4 can activate the NLRP3 inflammasome and coordinate the integration of IRF3 and NF-κB in atypical STING-PERK-mediated vascular endothelial inflammation. Therefore, subsequent studies showed that PT@hARV825 treatment significantly inhibited the activation of the STING / NF-κB pathway, manifested as a decrease in P-STING and P-IκBα levels (Figure 9, IK). Furthermore, BRD4 can promote the expression of inflammatory factors. RT-qPCR results showed that PT@hARV825 reduced the mRNA expression of Ang II-induced pro-inflammatory cytokines IL-1β, TNF-α, and IL-6. Figure 9 (L).

[0042] Finally, its classic functions in regulating abnormal proliferation and migration of smooth muscle cells (HASMCs) and senescence were examined. Ki67 immunofluorescence staining and cell scratch assays showed that Ang II stimulation significantly promoted the proliferation and migration of HASMCs, while PT@hARV825 treatment effectively inhibited this abnormally active cellular behavior (AE in Figure 10). Western blotting results from PCNA also confirmed this finding (F, G in Figure 10). Regarding cellular senescence, PT@hARV825 effectively inhibited the expression of senescence-related markers such as P53 / P21 / P16, which were upregulated by Ang II treatment (HK in Figure 10). In summary, PT@hARV825, through efficient degradation of BRD4, improved the dysfunction of HASMCs in multiple dimensions, including apoptosis, phenotypic transformation, proliferation and migration, inflammatory response, and cellular senescence, providing strong cellular-level evidence for the treatment of Alzheimer's disease (AD).

[0043] Example 9 This embodiment is a study on the physiological function of PT@hARV825 in protecting RAW264.7: Macrophage-mediated inflammatory responses also play a crucial role in the development and progression of aortic dissection. To comprehensively evaluate the therapeutic potential of PT@hARV825, its functional regulatory effect on LPS-stimulated RAW264.7 macrophages was further investigated. Similarly, LPS stimulation for 24 hours significantly upregulated BRD4 protein expression in macrophages, and it remained localized in the nucleus (Figure 8, B), while PT@hARV825 treatment effectively reversed this high expression (Figure 8, E, F).

[0044] At the level of inflammatory cytokine expression, RT-qPCR results showed that PT@hARV825 treatment significantly inhibited the mRNA levels of pro-inflammatory cytokines IL-1β, TNF-α, IL-6, and BRD4 itself, which were upregulated by LPS stimulation, demonstrating a strong anti-inflammatory transcriptional regulatory capacity (Figure 11, A). At the level of inflammatory signaling pathways, Western blot results showed that LPS stimulation activated the STING / NF-κB innate immune pathway, manifested by a significant increase in the protein levels of P-STING and P-IκBα, as well as increased protein expression of MMP9 and MMP2. The intervention of PT@hARV825 effectively blocked the expression of these markers (Figure 11, BG), suggesting that its anti-inflammatory effect may be related to its inhibition of BRD4-mediated immune pathway activation. Finally, the effect of PT@hARV825 on macrophage polarization was evaluated. By detecting the expression of the M1 marker iNOS and the M2 marker CD206, it was found that PT@hARV825 treatment not only significantly reduced the level of iNOS, but also upregulated the expression of CD206 (HJ in Figure 11). This bidirectional regulatory effect on polarization markers strongly suggests that PT@hARV825-mediated BRD4 degradation can not only inhibit pro-inflammatory M1 polarization, but may also promote the transformation of macrophages into M2 cells with anti-inflammatory and tissue repair functions, thereby playing a more comprehensive protective role at the level of immune inflammation.

[0045] Example 10 This example demonstrates the targeted application of PT@hARV825 to AD sites: Whether nanomedicines can remain stable in circulation and effectively accumulate at the lesion site is crucial to their in vivo efficacy. To evaluate the in vivo delivery performance of PT@hARV825, a series of studies were conducted using small animal in vivo imaging technology. Figure 12(Figure A) First, the far-infrared fluorescent probe DiD was used instead of ARV825 to evaluate its pharmacokinetic characteristics in AD model mice. The results showed that the fluorescence signal in each group gradually decayed. Notably, the PT@hDiD group exhibited the best blood circulation stability, with its fluorescence signal essentially metabolized after 48 hours (Figure 12, B, C). This is attributed to the "invisible" protection provided by the PEG shell, which effectively prevented the nanoparticles from being rapidly cleared by the reticuloendothelial system. Based on this, subsequent in vivo treatment experiments determined that the drug should be administered once every 48 hours.

[0046] Next, the targeting ability of the nanosystem to AD lesions was evaluated. In in vitro aortic imaging, the aorta of mice in the PT@hDiD group showed the strongest fluorescence signal accumulation, especially in the dissection lesion area, compared to the DiD and hDiD groups (Figure 12, D, E). This indicates that the PEG shell is stable in the blood, and when the nanoparticles reach the slightly acidic environment of the AD lesion site, the acid-unstable Schiff base bonds break, the PEG shell detaches, and the exposed TAT peptide mediates the enhanced retention and uptake of nanoparticles at the lesion site. To further clarify the cellular fate of nanoparticles in the blood vessel wall, we performed frozen sections of the aorta and co-stained with immunofluorescence. The results showed that PT@hDiD co-localized with the vascular smooth muscle cell marker α-SMA and the macrophage marker CD68, and the signals were stronger than those in other groups (Figure 12, F, G), providing direct morphological evidence that the nanoparticles can be effectively internalized by these two key cell types in the diseased blood vessels and precisely deliver ARV825. Finally, in vivo imaging and ex vivo imaging of major organs showed that PT@hDiD accumulation in the liver was relatively low, and its distribution in non-target organs was also maintained at a low level (Figure 13, A, B). This optimized tissue distribution pattern is expected to reduce potential systemic side effects. In summary, the PT@hARV825 nanosystem possesses excellent cyclic stability, lesion targeting capability, and precise delivery characteristics at the cellular level.

[0047] Example 11 This example is a study on the prevention of AD by PT@hARV825: Building upon the aforementioned in vitro and targeted studies, the in vivo therapeutic potential of PT@hARV825 was further evaluated in a classic BAPN-induced AD mouse model. Figure 14 As shown in Figure A, 3-week-old C57BL / 6 mice were administered BAPN by gavage for 28 consecutive days, with Ang II injected intraperitoneally for the last three days. Treatment groups received Ang II via tail vein injection every 48 hours starting the day before model establishment.

[0048] Survival analysis (Figure 14, B) showed a sharp decline in survival rate in the AD model group mice, while the survival rate in the treatment group improved, with PT@hARV825 showing the best effect. Consistent with this conclusion, the high mortality rate and AD incidence rate at the experimental endpoint in the model group were significantly lower in the PT@hARV825 treatment group (Figure 14, C, D). Furthermore, significant dissecting hematoma and aneurysm formation were observed in the model group. Although the severity of lesions was reduced in the free ARV825 treatment group, the aorta in the PT@hARV825 treatment group was macroscopically closest to the normal group, visually demonstrating its superior vascular protective effect (Figure 14, E). Ultrasound imaging on day 25 showed that the diameters of both the thoracic and abdominal aortas in the AD model group were significantly increased, while PT@hARV825 treatment more effectively inhibited this pathological vasodilation, maintaining the aortic diameter close to the normal physiological range (Figure 14, FH). In summary, PT@hARV825 can effectively prevent the occurrence of Alzheimer's disease (AD) and fatal ruptures, significantly reducing the overall disease burden and demonstrating good application value.

[0049] Example 12 This example is a study on the protection of the aortic morphology integrity by PT@hARV825: To understand the extent of vascular lesions and the therapeutic effect of PT@hARV825, blood vessels from each group were collected and paraffin sections were prepared. Morphological staining was used to provide microscopic evidence. First, HE staining revealed typical vascular wall structural disorder and thickening in the AD model group. Second, Masson's trichrome staining showed abnormally large amounts of collagen deposition in the vascular wall of the AD model group, indicating pathological remodeling. Finally, EVG staining showed widespread breakage and fragmentation of membrane elastic fibers in the AD model group, while PT@hARV825 treatment significantly alleviated these pathological changes and maintained the normal layered structure of the vascular wall (AC in Figure 15). To further verify these findings, immunohistochemical analysis confirmed that PT@hARV825 treatment reversed the increased expression of BRD4 in AD tissue, accompanied by the loss of αSMA and the increase of CD68 and MMP9, effectively transforming the phenotype of HASMCs to a contractile state, reducing the infiltration of inflammatory cells, and inhibiting matrix degradation. Figure 15 (Hydrogen thromboembolism). In summary, these changes indicate that PT@hARV825 demonstrates remarkable efficacy in inhibiting pathological vascular remodeling, laying the foundation for further research into its molecular mechanisms and safety.

[0050] Example 13 This example illustrates the molecular mechanism by which PT@hARV825 prevents the occurrence of Alzheimer's disease (AD). To further elucidate the molecular mechanism by which PT@hARV825 delays AD progression in vivo, aortic tissues from mice in each group were collected for Western blotting and RT-qPCR analysis. First, the targeted degradation effect of the nanoparticles was verified. The results showed that the protein level of BRD4 in the aortic tissue of the PT@hARV825-treated group was significantly reduced (Figure 16, A, B), laying the foundation for downstream functional validation. Similarly, it was found that after PT@hARV825 treatment, the expression of contractile HASMC markers α-SMA and CNN1 was re-upregulated, while the expression of anabolic markers OPN and matrix metalloproteinases MMP2 and MMP9 was inhibited (Figure 16, CH). Regarding apoptosis regulation, PT@hARV825 treatment inhibited the increase in cleaved-Caspase-3 levels and increased the Bcl-2 / Bax ratio (Figure 16, IK). Regarding inflammatory signaling pathways, PT@hARV825 treatment effectively inhibited the phosphorylation levels of STING and IκBα (6L-N in Figure 1). Furthermore, PT@hARV825 significantly reduced the mRNA levels of inflammatory cytokines IL-1β, TNF-α, and IL-6 (O in Figure 16). Interestingly, the decrease in BRD4 mRNA levels in the aortic tissue of AD mice was reversed after PT@hARV825 treatment, which is attributed to the body's negative feedback regulatory mechanism. In conclusion, the outstanding efficacy demonstrated by PT@hARV825 provides another important strategy for the prevention and treatment of AD.

[0051] Example 14 This example is a study on the in vivo safety of PT@hARV825: To ensure the feasibility of the developed nanotherapy strategy in future clinical translation, its systemic toxicity in mice at therapeutic doses was systematically evaluated. First, HE staining of the heart, liver, spleen, lungs, and kidneys showed that the organ structures of all experimental groups were clear and the cell morphology was normal (Figure 17A). Second, serum samples were collected from mice for a series of serum biochemical metabolic analyses. Lipid metabolism-related indicators, including total cholesterol (T-CHO), triglycerides (TG), and high-density lipoprotein (HDL); core liver function indicators, aspartate aminotransferase (AST) and alanine aminotransferase (ALT); and key markers of kidney function, serum creatinine (CREA) and urea (UREA), showed no statistically significant differences among all experimental groups (Figure 17BD). In conclusion, the PT@hARV825 delivery system exhibits good biosafety, providing important safety evidence for its further development and translational applications.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. Application of BRD4 inhibitors in the preparation of products for the prevention and treatment of aortic dissection.

2. The application according to claim 1, characterized in that, The BRD4 inhibitor is ARV825.

3. A product for preventing and treating aortic dissection, characterized in that, This includes therapeutically effective doses of BRD4 inhibitors.

4. A method for constructing a multi-level nanodelivery system for preventing aortic dissection, characterized in that, The construction method includes the following steps: (a) A solution containing BRD4 inhibitor was added dropwise to a PBS solution containing histones to obtain a nanocomposite. (b) The TAT membrane-penetrating peptide was coupled to the surface of the nanocomposite to obtain a nanocomposite coupled with the TAT membrane-penetrating peptide. (c) The aldehyde-modified polyethylene glycol and the nanocomposite coupled with TAT membrane-penetrating peptide are subjected to a Schiff base reaction to obtain the multi-level nanodelivery system with both nuclear targeting and long circulation functions.

5. The construction method according to claim 4, characterized in that, In step (a), the BRD4 inhibitor is ARV825; the molar ratio of the histone to the BRD4 inhibitor is 1:(2~4).

6. The construction method according to claim 1, characterized in that, In step (b), the coupling of the TAT transmembrane peptide to the surface of the nanocomposite specifically includes: (1) Histones were added to a cross-linking agent DMSO solution and reacted in the dark, followed by dialysis to obtain activated histones; (2) DTT was added to the PBS solution of TAT membrane-penetrating peptide for reduction treatment, and then permeabilized to obtain reduced TAT membrane-penetrating peptide. (3) The activated histone and reduced TAT membrane-penetrating peptide were subjected to a light-protected reaction, followed by dialysis to obtain a nanocomposite of TAT membrane-penetrating peptide.

7. The construction method according to claim 6, characterized in that, In step (1), the crosslinking agent is succinimide 3-(2-pyridyldithio)-propionate; the molar ratio of crosslinking agent to histone is (2~4):1; the reaction temperature is room temperature and the time is 50~70 min.

8. The construction method according to claim 6, characterized in that, In step (2), the molar ratio of TAT membrane-penetrating peptide to DTT is 1:(0.8~1.2); the reduction treatment temperature is room temperature and the time is 20~40 min; In step (3), the molar ratio of activated histone to reduced TAT transmembrane peptide is 1:(2~4); the reaction temperature is room temperature and the time is 6~10h.

9. The construction method according to claim 4, characterized in that, In step (c), the molar ratio of aldehyde-modified polyethylene glycol to the nanocomposite of coupled TAT membrane-penetrating peptide is (8~10):1; the Schiff base reaction is carried out at room temperature and the pH value is 7.5~8.

5.

10. The multi-level nanodelivery system for preventing aortic dissection constructed by the construction method of any one of claims 4 to 9.

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