PMX-53 nano drug-loaded particle and application thereof in treatment of thoracic aortic diseases

Abstract

The invention relates to PMX-53 nano drug-loaded particles and application thereof in treatment of thoracic aorta diseases, and belongs to the technical field of biological medicines. The Fe3O4 (at) PDA-PMX-53 nano drug-loaded particle disclosed by the invention comprises a superparamagnetic ferroferric oxide nano particle, a polydopamine shell and a complement C5a receptor inhibitor PMX-53, wherein the superparamagnetic ferroferric oxide nano particle is coated with the polydopamine shell, and the complement C5a receptor inhibitor PMX-53 is covalently connected to the polydopamine shell. According to the PMX-53 nano drug-loaded particle disclosed by the invention, a drug is guided to be specifically enriched on the inner surface of a blood vessel of a thoracic aorta by utilizing a magnetic targeting technology on the premise of overcoming a blood flow shearing force, so that the drug deeply permeates into a middle membrane layer and is used for treating thoracic aortic diseases, particularly C5a-mediated thoracic aortic diseases, such as thoracic aortic aneurysm and (TAAD); while the treatment effect is improved, the distribution of the medicine in the aorta abdominalis is effectively reduced, so that the side effects are avoided.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a PMX-53 nanoparticle drug-loaded particle and its application in the treatment of thoracic aortic diseases. Background Technology

[0002] Thoracic aortic diseases, including atherosclerosis, calcification, aneurysms, and dissections, are a group of serious cardiovascular diseases that threaten life and health. One of their common pathological mechanisms involves abnormal activation of the complement system, especially the chronic inflammatory response driven by the binding of C5a to its receptor (C5aR). This process can lead to persistent damage and structural remodeling of the vessel wall. A paper with DOI number 10.1007 / s11427-014-4711-5 points out that blocking C5a-C5aR signaling can effectively inhibit vascular lesions. Among them, thoracic aortic aneurysm and dissection (TAAD) is a serious cardiovascular disease with rapid onset and high mortality. Current clinical treatment options mainly include open surgery and endovascular repair, but these treatments are highly invasive and have a narrow range of indications. Drug therapy often uses systemic administration, which has limitations such as poor targeting and insufficient local effective concentration.

[0003] To address the aforementioned inflammatory mechanisms, complement C5a receptor inhibitors (such as PMX-53) have demonstrated significant potential in suppressing inflammatory responses. A paper with DOI number 10.1096 / fj.10-174284 indicates that PMX-53 can effectively reduce inflammatory cell infiltration in the vascular wall and delay the progression of atherosclerosis. However, previous research suggests that PMX-53 exhibits site-specific heterogeneity in treating TAAD: if it acts on the abdominal aorta, it may actually promote the development of abdominal aortic aneurysm (AAA). Therefore, achieving precise drug enrichment in thoracic aortic lesions while avoiding side effects in the abdominal aorta and other non-target organs remains a pressing technical challenge.

[0004] In recent years, the rapid development of nanomedicine and targeted delivery technology has opened up new research directions for the treatment of TAAD. To address the drug delivery problem, existing strategies often encapsulate drugs in liposomes or polymeric nanoparticles (such as PLGA), attempting to utilize the increased permeability of the vascular wall at the lesion site (i.e., enhanced permeability and retention effect, EPR effect) to achieve passive enrichment. However, this strategy faces serious challenges in the treatment of TAAD. A paper with DOI number 10.1021 / acsnano.8b08875 points out that the endothelial barrier in atherosclerotic or vascular lesion areas is highly heterogeneous. In particular, as the disease progresses, the endothelial junctions at the lesion site may undergo structural remodeling and stabilization (i.e., endothelial normalization), leading to a significant decrease in its passive permeability to nanoparticles. At the same time, this study also found that the uptake of nanoparticles by late-stage plaques is significantly reduced compared to early-stage lesions, and passive targeting mainly depends on superficial vascular leakage. For TAAD, a disease whose core lesion is located deep within the aorta (media layer), passive targeting strategies relying solely on the EPR effect are insufficient to ensure effective drug penetration of the endothelium and accumulation in this crucial media layer. Furthermore, the hemodynamic environment of the thoracic aorta is extremely complex, characterized primarily by extremely high wall shear stress. A paper with Doi number 10.1096 / fj.10-174284 points out that when vascular endothelial cells are subjected to high shear forces, fluid erosion becomes a significant obstacle to nanoparticle adhesion and endocytosis; if the external traction force on the particles is insufficient to overcome fluid resistance, they are cleared upon contact with the vessel wall. Therefore, a nanodelivery system loaded with PMX-53 must not only overcome the extreme blood flow shear forces of the thoracic aorta but also precisely penetrate deep into the thoracic aorta to improve therapeutic efficacy. Existing passive nanodelivery technologies cannot avoid the potential side effects of PMX-53 on the abdominal aorta, nor can they achieve deep penetration into the media layer of the thoracic aorta. Therefore, there is an urgent need to develop a precision delivery system that can actively overcome the heterogeneous response of PMX-53 sites and drive deep drug penetration. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem that the PMX-53 drug delivery system in the prior art cannot accurately target the deep layers of the thoracic aorta.

[0006] To address the aforementioned technical problems, this invention provides a PMX-53 drug-loaded nanoparticle and its application in the treatment of thoracic aortic diseases. This invention provides a Fe3O4@PDA-PMX-53 drug-loaded nanoparticle with magnetic targeting capability. The Fe3O4@PDA-PMX-53 drug-loaded nanoparticle comprises superparamagnetic iron oxide nanoparticles, a polydopamine shell coating the superparamagnetic iron oxide nanoparticles, and PMX-53 covalently linked to the polydopamine shell. Utilizing magnetic targeting technology, the PMX-53 drug-loaded nanoparticles of this invention guide the drug to specifically accumulate on the inner surface of the thoracic aorta while overcoming blood flow shear forces, thereby penetrating deeply into the tunica media for the treatment of thoracic aortic diseases, particularly those mediated by abnormal C5a activation, such as thoracic aortic aneurysms and aortic dissections (TAAD). This improves therapeutic efficacy while effectively reducing drug distribution in the abdominal aorta, thus avoiding side effects.

[0007] The first objective of this invention is to provide a PMX-53 nanoparticle drug-carrying particle, the PMX-53 nanoparticle drug-carrying particle comprising superparamagnetic iron oxide nanoparticles, a polydopamine shell coating the superparamagnetic iron oxide nanoparticles, and PMX-53 covalently linked to the polydopamine shell.

[0008] Furthermore, the PMX-53 drug-loaded nanoparticles of this invention can overcome the wall shear stress of the thoracic aorta and penetrate the media. The hemodynamic environment of the thoracic aorta is extremely complex, mainly characterized by extremely high wall shear stress. The paper with Doi number 10.1096 / fj.10-174284 points out that when vascular endothelial cells are subjected to high shear forces, fluid scouring becomes a significant obstacle to the adhesion and endocytosis of nanoparticles; if the external traction force on the particles is insufficient to overcome fluid resistance, they will be removed the instant they contact the vessel wall. Commonly used α-Fe2O3 (hematite) is a parasitic weakly ferromagnetic material, with a saturation magnetization typically only 0.2-0.4 emu / g. The Fe3O4 (magnetite) selected in this invention is a ferrimagnetic / superparamagnetic material, with a saturation magnetization typically as high as 90-92 emu / g. Based on the magnetophoretic force formula, under the same external magnetic field gradient and similar particle size, the magnetic traction force on α-Fe₂O₃ is only about 1 / 200 that on Fe₃O₄. Such a weak magnetic force is completely insufficient to resist the aorta's high magnetic field strength of 100 dyne / cm². 2 The above refers to the blood flow shear force. Therefore, if a weakly ferromagnetic material such as α-Fe2O3 is used as the core, even with the application of a magnetic field, the drug cannot achieve initial anchoring at the lesion site.

[0009] Besides magnetic strength, the magnetic domain structure of particles (whether they are superparamagnetic) directly determines their microscopic distribution behavior within the blood vessel wall. Non-superparamagnetic (i.e., remanent) magnetic particles often exhibit drastically different behaviors under a magnetic field: first, aggregation effect, due to magnetic dipole interactions, non-superparamagnetic particles easily form chain-like or cluster-like aggregates on the inner surface of the blood vessel wall; second, permeation blockage, these aggregates are much larger than the interendothelial space, causing materials that could originally pass through the endothelium to ultimately only "accumulate" on the inner surface of the blood vessel near the magnet, unable to penetrate deep into the media, let alone achieve uniform distribution. The PMX-53 drug-loaded nanoparticles of this invention overcome these difficulties, not only overcoming blood flow shear forces but also penetrating the media to achieve deep targeting of thoracic aortic aneurysms and dissections.

[0010] Furthermore, it is covalently linked via CN single bonds.

[0011] Furthermore, the hydrodynamic particle size of the PMX-53 nanoparticles is 100-150 nanometers.

[0012] Furthermore, the CAS number of the PMX-53 is 219639-75-5.

[0013] A second objective of this invention is to provide a method for preparing the above-mentioned PMX-53 drug-loaded nanoparticles, comprising the following steps:

[0014] S1. Superparamagnetic iron oxide (Fe3O4) was mixed and reacted with dopamine to obtain Fe3O4@PDA nanoparticles;

[0015] S2. PMX-53 and Fe3O4@PDA nanoparticles are co-incubated to obtain the magnetic drug-carrying nanoparticles.

[0016] Furthermore, the mass ratio of the Fe3O4@PDA nanoparticles to PMX-53 is (1-5):1.

[0017] Furthermore, the co-incubation temperature is 1-10℃.

[0018] A third objective of this invention is to provide the application of the above-described PMX-53 drug-loaded nanoparticles or the PMX-53 drug-loaded nanoparticles prepared by the above-described preparation method in the preparation of drugs for treating thoracic aortic diseases, wherein the thoracic aortic diseases refer to thoracic aortic diseases mediated by abnormal activation of C5a.

[0019] Furthermore, the thoracic aortic disease includes thoracic aortic aneurysm / dissection.

[0020] A fourth objective of this invention is to provide a therapeutic product targeting thoracic aortic disease, comprising the above-described PMX-53 drug-loaded nanoparticles or PMX-53 drug-loaded nanoparticles prepared by the above-described preparation method, wherein the thoracic aortic disease refers to thoracic aortic disease mediated by abnormal activation of C5a.

[0021] Furthermore, the therapeutic product is administered via intravascular injection.

[0022] Furthermore, the dosage forms of the therapeutic drugs include tablets, powders, suspensions, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops, or pills.

[0023] Furthermore, the therapeutic product also includes a device capable of emitting a magnetic field.

[0024] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0025] The Fe3O4@PDA-PMX-53 drug-carrying nanoparticles of this invention comprise superparamagnetic iron oxide nanoparticles, a polydopamine shell coating the superparamagnetic iron oxide nanoparticles, and PMX-53 covalently linked to the polydopamine shell. Utilizing magnetic targeting technology, these PMX-53 drug-carrying nanoparticles guide the drug to specifically accumulate on the inner surface of the thoracic aorta while overcoming blood flow shear forces, thereby penetrating deep into the tunica media for the treatment of thoracic aortic diseases, particularly those mediated by abnormal C5a activation. This improves therapeutic efficacy while effectively reducing drug distribution in the abdominal aorta, thus avoiding side effects. Attached Figure Description

[0026] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0027] Figure 1 This is a schematic diagram of the synthesis of Fe3O4@PDA-PMX-53;

[0028] Figure 2 These are scanning electron microscope images of Fe3O4, Fe3O4@PDA, and Fe3O4@PDA-PMX-53;

[0029] Figure 3 This is the elemental distribution diagram of Fe3O4@PDA-PMX-53;

[0030] Figure 4 This is the Fourier transform infrared spectrum of Fe3O4@PDA-PMX-53, where the solid lines are the reference spectral lines of PMX-53 and the dashed lines are the reference spectral lines of Fe3O4@PDA.

[0031] Figure 5 These are the hydrodynamic particle size distribution diagrams for Fe3O4, Fe3O4@PDA, and Fe3O4@PDA-PMX-53.

[0032] Figure 6 This is a diagram showing the results of an erythrocyte compatibility test;

[0033] Figure 7 The fluorescence images and corresponding quantitative analysis after rhodamine and DAPI staining in the cell uptake experiment;

[0034] Figure 8 This is an in vivo imaging image of the mouse thoracic aorta after a magnetic field has been applied.

[0035] Figure 9 This is a diagram showing the results of Prussian blue staining;

[0036] Figure 10 These are the survival curves of BAPN-induced TAAD model mice after receiving different treatments;

[0037] Figure 11 This refers to the incidence of events in BAPN-induced TAAD model mice after different treatments.

[0038] Figure 12 These are aortic ultrasound images of BAPN-induced TAAD model mice on day 28 after receiving different treatments;

[0039] Figure 13 It is an ex vivo organ imaging technology using BTO@PDA and BTO@PDA-RGD. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0041] Example 1: Preparation of Fe3O4@PDA-PMX-53 (FPP)

[0042] 1. Synthesis of Fe3O4@PDA

[0043] First, a 2 mg / mL Fe3O4 stock solution was vortexed for 1 minute to ensure a homogeneous dispersion. 2.5 mL of this dispersion (containing 5 mg Fe3O4) was then accurately measured and placed in a reaction vessel, and 40 mL of a pre-prepared Tris-HCl buffer system (10 mmol / L, pH adjusted to 8.5) was added.

[0044] The mixture was placed in an ice-water bath and dispersed using an ultrasonic disruptor with the following parameters: 30% power, 5 seconds of operation followed by a 10-second interval, for a total ultrasonic treatment time of 30 minutes. Subsequently, 10 mg of dopamine hydrochloride was dissolved in 10 mL of deionized water and rapidly injected into the ultrasonically dispersed magnetic suspension. A magnetic stirrer was immediately started and set to 500 rpm. The reaction was carried out continuously for 6 hours at room temperature and under strict light protection to initiate in-situ self-polymerization of dopamine on the Fe3O4 surface.

[0045] After the reaction was completed, the product was separated by magnetic adsorption using an external magnetic field, and the supernatant was removed. The resulting precipitate was resuspended in deionized water and vortexed twice. Finally, the purified Fe3O4@PDA nanoparticles were dispersed in 2.5 mL of deionized water (final concentration approximately 2 mg / mL) and stored at 4 °C for later use.

[0046] 2. Load of PMX-53

[0047] Accurately weigh an appropriate amount of PMX-53 dry powder, dissolve it completely in deionized water, and prepare a drug stock solution with a concentration of 1 mg / mL. Take the prepared Fe3O4@PDA dispersion and add it to a sterile centrifuge tube according to a carrier-to-drug mass ratio (Fe3O4@PDA:PMX-53) of 2:1. After vortexing to mix the system evenly, wrap the container with aluminum foil to isolate it from light. Place the mixture on a vertical rotary mixer and incubate it at 4°C for 12 hours to achieve sufficient drug loading through the physical adsorption and functional group interaction of the PDA shell. After the reaction endpoint is reached, collect the solid product Fe3O4@PDA-PMX-53 by magnetic response and gently wash it with deionized water to remove unbound free drug, thus obtaining the target nanocomposite (e.g., ...). Figure 1 (As shown).

[0048] Example 2: Characterization of Fe3O4@PDA-PMX-53 (FPP) material

[0049] 1. Morphology and elemental analysis by scanning electron microscopy (SEM)

[0050] Scanning electron microscopy (SEM) provides a direct record of the material's structural evolution through observation of its microstructure. Microscopic images are shown below. Figure 2As shown, the initial Fe3O4 magnetic core exhibits a smooth and neatly arranged spherical particle morphology with uniform particle size. After in-situ polymerization with the introduction of dopamine, the particle volume slightly increases and the surface roughness changes, forming a dense polymer coating layer, confirming the initial construction of the core-shell structure. Furthermore, after further loading with PMX-53, the resulting Fe3O4@PDA-PMX-53 composite still maintains good dispersibility and a spherical appearance, with no obvious aggregation observed. This indicates that the polydopamine intermediate layer not only provides drug binding sites but also effectively enhances the colloidal stability of the nanosystem. Simultaneous energy-dispersive X-ray spectroscopy (EDS) elemental mapping scans further reveal (…). Figure 3 The Fe element, representing the magnetic core, is highly concentrated at the center of the particle, while the N element, representing the organic components (PDA and PMX-53 peptide chain), is uniformly distributed around the periphery of the particle, with a high degree of overlap in their spatial distribution. This elemental distribution characteristic definitively proves that a multi-layered core-shell structure with Fe3O4 as the core, PDA as the middle layer, and PMX-53 as the outer functional molecule has been successfully constructed, laying a solid material foundation for subsequent magnetically responsive guided therapy.

[0051] 2. Fourier Transform Infrared (FTIR) Spectral Analysis

[0052] Functional groups in the stepwise synthesized nanoparticles were detected using Fourier transform infrared spectroscopy. Spectral analysis is shown below. Figure 4 As shown, the final product Fe3O4@PDA-PMX-53 exhibits significantly different characteristic peaks in its infrared spectrum compared to the precursor. Specifically, at 1650 cm⁻¹... -1 and 1540 cm -1 A distinct absorption band was detected nearby, corresponding to the characteristic signals of the amide I band (C=O stretching vibration) and amide II band (NH bending vibration) in the peptide chain structure, respectively. Given that PMX-53 has a cyclic hexapeptide structure, the presence of the aforementioned amide bond characteristic signals strongly confirms that the -NH2 group in the PMX-53 molecule has reacted with the quinone group on the surface of the polydopamine shell, ultimately linked by a stable CN single bond, demonstrating the effectiveness of the drug loading process.

[0053] 3. Dynamic Light Scattering (DLS) Particle Size Distribution Analysis

[0054] The hydrodynamic diameter and dispersion state of the Fe3O4@PDA-PMX-53 composite material in an aqueous environment were determined using dynamic light scattering (DLS) technology. The test results are as follows: Figure 5As shown, this composite nanomaterial exhibits a monodisperse distribution pattern, with its average hydrodynamic particle size concentrated in the 100-150 nanometer range. This specific nanoscale size range is a key physical prerequisite for the material to penetrate deep into aortic lesions. On the one hand, its particle size is much smaller than the interendothelial space, providing a physical basis for the material to cross the endothelial barrier and diffuse into deep tissues of the vascular wall (such as the media). On the other hand, compared with large-sized particles, this size range can effectively reduce the non-specific clearance of the reticuloendothelial system, thereby prolonging the in vivo circulation half-life. Combined with the internal magnetic core, it is conducive to achieving efficient directional capture and targeted enrichment under the action of an external magnetic field.

[0055] Example 3: Biosafety assessment of Fe3O4@PDA-PMX-53

[0056] This embodiment systematically evaluated the biosafety of the nanocomposite in blood circulation through an in vitro erythrocyte hemolysis experiment. The specific test procedure and results are as follows:

[0057] 1. Experimental Methods

[0058] First, fresh whole blood samples were collected from healthy mice and immediately injected into a sterile container containing an anticoagulant (sodium heparin, final concentration 25 U / mL) to prevent blood clotting. Then, the supernatant plasma components were removed by low-speed centrifugation (300 g, 5 minutes). The precipitated red blood cells were repeatedly washed with sterile saline (repeated 3 times) until the supernatant was clear and transparent, thus obtaining a high-purity red blood cell concentrate for later use.

[0059] The prepared erythrocyte resuspension was mixed with the test sample at a certain ratio. Different concentration gradients of Fe3O4@PDA carrier and Fe3O4@PDA-PMX-53 nanocomposite were set up for the experiment, with final concentrations of 50, 100, 200, and 400 μg / mL, respectively. To ensure the validity of the experimental results, two control groups were simultaneously set up: a negative control group using physiological saline as the medium to simulate a non-hemolytic environment; and a positive control group using 0.2% Triton X-100 solution as the medium to simulate a 100% complete hemolytic environment. All experimental groups were incubated in a 37°C constant temperature water bath for 90 minutes to simulate material-blood interactions under body temperature conditions.

[0060] After incubation, centrifuge again at 300 g for 5 minutes to settle unruptured red blood cells and nanomaterials. Carefully aspirate 100 μL of supernatant from each well and transfer it to a new 96-well ELISA plate. Measure the absorbance (OD value) at a characteristic wavelength of 540 nm using a microplate reader. Based on the absorbance data measured for each group, quantify the degree of hemolysis using the following formula:

[0061] Hemolysis rate (%) = (As - Anc) / (Apc - Anc) × 100%.

[0062] 2. Results Analysis

[0063] The experimental data showed that, firstly, the absorbance value of the negative control group was extremely low, and the calculated hemolysis rate was close to zero, confirming that the experimental operation was gentle and did not cause mechanical damage to the red blood cells; while the positive control group showed the expected complete hemolysis state, verifying the sensitivity and reliability of the detection system.

[0064] Analysis of the materials group showed that, across a wide concentration range of 50 to 400 μg / mL, both the unloaded Fe3O4@PDA carrier and the drug-loaded Fe3O4@PDA-PMX-53 complex maintained extremely low hemolysis rates, significantly below the ISO's hemolysis safety threshold (5%) for biomaterials. Even at the highest tested concentration (400 μg / mL), no significant signs of erythrocyte rupture and hemoglobin release were observed. Figure 6 ).

[0065] In summary, this nanocomposite exhibits excellent blood compatibility, meeting the safety requirements for intravenous administration.

[0066] Example 4: Evaluation of in vitro cellular uptake efficiency

[0067] This embodiment aims to investigate the influence of exogenous magnetic field and serum environment on the uptake of the nanocomposite by endothelial cells using an in vitro cell co-culture model.

[0068] 1. Experimental materials and cell model establishment

[0069] To achieve visualized tracking within cells, a strategy combining physical adsorption and chemical coupling was employed to load the Rhodamine B fluorescent probe onto the surface of a Fe3O4@PDA-PMX-53 nanocomposite, resulting in tracer nanoparticles exhibiting red fluorescence. Mouse microarterial endothelial cells were selected as a mimicry of the blood-brain barrier / vascular endothelium in vitro.

[0070] 2. Experimental grouping and intervention

[0071] Endothelial cells in the logarithmic growth phase were seeded into laser confocal microscopy culture dishes. Once the cells reached approximately 50%-60% confluence and exhibited well-developed morphology, the drug intervention experiment was initiated. The experimental design was based on two variables: "magnetic field" and "serum," constructing the following experimental systems: a magnetic targeting intervention group, in which neodymium iron boron permanent magnets were pre-placed at the bottom of the culture dish to provide a continuous and stable gradient magnetic field environment; and a passive uptake control group, which underwent routine culture without any external magnetic field. Within these two main groups, further subdivisions were made into a complete culture medium subgroup (containing serum to simulate a physiological blood environment) and a serum-free culture medium subgroup (excluding protein interference). An equal amount of rhodamine-labeled Fe3O4@PDA-PMX-53 complex (calculated as Fe3O4, with a final constant mass of 25 μg) was added to all experimental wells.

[0072] 3. Sample preparation and fluorescence imaging

[0073] The experiment included three kinetic observation time points: 10 minutes, 2 hours, and 4 hours after incubation began. Upon reaching the predetermined time points, the culture was immediately terminated, and the drug-containing culture medium was aspirated. The monolayer of cells was gently washed three times with pre-cooled phosphate-buffered saline (PBS) to thoroughly remove free nanoparticles adhering to the cell surface but not internalized. Subsequently, the cells were fixed with 4% paraformaldehyde solution at room temperature for 15 minutes, and the nuclei were counterstained and located using DAPI staining. Image acquisition was performed using a laser scanning confocal microscope (CLSM): the rhodamine channel (red fluorescence) was activated for qualitative and semi-quantitative analysis of the intracellular enrichment of nanoparticles; the DAPI channel (blue fluorescence) was activated to determine the morphology and location of the nuclei.

[0074] 4. Results Analysis

[0075] Confocal microscopy and fluorescence intensity quantification results are as follows: Figure 7 As shown, firstly, under the same incubation time and serum conditions, the intracellular red fluorescence intensity of the magnetic targeting intervention group was significantly better than that of the passive uptake control group. This phenomenon strongly confirms that the external magnetic field can overcome fluid resistance, endow nanoparticles with directional driving force, thereby significantly promoting the capture and endocytosis efficiency of Fe3O4@PDA-PMX-53 by endothelial cells.

[0076] Longitudinal comparison of data at three time points showed that the intracellular fluorescence signal in all experimental groups exhibited a clear gradient increase with prolonged incubation time. This indicates that the composite material did not induce cellular rejection, but rather could be continuously and efficiently internalized by endothelial cells via a time-dependent endocytic pathway.

[0077] Example 5: In vivo functional verification and magnetic targeting assessment

[0078] This embodiment, based on a BAPN (β-aminopropionitrile)-induced thoracic aortic aneurysm / dissection (TAAD) mouse model, systematically evaluated the in vivo distribution characteristics, magnetic targeting enrichment patterns, and therapeutic efficacy of the nanocomposite. TAAD is a clinically severe form of thoracic aortic disease, but its pathological process is closely related to the inflammatory response mediated by abnormal C5a activation. Therefore, this embodiment can serve as a validation of the effectiveness of the present invention's technical solution in treating thoracic aortic diseases mediated by abnormal C5a activation.

[0079] 1. Animal model construction and experimental grouping

[0080] A mouse model that highly replicates the pathological features of human TAAD was established using the BAPN induction method. Successfully modeled animals were randomly assigned to four groups for a control study: a model control group (BAPN group), receiving only solvent control; a treatment group (PMX-53 group), receiving intracardiac injection of PMX-53; a passive treatment group, receiving intracardiac injection of Fe3O4@PDA-PMX-53 without external magnetic field application; and a magnetic-targeted treatment group, receiving intracardiac injection of Fe3O4@PDA-PMX-53 with an external magnetic field applied to the thoracic aortic region. The dosage was set at 0.02 mg / 10 g body weight (i.e., 2 mg / kg) of PMX-53, with a volume of 0.05 mL.

[0081] 2. In vivo magnetic targeting assessment

[0082] Fluorescence imaging of isolated thoracic aortas showed that, compared to the control group and the untreated material group (Fe3O4@PDA-PMX-53), the treated group (Fe3O4@PDA-PMX-53+MF) with an external magnetic field (MF) exhibited significantly enhanced fluorescence signal in the vascular tissue. Figure 8 ).

[0083] exist Figure 8 As can be seen, under the influence of a magnetic field, the total radiant efficiency of the material on the blood vessel wall is significantly higher than that of the group without a magnetic field (P<0.01). This result indicates that the Fe3O4@PDA-PMX-53 nanoplatform possesses excellent magnetic response characteristics. Utilizing the magnetism of Fe3O4 in the nanoparticle core, under the precise guidance of an applied magnetic field, this material can effectively resist the high-speed blood flow in the aortic environment, thereby significantly increasing its specific accumulation and long-term retention in the thoracic aorta, achieving highly efficient magnetically targeted drug delivery.

[0084] Prussian blue staining (for iron detection) was performed on sections of the mouse thoracic aorta subjected to different magnetic field exposure times. The results are as follows: Figure 9As shown, drug accumulation in the blood vessel wall exhibits a clear time-dependent effect. With the magnetic field adsorption time increasing from 2 hours to 4 hours, the positive staining area in the aortic medial layer significantly expanded and deepened in color.

[0085] After intervention according to the above optimized dosing regimen (once a week, with magnetic field assistance for 6 hours), the treatment effect was evaluated from multiple dimensions.

[0086] The results are as follows Figures 10-11 As shown in Table 1, compared with the mortality rate of the BAPN control group, the survival period of mice in the magnetic targeting treatment group was significantly prolonged and the survival rate was significantly improved. The application of magnetic targeting successfully avoided the promoting effect of PMX-53 on AAA.

[0087] Table 1. Incidence rates in different treatment groups

[0088]

[0089] Non-invasive examination of aortic morphology was performed using high-resolution small animal ultrasound. Image data are as follows: Figure 12 As shown, the aortic arch diameter dilation in mice treated with magnetic targeting was significantly inhibited, with no obvious signs of dissecting hematoma or aneurysm rupture, and the vascular morphology of the abdominal aorta tended to be normal. Although the examples were validated using the TAAD model, since this model embodies the core inflammatory mechanism mediated by abnormal C5aR activation, the technical solution of this invention also has the potential to be applied to other thoracic aortic diseases with the same pathological mechanism.

[0090] Comparative Example 1: Preparation and Limitation Verification of Actively Targeted Nanoparticles BTO@PDA-RGD

[0091] 1. Experimental Design and Scientific Hypothesis

[0092] This comparative study aims to validate the effectiveness of an active targeting strategy relying solely on biological ligands in the thoracic aortic environment. Previous research suggests that, through transcriptomic analysis and immunohistochemical verification, we found a significant overexpression of integrin ItgaV (Integrin alpha-V) on the surface of vascular endothelial cells in the lesion region during the pathological progression of thoracic aortic aneurysm / dissection (TAAD). Based on these findings, we propose a biological active targeting strategy: utilizing an arginine-glycine-aspartic acid (RGD) short peptide as a high-affinity ligand to specifically recognize and anchor to ItgaV receptors on the endothelial surface. Theoretically, this strategy can achieve site-specific enrichment of nanocarriers, thereby releasing therapeutic drugs (such as nitric oxide (NO) donors or inhibitors) to delay dissection progression.

[0093] 2. Preparation of BTO@PDA-RGD materials

[0094] To verify the above hypothesis, we prepared RGD-modified nanoprobes: using barium titanate (BTO) nanoparticles (approximately 100 nm in diameter) as the core and coating the surface with a polydopamine (PDA) layer, we obtained BTO@PDA. We mixed the RGD targeting peptide with BTO@PDA at a 1:1 mass ratio and reacted it in Tris-HCl buffer (pH 8.5) for 12 hours. Utilizing the chemical activity of the PDA surface, the RGD peptide was covalently coupled or physically adsorbed onto the particle surface, thus obtaining BTO@PDA-RGD.

[0095] 3. In vivo distribution and residence verification

[0096] The above materials were injected into TAAD model mice via intracardiac injection, without the application of an external magnetic field, relying solely on the bioaffinity between RGD and ItgaV for targeting. Their distribution was monitored using in vivo imaging in small animals and imaging of ex vivo organs.

[0097] 4. Results Analysis

[0098] The experimental results showed a significant discrepancy with the expected hypothesis: despite high expression of ItgaV in the endothelial cells of the lesions, the fluorescence signal in the thoracic aorta region of the BTO@PDA-RGD group was not significantly different from that of the untargeted BTO@PDA group. The vast majority of nanoparticles were rapidly captured by the liver and spleen after injection. Figure 13 This result reveals the limitations of active biological targeting in TAAD therapy. The thoracic aorta, as the blood vessel segment with the fastest blood flow and the greatest shear force in the human body, has a physical scouring force that is far greater than the non-covalent chemical binding force between the RGD peptide and the ItgaV receptor. Nanoparticles are swept away by the high-speed blood flow before or at the moment of binding to the receptor, and cannot complete the "recognition-adhesion-endocytosis" process.

[0099] 5. Conclusion

[0100] This comparative example powerfully demonstrates that, under the unique hemodynamic environment of the thoracic aorta, ligand-targeting strategies that solely target endothelial molecular markers (such as ItgaV) are insufficient for achieving long-term retention. This, in turn, confirms the necessity and superiority of the magnetic targeting strategy employed in this invention—that is, introducing a strong external physical field (magnetic field force) to forcibly retain and fix nanoparticles to the vessel wall, overcoming blood flow erosion. This approach can effectively achieve truly precise enrichment and deep penetration.

[0101] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A PMX-53 nanoparticle drug-loaded particle, characterized in that, The PMX-53 nanoparticles include superparamagnetic iron oxide nanoparticles, a polydopamine shell covering the superparamagnetic iron oxide nanoparticles, and PMX-53 covalently linked to the polydopamine shell.

2. The PMX-53 drug-loaded nanoparticles according to claim 1, characterized in that, The hydrodynamic particle size of the PMX-53 nanoparticles is 100-150 nanometers.

3. The method for preparing PMX-53 drug-loaded nanoparticles according to claim 1 or 2, characterized in that, Includes the following steps: S1. Superparamagnetic iron oxide (Fe3O4) was mixed and reacted with dopamine to obtain Fe3O4@PDA nanoparticles; S2. PMX-53 and Fe3O4@PDA nanoparticles are co-incubated to obtain the magnetic drug-carrying nanoparticles.

4. The preparation method according to claim 3, characterized in that, The mass ratio of Fe3O4@PDA nanoparticles to PMX-53 is (1-5):

1.

5. The preparation method according to claim 3, characterized in that, The co-incubation temperature is 1-10℃.

6. The use of the PMX-53 drug-loaded nanoparticles according to claim 1 or 2, or the PMX-53 drug-loaded nanoparticles prepared by the preparation method according to any one of claims 3-5, in the preparation of drugs for treating thoracic aortic diseases, characterized in that, The aforementioned thoracic aortic disease refers to thoracic aortic disease mediated by abnormal activation of complement C5a.

7. A therapeutic product targeting thoracic aortic disease, characterized in that, The invention comprises the PMX-53 nanoparticles as described in claim 1 or 2, or the PMX-53 nanoparticles prepared by the preparation method described in any one of claims 3-5, characterized in that the thoracic aortic disease refers to thoracic aortic disease mediated by abnormal activation of complement C5a.

8. The therapeutic product according to claim 7, characterized in that, The therapeutic product is administered via intravascular injection.

9. The therapeutic product according to claim 7, characterized in that, The dosage forms of the therapeutic drugs include tablets, powders, suspensions, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops, or pills.

10. The therapeutic product according to claim 7, characterized in that, The therapeutic product also includes a device capable of emitting magnetic fields.

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