Calpeptin magnetic nano drug-loaded particle for targeted enrichment of concentrated membrane layer and application of Calpeptin magnetic nano drug-loaded particle

Abstract

The invention relates to Calpeptin magnetic nano drug-loaded particles of a targeted enrichment membrane layer and application, and belongs to the technical field of biological medicines. The invention designs a Calpeptin magnetic nano drug-loaded particle for targeting a middle membrane layer, the Calpeptin magnetic nano drug-loaded particle adopts a core-shell structure, a magnetic core is composed of superparamagnetic ferroferric oxide nano particles, and the core endows the material with response ability to an external magnetic field, so that the material can be used for preparing the drug-loaded particle. The polymer shell layer is formed by wrapping the surface of the magnetic core with polydopamine (PDA), and Calpeptin is covalently connected to a polydopamine shell. The Calpeptin magnetic nano drug-loaded particle disclosed by the invention solves the technical problem that a composite material cannot efficiently penetrate through an aorta wall and is specifically enriched in a middle membrane layer, so that a reliable directional intervention scheme with in-vivo experimental evidence support is provided for accurate treatment of TAAD.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing Calpeptin magnetic nanoparticles with targeted enrichment of the film layer and their application. Background Technology

[0002] Thoracic aortic aneurysm / dissection (TAAD) is an acute and critical condition in which a tear in the aortic intima allows blood to enter the media, forming a dissecting hematoma. It is a type of cardiovascular disease with a high mortality rate and severe disease progression. Current treatment strategies mainly include open surgery and endovascular repair; however, these methods are highly invasive, have strict indications, and lack effective early intervention methods.

[0003] Drug therapy often employs systemic administration, such as directly using Calpeptin inhibitors. Calpeptin, a cell-permeable protease inhibitor, is administered systemically via intravenous or intraperitoneal injection in animal models. However, this systemic administration means the drug is distributed throughout the body via the bloodstream, failing to achieve effective accumulation at specific lesion sites (such as the aortic wall of aortic aneurysms / dissections). This results in insufficient local drug concentration at the lesion site, while other normal tissues are exposed to the drug, leading to a high risk of off-target effects. Due to the lack of targeted delivery and widespread distribution throughout the systemic circulation, high single-dose administration is necessary to achieve effective therapeutic concentrations at the lesion site. Furthermore, Calpain inhibitors are metabolically unstable in vivo with short half-lives, requiring frequent dosing to maintain effective blood drug concentrations. This not only reduces patient adherence but also complicates long-term treatment management.

[0004] In recent years, the development of nanomedicine and targeted delivery technology has provided new ideas for the treatment of TAAD (Transcatheter Aortic Injury). A paper with DOI number 10.7150 / thno.109325 points out that NPCAs nanoparticles with sizes of 80 nm, 150 nm, and 240 nm can all penetrate the damaged endothelial barrier and achieve significant enrichment in the aortic medial layer. Therefore, nanoparticles can be used to achieve deep delivery of Calpain inhibitors. For example, therapeutic drugs such as Calpain inhibitors can be encapsulated in liposomes or polymeric nanoparticles (such as PLGA). After intravenous injection, the nanoparticles circulate in the bloodstream. Due to the increased permeability of the diseased aortic wall, the particles passively accumulate to a certain extent in the lesion area, thus achieving a certain degree of targeted drug delivery. However, this technology utilizes the enhanced permeability and retention effect (EPR effect) to achieve nanoparticle enrichment in the lesion area, which largely depends on the pathological state of the lesion area and is subject to significant individual differences, resulting in limited enrichment and failing to achieve precise and efficient drug enrichment. Difficulty in penetrating deep into the lesion core: Passive targeting mainly relies on leaking blood vessels. For treatments requiring action on the aortic media, the simple EPR effect is insufficient to ensure effective drug penetration and accumulation in this critical structure. Therefore, further improvements are needed to enhance the delivery of Calpain inhibitors by nanoparticles to achieve deeper targeting. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem that drug-loaded nanoparticles in the prior art are difficult to penetrate the middle membrane layer and thus cannot achieve deep drug targeting.

[0006] To address the aforementioned technical problems, this invention provides a method for preparing Calpeptin magnetic nanoparticles targeting and enriching the aortic media and their applications. The aortic media is composed of dozens of layers of ring-arranged elastic fibers, collagen, and smooth muscle cells, exhibiting a dense and elastic structure that is the core for maintaining the structural integrity of the aortic wall. The core pathological processes of TAAD (such as smooth muscle cell apoptosis and elastic fiber rupture) occur precisely in the media. However, due to the dense structure of the media, conventional drug delivery systems cannot penetrate it. Therefore, this invention designs a Calpeptin magnetic nanoparticle targeting the media. The Calpeptin magnetic nanoparticle of this invention adopts a core-shell structure. The magnetic core is composed of superparamagnetic iron(III) oxide nanoparticles, which endow the material with the ability to respond to external magnetic fields. The polymer shell is formed by encapsulating polydopamine (PDA) on the surface of the magnetic core, which is deposited in situ on the Fe3O4 core surface through the self-polymerization reaction of dopamine under weakly alkaline conditions. The Calpeptin inhibitor is covalently linked to the polymer shell. The Calpeptin magnetic nanoparticles of this invention solve the technical problem that composite materials cannot efficiently penetrate the aortic wall and specifically accumulate in the media, thus providing a reliable and in vivo experimentally supported targeted intervention scheme for the precise treatment of TAAD.

[0007] The first objective of this invention is to provide a Calpeptin magnetic nanoparticle targeting the middle film layer, the Calpeptin magnetic nanoparticle comprising superparamagnetic iron oxide nanoparticles, a polydopamine shell encapsulating the superparamagnetic iron oxide nanoparticles, and Calpeptin covalently attached to the surface of the polydopamine shell.

[0008] Furthermore, Calpeptin is immobilized by covalent bonding between its terminal amino group and the quinone group on the surface of PDA.

[0009] Furthermore, Calpeptin's CAS number is 117591-20-5.

[0010] A second objective of this invention is to provide a method for preparing the above-mentioned Calpeptin magnetic nanoparticles, comprising the following steps:

[0011] S1. Reaction of superparamagnetic iron oxide with dopamine yields Fe3O4@PDA nanoparticles;

[0012] S2. Calpeptin inhibitor was co-incubated with Fe3O4@PDA nanoparticles to obtain the magnetic drug-loaded nanoparticles.

[0013] Furthermore, the mass ratio of the Fe3O4@PDA nanoparticles to Calpeptin is (1-2):(1-2).

[0014] Furthermore, in step S2, the co-incubation is carried out under light-protected conditions, and the co-incubation temperature is 3-10℃.

[0015] Furthermore, in step S1, the pH of the reaction is 7.5-9.

[0016] A third objective of this invention is to provide an application of the above-described Calpeptin magnetic nanoparticles or the Calpeptin magnetic nanoparticles prepared by the above-described preparation method in the preparation of drugs for the treatment of aortic aneurysm / dissection.

[0017] A fourth objective of this invention is to provide a therapeutic agent for aortic aneurysm / dissection, the therapeutic agent comprising the above-described Calpeptin magnetic nanoparticles or Calpeptin magnetic nanoparticles prepared by the above-described preparation method.

[0018] 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.

[0019] Furthermore, the routes of administration of the drug include intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

[0020] The fifth objective of this invention is to provide an application of the above-described Calpeptin magnetic nanoparticles or the Calpeptin magnetic nanoparticles prepared by the above-described preparation method in the preparation of an aortic aneurysm / dissection treatment device, wherein the treatment device further includes a device capable of emitting a magnetic field.

[0021] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0022] The Calpeptin magnetic drug-carrying nanoparticles of this invention employ a core-shell structure, with the magnetic core composed of superparamagnetic iron oxide nanoparticles. This core endows the material with the ability to respond to external magnetic fields. The polymer shell is formed by polydopamine (PDA) encapsulating the surface of the magnetic core. The Calpeptin magnetic drug-carrying nanoparticles of this invention solve the technical challenge of composite materials failing to efficiently penetrate the aortic wall and specifically accumulate in the media, thus providing a reliable and in vivo experimentally supported targeted intervention strategy for the precise treatment of TAAD. Attached Figure Description

[0023] 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.

[0024] Figure 1 This is a schematic diagram of the synthesis of Fe3O4@PDA-Calpeptin;

[0025] Figure 2 This is the Fourier transform infrared spectrum of Fe3O4@PDA-Calpeptin, where the solid line is the reference spectral line of Calpeptin and the dashed line is the reference spectral line of Fe3O4@PDA.

[0026] Figure 3 This is a hydrodynamic particle size distribution diagram of Fe3O4@PDA and Fe3O4@PDA-Calpeptin;

[0027] Figure 4 These are scanning electron microscope images and elemental distribution maps of Fe3O4, Fe3O4@PDA, and Fe3O4@PDA-Calpeptin;

[0028] Figure 5 This is the result of an erythrocyte compatibility test;

[0029] Figure 6 These are fluorescence images after rhodamine and DAPI staining in a cell uptake experiment;

[0030] Figure 7 This is an in vivo imaging image of the mouse aorta before the application of a magnetic field;

[0031] Figure 8 This is an ultrasound image of the mouse thoracic aorta after a magnetic field has been applied.

[0032] Figure 9 These are HE staining and EVG staining of the mouse aorta;

[0033] Figure 10 This is a diagram showing the enrichment of Fe3O4@PDA-Calpeptin composite material in the thoracic aorta under the guidance of an external magnetic field.

[0034] Figure 11 These are images of the staining results of the membrane layer in mice, where A is the Prussian blue staining result and B is the HE EVG staining result.

[0035] Figure 12 This is a scanning electron microscope image of the area in which a magnetic field was applied to the thoracic aorta of a mouse in Comparative Example 1. Detailed Implementation

[0036] 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.

[0037] The experimental methods and procedures involved in the following embodiments are as follows:

[0038] I. Prussian Blue Staining:

[0039] 1. Laboratory animals and materials

[0040] Experimental animals: Six healthy C57BL / 6 mice were selected.

[0041] Test material: The prepared Fe3O4@PDA-Calpeptin composite material.

[0042] 2. Experimental Methods

[0043] Material injection and magnetic field intervention: All six mice were injected with an equal dose of Fe3O4@PDA-Calpeptin composite material via intracardiac injection. Immediately after injection, a stable external magnetic field was applied to the surface of the aortic region of the mice and maintained continuously.

[0044] Tissue sampling: Tissue samples were collected at three predetermined time points: 2 hours, 4 hours, and 6 hours after material injection. Two mice were sacrificed at each time point, and their thoracic aortic tissue was quickly separated and removed.

[0045] Histological processing and staining: The obtained aortic tissue was fixed in 4% paraformaldehyde and embedded in paraffin to prepare serial sections. The sections were then stained with Prussian blue to specifically label the iron oxide component in the composite material.

[0046] Observation and Imaging: The staining of aortic sections was observed under an optical microscope, and images were acquired.

[0047] Example 1: Preparation method of Fe3O4@PDA-Calpeptin composite material

[0048] Step 1: Prepare Fe3O4@PDA nanoparticles.

[0049] After vortexing the Fe3O4 stock solution (2 mg / mL) for 1 minute, 5 mg of Fe3O4 was weighed and added to 40 mL of Tris-HCl buffer (10 mM, pH 8.5). The mixture was then ultrasonically dispersed at 30% power under ice bath conditions, with a working time of 5 seconds and an interval of 10 seconds, for a total ultrasonic time of 30 minutes. Next, 10 mg of dopamine hydrochloride was weighed and dissolved in 10 mL of ultrapure water (ddH2O). This solution was then rapidly added to the above system and immediately stirred at 500 RPM at room temperature in the dark for 6 hours. The product was then collected by magnetic separation, resuspended in ultrapure water, and vortexed. This operation was repeated twice. Finally, the mixture was resuspended in 2.5 mL of ultrapure water to obtain a Fe3O4@PDA dispersion with a concentration of 2 mg / mL.

[0050] Step 2: Calpeptin loading

[0051] Fe3O4@PDA has been prepared at a concentration of 2 mg / mL; the calpeptin stock solution has a concentration of 10 mM (i.e., 36.246 mg / mL), aliquoted into 20 μL tubes and stored at -80°C, and diluted to 1 mg / mL. For the synthesis of Fe3O4@PDA-calpeptin material, Fe3O4@PDA is first removed from the solution at 4°C and vortexed to evenly disperse the powder. The required dose is added to a 2 mL centrifuge tube. Calpeptin stock solution (e.g., 1 mg / mL in ddH2O) is then added at a predetermined mass ratio (Fe3O4@PDA:Calpeptin = 1:1), and the mixture is vortexed to mix.

[0052] Wrap the mixture in aluminum foil to protect it from light, place it on a rotary mixer, and react at 4°C for 12 hours to allow Calpeptin to fully load. Attach a magnet to the tube wall and allow it to stand until the particles are completely adsorbed; discard the supernatant and add the same volume of ultrapure water as the initial solution for washing; repeat the washing step several times until the supernatant is clear. Discard the supernatant, add PBS to the tube, and bring the solution to a final volume of 0.2 mg / mL.

[0053] Finally, the final product Fe3O4@PDA-Calpeptin was collected by magnetic separation and washed with ddH2O to remove unbound drug, thus obtaining the target composite material.

[0054] Example 2: Characterization Analysis of Composite Materials

[0055] 1. Fourier transform infrared spectroscopy analysis

[0056] Fourier transform infrared spectroscopy analysis was performed on the synthesized Fe3O4@PDA-Calpeptin composite material. The results showed that vibrational absorption peaks of characteristic functional groups in the Calpeptin molecule could be observed in the infrared spectrum of Fe3O4@PDA-Calpeptin, including but not limited to the stretching vibration peak of the amide bond (-CONH-) (approximately 1640 cm⁻¹). -1 The characteristic absorptions of Calpeptin and other specific functional groups in the Calpeptin structure (such as ester groups and aromatic rings) were observed. The appearance of these characteristic peaks confirmed that Calpeptin had been successfully modified onto the surface of Fe3O4@PDA nanoparticles via covalent linkage, achieving effective loading of Calpeptin.

[0057] exist Figure 2 In the middle, located at approximately 3300 cm -1 The broad absorption peak at this point is attributed to the stretching vibrations of the NH and OH bonds. This peak is maintained or enhanced after loading Calpeptin, which includes the contribution of NH vibrations from the peptide chain and terminal amino acid residues in the Calpeptin molecule, indicating that nitrogen-containing functional groups are introduced into the system.

[0058] Located at approximately 1650 cm -1 Approximately 1530 cm -1 The two strong absorption peaks at the position are attributed to the amide I band (C=O stretching vibration) and the amide II band (coupling of NH bending vibration and CN stretching vibration), respectively. These two peaks are typical characteristics of polypeptides (peptide bonds), and their significant enhancement directly confirms that the Calpeptin molecular backbone structure has been successfully attached to the composite material.

[0059] Located at approximately 1160 cm -1 Approximately 1115 cm -1 The absorption peaks at this location are attributed to the stretching vibrations of aliphatic CN bonds and CO bonds. These characteristic peaks in the fingerprint region further confirm the presence of specific amino acid side chain structures in the Calpeptin molecule. Located at approximately 1250 cm⁻¹ -1 The absorption peak at this location is clearly attributed to the CN bond stretching vibration of the aromatic amine structure in the polydopamine (PDA) coating. The presence of this characteristic peak is a clear indication of the successful construction and retention of the PDA substrate.

[0060] 2. DLS particle size analysis

[0061] The prepared Fe3O4@PDA-Calpeptin composite material was systematically characterized. Dynamic light scattering analysis results showed (e.g.) Figure 3As shown in the figure, the hydrodynamic particle size of the composite material in the aqueous medium is mainly distributed in the range of 120-180 nanometers, and its particle size distribution curve shows a single, narrow peak shape.

[0062] This particle size distribution is crucial for the efficient penetration of the material into the membrane layer of the aorta. First, these nanoscale particles can utilize the endothelial spaces, providing physical feasibility for initial extravasation from the vascular lumen into the vascular wall tissue. Second, this size range optimizes the material's circulation time in vivo: it is small enough to avoid premature clearance by the body, yet large enough to achieve efficient capture and enrichment under the guidance of an external magnetic field.

[0063] 3. Analysis of Scanning Electron Microscopy Results

[0064] The morphology and structure of the Fe3O4@PDA-Calpeptin composite material were characterized by scanning electron microscopy (SEM). The results clearly demonstrate the successful preparation of the material and its structural evolution (e.g., Figure 4 (As shown).

[0065] Unmodified Fe3O4 nanoparticles are regularly spherical or near-spherical with relatively smooth surfaces and uniform particle size distribution. After coating with polydopamine (PDA), the resulting Fe3O4@PDA particles, while maintaining their original spherical shape, exhibit significantly increased surface roughness, displaying typical PDA encapsulation characteristics. This indicates that PDA has successfully formed a complete shell on the surface of the Fe3O4 magnetic core through in-situ polymerization. Further modification with Calpeptin resulted in the Fe3O4@PDA-Calpeptin composite material maintaining good particle morphology, without significant agglomeration, and exhibiting excellent dispersibility. This suggests that the PDA interlayer effectively improves the colloidal stability of the material and provides a good interface for subsequent Calpeptin bonding.

[0066] Furthermore, by mapping images through element distribution (such as...) Figure 4 As shown in the diagram, Fe elements (representing the Fe3O4 core) and N elements (representing the PDA shell and Calpeptin molecules) exhibit a high degree of overlap and uniform distribution on the particle. This result strongly confirms that the present invention has successfully constructed a complete core-shell structure with Fe3O4 as the core, PDA as the intermediate shell, and Calpeptin as the outermost layer. This uniform and stable structure is a crucial foundation for ensuring efficient magnetic targeting and controllable drug delivery in subsequent applications.

[0067] Example 3: Investigation into the biosafety of composite materials

[0068] Red blood cell hemolysis test:

[0069] 1. Experimental Methods

[0070] Preparation of red blood cell suspension: Fresh blood from healthy mice was collected and anticoagulated with physiological saline containing heparin sodium (25 U / mL). The mixture was centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the red blood cells (RBCs) were washed repeatedly with physiological saline 2-3 times until the supernatant was clear and colorless, thus obtaining a pure red blood cell precipitate.

[0071] Experimental grouping and incubation: Washed red blood cells were co-incubated with different concentrations of Fe3O4@PDA and Fe3O4@PDA-calpeptin composite solutions. The final concentration gradients of the materials were set at 50, 100, 200, and 400 μg / mL. A positive control was set up: 0.2% Triton X-100 solution (which can cause 100% hemolysis). A negative control was set up: physiological saline (theoretically, it does not cause hemolysis).

[0072] Detection and Calculation: After incubating all samples at 37°C for 90 minutes, centrifuge at 300 g for 5 minutes. Carefully aspirate 100 μL of supernatant from each tube and transfer it to a 96-well plate. Measure the absorbance (OD value) of hemoglobin at 540 nm using a microplate reader.

[0073] Hemolysis rate calculation: The percentage of hemolysis is calculated using the following formula:

[0074] Hemolysis rate (%) = (As - Anc) / (Apc - Anc) × 100%

[0075] Where As is the absorbance of the sample, Anc is the absorbance of the negative control, and Apc is the absorbance of the positive control.

[0076] 2. Experimental Results

[0077] Experimental results are as follows Figure 5 As shown, the negative control group exhibited extremely low absorbance and a hemolysis rate close to 0%, indicating the reliability of the experimental system. The positive control group showed the highest absorbance and a hemolysis rate of 100%, verifying the effectiveness of the experiment. At all tested concentrations (50 to 400 μg / mL), the hemolysis rates of both the Fe3O4@PDA and Fe3O4@PDA-Calpeptin groups were well below the international standard safety threshold of 5%. Even at the highest concentration of 400 μg / mL, no significant increase in hemolysis rate was observed. There was no significant difference in hemolysis rates between the Fe3O4@PDA and Fe3O4@PDA-Calpeptin groups, indicating that the loading of Calpeptin did not introduce any additional risk of hemolysis.

[0078] Example 4: Cellular uptake experiment of composite materials

[0079] Cellular uptake experiment:

[0080] 1. Material Preparation

[0081] Test material: Fe3O4@PDA-Calpeptin nanoparticles, labeled with rhodamine fluorescent dye via covalent linkage or physical adsorption.

[0082] Cell line: Mouse microartery endothelial cells were selected.

[0083] 2. Experimental Design

[0084] Cells were seeded at a specific density in confocal culture plates. After cell attachment, they were cultured in serum-containing complete medium and treated as follows: A. Magnetic field intervention group (MF+): A stable external magnetic field was applied to the bottom of the culture plate during cell culture; B. No magnetic field control group (MF-): No external magnetic field was applied. An equal amount of Fe3O4@PDA-Calpeptin-Rhodamine composite material was added to each well to achieve a final iron(III) oxide mass of 25 μg. Each group had three replicates, and observations were performed at 10 minutes, 2 hours, and 4 hours after drug addition.

[0085] 3. Detection Method

[0086] Culture was terminated at the three time points mentioned above. Cells were gently washed three times with PBS to thoroughly remove any untaken nanomaterials. Cells were fixed with 4% paraformaldehyde. Cell nuclei were stained with DAPI staining solution to mark cell locations.

[0087] Observation and image acquisition were performed using fluorescence microscopy or confocal microscopy. The level of nanomaterial uptake by cells was semi-quantitatively assessed using the fluorescence intensity of the rhodamine channel (red fluorescence); the location of the cell nucleus and cell localization were determined using the DAPI channel (blue fluorescence) (results are shown in Figure 1). Figure 6 (As shown).

[0088] 4. Experimental Results

[0089] At the same time point, the intracellular fluorescence intensity of rhodamine in the magnetic field intervention group was significantly higher than that in the control group without magnetic field, demonstrating that the external magnetic field can effectively promote the uptake of the composite material by cells. With the extension of time (from 10 minutes to 4 hours), the intracellular fluorescence intensity of all experimental groups showed an increasing trend, indicating that the composite material can be effectively internalized by cells, and the uptake process is time-dependent.

[0090] Example 5: Application of composite materials in the preparation of drugs for treating thoracic aortic aneurysms / dissections

[0091] In animal model experiments, a TAA / D mouse model was established using BAPN induction to simulate the pathological process of human aortic aneurysm / dissection. The experiment was divided into three groups: a BAPN control group and a Fe3O4@PDA-Calpeptin treatment group. The Fe3O4@PDA-Calpeptin+BAPN group received Fe3O4@PDA-Calpeptin injections once a week at a dose of 0.02 mg / 0.1 mL / 10 g; the Fe3O4@PDA-Calpeptin+BAPN+MF group received Fe3O4@PDA-Calpeptin injections once a week at a dose of 0.02 mg / 0.1 mL / 10 g, followed by application of a magnetic field for 4-6 hours after injection.

[0092] Step 1: Drug administration. A therapeutically effective dose of the Fe3O4@PDA-Calpeptin composite material was delivered to animal models with thoracic aortic aneurysm / dissection. To verify the distribution and retention characteristics of the composite material in vivo, mouse models injected with the rhodamine-conjugated composite material were monitored using an in vivo imaging system.

[0093] The results are as follows Figure 7 As shown, the imaging results indicate that significant fluorescence signal enrichment was observed in the thoracic aorta region 24 hours after injection, indicating that the composite material can be efficiently targeted to the lesion site; a clear fluorescence signal was still detected in the region 72 hours after injection, proving that the material has good in-situ retention ability; and by the seventh day after injection, the fluorescence signal in the thoracic aorta region had decayed to the background level, indicating that the material had been basically metabolized and cleared by the body.

[0094] The in vivo kinetic data clearly demonstrate that the targeted material can maintain an effective concentration at the lesion site for approximately 3 days. To ensure that the therapeutic drug maintains an effective concentration in the lesion area and thus achieves a stable therapeutic effect, weekly (i.e., every 7 days) administration is a necessary and reasonable frequency for maintaining the therapeutic window.

[0095] Step 2: Magnetic directional enrichment. An external magnetic field is applied to the surface of the patient's thoracic aortic arch lesion area immediately during or after drug administration.

[0096] The results showed that compared with the BAPN control group, the Fe3O4@PDA-Calpeptin treatment group (with magnetic field applied) showed significantly reduced aortic dilation and improved vascular wall integrity, as detected by small animal ultrasound. Figure 8 (As shown). Histopathological examination (HE staining, EVG staining, such as...) Figure 9(As shown) further confirmed that the pathological changes such as necrosis of the aortic wall media and rupture of elastic fibers were significantly reduced in the treated mice. Furthermore, fluorescent labeling and in vivo animal imaging confirmed that, under the guidance of an external magnetic field, the Fe3O4@PDA-Calpeptin composite material could achieve specific enrichment in the lesion area of ​​the thoracic aortic arch, demonstrating excellent directional delivery performance (e.g., Figure 10 (As shown).

[0097] Prussian blue staining results of mouse thoracic aortic sections showed that the accumulation of the composite material in the aortic media increased continuously with time from 2 hours to 6 hours (e.g. Figure 11 (As shown). Therefore, a magnetic field was continuously applied for 6 hours to guide the composite material to achieve specific enrichment in the aortic lesion area, especially the medial layer.

[0098] Comparative Example 1: Preparation of KNN@PDA-Fe3O4 (magnetic particles with potassium sodium niobate (KNN) as the core, PDA coated and Fe3O4 connected)

[0099] Step 1: Synthesis and Activation of Fe3O4 Nanoparticles

[0100] Superparamagnetic Fe3O4 nanoparticles were synthesized using a coprecipitation method. Specifically, FeCl3·6H2O and FeSO4·7H2O in a molar ratio of 2:1 were dissolved in deoxygenated ultrapure water. Under nitrogen protection and mechanical stirring, concentrated ammonia was added dropwise until the pH of the solution reached 10-11, and the reaction was continued for 1 hour.

[0101] After the reaction was completed, the product was separated by an external magnetic field and washed three times alternately with ultrapure water and anhydrous ethanol to thoroughly remove residual reactants and byproducts. Finally, the obtained Fe3O4 nanoparticles were redispersed in ultrapure water to prepare a dispersion with a concentration of 1 mg / mL and stored at 4°C for later use.

[0102] Step 2: Polydopamine coating to construct Fe3O4@PDA core-shell structure

[0103] Take an appropriate amount of the Fe3O4 dispersion obtained in step one and place it in a 10 mM Tris-HCl buffer solution with pH = 8.5. Sonicate for 30 minutes to ensure that the particles are fully dispersed.

[0104] Dopamine hydrochloride was added to the above system at a mass ratio of Fe3O4 to dopamine hydrochloride of 1:2. The mixture was stirred continuously at room temperature in the dark for 12 hours to allow dopamine to self-polymerize on the Fe3O4 surface, forming a uniform polydopamine shell.

[0105] After the reaction was complete, the Fe3O4@PDA composite material was obtained again by magnetic field separation and washed with ultrapure water until the supernatant was clear to remove unreacted monomers and homopolymers. Finally, the product was redispersed in ultrapure water and the concentration was adjusted to 1 mg / mL.

[0106] Step 3: Loading of KNN

[0107] Preparation of KNN solution: Dissolve KNN in dimethyl sulfoxide to prepare a 10 mM stock solution and store at -20°C protected from light. Before use, dilute with ultrapure water to a working concentration of 1 mg / mL.

[0108] Loading process: Take the Fe3O4@PDA dispersion prepared in step two, discard the supernatant after magnetic separation, and add the above-mentioned KNN working solution at a mass ratio of Fe3O4@PDA to KNN of 1:1. Transfer the mixture to a rotary mixer, wrap it with aluminum foil to protect it from light, and continue the reaction at room temperature for 12 hours. During this process, KNN molecules are stably loaded onto the PDA shell through various interactions such as π-π stacking, Michael addition, and Schiff base reaction.

[0109] Purification: After the reaction, the final product Fe3O4@PDA-KNN was separated by an external magnetic field and washed three times with ultrapure water to completely remove unbound KNN molecules, thus obtaining the pure target composite material. The hydrodynamic particle size, determined by DLS, was approximately 200-300 nm.

[0110] The KNN@PDA-Fe3O4 material was injected into TAAD model mice using the same administration method and dosage as in Example 2, and an external magnetic field of the same intensity was applied to the thoracic aortic region. The material retention was monitored using scanning electron microscopy.

[0111] The results showed that no significant signal of KNN core particles was detected in the thoracic aortic tissue of mice on day 7 post-injection. Scanning electron microscopy revealed that the material was blocked outside the endothelial barrier (e.g., Figure 12 (As shown). This indicates that the composite material cannot achieve long-term adhesion and retention in the hemodynamically complex thoracic aortic lesion area. It is speculated that this may be due to the differences in the structure and properties of the nanoparticles themselves. For example, the KNN core particle and the Fe3O4 outer shell of the PDA alter the properties of the composite material, making it unable to adhere to the vascular intima. Factors such as potential and particle size (200-250nm) cause the particles to be subjected to huge shear forces in high-speed blood flow, making them easy to be washed away. At the same time, the large size also seriously hinders its penetration and retention from the vascular lumen through the intima to the vascular wall media.

[0112] This comparative example demonstrates that not all magnetic nanocomposites can achieve the targeting and retention effects described in this invention. The properties of the composite material are a key factor determining whether the material can effectively accumulate and exert a long-term effect in the unique environment of the thoracic aorta. This invention solves the problem of blood flow erosion that large-particle-size materials cannot overcome by precisely controlling the particle size of Fe3O4@PDA-Calpeptin to 120-180 nm, achieving stable retention and effective penetration at the lesion site.

[0113] 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 Calpeptin magnetic nanoparticle targeting the middle membrane layer, characterized in that, The Calpeptin magnetic nanoparticles comprise superparamagnetic iron oxide nanoparticles, a polydopamine shell encapsulating the superparamagnetic iron oxide nanoparticles, and Calpeptin covalently linked to the polydopamine shell.

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

3. The preparation method according to claim 2, characterized in that: The mass ratio of Fe3O4@PDA nanoparticles to Calpeptin is (1-2):(1-2).

4. The preparation method according to claim 2, characterized in that: In step S2, the co-incubation is carried out under light-protected conditions, and the co-incubation temperature is 1-10℃.

5. The preparation method according to claim 2, characterized in that: In step S1, the pH of the reaction is 7.5-9.

6. The use of the Calpeptin magnetic nanoparticles according to claim 1 or the Calpeptin magnetic nanoparticles prepared by the preparation method according to any one of claims 2-5 in the preparation of drugs for the treatment of aortic aneurysm / dissection.

7. A therapeutic agent for aortic aneurysm / dissection, characterized in that, The therapeutic drug includes the Calpeptin magnetic nanoparticles according to claim 1 or the Calpeptin magnetic nanoparticles prepared by the preparation method according to any one of claims 2-5.

8. The therapeutic drug 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.

9. The therapeutic drug according to claim 7, characterized in that, The routes of administration of the therapeutic drugs include intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

10. The application of the Calpeptin magnetic drug-carrying nanoparticles according to claim 1 or the Calpeptin magnetic drug-carrying nanoparticles prepared by the preparation method according to any one of claims 2-5 in the preparation of aortic aneurysm / dissection treatment devices, characterized in that, The treatment device also includes a device capable of emitting a magnetic field.

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