Polydopamine nano-particles, targeted polydopamine nano-particles and preparation method and application of targeted polydopamine nano-particles

Abstract

The invention discloses a polydopamine nano-particle, a targeted polydopamine nano-particle and a preparation method and application of the targeted polydopamine nano-particle. The polydopamine nano-particles are used for treating atherosclerosis, the defects of an existing nano-therapy in the aspects of synthesis route, biocompatibility, treatment efficiency, disease site enrichment and the like are overcome, and a safer, more accurate and more efficient comprehensive treatment strategy is provided for early atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a polydopamine (PDA) nanoparticle, a targeted polydopamine nanoparticle, and a preparation method and application thereof. Background Art

[0002] Atherosclerosis (AS) is the most common cardiovascular disease, accounting for more than 60% of global cardiovascular disease-related deaths. It is the main cause of death from cardiovascular diseases. The exact mechanism of AS is still unclear, but its formation and progression are characterized by the accumulation of inflammatory cells at local inflammatory sites, an increase in the level of reactive oxygen species (ROS), the formation of foam cells, and the development of advanced unstable plaques. The clinical treatment of AS mainly relies on drugs such as non-steroidal anti-inflammatory drugs and vasodilators. These drugs aim to reduce cholesterol, lower blood lipids, and relieve AS-related inflammation. However, traditional drug delivery methods have limitations in terms of stability, bioavailability, and targeting efficiency. In addition, in the middle and late stages of AS, factors such as plaque sclerosis, calcification, and smooth muscle cell proliferation lead to vascular wall stiffness and impaired blood flow, and endothelial injury and persistent inflammation further exacerbate the condition. These changes make it difficult for blood vessels to repair and restore normal function. Therefore, there is an urgent need to explore a new multi-faceted strategy to more effectively reduce cholesterol accumulation, inhibit the inflammatory cytokine storm caused by excessive ROS production, and provide a comprehensive treatment method for early AS.

[0003] Mild photothermal therapy (PTT) is an innovative treatment strategy that mediates treatment through nanoparticles and maintains the temperature within a mild range of 42 - 45 °C. This temperature range mimics the "hot spring" effect and can multi-channel regulate the pathophysiological process of atherosclerosis. This therapy not only regulates reverse cholesterol transport, reduces inflammation, and promotes endothelial repair, but also avoids the local inflammatory response and excessive foam cell apoptosis that may be caused by traditional photothermal therapy, thereby reducing the risk of plaque rupture. The excessive production of ROS is another key factor in the progression of AS. Pathological changes in macrophages can induce apoptosis and trigger an inflammatory response by increasing oxidative stress. In addition, the local inflammation produced by pro-inflammatory macrophages is closely related to the excessive production of ROS. Since inflammation is a pathogenic factor for the excessive production of ROS during the development of AS, resolving inflammation and inhibiting the production of ROS in affected tissues have become a key research direction for improving treatment effects. Although preclinical studies have shown that some nanotherapies have dual therapeutic functions, challenges such as poor accumulation at the disease site, complex synthesis pathways, and potential toxicity have hindered their clinical application. Summary of the Invention

[0004] The technical problem solved by the present invention is to overcome the defects of poor accumulation at the disease site, complex synthesis route, and potential toxicity in the existing nano-therapies with dual therapeutic functions for atherosclerosis, and to provide a polydopamine nanoparticle, a targeted polydopamine nanoparticle, and their preparation methods and applications. The present invention uses polydopamine nanoparticles for the treatment of atherosclerosis, overcomes the deficiencies of the existing nano-therapies in terms of synthesis route, biocompatibility, treatment efficiency, and enrichment at the disease site, and provides a safer, more precise, and efficient comprehensive treatment strategy for early atherosclerosis.

[0005] In the first aspect, the present invention provides a method for preparing polydopamine nanoparticles, which includes the following steps:

[0006] Adding an alkaline substance to a mixture containing a dopamine monomer, a template agent, a pore-forming agent, and a solvent, adjusting the pH, and reacting to obtain polydopamine nanoparticles;

[0007] The temperature of the reaction is 20 - 25 °C;

[0008] The time of the reaction is 20 - 30 h.

[0009] In the present invention, the dopamine monomer can be dopamine hydrochloride.

[0010] In the present invention, the pore-forming agent can be poloxamer (F127), and the molecular weight of the poloxamer is preferably 10,000 - 15,000 g / mol.

[0011] In the present invention, the template agent can be 1,3,5-trimethylbenzene (TMB).

[0012] In the present invention, the solvent can be a mixture of water and ethanol, and the ratio of water to ethanol is preferably 1:1.

[0013] In the present invention, before adding the alkaline substance, ultrasonic treatment of the mixture can also be included; wherein, the frequency of the ultrasonic treatment is preferably 50 - 100 Hz, and the time is preferably 30 - 45 min.

[0014] In the present invention, the alkaline substance can be tris(hydroxymethyl)aminomethane or ammonia water.

[0015] In the present invention, the addition amount of the alkaline substance can be 3.8 - 4.5 mg.

[0016] In the present invention, the addition method of the alkaline substance can be dropwise addition.

[0017] In the present invention, the temperature of the reaction is preferably room temperature.

[0018] In the present invention, the pH is adjusted to 7.5 - 8.5.

[0019] In the present invention, the reaction time can be 24 h.

[0020] In the present invention, after the reaction, it may further include a post-treatment step;

[0021] Among them, the post-treatment preferably includes centrifugation and washing;

[0022] The rotation speed of the centrifugation is preferably 10,000 - 12,000 rpm; the time of the centrifugation is preferably 20 - 30 min;

[0023] The washing is preferably carried out with an organic solvent, the organic solvent is preferably a mixture of ethanol and acetone, and the ratio of ethanol to acetone is preferably 2:1.

[0024] In a second aspect, the present invention provides a polydopamine nanoparticle, which is prepared according to the preparation method of the polydopamine nanoparticle as described above.

[0025] In a third aspect, the present invention provides a polydopamine nanoparticle, the polydopamine nanoparticle contains mesopores, the pore diameter of the mesopores is 15 - 35 nm; the volume of the mesopores is 10.5 - 38.0 cm³ / g; the proportion of the mesopore volume is 60 - 95%.

[0026] In the present invention, the specific surface area of the polydopamine nanoparticle can be 50 - 500 m² / g.

[0027] In the present invention, the porosity of the polydopamine nanoparticle can be 30 - 80%.

[0028] In the present invention, the polydopamine nanoparticle can be a spherical nanoparticle, and its average diameter is preferably 50 - 200 nm.

[0029] In the present invention, the zeta potential of the polydopamine nanoparticle can be -40 mV to -20 mV.

[0030] In the present invention, the number-average molecular weight of the polydopamine nanoparticle can be 5,000 - 200,000 g / mol.

[0031] In a fourth aspect, the present invention provides a preparation method of a targeted polydopamine nanoparticle, which is characterized in that it includes the following steps:

[0032] Mix a polydopamine nanoparticle solution and a targeting agent solution, react to obtain a targeted polydopamine nanoparticle; wherein, the polydopamine nanoparticle solution includes the polydopamine nanoparticle as described in the second aspect and the third aspect.

[0033] In the present invention, the concentration of the targeting agent solution may be 1 - 20 mg / mL.

[0034] In the present invention, the volume ratio of the targeting agent solution to the polydopamine nanoparticle solution may be 1:(1 - 10).

[0035] In the present invention, the mass ratio of the targeting agent to the polydopamine nanoparticles may be 0.5% - 15%.

[0036] In the present invention, the targeting agent may be mannose, and preferably the mannose is D - mannose.

[0037] In the present invention, the temperature of the reaction may be 4 - 40 °C.

[0038] In the present invention, the time of the reaction may be 12 - 24 h.

[0039] In the present invention, after the reaction, washing may also be included;

[0040] Among them, the washing preferably uses deionized water;

[0041] Among them, the temperature of the washing is preferably 4 - 25 °C; for example, 4 °C.

[0042] In the fifth aspect, the present invention provides a targeted polydopamine nanoparticle, which is prepared by the preparation method of the targeted polydopamine nanoparticle as described above.

[0043] In the sixth aspect, the present invention provides a targeted polydopamine nanoparticle, which includes a polydopamine nanoparticle matrix and a targeting agent modified on the surface of the polydopamine nanoparticle matrix; wherein, the targeted polydopamine nanoparticle contains mesopores, and the pore diameter of the mesopores is 2 - 28 nm;

[0044] The mass ratio of the targeting agent to the polydopamine nanoparticles is 1:10.

[0045] In the present invention, the mesopore volume of the targeted polydopamine nanoparticle may be 0.3 - 2.8 cm³ / g.

[0046] In the present invention, the mesopore volume ratio of the targeted polydopamine nanoparticle may be 50 - 90%.

[0047] In the present invention, the specific surface area of the targeted polydopamine nanoparticle may be 40 - 450 m² / g.

[0048] In the present invention, the porosity of the targeted polydopamine nanoparticle may be 25 - 75%.

[0049] In the present invention, the targeted polydopamine nanoparticles may be spherical nanoparticles, and the average diameter thereof may be 60-220 nm.

[0050] In the present invention, the zeta potential of the targeted polydopamine nanoparticles may be -50 mV to -10 mV.

[0051] In the present invention, the molecular weight of the targeted polydopamine nanoparticles may be 5,000-250,000 g / mol.

[0052] In the present invention, the targeting agent may be mannose, and the mannose is preferably D-mannose.

[0053] In a seventh aspect, the present invention provides a use of polydopamine nanoparticles in the preparation of a drug for treating atherosclerosis.

[0054] In the present invention, the polydopamine nanoparticles include the polydopamine nanoparticles described above and / or the targeted polydopamine nanoparticles described above.

[0055] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0056] The reagents and raw materials used in the present invention are commercially available.

[0057] The positive and progressive effects of the present invention are:

[0058] 1. The present invention uses polydopamine nanoparticles for the treatment of atherosclerosis. Polydopamine nanoparticles can effectively reduce the level of oxidative stress due to their excellent antioxidant properties, and promote cholesterol efflux and plaque stabilization through mild photothermal effects, providing a safer, more accurate and efficient comprehensive treatment strategy for early atherosclerosis.

[0059] 2. Furthermore, by modifying polydopamine nanoparticles with targeting agents, the targeted accumulation of nanoparticles in macrophage-rich areas was enhanced, improving the therapeutic efficiency while reducing off-target effects. In vitro and in vivo experiments showed that the nanocarrier can alleviate mitochondrial damage, inhibit inflammation, reduce lipid deposition, and enhance the stability of vulnerable plaques. Compared with traditional treatments, it exhibits better biocompatibility and therapeutic effects, providing a new strategy for nanotherapy of cardiovascular diseases.

[0060] 3. The present invention also provides polydopamine nanoparticles with specific mesoporous structures and targeted polydopamine nanoparticles, which have particularly outstanding therapeutic effects on atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1Transmission electron microscopy (TEM) images of PDA NPs, hPDA NPs, and mPDA NPs.

[0062] Figure 2 Transmission electron microscopy (TEM) image of D-mPDA NPs.

[0063] Figure 3 Zeta potentials of PDA NPs, hPDA NPs, and mPDA NPs.

[0064] Figure 4 Zeta potential of D-mPDA NPs.

[0065] Figure 5 Particle size distributions of PDA NPs, hPDA NPs, and mPDA NPs.

[0066] Figure 6 Particle size distribution of D-mPDA NPs.

[0067] Figure 7 Characterization of DPPH scavenging ability of PDA NPs, hPDA NPs, mPDA NPs, and D-mPDA NPs.

[0068] Figure 8 Characterization of ABTS scavenging ability of PDA NPs, hPDA NPs, mPDA NPs, and D-mPDA NPs.

[0069] Figure 9 In vitro photothermal images of D-mPDA NPs under irradiation at different conditions.

[0070] Figure 10 Temperature change over time of different concentrations of D-mPDA NPs under laser irradiation.

[0071] Figure 11 Effects of mPDA NPs, D-mPDA NPs, and combined laser irradiation of D-mPDA NPs on cell viability.

[0072] Figure 12 Localization of mPDA NPs and D-mPDA NPs in cells.

[0073] Figure 13 、 14 Regulation of mitochondrial function by D-mPDA NPs at 15 and 16.

[0074] Figure 17A is a schematic diagram of the treatment process of atherosclerotic model mice, B is the in vivo photothermal performance evaluation of polydopamine, and C and D are the in vivo targeting verification of mPDA NPs and D-mPDA NPs.

[0075] Figure 18 A is a typical image of tissue staining of the cross-section of the aortic root of ApoE− / − mice in different treatment groups, and B, C, D, and E are quantitative analyses of plaque area.

[0076] Figure 19 It is a schematic diagram of the mechanism after phototherapy of D-mPDA NPs under 808 nm NIR laser irradiation. Specific implementation manners

[0077] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0078] In the following examples and comparative examples, the raw materials used are as follows:

[0079] 3-Hydroxytyramine hydrochloride was purchased from Adamas-beta (Shanghai, China). D-(+)-Mannose was purchased from Aladdin (Shanghai, China). Pluronic® F-127 was purchased from Meryea (Shanghai, China). 1,3,5-Trimethylbenzene (98%) was purchased from sigma-Aldrich (USA). Tris(hydroxymethyl)aminomethane was purchased from Adamas-beta (Shanghai, China). Acetone and ethanol (99.9%) were purchased from General-reagrent (Changshu, China). Rhodamine B was purchased from Energy Chemical (Anhui, China). Total antioxidant capacity (T-AOC) detection kit (ABTS, microplate method) and DPPH free radical scavenging detection kit were purchased from Yuanye (Shanghai, China). JC-1 mitochondrial membrane potential detection kit was purchased from MedChemExpress (USA). MitoSOX Red mitochondrial superoxide indicator was purchased from Yeasen (Shanghai, China). Mito-Tracker Red CMXRos was purchased from Beyotime (Shanghai, China).

[0080] Example 1 Preparation of mPDA NPs.

[0081] mPDA NPs were synthesized by a one-pot method. 0.1 g of F127, 0.15 g of dopamine hydrochloride and 0.16 mL of TMB were dispersed in a mixture of 10 mL of water and ethanol (5 mL each), and sonicated for 2 minutes to form an emulsion. Then, 0.375 mL of ammonia water was added dropwise to the reaction mixture under stirring. The mixture was stirred at room temperature for 24 hours, and then the obtained particles were collected by centrifugation (12,000 rpm, 10 minutes). Finally, the templates were removed using ethanol and acetone.

[0082] Example 2 Preparation of D-mPDA NPs.

[0083] D-Mannose (0.5 g) was dissolved in 10 mL of sodium acetate buffer (pH 4.5) and heated at 60 °C for 1 hour. After cooling, 25 mL of mPDA NPs solution was added to the mannose solution and stirred overnight. Subsequently, D-mPDA NPs were washed three times with deionized water (14,000 rpm, 10 minutes, 4 °C) and dispersed in deionized water for further use.

[0084] Example 3 Preparation of PDA NPs.

[0085] PDA NPs were synthesized by an oxidation reaction under alkaline conditions. First, 50 mg of dopamine hydrochloride was dissolved in 1 mL of deionized water, and then this solution was injected into a mixture of 4 mL of ethanol and 9 mL of deionized water. While gently stirring, 0.25 mL of ammonia water solution (28%) was added. The initially colorless solution quickly turned light yellow and gradually turned brown. The reaction continued for 24 hours at room temperature. After that, PDA nanoparticles were collected by centrifugation and washed three times with deionized water.

[0086] Example 4 Preparation of hPDA NPs.

[0087] hPDA NPs were synthesized by a one-pot method. 0.2 g of F127 and 0.2 g of TMB were dissolved in a mixed solution of 50 mL of deionized water and ethanol while stirring. Next, a solution of 10 mg of TRIS dissolved in 10 mL of water was added to the mixture, and then 60 mg of dopamine hydrochloride was added. After reacting at room temperature for 30 hours, hPDANPs were obtained by centrifugation (16,000 rpm), washed several times with ethanol and acetone, and then suspended in ethanol for further use.

[0088] Effect Example 1 Determination of physicochemical parameters of nanoparticles.

[0089] 1. Test objects: PDA NPs, hPDA NPs, mPDA NPs and D-mPDA NPs.

[0090] 2. Test method:

[0091] (1) Use a specific surface area and pore size analyzer (instrument model: ASAP 2460) to test the mesopore diameter, mesopore volume, specific surface area, and porosity;

[0092] (2) Use a Malvern particle size analyzer (instrument model: Mastersizer 3000) to test the average diameter and zeta potential;

[0093] (3) Use a gel permeation chromatograph (instrument model: Alliance e2695) to test the number-average molecular weight.

[0094] 3. Test results

[0095] Table 1 Physicochemical parameters of nanoparticles

[0096]

[0097] The synthesis of PDA NPs, hPDA NPs, and mPDA NPs was confirmed by transmission electron microscopy (TEM) ( Figure 1 ). As Figure 5 shown, the size ranges of the three PDA NPs were similar, approximately 140 - 200 nm. Zeta potential analysis indicated that all three PDA nanoparticles had a negative surface charge ( Figure 3 ). PDA NPs had a solid structure, and hPDA NPs had a hollow structure. Therefore, PDA NPs had no mesopore structure, and the mesopore volume, specific surface area, and porosity of hPDA NPs were lower than those of mPDA NPs and D-mPDA NPs.

[0098] TEM analysis showed that the morphology of D-mPDA NPs was black spherical ( Figure 2 ). When dispersed in water, its hydrodynamic size was approximately 270 nm ( Figure 6 ). In addition, after functionalizing with mannose, the zeta potential of the NPs increased from -19.6 mV to -8.52 mV ( Figure 4 ).

[0099] Effect Example 2 In vitro ROS scavenging performance characterization of PDA NPs, hPDA NPs, and mPDA NPs.

[0100] DPPH is a purple free radical with a stable absorption peak, usually having a strong absorption at 517 nm. When an antioxidant interacts with the DPPH free radical, DPPH is reduced, resulting in the fading of its purple color and a decrease in absorbance. The stronger the reducing ability of the antioxidant, the more obvious the decrease in absorbance, indicating a higher antioxidant activity.

[0101] The ABTS method is commonly used to evaluate the in vitro antioxidant capacity of substances. ABTS+· is a stable free radical that forms a blue-green solution with a maximum absorption wavelength of 734 nm.

[0102] 1. Test objects: PDA NPs treatment group, hPDA NPs treatment group, mPDA NPs treatment group, and control group (Control).

[0103] 2. Test method:

[0104] The DPPH stock solution was prepared in methanol. PDA NPs were mixed with the DPPH solution, and the reaction was carried out in the dark for 30 minutes. Then the absorbance of the mixture was measured at 517 nm using a microplate reader.

[0105] To prepare the ABTS+ working solution, 3.2 mg of ABTS and 1 mg of potassium persulfate were mixed and incubated in the dark for 12 hours. Then the working solution was incubated with PDA NPs at room temperature for 6 minutes, and the absorbance was measured at 734 nm using a microplate reader.

[0106] 3. Test results: As Figure 7 shown, among the three PDA NPs, mPDA NPs showed the highest ROS scavenging efficiency. Similar results were also observed in the ABTS assay. The cationic free radical ABTS+· is blue-green and has an absorption at 734 nm, which can be reduced by antioxidants. After incubation with PDA NPs, the solution gradually turned colorless, and the absorbance of the mPDA NPs group decreased most significantly ( Figure 8 ).

[0107] Effect Example 3 Characterization of the in vitro photothermal performance of D-mPDA NPs.

[0108] 1. Test objects: D-mPDA NPs treatment group.

[0109] 2. Test method: To evaluate the photothermal performance of D-mPDA NPs, 1.0 mL of aqueous solutions of m-PDA NPs with different concentrations (100, 150, and 250 μg·ml⁻¹) were placed in petri dishes and irradiated with an 808 nm laser (1.0 W·cm⁻²) for 10 minutes. An infrared thermal imaging camera was used to record the temperature changes and thermal imaging images.

[0110] 3. Test results: As Figure 9 and Figure 10As shown, the temperatures of D-mPDA nanoparticles increased by 10.5 °C, 14.4 °C, and 18.2 °C (at concentrations of 100, 150, and 250 μg·ml⁻¹, respectively). Considering the mild photothermal requirements, 150 μg / ml⁻¹ of D-mPDA NPs was selected as the optimal concentration for subsequent cell and animal studies.

[0111] Effect Example 4 Cytotoxicity experiment (CCK-8) of mPDA NPs and D-mPDA NPs.

[0112] 1. Test subjects: mPDA NPs treatment group, D-mPDA NPs treatment group, and D-mPDA NPs + NIR treatment group.

[0113] 2. Test method: RAW264.7 cells were seeded into 96-well plates and allowed to adhere for 24 hours. After exposing the cells to different concentrations of D-mPDA NPs for 24 hours, cell viability was evaluated using a CCK-8 kit (Dojindo Chemical Technology, Beijing, China). According to the manufacturer's instructions, absorbance was measured at 450 nm using a microplate reader.

[0114] 3. Test results: The CCK-8 assay results showed that 160 μg·ml⁻¹ of D-mPDA NPs had no significant effect on the viability of RAW264.7 cells. In addition, as Figure 11 shown, the cell viability in the D-mPDA NPs group remained above 80% after 808 nm laser irradiation.

[0115] Effect Example 5 Cellular uptake experiment of mPDA NPs and D-mPDA NPs.

[0116] 1. Test subjects: mPDA NPs treatment group, D-mPDA NPs treatment group.

[0117] 2. Test method:

[0118] Rhodamine B (Rho-B) at 100 μg / mL was stirred with nanoparticles (mPDA NPs or D-mPDA NPs) at 1 mg / mL for 24 hours, centrifuged, and washed to obtain Rho-B-loaded nanoparticles. After co-incubating the Rho-B-loaded nanoparticles with cells, they were stained with the fluorescent dye DAPI, and the staining results were observed under a microscope.

[0119] 3. Test results: As Figure 12As shown, there were no significant changes in nuclear morphology before and after co-incubation with nanoparticles. Red fluorescence indicated that mPDA NPs and D-mPDA NPs were mainly localized in the cytoplasm after being taken up by cells. Notably, the D-mPDA NPs group showed the highest red fluorescence accumulation and intensity, demonstrating the excellent targeting ability of D-mPDA NPs to RAW264.7 cells and enhancing the potential of its subsequent therapeutic applications.

[0120] Effect Example 6 D-mPDA NPs regulate mitochondrial dysfunction and maintain cell homeostasis.

[0121] Mitochondria are important organelles that generate cellular energy through oxidative phosphorylation, producing more than 80% of the energy required for cell function. Therefore, these organelles play a crucial role in regulating cell signaling and fate and are an important source of ROS. Excess production of superoxide radicals can lead to oxidative stress, mitochondrial dysfunction, and ultimately cell death, triggering various other pathologies. With its strong free radical scavenging ability, D-mPDA NPs are expected to regulate mitochondrial dysfunction and maintain cell homeostasis.

[0122] 1. Test subjects: Control group (Control), LPS-treated group, and D-mPDA NPs-treated group.

[0123] 2. Test method: 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) was used to evaluate the mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates into polymers in the mitochondrial matrix, emitting red fluorescence. Conversely, when the mitochondrial membrane potential is low, JC-1 exists as monomers, producing green fluorescence. Cells were treated with an equal volume of JC-1 staining solution, mixed well, and incubated at 37°C for 20 minutes. After washing twice with PBS, cells were examined using a laser confocal microscope.

[0124] 3. Test results: Figure 13 and Figure 14 showed that compared with the untreated control group, the production of mitochondrial superoxide anions in LPS-stimulated cells increased, quantified by the MitoSOX Red fluorescence intensity. Notably, D-mPDA NPs treatment significantly attenuated the oxidative burst compared with the LPS group, indicating the ROS scavenging ability of the nanoparticles. Figure 15 Morphological analysis in Figure 16showed that the red / green fluorescence ratio decreased in LPS-treated cells, indicating mitochondrial depolarization. Co-treatment with D-mPDA NPs reversed this effect, confirming its cytoprotective effect by protecting mitochondrial bioenergetics.

[0125] Effect Example 7 Evaluation of in vivo photothermal and targeting properties of D-mPDA NPs.

[0126] 1. Test subjects: Mice injected with D-mPDA NPs and mice injected with PBS.

[0127] 2. Test method: Irradiation with an 808 nm laser.

[0128] 3. Test results: Figure 17 B showed that the aortic wall temperature in the D-mPDA NPs group increased from 33.2 °C to 42.2 °C after irradiation with an 808 nm laser (1.0 W·cm⁻², 10 minutes). Then, the active targeting ability of mannose-modified D-mPDA NPs was compared with that of non-targeted mPDA NPs ( Figure 17 C and D). Due to the disruption of the plaque endothelial barrier, mPDA NPs showed significant accumulation in the aorta 12 hours after injection. Mannose-targeted D-mPDA NPs were able to target plaques more effectively through mannose receptor-mediated active targeting.

[0129] Effect Example 8 In vivo therapeutic effect of nanoparticles on atherosclerosis.

[0130] 1. Test subjects: Atherosclerotic mice were randomly divided into five groups: (i) PBS, (ii) mPDA NPs, (iii) D-mPDA NPs, (iv) D-mPDA NPs, and (v) D-mPDA NPs + NIR.

[0131] 2. Test method:

[0132] Animal model: All animal experiments were conducted in accordance with the guidelines of the Experimental Animal Welfare and Ethics Committee of Shanghai University (ECSHU2024-113) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. ApoE^(- / -)^ mice were then fed a high-fat diet for 14 weeks to induce an atherosclerosis model. The diet consisted of 4% milk powder, 10% lard, 1.5% cholesterol, and 0.5% sodium cholate. During the feeding period, the mice were housed in an animal facility with controlled temperature and a 12-hour light / dark cycle.

[0133] As Figure 17As shown in Figure A, 6-week-old ApoE^(- / -)^ mice were fed a high-fat diet for 8 weeks to induce the formation of atherosclerotic plaques. Then the mice were randomly assigned to different treatment groups: PBS, mPDA NPs, D-mPDA NPs, and D-mPDA NPs + NIR. Each group was injected with PBS or nanoparticles via the tail vein, once every 3 days for 4 weeks. After intravenous injection via the tail vein, the aortic region of each group was irradiated once every 3 days (808 nm, 10 minutes, 1.0 W⋅cm −2 ), for a total of 8 hours. During mild photothermal therapy, the temperature changes at the aortic lesion site were continuously monitored using an infrared thermal imaging system. After treatment, the entire aorta was dissected and the surrounding perivascular tissues were removed. Then the aorta was longitudinally incised and stained with Oil Red O solution for 15 minutes, followed by rinsing with PBS. The aorta was fully unfolded to expose the intima, and plaque imaging was performed using an optical microscope. In addition, the aortic root was sectioned for histological analysis. Hematoxylin and eosin (H&E) staining was used for tissue examination, while Oil Red O staining was used to evaluate the lipid content of the plaque area. Masson's trichrome staining was used to evaluate the collagen fiber content within the plaque. ImageJ software was used to quantify the percentage of plaque area and collagen fiber area.

[0134] 3. Test results: As Figure 18 shown in Figure A, the H&E and Oil Red O staining results showed a significant increase in plaque area and lipid accumulation in the control group. In contrast, the D-mPDA NPs combined with NIR irradiation group showed significant decreases in both plaque area and lipid content. As Figure 18 shown in Figure E, three aortas were selected from each group for Oil Red O staining, and the red areas represent atherosclerotic plaques. The number of plaques in the D-mPDA NPs + NIR group was significantly reduced. Quantitative analysis confirmed these observations, showing that the plaque areas in the PBS, PDANPs, and D-mPDA NPs groups were 37.2%, 36.6%, and 23.8% respectively ( Figure 18 Figure B). Notably, mild photothermal therapy mediated by D-mPDA NPs further reduced the plaque area to 19.5%, highlighting the potential of this treatment strategy in reducing atherosclerotic plaque formation. The Masson's trichrome staining results showed that mild photothermal therapy mediated by D-mPDA NPs significantly enhanced plaque stability. In the PBS group, mPDA NPs group, and D-mPDA NPs group, the collagen areas were 14.1%, 17.4%, and 35.9% respectively. Mild PTT mediated by D-mPDA nanoparticles increased the collagen area to 46.0% ( Figure 18 Figure D). The staining results further confirmed that mild PTT mediated by D-mPDA NPs effectively inhibited atherosclerosis and stabilized plaques.

Claims

1. A method for preparing polydopamine nanoparticles, characterized in that, It includes the following steps: Add an alkaline substance to a mixture containing dopamine monomer, template agent, pore-forming agent and solvent, adjust the pH, and react to obtain polydopamine nanoparticles; The temperature of the reaction is 20 - 25 °C; The time of the reaction is 20 - 30 h.

2. The preparation method of the polydopamine nanoparticles according to claim 1, characterized in that, It meets one or more of the following conditions: (1) The dopamine monomer is dopamine hydrochloride; (2) The pore-forming agent is poloxamer, and the molecular weight of the poloxamer is preferably 10,000 - 15,000 g / mol; (3) The template agent is 1,3,5-trimethylbenzene; (4) The solvent is a mixture of water and ethanol, and the ratio of water to ethanol is preferably 1:1; (5) Before adding the alkaline substance, ultrasonication of the mixture is also included; wherein, the frequency of the ultrasonication is preferably 50 - 100 Hz, and the time is preferably 30 - 45 min; (6) The alkaline substance is tris(hydroxymethyl)aminomethane or ammonia water; (7) The addition amount of the alkaline substance is 3.8 - 4.5 mg; (8) The addition method of the alkaline substance is dropwise addition; (9) The adjusted pH is 7.5 - 8.5; (10) The temperature of the reaction is preferably room temperature; (11) The time of the reaction is 24 h; (12) After the reaction, a post-treatment step is also included; Among them, the post-treatment preferably includes centrifugation and washing; The rotation speed of the centrifugation is preferably 10,000 - 12,000 rpm; the time of the centrifugation is preferably 20 - 30 min; The washing is preferably carried out with an organic solvent, and the organic solvent is preferably a mixture of ethanol and acetone, and the ratio of ethanol to acetone is preferably 2:

1.

3. A polydopamine nanoparticle, characterized in that, It is prepared according to the preparation method of the polydopamine nanoparticles as described in Claim 1 or 2.

4. A polydopamine nanoparticle, characterized in that, The polydopamine nanoparticles contain mesopores, and the pore diameter of the mesopores is 15 - 35 nm The volume of the mesopores is 10.5 - 38.0 cm³ / g; The proportion of the mesopore volume is 60 - 95%; The specific surface area of the polydopamine nanoparticles is 50 - 500 m² / g; The porosity of the polydopamine nanoparticles is 30 - 80%; The polydopamine nanoparticles are spherical nanoparticles, and their average diameter is preferably 50 - 200 nm; The zeta potential of the polydopamine nanoparticles is -40 mV to -20 mV; The number-average molecular weight of the polydopamine nanoparticles is 5,000 - 200,000 g / mol.

5. A preparation method of targeted polydopamine nanoparticles, characterized in that, It includes the following steps: Mix the polydopamine nanoparticle solution and the targeting agent solution, and react to obtain targeted polydopamine nanoparticles; wherein, the polydopamine nanoparticle solution includes the polydopamine nanoparticles as described in Claim 3 or 4.

6. The preparation method of the targeted polydopamine nanoparticles according to claim 5, wherein, It meets one or more of the following conditions: (1) The concentration of the targeting agent solution is 1 - 20 mg / mL; (2) The volume ratio of the targeting agent solution to the polydopamine nanoparticle solution is 1:(1 - 10); (3) The mass ratio of the targeting agent to the polydopamine nanoparticles is 0.5% - 15%; (4) The targeting agent is mannose, and the mannose is preferably D-mannose; (5) The temperature of the reaction is 4 - 40 °C; (6) The time of the reaction is 12 - 24 h; (7) After the reaction, washing is further included; Among them, the washing preferably uses deionized water; Among them, the temperature of the washing is preferably 4 - 25 °C; for example, 4 °C.

7. A targeted polydopamine nanoparticle, characterized in that, It is prepared according to the preparation method of the targeted polydopamine nanoparticles as described in claim 5 or 6.

8. A targeted polydopamine nanoparticle, characterized in that It includes a polydopamine nanoparticle matrix and a targeting agent modified on the surface of the polydopamine nanoparticle matrix; among them, the targeted polydopamine nanoparticle contains mesopores, and the pore diameter of the mesopores is 2 - 28 nm; The mass ratio of the targeting agent to the polydopamine nanoparticle is 1:10; The mesopore volume of the targeted polydopamine nanoparticle is 0.3 - 2.8 cm³ / g; The proportion of the mesopore volume of the targeted polydopamine nanoparticle is 50 - 90%; The specific surface area of the targeted polydopamine nanoparticle is 40 - 450 m² / g; The porosity of the targeted polydopamine nanoparticle is 25 - 75%; The targeted polydopamine nanoparticle is a spherical nanoparticle, and its average diameter is 60 - 220 nm; The zeta potential of the targeted polydopamine nanoparticle is -50 mV to -10 mV; The molecular weight of the targeted polydopamine nanoparticle is 5,000 - 250,000 g / mol; The targeting agent is mannose, and the mannose is preferably D-mannose.

9. Application of polydopamine nanoparticles in the preparation of drugs for treating atherosclerosis.

10. The use of the polydopamine nanoparticles according to claim 9 in the preparation of a medicament for treating atherosclerosis, characterized in that, The polydopamine nanoparticles include the polydopamine nanoparticles as described in claim 3 or 4 and / or the targeted polydopamine nanoparticles as described in claim 7 or 8.

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