A nanoparticle with plaque targeting function, its preparation method and application in preparing anti-atherosclerosis drugs

Abstract

This invention provides nanoparticles with plaque-targeting function, their preparation method, and their application in the preparation of anti-atherosclerotic drugs, belonging to the field of composite nanomaterials technology. The plaque-targeting nanoparticles provided by this invention comprise a core and a shell. The core is ZIF-8 nanoparticles loaded with neoindocyanine green, and the shell is arginine-modified polydopamine; the surface of the shell is modified with hyaluronic acid. The plaque-targeting nanoparticles provided by this invention can regulate lipid levels by simultaneously intervening in multiple pathways, providing anti-inflammatory effects, protecting damaged endothelial cells, and enabling low-temperature (42-45℃) photothermal therapy combined with anti-nlrp3 therapy. This treats atherosclerosis by regulating PCSK9 pathway-induced lipid accumulation and repairing damaged endothelial cells.

Description

Technical Field

[0001] This invention relates to the field of composite nanomaterials technology, and in particular to a nanoparticle with plaque-targeting function, its preparation method, and its application in the preparation of anti-atherosclerotic drugs. Background Technology

[0002] Atherosclerotic cardiovascular disease (AS) is a significant global public health problem. In clinical practice, lipid-lowering remains the primary focus of drug treatment for atherosclerosis. Among these drugs, statins are the most widely used lipid-lowering agents; they are HMG-CoA reductase inhibitors. While statin therapy can slow the progression of AS by lowering plasma LDL levels, it is insufficient to reduce AS plaque.

[0003] Protoplasmic subtilisin / kexin9 (PCSK9) is widely expressed in the liver and ankylosing spondylitis (AS) plaques. It promotes cholesterol accumulation by degrading low-density lipoprotein receptor (LDLr), thereby affecting cholesterol homeostasis. Compared to statins that inhibit HMB-CoA reductase, PCSK9 inhibitors work by preventing LDLr degradation, achieving promising results in clinical practice. For example, PCSK9 monoclonal antibodies (mABs) have been approved for clinical use by the U.S. Food and Drug Administration (FDA). The use of small interfering RNA (siRNA) as a PCSK9 inhibitor has also shown high application value in phase 3 clinical trials.

[0004] PCSK9 is regulated by multiple pathways, the most extensively studied being those related to metabolism and inflammation. Nanoparticles are an effective tool for modulating cell signaling pathways with high spatiotemporal precision. Due to the unique physical and chemical properties of cells, optical, electrical, and magnetic methods have been developed to modulate cell signals. Optical stimulation, particularly stimulation using near-infrared (NIR) light, offers significant advantages by penetrating deep into biological tissues with minimal attenuation and photodamage to cells.

[0005] Besides regulating lipid metabolism, the relationship between inflammation and lipids has recently attracted significant attention from researchers. The specific physiological and pathological process involves macrophages being recruited by damaged vascular endothelium, engulfing lipids to form foam cells, which further release inflammatory factors, creating a vicious cycle that exacerbates atherosclerosis. The NOD-like receptor pyrin domain containing protein 3 (NLRP3) inflammasome is a classic receptor for intracellular innate immunity, regulating inflammatory responses and playing a crucial role in the development and progression of atherosclerosis. Recent studies have also demonstrated an independent correlation between SREBP2 / PCSK9 and NLRP3. Therefore, reducing NLRP3 expression can effectively reduce PCSK9 expression.

[0006] Furthermore, endothelial cell (EC) dysfunction is the initiating step in the development of AS. Dysfunction of endothelial nitric oxide synthase (eNOS) and the resulting reduction in nitric oxide (NO) are the main causes of EC dysfunction.

[0007] In recent years, nanoparticle-based photothermal therapy (PTT) has attracted considerable attention as a potential treatment for atherosclerosis. PTT converts light into localized high temperatures (T > 50°C), inducing apoptosis or necrosis of macrophages and foam cells, ultimately leading to plaque ablation. However, the high temperatures (T > 50°C) can cause irreversible damage to surrounding tissues and blood vessels. Excessive apoptosis of foam cells induced by high-temperature PTT can lead to enlargement of the necrotic core, ultimately destabilizing the plaque and promoting acute cardiovascular events. Summary of the Invention

[0008] In view of this, the present invention aims to provide a plaque-targeting nanoparticle, its preparation method, and its application in the preparation of anti-atherosclerotic drugs. The plaque-targeting nanoparticle provided by the present invention can achieve low-temperature (42-45℃) photothermal therapy combined with anti-nlrp3, treating atherosclerosis by regulating lipid accumulation induced by the PCSK9 pathway and repairing damaged endothelial cells.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a nanoparticle with plaque-targeting function, comprising a core and a shell, wherein the core is a ZIF-8 nanoparticle loaded with neoindocyanine green, the shell is arginine-modified polydopamine, and the surface of the shell is modified with hyaluronic acid.

[0011] Preferably, the nanoparticles with plaque-targeting function have a particle size of 140–180 nm.

[0012] Preferably, the nanoparticles with plaque-targeting function contain 10-40% neoindocyanine green, 25-35% polydopamine, and 25-35% arginine by mass.

[0013] This invention provides a method for preparing the above-mentioned nanoparticles with plaque-targeting function, comprising the following steps:

[0014] Neoindocyanine green, water, soluble zinc salt and 2-methylimidazole were mixed and a coordination reaction was carried out to obtain ZIF-8 nanoparticles loaded with neoindocyanine green.

[0015] The ZIF-8 nanoparticles loaded with new indocyanine green were mixed with dopamine hydrochloride and a buffer solution to carry out a self-polymerization reaction, thereby obtaining polydopamine-encapsulated nanoparticles.

[0016] The polydopamine-coated nanoparticles were mixed with arginine and a buffer solution to carry out a first coupling reaction, thereby obtaining arginine-modified nanoparticles.

[0017] The modified arginine nanoparticles were mixed with hyaluronic acid and a buffer solution to carry out a second coupling reaction, thereby obtaining nanoparticles with plaque-targeting function.

[0018] Preferably, the mass ratio of the soluble zinc salt to 2-methylimidazole is 0.05 to 0.15:1;

[0019] The mass ratio of the new indocyanine green to 2-methylimidazole is 0.01 to 0.04:1.

[0020] Preferably, the mass ratio of dopamine hydrochloride to ZIF-8 nanoparticles loaded with neoindocyanine green is 5-10:5-10.

[0021] Preferably, the mass ratio of arginine to polydopamine-coated nanoparticles is 5-10:5-10.

[0022] Preferably, the mass ratio of the hyaluronic acid to the arginine-modified nanoparticles is 5-10:5-10.

[0023] This invention provides the application of the above-mentioned plaque-targeting nanoparticles in the preparation of anti-atherosclerotic drugs.

[0024] Preferably, the operating temperature of the nanoparticles with plaque targeting function is 42–45°C.

[0025] This invention provides a plaque-targeting nanoparticle (HA-PLIZ, abbreviated as HPLIZ), comprising a core and a shell. The core is a ZIF-8 nanoparticle loaded with neoindocyanine green (IR820), and the shell is arginine (LA)-modified polydopamine (PDA). The surface of the shell is modified with hyaluronic acid (HA). The plaque-targeting nanoparticle provided by this invention can regulate lipid levels by simultaneously intervening in multiple pathways, provide anti-inflammatory effects, and protect damaged endothelial cells (ECs). Specifically, damaged endothelial cells and foam cells within plaques highly express CD44, which can specifically bind to HA. Therefore, the HPLIZ nanoparticles provided in this invention can specifically bind to plaque areas. Arginine-modified polydopamine has a strong antioxidant effect, eliminating local ROS and reducing NLRP3 expression. The reduction in NLRP3 levels affects lipid regulation, ultimately leading to a decrease in PCSK9 expression in plaque areas. HPLIZ contains IR820 and PDA, which act as photothermal converters. Under near-infrared light stimulation, the temperature of foam cells rises to 42–45°C, TRPV1 channels open, leading to Ca2+ release. 2+ Influx occurs, followed by activation of the AMPK / SREBP2 / PCSK9 pathway. Both pathways lead to downregulation of PCSK9 expression, ultimately resulting in lipid clearance.

[0026] Furthermore, the HPLIZ provided by this invention is doped with LA, which can generate NO under the excitation of reactive oxygen species and near-infrared radiation. At a mild temperature of 42-45°C, HPLIZ releases NO, thereby activating the eNOS / NO pathway to protect endothelial cells. In addition, the mild temperature leads to high expression of HSP90, which helps maintain eNOS expression. Compared to the traditional PTT method, which alleviates AS by promoting foam cell apoptosis and thus easily leads to local inflammation and plaque rupture, this invention's HPLIZ-based mild PTT strategy can effectively combat AS by regulating lipid metabolism and repairing damaged endothelial cells, achieving a powerful effect of simultaneously lowering lipids, reducing inflammation, and protecting damaged endothelium.

[0027] This invention provides a method for preparing the above-mentioned nanoparticles with plaque targeting function. This method is simple to operate, low in cost, and easy to achieve industrial mass production. Attached Figure Description

[0028] Figure 1 A schematic diagram illustrating the preparation process and application of nanoparticles with plaque-targeting function;

[0029] Figure 2 TEM images of several types of nanoparticles;

[0030] Figure 3 These are the particle sizes of several nanoparticles;

[0031] Figure 4 The zeta potentials of several nanoparticles;

[0032] Figure 5 The images show the ultraviolet-visible spectra of several nanoparticles.

[0033] Figure 6 PXRD results for several types of nanoparticles;

[0034] Figure 7 The weight percentage results for IR820 in IZ, PIZ, PLIZ, and HPLIZ are shown.

[0035] Figure 8 The weight percentage of LA in PLIZ and HPLIZ;

[0036] Figure 9 For water, IZ, PIZ, PLIZ and HPLIZ at 808nm, 0.5W / cm 2 Thermal images and corresponding temperature change curves under near-infrared light irradiation;

[0037] Figure 10 Thermographs of RAW 264.7 cells under 808nm irradiation and their corresponding temperature change curves are shown.

[0038] Figure 11 Representative confocal images of ROS levels in RAW 264.7 cells after different treatments, measured on a DCFH-DA.

[0039] Figure 12 Representative photographs of RAW 264.7 cells after ORO staining following different treatments;

[0040] Figure 13 Representative confocal images of Dil-ox-LDL uptake in RAW 264.7 cells after different treatments;

[0041] Figure 14 To detect Ca2+ in foam cells under different treatment conditions using Fluo-4 AM flow cytometry 2+ Level concentration;

[0042] Figure 15 Western blot images of PCSK9, SREBP2, and NLRP3 expression in RAW 264.7 cells after different treatments;

[0043] Figure 16 Representative results of scratch experiments on HUVECs treated in different ways;

[0044] Figure 17 NO flow cytometry obtained by DAF-FM DA method;

[0045] Figure 18 WB results for HSP90 and eNOS;

[0046] Figure 19 In vitro fluorescence images of the mouse aorta at different time points after injection of three nanoparticles;

[0047] Figure 20 The staining results are for mice with atherosclerosis.

[0048] Figure 21 Representative immunohistochemical images of the aortic arch root stained with NLRP3, IL-1β, PCSK9, ABCA1 and eNOS antibodies from different groups;

[0049] Figure 22 Representative immunofluorescence images of the aortic arch root stained with anti-CD68 and CD31. Detailed Implementation

[0050] This invention provides a nanoparticle with plaque-targeting function, comprising a core and a shell, wherein the core is a ZIF-8 nanoparticle loaded with neoindocyanine green, and the shell is arginine-modified polydopamine; the surface of the shell is modified with hyaluronic acid.

[0051] In this invention, the particle size of the core is preferably 100–120 nm. In this invention, the particle size of the nanoparticles with plaque-targeting function is preferably 140–180 nm, more preferably 150–160 nm.

[0052] In this invention, the nanoparticles with plaque-targeting function preferably contain 10-40% neoindocyanine green by mass, more preferably 20-30%, the content of which is determined by elemental analysis of sulfur by mass percentage; the content of polydopamine is preferably 25-35% by mass, more preferably 30%, the content of which is determined by ultraviolet light; and the content of arginine is preferably 25-35% by mass, more preferably 30%, the content of which is determined by ninhydrin reaction.

[0053] In this invention, IR820 is a derivative of indocyanine green, which can be used as a photothermal and fluorescent agent and has been approved by the FDA for clinical use. ZIF-8 is a metal-organic framework material that can encapsulate IR820 (referred to as IZ). Compared with IR820 alone, IZ can improve photothermal conversion efficiency. Furthermore, ZIF-8 itself can induce foam cell autophagy, thereby activating ABCA1-dependent cholesterol efflux and reducing lipid levels.

[0054] Polydopamine (PDA) is a natural melanin analogue with good biocompatibility. This invention uses PDA as a shell material, which exhibits anti-NLRP3 activity and further leads to a decrease in PCSK9 expression. Furthermore, the PDA coating on the isocyanate layer (IZ) enhances its biocompatibility and, as another photothermal agent, improves the photothermal conversion efficiency of the IZ. In addition, PDA possesses numerous functional groups, allowing for extensive chemical modification and enhanced adhesion to various surfaces.

[0055] L-arginine (LA) has been shown to maintain eNOS and produce NO after reacting with reactive oxygen species (ROS) and nitric oxide synthase, thereby improving endothelial function and reducing oxidative stress.

[0056] Hyaluronic acid (HA) can specifically bind to CD44, which is highly expressed in damaged endothelial cells and foam cells within plaques, thereby enabling HPLIZ nanoparticles to specifically bind to plaque areas.

[0057] This invention provides a method for preparing the above-mentioned nanoparticles with plaque-targeting function, comprising the following steps:

[0058] Neoindocyanine green, water, soluble zinc salt and 2-methylimidazole were mixed and a coordination reaction was carried out to obtain ZIF-8 nanoparticles loaded with neoindocyanine green.

[0059] The ZIF-8 nanoparticles loaded with new indocyanine green were mixed with dopamine hydrochloride and a buffer solution to carry out a self-polymerization reaction, thereby obtaining polydopamine-encapsulated nanoparticles.

[0060] The polydopamine-coated nanoparticles were mixed with arginine and a buffer solution to carry out a first coupling reaction, thereby obtaining arginine-modified nanoparticles.

[0061] The modified arginine nanoparticles were mixed with hyaluronic acid and a buffer solution to carry out a second coupling reaction, thereby obtaining nanoparticles with plaque-targeting function.

[0062] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0063] This invention involves mixing neoindocyanine green, water, a soluble zinc salt, and 2-methylimidazole, and performing a coordination reaction to obtain ZIF-8 nanoparticles (IR820@ZIF-8, abbreviated as IZ) loaded with neoindocyanine green. In this invention, the soluble zinc salt is preferably Zn(NO3)3, more preferably Zn(NO3)3·6H2O. In this invention, the mass ratio of the soluble zinc salt to 2-methylimidazole is preferably 0.05–0.15:1, more preferably 0.1:1; the mass ratio of neoindocyanine green to 2-methylimidazole is 0.01–0.04:1, more preferably 0.02–0.03:1.

[0064] In this invention, the preferred mixing method is to add a new indocyanine green aqueous solution to a soluble zinc salt aqueous solution and stir to mix, followed by the addition of a 2-methylimidazole aqueous solution. In this invention, the preferred stirring and mixing time is 5–10 minutes.

[0065] In this invention, the coordination reaction is preferably carried out at room temperature and for a time of 10–15 min, more preferably 12–14 min. During the coordination reaction, soluble zinc salt reacts with 2-methylimidazole to form zeolite imidazole acid framework-8 (ZIF-8) nanoparticles, with neoindocyanine green loaded into the internal pores of the ZIF-8 nanoparticles.

[0066] In this invention, after the coordination reaction, the resulting reaction product is preferably centrifuged, washed, and dried. The centrifugation rate is preferably 10,000–20,000 rpm, more preferably 15,000 rpm; the centrifugation time is preferably 30–60 min, more preferably 40–60 min. The washing agent used is preferably water or methanol, and the washing is preferably performed 3–5 times. The drying temperature is preferably 60–180°C, more preferably 100–150°C, and the drying time is preferably 6–24 h, more preferably 12–18 h.

[0067] After obtaining the ZIF-8 nanoparticles loaded with neoindocyanine green, the present invention mixes the ZIF-8 nanoparticles loaded with neoindocyanine green with dopamine hydrochloride and a buffer solution to carry out a self-polymerization reaction, thereby obtaining polydopamine-encapsulated nanoparticles (PDA@IZ, abbreviated as PIZ). In the present invention, the buffer solution is preferably Triils-HCl buffer solution, and the pH of the buffer solution is preferably 8.5.

[0068] In this invention, the mass ratio of dopamine hydrochloride to ZIF-8 nanoparticles loaded with neoindocyanine green is 5–10:5–10, more preferably 6–8:5–10. In this invention, the mass ratio of the ZIF-8 nanoparticles loaded with neoindocyanine green to the volume ratio of the buffer solution is preferably 5–10 mg:5–10 mL.

[0069] In this invention, the self-polymerization reaction is preferably carried out under ultrasonic conditions, and the power of the ultrasonic waves is preferably 80-300W, more preferably 100-200W.

[0070] In this invention, the temperature of the self-polymerization reaction is preferably room temperature, and the time is preferably 40–120 min, more preferably 60–100 min. During the self-polymerization reaction, dopamine hydrochloride self-polymerizes into polydopamine, and simultaneously binds to Zn ions through coordination interactions, coating the surface of ZIF-8 nanoparticles loaded with neoindocyanine green.

[0071] Following the self-polymerization reaction, the resulting reaction product is preferably centrifuged, washed, and dried. In this invention, the centrifugation rate is preferably 10,000–20,000 rpm, more preferably 15,000 rpm; the centrifugation time is preferably 30–60 min, more preferably 40–60 min. In this invention, the washing agent is preferably water or methanol, and the washing is preferably performed 3–5 times. In this invention, the drying temperature is preferably 60–180°C, more preferably 100–150°C, and the drying time is preferably 6–24 h, more preferably 12–18 h.

[0072] After obtaining the polydopamine-coated nanoparticles, the present invention mixes the polydopamine-coated nanoparticles with arginine and a buffer solution to perform a first coupling reaction, thereby obtaining arginine-modified nanoparticles (LA@PIZ, abbreviated as PLIZ). In the present invention, the buffer solution is preferably Triss-HCl buffer solution, and the pH of the buffer solution is preferably 8.5.

[0073] In this invention, the preferred mass ratio of arginine to polydopamine-coated nanoparticles is 5–10:5–10, more preferably 6–8:5–10. In this invention, the preferred mass ratio of the polydopamine-coated nanoparticles to the buffer solution is 5–10 mg:5–10 mL.

[0074] In this invention, the temperature of the first coupling reaction is preferably room temperature, and the time is preferably 24 hours. During the first coupling reaction, the amino group of arginine undergoes a Schiff base and / or Michael addition reaction with polydopamine, thereby modifying the polydopamine shell surface with arginine.

[0075] Following the first coupling reaction, the resulting reaction product is preferably centrifuged, washed, and dried. In this invention, the centrifugation rate is preferably 10,000–20,000 rpm, more preferably 15,000 rpm; the centrifugation time is preferably 30–60 min, more preferably 40–60 min. In this invention, the washing agent is preferably water or methanol, and the washing is preferably performed 3–5 times. In this invention, the drying temperature is preferably 60–180°C, more preferably 100–150°C, and the drying time is preferably 6–24 h, more preferably 12–18 h.

[0076] After obtaining the modified arginine nanoparticles, the present invention mixes the modified arginine nanoparticles with hyaluronic acid and a buffer solution to carry out a second coupling reaction, thereby obtaining nanoparticles with plaque-targeting function (HA-PLIZ, abbreviated as HPLIZ). In the present invention, the buffer solution is preferably Triils-HCl buffer solution, and the pH of the buffer solution is preferably 8.5.

[0077] In this invention, the preferred mass ratio of hyaluronic acid to arginine-modified nanoparticles is 5–10:5–10, more preferably 6–8:5–10. In this invention, the preferred mass ratio of the arginine-modified nanoparticles to the buffer solution is 5–10 mg:5–10 mL.

[0078] In this invention, the temperature of the second coupling reaction is preferably room temperature, and the time is preferably 6–36 h, more preferably 12–24 h. During the second coupling reaction, hyaluronic acid interacts with polydopamine and arginine via hydrogen bonds, thereby modifying the surface of the arginine-modified polydopamine shell.

[0079] Following the second coupling reaction, the resulting reaction product is preferably centrifuged, washed, and dried. In this invention, the centrifugation rate is preferably 10,000–20,000 rpm, more preferably 15,000 rpm; the centrifugation time is preferably 30–60 min, more preferably 40–60 min. In this invention, the washing agent is preferably water or methanol, and the washing is preferably performed 3–5 times. In this invention, the drying temperature is preferably 60–180°C, more preferably 100–150°C, and the drying time is preferably 6–24 h, more preferably 12–18 h.

[0080] This invention provides the application of the above-mentioned plaque-targeting nanoparticles in the preparation of anti-atherosclerotic drugs. The core of the plaque-targeting nanoparticles provided by this invention is a zeolite imidazole acid framework-8 (ZIF-8) nanoparticle loaded with novel indocyanine green (IR820), and the outer shell is L-arginine (LA)-modified polydopamine (PDA), which is then modified with hyaluronic acid (HA) to target CD44, which is highly expressed in foam cells and damaged endothelial cells.

[0081] For foam cells, the material was given and, under NIR irradiation, the temperature was increased to 42–45 °C, and the TRPV1 channels were opened, leading to Ca2+ ionization. 2+ The influx of PDA subsequently activated the AMPK / SREBP2 / PCSK9 pathway, reducing PCSK9 expression. Furthermore, PDA exhibited potent anti-inflammatory properties by reducing NLRP3 expression, while NLRP3 simultaneously downregulated PCSK9 levels.

[0082] In damaged endothelial cells, HPLIZ accumulates and activates the eNOS / NO pathway due to LA doping, thereby repairing damaged ECs. Furthermore, mild PTT increases HSP90 expression, which in turn helps promote stable eNOS expression.

[0083] In this invention, the working temperature of the nanoparticles with plaque targeting function is preferably 42-45°C.

[0084] In this invention, the preparation process and application diagram of nanoparticles with plaque-targeting function are shown below. Figure 1 As shown.

[0085] The following examples illustrate in detail the plaque-targeting nanoparticles provided by the present invention, their preparation method, and their application in the preparation of anti-atherosclerotic drugs. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0086] Example 1

[0087] Synthesis of IR820@ZIF-8(IZ) nanoparticles: 32 mg of IR820 was first dissolved in 4 mL of deionized water. Then, the IR820 stock solution was added dropwise to 0.1 g of Zn(NO3)3·6H2O solution. After stirring for 5 min, the mixture was added dropwise to 1 g of 2-MI solution, and then stirred vigorously for 10 min. The mixture was then centrifuged (10000 rpm, 30 min), washed three times with water or methanol, and dried under vacuum at 65 °C for 24 h.

[0088] Synthesis of PDA@IZ (PIZ) nanoparticles: Following the method reported in the literature, 8 mg of the synthesized IZ from the previous step was dispersed in 8 mL of Trils-HCl buffer (pH = 8.5). Then, 8 mg of dopamine hydrochloride was weighed and added to the above solution. After sonication for 60 min, the mixture was centrifuged (10000 rpm, 30 min), washed three times with water or methanol, and vacuum dried at 65 °C for 24 h.

[0089] Synthesis of LA@PIZ (PLIZ) nanoparticles: 8 mg of PIZ was dispersed in 8 mL of Tris-HCl buffer (pH = 8.5). 8 mg of LA was weighed and added to the above solution. After stirring for 24 h, the mixture was centrifuged (10000 rpm, 30 min), washed three times with water or methanol, and dried under vacuum at 65 °C for 24 h.

[0090] Synthesis of HA-PLIZ (HPLIZ) nanoparticles: 10 mg of PLIZ was weighed and dispersed in 10 mL of Tris-HCl buffer (pH = 8.5). 10 mg of HA was weighed and added to the above solution. After stirring for 24 h, the mixture was centrifuged (10000 rpm, 30 min), washed three times with water or methanol, and dried under vacuum at 65 °C for 24 h.

[0091] Comparative Example 1

[0092] Synthesis of ZIF-8 nanoparticles: First, 0.1 g of Zn(NO3)3·6H2O was dissolved in 0.1 mL of deionized water, and 1 g of 2-MI was dissolved in 8 mL of deionized water. Then, the Zn(NO3)3·6H2O stock solution was added dropwise to the 2-MI solution, and the mixture was stirred for 10 min. The prepared ZIF-8 nanoparticles were centrifuged at 10000 rpm for 30 min, washed three times with water or methanol, and dried under vacuum at 65 °C for 24 h.

[0093] TEM images of several nanoparticles are shown below. Figure 2 As shown. Figure 2 In the image, (a) to (e) are TEM images of ZIF-8, IZ, PIZ, PLIZ, and HPLIZ, respectively. Figure 2 The scale bar is 200 nm.

[0094] The particle size of several nanoparticles is as follows Figure 3 As shown, the average dimensions of ZIF-8, IZ, PIZ, PLIZ, and HPLIZ are 131.6 nm, 137 nm, 143 nm, 153.4 nm, and 160.8 nm, respectively.

[0095] zeta potentials of several nanoparticles, such as Figure 4As shown, ZIF-8 and IZ exhibit positive zeta potentials in pure water, at 21.69 mV and 19.68 mV, respectively. The surface zeta potentials of PIZ, PLIZ, and HPLIZ are -32.81 mV, -20.025 mV, and -21.208 mV, respectively.

[0096] UV-Vis spectra of several nanoparticles are as follows Figure 5 As shown, the UV-Vis absorption spectrum indicates that the absorption peaks of IZ, PIZ, PLIZ, and HPLIZ are around 860 nm, indicating the successful loading of IR820.

[0097] The crystal structures of five nanoparticles were measured using powder X-ray diffraction (PXRD). The PXRD results for the various nanoparticles are shown in the figure below. Figure 6 As shown, all particles exhibit high crystallinity, consistent with published crystal structure data.

[0098] The weight percentage of IR820 in IZ, PIZ, PLIZ, and HPLIZ was analyzed by elemental analysis of the specific sulfur element. The results of the weight percentage of IR820 in IZ, PIZ, PLIZ, and HPLIZ are as follows: Figure 7 As shown in the figure, the results indicate that the weight percentages of IR820 are 23.1%, 20.0%, 12.4%, and 10.8%, respectively.

[0099] The qualitative and quantitative analysis of LA doping in PLIZ and HPLIZ was performed using the ninhydrin colorimetric method. The weight percentage of LA in PLIZ and HPLIZ is shown in the figure below. Figure 8 As shown, their weight percentages are 34.5% and 28.5%, respectively.

[0100] Example 2: Photothermal conversion performance of nanoparticles

[0101] First, 180 μL of each of IR820, IZ, PIZ, PLIZ, and HPLIZ (all at a concentration of 50 μg / mL) were added to 200 μL EP tubes. Deionized water (DI water) was used as a negative control. Then, the solution was incubated at 808 nm at 0.5 W / cm². 2 The solution was irradiated for 5 minutes. A thermal infrared imager was used to monitor temperature changes during this 5-minute period.

[0102] Water, IZ, PIZ, PLIZ, and HPLIZ at 808nm, 0.5W / cm 2 The thermal map and corresponding temperature change curve under near-infrared light irradiation are as follows: Figure 9As shown, the temperatures of all experimental groups increased over time compared to DI water. The temperatures of IZ, PIZ, PLIZ, and HPLIZ increased by 20.5, 27.8, 38.8, and 32.3 °C, respectively. The PDA coating improved the photothermal conversion efficiency of IZ. Furthermore, the presence of LA in PLIZ also significantly increased the temperature, due to the construction of the donor-acceptor microstructure in PDA-LA, which reduces non-thermal radiative transition processes by increasing the free radical concentration. Compared to PLIZ, HA modification slightly reduced the photothermal conversion efficiency of HPLIZ, but it was still higher than that of PIZ. These results indicate that HPLIZ can serve as an excellent photothermal conversion agent for further PTT studies in vitro and in vivo.

[0103] Example 3: Cell Temperature Control Results

[0104] RAW 264.7 cells were fed at a rate of 1×10⁻⁶. 6 Cells were seeded at a density of [number] cells / mL in six-well plates and cultured for 24 h. Then, 50 μg / mL of PIZ, PLIZ, and HPLIZ were added to each well, and the cells were cultured for another 24 h. Subsequently, the cells were washed three times with PBS, digested, and resuspended in 250 μL of PBS to form a cell suspension. Then, the cells were incubated at 808 nm at 0.5 W / cm². 2 The solution was irradiated for 5 minutes. A thermal infrared imager was used to monitor temperature changes during this 5-minute period.

[0105] The thermograms of RAW 264.7 cells collected under 808nm irradiation and their corresponding temperature change curves are shown below. Figure 10 As shown in the figure, the cell temperature treated with all three nanoparticles reached 42℃, and the cell temperature treated with PLIZ and HPLIZ even reached 45℃, indicating that the cell temperature can achieve good in vitro mild PTT effect.

[0106] Example 4: Effect of photothermal nanomedicines on reactive oxygen species in foam cells

[0107] ROS clearance was detected using a 2,7-dichlorofluorescein diacetate (DCFH-DA) assay kit. RAW 264.7 cells were cultured at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of 100–500 ng / mL in 6-well plates, with 6 replicates per group. Cells were then stimulated with lipopolysaccharide (LPS) at a concentration of 100–500 ng / mL (or without LPS as a negative control), followed by treatment with PIZ, PLIZ, or HPLIZ for 24 h. After 24 h, 3 samples from each group were randomly selected for illumination under the same conditions as above. Cells were then observed and photographed under a confocal microscope.

[0108] Representative confocal images of ROS levels in RAW 264.7 cells after different treatments, measured on a DCFH-DA, are shown below. Figure 11 As shown in the figure. The experimental results indicate that HPLIZ can effectively scavenge reactive oxygen species (ROS) produced by LPS-stimulated macrophages. Furthermore, increasing light exposure does not stimulate cells to produce more ROS, suggesting that low-temperature light exposure is safe for cells.

[0109] Example 5: Effect of photothermal nanomedicines on lipid content in foam cells

[0110] To determine lipid deposition in macrophages after different treatments, this invention performed ORO staining and simultaneously detected Dil-ox-LDL uptake. The experimental procedure for ORO staining is as follows: RAW264.7 cells (2.0 mL, 5 × 10⁻⁶ cells) were... 5 Cells were seeded in 6-well plates. RAW 264.7 cells were used as a negative control. RAW 264.7 cells treated with ox-LDL (80 μg / mL) and LPS (500 ng / mL) for 24 h served as positive controls. Other groups were treated with ox-LDL (80 μg / mL) and LPS (500 ng / mL), and PIZ, PLIZ, and HPLIZ (50 μg / mL) for 24 h, respectively. Two wells were set up for each of the three treatment groups. After 24 h, one well from each of PIZ, PLIZ, and HPLIZ was randomly selected and irradiated with a NIR laser (0.5 W / cm², 808 nm) for 5 min. Cells were then washed with PBS, fixed with 4% paraformaldehyde for 15 min, washed with 60% isopropanol, and stained with Oil Red O. Foam cells were observed and photographed using a fluorescence microscope (Olympus, Japan). The results are shown below. Figure 12 As shown ( Figure 12 (Medium scale bar is 50 μm). The results showed that HPLIZ+NIR could reduce the area of ​​red staining in cells, i.e., clear lipids from foam cells.

[0111] The Dil-ox-LDL uptake assay was performed as follows: RAW 264.7 cells were incubated at 2 × 10⁻⁶ cells per cell line. 5 Cells were seeded at 1 mL / mL in a confocal dish (35 mm). RAW 264.7 cells were used as a negative control. RAW 264.7 cells treated with LPS (500 ng / mL) served as a positive control. Then, cells were treated with LPS (500 ng / mL) and different nanoparticles (PIZ, PLIZ, and HPLIZ, 50 μg / mL) as experimental groups, with two wells in each group. After 24 h of culture, one well each of PIZ, PLIZ, and HPLIZ was randomly selected and treated with a NIR laser (0.5 W / cm²). 2The cells were irradiated with 808nm for 5 minutes. Then, they were treated with DiI-ox-LDL (40μg / mL) for 4 hours. Subsequently, the cells were observed and photographed under a confocal microscope. The results are as follows: Figure 13 As shown ( Figure 13 (Medium scale bar is 50 μm). The results showed that HPLIZ+NIR could effectively inhibit the uptake of ox-LDL, that is, inhibit the formation of foam cells.

[0112] Example 6: Study on the mechanism of nanomedicine regulating inflammation and lipids

[0113] This invention measures Ca 2+ The presence or absence of TRPV1 channels was determined by the concentration of FITC. Cell treatment was as described above. After treatment, cells were stained with Fluo-4AM staining agent, and then FITC channels were detected by flow cytometry. Ca2+ levels in foam cells under different treatment conditions were detected using Fluo-4AM flow cytometry. 2+ Level concentration such as Figure 14 As shown. The results show that Ca in the three illumination groups 2+ The content was significantly higher than that in the non-illuminated group, suggesting that low-temperature photothermal activity can indeed open the TRPV1 channel.

[0114] Western blot analysis was used to detect the expression levels of NLRP3, PCSK9, and SREBP2. Cell treatment was as described above, followed by lysis of RAW 264.7 cells using RIPA. The total protein concentration of each sample was then determined using a standard BCA assay. Protein samples of the same concentration were separated by 4–12% Bis-Tris gel electrophoresis and transferred to 0.22 μm PVDF membranes. These membranes were then incubated overnight at 4°C with different antibodies (anti-NLRP3, PCSK9, SREBP2). The resulting PVDF membranes were then incubated with the corresponding secondary antibodies for 2 hours. The target proteins were detected using a chemiluminescence imaging system (Tanon, China). Western blot images of PCSK9, SREBP2, and NLRP3 expression in RAW 264.7 cells after different treatments are shown below. Figure 15 As shown, LPS and ox-LDL treatment significantly increased the levels of NLRP3, SREBP2, and PCSK9 in RAW264.7 cells. The levels of NLRP3, SREBP2, and PCSK9 in all experimental groups were lower than those in the LPS and ox-LDL treatment groups. Furthermore, the HPLIZ+NIR treatment group showed the highest expression levels of NLRP3, SREBP2, and PCSK9. This suggests that HPLIZ+NIR cryophotothermal therapy can reduce NLRP3, PCSK9, and SREBP2, thereby achieving anti-inflammatory and lipid-lowering effects.

[0115] Example 7: Nanomedicine improves endothelial cell function through the eNOS / NO pathway

[0116] To verify the protective function of nanoparticles on endothelial cells, we conducted a cell scratch assay. 2 × 10⁶ nanoparticles were used. 5 HUVEC cells were cultured in 6-well plates for 24 h. A 200 μL pipette tip was used to run the cells through the plate, and the damaged area was photographed using a microscope (Olympus). The cell culture medium in the positive control group was then replaced with fresh medium without LPS (500 ng / mL). The cell culture medium in the other groups was replaced with fresh medium containing LPS (500 ng / mL) and different nanomedicines (50 μg / mL PIZ, PLIZ, and HPLIZ), two wells per group. After another 24 h of culture, one well each of PIZ, PLIZ, and HPLIZ was randomly selected and screened under near-infrared light (0.5 W / cm²). 2 Irradiated with 808 nm for 5 min. After 4 hours, images were taken using a microscope. Representative results of the scratch experiment on HUVECs treated with different methods are shown in the figure below. Figure 16 As shown in the figure, HUVECs healing was observed in all experimental groups, with the HPLIZ+NIR group exhibiting the highest degree of wound healing, indicating that HPLIZ+NIR can effectively promote endothelial cell repair.

[0117] NO production capacity was determined using the standard DAF-FM DA method. 2 × 10 5 HUVEC cells were seeded at 500 ng / mL in 6-well plates and cultured for 24 h. Then, the medium in the positive control group was replaced with fresh medium containing LPS (500 ng / mL). Simultaneously, the medium in the other groups was replaced with fresh medium containing LPS (500 ng / mL) and different nanomedicines (50 μg / mL PIZ, PLIZ, and HPLIZ), with 2 wells per group. After another 24 hours, the PIZ, PLIZ, and HPLIZ groups were randomly selected for NIR irradiation (0.5 W / cm², 5 min). Four hours after irradiation, cells were stained with the DAF-FM DA probe for flow cytometry analysis. The NO flow cytometry results obtained by the DAF-FM DA method are shown below. Figure 17 As shown in the figure. The results showed that the NO levels in the PLIZ+NIR and HPLIZ+NIR treatment groups were significantly higher than those in the LPS group, suggesting that doped LA can be excited into NO by ROS and NO, promoting endothelial cell repair.

[0118] Western blot (WB) was used to detect eNOS and HSP90 expression levels. Cell treatment was as described above, followed by lysis of HUVECs using RIPA. The total protein concentration of each sample was then determined using a standard BCA assay. Protein samples of the same concentration were separated by 4–12% Bis-Tris gel electrophoresis and transferred to 0.22 μm PVDF membranes. These membranes were then incubated overnight at 4°C with different antibodies (anti-enos, anti-hsp90). The resulting PVDF membranes were then incubated with the corresponding secondary antibodies for 2 hours. The target proteins were detected using a chemiluminescence imaging system (Tanon, China). Results indicated that LPS treatment significantly reduced eNOS levels in HUVECs. The eNOS levels in all experimental groups were higher than those in the LPS-treated groups. Furthermore, the PLIZ+NIR and HPLIZ+NIR treatment groups showed the highest eNOS expression levels, consistent with NO release levels. Interestingly, we also observed that even in the absence of LA, which typically produces eNOS and NO, PIZ+NIR-treated cells exhibited higher NO levels and eNOS expression than PIZ-treated cells. Therefore, this invention hypothesizes that a slight increase in temperature induced by NIR irradiation leads to upregulation of HSP90 expression, and HSP90 is a key intermolecular chaperone protein for stabilizing eNOS. Western blot results for HSP90 and eNOS are shown below. Figure 18 As shown in the figure. The results showed that the HSP90 expression level in the NIR irradiation group was significantly higher than that in the non-irradiation group. Therefore, it is demonstrated that mild PTT can induce HSP90 upregulation, further increasing and maintaining eNOS and NO levels.

[0119] Example 8: Specific uptake of nanomedicines for anti-inflammatory effects in atherosclerotic mice

[0120] Feeding ApoE on a high-fat diet - / - A mouse model of atherosclerosis was established. Wild-type mice were fed a normal diet as a healthy control group. PIZ, PLIZ, and HPLIZ were injected via tail vein, each containing 1 mg / kg of IR820 (neoindocyanine green, a fluorescent dye used to visualize the enrichment area and amount of material in the aorta). Mice were sacrificed 6 h and 24 h after injection, and the aorta was collected for in vitro fluorescence imaging. After imaging, the aorta was fixed in 4% paraformaldehyde and stained with Oil Red O to determine plaque areas. In vitro fluorescence images of the mouse aorta at different time points after injection of the three nanoparticles are shown below. Figure 19 As shown in the figure above, it can be seen that at 6 h and 24 h after injection, the active fluorescence signal in the WT mice injected with HPLIZ was negligible, while the aorta of the AS mice showed obvious red fluorescence. At all time points, the aorta fluorescence signal of the HPLIZ-injected mice was the strongest, followed by PLIZ, and PIZ was the weakest.

[0121] To determine whether there was a one-to-one correspondence between plaque locations and fluorescence locations, the extracted aorta was stained with ORO. The results showed that no plaques were formed in the WT group mice, while there was no statistically significant difference in plaque area among the three AS groups of mice. The ORO staining photograph (scale bar: 3mm) taken after in vitro fluorescence is shown in the image above. Figure 19 As shown in the figure below, the fluorescence intensity and plaque area of ​​AS mice correspond one-to-one. Among them, the HPLIZ group had the highest fluorescence intensity / plaque area 24 hours after injection, proving that it has the strongest targeting performance.

[0122] Example 9: Effects of nanomedicines on total lipids, total collagen, and plaque in atherosclerotic mice.

[0123] High-fat feeding ApoE - / - A 12-week-old mouse model of atherosclerosis was established. The atherosclerotic mice were then randomly divided into four groups (n=8 per group) to study the therapeutic effects: (1) PBS treatment (blank control group); (2) oral atorvastatin group (ATST); (3) tail vein injection of HPLIZ group; (4) tail vein injection of HPLIZ combined with 808nm (1W, 5min) laser irradiation group (HPLIZ+NIR group). Treatment was administered every 3 days for 3 weeks, during which a high-fat diet was maintained. At the end of treatment, the mice were sacrificed. Four mice were randomly selected for gross oil red staining to reflect the overall therapeutic effect. The remaining four mice underwent aortic sections after treatment, and ORO, HE, and Masson trichrome staining were performed to reflect lipid content, total plaque area, and collagen fiber content, respectively. The staining results of the atherosclerotic mice are shown below. Figure 20 As shown in the diagram. A schematic diagram of the gross Oil Red staining results is shown below. Figure 20 The above figure is shown (scale bar: 2mm). The results show that the HPLIZ+NIR group had the largest reduction in plaque area as a percentage of aortic area compared to the other groups.

[0124] A schematic diagram of the HE, ORO, and Masson's triple staining results after treatment is shown below. Figure 20 The figure below (scale bar: 100μm) shows that HE staining revealed a reduction in plaque area and a smaller necrotic core in the HPLIZ+NIR treatment group compared to other groups. ORO staining showed that the HPLIZ+NIR treatment group reduced lipid content in atherosclerotic mice compared to other groups. Masson's trichrome staining results showed that the HPLIZ+NIR treatment group had increased collagen content and more stable plaques compared to other groups.

[0125] Example 10: Effects of nanomedicines on the therapeutic mechanism in mice with atherosclerosis

[0126] After treatment, mouse aortas were fixed with 4% paraformaldehyde, sectioned, and incubated with antibodies against NLRP3, IL-1β, PCSK9, ABCA1, and eNOS for immunohistochemical analysis. Simultaneously, other aortic sections were incubated with antibodies against CD68 and CD31 for immunofluorescence staining. Representative immunohistochemical images of the aortic arch root stained with NLRP3, IL-1β, PCSK9, ABCA1, and eNOS antibodies from different groups are shown below. Figure 21 As shown (scale bar: 100 μm), representative immunofluorescence images of the aortic arch root stained with anti-CD68 and CD31 are as follows. Figure 22 As shown (scale bar: 100μm).

[0127] The results showed that in the HPLIZ+NIR group, indicators related to inflammation and lipid accumulation, such as NLRP3, IL-1β, PCSK9, and CD68, were decreased; while indicators related to cholesterol transport, ABCA1, were increased, and indicators related to endothelial cell function, eNOS and CD31, were increased. This indicates that HPLIZ+NIR reduces inflammation and lipid accumulation and repairs endothelial cells through the NLRP3-IL1β, PCSK9, and eNOS pathways, thereby achieving an anti-atherosclerotic effect.

[0128] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a plaque-targeting nanoparticle in the preparation of an anti-atherosclerotic drug, wherein the plaque-targeting nanoparticle comprises a core and a shell, wherein the core is a ZIF-8 nanoparticle loaded with neoindocyanine green, and the shell is arginine-modified polydopamine; the surface of the shell is modified with hyaluronic acid. The nanoparticles with patch-targeting function have a particle size of 140~180 nm; The nanoparticles with plaque-targeting function contain 10-40% neoindocyanine green, 25-35% polydopamine, and 25-35% arginine by mass. The preparation method of the nanoparticles with plaque targeting function includes the following steps: Neoindocyanine green, water, soluble zinc salt and 2-methylimidazole were mixed and a coordination reaction was carried out to obtain ZIF-8 nanoparticles loaded with neoindocyanine green. The ZIF-8 nanoparticles loaded with new indocyanine green were mixed with dopamine hydrochloride and a buffer solution to carry out a self-polymerization reaction, thereby obtaining polydopamine-encapsulated nanoparticles. The polydopamine-coated nanoparticles were mixed with arginine and a buffer solution to carry out a first coupling reaction, thereby obtaining arginine-modified nanoparticles. The modified arginine nanoparticles were mixed with hyaluronic acid and a buffer solution to carry out a second coupling reaction, thereby obtaining nanoparticles with plaque-targeting function.

2. The application according to claim 1, characterized in that, The mass ratio of the soluble zinc salt to 2-methylimidazole is 0.05~0.15:1; The mass ratio of the new indocyanine green to 2-methylimidazole is 0.01~0.04:

1.

3. The application according to claim 1, characterized in that, The mass ratio of dopamine hydrochloride to ZIF-8 nanoparticles loaded with neoindocyanine green is 5~10:5~10.

4. The application according to claim 1, characterized in that, The mass ratio of arginine to polydopamine-encapsulated nanoparticles is 5~10:5~10.

5. The application according to claim 1, characterized in that, The mass ratio of the hyaluronic acid to the modified arginine nanoparticles is 5~10:5~10.

6. The application according to claim 1, characterized in that, The nanoparticles with plaque-targeting function operate at a temperature of 42~45℃.

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