Nanometer lipid particles for treating atherosclerosis and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a kind of nano-lipid granules for treating atherosclerosis and its preparation method and application, the nano-lipid granules take PLGA encapsulated PDHA1 protein and aggregation-induced emission molecule as core, foam cell membrane is coated on the surface of PLGA, the outermost layer is coated with active oxygen responsive liposome;Aggregation-induced emission molecule is the coupling product of diphenylamine donor unit and benzothiadiazole-thiophene acceptor unit.The present application co-delivers PDHA1 protein and AIE photothermal molecule, homologous targeting of foam cell membrane, active oxygen responsive liposome outer layer and near-infrared two-zone imaging / photothermal therapy function is integrated in a single nano platform, realizes the precise diagnosis of HHcy-AS, metabolic microenvironment repair and photothermal removal of foam cells multimodal synergistic therapy, and provides a new treatment strategy for atherosclerosis, especially high homocysteine related atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nanolipid particles for treating atherosclerosis, its preparation method, and its application. Background Technology

[0002] Atherosclerosis, or AS for short, is considered a lipid-driven chronic inflammatory disease in which low-density lipoprotein accumulates under the arterial intima, triggering inflammation and plaque formation. Recent studies have found that metabolic disorders, particularly alterations in macrophage function, play a crucial role in disease progression beyond lipid accumulation.

[0003] Hyperhomocysteinemia, or HHcy for short, has been identified as an independent risk factor for ankylosing spondylitis (AS). Unlike classic AS, HHcy-associated AS (HHcy-AS) has a unique pathogenic mechanism: high homocysteine ​​levels not only exacerbate oxidative stress but also significantly induce profound metabolic reprogramming in macrophages, causing them to persistently favor glycolytic metabolism rather than mitochondrial oxidative phosphorylation under both baseline and inflammatory conditions. This metabolic shift is a key driver of chronic inflammation, accelerating the progression of AS.

[0004] Currently, clinical interventions for HHcy-AS are extremely limited, mainly relying on B vitamins and folic acid supplementation to lower homocysteine ​​levels. However, numerous clinical studies have shown that such regimens are ineffective in slowing plaque progression and reducing cardiovascular events, and cannot reverse existing macrophage metabolic reprogramming and inflammatory phenotypes. While traditional lipid-lowering drugs such as statins can lower LDL cholesterol, they have little effect on repairing HHcy-specific metabolic disorders, and long-term use can lead to side effects such as muscle toxicity and elevated liver enzymes.

[0005] More importantly, none of the aforementioned existing treatments possess the ability to actively target atherosclerotic plaques. Whether it's vitamins / folic acid or statins, their distribution in the body lacks specificity, failing to effectively accumulate locally on the plaque, resulting in insufficient drug concentration at the lesion site, limited efficacy, and increased risk of systemic toxicity. Therefore, there is an urgent need to develop a new treatment strategy for atherosclerosis with superior plaque-targeting capabilities to overcome the limitations of existing technologies. Summary of the Invention

[0006] The purpose of this invention is to provide nanolipid particles for treating atherosclerosis, thereby solving the problems existing in the prior art.

[0007] The technical solution adopted in this invention is: This invention provides a nanolipid particle for treating atherosclerosis. The nanolipid particle has PDHA1 protein encapsulated in PLGA and aggregation-induced luminescent molecules as its core, foam cell membranes covering the PLGA surface, and reactive oxygen species responsive liposomes as its outermost layer. The amino acid sequence of PDHA1 protein is shown in SEQ ID NO.1; The aggregation-induced emission molecule is a conjugate of a diphenylamine donor unit and a benzothiadiazole-thiophene acceptor unit; the molar ratio of the diphenylamine donor unit to the benzothiadiazole-thiophene acceptor unit is 1:0.2~0.5.

[0008] Preferably, the molar ratio of the diphenylamine donor unit to the benzothiadiazole-thiophene acceptor unit is 1:0.3.

[0009] Preferably, the diphenylamine donor unit is 4-(diphenylamino)phenylboronic acid; The benzothiadiazole-thiophene acceptor unit is 4,8-bis(5-bromo-3-(2-octyldodecyl)-2-thiophene)-2λ 4 δ²-benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole).

[0010] Preferably, the mass ratio of PDHA1 protein to aggregation-induced luminescence molecules is 0.4:200~500.

[0011] Preferably, the particle size of the nanolipid particles is 150nm~200nm.

[0012] A second aspect of the present invention provides a method for preparing the aforementioned nanolipid particles, comprising the following steps: Preparation of foam cell membranes; By utilizing the Suzuki cross-coupling reaction, a diphenylamine donor unit was linked to a benzothiadiazole-thiophene acceptor unit in the presence of a palladium catalyst to construct an aggregation-induced emission molecule. PDHA1 protein and aggregation-induced luminescence molecules were encapsulated in PLGA to obtain the PLGA core; The surface of the PLGA core is coated with a foam cell membrane, and then reactive oxygen species responsive liposomes are further coated on the surface of the foam cell membrane to obtain the nanolipid particles.

[0013] Preferably, the palladium catalyst is Pd(PPh3)4.

[0014] Preferably, the reaction conditions for the Suzuki cross-coupling reaction are 80°C and 12 h under nitrogen protection.

[0015] Preferably, the molar ratio of soybean phosphatidylcholine, cholesterol, and DSPE-SeSe-PEG2000 in the reactive oxygen species responsive liposomes is 8:1:1.

[0016] A third aspect of the present invention provides an application of the aforementioned nanolipid particles, wherein the nanolipid particles are used to prepare a drug for treating atherosclerosis.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention provides nanolipid particles for treating atherosclerosis. The nanolipid particles have a core of PLGA-encapsulated PDHA1 protein and aggregation-induced emission molecules, with a foam cell membrane coating the PLGA surface and an outermost layer of reactive oxygen species (ROS)-responsive liposomes. The amino acid sequence of the PDHA1 protein is shown in SEQ ID NO.1. The aggregation-induced emission molecules are conjugates of diphenylamine donor units and benzothiadiazole-thiophene acceptor units; the molar ratio of diphenylamine donor units to benzothiadiazole-thiophene acceptor units is 1:0.2~0.5. The nanolipid particles of this invention employ a multi-layered core-shell structure design, with a core of PLGA co-encapsulated PDHA1 protein and the near-infrared II aggregation-induced emission molecule TPAT12.8-B, an intermediate layer of foam cell membrane, and an outermost layer of ROS-responsive liposomes containing diselenyl bonds. This structure addresses the key issue of lacking active plaque targeting: the highly expressed adhesion receptors such as CD36 and MSR1 on the foam cell membrane surface mediate homologous targeting, endowing nanoparticles with the ability to specifically recognize and efficiently uptake foam cells, thereby achieving precise enrichment of atherosclerotic plaques. Simultaneously, the foam cell membrane coating also possesses immune escape capabilities, prolonging the half-life of nanoparticles in the bloodstream and further increasing drug accumulation at the lesion site. The outermost ROS-responsive liposomes ensure that dissociation is triggered under the highly reactive oxygen species conditions of the plaque microenvironment, achieving locally controllable release of PDHA1 protein and TPAT12.8-B, avoiding the problems of insufficient efficacy and increased toxicity caused by systemic distribution of traditional drugs.

[0018] Based on targeted delivery, this invention achieves synergistic intervention on the unique pathological mechanisms of HHcy-AS. On the one hand, the PDHA1 protein delivered to the lesion can repair the metabolic reprogramming of macrophages induced by high homocysteine, restore mitochondrial oxidative phosphorylation function, inhibit foam cell formation, and promote macrophage polarization towards the anti-inflammatory M2 phenotype, reversing the chronic inflammatory state from the metabolic root. On the other hand, the co-delivered TPAT12.8-B can generate a mild photothermal effect under near-infrared II laser irradiation, precisely ablate foam cells in the plaque, and its aggregation-induced emission properties can realize high-resolution NIR-II fluorescence imaging of deep tissues, integrating diagnostic and therapeutic functions on the same nanoplatform, breaking through the single-function limitation of traditional drugs that are "only for treatment and not for diagnosis".

[0019] In summary, this invention significantly improves the therapeutic effect of anti-atherosclerosis through the synergistic effects of multiple mechanisms, including biomimetic membrane-mediated homologous active targeting, microenvironment-responsive drug release, metabolic reprogramming repair, and NIR-II imaging-guided photothermal therapy, while reducing systemic toxicity. This provides a novel technical solution for the integrated precision diagnosis and treatment of HHcy-AS. Compared with existing interventions such as vitamin supplementation and statins, which are passively distributed and have limited efficacy, this invention demonstrates significant advancements in targeting, therapeutic efficiency, safety, and functional integration. Furthermore, its modular design can be extended to the diagnosis and treatment of other inflammatory or metabolic diseases, possessing significant scientific value and clinical translational potential. Attached Figure Description

[0020] Figure 1 These are representative transmission electron microscope (TEM) images of the molecular orbital distribution of the AIE molecule TPAT12.8 in this invention and the products of different steps after negative staining with phosphotungstic acid. A: Amplitude diagram of the highest occupied molecular orbital (HOMO); B: Amplitude plot of the lowest unoccupied molecular orbital (LUMO); C: Energy gap result; D: Transmission electron microscope images of products from different steps.

[0021] Figure 2 The images show Coomassie Brilliant Blue staining and Western blot images of P1-TPA@FL NPs prepared in this invention; A is Coomassie Brilliant Blue staining, and B is Western blot.

[0022] Figure 3 The P1-TPA@FL NPs prepared for this invention can efficiently target foam cells and effectively remove them; A: Infrared thermograph of foam cells after laser irradiation following incubation with P1-TPA@FL NPs; B: Temperature-time curves of P1-TPA@FL treated cells and control cells; C: Live / dead staining results of foam cells under different treatments; D: Statistical results of graph C; E: Representative flow cytometry images of foam cells after different treatments; F: Statistical results of graph E; G: Schematic diagram of P1-TPA@FL-mediated in vitro synergistic photothermal therapy (PTT) and enhanced phagocytosis; H: Flow cytometry analysis of macrophage phagocytosis after treatment with different substances. Cell tracking red: apoptotic foam cells / debris; Cell tracking green: macrophages;

[0023] Statistical results of the I:H plot.

[0024] Figure 3 In the study, G1: foam cells (control group); G2: control group + laser (808 nm, 0.8 W / cm², 10 min); G3: P1-TPA@FL; G4: P1-TPA@ML + laser (808 nm, 0.8 W / cm², 10 min); G5: P1-TPA@FL + laser (808 nm, 0.8 W / cm², 10 min).

[0025] Figure 4 The P1-TPA@FLNPs prepared for this invention can inhibit lipid uptake in homocysteine-treated macrophages and promote their conversion to M2 polarization. A: Representative optical images of RAW264.7 macrophages after stimulation with homocysteine ​​and oxidized low-density lipoprotein, followed by different treatments. Cells were stained with Oil Red O (ORO), and positively stained cells were identified as foam cells. Scale bar: 50µm;

[0026] B: Statistical results of graph A; C: Immunofluorescence staining results of CD86 fluorescence, scale bar: 40µm; D: Statistical results of graph C; E: Immunofluorescence staining results of CD206; F: Statistical results of graph E; G1~G5 are the control, model, rPDHA1, P1-TPA@F and P1-TPA@FL processes, respectively.

[0027] Figure 5 The P1-TPA@FLNPs prepared for this invention are used to counteract ApoE - / - Near-infrared two-zone imaging of atherosclerotic plaques in mice; A: In mice with atherosclerosis, near-infrared fluorescence imaging of the carotid artery was performed before and 30 minutes after intravenous injection of PBS, TPA@PLGA, or TPA@FL. The magnified area highlights the fluorescence distribution within the plaque region;

[0028] B: Near-infrared fluorescence imaging of the iliac artery was performed 30 minutes after injection of PBS, TPA@PLGA, or TPA@FL.

[0029] Figure 6 Gross stained images of blood vessels in different treatment groups for atherosclerosis according to the present invention; A: Representative images of the aortic arch obtained from each group, with red arrows indicating areas of lipid deposition; B: Macroscopic staining results of the aortic arch; C: Statistical results of graph B; D: Oro staining image of the area of ​​atherosclerotic plaques in a section of the arterial root; E: Statistical results of graph D; G1: Control group (normal diet); G2: Model group (high methionine diet); G3: rPDHA1; G4: P1-TPA@FL; G5: P1-TPA@FL + laser.

[0030] Figure 7 The images show H&E staining of the heart, liver, spleen, lungs, and kidneys of mice treated with different treatment groups according to the present invention. Detailed Implementation

[0031] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0032] The inventive concept of this invention is as follows: Targeting the unique pathological mechanism of macrophage metabolic reprogramming in hyperhomocysteinemia-related atherosclerosis, this invention, for the first time, co-encapsulates the pyruvate dehydrogenase E1α subunit (PDHA1) protein and the near-infrared II aggregation-induced emission photothermal molecule TPAT12.8-B within a PLGA core. The outer layers are successively disguised as foam cell membranes (endowing them with homologous targeting and immune escape capabilities) and reactive oxygen species responsive liposomes containing diselenyl bonds, constructing a multi-layered core-shell structured biomimetic nanoparticle. After intravenous injection, the particle utilizes receptors on the surface of foam cell membranes to mediate plaque-specific enrichment and triggers the degradation of outer liposomes in the highly reactive oxygen microenvironment of the plaque, releasing PDHA1 to repair macrophage oxidative phosphorylation function, inhibit foam cell formation, and promote M2-type anti-inflammatory polarization. Simultaneously, TPAT12.8-B achieves high-resolution imaging of deep tissues and local photothermal ablation of foam cells under near-infrared II laser irradiation, thus integrating metabolic microenvironment repair, precise photothermal ablation, and imaging diagnosis into a single platform to achieve multimodal synergistic treatment. Furthermore, this modular design can be extended to other inflammatory diseases.

[0033] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments. In the description of the present invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0034] Example 1 A nanolipid particle for treating atherosclerosis, the specific preparation method of which is as follows: S1. Prepare foam cell membranes.

[0035] Objective: To obtain foam cell membranes that highly express adhesion receptors such as CD36 and MSR1.

[0036] Specific operations: (1) Cell culture: RAW 264.7 macrophages were cultured in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin) and cultured in T175 culture flasks at 37°C in a 5% CO2 incubator to about 80% confluence.

[0037] (2) Foam cell induction: Discard the old culture medium, add fresh DMEM complete culture medium containing 100 μmol / L homocysteine ​​and 80 μg / mL oxidized low-density lipoprotein (ox-LDL), and continue to culture for 24 h.

[0038] (3) Cell collection: Discard the culture medium, wash three times with pre-cooled PBS, add 0.25% (w / v) trypsin to digest, centrifuge (700×g, 5min) to collect the cell pellet.

[0039] (4) Hypotonic lysis: The cell pellet was resuspended in hypotonic lysis buffer (formulation: 100 mmol / L sucrose, 15 mmol / L Tris-HCl pH 7.5, 2 mmol / L MgCl2, 10 mmol / L KCl, 20 mmol / L PMSF) in a volume 10 times that of the cell pellet, and the cells were mechanically disrupted using a Dounce homogenizer.

[0040] (5) Differential centrifugation extraction membrane: Centrifuge the homogenate at 6000×g, 4℃ for 30 min, and discard the precipitate (cell nuclei and large fragments).

[0041] The supernatant was transferred to an ultracentrifuge tube and centrifuged at 150,000 × g at 4 °C for 60 min. The supernatant was discarded, and a pale white foamy cell membrane precipitate was obtained.

[0042] (6) Preservation: The membrane precipitate was resuspended in PBS to a protein concentration of approximately 2 mg / mL, aliquoted, and stored at -80°C for later use. Before use, it was gently dispersed by ultrasonic water bath (40 kHz, 30 s).

[0043] S2. Synthesis of NIR-II region AIE photothermal molecule TPAT12.8-B: Using the Suzuki cross-coupling reaction, the diphenylamine donor unit was linked to the benzothiadiazole-thiophene acceptor unit under palladium catalyst to construct the aggregation-induced emission molecule.

[0044] (1) Under nitrogen protection, add the following ingredients sequentially to a dry 50 mL double-necked round-bottom flask: 0.30 g of 4-(diphenylamino)phenylboronic acid, 4,8-bis(5-bromo-3-(2-octyldodecyl)-2-thienyl)-2λ 4 δ²-benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) 0.32g, Pd(PPh3) 430mg, K2CO3 0.41g, the molar ratio of each substance is 1:0.3:0.025:3; then add 10mL of a mixed solvent of tetrahydrofuran (THF) / water with a volume ratio of 5:1 to make the system uniformly dispersed.

[0045] The reaction mixture was stirred and refluxed in an oil bath at 80°C under a nitrogen atmosphere for 12 hours. The reaction progress was monitored by thin-layer chromatography (TLC) during the reaction (disappearance of the starting material spot was considered the complete reaction). After the reaction was complete, the mixture was cooled to room temperature. 30 mL of dichloromethane (DCM) and 20 mL of water were added, and the mixture was separated. The aqueous phase was extracted twice with DCM (20 mL each time). The organic phases were combined and dried over anhydrous Na₂SO₄. After filtration, the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / petroleum ether (v / v 1:3). The target fraction was collected, concentrated, and a green solid was obtained in approximately 25% yield. This product is TPAT12.8-B, denoted as TPA.

[0046] (2) Structure confirmation.

[0047] pass 1 H NMR (600MHz, CDCl3), 13 The product structure was characterized by C10 NMR (150 MHz, CDCl3) and high-resolution mass spectrometry (HRMS). Chemical shifts (δ) were obtained using TMS as an internal standard, and the data were consistent with those of the target molecule TPAT12.8-B.

[0048] S3. Recombinant protein PDHA1 (P1) and NIR-II region AIE photothermal molecule TPAT12.8-B (TPA) were co-encapsulated in PLGA by nanoprecipitation.

[0049] (1) Organic phase preparation.

[0050] Weigh 50 mg of PLGA (50:50) and dissolve it completely in 2 mL of acetone. Continue adding TPAT12.8-B to a final concentration of 150 μg / mL, i.e., add 0.3 mg of TPAT12.8-B (calculated based on a subsequent volume of 2 mL). Add purified recombinant PDHA1 protein rPDHA1 to a final concentration of 200 ng / mL, i.e., add 0.4 μg of rPDHA1. The amino acid sequence of rPDHA1 is shown in SEQ ID NO. 1.

[0051] SEQ ID NO.1: ANDATFEIKKCDLHRLEEGPPVTTVLTREDGLKYYRMMQTVRRMELKADQLYKQKIIRGFCHLCDGQEACCVGLEAGINPTDHLITAYRAHGFTFTRGLSVREILAELTGRKGGCAKGKGGSMHMYAKNFYGGNGIVGAQVPLGAGIALACKYNGKDEVCLTLYGDGAANQGQIFEAYNM AALWKLPCIFICENNRYGMGTSVERAAASTDYYKRGDFIPGLRVDGMDILCVREATRFAAAYCRSGKGPILMELQTYRYHGHSMSDPGVSYRTREEIQEVRSKSDPIMLLKDRMVNSNLASVEELKEIDVEVRKEIEDAAQFATADPEPPLEELGYHIYSSDPPFEVRGANQWIKFKSVS.

[0052] (2) Aqueous phase preparation.

[0053] Take 8 mL of deionized water and add 1% (w / v) polyvinyl alcohol (PVA) as a stabilizer.

[0054] The polyvinyl alcohol used in this invention was purchased from Sigma-Aldrich, catalog number 363170, Mw 13000~23000.

[0055] (3) Nanoprecipitation.

[0056] The organic phase was slowly added dropwise to the aqueous phase, and the mixture was magnetically stirred (800 rpm) and kept at room temperature for 4 hours to allow the acetone to evaporate and the nanoparticles to self-assemble.

[0057] (4) Purification.

[0058] The nanoparticle suspension was transferred to an ultrafiltration centrifuge tube (MWCO 100kDa) and centrifuged at 3000×g for 20 min to remove free PDHA1, TPAT12.8-B and PVA. The suspension was washed twice with PBS and finally resuspended in 2 mL of PBS to obtain a PLGA core suspension loaded with PDHA1 protein and TPAT12.8-B, denoted as P1-TPA@PLGA.

[0059] S4. P1-TPA@PLGA was coated with foam cell membrane to prepare P1-TPA@F.

[0060] Objective: To coat the PLGA core surface with foam cell membranes to impart homologous targeting.

[0061] (1) Preparation of foam cell membrane vesicles.

[0062] Take 1 mL of the foam cell membrane suspension (protein concentration 2 mg / mL) prepared in S1 and pass it through a liposome extruder through a 400 nm and a 200 nm polycarbonate membrane 11 times each to form uniform membrane vesicles.

[0063] (2) Mixing and extrusion.

[0064] Mix 2 mL of P1-TPA@PLGA (containing approximately 50 mg of PLGA) with 1 mL of foam cell membrane vesicles (containing 2 mg of membrane protein) at a mass ratio of 25:1 (PLGA: membrane protein), and stir in a water bath at 37°C and 600 rpm for 30 min.

[0065] Using a liposome extruder, the material was extruded back and forth 11 times through a 200nm polycarbonate membrane.

[0066] (3) Purification.

[0067] After extrusion, the suspension was centrifuged at 10000×g, 4℃ for 20min, and the supernatant was discarded to remove excess uncoated membrane fragments.

[0068] The precipitate was resuspended in PBS to 2 mL to obtain P1-TPA@F NPs. The protein encapsulation efficiency was approximately 75%, and the drug loading was approximately 8.6% (based on PDHA1).

[0069] S5. Preparation of ROS-responsive liposomes.

[0070] Objective: To prepare functionalized liposomes containing diselenyl bonds for outermost coating.

[0071] Specific operations: (1) Lipid formulation (molar ratio) Soybean phosphatidylcholine:cholesterol:DSPE-SeSe-PEG2000=8:1:1.

[0072] (2) Thin film hydration method.

[0073] According to the above molar ratio formulation, weigh out each lipid and dissolve it in chloroform to achieve a total lipid concentration of 10 mg / mL. Evaporate the solvent in a rotary evaporator (40°C, 100 mbar vacuum) to form a uniform lipid film. Vacuum dry overnight to completely remove residual chloroform.

[0074] (3) Hydration.

[0075] PBS was added to the lipid membrane to achieve a final lipid concentration of 5 mg / mL. The membrane was then sonicated in a 37°C water bath for 10 min (40 kHz, 100 W) to form a milky white liposome suspension.

[0076] (4) Particle size uniformity.

[0077] Using a liposome extruder, the liposomes were passed sequentially through 400 nm and 200 nm polycarbonate membranes 11 times each. ROS-responsive liposomes (LPs) with an average particle size of approximately 120 nm were obtained and stored at 4°C for later use.

[0078] Final assembly of S6, P1-TPA@FL nanoparticles.

[0079] Objective: To encapsulate ROS-responsive liposomes on the surface of P1-TPA@F to form complete biomimetic nanoparticles.

[0080] (1) Mixing: Take 2 mL of P1-TPA@F suspension (containing approximately 50 mg of PLGA) and 1 mL of ROS-responsive liposome suspension (containing 5 mg of lipid) and mix them at a mass ratio of 10:1 (PLGA: lipid). Incubate at room temperature with magnetic stirring (300 rpm) for 20 min.

[0081] (2) Co-extrusion: The mixture was extruded 11 times using a liposome extruder and a 200nm polycarbonate membrane.

[0082] (3) Purification: After squeezing, the suspension was centrifuged at 10000×g, 4℃ for 20min, and the supernatant was discarded to remove excess liposomes. The precipitate was resuspended in PBS to a final volume of 2mL to obtain P1-TPA@FL NPs (denoted as NPs4).

[0083] (4) Storage: Store at 4℃ for short-term use (within 1 week), or freeze-dry and store at -20℃ for long-term storage.

[0084] Figure 1 This is a transmission electron microscope (TEM) image of the molecular orbital distribution of the AIE molecule TPAT12.8 in this invention and the products from different steps. (See also:) Figure 1 As shown, dynamic light scattering (DLS) revealed that the core particle size of PLGA was approximately 136.0 nm. The particle sizes of P1-TPA@F NPs and P1-TPA@FL NPs increased slightly sequentially, and the Zeta potential decreased from -26.2 mV to -43.5 mV. TEM images showed that P1-TPA@FL NPs had a clear core-shell-like structure with a particle size of approximately 150–200 nm.

[0085] Figure 2 Coomassie brilliant blue staining and Western blot images of the P1-TPA@FL NPs prepared in this invention. Figure 2As shown, SDS-PAGE revealed that the protein profile of P1-TPA@FL NPs was similar to but different from that of foam cell lysate. Western blotting confirmed that its surface carried characteristic proteins such as ABCA1, CD36, MSR1 and CD86, indicating that the foam cell membrane successfully disguised itself on the surface of nanoparticles.

[0086] Figure 2 G1 to G5 in A are, in order: PLGA, P1-TPA@PLGA NPs, P1-TPA@F NPs, P1-TPA@FL NPs, and foam cell lysate.

[0087] Example 2 The application of a type of nanolipid particles for treating atherosclerosis was investigated using P1-TPA@FLNPs prepared in Example 1, as detailed below: 1. Evaluation of the in vitro targeted and synergistic therapeutic effects of P1-TPA@FL NPs.

[0088] Different groups and dosages: Control group: culture medium only, no nanoparticles, no laser.

[0089] Control+Laser group: only culture medium was added, no nanoparticles were added, but the cells were irradiated with an 808nm laser.

[0090] P1-TPA@FL group: P1-TPA@FL nanoparticles (containing rPDHA1 200 ng / mL and TPAT12.8-B 150 μg / mL) were administered without laser treatment.

[0091] P1-TPA@ML+Laser group: P1-TPA@ML (macrophage membrane-encapsulated) nanoparticles (also containing rPDHA 1200 ng / mL and TPAT12.8-B 150 μg / mL) were administered and laser irradiation was performed.

[0092] P1-TPA@FL+Laser group: P1-TPA@FL nanoparticles (rPDHA1 200ng / mL + TPAT12.8-B 150μg / mL) were administered and subjected to laser irradiation.

[0093] (1) Extracorporeal photothermal therapy experiment.

[0094] Foam cells were pre-incubated with P1-TPA@FL NPs (TPAT12.8-B 150 μg / mL) and then irradiated with an 808 nm laser (0.8 W / cm²) for 10 min. Figure 3Figures A and B show that the temperature of foam cells in the P1-TPA@FL treatment group increased from 23.5℃ to approximately 45℃. Live / dead cell staining ( Figure 3 C and D) and Annexin V / PI apoptosis detection ( Figure 3 The results (E and F) showed that the apoptosis rate of foam cells in the P1-TPA@FL+Laser group reached 78.90%, while normal cells were less affected.

[0095] (2) Experiment on cell burial.

[0096] Foam cells were seeded into 6-well plates, treated with P1-TPA@FL, and then irradiated with an 808 nm laser (0.8 W / cm²). 2 Foam cells were incubated for 10 minutes. Cells were then labeled with CellTracker Red and incubated for another 12 hours (plate A). Simultaneously, macrophages were seeded into 6-well plates and stimulated with 100 μM Hcy, followed by treatment with recombinant PDHA1 (rPDHA1, 150 ng / mL), P1-TPA@F (containing 200 ng / mL rPDHA1 and 150 μg / mL TPAT12.8-B), or P1-TPA@FL (same component concentrations). After treatment, macrophages were labeled with CellTracker Green and incubated for 12 hours (plate B). Foam cells from plate A were transferred to the corresponding macrophage wells in plate B and incubated for a total of 2 hours. Unphagocytosed foam cells were removed by gentle washing. The remaining cells were centrifuged, resuspended in PBS, and immediately analyzed by flow cytometry.

[0097] according to Figure 3 As shown in scheme G, Cell Tracker Red-labeled apoptotic foam cells were co-incubated with Cell Tracker Green-labeled macrophages that had undergone different treatments. Flow cytometry analysis ( Figure 3 The H and I results showed that the proportion of double-positive macrophages in the P1-TPA@FL treatment group increased from 2.39% in the Hcy group to 34.9%, indicating that P1-TPA@FL NPs significantly enhanced the cytotoxicity of macrophages through metabolic reprogramming.

[0098] In summary, these results demonstrate that P1-TPA@FL nanoparticles can effectively enhance photothermal-induced apoptosis in foam cells and phagocytosis in macrophages, thus confirming their potential for synergistic therapy.

[0099] 2. In vitro evaluation of P1-TPA@FL NPs' inhibition of foam cell formation and promotion of M2 polarization.

[0100] (1) Oil Red O staining experiment.

[0101] RAW264.7 macrophages were stimulated with Hcy (100 μM) and ox-LDL (80 μg / mL) for 24 h, and then treated with PBS, rPDHA1, P1-TPA@F, or P1-TPA@FL, respectively. Oil Red O staining results ( Figure 4 As shown in A and B), intracellular lipid accumulation was significantly reduced and foam cell formation was significantly inhibited in the P1-TPA@FL treatment group.

[0102] (2) Immunofluorescence detection of macrophage polarization.

[0103] Macrophages were stimulated with Hcy and subjected to different treatments, followed by immunofluorescence staining for CD86 (M1 marker) and CD206 (M2 marker). Figure 4 C-F analysis showed that the fluorescence intensity of CD86 was significantly reduced and the fluorescence intensity of CD206 was significantly increased in the P1-TPA@FL treatment group, indicating that P1-TPA@FL NPs promoted the polarization of macrophages to the anti-inflammatory M2 phenotype.

[0104] In summary, these results indicate that P1-TPA@FL nanoparticles can inhibit the endocytosis of oxidized low-density lipoprotein and promote the transformation of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type under homocysteine-induced inflammatory conditions.

[0105] 3. NIR-II imaging of P1-TPA@FL NPs in ApoE⁻ / ⁻ mice.

[0106] P1-TPA@PLGA or P1-TPA@FL was diluted to a specific concentration using PBS, so that the administration volume for each mouse was 0.2 mL.

[0107] After establishing an atherosclerosis model in ApoE⁻ / ⁻ mice by feeding them a high-methionine diet (HMD) for 4 weeks, they were injected via tail vein with PBS, P1-TPA@PLGA, or P1-TPA@FL (5 mg / kg, calculated as TPAT12.8-B). NIR-II fluorescence imaging was performed 30 min after injection. Figure 5 The results showed that the P1-TPA @FL group exhibited clear, focal fluorescence signals in the carotid, iliac, and aortic regions, with signal intensity significantly higher than that of the PBS and P1-TPA @PLGA groups.

[0108] These results clearly demonstrate that P1-TPA@FL nanoparticles can specifically target atherosclerotic plaques and enable bright, deep tissue near-infrared II imaging in vivo.

[0109] 4. Evaluation of the in vivo anti-atherosclerotic efficacy of P1-TPA@FL NPs.

[0110] (1) Animal models and treatment plans.

[0111] Six-week-old male ApoE⁻ / ⁻ mice were fed a high-methionine diet (HMD) for 14 weeks to establish the HHcy-AS model. From week 11, mice were randomly divided into five groups (n=6): control group (normal diet), model group (Hcy), rPDHA1 group (200 μg / kg), P1-TPA@FL group (rPDHA1 200 μg / kg + TPAT12.8-B 5 mg / kg), and P1-TPA@FL+Laser group (same as P1-TPA@FL group, but irradiated with an 808 nm laser at 0.8 W / cm² for 10 min 4 h after injection). The drugs were administered intravenously every 3 days for 8 consecutive weeks. Mice were sacrificed at week 20 for further analysis.

[0112] (2) Gross Oil Red O staining of the aorta.

[0113] Next, to evaluate the therapeutic effect of P1-TPA@FL nanoparticles on atherosclerotic plaques in vivo, we first examined the plaque morphology at the aortic arch ( Figure 6 (A, indicated by the red arrow). In the P1-TPA@FL treatment group, plaque size was significantly reduced. At the end of treatment, the aorta from the heart to the iliac bifurcation was harvested and stained with Oil Red O (ORO). Macroscopic staining showed that P1-TPA@FL significantly inhibited the progression of atherosclerosis, while quantitative analysis of the lesion area relative to the total aortic area confirmed that both P1-TPA@FL and P1-TPA@FL+laser significantly reduced plaque burden (A). Figure 6 (B and C). Consistent with these results, ORO staining of the aortic root cross-section showed that the P1-TPA@FL+laser group exhibited the strongest anti-atherosclerotic effect among all treatments. Figure 6 The D and E values ​​are likely due to the targeted accumulation of nanoparticles in the plaque, as well as the synergistic anti-inflammatory and mild photothermal effects.

[0114] In summary, these findings indicate that the synergistic therapy of P1-TPA@FL is highly effective in preventing the progression of atherosclerotic plaques in vivo.

[0115] 5. In vivo biosafety evaluation of P1-TPA@FL NPs.

[0116] Healthy ApoE⁻ / ⁻ model mice were repeatedly injected with P1-TPA@FL NPs (5 mg / kg, twice a week for 8 weeks). Three days after the last administration, blood was collected for complete blood count and liver and kidney function tests. Major organs (heart, liver, spleen, lung, and kidney) were subjected to H&E staining. P1-TPA@FL NPs showed no significant hemolytic activity. There were no significant differences in complete blood count and liver and kidney function indicators compared to the PBS control group. H&E staining ( Figure 7 The results showed no obvious structural damage or inflammatory infiltration in any organ, confirming that P1-TPA@FL NPs have good in vivo biocompatibility.

[0117] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0118] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A nanolipid particle for treating atherosclerosis, characterized in that, The nanolipid particles are PLGA-encapsulated PDHA1 protein and aggregation-induced luminescent molecules as the core, foam cell membranes are coated on the PLGA surface, and the outermost layer is coated with reactive oxygen species responsive liposomes. The amino acid sequence of PDHA1 protein is shown in SEQ ID NO.1; The aggregation-induced emission molecule is a conjugate of a diphenylamine donor unit and a benzothiadiazole-thiophene acceptor unit; the molar ratio of the diphenylamine donor unit to the benzothiadiazole-thiophene acceptor unit is 1:0.2~0.

5.

2. The nanolipid particles as described in claim 1, characterized in that, The molar ratio of diphenylamine donor units to benzothiadiazole-thiophene acceptor units is 1:0.

3.

3. The nanolipid particles as described in claim 1, characterized in that, The diphenylamine donor unit is 4-(diphenylamino)phenylboronic acid; The benzothiadiazole-thiophene acceptor unit is 4,8-bis(5-bromo-3-(2-octyldodecyl)-2-thiophene)-2λ 4 δ²-benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole).

4. The nanolipid particles according to claim 1, characterized in that, The mass ratio of PDHA1 protein to aggregation-induced luminescence molecules is 0.4:200~500.

5. The nanolipid particles as described in claim 1, characterized in that, The particle size of the nanolipid particles is 150nm~200nm.

6. The method for preparing nanolipid particles as described in claim 1, characterized in that, The steps are as follows: Preparation of foam cell membranes; By utilizing the Suzuki cross-coupling reaction, a diphenylamine donor unit was linked to a benzothiadiazole-thiophene acceptor unit in the presence of a palladium catalyst to construct an aggregation-induced emission molecule. PDHA1 protein and aggregation-induced luminescence molecules were encapsulated in PLGA to obtain the PLGA core; The surface of the PLGA core is coated with a foam cell membrane, and then reactive oxygen species responsive liposomes are further coated on the surface of the foam cell membrane to obtain the nanolipid particles.

7. The preparation method according to claim 6, characterized in that, The palladium catalyst is Pd(PPh3)4.

8. The preparation method according to claim 6, characterized in that, The reaction conditions for the Suzuki cross-coupling reaction are 80°C and 12 h under nitrogen protection.

9. The preparation method according to claim 6, characterized in that, The molar ratio of soybean phosphatidylcholine, cholesterol, and DSPE-SeSe-PEG2000 in the reactive oxygen species responsive liposomes was 8:1:

1.

10. The application of the nanolipid particles as described in claim 1, characterized in that, The nanolipid particles are used to prepare drugs for treating atherosclerosis.

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