Water-soluble self-assembled nanoparticles of rhein and curcumin, and preparation method and application thereof

Abstract

The application discloses a water-soluble self-assembled nanoparticle of rhein and curcumin and a preparation method and application thereof, and relates to the biomedical technical field.The water-soluble self-assembled nanoparticle of rhein and curcumin is a nanoparticle composed of rhein and curcumin through intermolecular non-covalent bond combination.Rhein and curcumin are self-assembled into nanoscale crystal structures through intermolecular non-covalent bond action, the system does not need to use any exogenous carrier material, a drug loading capacity close to 100% is achieved, and the water solubility, the excellent biocompatibility and the preparation stability are significantly improved. Experimental researches prove that the self-assembled nanocrystal can effectively intervene in the pathological process of atherosclerosis through a multi-target point synergistic mechanism.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a water-soluble self-assembled nanoparticle of rhein and curcumin, its preparation method, and its application. Background Technology

[0002] Atherosclerosis is a chronic, progressive inflammatory disease of the arterial walls. It is the common pathological basis for various cardiovascular and cerebrovascular events, including coronary heart disease, ischemic stroke, and peripheral artery disease, and has become a leading cause of death and disability worldwide. Its core pathological processes involve abnormal lipid deposition, oxidative stress, inflammatory cell infiltration, and plaque formation.

[0003] Currently, first-line treatments in clinical practice mainly rely on chemically synthesized drugs, such as statins that inhibit endogenous cholesterol synthesis and ezetimibe that inhibits intestinal cholesterol absorption. However, these drugs typically have a single target and are difficult to systematically intervene in the complex pathological network of atherosclerosis, thus limiting their efficacy. Long-term use may also be accompanied by risks such as liver damage, muscle toxicity, and drug resistance.

[0004] In contrast, traditional Chinese medicine (TCM) demonstrates unique advantages in preventing and treating complex diseases through multi-target, multi-level intervention and holistic regulation, and often has the potential for lower toxicity and side effects, thus becoming an important direction for developing new strategies.

[0005] Based on this, this study aims to develop a traditional Chinese medicine composition that can effectively treat atherosclerosis, and to overcome the deficiencies in the solubility, stability and bioavailability of active ingredients by optimizing its dosage form, thereby significantly improving the therapeutic effect.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a water-soluble self-assembled nanoparticle of rhein and curcumin, its preparation method, and its application, thereby resolving the issues raised in the background section.

[0008] A water-soluble self-assembled nanoparticle of rhein and curcumin, which is composed of rhein and curcumin bound together by intermolecular non-covalent bonds.

[0009] Preferably, the mass ratio of rhein to curcumin is 2:1.

[0010] Preferably, the nanoparticles are in the form of solid powder, and the solid powder can be dispersed in an aqueous medium to form a nano-dispersion.

[0011] A method for preparing water-soluble self-assembled nanoparticles of rhein and curcumin as described above includes the following steps:

[0012] S41: A certain mass ratio of rhein and curcumin are dissolved together in an organic solvent to form an organic phase solution;

[0013] S42: Under stirring conditions, the organic phase solution is added dropwise to the aqueous phase medium until a uniform aqueous organic nanoparticle dispersion is formed;

[0014] S43: Freeze-dry the aqueous organic nanoparticle dispersion to obtain nanoparticles in solid powder form.

[0015] Preferably, the mass ratio of rhein to curcumin in step S41 is 2:1.

[0016] Preferably, the organic solvent in step S41 is dimethyl sulfoxide.

[0017] Preferably, the aqueous medium in step S42 is a phosphate buffer solution.

[0018] The application of water-soluble self-assembled nanoparticles of rhein and curcumin as described above in the preparation of drugs for the prevention or treatment of atherosclerosis.

[0019] Preferably, the drug can improve dyslipidemia, reduce serum levels of total cholesterol, triglycerides and low-density lipoprotein cholesterol, and increase high-density lipoprotein cholesterol levels.

[0020] Preferably, the main lipid component in foam cells was first identified as ceramide by lipid sequencing, and the drug can reduce aortic plaque area and decrease intracellular ceramide content.

[0021] A method for establishing an atherosclerosis model includes the following steps: stimulating macrophage culture systems with ox-LDL at intervals of 50 μg / ml, wherein the interval between each stimulation is 12 hours, and the cumulative stimulation time is 48 hours; and constructing ApoE cells through an early intermittent high-sugar, high-fat diet. - / - Mice were induced to develop atherosclerosis by alternating between a high-sugar, high-fat diet for one week and a normal diet for two weeks, for a total of 16 weeks.

[0022] The present invention provides a water-soluble self-assembled nanoparticle of rhein and curcumin, its preparation method, and its application, which have the following beneficial effects:

[0023] (1) The rhein-curcumin self-assembled nanocrystals constructed in this invention exhibit significant technical advantages and therapeutic potential. This nanosystem is formed autonomously through intermolecular non-covalent bonds (such as hydrogen bonds, π-π stacking, etc.), achieving near 100% drug loading efficiency without the need for any exogenous carrier materials, and successfully overcoming the delivery bottleneck of poor water solubility and low stability of rhein and curcumin themselves. The formed nanoparticles have uniform particle size, good dispersibility, and excellent biocompatibility and blood compatibility.

[0024] (2) In the atherosclerosis disease model, the self-assembled nanocrystals exhibited excellent multi-target therapeutic efficacy. It can not only effectively regulate lipid metabolism and significantly reduce serum total cholesterol, triglycerides and low-density lipoprotein cholesterol levels, but also increase high-density lipoprotein cholesterol. Furthermore, it can significantly inhibit the formation and development of aortic plaques from a pathological perspective and reduce plaque area.

[0025] (3) In-depth studies on the mechanism of action have shown that the therapeutic effect of this nanocrystal is closely related to its activation of intracellular lipophagy. It can upregulate the expression of the key autophagy protein LC3II, promoting the formation of autophagosomes; at the same time, it can reduce the level of p62 protein, accelerating the autophagic flux; and enhance the co-localization of LC3 and lysosomal membrane protein Lamp1 with intracellular lipid droplets, thereby promoting the efficient degradation of lipid droplets through the autophagy-lysosomal pathway. Non-targeted lipidomics analysis further confirmed that this nanocrystal can specifically reduce the abnormal accumulation of pathogenic lipids such as ceramides in foam cells.

[0026] (4) Most importantly, its therapeutic effect is significantly better than that of rhein, curcumin and their simple physical mixtures, which fully demonstrates the unique value of self-assembled nanostructures in achieving synergistic effects of multiple components and provides a new technical solution for the efficient treatment of atherosclerosis. Attached Figure Description

[0027] Figure 1 The morphology and particle size characterization results of rhein-curcumin self-assembled nanoparticles (RCNs);

[0028] in, Figure 1 A is a transmission electron microscope (TEM) image of RCNs;

[0029] Figure 1 B is a scanning electron microscope (SEM) image of RCNs;

[0030] Figure 1 C represents the dynamic light scattering (DLS) particle size distribution of RCNs;

[0031] Figure 1 D represents the zeta potential measurement result of RCNs.

[0032] Figure 2 The results are based on the spectroscopic and crystal structure analysis of RCNs;

[0033] in, Figure 2 A is the UV-Vis absorption spectrum of RCNs;

[0034] Figure 2 B is the Fourier transform infrared (FTIR) spectrum of RCNs;

[0035] Figure 2 C represents the X-ray diffraction (XRD) pattern of RCNs.

[0036] Figure 3 The results of the in vitro hemolytic activity evaluation experiment of RCNs;

[0037] Figure 4 The results of the in vivo pharmacodynamic evaluation of RCNs on atherosclerosis in ApoE- / - mice;

[0038] in, Figure 4 A- Figure 4 D represents the measurement results of TC, LDL-C, TG, and HDL-C in each group of mice;

[0039] Figure 4 E represents a representative image of the entire aorta of mice in each group stained with Oil Red O.

[0040] Figure 4 F shows microscopic images of frozen sections of the aorta of mice in each group stained with Oil Red O;

[0041] Figure 4 G represents HE-stained microscopic images of frozen sections of the aorta from each group of mice.

[0042] Figure 5 The optimal interval stimulation time for ox-LDL was used to simulate the fluctuating lipid microenvironment in vivo, thereby achieving the effect of RCN-regulated NRP1-lipophage pathway intervention in giant cell foaming.

[0043] in, Figure 5 A represents three experimental protocols for i-ox-LDL-induced macrophage foaming; Figure 5 B represents the quantitative result of the degree to which macrophage foaming is induced.

[0044] Figure 6 The results show the effects of RCNs on the expression of lipophage-related proteins in RAW264.7-derived foam cells;

[0045] in, Figure 6 A, Figure 6 C and Figure 6E represents the expression bands of LC3II, p62, and Plin2 proteins in each group of cells as detected by Western blot.

[0046] Figure 6 B Figure 6 D and Figure 6 F represents a semi-quantitative statistical analysis of the protein levels of LC3II, p62, and Plin2.

[0047] Figure 7 The results show the effect of RCNs on the colocalization of LC3 and lipid droplets (LD) in RAW264.7-derived foam cells;

[0048] in, Figure 7 A shows representative images of the co-localization of LC3 (red) and Bodipy-labeled LD (green) in each group of cells as shown by immunofluorescence staining;

[0049] Figure 7 B is a statistical analysis chart of the colocation coefficients of LC3 and LD.

[0050] Figure 8 The results show the effect of RCNs on the colocalization of Lamp1 and lipid droplets (LD) in RAW264.7-derived foam cells;

[0051] in, Figure 8 A shows representative images of the co-localization of Lamp1 (red) and Bodipy-labeled LD (green) in each group of cells as shown by immunofluorescence staining;

[0052] Figure 8 B is a statistical analysis chart of the colocation coefficients of Lamp1 and LD.

[0053] Figure 9 The results of non-targeted lipidomics analysis of the effects of RCNs on the lipid composition of RAW264.7-derived foam cells;

[0054] in, Figure 9 A represents the total number of lipids detected in all samples;

[0055] Figure 9 B shows the percentage of the top ten lipid components in terms of relative abundance in cells;

[0056] Figure 9 C represents the principal component analysis (PCA) score of cellular lipid metabolites in each group;

[0057] Figure 9 D is a heatmap of hierarchical clustering of differential lipid metabolites;

[0058] Figure 9E represents the lipid class that was significantly downregulated in the Control group and the RCNs (5 μg / mL) intervention group compared to the ox-LDL group. Detailed Implementation

[0059] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] To address the aforementioned technical problems, this invention provides a water-soluble self-assembled nanoparticle of rhein and curcumin, its preparation method, and its application, thereby resolving the issues raised in the background section.

[0061] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0062] I. Experimental Reagents and Instruments

[0063] Experimental reagents and consumables: Salvia miltiorrhiza slices (batch number: 240100791), Pueraria lobata slices (batch number: 240100109), 25PC Thick-walled Tube, 3% Uranium acetate, agarose, ethidium bromide, ACQUITY UPLC CSH C18, acetonitrile, isopropanol, methanol, QIAseq® miRNA Library Kit, high-sensitivity DNA clips, Nuclease-free Water, AHTSDNACleanBeads, 4% paraformaldehyde, angiotensin II, Alzet Ostomic Pumps, TUNEL staining kit, HE staining kit, elastic fiber staining kit, Oil Red O staining kit, ApoE- / - (male, 6 weeks old, 20-25g, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.)

[0064] Experimental instruments: electronic balance, rotary evaporator, transmission electron microscope, low-temperature high-speed centrifuge, low-temperature ultracentrifuge, nano-Coulter particle size analyzer, ultra-high performance liquid chromatograph, Q-Exactive Plus mass spectrometer, biosafety cabinet, Quantus Fluorometer, fully automated nucleic acid and protein analysis system, PCR instrument, ultraviolet spectrophotometer, small animal in vivo three-dimensional imaging system, VINNO 6 LAB, biological tissue slicer, cryostat, panoramic tissue scanner, gas anesthesia machine.

[0065] II. Experimental Procedure

[0066] 2.1 Synthesis of RCNs

[0067] Weigh out 2 mg of rhein and 1 mg of curcumin and dissolve them in 150 μL of DMSO. Then, add the solution dropwise to 15 mL of phosphate buffer while stirring. After the addition is complete, continue stirring for several minutes to form a homogeneous aqueous dispersion of self-assembled organic nanocrystals. Prepare the dispersion into a powder using freeze-drying technology and store it away from light. For immediate use, simply redisperse the powder in phosphate buffer to quickly obtain the liquid.

[0068] 2.2 Phenotypic Identification of RCNs

[0069] 2.2.1 The spectral characteristics of the rhein-curcumin self-assembled nanocrystals were analyzed using Fourier transform infrared spectroscopy. The test range was 4000-400 cm⁻¹. -1 To explore the characteristic functional groups and changes in chemical bonds between molecules.

[0070] 2.2.2 The absorption spectra of RCNs were determined using a UV-Vis spectrophotometer. The scanning range was 250-800 nm. The absorption characteristics in the UV-Vis region were analyzed to evaluate the assembly characteristics and molecular interactions of the rhein-curcumin self-assembled nanocrystals.

[0071] 2.2.3 X-ray diffraction was used to analyze the crystal structure of RCNs. The scanning range was 5°–80°, and the diffraction patterns of each sample were recorded to analyze their crystallinity and phase changes.

[0072] 2.3 Hemolytic activity of RCNs

[0073] Fresh anticoagulated venous blood was collected from mice and diluted with physiological saline to obtain a negative control group; the positive control group was prepared by adding single-distilled water. RCNs were prepared into experimental samples at concentrations of 0.3 mg / mL, 0.6 mg / mL, 1.2 mg / mL, and 2.4 mg / mL using the diluted anticoagulated solution. These solutions were incubated in a 37°C water bath for 1 h and 3 h, respectively, followed by centrifugation at 4°C and 3000 rpm for 15 min. The supernatant was collected. The absorbance (OD value) at 540 nm was measured using a microplate reader, and the hemolysis rate (%) was calculated.

[0074] 2.4 Pharmacodynamic evaluation of RCNs intervention in atherosclerosis

[0075] Early intermittent high-sugar, high-fat diet to build ApoE - / -Mice with atherosclerosis (iWD, fed a high-sugar, high-fat diet for one week followed by a normal diet for two weeks, for a total of 16 weeks) were injected via tail vein with rhein (R, 10 mg / kg), curcumin (C, 10 mg / kg), a common mixture of rhein and curcumin (R+C, non-self-assembled nanocrystal form, 10 mg / kg), simvastatin (Sim, 10 mg / kg), and RCNs (10 mg / kg) every 3 days. Two months later, serum was separated, and atherosclerotic lipid markers were detected. The thoracic aorta was separated, and plaque formation and the localization and expression of lipophage markers at the plaque sites were detected.

[0076] 2.5 Molecular mechanism of rhein-curcumin self-assembly of nanocrystals

[0077] In cell experiments, the degree of macrophage foaming was used as an evaluation index to screen for the optimal interval of ox-LDL (oxidized low-density lipoprotein) stimulation to simulate the fluctuating lipid microenvironment in vivo. The experiment used ox-LDL to simulate the fluctuating lipid microenvironment of an intermittent high-fat diet (ox-LDL administered every 12 hours for a total of 48 hours) to induce foaming in RAW246.7 macrophages. The study observed whether RCNs reduced the degree of macrophage foaming and elucidated the mechanism by which lipophage is involved.

[0078] 2.5.1 Effects of RCNs on the expression of lipophage-related proteins LC3II, p62, and plin2 in RAW264.7-derived foam cells. LC3 exists in two forms, LC3I and LC3II. In the cytoplasm, LC3I is converted to LC3II by enzymatic cleavage and binds to the autophagosome membrane; its level is positively correlated with autophagy. One of the most characteristic substrates of selective autophagy is p62, which can act as a bridge connecting LC3 and ubiquitinated substrates. After being incorporated into the autophagosome, it is degraded and is negatively correlated with autophagic flux. plin2 is mainly distributed on lipid droplets in mammalian cells, and its protein expression can reflect the accumulation of intracellular lipid droplets.

[0079] To further clarify the effect of RCNs on the lipophage level of RAW264.7-derived foam cells, the experiment used RAW264.7 cell suspension at 1×10⁻⁶. 5 / wells were seeded at a density of 6 wells. When the RAW264.7 confluence was about 60%, the cells were divided into 4 groups: (1) Control group (2) ox-LDL group (3) ox-LDL+1 μg / mL group (4) ox-LDL+5 μg / mL group. After 48 h, cell proteins were extracted and the expression of lipophage-related proteins LC3II, p62 and plin2 in each group was detected by Western blot.

[0080] 2.5.2 Effects of RCNs on LC3 and LD colocalization in RAW264.7-derived foam cells. Bodipy is commonly used to label intracellular lipid droplets, and LC3 fluorescence staining is used to label intracellular lipid droplets. Autologous image analysis software uses the Bodipy-LC3 colocalization coefficient to detect changes in intracellular lipid levels. In this experiment, RAW264.7 cell suspensions were subjected to a 2x10⁻¹⁰ staining process. 4 The cells were seeded at a density of / wells in 24-well plates. When the RAW264.7 confluence was about 60%, the cells were divided into 4 groups: (1) Control group (2) ox-LDL group (3) ox-LDL + 1 μg / mL group (4) ox-LDL + 5 μg / mL group. After 48 h, the culture medium was aspirated, the cells were washed with PBS, and then incubated with 1 μmol / L Bodipy staining solution in the dark for 10 min. The cells were fixed with 4% paraformaldehyde, blocked with serum for 2 h, and incubated with the corresponding primary and secondary antibodies. After that, the cells were stained with DAPI at room temperature in the dark for 5 min. After washing with PBS, the distribution of Bodipy and LC3 in each group of cells was observed under an inverted fluorescence microscope.

[0081] 2.5.3 Effects of RCNs on Lamp1 and LD colocalization in RAW246.7-derived foam cells. Bodipy is commonly used to label intracellular lipid droplets, and Lamp1 fluorescent staining is used to label intracellular lipid droplets. Image analysis software simulates the colocalization coefficients of Bodipy and Lamp1 to detect changes in intracellular lipid levels. In this experiment, RAW264.7 cell suspensions were subjected to a 2x10⁻¹⁰ staining. 4 The cells were seeded at a density of / wells in 24-well plates. When the RAW264.7 confluence was about 60%, the cells were divided into 4 groups: (1) Control group (2) ox-LDL group (3) ox-LDL + 1 μg / mL group (4) ox-LDL + 5 μg / mL group. After 48 h, the culture medium was aspirated, the cells were washed with PBS, and then incubated with 1 μmol / L Bodipy staining solution in the dark for 10 min. The cells were fixed with 4% paraformaldehyde, blocked with serum for 2 h, and incubated with the corresponding primary and secondary antibodies. After that, the cells were stained with DAPI at room temperature in the dark for 5 min. After washing with PBS, the distribution of Bodipy and Lamp1 in each group of cells was observed under an inverted fluorescence microscope.

[0082] 2.6 Effects of RCNs on the lipid composition of RAW246.7 source foam cells

[0083] The previous experimental results show that RCNs can reduce lipid accumulation in RAW246.7-derived foam cells. To further understand the types of lipid components reduced by RCNs in RAW246.7-derived foam cells and the specific regulatory mechanisms, non-target lipid metabolism was quantitatively analyzed using RCNs-treated RAW246.7-derived foam cells.

[0084] III. Experimental Results

[0085] 1. Characterization and identification of self-assembled synthetic RCNs

[0086] The morphology of RCNs was observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The results showed that RCNs were approximately spherical in appearance. Figure 1 AB); DLS analysis revealed that the average particle size of RCNs was approximately 182.75 nm ± 3.14 nm, and the polydispersity index was 0.172 ± 0.017 ( ). Figure 1 C) indicates that the formed nanoparticles have a uniform size and are consistent with the size distribution of SEM and TEM; the potential of RCNs is -37.15 mV ± 2.83, indicating that the formed nanoparticles have a stable size. Figure 1 D). This reflects the successful assembly of rhein and curcumin.

[0087] 2. Analysis of UV-Vis absorption spectra of RCNs

[0088] To investigate the structural characteristics of RCNs, we measured their UV-Vis absorption spectra. For example... Figure 2 As shown in Figure A, the characteristic absorption peaks of Rhe appear at 265 nm and 428 nm, respectively; while the absorption peak of Cur appears at 437 nm. The presence of these absorption peaks in RCNs indicates that Rhe and Cur have been integrated into the self-assembled structure of RCNs; as shown in Figure A. Figure 2 As shown in B, the typical peak of the Cur benzene ring is 715 cm⁻¹. -1 964 cm -1 and 1188 cm -1 ), Rhe at 1693 and 1630 cm -1 The typical carbonyl group is displayed at the 1693 and 1630 cm⁻¹, after the formation of Rhe-Cur NPs. -1 It shows a blue shift to 1664 and 1621 cm. -1 After self-assembly, these absorption peaks shift to lower wavenumbers, likely due to hydrogen bonding and conjugation effects. These effects may weaken the strength of the corresponding chemical bonds, leading to a decrease in the C=O stretching vibration frequency. These results indicate that RCNs form self-assemblies through hydrogen bonding and π-π interactions. Figure 2The results show that the typical XRD diffraction pattern of Cur exhibits a distinct peak at 17.72°, while the typical XRD diffraction pattern of Rhe shows a distinct peak at 28.24°, indicating that they possess crystalline properties. In contrast, the RCN powder did not show these typical diffraction peaks, indicating that it does not have a crystalline complex structure. These results support the hypothesis that Rhe and Cur constitute a self-assembled structure of RCNs.

[0089] 3. Security Evaluation of RCNs

[0090] Hemolysis test results as follows Figure 3 As shown, there was no significant change in hemolytic absorbance at different concentrations of RCNs at 1 h and 3 h. The hemolysis test results showed that no significant hemolysis was observed in mouse erythrocytes (RBCs) treated with RCNs at 1 h and 3 h, and the quantitative results were lower than the internationally recognized standard, confirming that RCNs injection does not cause hemolysis and has good blood compatibility.

[0091] 4. RCNs significantly reduced the area of ​​plaque in the aorta of ApoE- / - mice and regulated blood lipid balance.

[0092] The results are as follows Figure 4 As shown; where, Figure 4 A-4D showed the changes in serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in each group of mice.

[0093] Compared with the model group, the RCNs treatment group showed a significant lipid-regulating effect: TC, TG, and LDL-C levels were significantly reduced, indicating that RCNs can effectively inhibit the abnormal rise of blood lipids, especially reducing key lipid components that lead to atherosclerosis; HDL-C levels were significantly increased, which helps promote reverse cholesterol transport and further inhibits the development of atherosclerosis.

[0094] It is worth noting that RCNs have a significantly better lipid-regulating effect than rhein (R), curcumin (C) or a common mixture of the two (R+C) alone, indicating that self-assembled nanostructures have a significant advantage in synergistic regulation of lipid metabolism.

[0095] Figure 4EG results showed that in the model group, extensive and thick lipid plaques were visible on the aortic wall, with a significant plaque coverage area, indicating severe atherosclerotic lesions. In the RCNs treatment group, the aortic intima surface was relatively smooth, the plaque burden was significantly reduced, and the plaque area was significantly decreased. Compared with other treatment groups, RCNs showed the most significant therapeutic effect, far exceeding the effects of rhein (R), curcumin (C), and the common mixture of rhein and curcumin (R+C). Furthermore, the therapeutic effect of RCNs was also superior to that of the Sim treatment group.

[0096] 5. Determine the optimal interval stimulation time for ox-LDL to simulate the fluctuating lipid microenvironment in vivo.

[0097] In cell experiments, the degree of macrophage foaming was used as an evaluation index to screen for the optimal ox-LDL stimulation interval to simulate the fluctuating lipid microenvironment in vivo. Results are as follows: Figure 5 Figures A and B show the experimental conditions for determining fluctuating ox-LDL (i-ox-LDL): a stimulation concentration of 50 μg / ml, a stimulation interval of 12 h, and a cumulative stimulation time of 48 h. Under these conditions, the degree of macrophage foaming was over 80%. At the same concentration and cumulative stimulation time, continuous ox-LDL induced macrophage foaming to less than 50%, revealing that fluctuating blood lipids exacerbate the macrophage foaming process.

[0098] 6. Effects of RCNs on the expression of lipid droplet autophagy-related proteins LC3B, p62, and plin2

[0099] The results are as follows Figure 6 As shown, compared with the Control group, the ox-LDL group showed decreased LC3II protein levels (P<0.05) and increased p62 (P<0.05) and plin2 protein levels (P<0.01). Compared with the ox-LDL group, the RCNs (1 μg / mL, 5 μg / mL) groups showed significantly increased LC3II protein expression (P<0.01); the RCNs (1 μg / mL, 5 μg / mL) groups showed decreased p62 protein levels (P<0.001); compared with the ox-LDL group, the RCNs (1 μg / mL) group showed no significant decrease in plin2 protein levels, while the RCNs (5 μg / mL) group showed a significant decrease (P<0.01). This indicates that RCNs may enhance LC3II expression in autophagosome formation, promote autophagosome formation and lipid droplet phagocytosis, reduce p62 protein accumulation, and accelerate lipid droplet degradation in autolysosomes, thus inducing lipopyphagia in RAW264.7 cells.

[0100] 7. Effects of RCNs on LC3 and LD co-localization in RAW264.7 cells

[0101] The results are as follows Figure 7 As shown, compared with the Control group, the red fluorescence signal of LC3 in the ox-LDL group was weakened, while the green fluorescence signal of lipid droplets was increased, and the overlap coefficient between the two was significantly reduced (P<0.001). Compared with the ox-LDL group, the red fluorescence signal of LC3 in the RCNs (1 μg / mL, 5 μg / mL) groups was increased, and the overlap coefficient between the green fluorescence signals of LC3 and lipid droplets increased significantly in a concentration-dependent manner (P<0.001).

[0102] 8. Effects of RCNs on the co-localization of Lamp1 and LD in RAW246.7-derived foam cells

[0103] like Figure 8 As shown, compared with the Control group, the red fluorescence signal of Lamp1 in the ox-LDL group was weakened, while the green fluorescence signal of lipid droplets was increased, and the overlap coefficient between the two was significantly reduced (P<0.001). Compared with the ox-LDL group, the red fluorescence signal of Lamp1 in the RCNs (1 μg / mL, 5 μg / mL) groups was increased, while the green fluorescence signal of lipid droplets was weakened. The overlap coefficient of fluorescence signals of Lamp1 and lipid droplets increased significantly in a concentration-dependent manner (P<0.001).

[0104] 9. Effects of RCNs on the lipid composition of RAW246.7-derived foam cells

[0105] like Figure 9 As shown in A, a total of 3356 lipid components were detected in all cell groups; Figure 9 B represents the percentage of the top ten lipid components in the cell; Figure 9 Principal component analysis of cell metabolites showed small intra-group differences but inter-group differences, indicating a stable and reliable sample model. A heatmap was created to visualize the differentially abundant lipid metabolites based on their relative abundance. Figure 9 (DE) As can be seen from the figure, compared with the ox-LDL group, the main lipid component that was reduced in the Control group and the ox-LDL+5 μg / mL group was ceramide (Cer).

[0106] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of water-soluble self-assembled nanoparticles of rhein and curcumin in the preparation of drugs for the prevention or treatment of atherosclerosis; A method for preparing water-soluble self-assembled nanoparticles of rhein and curcumin includes the following steps: S11: A certain mass ratio of rhein and curcumin are dissolved together in an organic solvent to form an organic phase solution; S12: Under stirring conditions, the organic phase solution is added dropwise to the aqueous phase medium until a uniform aqueous organic nanoparticle dispersion is formed; S13: Freeze-dry the aqueous organic nanoparticle dispersion to obtain nanoparticles in solid powder form; The mass ratio of rhein to curcumin in step S11 is 2:

1.

2. The application of the water-soluble self-assembled nanoparticles of rhein and curcumin according to claim 1 in the preparation of drugs for the prevention or treatment of atherosclerosis, characterized in that, The organic solvent in step S11 is dimethyl sulfoxide; the aqueous medium in step S12 is phosphate buffer.

3. The application of the water-soluble self-assembled nanoparticles of rhein and curcumin according to claim 1 in the preparation of drugs for the prevention or treatment of atherosclerosis, characterized in that, The drug can improve dyslipidemia, reduce serum levels of total cholesterol, triglycerides and low-density lipoprotein cholesterol, and increase high-density lipoprotein cholesterol levels.

4. The application of the water-soluble self-assembled nanoparticles of rhein and curcumin according to claim 1 in the preparation of drugs for the prevention or treatment of atherosclerosis, characterized in that, For the first time, lipid sequencing has identified ceramide as the main lipid component in foam cells. The drug can reduce aortic plaque area and decrease intracellular ceramide content.

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