Nano-motor based on rice bran protein and application of nano-motor in preparation of anti-atherosclerosis medicine

Abstract

The invention provides a nano motor based on rice bran protein and application of the nano motor in preparation of an anti-atherosclerosis medicine, and belongs to the technical field of biological medicine. The nano motor based on the rice bran protein is prepared by adopting a method comprising the following steps: (1) dropwise adding an ethanol solution of dihydroquercetin into a rice bran protein solution, homogenizing, performing ultrasonic treatment, centrifuging, taking precipitate, and performing freeze drying to obtain RBP-DHQ core nano particles; (2) sequentially coupling L-arginine and D-mannose on the surface of the RBP-DHQ core nano-particle to obtain a functional nano-particle; and (3) mixing the functionalized nanoparticle suspension with the spirulina dispersion liquid, and stirring to obtain the rice bran protein-based nano motor RDHQ-Arg at Man / SP. The nano motor has ROS response self-driving capability and macrophage targeting property, is free from gastrointestinal tract environment damage, and is high in oral bioavailability and outstanding in anti-atherosclerosis curative effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a rice bran protein-based nanomotor and its application in the preparation of drugs for treating atherosclerosis. Background Technology

[0002] Atherosclerosis is the leading cause of cardiovascular disease worldwide. Its core pathological feature is the abnormal deposition of lipids and immune cells in the arterial intima, forming easily ruptured atherosclerotic plaques. Macrophages transform into foam cells through the internalization of oxidized low-density lipoprotein (ox-LDL), which is a key marker of disease progression. While currently the mainstream clinical statins can lower lipids, they generally have systemic side effects such as muscle damage and elevated liver enzymes, and they are difficult to precisely target atherosclerotic plaque sites, thus failing to achieve efficient disease intervention. There is an urgent need to develop safe and highly targeted new treatment options.

[0003] Dihydroquercetin (DHQ) is a natural flavonoid compound with excellent antioxidant, anti-inflammatory, and lipid-regulating activities. It can scavenge reactive oxygen species (ROS) and inhibit inflammatory pathways, making it a potential substance for combating atherosclerosis. However, the clinical translation of DHQ faces significant bottlenecks: its poor water solubility leads to low oral bioavailability; the acidic environment of the gastrointestinal tract and enzymatic hydrolysis easily destroy its chemical structure; and the lack of a targeting mechanism makes it difficult to accumulate in plaque sites. Essentially, there is an inherent contradiction between its physicochemical properties and the physiological barriers in vivo. Summary of the Invention

[0004] The purpose of this invention is to provide a nanomotor based on rice bran protein, which has both ROS-responsive self-driving ability and macrophage targeting capability, is protected from gastrointestinal environment damage, has high oral bioavailability, and has outstanding anti-atherosclerotic efficacy.

[0005] Another object of the present invention is to provide the application of the above-mentioned nanomotor in the preparation of drugs for treating atherosclerosis.

[0006] The objective of this invention is achieved through the following technical solution: A nanomotor based on rice bran protein was prepared using a method comprising the following steps: (1) Add the ethanol solution of dihydroquercetin dropwise to the rice bran protein solution, homogenize, sonicate, centrifuge, collect the precipitate, freeze dry, and obtain RBP-DHQ core nanoparticles. (2) L-arginine and D-mannose were sequentially coupled to the surface of RBP-DHQ core nanoparticles to obtain functionalized nanoparticles; (3) The functionalized nanoparticle suspension was mixed with the spirulina dispersion and stirred to obtain the rice bran protein-based nanomotor RDHQ-Arg@Man / SP.

[0007] In the preferred technical solution, the mass ratio of rice bran protein to dihydroquercetin in step (1) is 18-22:1.

[0008] In the preferred technical solution, in step (2), L-arginine and D-mannose are sequentially coupled to the surface of RBP-DHQ core nanoparticles by carbodiimide chemical method.

[0009] In a preferred embodiment, RBP-DHQ core nanoparticles and L-arginine are coupled at a mass ratio of 0.8-1.2:0.5 to obtain L-arginine-modified particles; the L-arginine-modified particles and D-mannose are coupled at a mass ratio of 0.8-1.2:0.2 to obtain functionalized nanoparticles.

[0010] In the preferred technical solution, the mass ratio of functionalized nanoparticle suspension to spirulina in step (3) is 0.8-1.2:1.

[0011] The present invention also provides the application of the nanomotor in the preparation of drugs for treating atherosclerosis.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The nanomotor provided by the present invention uses rice bran protein as the core matrix and spirulina as the coating material. Both are natural edible components with excellent biocompatibility and meet the requirements of food-grade delivery systems.

[0013] (2) The nanomotor provided by the present invention has both ROS-responsive self-driving capability and macrophage targeting capability, and can efficiently penetrate the matrix of atherosclerotic plaques and accumulate in the lesion site.

[0014] (3) The spirulina coating of the nanomotor provided by the present invention can effectively protect DHQ from the damage of the gastrointestinal environment, significantly improve the oral bioavailability of DHQ, and release a large amount in simulated intestinal fluid.

[0015] (4) The nanomotor provided by this invention can synergistically exert the antioxidant and anti-inflammatory effects of DHQ and the ROS consumption function of the self-driving unit, significantly improving the pathological state of inflammatory macrophages. In terms of cell viability, the cell viability of the NM group basically recovered to normal cell viability, which can efficiently clear ROS, significantly reduce lipid accumulation, and has outstanding anti-atherosclerotic efficacy. Attached Figure Description

[0016] Figure 1 Transmission electron microscopy (TEM) images of particles at different preparation stages.

[0017] Figure 2The graph shows the changes in particle size and zeta potential during the functionalization process. The horizontal axis represents the sample name, and the vertical axis represents the zeta potential. The bar chart represents the zeta potential, and the line chart represents the particle size. Data are expressed as mean ± error; different lowercase letters in the graph indicate significant differences between groups (P < 0.05).

[0018] Figure 3 Fourier transform infrared (FTIR) spectra of each component and particles at different stages, with wavenumber on the horizontal axis and transmittance on the vertical axis.

[0019] Figure 4 The in vitro drug release and self-driving performance of nanomotors are shown. (a) The trajectory of nanomotors at different H2O2 concentrations, where the x-axis (X / μm) and y-axis (Y / μm) represent spatial distances in micrometers (μm). These coordinates form a two-dimensional coordinate system to record the spatial movement path of the nanomotors over time in different H2O2 concentration environments (different colored curves in the figure correspond to the trajectory of the nanomotors in two-dimensional space at different H2O2 concentrations). (b) The mean square displacement (MSD) at different H2O2 concentrations varies with time lag, where the x-axis represents the time interval (Δt) in seconds (s); (c) The average velocity at different H2O2 concentrations, where the x-axis represents H2O2 concentration and the y-axis represents average velocity; (d) The effective diffusion coefficient at different H2O2 concentrations, where the x-axis represents H2O2 concentration and the y-axis represents diffusion coefficient. Data are expressed as mean ± error; different lowercase letters in the figure indicate significant differences between groups (P < 0.05).

[0020] Figure 5 This is a cumulative DHQ release curve simulating the gastrointestinal environment. The horizontal axis represents digestive fluid versus total digestion time; for example, SGF-2 indicates that the digestive fluid is SGF and the total digestion time is 2 hours. The vertical axis of the bar chart represents particle size, and the vertical axis of the line graph represents the DHQ release rate. Data are expressed as mean ± error; different lowercase letters in the figure indicate significant differences between groups (P < 0.05).

[0021] Figure 6 This is a comparison chart of the bioavailability of nanomotors and dihydroquercetin. The horizontal axis represents the sample name, and the vertical axis represents the bioavailability. "***" indicates that the difference in inflammatory factor concentration between the DHQ group and the NM group reached a statistically significant level (P<0.001).

[0022] Figure 7 The changes in the secretion levels of inflammatory factors (IL-1β, IL-6, TNF-α) after LPS treatment are shown. The horizontal axis represents the sample names of the blank control group and the LPS-treated group, and the vertical axis represents the concentration. "***" indicates that the difference in inflammatory factor concentrations between the control group and the LPS group reached a statistically significant level (P<0.001).

[0023] Figure 8 Laser confocal image of macrophage uptake of NM and DHQ.

[0024] Figure 9 The results of quantitative analysis of cell uptake are shown, with the horizontal axis representing uptake time and the vertical axis representing average fluorescence intensity. "***" indicates that the difference in cell uptake between the NM group and the DHQ group at the same time point reached a statistically significant level (P<0.001).

[0025] Figure 10 The effect of NM on cell viability is shown in (a) laser confocal micrographs of live and dead cells in each group and (b) quantitative analysis of cell viability. The horizontal axis represents the sample name, and the vertical axis represents the survival rate. Different lowercase letters in the figure indicate significant differences between groups (P<0.05).

[0026] Figure 11 Intracellular reactive oxygen species (ROS) scavenging assay: (a) laser confocal images of ROS staining in each group; (b) quantitative analysis of cell viability. The horizontal axis represents the sample name, and the vertical axis represents the average fluorescence intensity. Different lowercase letters in the figure indicate significant differences between groups (P<0.05).

[0027] Figure 12 The images show intracellular lipid accumulation: (a) laser confocal micrographs of lipid accumulation in each group; (b) quantitative analysis of lipid accumulation in each group. The horizontal axis represents the sample name, and the vertical axis represents the red / green fluorescence intensity ratio. Different lowercase letters in the figures indicate significant differences between groups (P<0.05).

[0028] Figure 13 The images show colocalization imaging of lipids and lysosomes: (a) colocalization laser confocal images of lysosomes in each group; (b) quantitative analysis of colocalization of lipids and lysosomes. The horizontal axis represents the sample name, and the vertical axis represents the average fluorescence intensity. Different lowercase letters in the figures indicate significant differences between groups (P<0.05). Detailed Implementation

[0029] The present invention will be further illustrated below with reference to specific examples and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0030] Example 1: A method for preparing a rice bran protein-based nanomotor and its physicochemical characterization. 1. A method for preparing nanomotors based on rice bran protein A method for preparing a rice bran protein-based nanomotor includes the following steps: (1) Preparation of RBP-DHQ core nanoparticles First, rice bran protein (RBP, purchased from Guangdong Wengjiang Chemical Reagent Co., Ltd., product number PA93136) was dissolved in 10mM PBS buffer (pH 8.5) and magnetically stirred for 2 hours until fully hydrated to prepare a 2 mg / mL RBP solution. Simultaneously, dihydroquercetin (DHQ) was dissolved in anhydrous ethanol to prepare a 1 mg / mL DHQ ethanol solution. Using a 1 ml syringe, the DHQ ethanol solution was slowly added dropwise to the RBP solution at a rate of 1 drop per 5 seconds, with the solution being homogenized simultaneously using a high-speed shear homogenizer at 10,000 rpm. After the addition was complete, homogenization continued for 5 minutes to form a preliminary suspension. The initial suspension was transferred to an ice bath and sonicated at 300W for 10 min using a probe sonicator. Then it was centrifuged at 12000g for 20 min to remove unencapsulated free DHQ and large particle aggregates. The precipitate was washed twice with deionized water and redispersed in 10mM PBS buffer (pH 8.5). After freeze-drying, RBP-DHQ core nanoparticles (denoted as RDHQ) were obtained.

[0031] (2) Preparation of functionalized nanoparticles L-arginine and D-mannose (targeting ligands) were sequentially coupled onto the surface of RDHQ using an EDC / NHS-mediated carbodiimide chemical method. In the first step, 20 mg of RDHQ was dispersed in 10 mL of MES buffer (pH 6.0), and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) was added to a final concentration of 50 mM. NHS (N-hydroxysuccinimide) was added to a final concentration of 25 mM. The mixture was gently stirred (400 rpm) for 30 min to activate the carboxyl groups on the RBP surface. L-arginine was added at a mass ratio of RDHQ to L-arginine of 1:0.5, and the reaction was carried out at room temperature in the dark for 4 h. After the reaction, the product was placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed against deionized water for 24 h to remove unreacted reagents. The precipitate was collected by centrifugation at 8000 g for 15 min and then freeze-dried to obtain L-arginine-modified particles (RDHQ-Arg). In the second step, 20 mg of RDHQ-Arg was dispersed in 10 ml of MES buffer (pH 6.0), EDC was added to a final concentration of 50 mM, and NHS was added to a final concentration of 25 mM. The mixture was gently stirred (400 rpm) for 30 min to activate the carboxyl groups on the RBP surface. D-mannose was added at a mass ratio of 1:0.2 of RDHQ-Arg to D-mannose. The reaction was carried out at room temperature in the dark for 6 h. After the reaction was completed, the mixture was centrifuged at 12000 g for 20 min. The precipitate was washed three times with deionized water and then freeze-dried to obtain functionalized nanoparticles (denoted as RDHQ-Arg@Man).

[0032] (3) Spirulina coating: Spirulina was coated onto the surface of RDHQ-Arg@Man using an adsorption method to achieve gastrointestinal protection. Spirulina powder was dispersed in deionized water and sonicated for 5 min at 200W using a probe sonicator to prepare a 1 mg / mL spirulina dispersion. 20 mg of RDHQ-Arg@Man was taken and resuspended in 10 mL of deionized water to form a functionalized nanoparticle suspension. The functionalized nanoparticle suspension and the spirulina dispersion were mixed at a mass ratio of 1:1 (RDHQ-Arg@Man to spirulina). The mixture was gently stirred in a 30℃ constant temperature water bath (400 rpm) for 2 h to allow the spirulina components to be adsorbed onto the particle surface through electrostatic and hydrophobic interactions, resulting in rice bran protein-based nanomotors RDHQ-Arg@Man / SP (denoted as NM).

[0033] Encapsulation efficiency (EE) of dihydroquercetin: 92.8 ± 0.8%, drug loading (DL): 4.43 ± 0.04%.

[0034] .

[0035] 2. Physicochemical characterization of rice bran protein-based nanomotors Using the RBP-DHQ core nanoparticles (RDHQ), RDHQ-Arg, and RDHQ-Arg@Man prepared in Title 1 of this embodiment as controls, the particle size, dispersibility, morphology, and chemical structure of RDHQ-Arg@Man / SP (NM) were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR). Their colloidal stability was also evaluated. The results are as follows: (1) Particle size and dispersibility analysis: The particle size, polydispersity index (PDI) and zeta potential of the particles at each stage were measured using a dynamic light scattering instrument at 25℃. The results showed that the particle size of RBP-DHQ was 158.5±8.1nm and the zeta potential was -30.5±0.8mV; the particle size of RDHQ-Arg increased to 168.7±8.5nm due to surface coupling with L-arginine and the zeta potential increased to -15.2±1.2mV; the particle size of RDHQ-Arg@Man continued to increase to 182.3±10.1nm and the zeta potential was -17.8±1.0mV; finally, the particle size of NM (RDHQ-Arg@Man / SP) reached 498.6±7.5nm due to the coating of Spirulina and the zeta potential decreased to -41.3±0.9mV. The PDI of all stages of particles was <0.2, indicating uniform dispersion. The absolute value of the zeta potential was >15mV, indicating good colloidal stability. The particle size and zeta potential detection results are shown below. Figure 2 .

[0036] Morphology and Microstructure Analysis: The morphology of particles at each stage was observed using transmission electron microscopy. A particle suspension with a concentration of 0.1 mg / mL was dropped onto a copper grid supported by a carbon film, negatively stained with 2% (w / v) phosphotungstic acid solution for 10 s, and imaged after air-drying at room temperature. The results are as follows: Figure 1 RBP-DHQ (RDHQ) is a regular sphere with a smooth surface and a particle size of about 150 nm, without obvious aggregation. RDHQ-Arg has a slightly rough surface and a thicker profile than RBP, indicating that L-arginine was successfully coupled. RDHQ-Arg@Man still maintains a spherical structure, but the surface density is increased, which is presumably due to the formation of hydrogen bonds between D-mannose and the particle surface. NM exhibits a clear core-shell structure, with RDHQ-Arg@Man as the core and a spirulina coating with a thickness of about 20 nm as the outer layer, confirming that the spirulina coating was successful.

[0037] (3) Chemical structure verification: Fourier transform infrared spectroscopy was used to analyze each component and particles at different stages, with a scanning range of 4000-500 cm⁻¹. -1 4cm resolution -1 The results are shown below. Figure 3 In the RBP-DHQ (RDHQ) spectrum, the amide I band of RBP starts from 1655 cm⁻¹. -1 Offset to 1648cm -1 And 3300cm -1 The broadening of the OH stretching peak indicates the formation of hydrogen bonds between RBP and DHQ; the RDHQ-Arg spectrum shows an increase of 1580 cm⁻¹. -1 (NH bending vibration) and 1640cm -1 The characteristic peak of (C=N stretching vibration) corresponds to the vibrational signal of the L-arginine guanidinyl group; RDHQ-Arg@Man at 1020 cm⁻¹ -1 A characteristic peak appears at 1000–1150 cm⁻¹, corresponding to the COC stretching vibration of the D-mannose pyran ring; the final RDHQ-Arg@Man / SP peaks at 1000–1150 cm⁻¹. -1 A broad absorption band appeared in the region, corresponding to the COC stretching vibration of spirulina polysaccharide. The above results verify that each step of the modification was successful.

[0038] Example 2: Self-driving performance and in vitro drug release behavior of rice bran protein-based nanomotors 1. Self-driving performance measurement The movement behavior of NM (prepared in title 1 of this embodiment) under different ROS (reactive oxygen species) concentrations was evaluated using a nanoparticle tracking and analysis (NTA) system. H2O2 was used to simulate ROS in the microenvironment of atherosclerotic plaques. The specific operation and results are as follows: (1) Sample preparation and determination: NM was dispersed in PBS buffer (pH=7.4) containing 1 mM, 5 mM, and 10 mM H2O2 to prepare suspensions with a concentration of 0.1 mg / mL. After incubation in the dark for 10 min, the suspensions were injected into the NTA sample cell. The motion trajectory of the nanomotor was recorded at 25 °C. The mean square displacement (MSD), mean velocity, and effective diffusion coefficient were analyzed using NTA software. Each concentration was measured three times.

[0039] (2) Results analysis: motion trajectory ( Figure 4 (a) shows that in a 1 mM H2O2 environment, the NM trajectory is short and irregular, mainly exhibiting random diffusion; in a 5 mM H2O2 environment, the trajectory length increases significantly and the directionality strengthens; in a 10 mM H2O2 environment, the trajectory shows long-distance linear extension. Quantitative analysis indicates that, for the same time lag, the MSD (mean square displacement) increases with increasing H2O2 concentration. Figure 4 (b)); average speed ( Figure 4 The effective diffusion coefficient (c) increased from 21.79 ± 1.88 μm / s in a 1 mM H₂O₂ environment to 25.11 ± 1.81 μm / s in a 5 mM environment, and further increased to 28.31 ± 2.22 μm / s in a 10 mM H₂O₂ environment; Figure 4 In the middle (d) environment, the concentration of NM increased from 1.10±0.39 μm² / s in the 1 mM H2O2 environment to 16.07±0.96 μm² / s in the 5 mM environment, and further increased to 20.67±1.83 μm² / s in the 10 mM environment. The differences between the groups were statistically significant (p<0.05), which confirmed that the self-driving ability of NM is ROS concentration-dependent and can achieve efficient movement in the high ROS microenvironment of plaques.

[0040] 2. In vitro drug release behavior and bioavailability determination (1) Determination of in vitro drug release behavior The release behavior of NM into DHQ in a simulated gastrointestinal environment was evaluated using the dialysis bag method, while particle size changes during the release process were monitored, as follows: Simulated digestion system and release experiment: Simulated gastric juice (SGF, pH 1.2, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was an HCl solution containing 0.32% pepsin, and simulated intestinal juice (SIF, pH 6.8, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was a phosphate buffer containing 0.1% trypsin. Accurately weigh NM containing 5 mg DHQ, disperse it in 20 mL of SGF, and incubate at 37°C in a water bath at 100 rpm for 2 h. After 2 h, transfer the suspension to a dialysis bag with a molecular weight cutoff of 30 kDa, immerse it in 20 mL of SIF, and continue incubation at 37°C and 100 rpm until the total digestion time is 8 h. Samples were taken at 1 mL after incubation in SGF for 0, 0.5, 1, and 2 h, and in SIF for 3, 4, 6, and 8 h, with an equal amount of fresh SGF or SIF added. After filtration through a 0.22 μm filter membrane, the absorbance was measured at 290 nm using a full-wavelength microplate reader to calculate the DHQ release. The particle size of NM at each time point was also measured.

[0041] Results Analysis: DHQ Release Curve Figure 5 The results showed that the release rate of DHQ in SGF was only 5.8±0.8% within 2 hours, indicating that the spirulina coating could effectively block the acidic environment in the stomach and the destruction of DHQ by pepsin. After being transferred to SIF, the release rate of NM increased significantly, reaching 82.6±2.5% at 4 hours and 96.2±1.3% at 8 hours, exhibiting a pH-responsive characteristic of "low release in the stomach and continuous release in the intestine". Particle size change monitoring showed that the particle size of NM in SGF increased from 498.6±7.5 nm to 512.3±9.1 nm, possibly due to slight swelling of the spirulina coating. After being transferred to SIF, as the coating gradually degraded, the particle size dropped back to 168.9±7.2 nm, indicating that NM can maintain structural stability in the intestine and ensure continuous release of DHQ.

[0042] (2) Bioavailability determination The in vitro bioavailability of dihydroquercetin is defined as the amount of dihydroquercetin released into the mixed micelles formed by phospholipids and bile salts after digestion in the intestine. The intestinal digestive fluid from (1) in Title 2 of this embodiment, after 8 hours of digestion, was centrifuged at 3000 × g for 15 min. The intermediate layer sample formed after centrifugation was then collected, the upper phase was diluted with methanol, and its absorbance was measured at 375 nm using an ELISA reader. The formula for calculating the in vitro bioavailability of dihydroquercetin is as follows: .

[0043] Among them, M 胶束 It is the weight of dihydroquercetin in micelle form, M 消化 This is the weight of dihydroquercetin before digestion.

[0044] The results are as follows Figure 6The bioavailability of free DHQ was 1.8%, while that of DHQ delivered by nanomotors (NM) reached 18.4%, which was 16.6% higher than that of the free form.

[0045] Example 3: Evaluation of the in vitro anti-atherosclerotic activity of rice bran protein-based nanomotors 1. Cell model construction An inflammation model was constructed using RAW 264.7 mouse macrophages (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) to simulate the macrophage microenvironment in atherosclerotic plaques. (1) Cell culture: RAW 264.7 mouse macrophages were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and passaged for two generations in a constant temperature incubator at 37℃ and 5% CO2. Subsequent experiments were carried out after the cells were in stable growth state.

[0046] (2) Induction of inflammation model: RAW 264.7 mouse macrophages were injected at a rate of 5 × 10⁻⁶ mcg / mL. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin for 24 hours until cell adhesion. The original medium was then removed, and fresh medium (DMEM medium containing 10% FBS and 1% penicillin-streptomycin) containing 1 μg / mL lipopolysaccharide (LPS) was added, followed by incubation for another 24 hours. Normal cells not treated with LPS were used as a blank control group. The secretion levels of IL-1β, IL-6, and TNF-α in the cell supernatant were detected using ELISA kits (Mouse IL-1β ELISA Kit, Mouse IL-6 ELISA Kit, Mouse TNF-α ELISA Kit, Shanghai Beyotime Biotechnology Co., Ltd.). Results are as follows: Figure 7 The difference in the secretion of inflammatory factors between the LPS-treated group and the blank control group confirmed the successful construction of the inflammation model.

[0047] 2. Cell uptake experiment The targeting uptake efficiency of NM in inflammatory macrophages (the inflammation model in title 1 of this embodiment) was observed using confocal laser scanning microscopy (CLSM): (1) Fluorescent labeling and cell incubation: Following standard methods, NM and RBP (prepared in Example 1) were fluorescently labeled with Cy5-NHS ester (Cyanine 5-N-hydroxysuccinimide ester, purchased from Shanghai Pengshuo Biotechnology Co., Ltd.) to obtain Cy5-labeled NM and Cy5-labeled DHQ. Inflammatory macrophages were incubated with Cy5-labeled NM or DHQ (final concentration 50 μg / mL) for 5, 10, 20, and 30 min, respectively. After incubation, the cells were washed three times with PBS, and the cell nuclei were stained with Hoechst 33258 for 10 min.

[0048] (2) Imaging and Result Analysis: Intracellular Cy5 fluorescence signal was observed using CLSM, and fluorescence intensity was quantified using ImageJ software. Results are shown below. Figure 8 and 9 Under the same incubation time, the intracellular fluorescence signal in the NM-treated group was significantly stronger than that in the DHQ group. Quantitative data showed that the average fluorescence intensity of NM after 30 min of incubation was 7.787±0.248, which was 1.95 times that of the DHQ group (4.001±0.209) at the same time. In terms of uptake rate, the fluorescence intensity of NM after 10 min of incubation (5.275±0.256) reached 67.7% of its maximum uptake at 30 min; the fluorescence intensity of DHQ after 10 min of incubation (3.286±0.213) was 82.1% of its uptake at 30 min, further confirming that NM can be taken up by macrophages in atherosclerotic plaques more efficiently.

[0049] 3. Anti-atherosclerotic activity assay The effect of NM on improving the pathological phenotype of inflammatory macrophages was evaluated using three indicators: cell viability, intracellular ROS, and lipid accumulation.

[0050] First, RBP-DHQ core nanoparticles (RDHQ) were prepared according to step (1) of Example 1. RDHQ was modified with only L-arginine (same as the first step of step (2), without freeze-drying after dialysis and centrifugation) and only D-mannose (without L-arginine modification, D-mannose was directly coupled to the RDHQ surface after EDC / NHS activation, the method is the same as the second step in step (2), the only difference is that the same mass of RDHQ was used to replace RDHQ-Arg, and after centrifugation and washing, it was not freeze-dried) to obtain RDHQ-Arg and RDHQ-Man. Then, according to the Spirulina coating method in step (3), RDHQ-Arg and RDHQ-Man were mixed with Spirulina dispersion at a mass ratio of 1:1 and stirred at 30℃ for 2h to obtain RDHQ-Arg@SP group (only L-arginine modification + Spirulina coating) and RDHQ-Man@SP group (only D-mannose modification + Spirulina coating).

[0051] The experiment was divided into a blank control group, an LPS inflammation group, a free DHQ group, an NM group, an RDHQ-Arg@SP group, and an RDHQ-Man@SP group. Inflammatory macrophages (prepared according to the method in heading 1 (2) of this embodiment) were divided into the LPS inflammation group, the free DHQ group, the NM group, the RDHQ-Arg@SP group, the RDHQ-Man@SP group, the free DHQ group, the NM group, the RDHQ-Arg@SP group, and the RDHQ-Man@SP group, respectively, with a final concentration of 50 μM of DHQ, NM, RDHQ-Arg@SP, and RDHQ-Man@SP. The LPS inflammation group was replaced with the same volume of PBS. Blank control group: The same volume of PBS was added to normal macrophages to replace the drug.

[0052] Cell viability assay: Cell viability was assessed using a double staining method with Calcein-AM (calcein methyl ester, Shanghai Beyotime Biotechnology Co., Ltd.) and Propidium Iodide (PI, Shanghai Beyotime Biotechnology Co., Ltd.). Twenty-four hours after drug intervention, the original culture medium was discarded, and fresh culture medium containing 2 μM Calcein-AM and 5 μM PI was added. Cells were incubated at 37°C in the dark for 30 minutes. Subsequently, the cells were gently washed twice with phosphate-buffered saline (PBS) to remove excess dye. Finally, fluorescence images were observed and acquired using a confocal laser scanning microscope (CLSM). Live cells showed green fluorescence (Calcein-AM), and dead cells showed red fluorescence (PI). ImageJ software was used to analyze the images and calculate cell viability. Cell viability results are shown below. Figure 10 As shown in the figure, compared with the LPS stimulation group (71.01 ± 5.82%), the cell viability of the NM group recovered to 90.73 ± 0.89%, a relative increase of approximately 19.72%; the RDHQ-Arg@SP group recovered to 75.46 ± 6.14%, an increase of approximately 4.45% compared with the LPS group; the RDHQ-Man@SP group recovered to 72.67 ± 5.33%, an increase of approximately 1.66% compared with the LPS group; and the free DHQ group recovered to 79.98 ± 7.95%, an increase of approximately 8.97% compared with the LPS group. The recovery effect of the NM group was 4.43 times that of the RDHQ-Arg@SP group, 11.87 times that of the RDHQ-Man@SP group, and 2.20 times that of the free DHQ group, respectively. These results indicate that NM can significantly enhance the protective effect of DHQ on macrophages under inflammatory conditions.

[0053] Intracellular ROS clearance assay: Intracellular ROS levels in each group of cells were assessed using the DCFH-DA probe (2′,7′-dichlorodihydrofluorescein diacetate, Shanghai Beyotime Biotechnology Co., Ltd.). After 24 hours of drug intervention, the original culture medium was discarded, and serum-free medium (DMEM medium containing 1% penicillin-streptomycin) containing 10 μM DCFH-DA was added. Cells were incubated at 37°C in the dark for 30 minutes. Cells were then gently washed three times with pre-cooled PBS to thoroughly remove any probes that had not entered the cells. Green fluorescence images were observed and acquired using a confocal laser scanning microscope (CLSM), and the average fluorescence intensity was quantitatively analyzed using ImageJ software. The results showed ( Figure 11 Using the LPS group as a baseline (with its ROS level set at 100%), the relative ROS levels and scavenging rates of each group were calculated. The fluorescence intensity of the NM group was 1.401 ± 0.695, equivalent to 16.6% of the LPS group, indicating a ROS scavenging rate of 83.4%. The fluorescence intensity of the free DHQ group was 4.935 ± 0.584, equivalent to 58.7% of the LPS group, with a scavenging rate of 41.3%. The fluorescence intensities of the RDHQ-Arg@SP group (8.205 ± 0.612) and the RDHQ-Man@SP group (8.083 ± 0.587) were similar to those of the LPS group, with scavenging rates of only 2.5% and 3.9%, respectively, indicating virtually no scavenging effect. The scavenging effect of the NM group was 2.02 times that of the free DHQ group, 33.4 times that of the RDHQ-Arg@SP group, and 21.4 times that of the RDHQ-Man@SP group, respectively. These results demonstrate that NM possesses significant ROS scavenging ability.

[0054] Lipid Degradation Assay and Lysosomal Colocalization Analysis: To evaluate the promoting effect of nanomotors on lipid degradation, double staining was performed using Nile Red (Shanghai Beyotime Biotechnology Co., Ltd.) and LysoTracker Green (Shanghai Beyotime Biotechnology Co., Ltd.). After 24 hours of drug intervention, cells in each group were incubated at 37°C for 15 minutes with 1 μM Nile Red (for neutral lipids, red fluorescence) and 50 nM LysoTracker Green (for acidic lysosomes, green fluorescence), respectively, in the dark. After washing with PBS, the spatial distribution of lipid droplets and lysosomes was observed using CLSM, and intracellular lipid accumulation was quantitatively analyzed using ImageJ software. Figure 12 ) and the degree of lipid droplet-lysosome colocalization ( Figure 13The lipid clearance efficiency was assessed. Lipid accumulation results showed that, compared to the LPS model group, NM treatment reduced lipid accumulation by 93.5%, restoring it to near-normal levels. In contrast, the lipid accumulation reduction rates in the free DHQ group, RDHQ-Arg@SP group, and RDHQ-Man@SP group were 58.9%, 65.6%, and 67.7%, respectively. The lipid-lowering effect of NM was 1.42 times and 1.38 times that of the RDHQ-Arg@SP group and the RDHQ-Man@SP group, respectively. Colocalization analysis further revealed that the colocalization coefficient of the NM group was as high as 7.2 ± 0.92, which was 7.2 times that of the control group and significantly higher than that of the DHQ group (4.8 times), the RDHQ-Arg@SP group (5.1 times), and the RDHQ-Man@SP group (5.9 times). These results confirm that NM can efficiently promote the transport of lipid droplets to lysosomes, and its mechanism may be related to the activation of the autophagy-lysosome pathway and the acceleration of lipid degradation, thereby effectively alleviating lipid deposition under inflammatory conditions.

Claims

1. A nanomotor based on rice bran protein, characterized in that... Prepared using a method including the following steps: (1) Add the ethanol solution of dihydroquercetin dropwise to the rice bran protein solution, homogenize, sonicate, centrifuge, collect the precipitate, freeze dry, and obtain RBP-DHQ core nanoparticles. (2) L-arginine and D-mannose were sequentially coupled to the surface of RBP-DHQ core nanoparticles to obtain functionalized nanoparticles. (3) The functionalized nanoparticle suspension was mixed with the spirulina dispersion and stirred to obtain the rice bran protein-based nanomotor RDHQ-Arg@Man / SP.

2. The nanomotor according to claim 1, characterized in that... In step (1), the mass ratio of rice bran protein to dihydroquercetin is 18-22:

1.

3. The nanomotor according to claim 2, characterized in that... In step (2), L-arginine and D-mannose are sequentially coupled onto the surface of RBP-DHQ core nanoparticles using a carbodiimide chemical method.

4. The nanomotor according to claim 3, characterized in that... RBP-DHQ core nanoparticles and L-arginine were coupled at a mass ratio of 0.8-1.2:0.5 to obtain L-arginine-modified particles; the L-arginine-modified particles and D-mannose were coupled at a mass ratio of 0.8-1.2:0.2 to obtain functionalized nanoparticles.

5. The nanomotor according to claim 4, characterized in that... In step (3), the mass ratio of functionalized nanoparticle suspension to spirulina is 0.8-1.2:

1.

6. The use of the nanomotor described in any one of claims 1-5 in the preparation of a drug for treating atherosclerosis.

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