Puerarin-loaded nano preparation as well as preparation method and application thereof

Abstract

The invention provides a puerarin-loaded nano preparation as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The preparation method of the puerarin-loaded nano preparation comprises the following steps: dissolving cinnamyl aldehyde in ethyl acetate, adding methyl 3-mercaptopropionate and trifluoroacetic acid, and stirring in a water bath to prepare cinnamyl aldehyde thioacetal methyl propionate; the preparation method comprises the following steps: dissolving cinnamyl aldehyde thioacetal methyl propionate in ethanol, and adding hydrazine hydrate to prepare cinnamyl aldehyde thioacetal propionyl hydrazine; the preparation method comprises the following steps: dissolving puerarin and cinnamyl thiohydrazide in dimethyl sulfoxide, dropwise adding into a PBS (Phosphate Buffer Solution), stirring and reacting in a dark place, dialyzing, and freeze-drying to obtain the puerarin-cinnamyl thiohydrazide compound. The invention aims to provide a novel PU (polyurethane)-loaded ROS (reactive oxygen species) responsive CA polymer nanoparticle, which shows good form and physiological stability, not only obviously inhibits oxidative stress and inflammation level in vitro, but also shows good biocompatibility in vivo.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a puerarin-loaded nano preparation, a preparation method and an application thereof. Background Art

[0002] Pueraria lobata (Willd.) Ohwi, a traditional Chinese medicine, is known for its remarkable effectiveness in promoting blood circulation and increasing blood flow. It is commonly used clinically for the prevention and treatment of cardiovascular disease and diabetes. Puerarin (PU), the primary active ingredient in pueraria that exerts pharmacological effects on the cardiovascular system, has been extensively studied since its initial isolation in the late 1950s for its therapeutic efficacy and diverse pharmacological activities. Modern pharmacological research confirms that PU possesses multiple antioxidant, anti-inflammatory, anti-tumor, anti-hypercholesterolemia, anti-hyperglycemia, and anti-hypertensive effects, and is used to treat a variety of conditions, including fever, diarrhea, diabetes, cardiovascular disease, and cerebrovascular disease.

[0003] Numerous studies have demonstrated that polyurethane (PU) effectively inhibits the progression of atherosclerosis (AS). During the development of AS, PU can reduce and control its deleterious effects, specifically by ameliorating endothelial damage, alleviating inflammation, inhibiting the migration and proliferation of vascular smooth muscle cells, lowering lipids, inhibiting foam cell formation, and inhibiting platelet aggregation and anticoagulation. Furthermore, PU effectively inhibits oxidized LDL (oxLDL)-induced macrophage activation and foam cell formation in macrophages. These findings suggest that PU is a potential therapeutic agent for AS.

[0004] However, the chemical structure of PU leads to low solubility and bioavailability (oral bioavailability is approximately 7%), which greatly limits its widespread clinical application. Therefore, how to improve the bioavailability of PU has become a key issue to fully realize its pharmacological effects and promote its application in clinical treatment.

[0005] In addition, nano-drug carriers have shown great potential in the precision treatment of AS. However, in practical applications, in order to effectively target pathological macrophages and foam cells in AS plaques, nano-drug carriers need to penetrate the thick fibrous cap composed of vascular smooth muscle cells and collagen fibers, which places high demands on the performance of nano-carriers.

[0006] In recent years, stimuli-responsive polymers have rapidly developed in the field of targeted delivery and site-specific release of therapeutic drugs. These polymers can respond to external stimuli (such as light and magnetic fields) or internal stimuli (such as reactive oxygen species (ROS), pH, temperature, or enzymes), thereby enabling precise drug release at specific locations. Studies have shown that ROS levels are significantly elevated in AS lesions, especially in vulnerable plaques. Large amounts of superoxide anions and hydrogen peroxide exacerbate the inflammatory response and induce cell apoptosis, causing irreversible damage to intracellular lipids, carbohydrates, proteins, and DNA. Therefore, excessive ROS in AS plaques can serve as a precise trigger for efficient drug release within the lesion, providing new insights into the treatment of AS.

[0007] Traditional stimuli-responsive polymers typically degrade in response to a single stimulus, but their degradation rate may slow after the stimulus subsides. This can lead to inadequate and inefficient drug release at the lesion site, compromising therapeutic efficacy. Innovative cascade-degradable polymers, however, trigger a self-destructive reaction through the cleavage of a susceptible end-capping agent, generating additional triggers and forming a self-amplifying degradation mechanism. Among various ROS-responsive polymers, novel self-amplifying, degradable polythioacetal polymers based on cinnamaldehyde (CA) have attracted considerable attention. CA, the primary bioactive component of cinnamon oil, has been approved as a food additive by the US Food and Drug Administration and exhibits excellent biosafety. However, the aldehyde group of CA is susceptible to oxidation, has poor solubility, and lacks selectivity for lesions. Its instability limits its clinical application. Based on this, the present invention provides a puerarin-loaded nanoformulation, its preparation method, and its application. Summary of the Invention

[0008] The purpose of the present invention is to provide a puerarin-loaded nanoformulation, a preparation method and application thereof, and to prepare a PU-loaded ROS-responsive CA polymer nanoparticle (RGCP@PU NPs), which exhibits good morphology and physiological stability, not only significantly inhibits oxidative stress and inflammation levels in vitro, but also shows good biocompatibility in vivo.

[0009] In one aspect, the present invention provides a method for preparing a puerarin-loaded nanoformulation, comprising the steps of: (1) Cinnamaldehyde was dissolved in ethyl acetate, methyl 3-mercaptopropionate and trifluoroacetic acid were added, and cinnamaldehyde-mercaptoacetal propionate methyl was prepared under stirring in a water bath; (2) dissolving cinnamaldehyde thioacetal propionate methyl ester in ethanol and adding hydrazine hydrate to prepare cinnamaldehyde thioacetal propionyl hydrazide; (3) Puerarin and cinnamaldehyde thioacetal propionyl hydrazide were dissolved in dimethyl sulfoxide, added dropwise to PBS buffer solution, stirred in the dark, dialyzed, and freeze-dried to obtain the product.

[0010] Furthermore, the usage ratio of puerarin, ethyl acetate, methyl 3-mercaptopropionate and trifluoroacetic acid is (11-15) g: (45-55) g: (20-30) g: (20-30) mg.

[0011] Furthermore, the weight ratio of the cinnamaldehyde methyl thioacetal propionate, ethanol and hydrazine hydrate is (9-10): (60-80): (7-9).

[0012] Furthermore, the molar ratio of the cinnamaldehyde methyl thioacetal propionate to hydrazine hydrate is 1:5-7.

[0013] Furthermore, the usage ratio of puerarin, cinnamaldehyde thioacetal propionyl hydrazide, dimethyl sulfoxide and PBS buffer is 1 mg: 11-12 mg: 9-11 mL: 90-110 mL.

[0014] Furthermore, the light-shielded stirring reaction is carried out at room temperature and a stirring speed of 350-450 rpm for 3.5-4.5 hours.

[0015] Furthermore, the dialysis bag has a molecular weight cut-off of 3.5 KD.

[0016] Furthermore, the freeze-drying step includes: pre-freezing the prepared RGCP@PU NPs solution at −80°C for at least 4 hours, ensuring complete solidification before transferring it into a freeze dryer, and performing primary drying in sequence: subliming free water at −40°C to −20°C and a vacuum of 0.1-0.01 mbar for 24-36 hours; secondary drying: gradually heating to 25°C and maintaining a vacuum of ≤0.01 mbar, and continuing drying for 12-24 hours to remove residual moisture.

[0017] On the other hand, the present invention also provides a puerarin-loaded nanoformulation, which is prepared using the aforementioned preparation method.

[0018] On the other hand, the puerarin-loaded nanoformulation prepared in the present invention and the preparation method of the puerarin-loaded nanoformulation provided in the present invention are used to prepare drugs for preventing and / or treating atherosclerosis and various acute and chronic cardiovascular diseases or oxidative stress-related diseases.

[0019] The beneficial effects of the present invention are: This study prepared a PU-loaded ROS-responsive cinnamaldehyde polymer nanoformulation, RGCP@PU NPs, for use in the preparation of drugs for the prevention and / or treatment of atherosclerosis, various acute and chronic cardiovascular diseases, or oxidative stress-related diseases. These NPs exhibit excellent physiological stability and biocompatibility, capable of cleaving and releasing PU in an environment with high levels of ROS, thereby enhancing the efficacy of PU. In vitro studies demonstrated that RGCP@PU NPs significantly inhibited ROS generation and the expression of inflammatory factors. Further in vivo experiments confirmed their effective anti-AS effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Characterization of the puerarin-loaded nanoformulation RGCP@PU NPs of the present invention. A, Synthesis process of RGCP. B, 1H-NMR spectrum of RGCP. C, 1H-NMR spectrum of RGCP. D, 1H-NMR spectrum of RGCP after incubation with H2O2. E, TEM images, bars = 200 nm. F, Particle size of RGCP@PU. G, Particle size of RGCP@PU under different H2O2 concentrations. H, Stability of RGCP@PU NPs.

[0021] Figure 2 Figure 2 shows the in vitro cytotoxicity and cellular uptake of the puerarin-loaded nanoformulation RGCP@PU NPs of the present invention. A, In vitro drug release curve. B, CCK-8 assay for the cytotoxicity of different concentrations of RGCP@PU NPs on HUVEC. B, CCK-8 assay for the cytotoxicity of different concentrations of RGCP@PU NPs on RAW264.7 cells. C, CCK-8 assay for the effect of RGCP@PU NPs co-incubated with 1 mM H2O2 on RAW264.7 cell viability. E, Effects of different exposure times of RGCP@PU NPs on HUVEC cellular uptake under ROS conditions. Bars = 100 μm. F, Effects of different exposure times of RGCP@PU NPs on RAW264.7 cellular uptake under ROS conditions. Bars = 100 μm. *P<0.05 vs 0 μg / mL, **P<0.01 vs 0 μg / mL, ***P<0.001 vs 0 μg / mL.

[0022] Figure 3The in vitro ROS scavenging and anti-inflammatory effects of the puerarin-loaded RGCP@PU NPs of the present invention. A, Fluorescence microscopy imaging of the effect of RGCP@PU NPs on ROS levels in RAW264.7 cells. Bars = 100 μm. B, ELISA assay for the expression of inflammatory cytokines (IL-6, TNF-α, and IL-1β). *** P<0.001 vs Control. # P<0.05vs ox-LDL, # P<0.001 vs ox-LDL, & P<0.05 vs ox-LDL+PU.

[0023] Figure 4 The in vivo therapeutic effect of puerarin-loaded RGCP@PU NPs on AS. A, Representative images of aortic root ORO staining. B, Quantitative analysis of aortic lesion area (n = 3). C, Representative images of HE, Masson's, and CD41 antibody staining (n = 3, bars = 200 μm). * P<0.05 vs PBS, ** P < 0.01 vs PBS. PBS, AS model mice. PU, AS mice treated with PU. RGCP, AS mice treated with RGCP. RGCP@PU, AS mice treated with RGCP@PU.

[0024] Figure 5 Biocompatibility analysis. A, Hemocompatibility test. B, Serum levels of AST, ALT, UREA, CRE, RBC, WBC, HGB, and PLT were measured using an automated biochemical analyzer (n = 5). C, HE staining of the heart, liver, spleen, lung, and kidney of each group of mice (n = 3). Control, C57BL / 6 mice injected with saline via the tail vein. RGCP, C57BL / 6 mice injected with RGCP via the tail vein. RGCP@PU, C57BL / 6 mice injected with RGCP@PU via the tail vein. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0026] It should be noted that all experiments in this invention were repeated at least three times. All data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 9 (GraphPad Inc, San Diego, CA, USA). Comparisons between multiple groups were performed using one-way analysis of variance, and statistical significance between groups was determined using the Turkey multiple comparison test. P < 0.05 was considered statistically significant.

[0027] It should also be noted that puerarin, ethyl acetate, methyl 3-mercaptopropionate, trifluoroacetic acid, ethanol and hydrazine hydrate in the present invention were all purchased from Aladdin Reagent.

[0028] Example 1 This embodiment provides a method for preparing a puerarin-loaded nanoformulation, comprising the following steps: (1) Preparation of cinnamaldehyde-thioacetal propionate (RGC) Dissolve 13.216 g of cinnamaldehyde in 50 g of ethyl acetate. Once completely dissolved, add 25 g of methyl 3-mercaptopropionate and 25 mg of trifluoroacetic acid. The mixture is then placed in a 30°C water bath and reacted for 4 hours with magnetic stirring at 500 rpm to produce cinnamaldehyde-methyl thioacetal propionate.

[0029] (2) Preparation of cinnamaldehyde-thioacetal propionylhydrazide (RGCP) 9.60 g of methyl cinnamaldehyde thioacetal propionate prepared in step (1) was dissolved in 70.00 g of ethanol and uniformly mixed under magnetic stirring at 500 rpm for 4 h; then, 7.96 g of hydrazine hydrate was added to the system, and the molar feed ratio of methyl cinnamaldehyde thioacetal propionate to hydrazine hydrate was 1:6. The reaction was carried out at 60°C for 8 h, and the reaction progress was monitored by the plate method. After the reaction, 70 g of the ethanol solution after the reaction was transferred to a rotary evaporation flask (liquid volume ≤ 50%), connected to a rotary evaporator, set the water bath temperature to 40°C and the vacuum pressure to 0.08 MPa, and rotated slowly at 80 rpm. The evaporation was gradually vacuumed until the solvent was steadily distilled out. The evaporation flask was stopped when the residue in the evaporation flask became viscous or solid precipitated; after turning off the vacuum, nitrogen was passed to balance the pressure, and the concentrate was immediately washed with cold ethanol. Finally, it was transferred to a vacuum drying oven at 42°C for drying for 24 h. During the operation, the vacuum gradient should be controlled to prevent violent boiling, and the condensed ethanol was recovered.

[0030] (3) Preparation of puerarin-loaded cinnamaldehyde-thioacetal propionylhydrazide nanoparticles (RGCP@PU NPs) 1 mg of puerarin and 11.4 mg of RGCP were dissolved in 10 mL of dimethyl sulfoxide (DMSO) and added dropwise to 100 mL of 0.1 mol / L PBS buffer with stirring at 400 rpm. The mixture was stirred for 30 min and then stirred in the dark at room temperature for 4 h. The solution was then dialyzed through a dialysis bag (molecular weight cutoff = 3.5 kD) for 24 h to remove the organic solvent and free drug. The prepared RGCP@PU NPs solution was pre-frozen at −80°C for at least 4 h to ensure complete solidification before being transferred to a freeze dryer. The solution was dried in sequence: free water was sublimated at −30°C under a vacuum of 0.05 mbar for 30 h; and the residual water was removed by drying in a secondary manner: the temperature was gradually increased to 25°C while maintaining a vacuum of ≤0.01 mbar for 18 h.

[0031] In order to explore the effects of different PU loadings, polymer ratios, and reaction conditions on the properties of nanoparticles, the following examples were designed in the present invention: It should be noted that the stirring speed and reaction time are the stirring speed and stirring time for mixing raw materials during the reaction in the preparation process of cinnamaldehyde thioacetal propionate methyl ester (RGC) and the preparation process of cinnamaldehyde thioacetal propionyl hydrazide (RGCP).

[0032] Based on the above results, a PU:RGCP ratio of 1:11.4 (Example 1) is the optimal ratio, balancing drug loading and encapsulation efficiency. Excessive PU loading (Example 2) can lead to decreased encapsulation efficiency and incomplete encapsulation, while low PU loading (Example 3) results in insufficient drug loading.

[0033] In order to explore and verify the necessity of puerarin, the present invention demonstrated the key role of PU by not loading the drug or replacing the drug, and designed the following comparative examples: In order to verify the superiority of cinnamaldehyde derivatives (RGCP), the present invention designed the following comparative examples: Based on the above results, PU is irreplaceable, and its anti-inflammatory and antioxidant properties are significantly better than other flavonoids (such as quercetin). In addition, ROS response is critical, while non-responsive carriers (PLGA) cannot achieve lesion-targeted drug release. The aldehyde group released by the cinnamaldehyde group (CA) can induce additional ROS generation, forming a self-amplifying degradation cycle ( Figure 5 ).

[0034] In summary, by comparing the drug-loaded and non-drug-loaded groups, it can be seen that PU inhibits the NF-κB pathway ( Figure 3 ) and upregulate collagen synthesis ( Figure 4), significantly enhancing plaque stability. The unloaded group only relied on the biocompatibility of the carrier and had no therapeutic effect. RGCP@PU released the drug rapidly in AS lesions (high ROS), while PLGA@PU released the drug without selectivity ( Figure 2 A). In addition, CA release induces mitochondrial damage → more ROS → accelerated carrier degradation (a positive feedback loop), which is not found in ordinary carriers.

[0035] Test Case 1. Characterization Transmission electron microscopy (TEM, FEI Talos F200X, USA) was used to observe the morphology of RGCP@PU NPs and their morphological changes after incubation with 1 mM H2O2. Dynamic light scattering (DLS) was used to measure the particle size of RGCP@PU NPs, and the changes in particle size after incubation with different concentrations of H2O2 (0, 0.1, 1, and 10 mM) were further analyzed.

[0036] The hydrated particle size of RGCP@PU NPs was measured using a nanoparticle size analyzer (Zetasizer Nano ZS, UK).

[0037] In addition, high-performance liquid chromatography (HPLC) was used to calculate the encapsulation efficiency (EE%) and loading efficiency (LE%) of RGCP@PU NPs.

[0038] Encapsulation efficiency (EE): EE (%) = [actual drug loading (m2 − m1) / total drug dosage (m2)] × 100%; Drug loading (DL): DL (%) = [actual drug loading (m2 − m1) / total mass of nanoparticles (mtotal)] × 100% Wherein, m1: the mass of free puerarin (PU) in the supernatant after centrifugation, representing the drug that was not successfully encapsulated; m2: the total mass of PU initially input into the experiment; mtotal: the dry weight of the nanoparticles, that is, the sum of the polymer carrier (RGCP) and the encapsulated drug (PU) (mtotal = mRGCP + mPU).

[0039] 2. In vitro drug release 100 mg / mL RGCP@PU NPs (containing 8 mg of PU) were suspended in 10 mL of PBS with or without 1.0 mM H₂O₂. The suspensions were placed in 10 mL centrifuge tubes and allowed to stand for 0, 1, 2, 4, 6, 8, 12, 18, 24, 48, and 72 hours. At each time point, the precipitate was filtered through a membrane, collected, and then refilled to 10 mL with PBS. PU concentrations at different time points were determined using high-performance liquid chromatography (HPLC, Agilent 1260, USA).

[0040] Through the above experiments, it can be seen that the synthesis flow chart of cinnamaldehyde-thioacetal propionyl hydrazide is as follows: Figure 1 As shown in A, and through 1 H-NMR confirmed the successful chemical bonding.

[0041] like Figure 1 As shown in B, 1 The H-NMR spectrum observed a single peak near 4.5-5.0 ppm, attributed to a hydrogen atom at the edge of the dithiol group. Simultaneously, multiple peaks appeared around 2.5-3.0 ppm, attributed to two hydrogen atoms adjacent to the dithiol group. These characteristic peaks were consistent with the structure of the reaction product, indicating a successful chemical reaction between cinnamaldehyde (CA) and methyl 3-mercaptopropionate.

[0042] In addition, the 1H-NMR spectrum of RGCP NPs ( Figure 1 C) A characteristic peak appears in the 4.0-4.5 ppm range, corresponding to the -NH2 proton signal in the hydrazide structure, further confirming the successful chemical binding.

[0043] The 1H-NMR spectrum after incubation of RGCP@PU NPs with H2O2 showed that the original characteristic peak of 4.5-5.0 ppm disappeared, and a new peak appeared in the range of 9.5-10.0 ppm, which was attributed to the aldehyde hydrogen signal of CA released after the cleavage of the thioacetal bond.

[0044] PU-loaded NPs (RGCP@PU NPs) were prepared via a self-assembly process, with a drug encapsulation efficiency (EE) of 88.68% and a drug loading of 8.06%.

[0045] Transmission electron microscopy (TEM) observations showed that ( Figure 1 E), RGCP@PU NPs are spherical and relatively evenly distributed.

[0046] Studies have shown that small-sized nanoparticles (about 50-100 nm) have a longer residence time in the blood circulation, enabling more efficient drug delivery. In this invention, the particle size of RGCP@PU NPs was analyzed by dynamic light scattering (DLS). The results showed that ( Figure 1 F), the diameter of RGCP@PU NPs is 66.62±1.71 nm, the PDI is 0.21, and the particle size distribution is between 50-100 nm, demonstrating its potential advantages in drug delivery.

[0047] TA-CA has been shown to be a ROS-sensitive structure, and compared with normal tissues, AS plaques have high levels of ROS. Therefore, to evaluate the ROS responsiveness of RGCP@PU NPs, they were co-incubated with H2O2 and their response characteristics were detected by particle size and morphology changes. Figure 1 As shown in E, 1G, after incubation with H2O2, the structure of RGCP@PU NPs was partially destroyed, generating fragments of different sizes, and the particle size increased significantly with the increase of H2O2 concentration.

[0048] As a drug delivery carrier, stability in the medium is also crucial. Therefore, the stability of RGCP@PU NPs in deionized water was tested. The results showed that ( Figure 1 H), RGCP@PU NPs can show good stability in water and maintain this stability for at least 7 days. This shows that RGCP@PU NPs have good stability.

[0049] In addition, the in vitro drug release curve results showed that ( Figure 2 A) RGCP@PU NPs release the drug slowly in PBS, but after incubation with H2O2, their nanostructure is disrupted, resulting in rapid and substantial release of PU. This is likely due to the cleavage of the TA structure in RGCP@PU NPs under ROS conditions, releasing CA. The released CA, in turn, induces more ROS generation through mitochondrial damage, accelerating the cleavage of RGCP@PU NPs and increasing PU release, demonstrating the ROS responsiveness of RGCP@PU NPs.

[0050] In summary, the present invention successfully prepared ROS-responsive RGCP@PU NPs with good morphology and stability.

[0051] 3. Cell culture Human umbilical vein endothelial cells (HUVECs) and mouse monocyte-macrophage RAW264.7 cells were purchased from the Chinese Academy of Sciences (Shanghai, China) and ATCC (USA), respectively. HUVECs and RAW264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and 1% penicillin / streptomycin. All cells were cultured at 37°C in a humidified atmosphere with 5% CO2.

[0052] 4. In vitro safety evaluation The CCK-8 assay was used to evaluate the in vitro cytotoxicity of RGCP@PU NPs against HUVEC and RAW264.7 cells. 5 Cells were seeded at a density of 1 μg / mL in 96-well plates, with at least six replicate wells per group. After 24 hours of incubation, the culture medium was replaced with RGCP@PU NPs at various concentrations (0, 10, 20, 50, 100, 200, and 400 μg / mL) with or without 1 mM H₂O₂, and the cells were treated for an additional 24 hours. Subsequently, the culture medium was discarded, 10 μL of CCK-8 solution was added to each well, and the 96-well plates were incubated in an incubator for an additional 2 hours. Finally, the absorbance of each well at 450 nm was measured to assess cell viability.

[0053] 5. Cellular Uptake In order to better observe the uptake of RGCP@PU NPs by cells, RGCP@PU NPs were labeled with Cy5 dye. The specific operation process is as follows: RGCP@PU NPs and Cy5 were incubated at 37 ° C for 1 h to prepare Cy5-labeled RGCP@PUNPs (RGCP@PU Cy5 ). After completing the marking, RGCP@PU Cy5 The cells were co-incubated with HUVEC and RAW264.7 cells in cell culture medium containing 0.02 mM or 1 mM H2O2 for 1, 2, and 4 hours, respectively. After incubation, the cells were washed with PBS, fixed with 4% paraformaldehyde, washed again with PBS, and counterstained with 4,6-diamidino-2-phenylindole (DAPI). Cellular uptake was observed using confocal microscopy.

[0054] Through the above experiments, in order to detect the potential toxicity of RGCP@PU NPs, endothelial cells (HUVEC) and macrophages (RAW264.7) were seeded in 96-well plates, respectively, and incubated with different concentrations of RGCP@PU NPs for 4 h. The cell viability was then detected using the CCK-8 kit.

[0055] The results show that ( Figure 2 BC), in HUVEC cells, even when the concentration of RGCP@PU NPs reached 400 μg / mL, the cell viability remained above 90%, indicating that RGCP@PU NPs had no obvious cytotoxicity to normal endothelial cells.

[0056] In RAW264.7 cells, cell viability gradually decreased with the increase of the concentration of RGCP@PU NPs, but even when the concentration of RGCP@PU NPs reached 400 μg / mL, the cell viability remained above 60%, indicating that RGCP@PU NPs had low cytotoxicity to RAW264.7 cells.

[0057] In the pathological environment of AS, a concentration of 1 mM H2O2 is considered an indicator of oxidative stress. RGCP@PUNPs were further incubated with 1 mM H2O2 to examine their effects on the viability of RAW264.7 cells.

[0058] The results show that ( Figure 2 D) Under H₂O₂ conditions, RGCP@PU NPs inhibited RAW264.7 cell viability in a dose-dependent manner. This may be because the TA-CA structure in RGCP@PU NPs cleaved under ROS conditions, releasing CA. The released CA induced mitochondrial damage and generated ROS, leading to RAW264.7 cell damage and inhibition of cell viability.

[0059] In addition, effective cellular uptake of drugs is also crucial to their efficacy. The present invention also tested the uptake of Cy5-labeled RGCP@PU NPs by HUVEC and RAW264.7 cells under different ROS concentrations (0.2 mM H2O2 and 1 mM H2O2).

[0060] The results show that ( Figure 2 EF), at two ROS concentrations, the uptake of RGCP@PU NPs by HUVEC and RAW264.7 cells was time-dependent, and under 1 mM H2O2 (ROS pathological condition), the uptake of RGCP@PU NPs by both cells was enhanced.

[0061] 6. ROS level detection RAW 264.7 cells were randomly divided into control group, ox-LDL group, ox-LDL+PU group, ox-LDL+RGCP group and ox-LDL+RGCP@PU group.

[0062] Each group of cells was divided into 1×10 5Cells were seeded at a density of 100 μg / mL in 24-well plates. The ox-LDL+PU, ox-LDL+RGCP, and ox-LDL+RGCP@PU groups were incubated with free PU, RGCP, and RGCP@PU NPs for 2 h. The control and ox-LDL groups did not receive any treatment. The equivalent PU concentration of free PU and RGCP@PU NPs was 60 μM, which was converted to the amount of RGCP in RGCP@PU NPs based on the corresponding encapsulation efficiency. Subsequently, cells in all four groups, except the control group, were treated with ox-LDL (20 μg / mL) for 24 h. Throughout the experiment, the control group remained incubated in normal culture medium without any treatment. After incubation, cells in each group were washed with PBS and treated with 10 μM DCFH-DA dye (dissolved in serum-free culture medium) for 30 minutes. Finally, fluorescence images of the cells were captured using a fluorescence imaging system (NIKON Ti-S, Japan) to observe ROS production.

[0063] 7. Enzyme-linked immunosorbent assay (ELISA) RAW264.7 cells were randomly divided into Control group, ox-LDL group, ox-LDL+PU group, ox-LDL+RGCP group and ox-LDL+RGCP@PU group.

[0064] First, cells from each group were seeded in the lower chamber of a Transwell. Except for the control group, all other groups were added with 20 μg / mL of ox-LDL and incubated for 24 hours. Subsequently, free PU (60 μM), RGCP, and RGCP@PU NPs were added to the upper chamber of the Transwell in the ox-LDL+PU, ox-LDL+RGCP, and ox-LDL+RGCP@PU groups, respectively, and cultured for another 24 hours. After incubation, the cells in the lower chamber of the Transwell were centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected. The expression levels of IL-1β, TNF-α, and IL-6 in the supernatant of each group were then assayed using ELISA kits.

[0065] Upon activation, M1 macrophages secrete proinflammatory cytokines, including the classic markers TNF-α, IL-1β, and IL-6. Furthermore, compared to normal tissue, AS pathology produces excessive amounts of ROS. These excess ROS induce oxidative stress, leading to cell damage and triggering an inflammatory cascade, exacerbating the progression of AS. Previous studies have demonstrated that PU can effectively inhibit oxidized low-density lipoprotein (oxLDL)-induced macrophage activation and foam cell formation, alleviating the occurrence and progression of AS by suppressing oxidative stress and inflammation. Therefore, RGCP@PU NPs may inhibit AS progression through a similar mechanism. During AS pathology, oxLDL induces monocyte-derived macrophages to produce ROS, further leading to inflammation and endothelial dysfunction, and promoting foam cell formation—processes that are crucial for the development and progression of AS.

[0066] In the present invention, oxLDL-treated macrophages (foam cell formation) were selected as an in vitro model to evaluate the effect of RGCP@PU NPs, and the effects of RGCP@PU NPs on the levels of ROS and inflammatory factors (IL-6, TNF-α, and IL-1β) were detected by ELISA analysis and immunofluorescence staining.

[0067] Immunofluorescence staining results showed that ( Figure 3 A) In the ox-LDL group, ROS levels were significantly increased. After intervention with PU and RGCP@PU, ROS levels were decreased, with RGCP@PU having a more significant inhibitory effect.

[0068] In addition, ELISA analysis results showed that ( Figure 3 B) Compared with the control group, the expression levels of IL-6, TNF-β, and IL-1β in the ox-LDL group were significantly increased (P < 0.001). After treatment with PU and RGCP@PU, these levels were significantly decreased (P < 0.05), with the decrease being greater in the RGCP@PU group (P < 0.05). This may be due to oxLDL-induced ROS generation, which cleaves the TA-CA structure in RGCP@PU NPs, releasing CA. The released CA, in turn, induces further ROS generation through mitochondrial damage, accelerating the cleavage of RGCP@PU NPs and increasing the release of PU, thereby exerting the antioxidant and anti-inflammatory effects of PU and reducing the levels of ROS and inflammatory factors. These results suggest that RGCP@PU NPs can enhance the effects of PU on AS and may hinder the progression of AS by reducing the levels of ROS and inflammatory factors.

[0069] 8. Animals All experiments used male ApoE - / -Mice (6 weeks old) and male BALB / c mice (6 weeks old) were purchased from SiPeiFu (Beijing, China). All animal experimental procedures in this paper complied with the Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Experimental Ethics Committee of Wenzhou Medical University (Approval No. wydw2024-0550).

[0070] 9. In vivo therapeutic effect of RGCP@PU NPs on AS Twenty-four six-week-old male ApoE - / - Mice were randomly divided into PBS, PU, RGCP, and RGCP@PU groups, with six mice per group. To establish a mouse model of atherosclerosis, each group was fed a high-fat diet (21.2% lard, 49.1% carbohydrates, 19.8% protein, and 0.2% cholesterol) for three months. After the first month of high-fat diet, each group of mice was injected via the tail vein with PBS, PU, RGCP, and RGCP@PU. The PU dose in the PU and RGCP@PU groups was equivalent to 30 mg / kg, and the corresponding RGCP and RGCP@PU doses were adjusted based on encapsulation efficiency. Injections were administered twice weekly, with intervals of 3-4 days, for two months. After two months of dosing, all mice were euthanized, and aortic tissues from each group were collected for subsequent experimental analysis.

[0071] 10. Atherosclerotic plaque detection The entire mouse aorta was isolated, excess fat removed, and fixed in 4% paraformaldehyde for 48 hours. The aorta was then rinsed with 60% isopropyl alcohol. The aorta was then treated with Oil Red O solution at 45°C for 10 minutes and rinsed with 60% isopropyl alcohol. The aorta was rinsed three times with distilled water and mounted on a coverslip with the endothelial surface facing up. Morphological lesions and collagen fibers were analyzed by hematoxylin and eosin (HE) staining and Masson's staining, and images were taken under an electron microscope.

[0072] 11. Immunohistochemical staining Paraffin sections of mouse aortic arches were deparaffinized with xylene and rehydrated with varying concentrations of ethanol. Sections were then immersed in 3% H₂O₂ to quench endogenous peroxidase and transferred to hot sodium citrate buffer (95°C) using microwave heating. After 30 minutes, slides were blocked with 1% BSA for 30 minutes at 37°C and incubated with a primary antibody against CD31 (Abcam) overnight at 4°C. Sections were then incubated with a secondary antibody at 37°C for 30 minutes. The sections were then developed with DAB. After counterstaining with hematoxylin, the slides were dehydrated, cleared with xylene, and mounted with neutral resin.

[0073] To confirm whether RGCP@PU NPs can inhibit the progression of AS in vivo, the present invention established ApoE - / - Mouse AS models were established and different treatment regimens were implemented. After the experiment, aortic tissues of mice in each group were collected and stained with Oil Red O (ORO).

[0074] The results show that ( Figure 4 Atherosclerotic lesions are primarily distributed in the bifurcation region (AB). Quantitative analysis of the aortic intimal surface lesion area showed that the PBS group had the highest plaque area, accounting for 21.11% of the total aortic area. Treatment with PU and RGCP@PU NPs significantly reduced the plaque area, with the RGCP@PU NPs group showing the most significant therapeutic effect, reducing the plaque area from 21.11% of the PBS group to approximately 11.62%.

[0075] In addition, HE staining analysis was performed on the aortic root ( Figure 4 C) Observed plaques in the PBS group contained a significant necrotic core. In contrast, the plaques and necrotic core areas in the PU and RGCP@PU NPs groups were significantly reduced, with the improvement in the RGCP@PU NPs group being more pronounced.

[0076] The key to plaque stability lies in a smaller necrotic core and a thicker fibrous cap, and the prognosis of AS lesions largely depends on plaque stability. Unstable plaques are more likely to rupture, leading to thrombosis and, consequently, serious complications such as myocardial infarction and stroke. Plaque stability is also closely linked to the formation of neovascularization within the plaque. Neovascularization can induce plaque rupture and bleeding, accelerating the transformation of stable plaques into unstable ones. Clinical symptoms gradually worsen as the number of neovascularization increases.

[0077] To further explore the effect of RGCP@PU NPs on plaque stability, the present invention used Masson staining and immunohistochemistry to detect the collagen level around the plaques in each group and the expression of the angiogenesis marker CD31.

[0078] The results show that ( Figure 4 C) RGCP@PU NPs significantly increased the collagen concentration and fibrous cap thickness around the plaque, while significantly reducing the number of CD31-positive neovascularization. These results indicate that RGCP@PU NPs can effectively stabilize plaques and inhibit their further deterioration, thereby delaying the progression of AS.

[0079] 12. In vitro hemolysis test The blood compatibility of RGCP@PU was evaluated using an in vitro hemolysis test. Fresh anticoagulated whole blood from healthy mice was taken, 3.8% sodium citrate anticoagulant solution was added, centrifuged and repeatedly washed with PBS to obtain red blood cells. The red blood cell suspension was diluted 10 times with PBS buffer (pH 7.4). 0.5 mL of diluted red blood cells was mixed with 0.5 mL of different concentrations of RGCP@PU (100, 200, 400, 600 μg / mL). Deionized water was used as the positive control and PBS solution was used as the negative control. Finally, all test samples were incubated at 37°C for 10 min, centrifuged at 10,000 rpm, and the supernatant was taken to detect the absorbance at 540 nm. The hemolysis rate was calculated as follows: .

[0080] 13. Biosafety Assessment of RGCP@PU Fifteen BALB / c mice, provided by SiPeiFu (Beijing, China), were randomly divided into a control group, an RGCP group, and an RGCP@PU group, with five mice in each group. The three groups were injected via the tail vein with saline, RGCP, or RGCP@PU, respectively. Two months after injection, all mice were euthanized, and blood samples were collected as described above. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), red blood cells (RBC), white blood cells (WBC), hemoglobin (HGB), and platelets (PLT) were measured using an automated chemical analyzer.

[0081] At the same time, heart, liver, spleen, lung and kidney tissue samples were collected from each group of mice, and HE staining was used to evaluate the histopathological changes of the heart, lung, liver, spleen and kidney sections of the mice.

[0082] Based on the above tests, the following conclusions are drawn.

[0083] 5. Biocompatibility of RGCP@PU NPs Materials or drugs used in living organisms must have good biocompatibility. When implanted or injected into the body, the red blood cell membrane may rupture, causing hemoglobin to dissolve, causing significant damage to the body. The hemolysis test is an in vitro evaluation method that can directly measure the degree of damage a material causes to red blood cells. According to the standards of the American Society for Testing and Materials (ASTM), a biomaterial is considered acceptable if the hemolysis rate is less than 5%.

[0084] In the present invention, the hemolysis test results showed that even when the concentration of RGCP@PU NPs was 600 μg / mL, the hemolysis rate did not exceed 5%, indicating that RGCP@PU NPs had good blood compatibility. Further blood-related analysis results showed that ( Figure 5B), the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), red blood cells (RBC), white blood cells (WBC), hemoglobin (HGB), and platelets (PLT) in the RGCP and RGCP@PU groups were within the normal range and comparable to those in the control group, indicating that RGCP@PU NPs had no blood, liver, or kidney toxicity.

[0085] In addition, HE staining was performed on sections of the mouse heart, liver, spleen, lung, and kidney to assess the overall health of these important organs after treatment. Figure 5 C) showed that neither the two treatment groups (RGCP and RGCP@PU NPs) nor the placebo group injected with PBS showed any organic lesions. These results indicate that RGCP@PU NPs have good biocompatibility.

[0086] In summary, the present invention provides PU-loaded ROS-responsive cinnamaldehyde polymer nanoparticles (RGCP@PUNPs) for use in the preparation of drugs for the prevention and / or treatment of atherosclerosis. These NPs exhibit excellent stability and biocompatibility in physiological environments and are capable of responding to ROS in the pathological environment of AS, thereby releasing the loaded PU. In vitro experiments demonstrated that these NPs significantly inhibited oxidative stress and inflammatory response levels. In vivo experiments demonstrated that RGCP@PU NPs effectively stabilized atherosclerotic plaques, inhibiting their further deterioration and thus delaying the progression of AS. Furthermore, compared with free PU, these NPs exhibited superior AS therapeutic efficacy. Therefore, RGCP@PU NPs, as a promising nanocarrier, not only effectively inhibit the progression of AS but also provide new research ideas for the design of AS nanotherapeutic systems based on responsive drug release.

[0087] Finally, it should be noted that the above embodiments and comparative examples are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a nanoparticle preparation loaded with puerarin, characterized in that the steps include: (1) Cinnamaldehyde was dissolved in ethyl acetate, methyl 3-mercaptopropionate and trifluoroacetic acid were added, and cinnamaldehyde-mercaptoacetal propionate methyl was prepared under stirring in a water bath; (2) dissolving cinnamaldehyde thioacetal propionate methyl ester in ethanol and adding hydrazine hydrate to prepare cinnamaldehyde thioacetal propionyl hydrazide; (3) Puerarin and cinnamaldehyde thioacetal propionyl hydrazide were dissolved in dimethyl sulfoxide, added dropwise to PBS buffer solution, stirred in the dark, dialyzed, and freeze-dried to obtain the product.

2. The method for preparing a puerarin-loaded nanoformulation according to claim 1, characterized in that: The usage ratio of puerarin, ethyl acetate, methyl 3-mercaptopropionate and trifluoroacetic acid is (11-15) g: (45-55) g: (20-30) g: (20-30) mg.

3. The method for preparing a puerarin-loaded nanoformulation according to claim 1, characterized in that: The weight ratio of the cinnamaldehyde methyl thioacetal propionate, ethanol and hydrazine hydrate is (9-10): (60-80): (7-9).

4. The method for preparing a puerarin-loaded nanoformulation according to claim 1, characterized in that: The molar ratio of the cinnamaldehyde methyl thioacetal propionate to hydrazine hydrate is 1:5-7.

5. The method for preparing a puerarin-loaded nanoformulation according to claim 1, characterized in that: The dosage ratio of puerarin, cinnamaldehyde thioacetal propionyl hydrazide, dimethyl sulfoxide and PBS buffer is 1 mg: 11-12 mg: 9-11 mL: 90-110 mL.

6. The method for preparing a puerarin-loaded nanoformulation according to claim 1, characterized in that: The light-shielded stirring reaction is carried out at room temperature and a stirring speed of 350-550 rpm for 3.5-4.5 hours.

7. The method for preparing a puerarin-loaded nanoformulation according to claim 1, characterized in that: The freeze-drying step includes: pre-freezing the prepared RGCP@PU NPs solution at −80°C for at least 4 hours, ensuring complete solidification before transferring it to a freeze dryer, and performing primary drying in sequence: subliming free water at −40°C to −20°C and a vacuum of 0.1-0.01 mbar for 24-36 hours; and secondary drying: gradually increasing the temperature to 25°C and maintaining a vacuum of ≤0.01 mbar for another 12-24 hours to remove residual moisture.

8. A puerarin-loaded nanoformulation, characterized in that: The puerarin-loaded nanoparticle preparation is prepared by the preparation method of any one of claims 1 to 7.

9. Use of the method for preparing the puerarin-loaded nanoformulation according to any one of claims 1 to 7 in preparing drugs for preventing and / or treating atherosclerosis and various acute and chronic cardiovascular diseases or oxidative stress-related diseases.

10. Use of the puerarin-loaded nanoformulation according to claim 8 in the preparation of a drug for preventing and / or treating atherosclerosis and various acute and chronic cardiovascular diseases or oxidative stress-related diseases.

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