A yolk low-density lipoprotein biomimetic liposome based on structure remodeling and application thereof in regulating cholesterol metabolism

Abstract

This invention belongs to the fields of biomedical materials and colloid chemistry, and discloses a structurally remodeled egg yolk low-density lipoprotein biomimetic liposome and its application in regulating cholesterol metabolism. The method includes extracting and purifying natural egg yolk low-density lipoprotein; selectively removing endogenous cholesterol by adding β-cyclodextrin under alkaline conditions; adding a phytosterol ethanol solution to the cholesterol-removed low-density lipoprotein dispersion, followed by incubation and dialysis purification to obtain the biomimetic liposome. The obtained biomimetic liposome uses partially cholesterol-removed egg yolk low-density lipoprotein as its structural framework, with phytosterols embedded in its phospholipid monolayer and lipid core interface. This biomimetic liposome can inhibit cholesterol micelle formation, reduce cholesterol absorption, promote macrophage cholesterol efflux, and inhibit macrophage foam formation. It can be used to prepare cholesterol metabolism regulating materials, functional delivery systems, and dietary supplement products.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and functional food technology, specifically to a structurally remodeled egg yolk low-density lipoprotein biomimetic liposome and its application in regulating cholesterol metabolism. Background Technology

[0002] Cardiovascular health is a major public health concern worldwide, and hypercholesterolemia is one of its clearly identified major risk factors. Current mainstream drug interventions have certain limitations, while dietary regulation faces challenges such as low bioavailability of active ingredients, limited efficacy, and a lack of efficient delivery systems.

[0003] It is noteworthy that modern nutritional studies have revealed that moderate egg consumption does not significantly increase the risk of cardiovascular disease in the population as expected. This suggests that the overall biological effect of egg yolk is the result of the synergistic effect of its various components. Among them, the abundant phospholipids (such as phosphatidylcholine) in egg yolk are considered key beneficial components. These high-quality lipids in egg yolk mainly exist in the form of lipoproteins. Among them, egg yolk low-density lipoprotein (LDL), as a natural nano-lipoprotein particle, is considered a highly promising oral delivery carrier due to its unique core-shell structure, excellent biocompatibility, and oral targeting potential. Current technologies for the development and utilization of egg yolk LDL mainly focus on two categories: one approach is to modify it through physical methods to optimize its performance as a universal carrier. For example, some studies have used ultrasound-assisted pH shifting to treat LDL, successfully improving its vitamin D3 loading and solution stability by changing its particle size and surface properties. The other approach is to use unmodified natural LDL directly as a building block for delivery systems. For example, it can be combined with high-density lipoprotein as a stabilizer to construct high internal phase Pickering emulsions for delivering phytosterol esters. The above methods can improve some of its performance as a carrier, but do not change its inherent characteristic of high cholesterol content.

[0004] Furthermore, the phytosterol-egg yolk lipoprotein complex disclosed in prior art CN1618320B mainly improves the water dispersibility and food applicability of phytosterols by directly mixing them in an aqueous medium, allowing the egg yolk lipoprotein to cover or stabilize the hydrophobic particles of phytosterols. This method does not selectively remove endogenous cholesterol from egg yolk low-density lipoprotein, nor does it utilize the phospholipid monolayer and lipid core interface vacancies formed after cholesterol removal to guide the insertion of phytosterols. Therefore, this prior art belongs to a direct mixing or surface complex system of phytosterols and egg yolk lipoproteins, and is not a lipoprotein structural reconstruction system based on cholesterol substitution insertion. Therefore, how to remove endogenous cholesterol from egg yolk low-density lipoprotein while maintaining its basic structure, and introduce components that have both structural substitution and cholesterol metabolism regulation functions, is a technical problem that needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a structurally remodeled egg yolk low-density lipoprotein (LDL) biomimetic liposome and its application in regulating cholesterol metabolism. The invention focuses on efficiently and selectively removing endogenous cholesterol from the natural lipoprotein without completely disintegrating its structure. It involves the in-situ and stable embedding of phytosterols into the interface of the cholesterol-removed LDL, achieving structural reconstruction and performance optimization, rather than simple physical mixing. This process endows the remodeled lipoprotein particles with stronger colloidal stability, digestive tolerance, and a clearly defined cholesterol metabolism regulatory function. The process achieves the substitutional embedding of phytosterols into the endogenous cholesterol sites of LDL and the reconstruction of the interface structure, rather than a simple physical mixing of phytosterols and egg yolk lipoprotein.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A method for preparing a biomimetic liposome based on structural remodeling of egg yolk low-density lipoprotein, wherein the biomimetic liposome is formed by structural remodeling of egg yolk low-density lipoprotein with endogenous cholesterol removed as the structural framework, through the embedding of phytosterol molecules into the interface of phospholipid monolayer and lipid core. The preparation method includes the following steps: S1. Extraction and purification of egg yolk low-density lipoprotein: Egg yolk low-density lipoprotein was obtained by centrifugation from egg yolk, purified by ultracentrifugation, and dispersed in neutral phosphate buffer. S2. Selective cholesterol removal treatment: After adjusting the pH value of the egg yolk low-density lipoprotein dispersion obtained in step S1, β-cyclodextrin is added; the mixture is ultrasonically treated, and then the supernatant is collected by centrifugation to obtain a cholesterol-free egg yolk low-density lipoprotein dispersion. S3. Phytosterol embedding and structural reconstruction: Add an ethanol solution containing phytosterols to the cholesterol-free egg yolk low-density lipoprotein dispersion obtained in step S2, incubate, and perform dialysis purification to obtain the biomimetic liposomes.

[0008] Preferably, in step S2, the pH value is adjusted to 8.0-10.0, and the molar ratio of β-cyclodextrin to cholesterol in egg yolk low-density lipoprotein is (0.8-1.2):1.

[0009] Preferably, in step S2, the conditions for ultrasonic treatment are as follows: ultrasonic treatment is performed at 40W-100W under ice bath cooling conditions for 5-15 minutes, in a pulse mode with 2-3 seconds of operation and 3-4 seconds of intermittent operation, and centrifugation is performed at 4°C and 6000-10000 r / min for 30-60 minutes. After step S2, the cholesterol content in the egg yolk low-density lipoprotein dispersion is reduced by more than 70% compared with that of natural egg yolk low-density lipoprotein.

[0010] Preferably, in step S3, the ethanol solution of phytosterols is added dropwise to the continuously stirred cholesterol-free egg yolk low-density lipoprotein dispersion at a rate of 1-1.5 mL / hour, and the concentration of the ethanol solution of phytosterols is 20-30 mg / mL.

[0011] Preferably, in step S3, the molar ratio of the phytosterol to the cholesterol removed in step S2 is (1.0-1.5):1; more preferably, the molar ratio of the phytosterol to the cholesterol removed in step S2 is 1.2:1.

[0012] Preferably, in step S3, the incubation is carried out at 37°C under dark conditions for 1-3 hours.

[0013] Preferably, in step S3, the dialysis purification is performed by dialyzing with a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzing with phosphate buffer at pH 7.4 for 24-48 hours.

[0014] The biomimetic liposomes obtained by the method are spherical nanoparticles with an average particle size ≤120nm and a polydispersity index of less than 0.5; the generalized polarization value corresponding to the membrane order is ≥0.5.

[0015] The biomimetic liposomes obtained by the aforementioned preparation method are used in the preparation of health food or dietary supplement products for regulating cholesterol metabolism.

[0016] The application of the biomimetic liposomes obtained by the aforementioned preparation method in drug delivery materials.

[0017] In the above-described scheme, firstly, the present invention employs a fine and selective cholesterol removal process using β-cyclodextrin as the removal agent. The LDL dispersion is adjusted to alkaline conditions, controlling the pH at 8.0-10. The molar ratio of β-cyclodextrin to cholesterol in the LDL is 0.8:1 to 1.2:1. Ultrasonic treatment is then performed under ice bath conditions at a power of 40-100 W for 5-10 minutes. After this step, the cholesterol removal rate from the LDL can reach 70%-80%. The attached figures show that the main protein bands of the cholesterol-free LDL are still retained. Although the particle morphology changes, it is not completely dispersed, indicating that its basic structure is still maintained.

[0018] Secondly, this invention achieves in-situ embedding and interfacial structure reconstruction through phytosterol embedding and structural reconstruction steps. Specifically, an ethanol solution of phytosterols is slowly added dropwise to a cholesterol-reduced LDL dispersion, controlling the molar ratio of phytosterols to cholesterol removed in step S2 to be 1.0:1 to 1.5:1. The mixture is incubated at 37°C in the dark for 1-3 h, followed by dialyzing for 24-48 h to remove unbound phytosterols, ethanol, and residual β-cyclodextrin, yielding reconstructed liposomes. The accompanying figures show that the reconstructed biomimetic liposomes are spherical nanoparticles with a relatively concentrated particle size distribution and higher membrane order than natural LDL.

[0019] Thirdly, the reconstructed biomimetic liposomes (rLDL) exhibit improved performance due to their novel structure: their particle size and polydispersity index show minimal changes under storage, different ionic strengths, and different pH conditions, and they exhibit low levels of lipid oxidation products. During in vitro simulated gastrointestinal digestion, the biomimetic liposome particles maintain their position for a longer period; when loaded with curcumin, their release in the gastric phase is low, exhibiting sustained-release characteristics in the intestinal phase, and their bioavailability is improved. Furthermore, these biomimetic liposomes can inhibit cholesterol micelle formation, suppress cholesterol uptake by Caco-2 cells, and promote cholesterol efflux from RAW264.7 macrophages, thus making them suitable for cholesterol metabolism regulation-related products and delivery systems.

[0020] Compared with the prior art, the present invention has the following advantages: 1. This invention selectively removes endogenous cholesterol from low-density lipoprotein in egg yolks and introduces phytosterols for structural reconstruction, thereby reducing cholesterol content while preserving the basic structure of the particles.

[0021] 2. The biomimetic liposomes prepared by this invention have an average particle size of no more than 120 nm, improved membrane order, and good colloidal stability, oxidative stability, and digestive stability.

[0022] 3. The biomimetic liposomes prepared by this invention can effectively inhibit the micellization and cell absorption of intestinal cholesterol, while promoting the efflux of cholesterol from macrophages, reducing lipid accumulation in RAW264.7 macrophages, and inhibiting the formation of macrophage foam. It has application value in regulating cholesterol metabolism and improving abnormal cholesterol accumulation.

[0023] 4. The main raw materials used in this invention are egg yolk low-density lipoprotein and phytosterols. The preparation process is relatively mild and can be used in functional delivery materials, health foods, dietary supplements and related delivery systems. Attached Figure Description

[0024] Figure 1 SDS-PAGE images of natural LDL (Comparative Example 1), cholesterol-free LDL (Comparative Example 2), and the biomimetic liposome of the present invention (Example 1).

[0025] Figure 2 The images show the transmission electron microscopy (TEM) morphology comparison of natural LDL (Comparative Example 1).

[0026] Figure 3 Comparative images of transmission electron microscopy morphology of decholesterol LDL (Comparative Example 2).

[0027] Figure 4 This is a transmission electron microscope (TEM) morphology comparison of the biomimetic liposomes (Example 1) of the present invention.

[0028] Figure 5 The bar chart shows the comparison of particle size and PDI of the samples.

[0029] Figure 6 The membrane properties (hydrophobicity, micropolarity, and flowability) of the three samples are shown.

[0030] Figure 7 AFM images of samples with different cholesterol removal rates.

[0031] Figure 8 This is a spectrum of structural changes characterized by FTIR.

[0032] Figure 9 This is a spectrum of membrane order changes characterized by XRD.

[0033] Figure 10 The following are comparative graphs showing the stability of samples under storage, different ionic strengths, and pH conditions: (A) Storage stability; (B) Ion concentration stability; (C) pH stability; (D) MDA content; (E) POV heatmap under different pH conditions; (F) POV heatmap under salt concentration conditions.

[0034] Figure 11 The images show the CLSM images and active substance release curves of the samples in simulated gastrointestinal digestion; (A) CLSM image after LDL gastrointestinal digestion; (B) CLSM image after rLDL gastrointestinal digestion; (C) Release curves and bioavailability of curcumin encapsulated in LDL and rLDL; (D) Gastrointestinal digestibility of curcumin encapsulated in LDL and rLDL.

[0035] Figure 12 The in vitro cholesterol micelle inhibition rate of the sample is shown.

[0036] Figure 13 Comparison of cholesterol uptake inhibition effects in Caco-2 cells; (A) Caco-2 cytotoxicity; (B) Cholesterol uptake inhibition effect in the Caco-2 model.

[0037] Figure 14Comparison of cholesterol efflux effects in RAW264.7 cells; (A) RAW264.7 cytotoxicity; (B) Cholesterol efflux promotion effect in the RAW264.7 model.

[0038] Figure 15 Comparison of RAW264.7 cell foaming inhibition effect; (A) Pathological image of RAW264.7 cell foaming stained with Oil Red; (B) RAW264.7 cytotoxicity; (C) RAW264.7 cell foaming inhibition effect. Detailed Implementation

[0039] To better understand the present invention, the following description, in conjunction with embodiments and accompanying drawings, further clarifies the content of the present invention. However, in order to enable those skilled in the art to fully understand the technical solutions and beneficial effects of the present invention, the following description, in conjunction with specific embodiments, provides further explanation. The embodiments are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

[0040] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0041] Example 1: Preparation of Egg Yolk LDL Bionic Liposomes a. Extraction of natural egg yolk LDL: Take 20g of fresh egg yolk and add 4 times the volume (80 mL) of pre-cooled 0.15 M NaCl solution. Homogenize at 10,000 r / min for 2 minutes at 4°C using a tissue homogenizer to form a homogeneous slurry. Centrifuge the slurry at 10,000 g for 30 minutes at 4°C. Carefully aspirate the middle layer of yellow supernatant, discarding the bottom precipitate and the top lipid droplets. Add solid KBr to the supernatant to adjust the solution density to 1.063 g / mL. Place this solution in an ultracentrifuge tube and ultracentrifuge at 100,000 g for 18 hours at 4°C. After centrifugation, carefully aspirate the milky white band appearing at the top of the tube using a syringe; this is the preliminarily purified egg yolk LDL. The LDL solution was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed against 0.01 M, pH 7.4 phosphate-buffered saline (PBS) at 4°C for 24 hours, with the dialysate changed three times during the process to completely remove KBr. Finally, the solution was filtered through a 0.22 μm filter for sterilization, and the protein concentration was determined (BCA method) and adjusted to approximately 6 mg / mL. The solution was then stored at 4°C for later use.

[0042] b. Selective Cholesterol Removal: Measure 10 mL of the above-mentioned natural LDL solution and place it in a 50 mL centrifuge tube. Under magnetic stirring, slowly add 0.1 M NaOH solution dropwise to precisely adjust the pH of the system to 10.0. Determine the cholesterol content in the LDL using a cholesterol kit. Add β-cyclodextrin to the LDL solution at a 1:1 molar ratio of β-cyclodextrin to cholesterol in the LDL, and continuously stir magnetically at 4°C for 30 minutes to ensure thorough mixing. Then, place the mixture in an ice-water bath and sonicate it for 10 minutes using an ultrasonic cell disruptor (6 mm probe diameter) at 40 W power in pulse mode (2 seconds on, 3 seconds off), maintaining the sample temperature below 10°C throughout the process. After treatment, centrifuge the sample at 4°C and 6000 r / min for 1 hour. Carefully collect the supernatant, which is the cholesterol-removed LDL (pLDL) dispersion. Analysis using a cholesterol kit showed that the cholesterol removal rate in this step was 76.9% ± 2.1%.

[0043] c. Phytosterol embedding and reconstruction: Take 5 mL of the pLDL dispersion obtained in step b and gently stir it in a 37°C water bath (300 r / min). The molar ratio of phytosterol to cholesterol removed in step b is 1.2:1. Dissolve 10 mg of phytosterol in 500 μL of anhydrous ethanol solution preheated to 37°C and vortex until completely dissolved. Using a microinjection pump, slowly and evenly add the sterol ethanol solution dropwise to the continuously stirred pLDL dispersion at a rate of 1 mL / h. After the addition is complete, continue to gently stir and incubate at 37°C in the dark for 2 hours. Transfer the mixture to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze against PBS buffer (pH 7.4) containing 0.01% EDTA at 4°C for 48 hours, changing the dialysate every 6-8 hours to completely remove unbound β-sitosterol, ethanol, and residual β-cyclodextrin. Collect the solution from the dialysis bag and centrifuge at 4°C, 10,000 g for 10 minutes to remove trace aggregates. The supernatant is the final prepared biomimetic liposome (rLDL), which can be used immediately for testing or stored after freeze-drying.

[0044] Comparative Example 1: Natural LDL The preparation method is the same as step a in Example 1, and a purified natural LDL dispersion is obtained, which is directly used in subsequent comparative experiments.

[0045] Comparative Example 2: No phytosterol reconstruction performed Based on Example 1, the preparation method is the same as step ab of Example 1 to obtain a cholesterol-free LDL dispersion. The phytosterol reconstruction treatment in step S3 is not performed, and it is directly used for subsequent comparative experiments.

[0046] Comparative Example 3: No cholesterol removal at all, phytosterols added directly. Based on Example 1, the purified natural egg yolk LDL dispersion was obtained by following the same steps as in Example 1 (a). The dispersion was then directly processed in step S3, with the same amount of phytosterols added as in step c of Example 1, for subsequent comparative experiments.

[0047] Comparative Example 4: Physical mixing of pLDL and phytosterols (non-structural reconstruction) Based on Example 1, following the same steps ab as in Example 1, a cholesterol-free LDL dispersion was obtained. An equivalent molar amount of phytosterol as in step c of Example 1 was directly added, and the mixture was vortexed and mixed evenly for subsequent comparative experiments.

[0048] Example 2: 30% Cholesterol Removal Based on Example 1, the difference lies in preparation method step b: pH=8.0, β-CD:cholesterol=0.6:1, sonication at 40 W for 5 min. All other steps are the same as in Example 1.

[0049] Example 3: 50% Cholesterol Removal Based on Example 1, the difference lies in preparation step b: pH=8.0, β-CD:cholesterol=0.8:1, sonication at 40 W for 5 min. All other steps are the same as in Example 1.

[0050] Example 4: 90% Cholesterol Removal Based on Example 1, the difference lies in preparation step b: pH=10.0, β-CD:cholesterol=1.4:1, sonication at 60 W for 10 min. All other steps are the same as in Example 1.

[0051] Structural characterization and physical stability To further verify the structural integrity of the biomimetic liposomes constructed in this invention and their sterol remodeling mechanism, multi-scale structural characterization was performed on natural LDL (obtained in Comparative Example 1), cholesterol-free LDL (pLDL) (obtained in Comparative Example 2), and reconstructed liposomes (rLDL) (obtained in Example 1).

[0052] (1) Protein backbone stability analysis Accurately measure 80 μL of LDL, pLDL, and rLDL samples and mix them with 5x protein loading buffer at a 4:1 volume ratio. After denaturation at 100°C for 10 min, equilibrate to 25°C. Inject 7 μL of the treatment solution into the loading channels of a precast gel (4-20% HEPES gradient, Beyotime). Electrophoresis parameters are set to a constant voltage of 150 V for 50 min, with migration calibration performed simultaneously using a protein molecular weight standard. After electrophoresis, stain with Coomassie Brilliant Blue (P0017F, Beyotime) for 30 min, followed by gradient destaining (replace with deionized water three times, 5 min each time) until the bands are clear.

[0053] (2) Morphology and particle size analysis: Morphology was observed using a transmission electron microscope (TEM, JEM-1400Flash). 10 μL of each sample (nLDL, pLDL, rLDL) was diluted 10 times with deionized water, dropped onto a copper grid, and negatively stained (2% phosphotungstic acid, pH 7.0) before observation.

[0054] LDL, pLDL, and rLDL were diluted 1:200 and filtered (0.45 μm). Particle size (Z-average), polydispersity index (PDI), and zeta potential were determined using dynamic light scattering at 25 °C.

[0055] (3) Membrane orderliness analysis (GP value determination): The environmentally sensitive fluorescent probe Laurdan was used for measurements. Each sample was diluted with PBS to a phospholipid concentration of 50 μM and incubated with 2 μM Laurdan probe at 37°C in the dark for 30 minutes. The emission spectrum (400-550 nm) was scanned using a fluorescence spectrophotometer (F-7000) at an excitation wavelength of 350 nm. The generalized polarization value (GP value) was calculated using the formula: GP = (I440 - I490) / (I440 + I490), where I440 and I490 are the fluorescence intensities at 440 nm and 490 nm, respectively.

[0056] (4) AFM structural analysis: To visually reveal the impact of cholesterol removal on the structure of LDL carriers, atomic force microscopy (AFM) was used to characterize samples at different treatment stages. The images clearly demonstrate the complete evolution of the LDL particle interface morphology from smooth to rough, reconstructed, collapsed, and aggregated as the cholesterol removal rate increased from 0% to 90%.

[0057] Sterol-driven membrane ordering structure analysis (1) Fourier Transform Infrared Spectroscopy (FTIR) LDL, pLDL, and rLDL powder samples were uniformly mixed with potassium bromide (1:100 mass ratio) and pressed into transparent sheets. Attenuated total reflectance-FTIR spectrometry was performed using a Nicolet 15 FTIR spectrometer (Thermo Fisher Scientific) at 4000-400 cm⁻¹. -1 Infrared absorption spectra were acquired within the wavenumber range, with the scanning parameters set to 4 cm⁻¹. -1 Resolution, 32 signal accumulations.

[0058] (2) X-ray diffraction (XRD) LDL, pLDL, and rLDL powder samples were freeze-dried for 48 hours using an LGJ-25C freeze dryer (Beijing Sihuan Scientific Instrument Factory, Beijing, China). The crystal structure of the freeze-dried liposomes was determined using an X-ray diffractometer (Miniflex 600, Rigaku Corporation, Japan). The scanning range (2θ) was 5° to 70°, and the scanning speed was 10° / min.

[0059] Storage stability Sodium chloride solutions of different concentrations were mixed with equal volumes of LDL, pLDL, and rLDL liposome samples, and then allowed to stand at room temperature for 2 hours to allow for sufficient interaction. To assess the effect of ionic strength, dynamic light scattering was used to characterize the zeta potential, particle size, and polydispersity index of the samples. Similarly, to investigate the pH stability of the nanoliposome carriers, buffer solutions with different pH values ​​were prepared in advance and mixed with the nanoliposome samples at a 1:1 volume ratio. After incubation at room temperature for 30 minutes, dynamic light scattering characterization was performed on the samples. All data were calculated by averaging at least three repeated measurements.

[0060] Peroxide value was determined according to the previous method. 0.2 mL of liposomes was mixed with 2.8 mL of methanol / butanol solution (2:1, v / v). Then, 15 μL of ferrous ion solution (prepared from 0.144 M FeSO4 and 0.132 M BaCl2 dissolved in 0.4 M HCl) and 15 μL of ammonium thiocyanate solution (3.94 M) were added sequentially. After reacting the mixture at room temperature in the dark for 20 minutes, the absorbance was measured at 510 nm using a spectrophotometer. The peroxide value was calculated based on a standard curve prepared using cumene hydrogen peroxide.

[0061] In vitro simulated digestion stability An in vitro simulated gastrointestinal digestion model was used. Equal volumes (based on phospholipids) of LDL and rLDL samples were placed in simulated gastric juice (containing pepsin, pH 3.0) and incubated at 37°C with shaking for 2 hours (gastric phase). Subsequently, the pH was adjusted to 6.8 with NaHCO3, and simulated intestinal juice (containing pancreatic enzymes and bile salts) was added, followed by incubation for another 2 hours (intestinal phase). Samples were taken at different time points, and particle morphology was observed using a laser confocal microscope (CLSM, using Nile Red to stain lipids), with particle size changes monitored by dynamic light scattering.

[0062] In vitro ability to inhibit cholesterol micelle formation The ability of samples to inhibit dietary cholesterol absorption was evaluated in a simulated intestinal fluid environment. A mixed solution containing sodium taurocholate, lecithin, monooleate glycerol, and cholesterol was prepared and incubated at 37°C to form mixed micelles. Equal volumes of LDL, pLDL, and rLDL samples (all at a final concentration of 1 mg / mL phospholipid) were added, and incubation continued for 2 hours. After incubation, the solution was ultracentrifuged at 100,000 g for 1 hour at 4°C. The supernatant was collected, and the cholesterol content dissolved in the micelles was determined using an enzymatic assay kit to calculate the cholesterol solubility rate.

[0063] Cellular level cholesterol metabolism regulation function a. Caco-2 cell cholesterol uptake inhibition experiment: Human colon adenocarcinoma cells (Caco-2) were seeded into Transwell plates and cultured for 21 days to form a complete monolayer. During the experiment, culture medium containing NBD-labeled cholesterol (5 μg / mL) and different samples (LDL, pLDL, rLDL, and phospholipids, all at 50 μM) was added to the top (intestinal lumen) side, while fresh culture medium was used to the bottom side. Wells with an equal volume of culture medium served as blank controls, and wells with 10 μM ezetimibe (a cholesterol uptake inhibitor) served as positive controls. After 4 hours of incubation, cells were collected, and intracellular fluorescence intensity was detected by flow cytometry to characterize cholesterol uptake. The cholesterol uptake inhibition rate (%) was calculated as: [1 - (sample group fluorescence intensity - background) / (control group fluorescence intensity - background)] × 100%.

[0064] b. RAW264.7 macrophage cholesterol efflux assay: A foam cell model was constructed by co-incubating mouse macrophages (RAW264.7) with acetylated low-density lipoprotein for 24 hours. After washing with PBS, the culture medium was replaced with serum-free medium containing apolipoprotein AI (apoA-I, 10 μg / mL) and different samples (LDL, rLDL, phospholipid concentration 25 μM). After another 24 hours of incubation, the culture medium was collected, and the cholesterol content released in the medium was measured using a cholesterol detection kit. The cholesterol efflux rate (%) was calculated as (cholesterol content in the culture medium / total cholesterol content in the cells) × 100%.

[0065] c. RAW264.7 macrophage cholesterol foaming inhibition experiment: RAW264.7 cells were seeded in cell culture plates. After the cells adhered and grew to a suitable density, they were incubated in medium containing oxidized low-density lipoprotein (ox-LDL) for 24 h to construct a macrophage foaming model. The culture medium was then discarded, and the cells were gently washed with PBS. Different samples were added to the medium for intervention treatment, and blank control, model control, and positive control groups were set up. After treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde, stained with Oil Red O, and the nuclei were counterstained with hematoxylin. Finally, the cells were observed and photographed under a microscope, and the area of ​​Oil Red O positive lipid droplets was calculated using image analysis software to evaluate the inhibitory effect of the samples on cholesterol foaming in RAW264.7 macrophages.

[0066] The following is a description of the determination methods and results for Examples 1-3 to Comparative Examples 1-4: 1. Protein structural integrity analysis SDS-PAGE was used to analyze the integrity of the protein backbone during sterol removal and re-intercalation to distinguish between structural rearrangement after cholesterol removal and structural damage caused by protein degradation. Figure 1 Electrophoresis results showed that the major protein bands of Comparative Example 1, Comparative Example 2, and Example 1 were generally consistent, indicating that the cholesterol removal and remodeling process did not lead to significant degradation of the main apolipoprotein components. Compared with Comparative Example 1, Comparative Example 2 showed enhanced bands in the 37-52 kDa region, suggesting that the particle interface structure changed after cholesterol removal treatment, exposing some protein components. In Example 1, the bands in this region weakened, indicating that the particle interface structure was somewhat restored after the introduction of phytosterols.

[0067] 2. Morphology and particle size analysis TEM images ( Figure 2 The results show that Comparative Example 1 exhibits typical, uniformly dispersed, nearly spherical particles with a relatively uniform particle size distribution, and localized observations of typical core-shell structure characteristics. In contrast, Figure 3Comparative Example 2 showed a significant morphological change, exhibiting marked aggregation and fusion, blurred boundaries, and a wider size distribution, directly demonstrating the disintegration and physical instability of the particle structure caused by cholesterol deficiency. Figure 4 Example 1 shows that the particles tend to become regular and round again, the boundary clarity is improved, the particle distribution is more uniform, and the morphology is close to or even better than that of Comparative Example 1, indicating that the particle structure is reorganized after the phytosterol is inserted.

[0068] Figure 5 The results showed that the particle size of Comparative Example 2 (148.07 nm) was significantly larger than that of the natural Comparative Example 1 (94.63 nm), and the polydispersity index (PDI) was significantly increased. This result is consistent with the increase in low molecular weight bands observed in SDS-PAGE and the aggregation morphology in TEM images, indicating that cholesterol removal disrupts the integrity of the particles, leading to the disintegration of the original particle structure and the formation of loose aggregates with uneven size and partial aggregation. The particle size of Example 1 (LDL) was significantly smaller than that of Comparative Example 2 (pLDL) after cholesterol removal and intermediate samples with different cholesterol removal rates (Examples 2, 3, and 4). To demonstrate that this invention differs from directly mixed complexes, Comparative Example 3, in which phytosterols were added directly without cholesterol removal, and Comparative Example 4, in which cholesterol-removed LDL and phytosterols were directly physically mixed, were also included. In these comparative examples, phytosterols and LDL were only placed in the same system; the phytosterols mainly exhibited external adsorption, free dispersion, or loose binding, making it difficult to form a stable embedded reconstructed structure. In contrast, Example 1, through a sequential process of first decholesterolizing and then embedding phytosterols, enabled phytosterols to enter the interfacial vacancies formed by decholesterolized LDL, resulting in a more stable and smaller biomimetic liposome. Although the LDL particle size was slightly larger than that of natural LDL (Comparative Example 1), its PDI recovered to a low level comparable to Comparative Example 1. This indicates that the embedding of phytosterols facilitates the recombination of the loose system after decholesterolization, forming a novel colloidal particle with a smaller size and more uniform distribution.

[0069] 3. Membrane Orderliness Analysis A higher GP value indicates a more ordered arrangement of membrane lipids, lower fluidity, and a more compact structure. Figure 6 The fluorescence measurements showed that the GP values ​​of Comparative Example 1, Comparative Example 2, and Example 1 were approximately 0.391, 0.318, and 0.593, respectively. This indicates that Example 1 exhibits the best membrane structure order and membrane rigidity.

[0070] 4. Morphological analysis of cholesterol removal like Figure 7As shown, from left to right, the AFM images are those of Comparative Example 1, Example 2, Example 3, Example 1, and Example 4. Natural LDL particles have relatively smooth surfaces and intact morphologies. At 30% removal, the particle surface shows slight roughness, but the overall outline is still maintained. At 50% to 70% removal, more obvious defect structures appear at the particle interface, indicating that cholesterol removal has significantly affected the interface arrangement while still preserving the basis for further reconstruction. At 90% removal, the particle structure collapses significantly and is accompanied by aggregation, indicating that excessive cholesterol removal will destroy particle integrity. These results indicate that there is an optimal range for cholesterol removal, and moderate removal is more conducive to subsequent phytosterol embedding and structural reconstruction.

[0071] 5. Structural Analysis FTIR further revealed the influence of sterol remodeling on liposome structure from the perspective of functional groups and intermolecular interactions. Figure 8 In the comparison, Example 1 is 1743.82 cm. - An absorption peak was observed at ¹. After cholesterol removal, the peak position of Comparative Example 2 shifted slightly to 1745.26 cm⁻¹. -1 Example 1, however, stably maintained this higher wavenumber. This was observed in the characteristic region (~722 cm⁻¹) characterizing the planar rocking vibration of the lipid chain methylene (-(CH₂)n-) group. -1 All three samples exhibited absorption peaks at their unchanged positions, indicating that the basic ordered arrangement of the hydrophobic regions of the lipid chains still exists. Simultaneously, absorption peaks were observed in the CH stretching vibration region (2800-3000 cm⁻¹). -1 The broadening of the band in Comparative Example 2 was restored to a sharper peak shape in rLDL, which is consistent with the planar rocking vibration peak of the lipid chain methylene group (~722 cm⁻¹). -1 The stability of the position indicates that although cholesterol removal temporarily reduces the stacking density and order of lipid chains, the stacking state of lipid chains is improved after phytosterol incorporation.

[0072] XRD is used to evaluate the ordered characteristics and phase information of lipid accumulation, and is an important piece of evidence for determining whether an ordered liquid phase has been formed. Figure 9 The results showed that Comparative Example 1 exhibited a broadened characteristic diffraction peak at 2θ ≈ 21.5°, while the diffraction peak intensity of Comparative Example 2 decreased. This change directly confirmed that the removal of cholesterol disrupted the tight packing of lipid molecules, resulting in a looser periodic structure and reduced order in the lipid layer. However, the diffraction peak of Example 1 not only failed to recover to its natural position but also shifted further to the left to 2θ ≈ 19.8°, with a sharper peak shape and significantly enhanced intensity, indicating that the reconstructed sample had a higher degree of ordered arrangement.

[0073] 6. Storage stability analysis A systematic analysis of particle size and polydispersity index under different storage times, ion concentrations, and pH conditions revealed the significant enhancing effect of phytosterol remodeling on liposome stability. In storage stability experiments, such as... Figure 10 A shows that Comparative Example 1 exhibits a significant increase in particle size and PDI with prolonged storage time, indicating its susceptibility to aggregation and aging. Comparative Example 2 shows even more pronounced particle size growth and wider distribution, reflecting that the system after sterol removal is more prone to rearrangement and aggregation during long-term storage. In contrast, Example 1 shows the smallest increase in particle size and maintains a low PDI level, indicating better storage stability. (The remaining text appears to be incomplete and requires further context.) Figure 10 In B), under conditions of increased salt concentration, both Comparative Example 1 and Comparative Example 2 showed significant aggregation, while the particle size change in Example 1 was smaller, indicating that it had a stronger resistance to salt disturbance. Figure 10 As shown in Figure C, under different pH conditions, Example 1 also maintained relatively stable particle size and PDI over a wide range.

[0074] Malondialdehyde (MDA) content and peroxide value (POV) reflect the levels of lipid oxidation end products and primary peroxide formation, respectively. Results showed that in the assessment of storage-induced oxidation endpoints ( Figure 10 (D) Comparative Example 2 showed the highest MDA content, Comparative Example 1 exhibited a moderate oxidation rate, while Example 1 consistently maintained the lowest MDA level. Peroxide value heatmaps under different pH conditions ( Figure 10 E) shows that, compared to Comparative Examples 1 and 2, Example 1 exhibits lower oxidation levels across a wide pH range from acidic to alkaline. This indicates that the phytosterol-reconstructed sample possesses good oxidative stability. Similarly, the oxidative stability heatmap at different NaCl concentrations ( Figure 10 F) Further confirmation shows that Example 1 can maintain a low peroxide value even under high ionic strength conditions. This indicates that phytosterols synergistically enhance the oxidative stability of LDL liposomes through remodeling, improving intrinsic antioxidant capacity, environmental pH tolerance, and ionic strength resistance.

[0075] 7. Analysis of gastrointestinal digestive characteristics and bioactive substance delivery behavior Using an in vitro simulated digestion model (oral-gastric-intestinal stage), particle morphology evolution was observed using confocal laser scanning microscopy (CLSM), and the in vitro release rate and bioavailability of curcumin after encapsulation were determined. CLSM results are as follows: Figure 11As shown in A and B, before digestion, both Comparative Example 1 and Example 1 exhibited scattered, bright yellow fluorescent spots, with clear spherical structures of green lipids and red proteins. During the gastric phase, the particles in Comparative Example 1 began to show loose structure and partial aggregation, with uneven distribution of red and green fluorescent signals, indicating initial separation at the lipid-protein interface. Upon entering the intestinal phase, the structure of Comparative Example 1 further depolymerized, and the dispersion of the fluorescent signal significantly increased, indicating rapid lipolysis of the particles under the action of bile salts and pancreatic lipase, forming a mixed micelle structure. In contrast, Example 1 maintained a more intact particle morphology in the gastric phase, and its depolymerization rate in the intestinal phase was slower than that of Comparative Example 1, indicating that the phytosterol-reconstructed sample had better digestive stability.

[0076] Quantitative analysis of curcumin release rates in different delivery systems revealed the decisive improvement in bioavailability and delivery kinetics of encapsulated active ingredients by phytosterol remodeling. For example... Figure 11 As shown in C and 11D, during the entire gastrointestinal digestion process, the final cumulative release rate of the delivery system of Example 1 was significantly higher than that of the preparation systems of Comparative Examples 1, 3, and 4. This indicates that phytosterol remodeling not only protects the active substances but also improves their overall bioavailability. During the gastric digestion stage, the release rate of curcumin in Example 1 was much lower than that of Comparative Examples 1, 3, and 4, indicating that Example 1 achieved strong protection and leakage prevention in the stomach. Upon entering the intestinal digestion stage, the system of Example 1 exhibited the dual advantages of sustained release and accelerated release: its release rate increased steadily, and the final intestinal stage release rate and cumulative release effect of curcumin were both the best.

[0077] 8. Ability to inhibit cholesterol micelle formation in vitro Experimental results of cholesterol micellization are as follows Figure 12 As shown, compared with Comparative Example 1, Comparative Example 2 exhibited significantly higher cholesterol micellization rates at different incubation times (1 h, 3 h, 5 h), indicating that the lipid components released after structural disintegration are more likely to participate in the formation of mixed micelles, which may actually promote cholesterol absorption. Compared with Comparative Examples 1 and 2, although Comparative Examples 3 and 4 reduced cholesterol micellization accordingly after the addition of phytosterols, indicating that phytosterols can inhibit cholesterol absorption, their effect was not as good as that of Example 1. Although Examples 2, 3, and 4 showed good inhibitory ability on cholesterol micellization formation, compared with them, the cholesterol micellization rate of Example 1 reached the lowest value at all time points and remained at a stable low value over time. This key result directly proves that the incorporation of phytosterols not only reversed the absorption-promoting tendency caused by decholesterol but also actively enhanced the ability of phytosterols to inhibit cholesterol micellization through in-situ incorporation.

[0078] 9. Analysis of Cholesterol Absorption Inhibition in Caco-2 Cells The inhibitory effects of Example 1 and Comparative Example 1 on the absorption of fluorescently labeled cholesterol (BODIPY-cholesterol) were determined using a Caco-2 cell monolayer model. Figure 13 As shown in Figures A and 13B, compared with the basal absorption control group, the positive control drug ezetimibe exhibited the strongest inhibitory effect, with an inhibition rate 2.0 times that of the control group. Comparative Example 1 showed some inhibitory ability, 1.29 times that of the control group; while Example 1 showed a significantly improved inhibitory effect, reaching 1.61 times that of the control group, with an efficacy of approximately 80% of the positive control drug. The inhibitory efficacy of Example 1 was approximately 25% higher than that of Comparative Example 1. This is consistent with the excellent inhibitory ability of rLDL shown in the aforementioned in vitro cholesterol micellization inhibition assay.

[0079] The promoting effect of Example 1 and Comparative Example 1 on the efflux of fluorescently labeled cholesterol (BODIPY-cholesterol) was determined using RAW264.7 cells. Figure 14 As shown in A and 14B, the positive control group, ezetimibe, exhibited a stronger inhibitory effect compared to the control group with basal cholesterol efflux. Comparative Example 1 did not demonstrate the ability to promote cholesterol efflux; however, Example 1 enhanced macrophage cholesterol efflux, achieving an efflux rate of 43.09%, which was superior to ezetimibe. These cellular results indicate that phytosterol replacement of cholesterol can effectively improve cholesterol metabolism by optimizing lipoprotein structure.

[0080] 10. Analysis of the inhibitory effect of RAW264.7 cells on foaming. Figure 15 The results showed that Figure 15 (A) Pathological image of foaming RAW264.7 cells stained with Oil Red chromatogram. After constructing a foam cell model using RAW264.7 macrophages, the number of Oil Red O positive lipid droplets in the cells of the natural LDL group was significantly increased and the lipid droplet area was expanded, indicating that it can promote lipid accumulation in macrophages and aggravate the foaming phenotype. However, after treatment with rLDL obtained in Example 1, the number of red lipid droplets in the cells was significantly reduced and the Oil Red O positive area was significantly reduced, which was significantly lower than that of the natural LDL group and the ezetimibe group. This indicates that after cholesterol removal and phytosterol embedding and reconstruction, rLDL can effectively inhibit foaming of RAW264.7 cells. Figure 15 (B) Further results from RAW264.7 cell viability analysis showed that neither LDL nor rLDL exhibited significant cytotoxicity in the range of 25–400 μg / mL, indicating that their foaming inhibition was not caused by cell damage or a decrease in cell number. Figure 15(C) Further quantitative analysis of the foaming effect showed that the natural LDL group in Example 1 exacerbated the foaming of RAW264.7 cells, but the rLDL inhibition rate in Example 1 was significantly reduced to approximately 0.73. Combined with the phenotypic changes shown in Figure A, this indicates that the example significantly reduced the degree of foaming. The ezetimibe group had the lowest inhibition rate, serving as a standard drug control and demonstrating the effectiveness of the experimental system. It also showed that Example 1 has good intervention potential. Based on the aforementioned cholesterol efflux results, it can be inferred that rLDL's reduction of lipid droplet accumulation may be related to its promotion of cholesterol efflux. The above results indicate that rLDL has the effect of reducing macrophage lipid deposition and inhibiting foaming at the cellular level, which can further support its application value in cholesterol metabolism regulation-related products.

[0081] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The embodiments and features described in these embodiments can be arbitrarily combined without conflict. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. A method for preparing egg yolk low-density lipoprotein biomimetic liposomes based on structural remodeling, characterized in that: The biomimetic liposome uses egg yolk low-density lipoprotein with endogenous cholesterol removed as its structural framework and is formed by structural reconstruction through the embedding of phytosterol molecules into the phospholipid monolayer and lipid core interface. The biomimetic liposome has the function of regulating cholesterol metabolism. The preparation method includes the following steps: S1. Extraction and purification of egg yolk low-density lipoprotein: Egg yolk low-density lipoprotein was obtained by centrifugation from egg yolk, purified by ultracentrifugation, and dispersed in neutral phosphate buffer. S2. Selective cholesterol removal treatment: After adjusting the pH value of the egg yolk low-density lipoprotein dispersion obtained in step S1, β-cyclodextrin is added; the mixture is ultrasonically treated, and then the supernatant is collected by centrifugation to obtain a cholesterol-free egg yolk low-density lipoprotein dispersion. S3. Phytosterol embedding and structural reconstruction: Add an ethanol solution containing phytosterols to the cholesterol-free egg yolk low-density lipoprotein dispersion obtained in step S2, incubate, and perform dialysis purification to obtain the biomimetic liposomes.

2. The production method according to claim 1, characterized by, In step S2, the pH value is adjusted to 8.0-10.0, and the molar ratio of β-cyclodextrin to cholesterol in egg yolk low-density lipoprotein is (0.8-1.2):

1.

3. The preparation method according to claim 1, characterized in that, In step S2, the ultrasonic treatment conditions are: 40W-100W, ice bath cooling, ultrasonic treatment for 5-15 minutes, performed in a pulse mode with 2-3 seconds of operation and 3-4 seconds of intermittent operation, and centrifugation conditions of 4°C and 6000-10000 r / min for 30-60 minutes. After step S2, the cholesterol content in the egg yolk low-density lipoprotein dispersion is reduced by more than 70% compared with that of natural egg yolk low-density lipoprotein.

4. The production method according to claim 1, characterized by, In step S3, the ethanol solution of phytosterols is added dropwise to the continuously stirred cholesterol-free egg yolk low-density lipoprotein dispersion at a rate of 1-1.5 mL / hour, and the concentration of the ethanol solution of phytosterols is 20-30 mg / mL.

5. The method of claim 1, wherein, In step S3, the molar ratio of the phytosterol to the cholesterol removed in step S2 is (1.0-1.5):

1.

6. The method of claim 1, wherein, In step S3, incubate at 37°C in the dark for 1-3 hours.

7. The preparation method according to claim 1, characterized in that, In step S3, dialysis purification is performed using a dialysis bag with a molecular weight cutoff of 10 kDa, and dialysis with phosphate buffer at pH 7.4 for 24-48 hours.

8. The method of any one of claims 1-7, wherein, The biomimetic liposomes obtained by the method are spherical nanoparticles with an average particle size ≤120nm and a polydispersity index of less than 0.

5. The generalized polarization value corresponding to the film's orderliness is ≥0.

5.

9. The use of the biomimetic liposomes obtained by the preparation method according to any one of claims 1-7 in the preparation of functional delivery materials, health foods or dietary supplements for regulating cholesterol metabolism.

10. The use of biomimetic liposomes obtained by the preparation method according to any one of claims 1-7 in drug delivery materials.

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