Curcumin-based multilayer modified nanoliposome and preparation method thereof

Abstract

The application discloses a curcumin-based multilayer modified nano-liposome and a preparation method thereof. The method assembles a mitochondrial specific ligand hydrophobicization (3-propylcarboxyl) triphenylphosphonium bromide, a lysosome pH responsive substance histidine, a TLR4 / CD44 targeting ligand hyaluronic acid and a polyelectrolyte complex whey protein nanofiber and pectin from the inside to the outside layer by layer to the outer layer of a conventional nano-liposome to construct a multilayer modified nano-liposome for embedding curcumin. The curcumin-based multilayer modified nano-liposome has high drug embedding rate and physical stability, stronger mitochondrial targeting capacity and stronger anti-tumor capacity, can better overcome multiple physiological barriers of the body and realize efficient targeted delivery of curcumin to tumor cell mitochondria.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery carriers and relates to a curcumin-based multilayer modified nanoliposome and its preparation method. Background Technology

[0002] Effective drug delivery within the body and efficient accumulation in tumor tissue are key factors in cancer treatment. Nanoparticle drug delivery systems are a promising strategy for cancer treatment, including lipid nanoparticles (such as solid lipid nanoparticles, nanostructured lipid carriers, and liposomes), polymer nanoparticles, micelles, nanogels, gold, and mesoporous silica nanoparticles (Maleki Dizaj S, Alipour M, Dalir Abdolahinia E, et al. Curcumin nanoformulations: Beneficial nanomedicine against cancer[J]. Phytotherapy Research, 2022, 36(3):1156-1181.). Nanoliposomes, as an effective nanodrug delivery carrier, have been widely used for the delivery of anticancer drugs. Nanoliposomes can enhance passive targeting of tumor tissues by improving permeability and retention effects. However, multiple physiological barriers, insufficient cellular uptake, and poor tumor targeting severely limit cancer treatment based on nanoliposome drug delivery systems. Developing multifunctional nanodrug delivery systems for efficient anticancer treatment is urgently needed.

[0003] Currently, parenteral administration is the most common route of delivery for nanoliposomes, primarily via injection. However, injected drugs are easily cleared by the body's immune system, and frequent, prolonged injections can lead to decreased patient comfort and compliance. Compared to injection, gastrointestinal administration can maintain drug concentrations in the blood and prolong the duration of action. Furthermore, it allows for flexible dosage selection and self-administration, easily customizable to meet patient needs, making it the most accepted route of delivery. However, oral ingestion of nanoliposomes must overcome many challenges, including the gastrointestinal digestive barrier, small intestinal epithelial cell and tumor cell membrane barriers, endosome phagocytosis, and target tissue targeting (Sosnik A, Augustine R. Challenges in oral drug delivery of antiretrovirals and the innovative strategies to overcome them[J]. Advanced drug delivery reviews, 2016, 103:105-120.). Traditional nanoliposomes become highly unstable when exposed to endogenous chemicals and enzymes in the gastrointestinal environment, leading to premature leakage of active substances and difficulty in reaching target tissues (Nguyen TX, Huang L, Gauthier M, et al. Recent advances in liposome surface modification for oral drug delivery[J]. Nanomedicine,2016,11(9):1169-1185.). Although surface modification with biopolymers can improve the stability of nanoliposomes in biological systems to some extent, their cellular uptake and target tissue targeting capabilities remain unsatisfactory (Aguilar-Pérez KM, Avilés-Castrillo JI, Medina DI, et al. Insight into nanoliposomes as smartnanocarriers for greening the 21st century biomedical settings[J]. Frontiers in Bioengineering and Biotechnology,2020,8:579536.). Therefore, it is necessary to take targeted measures to overcome the multiple physiological barriers of the body. Summary of the Invention

[0004] The present invention aims to provide a curcumin-based multilayer modified nanoliposome with highly efficient targeting of mitochondria in liver cancer cells and enhanced anti-tumor activity, as well as its preparation method. This method involves assembling mitochondrial-specific ligands (hydrophobicated (3-propanoyl)triphenylphosphine bromide), lysosomal pH-responsive substances (histidine), TLR4 / CD44 targeting ligands (hyaluronic acid), and polyelectrolyte complexes (whey protein isolate nanofibers and pectin) layer by layer onto the outer layer of a nanoliposome. The resulting curcumin-based multilayer modified nanoliposomes can overcome multiple physiological barriers, achieving highly efficient targeted delivery of curcumin to the mitochondria of tumor cells.

[0005] The technical solution to achieve the objective of this invention is as follows:

[0006] A method for preparing curcumin-based multilayer modified nanoliposomes, employing a thin-film dispersion-ultrasound-assisted combined electrostatic self-assembly method, specifically includes the following steps:

[0007] (1) Preparation of hydrophobic (3-propanoyl)triphenylphosphine bromide modified nanoliposomes:

[0008] Curcumin and soybean lecithin were dissolved in chloroform at a mass ratio of 1:45. Soybean lecithin and hydrophobic (3-propanoyl)triphenylphosphine bromide were added to the above solution at a mass ratio of 1:0.5-4. The mixture was thoroughly mixed, and the solution was rotary evaporated under water bath heating to form a film. The film was dried under nitrogen to remove the solvent. Then, phosphate buffer was added, and the mixture was stirred at room temperature until it swelled completely and the film detached. The film was then sonicated, filtered through a filter membrane for sterilization and granulation to obtain a solution of hydrophobic (3-propanoyl)triphenylphosphine bromide modified nanoliposomes.

[0009] (2) Modification with histidine-hyaluronic acid complex:

[0010] The hydrophobic (3-propanoyl)triphenylphosphine bromide-modified nanoliposome solution was added dropwise to an equal volume of histidine-hyaluronic acid complex solution at a mass ratio of 1:0.5-4 to the histidine-hyaluronic acid complex. The mixture was stirred while adding the solution and thoroughly mixed to obtain the histidine-hyaluronic acid complex-modified nanoliposome solution.

[0011] (3) Modification of whey protein isolate nanofibers:

[0012] The histidine-hyaluronic acid complex modified nanoliposome solution was added dropwise to an equal volume of whey protein isolate nanofiber solution at a mass ratio of 1:0.5 to 4. The mixture was stirred while adding the solution and thoroughly mixed to obtain a whey protein isolate nanoliposome solution modified with whey protein isolate nanofiber.

[0013] (4) Pectin modification:

[0014] The whey protein isolate nanofibers and pectin were mixed at a mass ratio of 1:0.5 to 4. The whey protein isolate nanofiber modified nanoliposome solution was added dropwise to an equal volume of pectin solution while stirring. After thorough mixing, curcumin-based multilayer modified nanoliposomes were obtained.

[0015] Preferably, in step (1), the phospholipid is selected from soybean phospholipid, soybean lecithin, egg yolk lecithin or sunflower seed phospholipid.

[0016] Preferably, in step (1), the pH of the phosphate buffer solution is 7.4.

[0017] Preferably, in step (1), the water bath heating temperature is 40°C.

[0018] Preferably, in step (1), the pore size of the filter membrane is 0.22 μm.

[0019] Preferably, in step (1), the ultrasonic power is 200W and the ultrasonic time is 5min.

[0020] Preferably, in step (1), the mass ratio of soybean lecithin to hydrophobic (3-propanoyl)triphenylphosphine bromide is 1:1.

[0021] Preferably, in step (2), the mass ratio of hydrophobic (3-propanoyl)triphenylphosphine bromide to histidine-hyaluronic acid complex is 1:1.

[0022] Preferably, in step (3), the mass ratio of histidine-hyaluronic acid complex to whey protein nanofiber is 1:2.

[0023] Preferably, in step (4), the mass ratio of whey protein isolate nanofibers to pectin is 1:1.

[0024] This invention utilizes whey protein isolate nanofibers and pectin to surface-modify nanoliposomes, preventing phospholipid degradation under low pH and enzymatic conditions in the gastrointestinal tract and improving the stability and mucosal adhesion of nanoliposomes in vivo. Hyaluronic acid is used to target specific receptors on intestinal epithelial cells and tumor cell membranes, promoting transmembrane transport of nanoliposomes. Furthermore, hyaluronidase in tumor cell cytoplasm can degrade the hyaluronic acid layer, changing the surface charge of nanoliposomes from negative to positive, exposing internal mitochondrial-specific ligands and promoting mitochondrial targeting. The imidazole group in histidine is reversibly protonated / deprotonated to construct a pH-responsive intelligent nanodelivery carrier, endowing nanoliposomes with multiple functions, such as promoting rapid drug release, enhancing formulation penetration, and improving lysosomal escape ability. The lipophilic cation (3-propanecarboxy)triphenylphosphine bromide is used to enhance the mitochondrial targeting ability of the nanoliposome carrier, promoting drug accumulation within the mitochondria and exerting antitumor activity.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) This invention uses a thin film dispersion-ultrasound-assisted electrostatic self-assembly method to prepare multilayer modified nanoliposomes. The modified layer consists of hydrophobic (3-propanoyl)triphenylphosphine bromide, histidine-hyaluronic acid complex, whey protein nanofibers and pectin from the inside out. The hydrophobic (3-propanoyl)triphenylphosphine bromide and histidine-hyaluronic acid complex, the histidine-hyaluronic acid complex and whey protein nanofibers, and the whey protein nanofibers and pectin are all combined through electrostatic interaction. The method is simple, green and environmentally friendly.

[0027] (2) The curcumin-based multilayer modified nanoliposomes prepared by this invention have uniform particle size, good stability and high encapsulation rate;

[0028] (3) The curcumin-based multilayer modified nanoliposomes prepared in this invention have the ability to highly enrich, rapidly penetrate and efficiently target the mitochondria of liver cancer cells.

[0029] (4) Compared with conventional single-layer / double-layer modification, the curcumin-based multilayer modified nanoliposomes prepared in this invention can effectively overcome multiple physiological barriers of the body, achieve efficient drug delivery and precise release, enhance the oral therapeutic effect of curcumin, and ultimately achieve safe and precise treatment of liver cancer. Attached Figure Description

[0030] Figure 1 The image shows the screening results for the concentration of hydrophobic (3-propanoyl)triphenylphosphine bromide in Example 1.

[0031] Figure 2 This is a graph showing the screening results of the concentration of the histidine-hyaluronic acid complex in Example 2.

[0032] Figure 3 This is a graph showing the screening results of whey protein isolate nanofiber concentration in Example 3.

[0033] Figure 4 This is a graph showing the screening results of pectin concentration in Example 4.

[0034] Figure 5 The particle size distribution of the nanoliposomes prepared in Comparative Example 1 and Example 5 is shown.

[0035] Figure 6 Potentiograms of nanoliposomes prepared in Comparative Example 1 and Example 5.

[0036] Figure 7 The diagram shows the encapsulation efficiency and loading rate of the nanoliposomes prepared in Comparative Example 1 and Example 5.

[0037] Figure 8 The graph shows the change in particle size of the nanoliposomes prepared in Comparative Example 1 and Example 5 after heat treatment for different times.

[0038] Figure 9 The graph shows the changes in mitochondrial membrane potential of the nanoliposomes prepared in Comparative Example 1 and Example 5.

[0039] Figure 10 The image shows the mitochondrial membrane potential fluorescence of the nanoliposomes prepared in Comparative Example 1 and Example 5.

[0040] Figure 11 The apoptosis morphology of the nanoliposomes prepared in Comparative Example 1 and Example 5 is shown. Detailed Implementation

[0041] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0042] In the following examples, the preparation of hydrophobic (3-propanoyl)triphenylphosphine bromide is described in reference [Guzman-Villanueva D, Mendiola MR, Nguyen HX, et al. Influence of triphenylphosphonium (TPP) cation hydrophobization with phospholipids on cellular toxicity and mitochondrial selectivity[J]. SOJ Pharm Pharm Sci, 2015, 2(1): 1-9.], and the preparation of histidine-hyaluronic acid complex is described in reference [LI J, YANG Y, YU Y, et al.]. nanoplatform functionalized with histidine modified oligomeric hyaluronic acid as an effective vehicle for the anticancer drug [J]. Journal of Materials Chemistry B, 2018. ], Reference for the preparation of whey protein nanofibers [Xu X, Zhang Z, Zhu J, et al. Whey Protein Isolate Nanofibers Prepared by Subcritical Water Stabilized High Internal Phase Pickering Emulsion to Deliver Curcumin[J]. Foods, 2022, 11(11): 1625. ]

[0043] Example 1

[0044] Curcumin and soybean lecithin were dissolved in chloroform at a mass ratio of 1:45. Hydrophobic (3-propanoyl)triphenylphosphine bromide was added to the solution at mass ratios of soybean lecithin to hydrophobic (3-propanoyl)triphenylphosphine bromide of 1:0, 1:0.5, 1:1, 1:2, and 1:4, and the mixture was thoroughly mixed. The mixture was then evaporated in a water bath at 40°C to form a film, which was then thoroughly dried under nitrogen to remove the solvent. 30 mL of phosphate buffer (pH = 7.4) preheated to 40°C was added, and the mixture was stirred at room temperature until it swelled and detached. The film was then sonicated (200 W, 5 min) and filtered through a 0.22 μm filter for sterilization and granulation to obtain nanoliposomes modified with different degrees of hydrophobic (3-propanoyl)triphenylphosphine bromide.

[0045] The particle size and potential of nanoliposomes modified with varying degrees of hydrophobicity (3-propanoyl)triphenylphosphine bromide were determined using a Malvern particle size analyzer (ES90 Nano). Figure 1 As shown, with the increase of the amount of hydrophobic (3-propanoic)triphenylphosphine bromide added, although the particle size of the nanoliposomes did not change much, the potential gradually increased. This is due to the positively charged hydrophobic (3-propanoic)triphenylphosphine bromide binding to the surface of the nanoliposomes. Furthermore, when the mass ratio of soybean lecithin to hydrophobic (3-propanoic)triphenylphosphine bromide was 1:1, the potential increased sharply, indicating that the binding effect of hydrophobic (3-propanoic)triphenylphosphine bromide with the nanoliposomes was optimal at this time.

[0046] Example 2

[0047] (1) Curcumin and soybean lecithin were dissolved in chloroform at a mass ratio of 1:45. Soybean lecithin and hydrophobic (3-propanoyl)triphenylphosphine bromide were added to the above solution at a mass ratio of 1:1 and mixed thoroughly. The mixture was then evaporated in a water bath at 40°C to form a film. The film was then thoroughly dried with nitrogen to remove the solvent. 30 mL of phosphate buffer (pH = 7.4) preheated to 40°C was added. The mixture was stirred at room temperature and allowed to swell until the film detached. The film was then sonicated (200 W, 5 min) and filtered through a 0.22 μm filter to remove bacteria and granulate, thus obtaining hydrophobic (3-propanoyl)triphenylphosphine bromide modified nanoliposomes.

[0048] (2) The hydrophobic (3-propanoyl)triphenylphosphine bromide modified nanoliposome solution was added dropwise to an equal volume of histidine-hyaluronic acid complex solution according to the mass ratio of hydrophobic (3-propanoyl)triphenylphosphine bromide to histidine-hyaluronic acid complex solution of 1:0, 1:0.5, 1:1, 1:2 and 1:4 respectively. The mixture was stirred while adding the solution and thoroughly mixed to obtain nanoliposomes modified with histidine-hyaluronic acid complex to different degrees.

[0049] The particle size and potential of nanoliposomes modified with histidine-hyaluronic acid complexes to varying degrees were determined using a Malvern particle size analyzer (ES90 Nano). Figure 2 As shown, the nanoliposomes exhibited the smallest particle size (236.93 nm) when the mass ratio of hydrophobic (3-propanoyl)triphenylphosphine bromide to the histidine-hyaluronic acid complex was 1:1. Furthermore, compared to the unmodified nanoliposomes, the potential of the modified nanoliposomes decreased from -32.03 mV to -39.63 mV and began to plateau. This indicates that the histidine-hyaluronic acid complex was successfully modified onto the surface of the nanoliposomes, and that the optimal state was achieved at a mass ratio of 1:1 for the hydrophobic (3-propanoyl)triphenylphosphine bromide to the histidine-hyaluronic acid complex.

[0050] Example 3

[0051] (1) Curcumin and soybean lecithin were dissolved in chloroform at a mass ratio of 1:45. Soybean lecithin and hydrophobic (3-propanoyl)triphenylphosphine bromide were added to the above solution at a mass ratio of 1:1 and mixed thoroughly. The mixture was then evaporated in a water bath at 40°C to form a film. The film was then thoroughly dried with nitrogen to remove the solvent. 30 mL of phosphate buffer (pH = 7.4) preheated to 40°C was added. The mixture was stirred at room temperature and allowed to swell until the film detached. The film was then sonicated (200 W, 5 min) and filtered through a 0.22 μm filter to remove bacteria and granulate, thus obtaining hydrophobic (3-propanoyl)triphenylphosphine bromide modified nanoliposomes.

[0052] (2) The hydrophobic (3-propanoyl)triphenylphosphine bromide-histidine-hyaluronic acid complex was added dropwise to an equal volume of histidine-hyaluronic acid complex solution at a mass ratio of 1:1. The mixture was stirred while adding the solution and thoroughly mixed to obtain the histidine-hyaluronic acid complex-modified nanoliposomes.

[0053] (3) The histidine-hyaluronic acid complex modified nanoliposome solution was added dropwise to an equal volume of whey protein isolate nanofiber solution according to the mass ratio of histidine-hyaluronic acid complex to whey protein isolate nanofiber solution as 1:0, 1:0.5, 1:1, 1:2 and 1:4 respectively. The mixture was stirred while adding the solution and thoroughly mixed to obtain nanoliposomes modified with whey protein isolate nanofiber to different degrees.

[0054] The particle size and potential of nanoliposomes modified with whey protein isolate nanofibers to varying degrees were determined using a Malvern particle size analyzer (ES90 Nano). Figure 3 As shown, when the mass ratio of histidine-hyaluronic acid complex to whey protein isolate nanofibers was 1:2, the particle size (460.63 nm) and potential (-28.76 mV) of the nanoliposomes began to plateau. The increase in particle size and potential was due to the binding of positively charged whey protein isolate nanofibers to the surface of negatively charged nanoliposomes, and this electrostatic binding effect was best at a mass ratio of histidine-hyaluronic acid complex to whey protein isolate nanofibers of 1:2.

[0055] Example 4

[0056] (1) Curcumin and soybean lecithin were dissolved in chloroform at a mass ratio of 1:45. Soybean lecithin and hydrophobic (3-propanoyl)triphenylphosphine bromide were added to the above solution at a mass ratio of 1:1 and mixed thoroughly. The mixture was then evaporated in a water bath at 40°C to form a film. The film was then thoroughly dried with nitrogen to remove the solvent. 30 mL of phosphate buffer (pH = 7.4) preheated to 40°C was added. The mixture was stirred at room temperature and allowed to swell until the film detached. The film was then sonicated (200 W, 5 min) and filtered through a 0.22 μm filter to remove bacteria and granulate, thus obtaining hydrophobic (3-propanoyl)triphenylphosphine bromide modified nanoliposomes.

[0057] (2) The hydrophobic (3-propanoyl)triphenylphosphine bromide-histidine-hyaluronic acid complex was added dropwise to an equal volume of histidine-hyaluronic acid complex solution at a mass ratio of 1:1. The mixture was stirred while adding the solution and thoroughly mixed to obtain the histidine-hyaluronic acid complex-modified nanoliposomes.

[0058] (3) The histidine-hyaluronic acid complex and whey protein nanofibers were added dropwise to an equal volume of whey protein nanofiber solution at a mass ratio of 1:1. The mixture was stirred while adding the mixture until it was fully mixed to obtain whey protein nanofiber modified nanoliposomes.

[0059] (4) The whey protein isolate nanofibers modified with pectin were added dropwise to an equal volume of pectin solution at mass ratios of 1:0, 1:0.5, 1:1, 1:2, and 1:4, respectively. The mixture was stirred while adding the solution and thoroughly mixed to obtain nanoliposomes modified with pectin to different degrees.

[0060] The particle size and potential of pectin-modified nanoliposomes with different degrees of modification were determined using a Malvern particle size analyzer (ES90 Nano). Figure 4 As shown, with the increase of pectin addition, the particle size of the nanoliposomes further increases, while the potential shows a decreasing trend. This is due to the anti-electrostatic interaction between the negatively charged pectin and the positively charged whey protein isolate nanofibers. When the mass ratio of whey protein isolate nanofibers to pectin is 1:1, the electrostatic interaction between the two reaches a relatively balanced state, and the particle size and potential of the nanoliposomes are also relatively stable.

[0061] Comparative Example 1

[0062] Preparation of curcumin-based conventional nanoliposomes

[0063] Curcumin and soybean lecithin were dissolved in chloroform at a mass ratio of 1:45 and mixed thoroughly. The mixture was then evaporated in a water bath at 40°C to form a film, which was then thoroughly dried with nitrogen to remove the solvent. 30 mL of phosphate buffer (pH = 7.4) preheated to 40°C was added, and the mixture was stirred at room temperature until it swelled and detached from the film. The film was then sonicated (200 W, 5 min) and filtered through a 0.22 μm filter to remove bacteria and granulate, thus obtaining curcumin-based conventional nanoliposomes.

[0064] Example 5

[0065] Preparation of curcumin-based multilayer modified nanoliposomes

[0066] (1) Curcumin and soybean lecithin were dissolved in chloroform at a mass ratio of 1:45. Soybean lecithin and hydrophobic (3-propanoyl)triphenylphosphine bromide were added to the above solution at a mass ratio of 1:1 and mixed thoroughly. The mixture was then evaporated in a water bath at 40°C to form a film. The film was then thoroughly dried with nitrogen to remove the solvent. 30 mL of phosphate buffer (pH = 7.4) preheated to 40°C was added. The mixture was stirred at room temperature and allowed to swell until the film detached. The film was then sonicated (200 W, 5 min) and filtered through a 0.22 μm filter to remove bacteria and granulate, thus obtaining hydrophobic (3-propanoyl)triphenylphosphine bromide modified nanoliposomes.

[0067] (2) The hydrophobic (3-propanoyl)triphenylphosphine bromide modified nanoliposomes were added dropwise to an equal volume of histidine-hyaluronic acid complex solution at a mass ratio of 1:1 to the hydrophobic (3-propanoyl)triphenylphosphine bromide modified nanoliposomes. The mixture was stirred while adding the nanoliposomes and thoroughly mixed to obtain the histidine-hyaluronic acid complex modified nanoliposomes.

[0068] (3) The histidine-hyaluronic acid complex and whey protein nanofibers were added dropwise to an equal volume of whey protein nanofiber solution at a mass ratio of 1:2. The mixture was stirred while adding the mixture until it was fully mixed to obtain whey protein nanofiber modified nanoliposomes.

[0069] (4) Add the whey protein isolate nanofibers modified with pectin dropwise to an equal volume of pectin solution at a mass ratio of 1:1. Stir while adding the nanofibers and mix thoroughly to obtain pectin-modified nanoliposomes, namely curcumin-based multilayer modified nanoliposomes.

[0070] Example 6

[0071] Performance test

[0072] Preparation and characterization of curcumin-based conventional nanoliposomes and curcumin-based multilayer modified nanoliposomes. Through repeated experiments, the curcumin-based multilayer modified nanoliposomes prepared in this invention possess suitable particle size, potential, drug loading, and good stability. Their apoptosis-inducing effect on liver cancer cells is significantly superior to that of conventional curcumin-based nanoliposomes. The relevant experimental results are as follows:

[0073] 1. Particle size and potential determination

[0074] The particle size and potential of curcumin-based conventional nanoliposomes and curcumin-based multilayer modified nanoliposomes were determined using a Malvern particle size analyzer (ES90 Nano). Samples were diluted 10-fold with distilled water, thoroughly mixed, and then the particle size and potential were determined using the Malvern particle size analyzer (ES90 Nano). Figure 5 and Figure 6 As shown, the average particle size of the traditional curcumin-based nanoliposomes was 208.8 nm, and the potential was -58.9 mV; the average particle size of the curcumin-based multilayer modified nanoliposomes was 308.3 nm, and the potential was -25.2 mV. The results indicate that the particle size of the modified nanoliposomes increased and the absolute value of the potential decreased due to the different modification layers binding to the surface of the nanoliposomes.

[0075] 2. Measurement of embedding rate and loading rate

[0076] The encapsulation efficiency and loading rate of curcumin in traditional nanoliposomes and multilayer modified nanoliposomes were determined by ultraviolet spectrophotometry. 2 mL of each sample was taken and diluted to 10 mL with anhydrous ethanol. After ultrasonic demulsification for 20 min, the curcumin concentration in the sample was detected at 425 nm using an ultraviolet spectrophotometer, thereby calculating the total mass of curcumin (m³). 总 ), and the total mass (m) of the drug and excipients.

[0077] Take another 2 mL of sample and centrifuge at 4500 rpm for 15 min to remove free curcumin. Dilute the supernatant to 10 mL with anhydrous ethanol, and sonicate for 20 min to break the emulsion. Detect the curcumin concentration in the sample at 425 nm using a UV spectrophotometer to calculate the mass (m) of the encapsulated curcumin. 包 ).

[0078] The encapsulation ratio (EE, %) and load factor (DL, %) are calculated using the following formulas:

[0079]

[0080]

[0081] like Figure 7 As shown, traditional nanoliposomes achieved an encapsulation rate of 72.24% and a loading rate of 1.59% for curcumin; while multilayer modified nanoliposomes achieved an encapsulation rate of 95.56% and a loading rate of 3.14% for curcumin. Compared with traditional nanoliposomes, the encapsulation rate and loading rate of curcumin in the modified nanoliposomes increased by 21.34% and 1.50%, respectively. This may be due to the binding of free curcumin to different modified layers or its encapsulation between different modified layers.

[0082] 3. Thermal stability test

[0083] Curcumin-based conventional nanoliposomes and curcumin-based multilayer modified nanoliposomes were placed in an 80℃ water bath, and samples were extracted at different time points (0, 5, 15, 30, 45, and 60 min) to determine the particle size. Figure 8 It can be seen that the particle size of traditional curcumin-based nanoliposomes decreases significantly over time, which may be due to the cleavage of the phospholipid bilayer of nanoliposomes under high temperature conditions for a long time; while the particle size of curcumin-based multilayer modified nanoliposomes remains relatively stable, indicating that the modified nanoliposomes have a certain resistance to high temperature.

[0084] 4. Evaluation of antioxidant activity after oral, gastrointestinal digestion

[0085] (1) Determination of the DPPH free radical scavenging capacity of samples after oral and gastrointestinal digestion

[0086] Take 1 mL of conventional curcumin-based nanoliposomes and curcumin-based multilayer modified nanoliposomes after simulating oral and intestinal digestion, respectively, dilute them 10 times with anhydrous ethanol, centrifuge at 5000 r / min for 5 min, take 2 mL of supernatant and mix it with 2 mL of freshly prepared 0.1 mmol / L DPPH ethanol solution, react at room temperature in the dark for 30 min, and then measure the absorbance at 517 nm using a UV spectrophotometer.

[0087] Calculate using the following formula:

[0088]

[0089] Where A0 is the absorbance value of the control group and A1 is the absorbance value of the sample group.

[0090] (2) Determination of total antioxidant capacity of samples after oral and gastrointestinal digestion

[0091] Acetate buffer (300 mmol / L), FeCl3 solution (20 mmol / L), and TPTZ solution (10 mmol / L TPTZ dissolved in 40 mmol / L HCl) were mixed in a volume ratio of 10:1:1 (v / v / v) to obtain the FRAP working solution. 0.1 mL of ferrous sulfate solutions of different concentrations (0.3-2.1 mmol / L) were added to 5 mL of the FRAP working solution, mixed well, and incubated at 37°C in the dark for 8 min. The absorbance was measured at 593 nm to plot a standard curve. The absorbance of the sample solutions (curcumin-based conventional nanoliposomes and curcumin-based multilayer modified nanoliposomes) after oral-gastric digestion was measured using the same method. The antioxidant capacity was expressed as the FeSO4·7H2O equivalent calculated based on the linear standard curve, i.e., 1 FRAP unit = 1 mmol / L FeSO4·7H2O.

[0092] Table 1 shows that after oral-gastric digestion, curcumin-based multilayer modified nanoliposomes exhibited a 3.44% higher DPPH free radical scavenging rate and a 0.0022 mmol / L higher total antioxidant capacity (FeSO4·7H2O equivalent) compared to conventional curcumin-based nanoliposomes. These results indicate that the addition of each modification layer enhanced the nanoliposomes' ability to overcome degradation in the oral-gastric region and its protective ability against curcumin.

[0093] Table 1. DPPH free radical scavenging capacity and total antioxidant capacity of samples after oral and gastrointestinal digestion.

[0094]

[0095] 5. Assessment of the ability to induce apoptosis in liver cancer cells

[0096] Hochest 33342 staining assay for apoptosis: Logarithmic growth phase HepG2 cells were subjected to a 1×10⁻⁶ staining assay. 5 Cells were seeded per well in 12-well plates. Culture medium containing 500 μL of 8 mg / mL curcumin-based conventional nanoliposomes and curcumin-based multilayer modified nanoliposomes were added to each well. After 24 h of incubation, the culture medium was discarded, and the cells were washed twice with PBS. 0.5 mL of 75% ethanol was added to each well, and the cells were fixed at 4°C for 10 min. The fixative was discarded, and the cells were washed three times with PBS. 0.5 mL of Hoechst 33342 staining solution (5 mg / L) was added to each well, and the cells were stained at 37°C in the dark for 5 min. The staining solution was discarded, and the cells were washed twice with PBS. 0.5 mL of PBS was added, and the apoptotic morphology of the cells was observed under a fluorescence microscope. Figure 9 and Figure 10 It can be seen that the JC-1 monomer in the curcumin-based multilayer modified nanoliposome group has higher absorbance and more and stronger green fluorescence, indicating that the modified liposomes can increase mitochondrial membrane permeability, reduce mitochondrial membrane potential, and induce apoptosis of more tumor cells.

[0097] 6. Effects on mitochondrial membrane potential and fluorescence imaging in liver cancer cells

[0098] HepG2 cells in logarithmic growth phase were fed at a rate of 1×10⁻⁶. 5Cells were seeded per well in 12-well plates, and culture medium containing 500 μL of 8 mg / mL curcumin-based conventional nanoliposomes and curcumin-based multilayer modified nanoliposomes were added. After culturing for 24 h, the culture medium was discarded, and the cells were washed twice with PBS. Pre-prepared JC-1 staining solution (1 ml staining solution = 5 μL JC-1 + 800 μL ultrapure water + 200 μL 5× staining buffer) was added, and the cells were incubated at 37 °C for 20 min. The staining solution was discarded, and the cells were washed twice with JC-1 staining buffer (1×), and 2 mL of cell culture medium was added. The absorbance of JC-1 monomer (excitation wavelength 490 nm, emission wavelength 530 nm) and JC-1 polymer (excitation wavelength 425 nm, emission wavelength 590 nm) was measured using a microplate reader, and photographs were taken under a fluorescence microscope. Figure 11 It can be seen that the fluorescence quantity and intensity of the curcumin-based multilayer modified nanoliposome group are higher, and the tumor cell nuclei show shrinkage and fragmentation, indicating that the modified liposomes can promote tumor cell apoptosis.

Claims

1. A method for preparing curcumin-based multilayer modified nanoliposomes, characterized in that, Specifically, the steps include the following: (1) Preparation of hydrophobic (3-propanoyl)triphenylphosphine bromide modified nanoliposomes: Curcumin and soybean lecithin were dissolved in chloroform at a mass ratio of 1:

45. Soybean lecithin and hydrophobic (3-propanoyl)triphenylphosphine bromide were added to the above solution at a mass ratio of 1:

1. The mixture was thoroughly mixed, and the solution was rotary evaporated under water bath heating to form a film. The film was dried under nitrogen to remove the solvent. Then, phosphate buffer was added, and the mixture was stirred at room temperature until it swelled completely and the film detached. The film was then sonicated, filtered through a filter membrane for sterilization and granulation to obtain a solution of hydrophobic (3-propanoyl)triphenylphosphine bromide modified nanoliposomes. (2) Modification by histidine-hyaluronic acid complex: The hydrophobic (3-propanoyl)triphenylphosphine bromide-modified nanoliposome solution was added dropwise to an equal volume of histidine-hyaluronic acid complex solution at a mass ratio of 1:1 to the hydrophobic (3-propanoyl)triphenylphosphine bromide-modified nanoliposome solution. The mixture was stirred while adding the solution and thoroughly mixed to obtain the histidine-hyaluronic acid complex-modified nanoliposome solution. (3) Modification of whey protein isolate nanofibers: The histidine-hyaluronic acid complex and whey protein nanofibers were mixed at a mass ratio of 1:

2. The histidine-hyaluronic acid complex modified nanoliposome solution was added dropwise to an equal volume of whey protein nanofiber solution while stirring. After thorough mixing, the whey protein nanofiber modified nanoliposome solution was obtained. (4) Pectin modification: The whey protein isolate nanofibers and pectin were mixed at a mass ratio of 1:

1. The whey protein isolate nanofiber modified nanoliposome solution was added dropwise to an equal volume of pectin solution while stirring. After thorough mixing, curcumin-based multilayer modified nanoliposomes were obtained.

2. The preparation method according to claim 1, characterized in that, In step (1), the phospholipids are selected from soybean phospholipids, soybean lecithin, egg yolk lecithin or sunflower seed phospholipids.

3. The preparation method according to claim 1, characterized in that, In step (1), the pH of the phosphate buffer solution is 7.4, and the water bath heating temperature is 40℃.

4. The preparation method according to claim 1, characterized in that, In step (1), the pore size of the filter membrane is 0.22 μm.

5. The preparation method according to claim 1, characterized in that, In step (1), the ultrasonic power is 200W and the ultrasonic time is 5 min.

6. Curcumin-based multilayer modified nanoliposomes prepared by any one of the preparation methods according to claims 1 to 5.

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