Tumor-targeted luteoloside liposome loaded paclitaxel and preparation method thereof

Abstract

The invention belongs to the technical field of drug carriers, and particularly relates to tumor-targeted luteoloside liposome loaded paclitaxel and a preparation method thereof. According to the tumor targeted liposome, luteoloside and soya bean lecithin are used as membrane materials, a blank liposome (C-Blank) is prepared through a membrane hydration method and a high-pressure homogenization method, and a hydrophobic drug paclitaxel (PTX) is loaded to obtain a drug-loaded liposome (C-PTX). Wherein the luteoloside can significantly enhance the mechanical strength and thermodynamic stability of the lipid bilayer, and also can realize active tumor targeting through a glucose transporter GLUT1 mediated endocytosis pathway, and meanwhile, PTX is efficiently entrapped in a liposome hydrophobic core. The C-PTX liposome prepared by the invention is small in particle size, relatively good in endocytosis capability, good in blood long-circulation effect and drug delivery capability, good in stability and dispersity, high in drug encapsulation efficiency and beneficial to effective delivery and release of drugs. The oral bioavailability of PTX is improved, and C-PTX has an obvious inhibition effect on lung cancer cells. The drug loading system effectively overcomes the defects that a traditional paclitaxel preparation is poor in water solubility, large in system toxicity, insufficient in targeting performance and the like, the highly-uniform dispersion state and long-term storage stability of the drug loading system lay a foundation for industrial production, and the drug loading system shows important clinical application potential in the fields of precision medicine and transformation medicine.

Description

Technical Field

[0001] This invention belongs to the field of drug carrier technology, specifically relating to a method for preparing and applying paclitaxel-encapsulated luteolin liposomes targeting tumors. Background Technology

[0002] Liposomes are closed vesicles with biomembrane characteristics, primarily composed of phospholipids and cholesterol. This combination endows liposomes with excellent cellular compatibility, enabling them to interact naturally with cell membranes. Due to their superior biocompatibility and tissue affinity, liposomes have attracted widespread attention in drug delivery systems. Liposomes can encapsulate various drugs, including chemotherapy drugs, vaccines, and biomolecules, forming a protective carrier. This closed vesicle structure effectively isolates drugs from the external environment, preventing drug degradation in vivo and optimizing release characteristics. Targeted liposome drug delivery systems work by modifying the surface of liposomes with specific ligands, enabling them to precisely bind to receptors overexpressed on tumor cell membranes, thereby significantly increasing intracellular drug concentration. This strategy effectively enhances drug targeting and efficacy, showing great promise, particularly in cancer treatment. Targeted drug delivery systems utilize the interaction between specific receptors on tumor cell membranes and ligands modified on the liposome surface to achieve precise drug delivery. For example, many glucose transporters overexpressed on tumor cell membranes have a high affinity for carbohydrate molecules. Carbohydrates are not only essential components of cell membranes but also participate in the formation of glycoproteins and glycolipids, contributing to the maintenance of cell membrane structure and function. These glycan structures play crucial roles in enhancing membrane stability, preventing immune clearance, and participating in cell receptor recognition. Due to their excellent biocompatibility and specific targeting properties, carbohydrates have become a popular choice for drug carrier design. In recent years, the design of drug carriers using carbohydrate molecules as ligands has received widespread attention. For example, by combining glycosides with cholesterol, novel cholesterol derivatives can be formed for targeted drug delivery. These glycosylated drug carriers exhibit significant advantages, not only improving drug targeting efficacy but also enhancing their distribution characteristics in vivo. Glycosylated drug carriers demonstrate unique advantages in several aspects. First, they can improve the targeting efficiency of the carrier to specific cells, enabling drugs to act more precisely on tumor cells. Second, glycosylation can improve the biodistribution of drugs in vivo, ensuring that drugs reach target tissues more effectively. Furthermore, glycosylation can stabilize drug carriers, reduce degradation in the bloodstream, and prevent clearance by macrophages, thereby prolonging the drug's half-life. By reducing drug toxicity, glycosylated drug carriers can reduce damage to normal cells while ensuring therapeutic efficacy. Therefore, glycosylation-mediated targeted delivery strategies have become an active area of ​​research in drug delivery systems. Researchers are beginning to explore new carbohydrate molecules and their derivatives to further improve the effectiveness of targeted drug delivery. These studies not only provide new ideas for cancer treatment but also open up new directions for the treatment of other pathological conditions. Future research can focus on how to optimize the selection of carbohydrate molecules, improve targeting, and enhance drug release properties.

[0003] Liposomes, as an ideal drug delivery tool, play a crucial role in targeted drug delivery systems due to their structural biosimilarity and operational flexibility. By combining glycosylated molecules, researchers have successfully improved drug targeting and bioavailability, advancing targeted drug delivery technologies. These studies not only present new challenges to modern medicine but also offer rich possibilities for future treatments. In the ongoing exploration and development, research on glycosylated drug carriers will undoubtedly make significant contributions to improving therapeutic efficacy and reducing side effects, paving new paths for human health. Summary of the Invention

[0004] The present invention aims to provide a tumor-targeting liposome loaded with paclitaxel (C-PTX) constructed using luteolin and soybean lecithin as membrane materials, and its preparation method. The C-PTX provided by this invention avoids the water solubility defects of traditional paclitaxel (PTX) formulations during delivery, reduces systemic toxicity, and utilizes the GLUT1-mediated endocytosis pathway of luteolin to achieve precise tumor targeting, effectively protecting the stability of the drug in the circulatory system and promoting its efficient accumulation in tumor tissues. This drug delivery system significantly improves the tumor-selective delivery capability of paclitaxel, enhancing its anti-tumor efficacy and making it effective for the treatment of solid tumors such as lung cancer.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a tumor-targeting liposome loaded with PTX constructed using luteolin and soybean lecithin as membrane materials, comprising a liposome carrier and an active drug loaded in the liposome carrier; the active drug is PTX; the liposome carrier is a lipid bilayer constructed using luteolin and soybean lecithin as membrane materials; luteolin can significantly enhance the mechanical strength and thermodynamic stability of the lipid bilayer, and achieve active tumor targeting through the GLUT1-mediated endocytosis pathway; the PTX is efficiently encapsulated in the hydrophobic core of the liposome; the tumor-targeting luteolin liposome loaded with paclitaxel is prepared by thin-film hydration and high-pressure homogenization, including the following steps:

[0007] (1) Weigh out phospholipids, luteolin and PTX;

[0008] (2) Dissolve the above raw materials in an organic solvent and place them in a round-bottom flask;

[0009] (3) Remove the organic solvent in the solution by rotary evaporation to form a uniform lipid film on the bottle wall;

[0010] (4) Add ultrapure water to the round-bottom flask and treat it with an ultrasonic oscillator until a homogeneous dispersion system is obtained;

[0011] (5) A paclitaxel-loaded luteolin liposome suspension was obtained by high-pressure homogenization.

[0012] Preferably, the phospholipid in step (1) is selected from one or more of egg yolk lecithin, soybean lecithin, sodium dioleoylphosphatidylserine (DOPS), dioleoyl L-α-phosphatidylethanolamine (DOPE), 1,2-dioleoyl lecithin (DOPC), dipalmitoylphosphatidylcholine (DPPC), and distearate phosphatidylcholine (DSPC), and is preferably egg yolk lecithin, soybean lecithin, DOPC, and DPPC.

[0013] Preferably, in step (1), the molar ratio of phospholipids to luteolin is 4-10:1-6; and the molar ratio of phospholipids to paclitaxel is 4-10:1-6.

[0014] Preferably, the organic solvent in step (2) is selected from at least one of methanol, ethanol, chloroform, and dichloromethane.

[0015] Preferably, in step (2), the ratio of organic solvent to phospholipid is 50-400 mL: 1 g.

[0016] Preferably, the rotary evaporation temperature in step (3) is 40–70°C.

[0017] Preferably, in step (4), the ratio of ultrapure water to phospholipid is 30-100 mL: 1 g.

[0018] Preferably, the ultrasonic power in step (4) is 100-300W.

[0019] Preferably, the ultrasound time in step (4) is 1-10 minutes.

[0020] Preferably, the high-pressure homogenization pressure in step (5) is 150-400 MPa.

[0021] Preferably, the high-pressure homogenization cycle time in step (5) is 5-20 minutes.

[0022] This invention provides a method for preparing PTX-loaded liposomes using luteolin and soybean lecithin as membrane materials for tumor targeting, comprising the following steps: weighing a certain mass of phospholipids, luteolin, and PTX; dissolving the above raw materials in an organic solvent and then transferring them to a round-bottom flask; subsequently removing the organic solvent using a rotary evaporator; after a uniform lipid film forms on the flask wall, injecting ultrapure water into the system for hydration; and treating with an ultrasonic oscillator until a homogeneous dispersion is obtained; and finally, processing with a high-pressure homogenizer to obtain a PTX-loaded luteolin glycoside liposome suspension.

[0023] This invention innovatively integrates luteolin into a lipid bilayer structure, combining the synergistic effects of thin-film hydration and high-pressure homogenization techniques to successfully construct a nanoscale drug carrier with multiple functional advantages. The prepared PTX-loaded luteolin liposomes exhibit a uniform nanoparticle size of 80-120 nm. The antioxidant properties of luteolin further ensure that the particle size remains largely unchanged after 72 hours. The paclitaxel-loaded luteolin liposome formulation exhibits significant sustained-release properties and effectively protects the drug molecules from the effects of gastric acid. In animal experiments, it demonstrates breakthrough long-circulation properties (half-life extended to 2.8 times that of conventional formulations), achieving highly efficient targeted accumulation in tumor tissues through enhanced penetration and retention effects. Luteolin itself, as a natural active ingredient, also enhances the mechanical strength of the lipid membrane (elastic modulus increased by 35% as measured by atomic force microscopy). This formulation, through its unique carrier system, effectively reduces the toxicity of drugs to major organs. This innovative formulation, which combines synergistic therapy and long-term stability, provides a novel solution for the treatment of solid tumors such as breast cancer and ovarian cancer, breaking through the limitations of traditional chemotherapy. It has significant clinical application value and industrialization prospects in the fields of precision medicine and translational medicine. Detailed Implementation

[0024] This invention provides a tumor-targeting liposome loaded with PTX constructed using luteolin and soybean lecithin as membrane materials, comprising a liposome carrier and an active drug loaded in the liposome carrier; the active drug is PTX; the liposome carrier is a lipid bilayer constructed using luteolin and soybean lecithin as membrane materials; luteolin can significantly enhance the mechanical strength and thermodynamic stability of the lipid bilayer, and achieve active tumor targeting through the GLUT1-mediated endocytosis pathway; the PTX is efficiently encapsulated in the hydrophobic core of the liposome; the tumor-targeting liposome loaded with PTX constructed using luteolin and soybean lecithin as membrane materials is prepared by thin-film hydration and high-pressure homogenization, including the following steps:

[0025] (1) Weigh out phospholipids, luteolin and PTX;

[0026] (2) Dissolve the raw material weighed in step (1) in an organic solvent and place it in a round-bottom flask;

[0027] (3) Remove the organic solvent from the solution in step (2) using a rotary evaporator to form a uniform lipid film on the bottle wall;

[0028] (4) After removing the organic solvent, add ultrapure water to the round-bottom flask in step (3) and treat it with an ultrasonic oscillator until a homogeneous dispersion system is obtained.

[0029] (5) The suspension obtained in step (4) is processed by a high-pressure homogenizer to obtain a paclitaxel-loaded luteolin liposome suspension.

[0030] In this invention, phospholipids, luteolin and PTX are weighed. The phospholipids in step (1) are selected from one or more of egg yolk lecithin, soybean lecithin, sodium dioleoylphosphatidylserine (DOPS), dioleoyl L-α-phosphatidylethanolamine (DOPE), 1,2-dioleoyl lecithin (DOPC), dipalmitoylphosphatidylcholine (DPPC), and distearate phosphatidylcholine (DSPC), preferably egg yolk lecithin, soybean lecithin, DOPC and DPPC, and most preferably soybean lecithin.

[0031] In this invention, phospholipids, luteolin and PTX are weighed. The preferred molar ratio of phospholipids to luteolin in step (1) is 4-10:1-6; the most preferred ratio is 10:4. The preferred molar ratio of phospholipids to PTX is 4-10:1-6, and the most preferred ratio is 10:4.

[0032] In this invention, the weighed raw materials are dissolved in an organic solvent and placed in a round-bottom flask. The organic solvent in step (2) is preferably at least one of methanol, ethanol, chloroform, and dichloromethane, with ethanol being the most preferred.

[0033] In this invention, the weighed raw materials are dissolved in an organic solvent and placed in a round-bottom flask. The preferred ratio of organic solvent to phospholipid in step (2) is 1-20 mL: 1 μmol, and the most preferred ratio is 4 mL: 1 μmol.

[0034] In this invention, the organic solvent is removed by a rotary evaporator, so that a uniform lipid film is formed on the bottle wall. The operating temperature of the rotary evaporator in step (3) is preferably 40-70°C, and most preferably 50°C.

[0035] In this invention, after the organic solvent is removed, ultrapure water is added to the round-bottom flask. The ratio of ultrapure water to phospholipid in step (4) is preferably 0.1-1 mL:1 μmol, and most preferably 0.6 mL:1 μmol.

[0036] In this invention, after adding ultrapure water to a round-bottom flask, the mixture is treated with an ultrasonic oscillator until a homogeneous dispersion system is obtained. The ultrasonic power in step (4) is preferably 100-300W, and most preferably 200W. The ultrasonic time is preferably 1-10 minutes, and most preferably 5 minutes.

[0037] In this invention, the obtained suspension is processed by a high-pressure homogenizer to obtain a paclitaxel-loaded luteolin liposome suspension. The pressure setting of the high-pressure homogenizer in step (5) is preferably 150-400 MPa, and most preferably 300 MPa; the circulation time setting of the high-pressure homogenizer is preferably 5-20 minutes, and most preferably 10 minutes.

[0038] The advantages of this invention are as follows:

[0039] (1) Excellent structural stability and high drug loading efficiency

[0040] By forming a dense hydrogen bond network between luteolin and phospholipid molecules, the mechanical strength and thermodynamic stability of the lipid bilayer are significantly enhanced. The complete phospholipid bilayer structure and uniform nanoparticle size are verified by multi-scale characterization (transmission electron microscopy, atomic force microscopy, etc.). The particle size distribution is stable within 72 hours, and the membrane elastic modulus is significantly improved.

[0041] (2) Highly effective and low-toxicity multidimensional anti-tumor mechanism

[0042] It has a synergistic effect of "drug loading enhancement and cell cycle arrest": luteolin significantly enhances intracellular drug accumulation; it simultaneously arrests tumor cell proliferation in the S phase (increasing the proportion of S phase to 43.01%), showing outstanding tumor inhibition rate and survival extension in vivo. After administration, no abnormalities were observed in HE staining sections of major organs of rats, demonstrating excellent biosafety.

[0043] (3) Precise active targeting and long-cycle performance

[0044] Surface glycosylation reduces protein corona adsorption and prolongs blood circulation time. The half-life is 18.7 hours, which is 2.3 times that of the traditional cholesterol liposome (L-PTX). It actively targets the tumor through the GLUT1 transporter. In vivo imaging showed that the tumor accumulation was 1.8 times that of L-PTX. The siRNA knockdown experiment confirmed that it achieves efficient cellular uptake by relying on the clathrin endocytosis pathway, which significantly improves the lesion targeting efficiency. Attached Figure Description

[0045] Figure 1 The image shows the fluorescence colocalization of paclitaxel-loaded luteolin liposomes (C-PTX) and a physical mixture of soybean lecithin and luteolin.

[0046] Figure 2 The images show transmission electron microscopy (TEM), atomic force microscopy (AFM), and cryo-transmission electron microscopy (Cryo-EM) images of C-PTX.

[0047] Figure 3The graph shows the particle size change of C-PTX over 72 hours, the Young's modulus of C-PTX and L-PTX, and the fluorescence anisotropy of C-PTX and L-PTX.

[0048] Figure 4 The dissolution curves of C-PTX, L-PTX and PTX in artificial gastric and intestinal fluids are shown.

[0049] Figure 5 The graph shows the blood concentration-time curves of C-PTX, L-PTX, and PTX in rats.

[0050] Figure 6 H&E staining images of major organ sections of mice in different drug administration groups;

[0051] Figure 7 Figure 1 shows the in vitro antitumor activity analysis of different drug administration groups on LLC, NCI-H460, NCI-H292, NCI-H157 and NCI-H522 cell lines;

[0052] Figure 8 Graph showing the in vivo antitumor activity analysis of different drug administration groups;

[0053] Figure 9 In vivo and in vitro fluorescence images of LLC tumor-bearing nude mice after treatment with different drug groups;

[0054] Figure 10 This is a diagram showing the distribution of different PTX formulations in LLC tumor-bearing mice.

[0055] Figure 11 Fluorescence colocalization map of GLUT1 and PTX preparation in LLC subcutaneous tumors;

[0056] Figure 12 Figure showing the expression of GLUT1 protein in LLC cells before and after siRNA transfection (*** indicates p<0.001).

[0057] Figure 13 The figure shows the effect of inhibiting GLUT1 transporter expression in LLC cells via siRNA transfection on formulation uptake.

[0058] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The specific embodiments described herein are only used to explain the present invention. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0059] Example 1

[0060] Weigh out 50 μmol of soybean lecithin, 20 μmol of luteolin, and 20 μmol of PTX. Dissolve the above raw materials in 200 mL of ethanol and transfer to a round-bottom flask. Evaporate under reduced pressure at 50 °C in a rotary evaporator to form a uniform lipid film. Add 30 mL of ultrapure water (hydration volume ratio of 0.6 mL / 1 μmol phospholipid) to the flask and sonicate at 200 W for 5 minutes to obtain a dispersion. Treat the dispersion with a high-pressure homogenizer at 300 MPa for 10 minutes to obtain a luteolin-loaded liposome suspension with a particle size of 96-121 nm and an encapsulation efficiency >96%.

[0061] Example 2

[0062] Soybean lecithin, DOPC (molar ratio 3:1, total 60 μmol), luteolin (20 μmol), and PTX (15 μmol) were weighed and dissolved in 300 mL of an ethanol / chloroform mixture (volume ratio 3:1). The mixture was evaporated under reduced pressure at 60 °C in a rotary evaporator to form a uniform lipid film. 48 mL of ultrapure water (hydration volume ratio 0.8 mL / 1 μmol phospholipid) was added to the flask, and the mixture was sonicated at 250 W for 8 minutes to obtain a dispersion. The dispersion was then homogenized using a high-pressure homogenizer at 350 MPa for 10 minutes. A luteolin-loaded liposome suspension with a particle size of 90-150 nm and an encapsulation efficiency of 88% was obtained.

[0063] Example 3:

[0064] Weigh out 50 μmol of soybean lecithin, 12 μmol of luteolin, and 12 μmol of PTX. Dissolve the above raw materials in 180 mL of chloroform / methanol mixed solvent (volume ratio 3:1) and transfer to a round-bottom flask. Evaporate under reduced pressure at 50 °C in a rotary evaporator to form a uniform lipid film. Add 27.5 mL of ultrapure water (hydration volume ratio of 0.55 mL / 1 μmol phospholipid) to the flask and sonicate at 220 W for 6 minutes to obtain a dispersion. Homogenize the dispersion at 220 MPa for 12 minutes to obtain a luteolin glycoside liposome suspension loaded with paclitaxel with a particle size of 75-115 nm and an encapsulation efficiency >91%.

[0065] Example 4:

[0066] Weigh out DOPC (35 μmol), DPPC (15 μmol), luteolin (8 μmol), and PTX (8 μmol). Dissolve the above raw materials in 120 mL of ethanol / chloroform mixed solvent (volume ratio 1:1) and transfer to a round-bottom flask. Evaporate under reduced pressure at 55 °C in a rotary evaporator to form a uniform lipid film. Add 22.5 mL of ultrapure water (hydration volume ratio of 0.45 mL / 1 μmol phospholipid) to the flask and sonicate at 180 W for 4 minutes to obtain a dispersion. Homogenize the dispersion in a high-pressure homogenizer at 180 MPa for 18 minutes to obtain a luteolin glycoside liposome suspension loaded with paclitaxel with a particle size of 85-130 nm and an encapsulation efficiency >89%.

[0067] Example 5:

[0068] Weigh out egg yolk lecithin (60 μmol), luteolin (15 μmol), and PTX (18 μmol). Dissolve the above raw materials in 220 mL of dichloromethane / methanol mixed solvent (volume ratio 4:1) and transfer to a round-bottom flask. Evaporate under reduced pressure at 65 °C in a rotary evaporator to form a uniform lipid film. Add 36 mL of ultrapure water (hydration volume ratio of 0.6 mL / 1 μmol phospholipid) to the flask and sonicate at 280 W for 7 minutes to obtain a dispersion system. Treat the dispersion system with a high-pressure homogenizer at a pressure of 380 MPa for 8 minutes to obtain a luteolin glycoside liposome suspension loaded with paclitaxel with a particle size of 95-140 nm and an encapsulation efficiency >87%.

[0069] Test Example 1

[0070] The integrity of the C-PTX structures prepared in Examples 1-5 was verified by fluorescence co-localization. The specific method is as follows:

[0071] Soybean lecithin was labeled using Neilred as a fluorescent marker to facilitate subsequent observation. C6 was used to label luteolin, allowing for clear differentiation of the two distinct fluorescent signals. C-PTX and its physical mixtures were prepared and observed using a high-content imaging system. During observation, the degree of overlap of the fluorescent signals was closely monitored to determine the interaction between luteolin and soybean lecithin and their localization within the liposomes.

[0072] Figure 1 (a) shows the fluorescence colocalization map of C-PTX. Figure 1 (b) is the fluorescence colocalization map of the physical mixture.

[0073] Figure 1It was observed that when soybean lecithin and luteolin were physically mixed, the red and green fluorescence signals were almost completely separated, demonstrating their spatial independence. However, when soybean lecithin and luteolin were prepared into C-PTX, a significant overlap between the red and green fluorescence signals was observed, resulting in yellow fluorescence. This overlap of fluorescence signals indicates that luteolin and soybean lecithin can effectively co-construct liposome structures.

[0074] Test Example 2

[0075] The physicochemical structures of C-PTX prepared in Examples 1-5 were characterized, specifically as follows: Figure 2 As shown:

[0076] Figure 2 (a) is a TEM image of C-PTX. Figure 2 (b) is the AFM plot of C-PTX; Figure 2 (c) is the Cryo-EM plot of C-PTX.

[0077] Figure 2 As can be seen from the TEM image of C-PTX, the liposomes of C-PTX are spherical and of suitable size. The AFM image of C-PTX shows that the distribution of C-PTX is relatively uniform, with no obvious aggregation. The Cryo-EM image of C-PTX shows that C-PTX has a distinct phospholipid bilayer structure.

[0078] Test Example 3

[0079] The stability of C-PTX prepared in Examples 1-5 was tested, and the specific method was as follows:

[0080] 1. Particle size variation, as detailed below:

[0081] The stability of C-PTX was monitored in a 4℃ constant temperature incubator. Samples were taken at the initial state of 0 h and at each preset time point (6, 12, 24, 48, 72 h), and the particle size change was measured using a dynamic light scattering nanoparticle size analyzer.

[0082] Figure 3 (a) It can be seen that the particle size of C-PTX did not change significantly within 72 hours after preparation, indicating that the formulation has good stability. This stability is not only beneficial to the storage and transportation of the drug, but also significantly improves the reliability of the drug's precise delivery to the target site.

[0083] 2. Young's Modulus test, details are as follows:

[0084] The effect of luteolin on the elastic modulus of liposomes was evaluated by measuring the Young's Modulus change of C-PTX using an autoclave-mass spectrometry (AFM). Mica substrates suitable for AFM were selected and coated with poly-L-lysine solution before analysis. The prepared liposome suspension was serially diluted with ultrapure water until the soybean lecithin concentration reached 2 mg / mL. 10 μL of the diluted solution was slowly spotted onto the coated mica substrate. After standing at room temperature for 10 minutes, the unfixed portions were gently removed using a low-pressure nitrogen stream. Once the sample preparation was complete, the Young's modulus was quantitatively determined using the mechanical mode of AFM.

[0085] Figure 3 (b) It can be seen that the Young's modulus of C-PTX is significantly higher than that of L-PTX, by approximately 72.7%. The lower Young's modulus of L-PTX indicates that it is relatively softer and more prone to deformation. C-PTX has stronger rigidity and resistance to deformation, exhibits better stability, and carries a lower risk of drug leakage in vivo. The smaller dispersion of the measurement data indicates that the measurement results have good reliability and repeatability.

[0086] 3. Fluorescence anisotropy test, details are as follows:

[0087] Liposome membrane fluidity was assessed using DPH fluorescent probes. C-PTX and L-PTX were fluorescently labeled with DPH. Samples were equilibrated for 20 minutes in a temperature-controlled water bath (20-50ºC) before analysis. Fluorescence intensity was then measured using a fluorescence spectrophotometer at an excitation wavelength of 362 nm and an emission wavelength of 432 nm. The fluorescence anisotropy (R) formula is shown below:

[0088]

[0089] I a I represents the intensity of emitted light parallel to the polarization direction of the excitation beam. b This represents the intensity of the emitted light that is perpendicular to the polarization direction of the excitation beam.

[0090] Figure 3 (c) It is evident that within a temperature gradient range of 25-45 °C, luteolin has a more significant inhibitory effect on phospholipid membrane fluidity. This indicates that luteolin is more effective than cholesterol in enhancing liposome stability. In this way, luteolin can more effectively maintain the structural integrity of liposomes, reduce drug loss, and thus improve liposome stability.

[0091] Test Example 4

[0092] The C-PTX prepared in Examples 1-5 were subjected to functional evaluation tests, and the specific methods were as follows:

[0093] 1. Solubility test in artificial gastric fluid and artificial intestinal fluid, the specific steps are as follows:

[0094] To better simulate the drug release process of liposomes in the gastrointestinal environment, simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 6.8) were selected as dissolution media to investigate the drug release behavior under different physiological conditions. The in vitro release behavior of C-PTX was investigated using dialysis. C-PTX, L-PTX, and PTX active pharmaceutical ingredients loaded with 1 mg of PTX were encapsulated in MWCO 8-14 kDa regenerated cellulose dialysis units, completely immersed in 50 mL of simulated gastric and intestinal fluid release media. The drug-loaded devices were placed in a 37℃ constant-temperature oscillating water bath system (100 rpm) to construct an in vitro release kinetic model. The release media were completely replaced at 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours. After each sampling, an isothermal and equal-volume supply of fresh media was simultaneously added to maintain the leakage conditions. After all samples were collected, the samples were centrifuged at 12000 r / min for 10 minutes. The supernatant was then collected to determine the PTX content released from each sample solution, and the corresponding PTX in vitro release curves were plotted.

[0095] Figure 4 (a) shows the dissolution curves of PTX, L-PTX, and C-PTX in artificial gastric fluid. Figure 4 (b) shows the dissolution curves of PTX, L-PTX and C-PTX in artificial intestinal fluid.

[0096] Figure 4 (a) It can be seen that in the artificial gastric fluid environment, the cumulative release rate of C-PTX formulation within 24 hours was 51.13%, which was significantly lower than that of L-PTX control group (86.12%) (P < 0.05). Figure 4 (b) As shown, under simulated intestinal fluid conditions, the 24-hour cumulative release rate of C-PTX was 66.13%, which was also lower than the 89.27% ​​of the L-PTX control group (P < 0.05). This indicates that the C-PTX formulation has significant sustained-release properties, and that C-PTX effectively protects the drug molecules from the effects of the gastric acid environment. This characteristic is of significant clinical importance for improving drug bioavailability and reducing gastrointestinal irritation.

[0097] 2. Bioavailability test, the specific steps are as follows:

[0098] SD rats were randomly divided into three groups (n=6), and administered C-PTX, L-PTX, and PTX suspension (equivalent dose 15 mg / kg) orally by gavage, respectively. Approximately 0.5 mL of blood was collected from the ocular vein at 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after administration. The whole blood was placed in anticoagulant EP tubes and centrifuged at 2000 r / min for 15 minutes at 4ºC. The plasma was then separated and stored at -80ºC. Before analysis, 3 mL of acetonitrile was added to dissolve the plasma, and the mixture was vortexed for 3 minutes. Immediately afterward, the mixture was centrifuged at 4000 r / min for 10 minutes at 4ºC. The upper organic phase was then transferred to a nitrogen blow-off tube and placed in a 40ºC constant-temperature water bath nitrogen blow-off apparatus to gently evaporate the organic solvent under nitrogen protection. After the sample was completely dry, 100 μL of acetonitrile was precisely added to the residue at the bottom of the tube to reconstitute the plasma. The PTX content in the plasma sample at each time point was calculated by HPLC.

[0099] Figure 5 The values ​​represent plasma concentrations over time for different PTX formulations. Table 1 shows the pharmacokinetic parameters of different PTX formulations.

[0100] Figure 5 It can be seen that C-PTX exhibits significant pharmacokinetic advantages compared to PTX and L-PTX: its area under the plasma concentration-time curve (AUC) is significantly increased, and the slope of the elimination phase is more gradual, indicating that this formulation has a longer systemic circulation time (t). 1 / 2 = 7.88 h) and higher plasma exposure (AUC) (0-∞) = 216.3 h·μg / mL). Table 1, a comparative analysis of pharmacokinetic parameters, further reveals that the elimination half-life (t) of C-PTX is 216.3 h·μg / mL. 1 / 2 The concentration of C-PTX was 1.89 times that of L-PTX and 3.32 times that of PTX. Meanwhile, the peak concentration of C-PTX (C...) was... max = 14.27 μg / mL) is 1.73-fold and 3.89-fold higher than L-PTX and PTX, respectively. This early high-concentration exposure may facilitate a rapid achievement of the therapeutic threshold. Regarding drug exposure, C-PTX's AUC is... (0-t) (184.5 h·μg / mL) and AUC (0-∞) (216.3 h·μg / mL) reached 2.54 times and 2.77 times that of L-PTX, and 8.55 times and 9.18 times that of PTX technical grade, respectively. The characteristic parameters of the drug-time curve showed that its mean residence time (MRT) was... (0-t) (9.00 hours) and MRT (0-∞)(12.86 hours) was 21% and 25% longer than L-PTX, respectively, confirming the sustained release characteristics of this formulation. Analysis of distribution and metabolism characteristics showed that the apparent clearance of C-PTX (CL / F = 69.3 mL / h / kg) was only 36% of L-PTX and 11% of PTX, while the apparent volume of distribution (Vd / F = 787 mL / kg) was 88% lower than PTX. These data indicate that: (1) the elimination rate of the drug from the central compartment was significantly reduced; (2) the formulation modification made the drug more likely to be distributed in the vascular compartment rather than peripheral tissues, a characteristic that may improve tumor targeting by enhancing the EPR effect. C-PTX reached an effective concentration (2.087 μg / mL) 0.5 hours after administration, T... max With a duration of 4 hours, it maintains rapid onset of action while avoiding the burst release phenomenon of traditional PTX.

[0101] Table 1. Pharmacokinetic parameters of different PTX formulations

[0102] Room parameters PTX L-PTX C-PTX <![CDATA[t 1 / 2 (h)]]> 2.37 4.17 7.88 <![CDATA[C max (μg / mL)]]> 3.67 8.27 14.27 <![CDATA[T max (h)]]> 1 4 4 <![CDATA[AUC (0-t) (h·μg / mL)]]> 21.57 72.69 184.5 <![CDATA[AUC (0-∞) (h·μg / mL)]]> 23.56 78.07 216.3 CL / F (L / h / kg) 0.637 0.192 0.069 Vd / F (L / kg) 6.78 1.16 0.79 <![CDATA[MRT (0-t) (h)]]> 8.21 7.45 9.00 <![CDATA[MRT (0-∞) (h)]]> 11.63 10.26 12.86

[0103] 3. In vivo safety testing, the specific steps are as follows:

[0104] SPF-grade SD rats were randomly divided into four experimental groups: luteolin group, PTX group, L-PTX group, and C-PTX group. Following the pre-determined dosing regimen, the corresponding formulations were administered via gavage for 14 consecutive days. Rats were euthanized by cervical dislocation, and vital organs such as the liver, spleen, kidneys, lungs, and heart were dissected and removed. Tissue samples were fixed in 4% paraformaldehyde solution for 24 hours, dehydrated with graded ethanol, cleared with xylene, and then prepared into paraffin blocks using a paraffin embedding machine. The tissues were sectioned into 5 μm thick sections using a rotary microtome, dewaxed, and then stained with hematoxylin and eosin (H&E) using standard methods. After stained sections were dehydrated and cleared, mounted with neutral resin, and finally, histopathological changes were observed under an optical microscope, with images acquired using a digital imaging system.

[0105] Figure 6 H&E staining images of major organ sections of mice in different drug administration groups.

[0106] Figure 6It was found that the liver tissue structure of rats in the experimental groups that were given luteolin or PTX alone was significantly abnormal, showing loose liver lobule structure, disordered hepatocyte arrangement, and scattered punctate necrotic foci, indicating that both administration methods caused certain degrees of damage to the liver tissue. In contrast, the major organ structures in the C-PTX group remained intact: the liver tissue showed clear lobular structure, distinct hepatocyte lobulation, and regular nucleus morphology; the kidney tissue showed clear demarcation between the cortex and medulla, intact glomerular structure, and neatly arranged proximal convoluted tubules, distal convoluted tubules, and collecting ducts, with no obvious pathological changes; the lung tissue showed intact alveolar structure, clear alveolar septa, neatly arranged bronchial mucosal epithelial cells, and normal vascular structure; the spleen tissue showed uniform distribution and clear boundaries of white pulp (composed of dense lymphocytes) and red pulp (rich in sinusoids and reticular cells), with no abnormal proliferation or destructive lesions; the myocardial tissue showed neatly arranged myocardial fibers, with regularly elliptical, uniformly sized, and evenly distributed nuclei, and normal cytoplasmic staining, showing no significant difference compared to the saline control group. These histological observations fully demonstrate that the C-PTX formulation effectively reduces the toxic effects of the drug on major organs through its unique carrier system.

[0107] 4. Cellular efficacy evaluation, the specific steps are as follows:

[0108] The cytotoxic effects of different PTX formulations were systematically evaluated using the MTT assay. LLC cells and non-small cell lung cancer cell lines NCI-H460, NCI-H292, NCI-H157, and NCI-H522 were selected as research subjects. The specific procedure was as follows: Cells in logarithmic growth phase were digested with 0.25% trypsin, and cell viability was confirmed to be >95% using trypan blue staining. Subsequently, cells were digested with 1×10⁻⁶ PTX formulations. 4 Cells / wells were evenly seeded at a density of 1:1 ratio in 96-well cell culture plates and incubated at 37ºC with 5% CO2 for 16 hours. The following day, the following treatment groups were administered: PTX, luteolin, a physical mixture of PTX / luteolin (molar ratio 1:4), C-Blank, L-PTX, and C-PTX, with six replicates for each concentration. An equal volume of complete culture medium was added to the blank control group, and culture medium containing 0.1% DMSO was added to the solvent control group. After 48 hours of drug treatment, the drug-containing culture medium was discarded, and 100 μL of freshly prepared MTT working solution was added to each well. Incubation was continued for 4 hours in the dark. The culture supernatant was then discarded, and 150 μL of DMSO (containing 10% SDS) was added to dissolve the formazan crystals. The plates were shaken at 150 rpm for 15 minutes on a horizontal shaker. The absorbance was measured at 490 nm using a multi-sensor microplate reader, with a reference wavelength of 630 nm. Cell viability was calculated using the following formula:

[0109]

[0110] The half-maximal inhibitory concentration (IC50) of each formulation was calculated using a nonlinear regression analysis with a four-parameter logistic equation (log(inhibitor) vs. response -- variable slope) in GraphPad Prism 9.0 software.

[0111] Figure 7 (a) shows the cell viability of LLC cell lines as detected by the MTT assay. Figure 7 (b) shows the cell viability of the NCI-H460 cell line as determined by the MTT assay. Figure 7 (c) shows the cell viability of the NCI-H157 cell line as determined by the MTT assay. Figure 7 (d) shows the cell viability of the NCI-H292 cell line as determined by the MTT assay. Figure 7 (e) shows the cell viability of the NCI-H522 cell line as determined by the MTT assay. Table 2 shows the IC50 values ​​of the PTX formulation in LLC, NCI-H460, NCI-H157, NCI-H292 and NCI-H522 cell lines.

[0112] Figure 7 As shown in Table 2, in LLC, NCI-H460, NCI-H292, NCI-H522, and NCI-H157 lung cancer cell lines, the combination of luteolin and PTX raw material exhibited significant cytotoxicity, with its inhibitory effect being significantly superior to the control group using free PTX alone. This result indicates that a simple drug combination strategy can effectively enhance the inhibitory effect on tumor cells, suggesting a possible synergistic mechanism between the two. Further analysis revealed that the C-PTX group showed stronger cytotoxicity against lung cancer cells than the L-PTX conventional liposome group, confirming that the novel C-PTX liposome, through structural optimization, can effectively enhance the inhibitory effect of PTX on tumor cell growth, which may be related to its unique nanocarrier properties. Luteolin alone also showed a certain inhibitory effect on the above-mentioned cell lines, suggesting that luteolin itself has certain anti-tumor activity.

[0113] Table 2 IC50 values ​​of PTX formulation in LLC, NCI-H460, NCI-H157, NCI-H292 and NCI-H522 cell lines

[0114] preparation LLCIC50 (ng / mL) NCI-H460IC50 (ng / mL) NCI-H292IC50 (ng / mL) NCI-H522IC50 (ng / mL) NCI-H157 IC50 (ng / mL) PTX 72.06±8.05 62.38±9.09 71.57±8.77 523.4±44.33 454.1±44.49 Cy+PTX 44.28±7.14 39.91±7.60 41.06±7.48 342.1±39.43 334.6±36.57 L-PTX 65.41±8.13 56.08±4.57 60.08±9.78 387.2±37.31 436.6±38.30 C-PTX 38.20±5.45 38.98±6.53 42.49±5.38 342.6±36.18 316.6±30.29

[0115] 5. In vivo efficacy evaluation, the specific steps are as follows:

[0116] LLC cell lines in the logarithmic proliferation phase were selected. When cell confluence reached 80%-90%, cell dissociation was performed using a digestion system containing 0.25% trypsin. After centrifugation at 2000 r / min for 5 minutes, the supernatant was removed, and the cell pellet was washed three times with pre-cooled phosphate-buffered saline to remove residual culture medium. Cell viability was assessed using the trypan blue rejection assay, and the cell density was precisely adjusted to 5 × 10⁶ cells / year using a serial dilution method. 7 A single-cell suspension of cells / mL was administered. 100 μL of the cell suspension was precisely injected into the subcutaneous tissue of the right axilla of 6-8 week old C57BL / 6 mice, which were then housed in a temperature- and humidity-controlled isolation system. Tumor formation was monitored daily. Pharmacodynamic studies were initiated when the subcutaneous tumor volume reached 1.0 ± 0.2 cm³ on day 7 post-transplantation. The formula for calculating tumor volume is shown below:

[0117]

[0118] The experiment employed a stratified randomized controlled trial (n = 8 / group), establishing the following treatment groups: negative control group (Model, 0.9% saline), experimental groups receiving 20 mg / kg PTX, 20 mg / kg L-PTX, and 20 mg / kg C-PTX (based on PTX equivalents), and blank carrier groups receiving C-Blank and luteolin (luteolin concentration equal to C-PTX carrier). Administered orally via gavage daily from 09:00 to 11:00 for 14 days. Tumor volume was monitored using a double-blind method, with the long and short diameters of the tumor measured every 48 hours, and the animal's body weight recorded simultaneously. Trend graphs of tumor volume and body weight changes were plotted to assess the antitumor activity and safety of the formulation.

[0119] Figure 8 In the middle (a), the tumor growth curve in vivo is shown. Figure 8 (b) shows the comparison of tumor weight. Figure 8 (c) shows the weight comparison of tumor-bearing mice.

[0120] Figure 8 (a) and Figure 8(b) It can be seen that the model group exhibited a typical exponential growth pattern (R²=0.983) during the 21-day observation period, consistent with the invasive growth characteristics of LLC tumors. The luteolin group and C-Blank group showed a slight growth inhibition effect compared to the model group, indicating that luteolin preparations have a weak inhibitory effect on tumor growth and only possess marginal anti-tumor activity. In contrast, under equivalent dose (20 mg / kg) PTX intervention, different drug delivery systems showed significantly differentiated therapeutic effects. The tumor volume changes in the L-PTX and C-PTX groups were significantly smaller than those in the PTX group. L-PTX treatment had a certain effect on inhibiting tumor growth, but the effect was not as significant as that in the C-PTX group. Figure 8 (c) It can be seen that the body weight of the model group mice increased due to compensatory increased food intake caused by tumor consumption. Later, due to cachexia syndrome, the final body weight decreased from the peak, showing a trend of first increasing and then decreasing. Unlike L-PTX and PTX, the C-PTX group reduced the fluctuation of body weight during the experimental period due to its tumor targeting and sustained-release characteristics. The mice in this group maintained their body weight better.

[0121] Test Example 5

[0122] The C-PTX prepared in Examples 1-5 were subjected to tumor targeting tests, and the specific methods were as follows:

[0123] 1. In vivo and ex vivo imaging tests, the specific steps are as follows:

[0124] C57BL / 6 mice were randomly divided into groups (n=6). LLC cells were starved in serum-free DMEM medium for 4 hours before tumor implantation, then digested from the culture flask with trypsin, centrifuged, and resuspended in PBS buffer. After counting, cells containing 1×10⁻⁶ cells were collected. 6 One LLC cell was injected subcutaneously into the right upper limb axilla of C57BL / 6 mice in PBS solution at a dose of 100 μL. The injection was continued until the subcutaneous tumor volume reached 1 cm². 3 The mice were used for subsequent experiments starting at approximately 1:00 PM. Nile Red-labeled C-PTX and L-PTX liposome solutions were administered to tumor-bearing mice via gavage, and observations were performed at specified time points (1, 2, 4, 6, 8, and 12 hours) using a small animal in vivo imaging system. The excitation wavelength was 488 nm, and the emission wavelength was 590 nm. After in vivo imaging, the tumor-bearing mice were euthanized by intraperitoneal injection of an excessive amount of sodium pentobarbital. The brain, heart, lungs, liver, spleen, kidneys, and tumors were then dissected for ex vivo imaging observation.

[0125] Figure 9It was found that after gavage administration, the C-PTX group showed significant drug distribution characteristics at 4, 6, and 8 hours in fluorescence imaging. Compared with the traditional L-PTX formulation, C-PTX was rapidly absorbed after administration, and its fluorescence signal was widely distributed in the abdominal region of the experimental mice, especially showing obvious accumulation in metabolic organs such as the liver and kidneys. A persistent and strong fluorescence signal was observed at the subcutaneous tumor site of LLC, a phenomenon corroborated by the results of in vitro tissue fluorescence imaging. Quantitative analysis of fluorescence intensity in the heart, liver, spleen, lung, kidney, and tumor tissues showed that the relative fluorescence intensity of C-PTX in tumor tissue was approximately 1.8 times higher than that of the L-PTX group, confirming that C-PTX has a significant tumor-targeting enrichment ability.

[0126] 2. Organizational distribution research, the specific steps are as follows:

[0127] After administration of the drug via gavage, mice were euthanized and placed abdomen-up on a dissection table to fully expose the thoracic and abdominal cavities. Heart, liver, spleen, lung, kidney, and tumor tissues were collected. The collected tissue samples were washed three times with physiological saline, blotted dry with filter paper, and weighed. Physiological saline was added to each tissue sample at a specific mass-to-volume ratio, and a homogenized suspension was prepared using an electric homogenizer. 300 μL of the tissue homogenate was extracted with 3 mL of acetonitrile, vortexed for 3 minutes, and immediately centrifuged at 4000 r / min for 10 minutes at 4ºC. The upper organic phase was then transferred to a nitrogen blow-off tube and placed in a 40ºC constant-temperature water bath nitrogen blow-off tube to gently evaporate the organic solvent under nitrogen protection. After the sample was completely dry, 100 μL of acetonitrile was precisely added to the residue at the bottom of the tube for redissolution. The PTX content in each tissue was calculated using HPLC.

[0128] Figure 10 It was found that the drug concentration in tumor tissue of the C-PTX group was significantly better than that of the L-PTX group and the PTX group at four time points: 2 hours, 4 hours, 6 hours, and 8 hours after administration. In particular, at 4 hours after administration, the drug accumulation in tumor tissue of the C-PTX group reached (6.28 ± 2.07) μg / g, which was 4.79 times and 1.47 times that of the PTX group (1.31 ± 0.66) μg / g and the L-PTX group (4.28 ± 1.86) μg / g, respectively.

[0129] 3. Fluorescence co-localization analysis of tumor accumulation, the specific steps are as follows:

[0130] 200 μL of Nile Red-labeled C-PTX and L-PTX liposomes were administered orally to tumor-bearing mouse models. Four hours after administration, the mice were sacrificed, and the tumor tissue was dissected and divided into 5 mm³ blocks. These blocks were placed in cryoembryoforming cassettes, and after adding an appropriate amount of OCT embedding medium, they were rapidly frozen in liquid nitrogen. A sample holder was pre-coated with an OCT substrate gel layer. The frozen tissue blocks were positioned and then covered with an additional OCT matrix. The blocks were then transferred to the cryostat sample chamber (-20 ℃) ​​for cryogenic equilibration, and serial sections were prepared with a section thickness of 5 μm. The frozen sections were attached to glass slides and allowed to stand at room temperature for 30 minutes, then fixed with 4% paraformaldehyde for 15 minutes. The sections were washed three times with PBS buffer (5 minutes each time), blocked with PBS solution containing 5% BSA at room temperature for 1 hour, and incubated overnight in a humidified chamber at 4℃ with GLUT1 primary antibody working solution. The next day, the samples were gently washed three times with PBS and incubated with Alexa Fluor 488-labeled secondary antibody at room temperature in the dark for 1 hour. After repeating the PBS rinsing process, nuclear staining with DAPI solution was performed for 5 minutes. After the final rinse, anti-quenching mounting medium was added, and multispectral imaging analysis was performed under a fluorescence microscope.

[0131] Figure 11 It was found that C-PTX exhibited a stronger fluorescence signal intensity in tumor tissue compared to L-PTX, and the overlap region with GLUT1 protein was significantly increased. This phenomenon indicates that C-PTX can not only effectively accumulate in subcutaneous tumor tissue, but also has the ability to deliver drugs to tumor sites in vivo, demonstrating excellent active targeting.

[0132] 4. The specific steps regarding the effect of siRNA silencing the GLUT1 gene in tumor cells on cellular uptake are as follows:

[0133] Log-proliferating LLC cells were enzymatically digested with a 0.25% trypsin-EDTA mixture, and the cell density was adjusted to 1×10⁻⁶ using a cell counter. 5Cells / wells were seeded in 24-well cell culture plates and cultured in an incubator at 37ºC for adherence. When cell confluence reached 70%, the medium was replaced with serum-free and antibiotic-free basal medium. GLUT1 siRNA transfection system construction: 1 OD siRNA oligo (5'-GGAAUUCAAUGCUGAUGAUTT-3' positive strand sequence) was reconstituted with DEPC water to prepare a stock solution (frozen at -20ºC for later use). Immediately before use, 1.25 μL of the stock solution was vortexed with 50 μL of Opti-MEM low-serum medium (antibiotic-free) and equilibrated at room temperature for 5 minutes; 1.25 μL of liposome transfection reagent was diluted using the same method. After mixing equal volumes of the two solutions, the mixture was incubated at room temperature for 20 minutes to form an siRNA-liposome nanocomplex. This complex was then slowly added dropwise to cell culture wells. After transfection at 37ºC for 6 hours, the medium was replaced with maintenance medium containing 10% FBS, and the cells were cultured for another 16 hours. The expression level of GLUT1 protein in LLC cells transfected with siRNA-GLUT1 was quantitatively analyzed according to the standardized Western blot procedure in section “5.3.8”. Nile red-labeled C-PTX and L-PTX were added to LLC cells, and co-incubated for 2 hours with either FAM-labeled siRNA-GLUT1 or FAM-labeled control siRNA. Subsequently, the cells were washed three times with PBS (5 min each time) and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Then, the cells were washed three more times with PBS (5 min each time), stained with DAPI for 5 minutes, and observed under a fluorescence microscope.

[0134] Figure 12 It was found that, compared with the untransfected group and the siRNA-NC group, the expression level of GLUT1 protein in the siRNA-GLUT1 transfected group was significantly reduced (P < 0.01), with an inhibition efficiency of over 75%. This result indicates that the designed siRNA-GLUT1 can effectively inhibit the expression of GLUT1 protein in LLC cells, providing a reliable experimental model for subsequent research on the biological function of GLUT1 in tumor cells (*** indicates p < 0.001). Figure 13 The siRNA was successfully transfected into the cells. Compared with the blank control group, the C-PTX uptake in the siRNA-GLUT1 transfection group was significantly reduced, while the C-PTX uptake in the FAM-labeled negative control siRNA group was not significantly different from that in the blank control group. This indicates that the cellular uptake of C-PTX is highly dependent on the expression level of GLUT1. C-PTX is mainly located in the cell membrane, which is consistent with the subcellular localization of GLUT1. The above experimental results fully confirm that C-PTX achieves its specific uptake in LLC tumor cells through a GLUT1-mediated active targeting mechanism.

[0135] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A tumor-targeting luteolin glycoside liposome loaded with paclitaxel, characterized in that, Using phospholipids and luteolin as carriers, luteolin-loaded liposomes were prepared by thin-film hydration and high-pressure homogenization. The preparation method includes the following steps: (1) The molar ratio of phospholipids to luteolin is 4-10:1-6; the molar ratio of phospholipids to paclitaxel is 4-10:1-6. (2) Dissolve the above raw materials in an organic solvent and place them in a round-bottom flask; (3) Remove the organic solvent in the solution by rotary evaporation to form a uniform lipid film on the bottle wall; (4) Add ultrapure water to the round-bottom flask and treat it with an ultrasonic oscillator until a homogeneous dispersion system is obtained; (5) The luteolin liposome suspension loaded with paclitaxel was obtained by high pressure homogenization.

2. The production process according to claim 1, characterized in that, The phospholipids in step (1) are selected from one or more of egg yolk lecithin, soybean lecithin, sodium dioleoylphosphatidylserine (DOPS), dioleoyl L-α-phosphatidylethanolamine (DOPE), 1,2-dioleoyl lecithin (DOPC), dipalmitoylphosphatidylcholine (DPPC), and distearate phosphatidylcholine (DSPC), preferably egg yolk lecithin, soybean lecithin, DOPC, and DPPC.

3. The preparation process according to claim 1, characterized in that, The organic solvent in step (2) is selected from at least one of methanol, ethanol, chloroform, and dichloromethane.

4. The production process according to claim 1, characterized in that, The evaporation temperature in step (3) is 40-70℃.

5. According to the production process of claim 1, the ultrasonic power in step (4) is 100-300W, preferably 100-200W; the ultrasonic time is 1-10 minutes, preferably 1-5 minutes.

6. According to the production process described in claim 1, the high-pressure homogenization pressure in step (5) is 150-400 MPa, and the high-pressure homogenization cycle time is 5-20 minutes.

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