Selenocysteine liposome nanomaterial based on cell membrane fusion and its anti-tumor application under cysteine deficiency

Abstract

The application belongs to the field of biological medicine and nanomaterials, and discloses selenocystine liposome nanomaterial based on cell membrane fusion and its anti-tumor application under cysteine deficiency conditions. The nanomaterial comprises selenocystine liposomes and tumor cell membrane proteins wrapped on the surface of the selenocystine liposomes. The selenocystine liposomes comprise selenocystine, phospholipids, cholesterol and DSPE-MPEG. The application fuses tumor cell membrane proteins with selenocystine-loaded liposomes to construct nanomaterials with tumor targeting and metabolic regulation functions, which can selectively kill tumor cells under cysteine deficiency conditions. The application proves that the selenocystine liposome nanomaterial wrapped with HeLa cell membranes has excellent anti-tumor effect and significantly reduced systemic toxicity in in vitro and in vivo experiments. The application has great clinical application potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomaterials, specifically relating to a liposome-encapsulated selenocystine nanomaterial based on cell membrane fusion and its anti-tumor application under cystine deficiency conditions. Background Technology

[0002] Metabolic reprogramming of tumor cells is a core characteristic that enables them to maintain rapid proliferation and resist cell death. Abnormal amino acid metabolism is a key aspect of tumor metabolism. Cystine (Cys2) is the main source of extracellular cysteine ​​and is crucial for maintaining intracellular glutathione (GSH) synthesis, redox homeostasis, and protein function. Selenium has shown significant potential in the field of chemotherapy for tumors, and studies have shown that several small organoselenic selenium molecules, such as methylselenoic acid and selenomethionine, possess good antitumor activity. Among them, selenocysteine ​​is an essential amino acid with low cytotoxicity and is considered a potential candidate antitumor molecule.

[0003] However, selenocysteine ​​exhibits limited stability in physiological environments and is difficult to target tumor tissues. Nanodelivery systems, particularly liposomes, offer the potential to improve the pharmacokinetics and targeting of amino acid drugs. However, traditional liposomes suffer from insufficient targeting, susceptibility to clearance by mononuclear phagocytic systems, and difficulty in achieving spatiotemporally controlled release of multiple payloads. In recent years, biomimetic nanotechnology, especially cell membrane fusion technology, has shown great research potential by coating nanoparticles with natural cell membranes (such as tumor cell membranes and erythrocyte membranes), endowing them with various biological functions of their source cells, such as immune escape, homologous targeting, and long circulation. Summary of the Invention

[0004] This invention discovers that selenocysteine ​​can effectively kill tumor cells under cystine-deficient conditions. This invention aims to provide a cell membrane fusion-based liposome-encapsulated selenocysteine ​​material (SeleCys2@Lip-CCM) for tumor treatment under cystine-deficient conditions and its preparation method. This material has high encapsulation efficiency and good stability.

[0005] The technical solution adopted in this invention is:

[0006] Application of selenocystine in the preparation of antitumor drugs under cystine deficiency conditions.

[0007] The tumor could be cervical cancer or liver cancer.

[0008] This invention has discovered that under cystine deprivation conditions, selenocystine can significantly increase tumor cell mortality and has a significant anti-tumor effect.

[0009] The present invention also provides a selenocysteine ​​liposome nanomaterial based on cell membrane fusion, the nanomaterial comprising selenocysteine ​​liposomes and tumor cell membrane proteins encapsulated on the surface of the selenocysteine ​​liposomes; the selenocysteine ​​liposomes comprising selenocysteine, phospholipids, cholesterol, and DSPE-MPEG.

[0010] DSPE-MPEG is distearate phosphatidylethanolamine-polyethylene glycol, with a preferred molecular weight of 2000.

[0011] Furthermore, phospholipids, cholesterol, and DSPE-MPEG form nanoliposomes, which encapsulate selenocysteine.

[0012] The preferred phospholipid is lecithin.

[0013] Furthermore, the tumor cell membrane protein is obtained by extracting membrane proteins from tumor cells.

[0014] Membrane proteins can be extracted using appropriate cell membrane protein extraction kits.

[0015] Furthermore, the tumor cell membrane protein is extracted from a cell line homologous to the target tumor. More specifically, when selenocysteine ​​liposome nanomaterials based on cell membrane fusion are used to prepare anti-cervical cancer drugs, the tumor cell membrane protein is extracted from the HeLa cell line.

[0016] The selenocysteine ​​liposome nanomaterial based on cell membrane fusion can be prepared by the following method:

[0017] (1) Selenocystine, phospholipids, cholesterol and DSPE-MPEG were used to prepare selenocystine liposomes by thin-film hydration method;

[0018] (2) Extraction of tumor cell membrane proteins from target tumor cells;

[0019] (3) The tumor cell membrane protein was coated onto the surface of selenocysteine ​​liposomes by ultrasonic fusion method to obtain the selenocysteine ​​liposome nanomaterial based on cell membrane fusion.

[0020] In step (1), the mass ratio of selenocysteine ​​to phospholipid is 1:1.5~3;

[0021] The mass ratio of phospholipids, cholesterol, and DSPE-MPEG is 5~6:2~3:1.

[0022] Furthermore, step (1) is preferably performed as follows:

[0023] Phospholipids, cholesterol, and DSPE-MPEG are dissolved in an organic solvent, and the organic solvent is removed by rotary evaporation to form a lipid film. An aqueous solution of selenocysteine ​​is added to the lipid film to hydrate it. Then, the film is ultrasonically treated with a probe to obtain selenocysteine ​​liposomes.

[0024] The preferred organic solvent is chloroform.

[0025] Step (2) can be performed using a cell membrane protein extraction kit to extract tumor cell membrane proteins.

[0026] The tumor cells can be HeLa cells.

[0027] In step (3), the total mass ratio of phospholipids, cholesterol, and DSPE-MPEG to the mass ratio of tumor cell membrane proteins is 20-30:1.

[0028] Furthermore, in step (3), the process of the ultrasonic fusion method is as follows: the amplitude output is set to 100%, the ultrasonic operation lasts for 2 seconds and stops for 3 seconds, the total ultrasonic operation time is 20 to 30 minutes, and it is carried out under ice bath conditions.

[0029] The present invention also provides the application of the cell membrane fusion-based selenocysteine ​​liposome nanomaterial in the preparation of antitumor drugs.

[0030] Furthermore, the application of the cell membrane fusion-based selenocystine liposome nanomaterials in the preparation of antitumor drugs under cystine deficiency conditions.

[0031] More preferably, the cell membrane is extracted from HeLa cells, and the tumor is cervical cancer.

[0032] The selenocystine liposome nanomaterial based on cell membrane fusion of the present invention can selectively kill tumor cells, inhibit tumor cell growth and proliferation, and inhibit the increase in weight and volume of solid tumors under cystine deficiency conditions.

[0033] The selenocysteine ​​liposome nanomaterials based on cell membrane fusion prepared in this invention have a three-layer structure of "core-layer-shell", which includes, from the inside out:

[0034] Drug-loaded lipid core: composed of a lipid bilayer, the internal aqueous phase of which encapsulates a therapeutically effective amount of selenocysteine. Preferably, the lipid is composed of phospholipids and cholesterol to provide a stable membrane structure and high encapsulation efficiency.

[0035] Functionalized modification layer: DSPE-PEG2000 is further modified on the outer surface of the drug-loaded lipid core to prolong blood circulation time;

[0036] Tumor cell membrane shell: Completely encapsulated on the outer surface through fusion technology, this cell membrane shell endows the entire material with homologous targeting capability.

[0037] This invention discovers that selenocysteine ​​induces tumor cell death under cystine deficiency conditions and can be used to prepare anti-tumor drugs. Based on this, a cell membrane-encapsulated selenocysteine ​​liposome nanomaterial was constructed. This nanodelivery system, by encapsulating the drug in liposomes and modifying the liposome surface, can improve the stability of selenocysteine ​​in vivo and prolong its time in blood circulation. Furthermore, the nanomaterial can target and bind to corresponding ligands on the surface of similar tumor cells through signaling molecules on the tumor cell membrane, thereby improving the enrichment efficiency of the loaded drug at the tumor site and enhancing its targeting at the tumor site. This invention fuses tumor cell membranes with selenocysteine-loaded liposomes to construct nanomaterials with tumor targeting and metabolic regulation functions, which can selectively kill tumor cells and inhibit tumor cell growth in the cystine-deficient tumor microenvironment. Animal experiments of this invention have demonstrated that under cystine deficiency conditions, the HeLa cell membrane-encapsulated selenocysteine ​​liposome nanomaterial can significantly reduce the tumor weight and volume in HeLa xenograft mice. This invention demonstrates in both in vitro and in vivo experiments that the selenocysteine ​​liposome nanomaterials encapsulated in HeLa cell membranes possess excellent anti-tumor effects and significantly reduced systemic toxicity.

[0038] The tumor cell membrane of this invention endows the material with homologous targeting capability and immune evasion. Selenocysteine ​​can significantly inhibit tumor growth and metabolism under cystine-deficient conditions, and the selenocysteine-loaded nanomaterials of this invention exhibit tumor microenvironment responsiveness, low toxicity to normal cells, and high therapeutic specificity and safety. This invention has good therapeutic potential, providing a new potential therapeutic strategy for cancer treatment, especially cervical cancer. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this application, and are not intended to limit this application.

[0040] Figure 1 This is a graph showing the effect of selenocystine on tumor cell mortality under cystine deficiency conditions.

[0041] (a) The figure shows the cell mortality rate after selenocystine treatment in the HeLa cell cystine deprivation model;

[0042] (b) The figure shows the cell mortality rate after selenocystine treatment in the HepG2 cell cystine deprivation model;

[0043] (c) The figure shows the cell mortality rate after selenocystine treatment in the Huh7 cell cystine deprivation model;

[0044] (e) The figure shows the changes in cell death rate of HeLa cells after treatment with different concentrations of cystine at 50 μM and 100 μM selenocystine concentrations.

[0045] (f) The figure shows the changes in cell death rate of HepG2 cells after treatment with different concentrations of cystine at 50 μM and 100 μM selenocystine.

[0046] Figure 2 This is a synthesis and characterization diagram of the liposome nanoparticles prepared in the embodiments of this application, wherein:

[0047] (a) The figure shows transmission electron micrographs of Lip, Lip-CCM, SeleCys2@Lip and SeleCys2@Lip-CCM;

[0048] (b) The figure shows the particle size distribution of Lip, Lip-CCM, SeleCys2@Lip and SeleCys2@Lip-CCM;

[0049] (c) The figure shows the particle size and PDI plots of Lip, Lip-CCM, SeleCys2@Lip and SeleCys2@Lip-CCM measured continuously for five days;

[0050] (d) The figure shows the Zeta potentials measured continuously for five days at Lip, Lip-CCM, SeleCys2@Lip and SeleCys2@Lip-CCM;

[0051] (e) The figure shows the drug release curves of SeleCys2@Lip-CCM after incubation in PBS buffer at pH 5.7 and pH 7.4 for 24 hours.

[0052] Figure 3 The figure shows the pharmacological performance verification results of the liposome nanoparticles prepared in the embodiments of this application, wherein:

[0053] (a) The figure shows the detection results of successful cell membrane coating on the lipid exfoliation layer verified by fluorescence resonance energy transfer technology;

[0054] (b) The figure shows the results of flow cytometry analysis of the uptake of Lip-CCM-FITC by six different cell types at 0, 1, 2 and 4 hours to demonstrate its targeting.

[0055] (c) The figure is for Figure 3 (b) A bar chart showing the statistical analysis of cell uptake data at 4-hour time points;

[0056] (d) The figure shows a comparison of the effects of different concentrations of blank liposomes, drug-loaded liposomes and single drugs on cell viability in HeLa cells;

[0057] (e) The figure shows a comparison of the effects of different concentrations of blank liposomes, drug-loaded liposomes and single drugs on cell viability in HUVEC cells.

[0058] Figure 4 The following is a diagram showing the in vivo efficacy evaluation results of the liposome nanoparticles prepared in the embodiments of this application in a HeLa cell xenograft tumor model, wherein:

[0059] (a) The figure shows the overall condition of the mouse after treatment and before dissection;

[0060] (b) The image shows a photograph of a tumor removed from a mouse.

[0061] (c) The figure shows a bar chart of statistical analysis of tumor weight in mice of each experimental group;

[0062] (d) The figure shows the statistical analysis curves of the changes in tumor volume of mice in each experimental group over time;

[0063] (e) Figure shows the curves of changes in the body weight of mice in each experimental group during the drug administration period.

[0064] Figure 5 The images show the results of pathological analysis of tumor tissue and major organs after animal experiments, including:

[0065] (a) The figure shows the Ki67 immunohistochemical staining results of tumor tissue sections from tumor-bearing mice;

[0066] (b) The figure is for Figure 5 (a) A bar chart showing the positive rate of Ki67 staining results after quantitative analysis;

[0067] (c) The figure shows the results of hematoxylin-eosin (HE) staining of heart and liver tissue sections from tumor-bearing mice. Detailed Implementation

[0068] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application.

[0069] Based on the embodiments described in this application, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this application. It is understood that, unless otherwise specified, some technical means of the various embodiments described herein can be substituted for or combined with each other.

[0070] This application successfully constructed a cell membrane-coated liposome with homologous targeting function. This system, by combining selenocysteine ​​with a cystine-deprived diet and utilizing biomimetic nanotechnology for precise delivery, demonstrated superior antitumor efficacy and significantly reduced systemic toxicity both in vitro and in vivo. The results of Examples 1-4 constitute a complete chain of evidence from mechanism validation, product preparation, functional characterization to efficacy evaluation, providing a practical approach for developing novel combination therapies for cancer.

[0071] Example 1: Verification of the synergistic effect of selenocystine and cystine deprivation

[0072] This embodiment aims to verify in vitro the core biological principle upon which the strategy of this invention is based: selenocystine can induce the death of tumor cells under cystine deprivation conditions.

[0073] 1. Materials and Methods

[0074] HeLa, HepG2, and Huh7 tumor cell lines were selected, and the cells were stored at 1×10⁶ cells per well. 5 Cells were seeded at a density of 100 μM / well in 24-well plates and cultured in fresh medium for 12 hours. The cells were then divided into four groups: Group 1 received normal medium; Group 2 received normal medium supplemented with 100 μM selenocysteine; Group 3 received cysteine-deficient medium; and Group 4 received cysteine-deficient medium supplemented with 100 μM selenocysteine. After 24 hours of treatment, 1 μg / mL propidium iodide (PI) was added to the medium and the cells were incubated for 30 minutes. The cells were then digested with trypsin, washed, and resuspended in 0.3 mL PBS supplemented with 5% fetal bovine serum for flow cytometry analysis. The proportion of PI-positive cells (i.e., dead cells) was calculated using flow cytometry software (such as FlowJo) to characterize cell death. HeLa and HepG2 tumor cell lines were also used, with cells seeded at a density of 1 × 10⁶ cells / well. 5 Cells were seeded at a density of 100 μM into 24-well plates and cultured in fresh medium for 12 hours. The medium was then replaced with cystine-deprived medium, and the cells were divided into two groups. Selenocysteine ​​at concentrations of 50 μM and 100 μM was added to the cells, respectively. Cystine at concentrations of 0, 1, 5, 10, 50, 100, and 200 μM was added to each group. After 24 hours of treatment, precise quantification was performed using propidium iodide (PI) staining combined with flow cytometry.

[0075] 2. Results

[0076] The results are as follows Figure 1 As shown.

[0077] Morphological observation: such as Figure 1As shown in Figures (a) to (c), under cystine deprivation conditions, the tumor cell mortality rate of the three types of tumor cells increased significantly after treatment with selenocystine.

[0078] Quantitative analysis: such as Figure 1 As shown in Figures (e) and (f), in HeLa and HepG2 cells, cell viability decreased in a dose-dependent manner with decreasing cysteine ​​concentration in the culture medium when selenocysteine ​​(50 μM or 100 μM) was present in the background. Quantitative analysis further demonstrated that selenocysteine ​​and amino acid deprivation have a clear synergistic lethal effect.

[0079] Example 2: Preparation and characterization of cell membrane-coated drug-loaded liposomes

[0080] This embodiment provides a specific preparation method for the biomimetic nanodelivery system and characterizes its key physicochemical properties.

[0081] 1. Preparation method

[0082] (a) Preparation of drug-loaded liposomes:

[0083] (1) Turn on the rotary evaporator and set the water bath to 50°C. Add 3 mL of anhydrous ethanol to a clean 25 mL single-necked round-bottom flask, connect the rotary evaporator, and evaporate for 30 min to ensure that the round-bottom flask is fully dry and clean.

[0084] (2) Weigh 3 mg of lecithin, 1.5 mg of cholesterol and 0.5 mg of DSPE-PEG2000 and dissolve them in 1 mL of chloroform solution. After vortexing, place the solution in an ultrasonic cleaner and mix thoroughly by ultrasonication.

[0085] (3) Add the mixed solution from (2) into the single-necked round-bottom flask from (1) that has been evaporated, and connect it to a rotary evaporator;

[0086] (4) Adjust the height of the round-bottomed spherical bottle so that the bottom of the bottle is submerged in the water bath. Turn on the rotary evaporator, close the vent valve, turn on the circulating water pump and the condensate pipe, and rotary evaporate for 30 minutes to allow the sample to adhere to the wall and form a milky white film.

[0087] (5) Remove the flask, add 1 ml ddH2O, and use an ultrasonic cleaner to hydrate the liposomes in the flask. Collect the solution in a 15 ml centrifuge tube and recover the liposomes.

[0088] (6) Place a 15 mL centrifuge tube containing 1 mL of liposomes into a 50 mL beaker, fill the beaker with an appropriate amount of crushed ice, place the ultrasonic disruptor probe into the 15 mL centrifuge tube, and adjust the probe depth so that it is submerged below the liquid surface.

[0089] (7) Perform sonication with a microprobe of an ultrasonic cell disruptor, set the amplitude output to 100%, with a sonication-on period of 2 s and a sonication-off period of 3 s, and the total effective sonication duration is 20 min;

[0090] (8) After the sonication is completed, collect the sample and store it in a refrigerator at 4°C; Lip is thus prepared;

[0091] (9) During the above process, 2 mg of selenocystine SeleCys2 powder is added in step (5), so as to prepare SeleCys2@Lip;

[0092] (b) Preparation of tumor cell membranes:

[0093] (1) Thaw the membrane protein extraction reagent A in the Membrane and Cytosol Protein Extraction Kit (Beyotime-P0033) at room temperature, and store it in an ice bath after thawing. For every 5×10 7 cells collected, 1 mL of membrane protein extraction reagent A is used, and PMSF is added within 5 minutes before use to make the final concentration of PMSF 1 mM;

[0094] (2) Count and collect 5×10 7 HeLa cells, centrifuge at 1000 rpm for 5 min to collect the cells, aspirate and discard the supernatant, and keep the cell pellet for later use;

[0095] (3) Gently resuspend the cell pellet with 1 mL of pre-cooled PBS in an ice bath, centrifuge at 4°C and 600 g for 5 min to precipitate the cells. Discard the supernatant, then centrifuge at 4°C and 600 g for 1 min to precipitate the residual liquid on the wall of the centrifuge tube and further precipitate the cells, and aspirate the residual liquid as thoroughly as possible;

[0096] (4) Add 1 mL of membrane protein extraction reagent A supplemented with PMSF to 5×10 7 cells, gently and fully suspend the cells, and place the mixture in an ice bath for 15 min;

[0097] (5) Transfer the HeLa cell suspension to a pre-cooled glass homogenizer in an ice bath, and homogenize for 50 strokes;

[0098] (6) Centrifuge at 4°C and 700 g for 10 min, and carefully collect the supernatant into a new centrifuge tube. Do not touch the pellet when aspirating the supernatant, and 50 μL of the supernatant can be left unaspirated to ensure the high purity of the aspirated supernatant;

[0099] (7) Centrifuge at 4°C and 14000 g for 30 min to precipitate cell membrane fragments;

[0100] (8) The supernatant is cytoplasmic protein and can be stored at -80℃ for later use. When aspirating the supernatant, 50 μL of supernatant residue is acceptable to avoid contact with the precipitate and contamination of the supernatant sample.

[0101] (9) Centrifuge at 14000 g for 10 s at 4℃, remove the supernatant as much as possible, retain the precipitate as membrane protein, and store in a -80℃ refrigerator;

[0102] (c) Fusion of cell membrane and liposomes:

[0103] (1) The collected membrane proteins were quantified by adding 0.25 mg of membrane protein required for the experiment into a 15 mL centrifuge tube containing 1 mL of liposome emulsion (containing 5 mg of liposome raw material); the liposomes were Lip and SeleCys2@Lip;

[0104] (2) Place a 15 mL centrifuge tube containing 1 mL of liposomes into a 50 mL beaker, fill the beaker with an appropriate amount of crushed ice, place the ultrasonic disruptor probe into the 15 mL centrifuge tube, and adjust the probe depth so that it is submerged below the liquid surface.

[0105] (3) Ultrasound was performed using a micro probe of an ultrasonic disruptor, with the amplitude output set to 100%. The ultrasonic operation lasted for 2 seconds and stopped for 3 seconds, with a total ultrasonic operation time of 20 minutes, resulting in HeLa cell membrane-coated targeted drug-loaded liposomes. Lip and SeleCys2@Lip were coated with HeLa cell membranes to prepare Lip-CCM and SeleCys2@Lip-CCM, respectively.

[0106] (4) After the ultrasound is completed, collect the sample and store it in a refrigerator at 4°C;

[0107] 2. Characterization Results

[0108] Systematically characterize the final product, such as Figure 2 As shown.

[0109] Appearance: Figure 2 Transmission electron microscopy (a) shows that the particles are spherical, uniform in size and shape, and have a stable structure.

[0110] Particle size: Figure 2 (b) The hydrated particle size of each liposome is shown, where Lip is 164 nm, Lip-CCM is 194.8 nm, SeleCys2@Lip is 122.9 nm, and SeleCys2@Lip is 152.4 nm.

[0111] Stability: Stored at 4°C for 5 days. Figure 2 Figure (c) shows the particle size versus PDI curve; Figure (d) shows the zeta potential curve. Both curves show no significant changes, indicating good formulation stability.

[0112] Drug release: such as Figure 2 As shown in Figure (e), SeleCys2@Lip-CCM achieved a cumulative drug release rate of over 65% in a simulated tumor microacidic environment (pH 5.7) over 24 hours, while the release was slow at physiological pH (7.4), exhibiting intelligent drug release characteristics.

[0113] Example 3: In vitro targeting, uptake, and pharmacodynamic evaluation of liposomes

[0114] This embodiment validates the targeting ability, uptake mechanism, and in vitro antitumor efficacy and safety of the liposomes at the cellular level.

[0115] 1. Validation of targeting and cellular uptake

[0116] Membrane fusion verification: Fluorescence resonance energy transfer (FRET) technology was employed. For example... Figure 3 As shown in Figure (a), a strong FRET signal was detected in the experimental group that was wrapped in the cell membrane, confirming that the liposomes were effectively fused with the cell membrane.

[0117] Specific uptake: such as Figure 3 As shown in Figures (b) and (c), flow cytometry results indicate that HeLa cells have a significantly higher uptake rate of homologous HeLa membrane-coated liposomes than the other five non-HeLa cell lines, demonstrating excellent homologous targeting.

[0118] 2. In vitro efficacy and safety

[0119] Antitumor activity: such as Figure 3 As shown in (d), in HeLa cells, the IC50 of SeleCys2@Lip is... 50 With a value of 10.30 μg / mL, SeleCys2@Lip-CCM exhibited stronger cytotoxicity, with an IC50 value of 10.30 μg / mL. 50 The value was further reduced to 8.35 μg / mL. The IC50 value of the selenocysteine-loaded liposomes (SeleCys2@Lip-CCM) was significantly lower than that of free selenocysteine, while the blank liposomes (Lip) showed no significant toxicity, proving that the nanocarrier is safe and can enhance drug efficacy.

[0120] Attenuation effect: such as Figure 3 As shown in Figure (e), in normal endothelial cells (HUVECs), the effect of free SeleCys2 on the IC50 of HUVEC cells... 50 The concentration was 10.11 μg / mL. SeleCys2@Lip-CCM significantly reduced the cytotoxicity of HUVEC cells, with an IC50 value of 10.11 μg / mL. 50The concentration rose to 15.17 μg / mL, indicating that the free drug group showed strong toxicity, while the drug-loaded liposome group showed significantly higher cell viability, confirming that the delivery system can reduce damage to normal tissues.

[0121] Example 4: Evaluation of the in vivo antitumor efficacy and safety of liposomes

[0122] This embodiment uses an animal model to comprehensively evaluate the therapeutic efficacy and systemic safety of the liposomes in vivo.

[0123] 1. Animal Experiment Design

[0124] A subcutaneous xenograft tumor model of BALB / c nude mice using HeLa cells was established. The tumor volume was expected to reach approximately 80 mm². 3 Mice were randomly divided into four groups (n=6): (1) normal diet blank liposome group; (2) normal diet HeLa membrane-coated targeted drug-loaded liposome group; (3) cystine-deficient diet blank liposome group; and (4) cystine-deficient diet HeLa membrane-coated targeted drug-loaded liposome group. The drugs were administered via intraperitoneal injection every three days for a total of seven times. Tumor volume and mouse weight were measured daily.

[0125] 2. In vivo efficacy results

[0126] The results are as follows Figure 4 As shown.

[0127] Tumor growth inhibition: After treatment, if Figure 4 As shown in Figures (a) and (b), the mice in the targeted drug-loaded liposome group had the smallest tumor volume. Figure 4 Quantitative data in (c) and (d) showed that the tumor weight and volume in the cystine-deficient diet HeLa membrane-coated targeted drug-loaded liposome group were significantly lower than those in other groups, indicating the best efficacy.

[0128] System security: Figure 4 Figure (e) shows that the body weight of mice in the cystine-deficient diet group with HeLa membrane-coated targeted drug-loaded liposomes remained stable throughout the treatment period, indicating that the treatment regimen was well-tolerated and safe.

[0129] 3. Histopathological analysis

[0130] After treatment, tumor samples and major organs were taken for pathological analysis, such as... Figure 5 As shown.

[0131] Tumor proliferation inhibition: Ki67 staining of tumor tissue yielded the following results: Figure 5 As shown in Figure (a). Quantitative analysis is as follows: Figure 5Figure (b) shows that the group with a cystine-deficient diet and HeLa membrane-coated targeted drug-loaded liposomes had the lowest rate of Ki67 positive cells, indicating that tumor cell proliferation was most effectively inhibited.

[0132] Organ safety: HE staining of heart and liver tissues ( Figure 5 Figure (c) shows that the major organs of all mice in the treatment group were structurally intact, with no obvious pathological damage such as inflammation, necrosis or vacuolar degeneration, which confirmed the safety of the treatment at the histological level.

Claims

1. A selenocysteine ​​liposome nanomaterial based on cell membrane fusion, characterized in that... The nanomaterials include selenocysteine ​​liposomes and tumor cell membrane proteins encapsulated on the surface of the selenocysteine ​​liposomes; the selenocysteine ​​liposomes include selenocysteine, phospholipids, cholesterol, and DSPE-MPEG; the tumor cell membrane proteins are extracted from cell lines homologous to the target tumor.

2. The selenocysteine ​​liposome nanomaterial based on cell membrane fusion as described in claim 1, characterized in that... The tumor cell membrane protein is obtained by extracting membrane proteins from tumor cells.

3. The selenocysteine ​​liposome nanomaterial based on cell membrane fusion as described in claim 1, characterized in that... The selenocysteine ​​liposome nanomaterial based on cell membrane fusion was prepared by the following method: (1) Selenocystine, phospholipids, cholesterol and DSPE-MPEG were used to prepare selenocystine liposomes by thin-film hydration method; (2) Extraction of tumor cell membrane proteins from target tumor cells; (3) The tumor cell membrane protein was coated onto the surface of selenocysteine ​​liposomes by ultrasonic fusion method to obtain the selenocysteine ​​liposome nanomaterial based on cell membrane fusion.

4. The selenocysteine ​​liposome nanomaterial based on cell membrane fusion as described in claim 3, characterized in that... In step (1), the mass ratio of selenocysteine ​​to phospholipid is 1:1.5~3; The mass ratio of phospholipids, cholesterol, and DSPE-MPEG is 5~6:2~3:

1.

5. The selenocysteine ​​liposome nanomaterial based on cell membrane fusion as described in claim 3, characterized in that... In step (3), the total mass ratio of phospholipids, cholesterol and DSPE-MPEG in the selenocysteine ​​liposome to the mass ratio of tumor cell membrane proteins is 20~30:

1.

6. The use of the selenocysteine ​​liposome nanomaterial based on cell membrane fusion as described in any one of claims 1 to 5 in the preparation of antitumor drugs.

7. The application as described in claim 6, characterized in that... The application of the cell membrane fusion-based selenocystine liposome nanomaterials in the preparation of antitumor drugs under cystine deficiency conditions.

8. The application as described in claim 7, characterized in that... The cell membrane was extracted from HeLa cells, and the tumor was cervical cancer.

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