Light-operated cell membrane chimeric multi-unit drug-loaded liposome and application thereof

Abstract

The invention discloses a light-operated cell membrane chimeric multi-unit drug-loaded liposome and an application thereof. The light-controlled multi-unit drug-loading liposome comprises a cell membrane, a light-sensitive element, a temperature-sensitive lipid material and a multi-unit drug-loading core, the multi-unit drug-loading core comprises a first drug-loading unit, a second drug-loading unit and a third drug-loading unit, wherein the first drug-loading unit is used for entrapping curcumin by taking vitamin E succinate-grafted-polylysine as a material, the second drug-loading unit is used for entrapping trabectedin by taking natural silk fibroin as a material, and the third drug-loading unit is used for compounding doxorubicin hydrochloride by taking dopamine-grafted-gamma-polyglutamic acid as a material. The light-operated cell membrane chimeric multi-unit drug-loaded liposome can reverse the immunosuppressive microenvironment of tumor tissues and improve the drug resistance problem of glioma after multiple chemotherapy, and has a wide application prospect in the aspect of tumor treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to a light-controlled cell membrane chimeric multi-unit drug-loaded liposome and application thereof. BACKGROUND

[0002] Glioma is a common intracranial malignant tumor, and surgical resection combined with radiotherapy and chemotherapy is the standard treatment in clinic. However, in the past 20 years, the average survival time of patients with malignant glioma has not changed much. Recent studies have found that chemotherapy drugs induce cancer cells into a senescent state, which also endows them with stem cell activity, making them have stronger proliferation and invasion ability. Senescent tumor cells eventually undergo apoptosis and necrosis, and also release danger-associated molecules to activate peripheral immune cells, which migrate and infiltrate tumor tissues. Many studies have shown that after radiotherapy and chemotherapy, a large number of tumor stem cells and macrophages infiltrate the glioma tissue, which is positively correlated with the malignancy of the tumor.

[0003] Nanocarriers can change the in vivo distribution of drugs, improve drug targeting, and reduce toxic side effects to some extent due to their nanoscale size, large surface-to-volume ratio, and easy functionalization modification on the surface. However, studies have found that due to the heterogeneity of tumor tissue cell populations, the expression of target receptors on the surface of different cells varies greatly, and the nanometer targeted delivery system based on receptor-ligand binding often has low targeting accuracy and serious "off-target" problems. In addition, PEG modification on the surface of nanocarriers is a common means to prolong their in vivo circulation time. Recent studies have found that nanomedicines with surface PEG modification are prone to activate B cells in the body to produce anti-PEG antibodies after multiple injections, causing the ABC phenomenon of accelerated clearance, ultimately leading to short circulation time and low targeting.

[0004] Therefore, it is crucial to construct a liposome nanomedicine that can precisely target tumor cells and deliver multiple drugs. SUMMARY

[0005] In order to make up for the shortcomings of the prior art, the purpose of the present application is to construct a new type of biomimetic cell nanocarrier to deliver multiple drugs, achieve multiple precise targeting of the immunosuppressive microenvironment of brain glioma and differentiated tumor cells, and improve the effect of brain glioma chemotherapy.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The present application provides a light-controlled cell membrane chimeric multi-unit drug-loaded liposome.

[0008] Further, the light-controlled multi-unit drug-loaded liposome comprises a cell membrane, a photosensitive element, a temperature-sensitive lipid material, and a multi-unit drug-loaded core.

[0009] Further, the multi-unit drug-loaded core comprises a first drug-loaded unit, a second drug-loaded unit, and a third drug-loaded unit.

[0010] Further, the first drug-loaded unit comprises a first shell layer, a therapeutic drug targeting tumor stem cells wrapped in the first shell layer.

[0011] Further, the second drug-loaded unit comprises a second shell layer, a therapeutic drug targeting tumor-associated macrophages wrapped in the second shell layer.

[0012] Further, the third drug-loaded unit comprises a third shell layer, a therapeutic drug targeting differentiated tumor cells wrapped in the third shell layer.

[0013] Further, the cell membrane is a cancer cell membrane.

[0014] Further, the cancer cell membrane is a glioma cell membrane.

[0015] Further, the cancer cell membrane is a C6 cell membrane.

[0016] In some embodiments, the cancer cell membrane comprises any cancer cell-derived cell membrane, including but not limited to U87, U251, LN-229, T98G, A172, SF295, U118 cell line-derived cell membranes.

[0017] Further, the photosensitive element is indocyanine green.

[0018] In some embodiments, the photosensitive element refers to a photosensitizer or an optical imaging probe. In addition to indocyanine green, one skilled in the art can select other photosensitive elements, including but not limited to porphyrin and its derivative photosensitizers, non-porphyrin photosensitizers, etc.

[0019] Further, the temperature-sensitive lipid material comprises one or more of dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, and 1-palmitoyl-2-hydroxy-glycerol-3-phosphatidylcholine.

[0020] Further, the temperature-sensitive lipid material is a combination of dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, and 1-palmitoyl-2-hydroxy-glycerol-3-phosphatidylcholine.

[0021] Further, the molar ratio of dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, and 1-palmitoyl-2-hydroxy-glycerol-3-phosphatidylcholine is 1:1:0.5.

[0022] In some embodiments, the therapeutic drug targeting tumor stem cells comprises one or more of curcumin, MG-132, idarubicin. In a specific embodiment of the present application, the therapeutic drug targeting tumor stem cells is curcumin.

[0023] In some embodiments, the therapeutic drug targeting tumor-associated macrophages comprises one or more of Triciribine, Pexidartinib, BLZ945, SNDX-6352. In a specific embodiment of the present application, the therapeutic drug targeting tumor-associated macrophages is Triciribine.

[0024] In some embodiments, the therapeutic drug targeting differentiated tumor cells comprises one or more of doxorubicin hydrochloride, temozolomide, lomustine, vincristine, irinotecan. In a specific embodiment of the present application, the therapeutic drug targeting differentiated tumor cells is doxorubicin hydrochloride.

[0025] Further, the first shell layer of the first drug-loaded unit is vitamin E succinate-graft-polylysine.

[0026] Further, the second shell layer of the second drug-loaded unit is natural silk fibroin.

[0027] Further, the third shell layer of the third drug-loaded unit is dopamine-graft-gamma-polyglutamic acid.

[0028] Further, the average particle size of the light-controlled cell membrane chimeric multi-unit drug-loaded liposome is 210 nm; and the Zeta potential of the light-controlled cell membrane chimeric multi-unit drug-loaded liposome is -26 mV.

[0029] The second aspect of the present application provides a method for preparing the light-controlled cell membrane chimeric multi-unit drug-loaded liposome of the first aspect of the present application.

[0030] Further, the method comprises:

[0031] a) dissolving the temperature-sensitive lipid material and the photosensitive element in an organic solvent to obtain an organic phase;

[0032] b) sequentially stacking the first drug-loaded unit, the second drug-loaded unit and the third drug-loaded unit in the water dispersion phase into the above-mentioned organic phase by reverse evaporation method to obtain a three-time stacked lipid film; adding distilled water to hydrate the film, ultrasonic dispersion, and then extruding to obtain a light-controlled multi-unit drug-loaded liposome;

[0033] c) coating the cancer cell membrane on the lipid bilayer of the light-controlled multi-unit drug-loaded liposome obtained above by using the biomembrane fusion technology and the extrusion method, thereby obtaining the light-controlled cell membrane chimeric multi-unit drug-loaded liposome.

[0034] Further, vitamin E succinate-graft-polylysine is used to encapsulate curcumin to construct a first drug-loaded unit.

[0035] Further, natural silk fibroin is used to encapsulate trabectidin to construct a second drug-loaded unit.

[0036] Further, dopamine-graft-gamma-polyglutamic acid is used to encapsulate doxorubicin hydrochloride to construct a third drug-loaded unit.

[0037] Further, the specific steps for obtaining the three times of stacked lipid films in step b) are as follows:

[0038] The first drug-loaded unit water dispersion phase is slowly dropped into the organic phase in step a), and then stirred, emulsified, ice-bathed, ultrasonicated, and rotary evaporated to obtain a first stacked lipid film.

[0039] The first stacked lipid film obtained above is dissolved in an organic solvent to obtain an organic phase; the second drug-loaded unit water dispersion phase is slowly dropped into the organic phase, and then stirred, emulsified, ice-bathed, ultrasonicated, and rotary evaporated to obtain a second stacked lipid film.

[0040] The second stacked lipid film obtained above is dissolved in an organic solvent to obtain an organic phase; the third drug-loaded unit water dispersion phase is slowly dropped into the organic phase, and then stirred, emulsified, ice-bathed, ultrasonicated, and rotary evaporated to obtain a third stacked lipid film.

[0041] Further, the specific steps for obtaining the light-controlled cell membrane chimeric multi-unit drug-loaded liposome in step c) are as follows: the cancer cell membrane is mixed with the light-controlled multi-unit drug-loaded liposome obtained in step b), and then incubated at low temperature; the incubated sample is loaded into a liposome micro-extruder, and then passed through 1 μm, 400 nm, and 100 nm polycarbonate membranes in sequence, and extruded back and forth 10 times each time to obtain a light-controlled cell membrane chimeric multi-unit liposome.

[0042] Further, the low temperature is 4℃.

[0043] Further, the time for low-temperature incubation is 2 h.

[0044] Further, the temperature-sensitive lipid material is a combination of dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, and 1-palmitoyl-2-hydroxy-glycerol-3-phosphatidylcholine.

[0045] Further, the molar ratio of dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, and 1-palmitoyl-2-hydroxy-glycerol-3-phosphatidylcholine is 1:1:0.5.

[0046] In some embodiments, the organic solvent is selected from a combination of any one or more of dichloromethane, methanol, ethanol, acetone, acetonitrile, diethyl ether. In a specific embodiment of the present application, the organic solvent is dichloromethane.

[0047] Further, the specific construction method of the first drug-loaded unit is as follows: vitamin E platinum succinate-graft-polylysine and curcumin are dissolved in DMSO to prepare an organic phase, the organic phase is added dropwise into distilled water under stirring to perform self-assembly, DMSO is removed by dialysis with distilled water, and un-loaded curcumin is removed by ultracentrifugation, so that the first drug-loaded unit is obtained.

[0048] Further, the grafting degree of the vitamin E platinum succinate-graft-polylysine is 50%, the dialysis time is 12 h, and the ultracentrifugation speed is 10,000 rpm.

[0049] Further, the specific construction method of the second drug-loaded unit is as follows: trilaciclib is dissolved in a silk fibroin solution as an aqueous phase, the aqueous phase is slowly added dropwise into acetone under stirring to perform self-assembly, acetone is removed by rotary evaporation, and larger particles are removed by ultracentrifugation, so that the second drug-loaded unit is obtained.

[0050] Further, the concentration of the silk fibroin solution is 2%, the speed of adding the aqueous phase into acetone is 50 μL / drop, and the ultracentrifugation speed is 10,000 rpm.

[0051] Further, the specific construction method of the third drug-loaded unit is as follows: dopamine-graft-gamma-polyglutamic acid and doxorubicin hydrochloride are dissolved in distilled water, NaOH solution is used to adjust the pH to 7.0, the compound is stirred at room temperature for a period of time, the compound is slowly added dropwise into a Tris base buffer solution to continue stirring and crosslinking, and doxorubicin hydrochloride that is not loaded is removed by dialysis with distilled water, so that the third drug-loaded unit is obtained.

[0052] Further, the stirring and compounding time at room temperature is 10 h.

[0053] The third aspect of the present application provides application of the light-controlled cell membrane chimeric multi-unit drug-loaded liposome in the preparation of an antitumor drug.

[0054] In some embodiments, the tumor includes glioma, gastric cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, colon cancer, rectal cancer, prostate cancer, blood cancer, lymphoma, cervical cancer, osteosarcoma, melanoma, pancreatic cancer, liver cancer, kidney cancer, gallbladder cancer or cholangiocarcinoma. In a specific embodiment of the present application, the tumor is glioma.

[0055] Advantages and beneficial effects of the present application:

[0056] (1) The present application adopts layer-by-layer accumulation technology to realize the simultaneous loading of different structural types of ultra-small nanometer drug-loaded units, solving the drug loading defect of current liposomes which can only load molecular drugs;

[0057] (2) The present application uses cell membrane modified multi-unit drug-loaded liposomes to imitate the circulating cell glioma invasion / targeting characteristics, promote the brain transport of multiple nanometer drugs, and overcome the technical bottleneck of low brain transport efficiency of current nanometer drugs;

[0058] (3) The present application uses temperature-sensitive lipid materials combined with photosensitive elements to realize the photosensitive release of multiple nanometer drug-loaded units of cell membrane chimeric liposomes, target tumor-associated macrophages, tumor stem cells and differentiated tumor cells, reverse the immunosuppressive microenvironment of tumor tissue, and improve the drug resistance problem after multiple chemotherapy of glioma. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 is a characterization diagram of the first drug-loaded unit, the second drug-loaded unit and the third drug-loaded unit; wherein Figure 1 A is a transmission electron microscope image and a particle size distribution diagram of the first drug-loaded unit (the upper image is a transmission electron microscope image, and the lower image is a particle size distribution diagram); Figure 1 B is a transmission electron microscope image and a particle size distribution diagram of the second drug-loaded unit (the upper image is a transmission electron microscope image, and the lower image is a particle size distribution diagram); Figure 1 C is a transmission electron microscope image and a particle size distribution diagram of the third drug-loaded unit (the upper image is a transmission electron microscope image, and the lower image is a particle size distribution diagram);

[0060] Figure 2 is a process flow diagram of the layer-by-layer accumulation method for preparing multi-unit drug-loaded liposomes;

[0061] Figure 3 is a characterization result diagram of the light-controlled cell membrane chimeric multi-unit drug-loaded liposome (LS-MCL);

[0062] Figure 4 is an MRI image detection result diagram;

[0063] Figure 5 is an HE staining result diagram;

[0064] Figure 6 is a result diagram of immunohistochemical detection of Caspase-3 expression;

[0065] Figure 7 is a result diagram of immunohistochemical detection of the proportion of Ki-67 positive cells;

[0066] Figure 8 is a cell ball experiment result. DETAILED DESCRIPTION

[0067] The technical solutions in the present application will be described clearly and completely below in combination with the embodiments in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without any creative work, fall within the protection scope of the present application.

[0068] The specific conditions not mentioned in the following examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not marked with the manufacturer, which are all conventional products that can be obtained by purchase.

[0069] Example 1 Preparation and characterization of drug-loading units

[0070] 1. Preparation of the first drug-loading unit, curcumin-loaded nanomicelles (Cur-NPs)

[0071] 100 mg of vitamin E succinate-grafted-polylysine (VES-g-ε-PLL) and a certain amount of curcumin (Cur) were dissolved in 2 ml of DMSO to prepare an organic phase. Under magnetic stirring, the organic phase was added dropwise into 3 ml of distilled water and continued to stir for 10 minutes for self-assembly. After dialysis of the self-assembled nanomicelles against distilled water for 12 hours to remove DMSO, and then removing the unloaded curcumin by ultracentrifugation at 10000 rpm, curcumin-loaded nanomicelles (abbreviated as: Cur-NPs) were obtained. Further freeze-drying can prepare a freeze-dried powder for storage. The grafting degree of the VES-g-ε-PLL polymer is 50%, and its synthesis method is referred to Therapeutic supermolecular micelles of vitamin E succinate-grafted ε-polylysine as potential carriers for curcumin: Enhancing tumour penetration and improving therapeutic effect on glioma (DOI: 10.1016 / j.colsurfb.2017.07.019), which is hereby incorporated in its entirety.

[0072] 2. Preparation of the second drug-loading unit, trabectidin-loaded silk fibroin nanoparticles (Tr-SFNPs)

[0073] Silk cocoon was used as raw material to extract 2% silk fibroin solution through alkaline water degumming, LiBr dissolution and dialysis process. Triclabendazole was dissolved in 2% silk fibroin solution as the aqueous phase. Under the condition of strong magnetic stirring, 5ml aqueous phase was slowly dropped into 10ml acetone at the speed of 50μL / drop for self-assembly. After maintaining stirring reaction at room temperature for 24h, the acetone was evaporated by rotary evaporator. The obtained nano-particle water dispersion was centrifuged at 10000rpm to remove larger particles, and triclabendazole-loaded silk fibroin ultra-small nanoparticles (Tr-SFNPs) were prepared.

[0074] 3. Preparation of the third drug-loaded unit, doxorubicin-loaded nanocomposite (DOX-NPs)

[0075] Dopa-γ-PGA was synthesized by EDC / NHS-mediated condensation reaction using the carboxyl group of γ-polyglutamic acid (γ-PGA) and the amino group of dopamine. 100mg Dopa-γ-PGA was dissolved in distilled water with a certain amount of doxorubicin hydrochloride (DOX). The pH of the solution was slowly adjusted to 7.0 using NaOH. After stirring at room temperature for 10h, the reaction nanocomposite was slowly dropped into Tris base (10mM, pH8.5) buffer solution for further stirring and crosslinking for 3h. The nanocomposite was dialyzed against distilled water to remove the unloaded doxorubicin hydrochloride and replace the medium, and doxorubicin-loaded nanocomposite (DOX-NPs) was prepared.

[0076] 4. Characterization of the nanodrug-loaded unit

[0077] ① Particle size and potential: The particle size and distribution of the nanoparticles were measured by dynamic light scattering (DLS) using a laser particle size analyzer (Marven Zetasizer NANO ZS) at a laser wavelength of 632.8nm and a scattering angle of 90°. Before DLS analysis, the nanoparticles were filtered through a Millipore membrane (0.8μm) filter to remove dust and the like. The concentration of the filtered polymer was 0.5mg / ml.

[0078] ② Morphology study: Transmission electron microscopy (TEM): 100μL of the above-mentioned nanodrug-loaded unit (1mg / ml) was deposited on a carbon film copper grid. After removing the excess sample solution, a drop of 2% phosphotungstic acid was added to the copper grid loaded with nanoparticles. After drying, the excess liquid was absorbed with filter paper, and the grid was stained at room temperature and left to dry naturally for 2 days. The grid was then observed under a JEOL JEM-2000EX transmission electron microscope at an acceleration voltage of 100kV.

[0079] 5. Experimental results

[0080] The characterization results of the first drug-loaded unit, the second drug-loaded unit and the third drug-loaded unit are as follows: Figure 1As shown, the average particle size of the first drug-loaded unit is 190 nm; the average particle size of the second drug-loaded unit is 200 nm; and the average particle size of the third drug-loaded unit is 210 nm. It is detected that the Zata potential of the first drug-loaded unit is -37 mV, the Zata potential of the second drug-loaded unit is -33 mV, and the Zata potential of the third drug-loaded unit is -28 mV.

[0081] Example 2 Extraction of cell membrane

[0082] Extraction of cancer cell membrane: C6 glioma cells were selected to extract cell membrane proteins, 1.4 x 10 6 cells were collected and suspended in 200 μL separation buffer (HB) (75 mM mannitol, 25 mM sucrose, 10 mM Tris-HCL, 1 mM MgCl2, 1 mM KCL, 20 μL / 4 mL protease inhibitor and 1% (v / v) phosphatase inhibitor) to avoid sample degradation. The cells were homogenized with an ultrasonic cell disruptor (40 W, three times with pauses on ice), and the cell suspension was centrifuged at 1000 rpm at 4°C for 10 minutes to remove the precipitate. The above homogenization and centrifugation steps should be repeated twice, and then the supernatant was collected and centrifuged at 4°C and 13000 rpm for 40 minutes. The precipitate was removed, the supernatant was collected, and the step was repeated, and the particles in the supernatant were examined under a light microscope. The isolated cell membrane was stored at -20°C or lyophilized for further use. For specific extraction method, refer to Tumor cellular membrane camouflaged liposomes as the non-invasive vehicle of gene: the specific targeting toward homologous glioma and the traversing blood brain barrier (DOI: 10.1039 / D0NR04212A), which is hereby incorporated in its entirety.

[0083] Example 3 Preparation and characterization of light-controlled cell membrane chimeric multi-unit drug-loaded liposomes

[0084] 1. Preparation of light-controlled multi-unit drug-loaded liposomes by layer-by-layer stacking method

[0085] The light-controlled multi-unit liposomes were prepared by layer-by-layer stacking method, and the process flow is as follows Figure 2The. 90 mg of temperature-sensitive lipid material (temperature-sensitive lipid material (DPPC / DPPG / MSPC, molar ratio 1:1:0.5) is weighed and dissolved in 4 ml of dichloromethane organic solvent. First, a proper amount of Cur-NPs unit aqueous dispersion is slowly dropped into the organic solvent and stirred to emulsify. Then, the emulsion is quickly placed in an ice bath ultrasonic cell disruptor and ultrasonically treated at 20% power for 15 s to obtain a stable emulsion (the emulsion does not separate for half an hour). The obtained emulsion is placed on a rotary evaporator to form a layer of colloidal primary accumulation solid film adhering to the bottom of the bottle. Then, dichloromethane is added to repeat the above steps, and the Tr-SFNP unit and the DOX-NPs unit are sequentially accumulated. After three times of accumulation, distilled water is added to hydrate the film to prepare an emulsion. After ultrasonic dispersion and liposome extrusion, the light-controlled multi-unit drug-loaded liposome (LS-Lip) is prepared.

[0086] 2. Preparation of light-controlled cell membrane chimeric multi-unit drug-loaded liposome by membrane fusion technology

[0087] The cell membrane and the liposome phospholipid bilayer are spontaneously fused by using the extrusion technology. The extracted cancer cell membrane is mixed with the previously prepared LS-Lips, and incubated at 4°C for 2 h. The incubated sample is loaded into a liposome micro-extruder (LiposoFast-Basic), and extruded through 1 μm, 400 nm and 100 nm polycarbonate membranes in sequence, 10 times back and forth each time. The light-controlled cell membrane chimeric multi-unit liposome (LS-MCL) is obtained.

[0088] 3. Performance characterization of light-controlled cell membrane chimeric multi-unit drug-loaded liposome (LS-MCL)

[0089] Particle size, potential and morphology: the particle size, particle size distribution and Zeta potential are measured by dynamic laser scattering (DLS) using a laser particle size analyzer (Marven Zetasizer NANO ZS) at a laser wavelength of 632.8 nm and a scattering angle of 90°. The fine morphology is observed by transmission electron microscopy (TEM).

[0090] 4. Experimental results

[0091] The characterization results of LS-MCL are shown in Table 1. Figure 3

[0092] Example 4 Animal experiment

[0093] 1. Establishment of microenvironment immunosuppressive glioma animal model

[0094] ​①Establishment of intracranial brain glioma rat model: a) Incision and fixation: Intraperitoneal injection of 1% sodium pentobarbital (0.3 ml / 100 g) to anesthetize the rat, cut the hair on the top of the head, make a 1 cm long incision on the scalp at the intersection of the internal boundary line and the sagittal midline of the head, and separate and expose the skull. Fix the head of the rat on the rat brain stereotaxic instrument, disinfect with iodine and alcohol, and lay a hole towel. b) Drilling: Select the right caudate nucleus region of the rat brain as the target, and determine the drilling position corresponding to the right caudate nucleus according to the rat head stereotactic atlas. Drill a small hole with a diameter of 1 mm using a dental drill, and drill to the surface of the dura mater without piercing the dura mater. c) Cell inoculation: Adjust the needle tip of the microsyringe on the positioning instrument to touch the dura mater, insert the needle 6 mm, and then withdraw 1 mm, so that the needle tip is 5 mm away from the dura mater. Slowly inject 10 μl of cell suspension (cell number is 1×10 6 / 10 μl) into the brain, the injection speed is 2.5 μl / min, after injection, leave the needle for 5 min, slowly withdraw the needle, immediately seal the bone hole with sterile bone wax, suture the skin, and disinfect the incision. All operations are performed under sterile conditions in the operating room. The control group of rats is replaced with an equal volume of cell culture solution instead of 1×10 6 / 10 μl of C6 cell suspension. After inoculation, by observing the physiological state changes of rats at different time periods, MRI images, brain tumor morphology and pathology evaluation, it has been proved that the intracranial brain glioma model is successfully established.

[0095] ②Establishment of temozolomide-induced immunosuppressive glioma model: In order to induce the formation of glioma immunosuppressive microenvironment, on the 9th day after C6 cell inoculation, chemotherapy was started, with a cycle of 5 days, intraperitoneal injection (left lower side), once a day, the experimental group (TMZ) was given a dose of 5 mg / (kg*d) of chemotherapeutic drug; the control group (CNT) was given an equal volume of 0.01 mol / L PBS solution. Three days after treatment, brain glioma tissue sections were stained for GSCs cells by CD133 / nestin double staining, TAMs by CD11b / F4 / CD80 staining, and microenvironment immunosuppression related markers by anti-Elastin, anti-GAG, anti-Fibronectin, anti-Collagen staining and IL-10, TGF-β detection, to confirm the formation of immunosuppressive microenvironment in tumor tissue.

[0096] 3, Pharmacodynamic study

[0097] ①Experimental grouping: The microenvironment immunosuppressive SD rats were evenly divided into 2 groups: model control group, simple near-infrared light intervention group (NIR); treatment group: NIR+light-controlled cell membrane chimeric multi-unit drug-loaded liposome group (LS-MCL).

[0098] ② NIR light intervention parameters: light transmission fiber inserted through the skull, deep into brain tumor tissue, in vivo / MRI imaging positioning fiber position, fiber end (diameter 272 nm) transmission light beam (652 nm, 50 mW, 8 min 40 s, 26 J).

[0099] ③ Dosing regimen: model control group tail vein injection of normal saline 0.4 ml / each; NIR+LS-MCL group of rats first tail vein injection of LS-MCL nano-preparation, immediately light transmission fiber intervention on tumor site. Each dose according to the grouping, the first dose after MRI detection screening from the 9th day of inoculation, every other week, a total of three doses. All the solution for administration was prepared with normal saline. After administration, the physiological state, survival status, body weight of rats were observed, and the tumor volume was detected by regular MRI imaging. Brain tissue sections were prepared by routine operation, and pathological evaluation was carried out by HE staining, stem cells, macrophages and tumor microenvironment immunosuppressive index detection.

[0100] The MRI imaging detection results are shown in Figure 4 Compared with the control group, the tumor in the brain of the rats injected with drug-loaded LS-MCL was significantly reduced. The HE staining results are shown in Figure 5 The control group tumor cells showed nuclear pleomorphism, i.e. the size of the nucleus was not uniform, the shape was irregular, the chromatin distribution was abnormal, the nucleolus was enlarged, the cell structure was dissolved and disappeared, and the tissue structure became blurred. After LS-MCL treatment, the rat brain tissue structure was significantly improved. The activation of Caspase-3 is often used as an indicator to evaluate the therapeutic effect. Immunohistochemical detection of Caspase-3 expression can be used to monitor the efficacy of the drug. The expression of Caspase-3 is shown in Figure 6 Compared with the control group, the Caspase-3 expression of the rats treated with LS-MCL was higher. The proportion of Ki-67 positive cells (Ki-67 index) detected by immunohistochemistry can quantify the proliferation activity of cell population. In tumor tissue, the higher the Ki-67 index, the more active the proliferation of tumor cells. The expression of Ki-67 is shown in Figure 7 Compared with the control group, the Ki-67 expression of the LS-MCL group was lower. Further, the control group and the treatment group were incubated with GSCs / TAMs cell spheres. After different time points of action, the growth of tumor spheres was observed under an inverted microscope. The results are shown in Figure 8 The volume of cell spheres in the drug-loaded LS-MCL treatment group was significantly inhibited. In summary, these results show that the light-controlled cell membrane chimeric multi-unit drug-loaded liposome has a significant effect on treating brain glioma.

[0101] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. A light-controlled cell membrane chimeric multi-unit drug-loaded liposome, characterized in that: The light-controlled multi-unit drug-loaded liposome comprises a cell membrane, a photosensitive element, a temperature-sensitive lipid material and a multi-unit drug-loaded core; The multi-unit drug-carrying core comprises a first drug-carrying unit, a second drug-carrying unit and a third drug-carrying unit; The first drug-carrying unit includes a first shell layer and a therapeutic drug targeting tumor stem cells encapsulated in the first shell layer; The second drug-carrying unit includes a second shell layer and a therapeutic drug targeting tumor-associated macrophages encapsulated in the second shell layer; The third drug-carrying unit includes a third shell layer and a therapeutic drug targeted at differentiating tumor cells encapsulated in the third shell layer.

2. The light-controlled cell membrane chimeric multi-unit drug-loaded liposome according to claim 1, characterized in that: The cell membrane is a cancer cell membrane; Preferably, the cancer cell membrane is a glioma cell membrane; More preferably, the cancer cell membrane is a C6 cell membrane; Preferably, the photosensitive element is indocyanine green; Preferably, the thermosensitive lipid material comprises one or more of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and 1-palmitoyl-2-hydroxy-glycero-3-phosphatidylcholine; Preferably, the thermosensitive lipid material is a combination of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol and 1-palmitoyl-2-hydroxy-glycero-3-phosphatidylcholine; More preferably, the molar ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol and 1-palmitoyl-2-hydroxy-glycero-3-phosphatidylcholine is 1:1:0.

5.

3. The light-controlled cell membrane chimeric multi-unit drug-loaded liposome according to claim 1, characterized in that: The therapeutic drugs targeting tumor stem cells include one or more of curcumin, MG-132, and idarubicin; Preferably, the therapeutic drug targeting tumor stem cells is curcumin; Preferably, the therapeutic drug targeting tumor-associated macrophages includes one or more of trabectedin, Pexidartinib, BLZ945, and SNDX-6352; Preferably, the therapeutic drug targeting tumor-associated macrophages is trabectedin; Preferably, the therapeutic drug targeting differentiated tumor cells comprises one or more of doxorubicin hydrochloride, temozolomide, lomustine, vincristine, and irinotecan; Preferably, the therapeutic drug targeting differentiated tumor cells is doxorubicin hydrochloride; Preferably, the first shell layer of the first drug-carrying unit is vitamin E succinate-grafted-polylysine; Preferably, the second shell layer of the second drug-carrying unit is natural silk fibroin; Preferably, the third shell layer of the third drug-carrying unit is dopamine-grafted-γ-polyglutamic acid.

4. The light-controlled cell membrane chimeric multi-unit drug-loaded liposome according to any one of claims 1 to 3, characterized in that: The average particle size of the light-controlled cell membrane chimeric multi-unit drug-loaded liposome is 210 nm; and the Zeta potential of the light-controlled cell membrane chimeric multi-unit drug-loaded liposome is -26 mV.

5. A method for preparing the light-controlled cell membrane chimeric multi-unit drug-loaded liposome according to any one of claims 1 to 4, characterized in that: The method comprises: a) dissolving the temperature-sensitive lipid material and the photosensor in an organic solvent to obtain an organic phase; b) sequentially stacking the aqueous dispersions of the first drug-loading unit, the second drug-loading unit, and the third drug-loading unit into the organic phase using a reverse evaporation method to obtain a tertiary stacked lipid film; adding distilled water to hydrate the film, ultrasonically dispersing the film, and then extruding the film to obtain a light-controlled multi-unit drug-loaded liposome; c) coating the cancer cell membrane within the lipid bilayer of the light-controlled multi-unit drug-loaded liposome obtained above using biomembrane fusion technology and extrusion method, thereby obtaining the light-controlled cell membrane chimeric multi-unit drug-loaded liposome; Preferably, vitamin E succinate-grafted-polylysine is used as the material to encapsulate curcumin to construct the first drug-carrying unit; Preferably, natural silk fibroin is used as a material to encapsulate trabectedin to construct the second drug-carrying unit; Preferably, dopamine-grafted-γ-polyglutamic acid is used as a material to compound doxorubicin hydrochloride to construct the third drug-carrying unit.

6. The method according to claim 5, characterized in that The specific steps of obtaining the triple stacked lipid film in step b) are: Slowly dripping the first drug-loaded unit aqueous dispersion into the organic phase described in step a), stirring and emulsifying, ultrasonicating in an ice bath, and rotary evaporating to obtain a primary lipid film; The primary stacked lipid film obtained above is dissolved in an organic solvent to obtain an organic phase; the second drug-loaded unit aqueous dispersion phase is then slowly dripped into the organic phase, stirred for emulsification, ultrasonicated in an ice bath, and rotary evaporated to obtain a secondary stacked lipid film; The secondary stacked lipid film obtained above is dissolved in an organic solvent to obtain an organic phase; the third drug-loaded unit aqueous dispersion phase is then slowly dripped into the organic phase, stirred for emulsification, ultrasonicated in an ice bath, and rotary evaporated to obtain a tertiary stacked lipid film.

7. The method according to claim 5, characterized in that The specific steps of obtaining the light-controlled cell membrane chimeric multi-unit drug-loaded liposomes in step c) are as follows: mixing the cancer cell membrane with the light-controlled multi-unit drug-loaded liposomes obtained in step b), incubating at low temperature, loading the incubated sample into a liposome micro-extruder, and passing it through 1 μm, 400 nm, and 100 nm polycarbonate membranes in sequence, extruding back and forth 10 times each time, to obtain the light-controlled cell membrane chimeric multi-unit drug-loaded liposomes; Preferably, the low temperature is 4°C; Preferably, the low-temperature incubation time is 2 hours.

8. The method according to claim 5, characterized in that The thermosensitive lipid material is a combination of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol and 1-palmitoyl-2-hydroxy-glycero-3-phosphatidylcholine; Preferably, the molar ratio of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol and 1-palmitoyl-2-hydroxy-glycero-3-phosphatidylcholine is 1:1:0.

5. Preferably, the organic solvent is selected from any one or more combinations of dichloromethane, methanol, ethanol, acetone, acetonitrile, and diethyl ether; Preferably, the organic solvent is dichloromethane.

9. The method according to any one of claims 5 to 8, characterized in that: The specific method for constructing the first drug-loading unit is as follows: vitamin E succinate-grafted-polylysine and curcumin are dissolved in DMSO to prepare an organic phase, the organic phase is added dropwise to distilled water under stirring for self-assembly, DMSO is removed by dialysis with distilled water, and unencapsulated curcumin is removed by ultracentrifugation to obtain the first drug-loading unit; Preferably, the grafting degree of the vitamin E succinate-grafted-polylysine is 50%, the dialysis time is 12 h, and the speed of the ultracentrifugation is 10000 rpm; Preferably, the specific method for constructing the second drug-carrying unit is: dissolving trabectedin in a silk fibroin solution as an aqueous phase, slowly dripping the aqueous phase into acetone under stirring for self-assembly, removing the acetone by rotary evaporation, and removing larger particles by ultracentrifugation, thereby obtaining the second drug-carrying unit; Preferably, the concentration of the silk fibroin solution is 2%; the speed of dripping the aqueous phase into acetone is 50 μL / drop; the speed of the ultracentrifugation is 10000 rpm; Preferably, the specific method for constructing the third drug-carrying unit is as follows: dopamine-grafted-γ-polyglutamic acid and doxorubicin hydrochloride are dissolved in distilled water, adjusted to pH 7.0 with NaOH solution, stirred at room temperature for a period of time, and then the complex is slowly dripped into Tris base buffer and continued to stir and cross-link, and unloaded doxorubicin hydrochloride is dialyzed with distilled water to obtain the third drug-carrying unit; Preferably, the time for the compounding under stirring at room temperature is 10 h.

10. Use of the light-controlled cell membrane chimeric multi-unit drug-loaded liposome according to any one of claims 1 to 4 in the preparation of anti-tumor drugs; Preferably, the tumor comprises glioma, gastric cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, colon cancer, rectal cancer, prostate cancer, blood cancer, lymphoma, cervical cancer, osteosarcoma, melanoma, pancreatic cancer, liver cancer, kidney cancer, gallbladder cancer or bile duct cancer; Preferably, the tumor is a glioma.

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