CN114903870B - Engineered cell membrane nano-particles and preparation method and application thereof - Google Patents

Abstract

The invention discloses an engineering cell membrane nanoparticle, a preparation method and application thereof, wherein functional proteins can be expressed on an engineering cell membrane through genetic engineering design, and small molecular drugs are entrapped in the engineering cell membrane, and the entrapped small molecules can be chemotherapeutic drugs or small molecular drugs cooperated with the proteins on the cell membrane. According to the invention, the fibroblast is selected as a source cell of a cell membrane vesicle, tumor therapeutic protein TRAIL is expressed on an engineering cell membrane, the expression of death receptor DR5 of TRAIL can be up-regulated through chloroquine entrapped by lactic acid-glycolic acid copolymer, and the capability of TRAIL for inducing tumor cell apoptosis is synergistically enhanced; the CQ can specifically inhibit the endocytosis of the macrophage on the nano-particles, further optimize the distribution of cell membrane encapsulation carriers at tumor sites, and is expected to be used for tumor treatment. The preparation method is simple, convenient and controllable, and has good application prospect.

Description

Engineered cell membrane nano-particles and preparation method and application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to an engineering cell membrane nanoparticle and a preparation method and application thereof.

Background

In recent years, nano-carriers have been developed into the field of clinical application of tumors, and a great number of carriers pass preclinical researches every year, but few nano-systems pass clinical experiments. The biggest obstacle limiting the clinical transformation of nanocarriers is the delivery efficiency, and nanocarriers that are injected into the blood intravenously face a variety of biological barriers such as mononuclear phagocyte systems and blood brain barriers during circulation, high interstitial pressures and extracellular matrix in the microenvironment, obstruction of cell membranes or nuclear membranes at the time of entry into the cell or nucleus, and the like. Among the barriers that have the greatest impact on delivery efficiency are the mononuclear phagocyte systems. The mononuclear phagocyte system includes monocytes present in the blood circulation and tissue resident macrophages distributed in the tissue, the primary physiological effect of which is the removal of foreign substances. For coping with the entrapment of the mononuclear phagocyte system on the nano-carrier, the delivery system can be designed from two angles, namely, the physical and chemical properties of the nano-particles are improved and the endocytosis capacity of the mononuclear phagocyte system is regulated. In general, to avoid rapid excretion based on surface properties, many nanoparticle formulations incorporate hydrophilic polymers as a stealth coating, such as zwitterionic polymers like polyethylene glycol, polyamino acids, polyvinylpyrrolidone, polyglycerol, etc. However, these modification methods lack targeting on the one hand, and on the other hand, hydrophilic polymers represented by PEG are immunogenic after multiple injections and still are rapidly cleared.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and provides an engineering cell membrane nanoparticle and a preparation method and application thereof.

The aim of the invention is realized by the following technical scheme:

in a first aspect, the present invention provides a method for preparing engineered cell membrane nanoparticles comprising the steps of:

(1) Preparation of LX2cell line stably expressing TRAIL protein: culturing LX2 cells in the logarithmic growth phase until the cells adhere to the wall; then, transfecting LX2 cells by using a culture medium containing Lv-TRAIL-zsgreen over-expression lentivirus, culturing for 24 hours at 37 ℃, then, replacing the culture medium containing the Lv-TRAIL-zsgreen over-expression lentivirus by using a DMEM culture medium for further culturing for 48 hours, and then, performing resistance screening by using a G418 culture medium containing 200 mu mol/L to obtain a LX2cell line which can stably express TRAIL protein by stable transfection;

(2) Preparation of engineered cell membranes: washing the LX2cell line which is prepared in the step (1) and stably expresses TRAIL protein with PBS for three times at 4 ℃, re-suspending with hypotonic cell lysate, then lysing for 4-6h on a shaker at 4 ℃, and then using a probe with output power of 195-260W for 1-3min; then, differential centrifugation is carried out to extract cell membrane precipitation; finally, re-suspending the proposed cell membrane sediment by using PBS, and extruding the cell membrane suspension back and forth from the polycarbonate membrane for 9-14 times by a micro extruder to obtain an engineering cell membrane;

(3) Preparing a nanoparticle core: the chloroquine and lactic acid-glycolic acid copolymer are mixed according to the mass ratio of 1-1.5:10 in dry dichloromethane, the aqueous phase is prepared by dissolving polyvinyl alcohol in deionized water, and 4g of polyvinyl alcohol is added into every 100ml of deionized water; then ultrasonic emulsification is carried out on ice, the ultrasonic condition is that the output power is 260W, and the duration is 10min; slowly dripping the white emulsion formed by ultrasonic emulsification into 12-14mL of single distilled water, and stirring for 4-5h at room temperature to volatilize dichloromethane and solidify nanoparticle cores; finally, collecting nanoparticle cores after ultrafiltration centrifugation at 3000rpm for 30min;

(4) Preparing engineered cell membrane nanoparticles: the engineering cell membrane prepared in the step (2) and the nanoparticle core prepared in the step (3) are mixed according to the mass ratio of 1:1, extruding the blend liquid back and forth from a 200 nm-aperture polycarbonate membrane for 9-13 times by a micro extruder to obtain the engineering cell membrane nano particles.

Further, the differential centrifugation in step (2) includes: firstly, centrifuging for 10-15min under the centrifugal force of 1,000-1500g and the centrifugal condition of 4 ℃, and taking supernatant; centrifuging supernatant at 10000-12000g centrifugal force and 4deg.C for 30-40min; finally, the mixture is centrifuged for 1 to 3 hours under the centrifugal force of 900000 to 100000g and the centrifugal condition of 4 ℃.

Further, the hypotonic cell lysate comprises 0.25 XPBS, a protease inhibitor, and a phosphatase inhibitor.

Further, the polycarbonate membrane in the step (2) has a pore size of 50nm, 100nm, 200nm or 400nm.

Further, the polycarbonate membrane pore size is preferably 200nm.

In a second aspect, the present invention provides an engineered cell membrane nanoparticle prepared by the above method.

In a third aspect, the invention provides an application of an engineered cell membrane nanoparticle in preparing a drug for treating tumor.

The beneficial effects of the invention are as follows: 1. the engineering cell membrane provided by the invention takes the fibroblast as a main source cell, and the fibroblast is taken as a normal somatic cell, so that the function of targeting tumor cells can be achieved due to the heterophilic adhesion protein expressed on the cell membrane; after stably transfecting a virus vector designed by genetic engineering, the fibroblast can express therapeutic protein on a cell membrane, so that the cell membrane nanovesicle can be endowed with greater therapeutic potential, and the protein is conveyed through a stable membrane structure, so that the half-life period of the protein in blood circulation can be prolonged, and the biological activity of the functional protein is effectively maintained;

2. the outer layer of the engineering cell membrane nanoparticle is wrapped by the engineering cell membrane, so that the nanoparticle has more optimized surface physicochemical properties, and opsonin and complement activation caused by exogenous nanoparticles are reduced to further cause the removal of the nanoparticle; meanwhile, the endocytic activity of the mononuclear phagocyte system on the nano-particles can be reduced by the small molecular medicine encapsulated by the inner core, so that the non-specific clearance of the nano-particles can be reduced on the other hand; in order to achieve better treatment purpose, the small molecular medicine encapsulated by the inner core is selected from medicines which can have synergistic effect with therapeutic proteins expressed on outer cell membranes; the introduction of the nano-particles not only greatly improves the conveying efficiency of the nano-particles, but also produces an addition effect on the therapeutic effect of the nano-particles.

Drawings

FIG. 1 is a schematic diagram of the preparation and structure of engineered cell membrane nanoparticles (TM-CQ/NP);

FIG. 2 is a view of confocal laser microscopy;

FIG. 3 is a flow cytometer detection diagram;

FIG. 4 is a Western blot assay;

FIG. 5 is a transmission electron microscope image of an engineered cell membrane;

FIG. 6 is a dynamic light scattering diagram of CQ/NPs and TM-CQ/NPs;

FIG. 7 is a graph showing the effect of detecting TM-CQ/NP and M-NP, TM-NP, CQ/NP, and M-CQ/NP on inducing apoptosis by crystal violet;

FIG. 8 is a graph showing endocytosis levels of NP, M-NP, and CQ+M-NP on Kupffer cells, FIG. 8 shows a confocal laser microscopy image, and FIG. 8 shows a flow cytometer measurement image;

FIG. 9 is a graph showing the distribution of NP and M-NP in each organ after 6h of tail vein injection detected by a small animal in vivo imaging method;

FIG. 10 is a graph showing the average fluorescence intensity of RhoB-positive cells in liver cancer cells and Kupffer cells, FIG. 10 a is a graph showing the average fluorescence intensity of RhoB-positive cells in liver cancer cells, and FIG. 10 b is a graph showing the average fluorescence intensity of RhoB-positive cells in Kupffer cells;

FIG. 11 is a graph showing the distribution of NP, M-NP, and CQ+M-NP in each organ after 6h of tail vein injection;

FIG. 12 is a graph showing the distribution of different liver cell populations of NP, M-NP, and CQ+M-NP on an in situ model of liver cancer, wherein FIG. 12, panel A, FIG. 12, panel B, FIG. 12, panel C, FIG. 12, and FIG. 12, respectively, is a flow scatter plot of the uptake of nanoparticles by Kupffer cells collected in the liver, a flow histogram of the uptake of nanoparticles by Kupffer cells collected in the liver, and a flow histogram of the uptake of nanoparticles by liver tumor cells;

FIG. 13 is a graph showing tumor inhibition of PBS group, TM-NP group, CQ/NP group, and TM-CQ/NP group on an in situ model of liver cancer;

FIG. 14 is a statistical plot of tumor weights of PBS group, TM-NP group, CQ/NP group, and TM-CQ/NP group on an in situ model of liver cancer.

Detailed Description

For the purposes of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and examples, it being understood that the specific examples described herein are for the purpose of illustrating the present invention only, and not all the examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are within the scope of the present invention.

FIG. 1 is a schematic diagram showing the preparation and structure of the engineered cell membrane nanoparticle (TM-CQ/NP) of the present invention.

The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.

Example preparation of engineered cell membrane nanoparticles (TM-CQ/NP)

(1) Preparation of LX2cell line (LX 2-TRAIL-zsgreen) stably expressing TRAIL protein

Inoculating LX2 cells in the logarithmic growth phase into a six-hole plate according to the number of 100000 cells/hole, and culturing for 12 hours until the cells adhere to the wall; then, LX2 cells are transfected by using a culture medium containing Lv-TRAIL-zsgreen over-expression lentivirus, after culturing for 24 hours at 37 ℃, the culture medium containing the Lv-TRAIL-zsgreen over-expression lentivirus is replaced by a fresh culture medium for further culturing for 48 hours, at the moment, the cell fusion degree in each hole reaches about 90%, and then, resistance screening is carried out by using a G418 culture medium containing 200 mu mol/L, so that a LX2cell line (LX 2-TRAIL-zsgreen) which can stably express TRAIL protein by stable transfection is obtained.

The fresh culture medium is DMEM culture medium.

The Lv-TRAIL-zsgreen over-expression slow virus is from Shanghai Ji Kai gene medical science and technology Co., ltd, comprises a gene fragment capable of expressing TRAIL protein and a gene fragment capable of expressing zsgreen fluorescent protein.

The G148 is an aminoglycoside antibiotic and is the most commonly used resistance screening reagent for stable transfection in molecular genetic tests; the G418 culture medium is prepared by adding G418 into fresh culture medium.

Fig. 2 is a view of confocal laser microscopy. Respectively inoculating the untransfected LX2 cells and the LX2cell line (LX 2-TRAIL-zsgreen) which is prepared in the step (1) and stably expresses TRAIL protein into a confocal imaging dish, and culturing for 12 hours until the cells adhere to the wall; then adding 2 mu mol/L blue fluorescent dye (Hoechst 33342) into a confocal imaging dish for incubation, changing a culture medium containing the blue fluorescent dye into a fresh culture medium after 30min, and then observing and recording the expression conditions of the zsgreen fluorescent proteins of the two cells by using a laser confocal microscope; as can be seen from FIG. 2, the untransfected LX2 cells were non-fluorescent, while the LX2cell line stably expressing TRAIL protein expressed a large amount of green fluorescence, indicating that the LX2cell line stably expressing TRAIL protein prepared in step (1) was successfully transfected.

FIG. 3 is a flow cytometer detection diagram. Culturing untransfected LX2 cells and the LX2cell line (LX 2-TRAIL-zsgreen) stably expressing TRAIL protein prepared in the step (1) for 12 hours respectively, and then allowing the cells to adhere to the wall; then, the cells are resuspended by pancreatin digestion, washed twice with PBS at 4 ℃, and then resuspended in 10-6 cells/mL of cell suspension in a flow cell tube; and (3) analyzing the positive rate of zsgreen positive cells in the untransfected LX2 cells and the LX2cell line stably expressing TRAIL protein prepared in the step (1) respectively through a FL2 channel of a flow cytometer. As can be seen from FIG. 3, the ratio of successful transfection in the total amount of cells was calculated by counting cells expressing zsgreen fluorescent protein by flow cytometry, and the positive rate of the LX2cell line (LX 2-TRAIL-zsgreen) stably expressing TRAIL protein prepared in step (1) was detected by flow cytometry to be 42.4% compared to the untransfected LX2 cells.

(2) Preparation of engineered cell membranes (LX 2-zsgreen-trail cell membrane)

Washing the LX2cell line (LX 2-TRAIL-zsgreen) which is prepared in the step (1) and stably expresses TRAIL protein in PBS (phosphate buffer solution) at 4 ℃ for three times, re-suspending the cell line by using hypotonic cell lysate added with protease inhibitor, then performing lysis on a shaker at 4 ℃ for 4 hours, and then performing ultrasonic treatment for 1min by using a probe with output power of 195W; then, differential centrifugation is carried out to extract cell membrane precipitation; the differential centrifugation includes: firstly, centrifuging for 10min under the centrifugal force of 1000g and the centrifugal condition of 4 ℃, and taking supernatant; centrifuging supernatant at 10000g centrifugal force and 4deg.C for 30min; finally, under the centrifugal force of 100000g and the centrifugal condition of 4 ℃, the transparent cell membrane sediment is obtained after centrifugation for 1 h; finally, the proposed cell membrane pellet was resuspended in PBS, and the cell membrane suspension was squeezed back and forth 11 times from a 200nm pore size polycarbonate membrane by a micro-extruder to obtain an engineered cell membrane (LX 2-zsgreen-trail cell membrane) having a relatively uniform particle size.

The hypotonic cell lysate comprises 0.25 XPBS, a protease inhibitor and a phosphatase inhibitor.

FIG. 4 is a Western blot analysis. The membrane protein concentrations of LX2cell lysate (LX 2cell lysate, CL), LX2cell line lysate stably expressing TRAIL protein (LX 2-TRAIL-zsgreen CL), LX2cell membrane (LX 2cell membrane) and engineered cell membrane (LX 2-zsgreen-TRAIL cell membrane) were measured using BCA kit, respectively, and as shown in fig. 4, it was revealed that LX2cell line lysate stably expressing TRAIL protein (LX 2-TRAIL-zsgreen CL) and engineered cell membrane (LX 2-zsgreen-TRAIL cell membrane) were all capable of expressing TRAIL protein.

FIG. 5 is a transmission electron microscope image of an engineered cell membrane; it can be seen from fig. 5 that the engineered cell membrane has a classical core-shell structure.

(3) Preparation of nanoparticle inner cores (CQ/NPs)

The preparation of the nanoparticle inner core (CQ/NP) adopts a single emulsification method, and the preparation process comprises the following steps: 10mg of Chloroquine (CQ) and 100mg of lactic-co-glycolic acid (PLGA) are blended in 1mL of anhydrous methylene chloride, and 4% polyvinyl alcohol (PVA) is used as an aqueous phase, wherein the aqueous phase is prepared by dissolving polyvinyl alcohol in deionized water, and 4g of polyvinyl alcohol is added to every 100mL of deionized water; then emulsifying on ice by using probe ultrasound under the condition that the output power is 260W for 10min; the white emulsion formed by phacoemulsification was slowly added dropwise to 14mL of single distilled water, stirred at room temperature for 5h to volatilize the dichloromethane and solidify the nanoparticle core (CQ/NP). Finally, collecting nanoparticle inner cores (CQ/NP) after ultrafiltration centrifugation at 3000rpm for 30min; the Chloroquine (CQ) is a small molecule drug.

(4) Preparation of engineered cell membrane nanoparticles (TM-CQ/NP)

Blending 1mg of the engineering cell membrane (LX 2-zsgreen-trail cell membrane) prepared in the step (2) and 1mg of the nanoparticle inner core (CQ/NP) prepared in the step (3), and extruding the blending liquid back and forth from the 200 nm-aperture polycarbonate membrane for 11 times through a micro extruder to obtain the engineering cell membrane nanoparticle (TM-CQ/NP).

FIG. 6 is a transmission electron microscopy image of nanoparticle cores (CQ/NPs) and engineered cell membrane nanoparticles (TM-CQ/NPs). Diluting the nanoparticle inner core (CQ/NP) obtained in the step (3) to a concentration of 1mg/mL, diluting the engineering cell membrane nanoparticle (TM-CQ/NP) obtained in the step (4) to a concentration of 1mg/mL, respectively detecting the particle size distribution of the CQ/NP and the TM-CQ/NP by using a dynamic light scattering instrument (DLS), and photographing a transmission electron microscope image as shown in figure 6. CQ/NP particle size was approximately 221.5nm as measured by dynamic light scattering, and polymer dispersion index (Polymer dispersity index, PDI) was 0.179; the TM-CQ/NP particle size was about 254 nm and the PDI was 0.268. It can be seen from FIG. 6 that CQ/NP prepared by single emulsification method is uniform round particles; the TM-CQ/NP can see a distinct core-shell structure and is uniform in particle size, indicating that the engineered cell membrane has been completely encapsulated outside the CQ/NP nanoparticle.

Application example 1

(1) Cytotoxicity test

The crystal violet dyeing detection process comprises the following steps: inoculating Huh7 cells into a 12-well plate according to the number of 5X 10-4 cells/well, culturing for 12 hours, and standing for cell attachment; huh7 cells were treated with medium containing cell membrane-coated empty nanoparticle (nanoparticles coated with fibroblast membrane, M-NP), TRAIL protein-expressing cell membrane-coated empty nanoparticle (nanoparticles coated with TRAIL-expressing fibroblast membrane, TM-NP), nanoparticle inner core (CQ-encapsulated nanoparticles, CQ/NP), cell membrane-coated CQ-loaded nanoparticle (CQ-encapsulated nanoparticles coated with fibroblast membrane, M-CQ/NP), engineered cell membrane nanoparticle (CQ-encapsulated nanoparticles coated with TRAIL-expressing fibroblast membrane, TM-CQ/NP) for 24h as experimental group, and Huh7 cells were treated with fresh medium for 24h as control group (control); then, respectively discarding the culture medium containing M-NP, TM-NP, CQ/NP, M-CQ/NP or TM-CQ/NP, washing twice with PBS solution, pouring the plates on filter paper, and draining; to each well of the 5 experimental groups (M-NP, TM-NP, CQ/NP, M-CQ/NP, and TM-CQ/NP) and 1 control group (control) was added 50. Mu.L of crystal violet staining solution and incubated on a shaker at room temperature for 20min at a frequency of 20 oscillations per minute. Discarding crystal violet staining solution in the plate, washing with PBS for four times, pouring the plate on filter paper, draining water, and observing cells under a microscope; the detection results are shown in FIG. 7.

FIG. 7 is a graph showing the effect of detecting the apoptosis induced by TM-CQ/NP and M-NP, TM-NP, CQ/NP, and M-CQ/NP by crystal violet staining solution. As can be seen from FIG. 7, the number of Huh7 cells in the M-NP treated group was significantly reduced compared to the control group (control) without any treatment, indicating that TRAIL cell membrane itself was toxic to tumor cells; meanwhile, the Huh7 cells treated by CQ/NP and M-CQ/NP are compared with a control group (control) which does not carry out any treatment, so that the number of the Huh7 cells in the CQ/NP and M-CQ/NP experimental groups is reduced to a certain extent, which indicates that the CQ/NP and M-CQ/NP can kill tumor cells to a small extent, and the toxicity of the CQ/NP and the M-CQ/NP are almost the same, which indicates that the LX2cell membrane wrapping CQ/NP has no effect of increasing killing effect; the minimum Huh7 cell number after treatment of the TM-CQ/NP experimental group shows that the cytotoxicity of the TM-CQ/NP is maximum, and also shows that Chloroquine (CQ) can synergistically enhance the tumor cell killing effect of TRAIL.

(2) Endocytosis assay of Chloroquine (CQ) on nanoparticle inner core (CQ/NP) on Kupffer cells

Observing endocytosis of the nano-carrier by using a laser confocal microscope: kupffer cells (macrophages in liver blood sinuses) are inoculated into a confocal imaging dish according to the quantity of 10 times per dish, and incubated for 12 hours until the cells adhere to the wall; after Kupffer cell attachment, the cells were divided into 3 groups, namely NP, M-NP and cq+m-NP; in the NP group, the attached Kupffer cells were first treated in a medium containing 50. Mu. Mol/L Chloroquine (CQ) at 37℃for 24 hours, followed by treatment with 1mg NP for 60 minutes; in the M-NP group, the Kupffer cells after adherence are firstly treated for 24 hours at 37 ℃ in a fresh culture medium, and then treated for 60 minutes after 1mg of M-NP is added; in the CQ+M-NP group, the attached Kupffer cells were first treated in a medium containing 50. Mu. Mol/L Chloroquine (CQ) at 37℃for 24 hours, followed by treatment with 1mg of M-NP for 60 minutes; the NP group, M-NP group and CQ+M-NP group were all treated with RhoB fluorochromes; after 60min of treatment with NP, M-NP, and CQ+M-NP, the cells were gently washed twice with PBS, and then endocytosis of the Kupffer cells in the NP, M-NP, and CQ+M-NP groups was observed and recorded using a laser confocal microscope.

Flow cytometry detects endocytosis of nanocarriers: inoculating Kupffer cells into a confocal imaging dish according to the quantity of 10 times of 5 cells per dish, and incubating for 12 hours until the cells adhere to the wall; after Kupffer cell attachment, the cells were divided into 3 groups, namely NP, M-NP and cq+m-NP; in the NP group, the attached Kupffer cells were first treated in a medium containing 50. Mu. Mol/L Chloroquine (CQ) at 37℃for 24 hours, followed by treatment with 1mg NP for 60 minutes; in the M-NP group, the Kupffer cells after adherence are firstly treated for 24 hours at 37 ℃ in a fresh culture medium, and then treated for 60 minutes after 1mg of M-NP is added; in the CQ+M-NP group, the attached Kupffer cells were first treated in a medium containing 50. Mu. Mol/L Chloroquine (CQ) at 37℃for 24 hours, followed by treatment with 1mg of M-NP for 60 minutes; the NP, M-NP, and CQ+M-NP groups were all treated with RhoB fluorochrome, cells were gently washed twice with PBS, and cells were resuspended in 400. Mu.L of pre-chilled PBS by trypsin digestion; the average fluorescence intensity of RhoB positive cells after the drug addition treatment of NP group, M-NP group and CQ+M-NP group was analyzed by FL2 channel of flow cytometer, and 1X 10≡4 cells in the living cell population were quantitatively selected for detection for each sample. Each set of data is the average of three independent experiments with the same cells treated in the same manner.

FIG. 8 is a graph showing endocytosis levels of NP, M-NP, and CQ+M-NP on Kupffer cells, FIG. 8 shows a graph of confocal laser microscopy, and FIG. 8 shows a graph of flow cytometry. As can be seen from FIG. 8, panel A shows that the fluorescence intensity of RhoB in Kupffer cells treated with M-NP group is significantly weaker than that of Kupffer cells treated with NP group; in the CQ+M-NP group, kupffer cells treated with the cell membrane-coated empty nanoparticle (CQ+M-NP) after CQ culture were weaker. As can be seen from FIG. 8, panel B, the fluorescence intensity (MFI) of RhoB in Kupffer cells treated with the M-NP group was significantly lower than that of the NP group; in the CQ+M-NP group, kupffer cells treated with the cell membrane-coated empty nanoparticle (CQ+M-NP) after CQ culture were weaker. It was demonstrated that the uptake of cell membrane-coated empty nanoparticles (CQ+M-NP) by Kupffer cells after CQ culture was further reduced.

(2) Organ distribution and liver cell population distribution experiment of nanocarriers

200. Mu.L of Nanoparticles (Nanoparticles, NP) or M-NP (NP and M-NP, respectively, were treated with DiR fluorochromes at a concentration of 4. Mu.g/min) were injected into ICR mice (female, 8 weeks old) via the tail vein, the mice were anesthetized at 6h via the orbital vein Cong Caixie (about 50. Mu.L), whole blood was collected in heparin sodium tubes dedicated to blood collection, and then sacrificed by neck-drawing. The Heart (Heart), liver (Liver), spleen (Spleen), lung (Lung) and Kidney (Kidney) were dissected, weighed, 10 μl of 5% phenylmethylsulfonyl fluoride (PMSF) in PBS was added to 1mg organ mass, 2-3 beads were added to each 1.5ml centrifuge tube, and the tubes were then milled for 5min at 4 ℃. The resulting tissue grinding fluid was added to a black 96-well plate together with blood, and the fluorescence intensity of each well sample in the plate was measured by in vivo imaging of a small animal, and the results are shown in FIG. 9. As can be seen from FIG. 9, the fluorescence intensity of the liver is significantly stronger than that of other organs, indicating that NP and M-NP are mainly concentrated in the liver.

The phenylmethylsulfonyl fluoride (PMSF) is an irreversible serine protease inhibitor.

After grouping nude mice two weeks after successful inoculation of liver cancer cells in situ, 200 mu L of PBS, nano Particles (NP) or empty nano particles (M-NP) coated by cell membranes are respectively injected through tail veins, wherein the PBS, the NP and the M-NP are respectively treated by RhoB fluorescent dye, and the concentration of the RhoB fluorescent dye is 1 mu g/mouse; PBS was injected as a control group and NP or M-NP was injected as an experimental group; anesthetizing a mouse by using hydrated chloral, injecting the mouse for 6 hours, separating the mouse by using magnetic beads after a dispersed liver cell group is proposed, respectively detecting the average fluorescence intensity (MFI) of RhoB positive cells by using liver cancer cells (tumor cells) and Kupffer cells obtained after separation through a flow cytometer FL2 channel (488 nm wavelength laser and a 585/42nm filter), quantitatively selecting 5 0000 cells in a living cell group for detection by using each sample, and detecting the detection results are shown in figure 10; panel A in FIG. 10 shows the mean fluorescence intensity of RhoB-positive cells in Kuffer cells, and panel B in FIG. 10 shows the mean fluorescence intensity of RhoB-positive cells in tumor cells.

As can be seen from FIG. 10, panels A and B, the average fluorescence intensity of RhoB-positive cells in the Kupffer and hepatoma cell groups was significantly higher in the experimental group injected with NP or M-NP than in the control group injected with PBS, indicating significant aggregation of NP and M-NP in both the Kupffer and hepatoma cells. The fluorescence intensity of M-NP group is weaker than that of NP group in Kupffer cell group, which shows that the nonspecific accumulation of the membrane-coated empty nanoparticle (M-NP) -coated nanoparticle in liver is reduced to a certain extent, while the difference in liver cancer cell group is not obvious

FIG. 11 shows the distribution of NP, M-NP, and CQ+M-NP in each organ after 6h of tail vein injection. As can be seen from fig. 11, the nonspecific accumulation of the empty nanoparticle (M-NP) coated with the cell membrane in the liver is significantly reduced, and the circulation time of the nanoparticle in the blood can be prolonged by the cell membrane coating; non-specific accumulation of cell membrane-coated empty nanoparticles (cq+m-NP) in the liver after CQ culture was further reduced.

FIG. 12 is a graph showing the distribution of NP, M-NP, and CQ+M-NP among different hepatocyte populations on an in situ model of liver cancer; FIG. 12A is a flow scattergram of the uptake of nanoparticles by Kupffer cells collected from the liver of tumor-bearing mice 6 hours after injection of PBS, nanoparticles (NP, M-NP); panel B is a flow histogram of the uptake of nanoparticles by Kupffer cells collected from the liver of tumor-bearing mice 6 hours after injection of PBS, nanoparticles (NP, M-NP). Panel C is a flow scattergram of nanoparticle uptake in tumor-bearing mouse liver tumor cells 6 hours after PBS, nanoparticle (NP, M-NP) injection; panel D is a flow histogram of nanoparticle uptake in tumor-bearing mouse liver tumor cells 6 hours after PBS, nanoparticle (NP, M-NP) injection. Graph a abscissa FSC is the forward angle scatter, whose value represents the cell size. The greater the cell volume, the greater the FSC value, and the ordinate represents the fluorescence intensity, and the Control group (Kuffer cells injected with PBS), the endocytosis of nanoparticles by Kuffer cells without CQ treatment (NP group and M-NP group), and the endocytosis of nanoparticles by Kuffer cells with CQ treatment (CQ+M-NP group) were compared by detecting the fluorescence intensity of RhoB carried by PBS, NP, and M-NP. The successful endocytosis of the nanoparticles of Control group to generate fluorescence resulted in a cell positive rate of 1.97%, 10.7% for NP group, 6.59% for M-NP group, 2.77% for cq+m-NP group, indicating that M-NP and cq+m-NP treatment significantly reduced the uptake of nanoparticles by Kupffer cells. Whereas, in panel B, the endocytosis of M-NP and CQ+M-NP was found to be significantly lower than in the NP group by more visual comparison of the histograms, there was a significant statistical difference. As can be seen from fig. 12, panels a and B, endocytosis of the nanoparticles wrapping the cell membrane by the Kupffer cells was significantly reduced, and endocytosis of the nanoparticles by the Kupffer cells after CQ treatment was further reduced. From fig. 12, panels C and D, tumor cells (Tumor cells) did not significantly increase M-NP, but after CQ treatment, tumor cells significantly increased endocytosis of nanoparticles, suggesting that CQ may further optimize the distribution of M-NP in the hepatocyte population, increasing accumulation in Tumor cells.

(3) Tumor inhibition experiment

Nude mice after two weeks of in situ inoculation of liver cancer cells were randomly divided into 4 groups, namely PBS group, TM-NP group, CQ/NP group and TM-CQ/NP group, each group of nude mice was inoculated with Huh7-luci cells, administration was started after 14 th day of Huh7-luci cell inoculation, and each group of tumor-bearing mice was respectively injected with 200. Mu.L of PBS-, TM-NP-, CQ/NP-or TM-CQ/NP-containing solution by tail vein, calculated according to the body weight of nude mice. The administration is carried out once every two days, and the whole treatment period is carried out five times, and the progress of the liver cancer of the nude mice is indirectly observed through a small animal living body imager. On day 15 of dosing, after the animals were sacrificed according to ethical requirements, the tumors were then peeled off and weighed separately.

FIG. 13 is a graph showing tumor inhibition of PBS group, TM-NP group, CQ/NP group, and TM-CQ/NP group on an in situ model of liver cancer. As can be seen from FIG. 13, the tumors of the PBS group increased significantly to about four times after 15 days, and the CQ/NP group and the TM-NP group increased 2-3 times, compared with the PBS group, the tumors of the TM-CQ/NP group showed little increase, indicating that the TM-CQ/NP group had significant anticancer activity relative to the PBS, CQ/NP and TM-NP groups.

FIG. 14 is a statistical plot of tumor weights of PBS group, TM-NP group, CQ/NP group, and TM-CQ/NP group on an in situ model of liver cancer. The body weight of each group of nude mice was counted and analyzed for statistical differences. From fig. 14, it can be seen that the tumor weight of the TM-CQ/NP group was significantly lower than that of the PBS, CQ/NP and TM-NP groups, and the tumor-inhibiting effect was significant. The PBS, CQ/NP and TM-NP groups did not differ significantly from the TM-CQ/NP groups, indicating that the therapeutic effect of the TM-CQ/NP group was significantly better than the other groups.

The engineering cell membrane nano-particles prepared by the engineering cell membrane nano-particles preparation method can be used for preparing medicines for treating tumors.

The invention adopts engineering cell membrane (LX 2-zsgreen-trail cell membrane) to wrap nanoparticle inner core (CQ/NP), and the engineering cell membrane can reduce activation of opsonin and complement caused by exogenous nano-carrier. The engineered cell membrane encapsulates the nanoparticle core providing a novel biomimetic platform that also mimics the function of the source cell in interacting with surrounding biological components. Fibroblasts express biologically active adhesion proteins on the cell membrane and can be associated with their metastasis by forming heterophilic linkages with adhesion proteins on tumor cells. When the nano particles are coated into a fiber cell membrane, the phospholipid bilayer structure is obtained, and meanwhile, the heterogeneous targeting property is possessed, so that the nano particles can be possibly used as a material for targeting tumor sites. In order to reduce the entrapment of the mononuclear phagocyte system on the nano-carrier, the endocytosis capability of the mononuclear phagocyte system is regulated by adopting empty carriers or small molecular drugs, so that the phagocytosis of the mononuclear phagocyte is saturated or the endocytosis capability is inhibited. CQ is a commonly used inhibitor of phagocytic activity of viruses by inhibiting the production of clathrin, which inhibits the major endocytic pathway of the virus into the cell. At the same time, CQ has also been shown to be an endocytosis inhibitor specific for macrophages. The fusion of the two design methods for nanocarriers and mononuclear phagocyte systems in the same vector was not attempted by previous studies.

Based on the cell membrane coating technology, the structure of the cell membrane is utilized, and some functional therapeutic proteins are expressed on the cell membrane through genetic engineering design, so that the application range of a cell membrane carrier platform can be further widened. TRAIL, a tumor necrosis factor-related apoptosis-inducing ligand, is a type two transmembrane protein that binds to TRAIL receptors (death receptors) in the form of trimers, causing activation of the apoptotic pathway downstream of the cell. The CQ can up-regulate the expression of death receptor, can achieve the apoptosis effect of sensitized TRAIL, can be used for being transported together with small molecules which are synergistic with TRAIL, and provides a new idea for tumor treatment.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather to enable any modification, equivalent replacement, improvement or the like to be made within the spirit and principles of the invention.

Claims (6)

1. A method for preparing engineered cell membrane nanoparticles, comprising the steps of:

(1) Preparation of LX2cell line stably expressing TRAIL protein: culturing LX2 cells in the logarithmic growth phase until the cells adhere to the wall; then, transfecting LX2 cells by using a culture medium containing Lv-TRAIL-zsgreen over-expression lentivirus, culturing for 24 hours at 37 ℃, then, exchanging a culture medium containing the Lv-TRAIL-zsgreen over-expression lentivirus by using a DMEM culture medium for further culturing for 48 hours, and then, carrying out resistance screening by using a G418 culture medium containing 200 mu mol/L to obtain a LX2cell line which can stably express TRAIL protein by stable transfection; the Lv-TRAIL-zsgreen over-expression slow virus comprises a gene fragment capable of expressing TRAIL protein and a gene fragment capable of expressing zsgreen fluorescent protein;

(2) Preparation of engineered cell membranes: washing the LX2cell line which is prepared in the step (1) and stably expresses TRAIL protein in PBS (phosphate buffered saline) at 4 ℃ for three times, re-suspending the cell line by using hypotonic cell lysate, then lysing the cell line on a shaker at 4 ℃ for 4-6h, and then performing ultrasonic treatment by using a probe with output power of 195-260W for 1-3min; then, differential centrifugation is carried out to extract cell membrane precipitation; finally, re-suspending the proposed cell membrane sediment by using PBS, and extruding the cell membrane suspension back and forth from the polycarbonate membrane for 9-14 times by a micro extruder to obtain an engineering cell membrane;

(3) Preparing a nanoparticle core: the chloroquine and lactic acid-glycolic acid copolymer are blended in anhydrous dichloromethane according to the mass ratio of 1-1.5:10, the water phase is made of polyvinyl alcohol, the water phase is prepared by dissolving the polyvinyl alcohol in deionized water, and 4g of polyvinyl alcohol is added into every 100ml of deionized water; then ultrasonic emulsification is carried out on ice, the ultrasonic condition is that the output power is 260W, and the duration is 10min; slowly dripping the white emulsion formed by ultrasonic emulsification into 12-14mL single distilled water, stirring for 4-5 hours at room temperature to volatilize dichloromethane and solidify nanoparticle cores; finally, collecting nanoparticle cores after ultrafiltration centrifugation at 3000rpm for 30min;

(4) Preparing engineered cell membrane nanoparticles: the engineering cell membrane prepared in the step (2) and the nanoparticle core prepared in the step (3) are mixed according to the mass ratio of 1:1, extruding the blend liquid back and forth from a polycarbonate membrane with the aperture of 200nm for 9-13 times by a micro extruder to obtain the engineering cell membrane nano particles.

2. The method of claim 1, wherein the hypotonic cell lysate comprises 0.25 x PBS, protease inhibitor and phosphatase inhibitor.

3. The method of claim 1, wherein the polycarbonate membrane in step (2) has a pore size of 50nm, 100nm, 200nm or 400nm.

4. A method of preparing an engineered cell membrane nanoparticle according to claim 3, wherein the polycarbonate membrane pore size is preferably 200nm.

5. An engineered cell membrane nanoparticle obtained by the method of any one of claims 1-4.

6. Use of the engineered cell membrane nanoparticle of claim 5 in the preparation of a medicament for treating a tumor, the tumor being liver cancer.