A liposome enzyme nanoreactor and its preparation method and application in anti-tumor treatment

Abstract

A liposome enzyme nanoreactor and its preparation method and application in anti-tumor treatment belong to the field of bio-nanomedicine technology. The outer layer of the liposome enzyme nanoreactor is a liposome with good biocompatibility, and the interior contains glucose oxidase and horseradish peroxidase. The liposome enzyme nanoreactor causes glucose oxidase and horseradish peroxidase to undergo a cascade reaction after compartmentalization, which can effectively inhibit tumor growth in vivo and in vitro by inhibiting tumor nutrient supply and generating highly cytotoxic hydroxyl free radicals (·OH), leading to cancer cell apoptosis. Enzyme co-compartmentalization in the liposome increases the intermediate product hydrogen peroxide (H 2 O 2 ) and the local concentration of acid, further accelerating the second step of the horseradish peroxidase-mediated reaction of producing cytotoxic OH, so that the entire cascade reaction reaches a higher reaction efficiency. The liposome enzyme nanoreactor disclosed in the present invention has a highly efficient anti-tumor ability and has clinical potential for treating tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of bio-nanomedicine, and specifically relates to a liposome enzyme nanoreactor, a preparation method and an application in anti-tumor treatment. Background Art

[0002] Enzymes play an indispensable role in biological reactions and metabolic processes by reducing activation energy and significantly increasing the rate of chemical reactions. Cascade reactions involving multiple enzymes are the basis of signal transduction and metabolism (such as oxidative phosphorylation) in organisms. In eukaryotic cells, enzymes are compartmentalized in specific organelles and biomolecular condensates, providing a closed environment for multi-step reactions to complete the desired biological process, with the characteristics of high reaction efficiency and strong independence.

[0003] Most studies have focused on mimicking nature's compartmentalization strategies with synthetic systems. A variety of artificial cells and subcellular analogs, such as artificial cells and nanoorganelles, have been developed to mimic basic cellular structures and reactions. This compartmentalized separation not only retains the activity and function of enzymes, but also enables the assembly of target enzymes in different ways to mimic complex natural catalytic processes. However, the combination of different enzymes and the quantitative compartmentalization of enzymes and the realization of spatiotemporal control of cascade reactions remain challenging. In addition, research on the use of these artificial synthetic systems for biomedical applications is currently rare. Mesoporous silica nanocontainers are often used to encapsulate enzymes or as carriers for other drugs. However, in situ enzyme encapsulation requires harsh synthetic conditions during the silica formation process, which usually leads to reduced enzyme activity. Polymers are another option for capturing enzymes in the water core of enzyme nanoreactors. In this case, the synthesis of block copolymers requires a multi-step process. In addition, the biocompatibility of synthetic polymers remains another major issue for in vivo applications.

[0004] In order to solve the current problems, it is of great research significance to develop an enzyme nanoreactor with efficient enzyme cascade reaction capabilities for use in biomedicine and other fields. Summary of the invention

[0005] The present invention aims at the existing problems and provides a liposome enzyme nanoreactor, a preparation method, and the application of the liposome enzyme nanoreactor for anti-tumor treatment. The present invention does not require a complicated synthesis process and adopts a thin film dispersion method and an extrusion method to prepare a liposome enzyme nanoreactor (LNRs). The nanoreactor has a high-efficiency enzyme cascade reaction capability and has a prospect for clinical anti-tumor treatment.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is: a liposome enzyme nanoreactor comprises liposome; and glucose oxidase and horseradish peroxidase, which are compounded with the liposome.

[0007] The liposomes include L-α-phosphatidylcholine, cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0008] The loading amounts of GOx (glucose oxidase) and HRP (horseradish peroxidase) in the liposome enzyme nanoreactor are 1 to 1000 μg mg -1 The loading amount is the loading rate of glucose oxidase or horseradish peroxidase on the liposome.

[0009] Wherein, the fluid dynamics radius of the liposome enzyme nanoreactor is 1 to 1000 nm.

[0010] Wherein, the electromotive force of the liposome enzyme nanoreactor is -100 to 100 mW.

[0011] In some specific embodiments, the hydrodynamic radius of the liposome enzyme nanoreactor is 10 to 300 nm.

[0012] In some specific embodiments, the hydrodynamic radius of the liposome enzyme nanoreactor is 20 to 220 nm.

[0013] In some specific embodiments, the loading amounts of GOx (glucose oxidase) and HRP (horseradish peroxidase) in the liposome enzyme nanoreactor are 1 to 300 μg mg -1 .

[0014] In some specific embodiments, the loading amounts of GOx (glucose oxidase) and HRP (horseradish peroxidase) in the liposome enzyme nanoreactor are 10 to 300 μg mg -1 .

[0015] The method for preparing the liposome enzyme nanoreactor adopts liposome, glucose oxidase and horseradish peroxidase to prepare the liposome enzyme nanoreactor through thin film dispersion method and extrusion method.

[0016] The specific preparation method of the liposome enzyme nanoreactor comprises the following steps:

[0017] 1) dissolving L-α-phosphatidylcholine, cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in an organic solvent;

[0018] 2) removing the solvent to obtain a uniform and transparent liposome film;

[0019] 3) mixing the liposome film, glucose oxidase and horseradish peroxidase in a solution to obtain a liposome suspension;

[0020] 4) using polycarbonate membranes with different pore sizes to squeeze and filter the suspension in step 3;

[0021] 5) The filtered filtrate is centrifuged for purification and resuspended in fresh PBS to obtain a suspension of the liposome enzyme nanoreactor.

[0022] In some specific embodiments, in the preparation method, the amount of L-α-phosphatidylcholine is 0 to 800 mg, the amount of cholesterol is 0 to 200 mg, and the amount of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is 0 to 200 mg.

[0023] In some specific embodiments, in the preparation method, the amount of glucose oxidase used is 1 to 10,000 μg, and the amount of horseradish peroxidase used is 1 to 10,000 μg.

[0024] In some specific embodiments, in the preparation method, polycarbonate membranes with pore sizes of 800 nm, 400 nm and 200 nm are used in sequence to squeeze and filter the suspension in step 3.

[0025] In the preparation method, the time for extruding and filtering the suspension in step 3 using a polycarbonate membrane varies according to the amount of the experimental substance fed, and the extrusion is complete. Specifically, the extrusion filtering time is 0-30 minutes.

[0026] The application of the liposome enzyme nanoreactor in the preparation of anti-tumor therapeutic materials or drugs.

[0027] Liposome-based enzyme nanoreactors (GOx / HRP-LNRs) were developed to mimic the structure and tandem reactions of eukaryotic organelles for anti-tumor therapy. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were co-loaded in the water core center of the liposomes. The co-compartmentalization of the two enzymes improved the overall efficiency of the tandem reaction. GOx in the nanoreactor can consume glucose in tumor cells to generate gluconic acid and hydrogen peroxide (H2O2). The generated gluconic acid leads to a lower pH value and an increase in the local H2O2 concentration, which can accelerate the catalytic efficiency of HRP and produce highly cytotoxic hydroxyl radicals (·OH), killing tumor cells. In vitro and in vivo studies have shown that the enzyme nanoreactors GOx / HRP-LNRs have good biocompatibility and significant anti-tumor effects in vitro and in vivo.

[0028] The outer layer of the liposome enzyme nanoreactor is a liposome with good biocompatibility, and the interior contains glucose oxidase and horseradish peroxidase. When the two enzymes are compartmentalized in the nanoreactor, an efficient cascade reaction is achieved.

[0029] Compared with the prior art, the present invention has the following characteristics and advantages: the principle of the present invention is novel, the preparation method of the liposome enzyme nanoreactor is simple, the enzyme activity is retained while having a high cascade reaction efficiency, and the liposome-based enzyme nanoreactor is used for catalytic anti-tumor treatment. The nanoreactor composed of glucose oxidase and horseradish peroxidase consumes glucose and produces ·OH with high cytotoxicity as the final product of the cascade reaction. The co-compartmentalization of the two enzymes in the liposome creates a restricted microenvironment, increases the local H2O2 concentration, and thus improves the efficiency of the cascade reaction. The liposome nanoreactor has good biocompatibility, can effectively inhibit tumor growth both in vivo and in vitro, and has broad development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a cryo-TEM image of the liposome enzyme nanoreactor GOx / HRP-LNRs.

[0031] Figure 2 Fluorescence images of BODIPY-labeled GOx (GOx-BDP) and Cy5-labeled HRP (HRP-Cy5) in liposomes.

[0032] Figure 3 This is the reaction kinetic test diagram of GOx / HRP-LNRs under different glucose concentrations.

[0033] Figure 4 This is the Michaelis-Menten kinetic test diagram of the GOx / HRP-LNRs cascade reaction.

[0034] Figure 5 This is an imaging diagram of cellular uptake of liposome enzyme nanoreactor.

[0035] Figure 6 This is the fluorescence image of GOx / HRP-LNRs producing hydroxyl radicals in cells.

[0036] Figure 7 These are the cytotoxicity experimental diagrams of liposome enzyme nanoreactors with different enzyme types under normoxic and hypoxic conditions.

[0037] Figure 8 This is a graph showing the effects of different types of liposome enzyme nanoreactors on the tumor volume of mice. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0039] In some specific embodiments, the liposome enzyme nanoreactor comprises liposomes; and glucose oxidase and horseradish peroxidase complexed with the liposomes. The liposomes comprise L-α-phosphatidylcholine, cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0040] Basic characterization of liposome enzyme nanoreactors: characterize the particle size, PDI, zeta potential and morphology of liposome enzyme nanoreactors, and measure the hydrodynamic radius (R h ). GOx / HRP-LNRs were labeled with fluorescent probes to verify the simultaneous encapsulation of the two enzymes. The Michaelis-Menten kinetics of the cascade reaction in the liposome enzyme nanoreaction and the enzyme activity of GOx and HRP after encapsulation were detected. For in vitro cells, MCF-7 cells were incubated with LNRs culture medium containing fluorescent probes, and the changes in fluorescence intensity in the cells over time were measured as an evaluation index for the uptake of LNRs by cells, and the ability of LNRs to produce hydroxyl radicals in cells was evaluated using confocal microscopy. Cytotoxicity experiments were used as an evaluation of in vitro anti-tumor ability. The nanoreactor was used to treat different cells and tumor-bearing mice by tail vein injection, and its anti-cancer ability was evaluated based on its inhibitory level on tumor cells in vivo and in vitro.

[0041] The enzyme activity and cascade reaction efficiency of the liposome enzyme nanoreactor in the solution were tested, and the GOx and HRP activities of the corresponding substrates were determined: each enzyme sample was added to a single well of a 96-well microplate, and then the corresponding enzyme nanoreactor was added. Then, the fluorescence intensity of the product reacting with resorufin was recorded. The experimental data were fitted into a hyperbolic equation through computer-assisted nonlinear regression analysis to determine the values ​​of the Michaelis constant (KM) and the maximum rate (Vmax).

[0042] The liposome enzyme nanoreactor was used for cell uptake experiments to evaluate its ability to enter cells: MCF-7 cells were seeded on a 6-well plate. After overnight culture, the old culture medium was replaced with a complete culture medium containing GOx / HRP-LNRs and incubated for 0, 1, 2, 3, 4, 5, 6, 8, 10 and 24 hours. Subsequently, the fluorescence intensity of the cells at different times after incubation was tested by flow cytometry. Confocal laser scanning microscopy (CLSM) was used to observe the fluorescence images of MCF-7 cells at different time points (0, 1, 2, 3, 4h).

[0043] Liposome enzyme nanoreactor for intracellular hydroxyl radical detection: ·OH-specific fluorescent probe hydroxyphenylfluorescein (HPF) was used to detect the generation of ·OH in MCF-7 cells. MCF-7 cells were cultured at 5×10 3The confocal plates were inoculated at a density of 100 μg / mL and cultured for 24 h. The medium containing empty LNRs, GOx-LNRs, HRP-LNRs and GOx / HRP-LNRs was added to each well to replace the old medium and incubated for 4 h. HPF and Hoechst 33342 were added for incubation. After washing with PBS, the cell morphology was observed by CLSM. The average fluorescence intensity of ·OH detected by HPF was measured.

[0044] The liposome enzyme nanoreactor was used for cytotoxicity experiments as an evaluation of anti-tumor ability. MCF-7 cells were seeded in 96-well plates and cultured under normoxic or hypoxic conditions for 24 h. Enzyme nanoreactors of different enzyme types with different enzyme concentrations were then added to the DMEM cells and incubated for another 24 h. Subsequently, MTT solution was added to each well and incubated again under the same conditions. The MTT solution was removed and DMSO was added. The OD value was measured using a microplate reader. The same experimental procedure was performed on A549 cells and 4T1 cells to evaluate cell viability.

[0045] Liposome enzyme nanoreactors are used for in vitro and in vivo anti-tumor therapeutic applications: liposomes, which have good biocompatibility and have been widely used in clinical practice, are used as enzyme carriers and administered to mice via tail vein injection. Liposome enzyme nanoreactors accumulate in areas with enhanced vascular permeability in the body through the EPR effect, enhancing tumor targeting. At the same time, liposomes are used to construct special chambers to simulate basic cell structures and reactions, retaining the activity and function of the enzyme, and being able to simulate complex natural catalytic processes. It has significant anti-tumor effects in vivo and in vitro.

[0046] The liposome enzyme nanoreactor was administered by tail vein injection to tumor-bearing mice, and the changes in tumor volume over time were recorded to evaluate its in vivo anti-tumor ability. The in vivo anti-tumor study was conducted on BALB / c mice (female, 6-7 weeks old) carrying the mouse breast cancer cell line 4T1. 4T1 cells were inoculated under the axilla of the right forelimb of the mice, and the tumor volume was monitored. When the tumor reached a certain volume, the mice were randomly divided into 5 groups (n=5). Each group of tumor-bearing mice was intravenously injected with an equal concentration of liposome nanoreactors. Tumor volume and body weight were monitored for 18 days.

[0047] Example 1

[0048] The liposome enzyme nanoreactor of the present invention is prepared in the following manner:

[0049] (1) L-α-phosphatidylcholine (egg PC) (40 mg), cholesterol (Chol) (10 mg) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (10 mg) (molar ratio = 55:30:15) were dissolved in anhydrous chloroform. DiD dye (0.2 mol% of total lipids) was added to anhydrous chloroform to label the liposome nanoreactor for subsequent cell experiments;

[0050] (2) The solvent in step 1 was evaporated to dryness on a rotary evaporator to obtain a uniform transparent film. 4 mL of aqueous phase (PBS, 0.1 M, pH = 7.4) was added to a round-bottom bottle and mixed with the film (stirring, no ultrasound) to make it fall off to form a blank liposome nanoreactor (LNRs) suspension without enzyme.

[0051] (3) Preparation of liposomes was the same as step 1. The solvent was evaporated to dryness on a rotary evaporator to obtain a uniform and transparent film. GOx (10 mg mL -1 ) was added with 4 mL of aqueous phosphate buffer (PBS, 0.1 M, pH = 7.4) and mixed with the membrane (stirring, no ultrasound) to cause it to fall off to form a suspension of liposome nanoreactors (GOx-LNRs) containing glucose oxidase.

[0052] (4) Preparation of liposomes was the same as step 1. The solvent was evaporated to dryness on a rotary evaporator to obtain a uniform and transparent film. HRP (10 mg mL -1 ) was added with 4 mL of aqueous phosphate buffer (PBS, 0.1 M, pH = 7.4) and mixed with the membrane (stirring, no ultrasound) to cause it to fall off to form a liposome nanoreactor (HRP-LNRs) suspension containing horseradish peroxidase.

[0053] (5) Preparation of liposomes was the same as step 1. The solvent was evaporated to dryness on a rotary evaporator to obtain a uniform and transparent film. GOx / HRP (the concentration of both enzymes was 10 mg mL) was added to a round-bottomed bottle. -1 ) was added with 4 mL of aqueous phosphate buffer (PBS, 0.1 M, pH = 7.4) and mixed with the membrane (stirring, no ultrasound) to cause it to fall off and form a liposome nanoreactor (GOx / HRP-LNRs) suspension containing glucose oxidase and horseradish peroxidase.

[0054] (6) using polycarbonate membranes with pore sizes of 800 nm, 400 nm and 200 nm to squeeze and filter the suspension in the above step for 5 min in sequence, and replacing the new filter membrane when squeezing stops;

[0055] (7) The LNRs were purified by centrifugation, resuspended in fresh PBS, and stored in a refrigerator at 4°C until use.

[0056] The loading amounts of the two enzymes in different liposome nanoreactors were obtained by UV spectroscopy test, where the loading amount of GOx relative to liposomes in the GOx-LNR suspension was 31.82 μg mg -1 The loading of GOx and HRP relative to liposomes in GOx / HRP-LNRs suspension was 43.18 μg mg -1 (where the molar ratio of GOx to HRP was 3.7:1), and the HRP loading relative to the liposomes in the HRP-LNRs suspension was 21.36 μg mg -1 The liposome content in GOx-LNR suspension, GOx / HRP-LNRs suspension, and HRP-LNRs suspension was 2.2 mg mL -1 .

[0057] Example 2

[0058] (1) L-α-phosphatidylcholine (egg PC) (800 mg), cholesterol (Chol) (200 mg) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (200 mg) were dissolved in anhydrous chloroform.

[0059] (2) The solvent was evaporated to dryness on a rotary evaporator to obtain a uniform and transparent film. GOx / HRP (the concentration of both enzymes was 10 mg mL -1 ) was added with 40 mL of aqueous phosphate buffer (PBS, 0.1 M, pH = 7.4) and mixed with the membrane (stirring, no ultrasound) to cause it to fall off and form a liposome nanoreactor (GOx / HRP-LNRs) suspension containing glucose oxidase and horseradish peroxidase.

[0060] (3) using polycarbonate membranes with pore sizes of 800 nm, 400 nm and 200 nm to squeeze and filter the suspension in the above step for 30 min in sequence, and replacing the new filter membrane when squeezing stops;

[0061] (4) The LNRs were purified by centrifugation, resuspended in fresh PBS, and stored in a refrigerator at 4°C for later use.

[0062] Example 3

[0063] (1) L-α-phosphatidylcholine (egg PC) (400 mg), cholesterol (Chol) (100 mg) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (100 mg) were dissolved in anhydrous chloroform.

[0064] (2) The solvent was evaporated to dryness on a rotary evaporator to obtain a uniform and transparent film. GOx / HRP (the concentration of both enzymes was 10 mg mL -1 ) was added with 20 mL of aqueous phosphate buffer (PBS, 0.1 M, pH = 7.4) and mixed with the membrane (stirring, no ultrasound) to cause it to fall off and form a liposome nanoreactor (GOx / HRP-LNRs) suspension containing glucose oxidase and horseradish peroxidase.

[0065] (3) using polycarbonate membranes with pore sizes of 800 nm, 400 nm and 200 nm to squeeze and filter the suspension in the above step for 15 min in sequence, and replacing the new filter membrane when squeezing stops;

[0066] (4) The LNRs were purified by centrifugation, resuspended in fresh PBS, and stored in a refrigerator at 4°C for later use.

[0067] Example 4

[0068] (1) L-α-phosphatidylcholine (egg PC) (200 mg), cholesterol (Chol) (50 mg) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (50 mg) were dissolved in anhydrous chloroform.

[0069] (2) The solvent was evaporated to dryness on a rotary evaporator to obtain a uniform and transparent film. GOx / HRP (the concentration of both enzymes was 10 mg mL -1 ) was added with 10 mL of aqueous phosphate buffer (PBS, 0.1 M, pH = 7.4) and mixed with the membrane (stirring, no ultrasound) to cause it to fall off and form a liposome nanoreactor (GOx / HRP-LNRs) suspension containing glucose oxidase and horseradish peroxidase.

[0070] (3) using polycarbonate membranes with pore sizes of 800 nm, 400 nm and 200 nm to squeeze and filter the suspension in the above step for 8 min, and replacing the new filter membrane when squeezing stops;

[0071] (4) The LNRs were purified by centrifugation, resuspended in fresh PBS, and stored in a refrigerator at 4°C for later use.

[0072] Example 5

[0073] (1) L-α-phosphatidylcholine (egg PC) (40 mg), cholesterol (Chol) (10 mg) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (10 mg) were dissolved in anhydrous chloroform.

[0074] (2) The solvent was evaporated to dryness on a rotary evaporator to obtain a uniform and transparent film. GOx / HRP (the concentration of both enzymes was 10 mg mL -1 ) was added with 1.5 mL of aqueous phosphate buffer (PBS, 0.1 M, pH = 7.4) and mixed with the membrane (stirring, no ultrasound) to cause it to fall off and form a liposome nanoreactor (GOx / HRP-LNRs) suspension containing glucose oxidase and horseradish peroxidase.

[0075] (3) using polycarbonate membranes with pore sizes of 800 nm, 400 nm and 200 nm to squeeze and filter the suspension in the above step for 5 min in sequence, and replacing the new filter membrane when squeezing stops;

[0076] (4) The LNRs were purified by centrifugation, resuspended in fresh PBS, and stored in a refrigerator at 4°C for later use.

[0077] Example 6: Characterization experiment of co-loading of glucose oxidase (GOx) and horseradish peroxidase (HRP).

[0078] The morphology of the liposome or liposome nanoreactor prepared in Example 1 was observed using a cryo-transmission electron microscope (JEOL JEM1400). Figure 1 As shown in the figure, the liposomes are spherical in shape and have a uniform size distribution. GOx (GOx-BDP) labeled with fluorescent probe BODIPY and HRP (HRP-Cy5) labeled with Cy5 were used, and GOx / HRP-LNRs were co-labeled with fluorescent probes BODIPY and Cy5, and co-localization analysis was performed using a laser confocal microscope. Figure 2 As shown, it was observed that the fluorescence signals of the two labeled dyes overlapped in GOx / HRP-LNRs ( Figure 2 The co-encapsulation of two enzymes was confirmed, with glucose oxidase (GOx) and horseradish peroxidase (HRP) co-loaded in the water core of the liposomes.

[0079] Example 7: Basic characterization experiments were performed on the liposome enzyme nanoreactor.

[0080] The particle size, PDI and zeta potential of the liposomes or liposome enzyme nanoreactors prepared in Example 1 were measured using a Zetasizer (Nano S90, Malvern Panalytical GmbH, Germany). The hydrodynamic radius (Rh) of the final dispersion was obtained using an ALV spectrometer (ALV Langen GmbH, Germany). As shown in Table 1, the hydrodynamic radius (Rh) of LNRs depends on its loading state, increases with the increase of enzyme loading, and the zeta potential does not change much.

[0081] Table 1 Basic characterization data of samples

[0082]

[0083] Example 8: Enzyme activity and cascade reaction efficiency of liposome enzyme nanoreactor in solution.

[0084] The AmplexTM Red method was used to determine the activity of GOx and HRP in the liposomes or liposome nanoreactors prepared in Example 1. The samples to be tested were prepared as follows: for samples containing GOx (the concentration of free GOx in the sample was 5 μg mL -1 , the concentration of GOx-LNR was 70 μg mL -1 ), the corresponding reaction mixture contained 940 μL sodium phosphate buffer (50 mM, pH 7.4), 20 μL glucose solution (different initial concentrations were added between 1.5 mM and 200 mM), 3.3 μL Amplex Red DMSO solution (1 mM), and 10 μL HRP solution (0.1 mg mL -1 ,300 units mL -1 ), the addition of HRP ensured that the mixture contained free HRP. For GOx / HRP-LNRs (the concentration of nanoreactors in the sample was 95 μg mL -1 ) samples, the corresponding reaction mixture contained 940 μL sodium phosphate buffer (50 mM, pH 7.4), 20 μL glucose solution (different initial concentrations), 3.3 μL Amplex Red DMSO solution (1 mM), and no free HRP was added, which was already provided by the liposome sample (HRP / GOx molar ratio: 3.7). On the other hand, the test samples containing HRP (free HRP in the sample, 5 μg ml -1 ,HRP-LNRs,47μg ml -1 ), the corresponding reaction mixture contained a DMSO mixture of 1 mL sodium phosphate buffer (10 mM, pH = 7.4), 3.3 μL 0.35 wt % H2O2 solution (0.1 M) and 3.3 μL Amplex Red solution (different initial concentrations were added in the range of 0.3 μM to 33 μM).

[0085] The cascade reaction efficiency of the liposome or liposome nanoreactor prepared in Example 1 was tested in a solution.

[0086] 100 μL of the above-prepared GOx-LNR, 100 μL of GOx / HRP-LNRs, 100 μL of HRP-LNRs reaction mixture samples, and 2.5 μL of the above-prepared two free enzyme samples were added to a single well of a 96-well microplate. Then, the fluorescence intensity (λem=595 nm) of the reaction between the product and resorufin was recorded every 15 seconds using a TECAN M1000 microplate reader to calculate the Michaelis-Menten kinetic data. The experimental data were fitted into a hyperbolic equation by computer-assisted nonlinear regression analysis, and the Michaelis constant (K M ) and the maximum rate (Vmax). Figure 3 As shown in Figure 2, for the GOx / HRP-LNRs sample, the fluorescence intensity of resorufin gradually increased over time after adding different concentrations of glucose, indicating that GOx / HRP-LNRs effectively carried out the enzyme cascade reaction. Figure 4 As shown, the catalytic efficiency of GOx / HRP-LNRs was increased by 4.4 times compared with that of GOx-LNRs sample.

[0087] The kinetic data of several samples are shown in Table 2. Both enzymes remain active after encapsulation, and the reaction substrate (glucose or H2O2) can contact the enzyme in the liposome. The Michaelis constant did not change significantly after GOx was encapsulated, and the maximum reaction rate constant did not decrease significantly after HRP was encapsulated, indicating that GOX and HRP still have good enzyme activity in the liposome. The cascade reaction efficiency of liposome nanoreactors with different loading amounts was tested, and the catalytic efficiency was significantly improved compared with the GOx-LNRs sample.

[0088] Table 2 Sample kinetic data

[0089]

[0090] Example 9: The liposomes or liposome nanoreactors prepared in Example 1 were subjected to a cell uptake experiment to evaluate their ability to enter cells.

[0091] MCF-7 cells were grown at 5×10 5 The cells were seeded on a 6-well plate at a density of 1.54 × 10 cells / well. After overnight culture, the cells were incubated with GOx / HRP-LNRs (75 μL, 2.2 mg mL -1The old medium was replaced with the complete culture medium containing 0.2 mol% DiD (containing 0.2 mol% of total liposome content) and incubated for 0, 1, 2, 3, 4, 5, 6, 8, 10 and 24 h. Subsequently, the fluorescence intensity of the cells at different times after incubation was tested by flow cytometry (Invitrogen TMAttune TMNxT). The fluorescence images of MCF-7 cells at different time points (0, 1, 2, 3, 4 h) were observed by confocal laser scanning microscopy (CLSM). Figure 5 As shown, a is the flow cytometry analysis of the absorption of GOx / HRP-LNRs by MCF-7 cells at different times. B is the quantification of the average fluorescence intensity at different times. C is the fluorescence image of the uptake of GOx / HRP-LNRs by MCF-7 cells at different time points. The fluorescence at 4 hours in Figures a and b is the strongest, indicating that GOx / HRP-LNRs are taken up by the cells in the largest amount. The cell fluorescence image in Figure c further shows that GOx / HRP-LNRs are taken up by cells.

[0092] Example 10: The liposome or liposome enzyme nanoreactor prepared in Example 1 is used to detect intracellular hydroxyl radicals.

[0093] The specific fluorescent probe hydroxyphenylfluorescein (HPF) was used to detect the generation of ·OH in MCF-7 cells. MCF-7 cells were cultured at 5×10 3 The confocal plate was inoculated at a density of 1.5 μg / mL and cultured for 24 h. Blank LNRs (60 μL, 2.2 mg mL -1 ), GOx-LNRs (60 μL, 2.2 mg mL -1 )、HRP-LNRs(60μL,2.2mg mL -1 ) and GOx / HRP-LNRs (60 μL, 2.2 mg mL -1 ) 1500μL culture medium was added to replace the old culture medium and incubated for 4h. 10μM HPF and 10μM Hoechst 33342 were added and incubated for 30min. After washing with PBS three times, the cell morphology was observed by CLSM. The average fluorescence intensity of HPF detection ·OH was determined by CLSM fluorescence intensity analysis. Figure 6 As shown, compared with blank LNRs, GOx-LNRs, HRP-LNRs, and GOx / HRP-LNRs showed stronger green fluorescence signals, indicating that the GOx / HRP-LNRs cascade reaction had the highest catalytic efficiency and the strongest ability to produce hydroxyl radicals.

[0094] Example 11: The liposomes or liposome enzyme nanoreactor prepared in Example 1 were used for cytotoxicity experiments.

[0095] MCF-7 cells were grown at 5×103 The cells were seeded in a 96-well plate at a density of 1:1 and cultured at 37°C, 21% O2 (normoxia) or 5% O2 (hypoxia) for 24 h. Then, 0, 4.4, 8.8, 17.6, 26.4, 35.2, 44, and 88 μg mL -1 Empty-LNRs (enzyme-free liposomes), GOx-LNRs, HRP-LNRs or GOx / HRP-LNRs at different liposome concentrations were incubated for another 24 h under normoxic or hypoxic conditions. Subsequently, MTT solution was added to each well and incubated for another 4 h under the same conditions. The MTT solution was removed and 100 μL DMSO was added to each well. The OD value was measured at 490 nm using a microplate reader. The cell viability calculation formula is: Cell viability (%) = (ODtreated / ODcontrol) × 100%. The same experimental procedures were performed on A549 cells and 4T1 cells to evaluate cell viability. Figure 7 As shown, GOx / HRP-LNRs had the highest antitumor efficacy against various tumor cells (A549, MCF-7, and 4T1 cells) under both normoxic and hypoxic conditions.

[0096] Example 12: The liposomes or liposome enzyme nanoreactors prepared in Example 1 were administered to tumor-bearing mice by tail vein injection, and the changes in tumor volume over time were then recorded to evaluate their in vivo anti-tumor ability. In vivo anti-tumor studies were conducted on BALB / c mice (female, 6-7 weeks old) carrying the mouse breast cancer cell line 4T1. Approximately 1×10 6 When the tumor volume reached 100 mm 3 At about 14 h, the mice were randomly divided into 5 groups: control group (normal saline), blank LNRs group, GOx-LNRs group, HRP-LNRs group, and GOx / HRP-LNRs group. Each group of tumor-bearing mice was intravenously injected with an equal amount of liposomes (0.011 mg / kg). The changes in tumor volume were monitored for 18 days. Figure 8 As shown, the tumor volume of mice treated with GOx / HRP-LNRs was significantly reduced, indicating that the enzyme nanoreactor GOx / HRP-LNRs has the best ability to inhibit tumor growth.

Claims

1. A liposome enzyme nanoreactor, characterized in that: The invention comprises liposomes; and glucose oxidase and horseradish peroxidase, which are complexed with the liposomes; the liposomes comprise L-α-phosphatidylcholine, cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; The preparation method of the liposome enzyme nanoreactor comprises the following steps: 1) dissolving L-α-phosphatidylcholine, cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in an organic solvent; 2) removing the solvent to obtain a uniform and transparent liposome film; 3) mixing the liposome film, glucose oxidase and horseradish peroxidase in a solution to obtain a liposome suspension; 4) using polycarbonate membranes with different pore sizes to squeeze and filter the suspension in step 3; 5) The filtered filtrate is centrifuged and purified, and resuspended in fresh PBS to obtain a suspension of the liposome enzyme nanoreactor; The loading amount of glucose oxidase and horseradish peroxidase in the liposome enzyme nanoreactor is independently 1 to 300 μg mg -1 .

2. A liposome enzyme nanoreactor according to claim 1, characterized in that: The fluid dynamics radius of the liposome enzyme nanoreactor is 1 to 1000 nm.

3. A liposome enzyme nanoreactor according to claim 1, characterized in that: The electromotive force of the liposome enzyme nanoreactor is -100 to 100 mW.

4. The method for preparing the liposome enzyme nanoreactor according to any one of claims 1 to 3, characterized in that: Liposome enzyme nanoreactor was prepared by using liposome, glucose oxidase and horseradish peroxidase through thin film dispersion method and extrusion method.

5. Use of a liposome enzyme nanoreactor according to any one of claims 1 to 3 in the preparation of anti-tumor therapeutic materials or drugs.

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