A pH-sensitive bionic drug lipid vesicle and its preparation method and application

Abstract

The present invention discloses a pH-sensitive bionic drug lipid vesicle, a preparation method and an application thereof. The bionic drug lipid vesicle is a drug-loaded lipid vesicle formed by self-assembly after mixing cell membrane vesicles and drug-loaded phospholipids; the drug-loaded phospholipids are obtained by connecting the drug and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) through a pH-sensitive linker aconitic anhydride. The bionic drug lipid vesicle prepared by the present invention has excellent pH responsiveness, can accurately respond to changes in the pH value of the tumor microenvironment, and realize tumor-targeted release of the drug. The bionic drug lipid vesicle of the present invention is prepared by a method of ultrasonic self-assembly after physical mixing, which is simple and easy to operate and has relatively low requirements for process conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a pH-sensitive bionic drug lipid vesicle and a preparation method and application thereof. Background Art

[0002] With the development of drug delivery system research, cell-derived nanocarriers have received extensive attention in biomimetic modification research in recent years. People coat different cell membranes on the surface of synthetic carriers, which can not only greatly improve the biocompatibility of the carriers, but also the characteristic proteins on the surface of different cell membranes can give the carriers different properties. For example, the red blood cell membrane can disguise the drug and escape the body's immune system, successfully pass the immune recognition, thereby extending the drug's circulation time in the blood and improving the drug's administration efficiency. The homologous targeting effect of tumor cell membranes can help improve the tumor targeting ability of drugs. The immune cell membrane gives the nanosystem immunomodulatory function. Therefore, it is of great significance for immune escape, improving drug targeting and biocompatibility.

[0003] At present, the research on bionic cell membrane-modified nanomedicines has occupied an important position in the field of anti-tumor application research. Researchers often use membrane wrapping, coating and other means to modify the cell membrane on the outer layer of nanomaterials or nanoparticles, thereby preparing various functional nanomedicines. The membrane-modified multifunctional nanoplatform is usually composed of an outer cell membrane and an inner core nanomaterial. The inner core nanomaterial can include metal or metal oxide inorganic nanoparticles, metal-organic framework materials, organic synthetic materials, etc. These materials usually have certain functions themselves, such as photoactivity, redox ability, etc. In addition, they can also carry therapeutic drugs through chemical coupling or physical encapsulation. After being coated with the outer cell membrane, they finally form an anti-tumor nanotherapy platform with multiple functions synergistic with each other. However, when designing these anti-tumor nanotherapy platforms, the more functions the inner core has, the greater the complexity of its preparation and the instability of the process. In addition, the drug is wrapped in the inner core, and the release behavior will also be affected by the external modification material. Summary of the invention

[0004] The purpose of the present invention is to overcome the disadvantage of the complex preparation process of the inner and outer layer structures of common cell membrane bionic drug carriers, and to provide a pH-sensitive bionic drug lipid vesicle and its preparation method and application. The bionic drug lipid vesicle can achieve pH-responsive drug release in response to the tumor microenvironment through simple mixing and self-assembly, improve the carrying efficiency, and also use the cell membrane to achieve different functions.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a pH-sensitive bionic drug lipid vesicle, wherein the bionic drug lipid vesicle is a drug-loaded lipid vesicle formed by self-assembly of a mixture of cell membrane vesicles and drug-loaded phospholipids; the drug-loaded phospholipid is obtained by connecting a drug and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) via a pH-sensitive linker aconitic anhydride.

[0007] As a preferred embodiment of the first aspect, the drug is Sotuletinib (BLZ-945), Camptothecin (CPT) or Docetaxel (DTX).

[0008] As a preferred embodiment of the first aspect, when the cell membrane vesicles and the drug-loaded phospholipids are mixed, the mass of the cell membrane protein: the mass of the drug is 10:1 to 1:10.

[0009] As a preference for the first aspect above, the cells from which the cell membrane vesicles are derived are 4T1 cells, 4T1 / SIRPα cells or M1 macrophages.

[0010] In a second aspect, the present invention provides a method for preparing the drug-loaded lipid vesicles as described in any one of the first aspects above, comprising:

[0011] S1. After the cells have been proliferated and cultured, they are washed several times with TM buffer solution and then suspended in TM buffer solution. The cells are then lysed by repeated freezing and thawing to obtain cell fragments. Sucrose solution is added to the cell fragments after lysis and the cell membrane fragments and other components are initially separated by centrifugation. The supernatant containing the cell membrane fragments is further centrifuged at high speed. The precipitate obtained by centrifugation is collected and washed and resuspended with PBS solution. The resuspended solution is passed through a polycarbonate membrane several times using a liposome extruder to obtain homogenized cell membrane vesicles.

[0012] S2, dissolving cis-aconitic anhydride in dry dichloromethane and adding 3-6 times molar equivalents of oxalyl chloride, adding N,N-dimethylformamide (DMF) after sufficient reaction on ice and reacting at room temperature for 1-2 h, removing the solvent and excess oxalyl chloride by rotary evaporation after the reaction, then dissolving the obtained reactant in dry dichloromethane, adding 0.2-0.5 times molar equivalents of the drug and 0.24-1.2 times molar equivalents of pyridine to react, separating and purifying to obtain the intermediate product after the reaction; then dissolving DSPE-PEG-NH2, 2-5 times molar equivalents of the intermediate product and 1-1.5 times molar equivalents of 4-dimethylaminopyridine (DMAP) in dry dimethyl sulfoxide (DMSO) and reacting under the protection of inert gas, after the reaction, the reaction solution is dialyzed and purified with deionized water and freeze-dried to obtain the drug-loaded phospholipid;

[0013] S3. According to the ratio of cell membrane protein mass to drug mass of 10:1 to 1:10, the cell membrane vesicles and the drug-loaded phospholipids are mixed, and self-assembled under the assistance of ultrasound or extrusion to obtain drug-loaded lipid vesicles.

[0014] As a preferred embodiment of the second aspect, the concentration of the sucrose solution is 0.45-0.55 M, and the amount added is 0.8-1.2 times the volume of the cell disruptor.

[0015] As a preferred embodiment of the second aspect, in S2, aconitic anhydride is dissolved in dry dichloromethane in a reaction container, and then the reaction container is placed on ice and oxalyl chloride is added while stirring. After the reaction is carried out on ice until no bubbles are generated, N,N-dimethylformamide (DMF) is added to react at room temperature.

[0016] As a preferred embodiment of the second aspect, in S2, the obtained reactant is dissolved in dry dichloromethane, and 0.2 to 0.5 times the equivalent of the drug and 0.24 to 1.2 times the molar equivalent of pyridine are added to carry out the reaction. The mixture is stirred at room temperature overnight, and the degree of reaction is monitored by thin layer chromatography. After the reaction is completed, a saturated potassium chloride solution is added to the reaction solution to terminate the reaction, and the organic layer is collected and separated by silica gel column chromatography to obtain the intermediate product.

[0017] As a preferred embodiment of the second aspect, in S2, DSPE-PEG-NH2, 2 to 5 times the molar equivalent of the intermediate product and 1 to 1.5 times the equivalent of 4-dimethylaminopyridine (DMAP) are dissolved in dry dimethyl sulfoxide (DMSO), and then stirred at room temperature to 36 ° C for 18 to 24 hours under nitrogen protection. After the reaction, the reaction solution is dialyzed with deionized water for 24 to 48 hours, and the dialysis molecular weight cutoff (MWCO) is 950 to 1050. The solution in the dialysis bag is collected and freeze-dried to obtain the drug-loaded phospholipid.

[0018] In a third aspect, the present invention provides a use of the pH-sensitive bionic drug lipid vesicles as described in any one of the first aspects above in the preparation of a tumor-targeted drug-loaded nanoplatform.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) In the pH-sensitive biomimetic drug lipid vesicles prepared by the present invention, aconitic anhydride is cleverly introduced as a pH-responsive linker, so that the vesicles can significantly enhance the drug release effect in an acidic environment. Therefore, this biomimetic drug lipid vesicle has excellent pH responsiveness and can accurately respond to changes in the pH value of the tumor microenvironment to achieve controlled release of the drug. At the same time, the vesicles also have good tumor targeting and can efficiently deliver drugs to the tumor site to exert a therapeutic effect.

[0021] (2) The biomimetic drug lipid vesicles of the present invention are prepared by a method of self-assembly under the assistance of ultrasound or extrusion after physical mixing, which is simple and easy to operate and has relatively low requirements for process conditions. In addition, the present invention has high flexibility and wide applicability, and can assemble and integrate various cell membranes with various drugs according to different functional requirements to meet diverse application needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of material preparation for pH-sensitive biomimetic drug lipid vesicles;

[0023] Figure 2 The synthetic route for connecting drugs to DSPE-PEG using cis-aconitic anhydride;

[0024] Figure 3 is the H NMR spectrum of BLZ-945;

[0025] Figure 4 is the H NMR spectrum of DSPE-PEG2000-CA-BLZ-945;

[0026] Figure 5 The co-localization relationship diagram of 4T1 / SIRPα lipid vesicles loaded with BLZ-945;

[0027] Figure 6 Transmission electron microscopy results of 4T1 / SIRPα lipid vesicles loaded with BLZ-945;

[0028] Figure 7 The in vitro release curve of drug BLZ-945 in 4T1 / SIRPα lipid vesicles loaded with BLZ-945 in release media with different pH values;

[0029] Figure 8 The in vivo fluorescence distribution results corresponding to different experiments in Example 2;

[0030] Fig. 9 H NMR spectra of CPT-CA and DSPE-PEG2000-CA-CPT;

[0031] Fig.10 This is a co-localization relationship diagram of CPT-loaded macrophage lipid vesicles;

[0032] Fig.11 This is the in vitro release curve of the drug CPT in CPT-loaded macrophage membrane lipid vesicles in release media with different pH values. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation mode of the present invention is described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0034] The present invention provides a pH-sensitive biomimetic drug lipid vesicle, such as Figure 1 As shown, the biomimetic drug lipid vesicle is a drug-loaded lipid vesicle formed by mixing cell membrane vesicles and drug-loaded phospholipids and then self-assembling under the assistance of ultrasound or extrusion. The drug-loaded phospholipids are obtained by connecting the drug and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) through a pH-sensitive linker aconitic anhydride (CA).

[0035] It should be noted that the molecular weight of the polyethylene glycol (PEG) in the above-mentioned distearoylphosphatidylethanolamine-polyethylene glycol is 1000-5000, preferably 1000, 2000, or 5000.

[0036] It should be noted that the drug loaded on the above-mentioned drug-loaded phospholipid can be selected according to the actual application needs. In the embodiment of the present invention, Sotuletinib (BLZ-945), camptothecin (CPT) or docetaxel (DTX) can be selected. The drug and DSPE-PEG are connected by cis-aconitic anhydride as a linker, and this connection method has pH sensitivity function.

[0037] It should be noted that during the process of self-assembly to form drug-loaded lipid vesicles, the ratio of cell membrane vesicles and drug-loaded phospholipids when mixed can be optimized and adjusted according to actual conditions. In the embodiments of the present invention, the ratio can be controlled based on the mass of cell membrane protein and the mass of the drug. The mass of cell membrane protein: the mass of the drug is preferably 10:1 to 1:10.

[0038] It should be noted that the above-mentioned cell membrane vesicles can be constructed based on selected cells. The cells selected in the embodiments of the present invention include 4T1 cells, 4T1 / SIRPα cells or M1 macrophages.

[0039] The present invention also provides a method for preparing the drug-loaded lipid vesicles, the specific steps of which are as follows:

[0040] S1. After cell proliferation and culture, the cells are washed several times with TM buffer solution and then suspended in TM buffer solution. The cells are then lysed by repeated freezing and thawing to obtain cell fragments. Sucrose solution is added to the cell fragments after lysis and the cell membrane fragments and other components are initially separated by centrifugation. The supernatant containing the cell membrane fragments is further centrifuged at high speed. The precipitate obtained by centrifugation is collected and washed and resuspended with PBS solution. The resuspended solution is passed through a polycarbonate membrane several times using a liposome extruder to obtain homogenized cell membrane vesicles.

[0041] In the S1 step, the main purpose is to prepare the cell membrane vesicles required for the subsequent steps based on the proliferating cultured cells, and the specific parameters can be reasonably adjusted and optimized according to the actual situation. In an embodiment of the present invention, the above S1 step can be implemented by the following specific method:

[0042] Collect cells from 3 to 8 culture dishes, wash them 2 to 5 times with TM buffer solution at 4°C, and then suspend them in TM buffer solution. Repeat freezing and thawing at -80°C and 37°C for 3 to 8 times to fully lyse the cells into cell fragments. Add 0.8 to 1.2 times the volume of 0.45 to 0.55 M sucrose solution (preferably 0.5 M) to the cell fragments, centrifuge (4°C, 1500 to 2500 rpm, 10 min), take the supernatant and centrifuge it at high speed (4°C, 10000 to 15000 rpm, 30 min), collect the precipitate, which is the cell membrane vesicles, wash it 3-5 times with PBS solution, and resuspend it. First, pass it through a 400 nm polycarbonate membrane with a liposome extruder 10 to 20 times, and then pass it through a 200 nm polycarbonate membrane with a liposome extruder 10 to 20 times, and then collect the final cell membrane vesicles, and store them at -80°C for later use.

[0043] S2. Dissolve cis-aconitic anhydride in dry dichloromethane and add 3-6 times the molar equivalent of oxalyl chloride. After sufficient reaction on ice, add N,N-dimethylformamide (DMF) and react at room temperature for 1-2 hours. h. After the reaction is completed, the solvent and excess oxalyl chloride are removed by rotary evaporation, and then the obtained reactant is dissolved in dry dichloromethane, and 0.2~0.5 times molar equivalent (based on the molar amount of the reactant dissolved in dry dichloromethane) of the drug and 0.24~1.2 times molar equivalent (based on the molar amount of the reactant dissolved in dry dichloromethane) of pyridine are added for reaction. After the reaction is completed, the intermediate product is separated and purified; DSPE-PEG-NH2, 2~5 times molar equivalent (based on the molar amount of DSPE-PEG-NH2) of the intermediate product and 1~1.5 times molar equivalent (based on the molar amount of DSPE-PEG-NH2) of 4-dimethylaminopyridine (DMAP) are dissolved in dry dimethyl sulfoxide (DMSO) and reacted under the protection of inert gas. After the reaction is completed, the reaction solution is dialyzed and purified with deionized water and freeze-dried to obtain the drug-loaded phospholipid.

[0044] In the S1 step, the main purpose is to use aconitic anhydride as a linker to connect the drug to DSPE-PEG, thereby obtaining the drug-loaded phospholipid required for the subsequent steps. The principle is as follows Figure 2 The specific parameters in this step can be reasonably adjusted and optimized according to actual conditions. In an embodiment of the present invention, the above step S2 can be implemented by the following specific method:

[0045] Dissolve cis-aconitic anhydride in dry dichloromethane, place the reaction bottle on ice, add 3-6 times molar equivalents (based on the molar amount of cis-aconitic anhydride) of oxalyl chloride while stirring, react on ice until no obvious bubbles are generated, add N,N-dimethylformamide (DMF) and react at room temperature for 1-2 h. After the reaction is completed, the obtained reactant is dissolved in dry dichloromethane by rotary evaporation, and 0.2-0.5 times molar equivalents (based on the molar amount of the reactant dissolved in dry dichloromethane obtained by rotary evaporation) of the drug and 0.24-1.2 times molar equivalents (based on the molar amount of the reactant dissolved in dry dichloromethane obtained by rotary evaporation) of pyridine are added, stirred at room temperature overnight, and the reaction degree is monitored by thin layer chromatography. After the reaction is completed, add saturated potassium chloride solution to the reaction solution to terminate the reaction, collect the organic layer, and separate the intermediate product by silica gel column chromatography. Then weigh a certain amount of DSPE-PEG-NH2, dissolve it in dry DMSO together with 2-5 times the molar equivalent of the intermediate product and 1-1.5 times the molar equivalent of 4-dimethylaminopyridine (DMAP), and stir the reaction at room temperature or 36 ° C for 18-24 hours under nitrogen protection. After the reaction is completed, dialyze with deionized water for 24-48 hours (MWCO=950-1050), collect the solution in the dialysis bag, freeze-dry to obtain the drug-loaded phospholipid, and store it at -20 ° C for use.

[0046] The type of phospholipid used in the above-mentioned drug-carrying phospholipid is DSPE-PEG, wherein the molecular weight of PEG includes 1000, 2000, and 5000. Preferably, end-amino-containing DSPE-PEG, i.e., DSPE-PEG-NH2, is used to improve the reaction activity. The drugs include Sotuletinib (BLZ-945), camptothecin (CPT), and docetaxel (DTX). Since the two are connected with cis-aconitic anhydride as a linker, this connection method has a pH-sensitive function.

[0047] S3. According to the ratio of cell membrane protein mass: drug mass of 10:1 to 1:10, the cell membrane vesicles prepared in S1 and the drug-loaded phospholipids prepared in S2 are mixed, and self-assembled under the assistance of ultrasound or extrusion to obtain drug-loaded lipid vesicles.

[0048] In the S3 step, the main purpose is to assemble the cell membrane vesicles with the drug-loaded phospholipids to obtain drug-loaded lipid vesicles, and the specific parameters can be reasonably adjusted and optimized according to actual conditions. In an embodiment of the present invention, the mass ratio of the cell membrane vesicles to the drug-loaded phospholipids (measured in terms of cell membrane protein mass: drug mass) can be optimized to 1:1, at which time the mixing uniformity of the two components is better.

[0049] The pH-sensitive bionic drug lipid vesicles prepared by the present invention have similar structures and properties to cell membranes, which enables them to effectively fuse with cell membranes; at the same time, they have pH responsiveness and can achieve pH-responsive drug release in tumor microenvironment. Therefore, the pH-sensitive bionic drug lipid vesicles prepared by the present invention can be used to further prepare tumor-targeted drug-loaded nanoplatforms, providing a new powerful tool for tumor treatment.

[0050] The actual construction process and technical effects of the pH-sensitive bionic drug lipid vesicles are demonstrated below through multiple examples. Among them, Examples 1 and 2 demonstrate the bionic drug lipid vesicles loaded with somatostatin BLZ-945 constructed based on 4T1 cells and 4T1 / SIRPα cells and their related properties, while Examples 3 and 4 demonstrate the bionic drug lipid vesicles loaded with camptothecin CPT constructed based on macrophages and their related properties.

[0051] Example 1

[0052] (1) Extraction of cancer cell 4T1 membranes and cancer cell 4T1 membranes stably transfected with SIRPα protein (4T1 / SIRPα)

[0053] 4T1 cells and 4T1 / SIRPα cells were cultured with DMEM high-glucose culture medium containing 10% fetal bovine serum, and then the 4T1 cells and 4T1 / SIRPα cells were collected respectively, and the following cell membrane vesicle extraction process was performed:

[0054] For each type of cells collected after culture, they were washed three times with TM buffer solution at 4°C and then suspended in TM buffer, and then repeatedly frozen and thawed 5 times at -80°C and 37°C to obtain cell fragments. A 0.5 M sucrose solution with an equal volume of TM buffer solution used for suspension was added to the cell fragments and then centrifuged (4°C, 2000 rpm, 10 min). The supernatant obtained by centrifugation was further subjected to high-speed centrifugation (4°C, 10000 rpm, 30 min). The precipitate obtained by high-speed centrifugation was collected, which was the cell membrane vesicles. The cell membrane vesicles were resuspended in PBS solution and further homogenized by liposome extrusion. The specific method was to first pass through a 400 nm polycarbonate membrane with a liposome extruder 15 times, and then continue to pass through a 200 nm polycarbonate membrane with a liposome extruder 15 times. The final homogenized cell membrane vesicles were collected and stored at -80°C for later use. Finally, the cell membrane vesicles obtained based on 4T1 cells were recorded as 4T1 cell membrane vesicles, and the cell membrane vesicles obtained based on 4T1 / SIRPα cells were recorded as 4T1 / SIRPα cell membrane vesicles. The protein content in the cell membrane vesicles was detected using a BCA protein analysis kit to facilitate subsequent mixing and proportioning.

[0055] (2) Synthesis of DSPE-PEG2000-BLZ-945

[0056] 0.267 g of cis-aconitic anhydride (CA) was weighed and dissolved in a reaction bottle containing 10 mL of dry dichloromethane. The reaction bottle was placed on ice, and 5 times the molar equivalent (based on the molar amount of cis-aconitic anhydride) of oxalyl chloride was added while stirring. The reaction was allowed to react on ice until no obvious bubbles were generated. 40 μL of N,N-dimethylformamide (DMF) was added as a catalyst, and the reaction bottle was moved to room temperature for 1.5 h. After the reaction was completed, the solvent and excess oxalyl chloride were removed by rotary evaporator to obtain the reactant. The obtained reactant was dissolved in 5 mL of dry dichloromethane, 0.199 g of BLZ-945 and 30 μL of pyridine were added, and the reaction was stirred at room temperature overnight. The reaction was monitored by thin layer chromatography. After the reaction was completed, 5 mL of saturated potassium chloride solution was added to the reaction solution to terminate the reaction. The organic layer was collected and separated by silica gel column chromatography to obtain BLZ-945-CA.

[0057] Then 10 mg BLZ-945-CA, 20 mg DSPE-PEG2000-NH2 and 0.8 mg DMAP were weighed and dissolved in 5 mL dry DMSO solution. Under nitrogen protection, the mixture was stirred at 36 °C for 24 hours. After the reaction was completed, the mixture was dialyzed with deionized water for 48 h (MWCO = 1000), and the solution in the dialysis bag was collected and freeze-dried to obtain DSPE-PEG2000-CA-BLZ-945, which was stored at -20 °C for future use. Figure 3 The H NMR spectrum of the raw material BLZ-945 is shown. Figure 4 The hydrogen nuclear magnetic resonance spectrum of DSPE-PEG2000-CA-BLZ-945 is shown, indicating that DSPE-PEG2000-CA-BLZ-945 is synthesized in the present invention.

[0058] (3) Preparation of BLZ-945-loaded lipid vesicles

[0059] Different masses of DSPE-PEG2000-CA-BLZ-945 were weighed and mixed with a certain amount of cell membrane vesicle extracts (4T1 cell membrane vesicles and 4T1 / SIRPα cell membrane vesicles were selected respectively) in an ice bath and ultrasonicated for 30 min. The mass ratio of the mixture was calculated according to the mass of cell membrane protein: the mass of the drug (referred to as mem:BLZ-945) and 7 groups of different mass ratios were selected in the range of 10:1 to 1:10, that is, lipid vesicles loaded with BLZ-945 at different mass ratios were obtained. Among them, the lipid vesicles loaded with BLZ-945 obtained when 4T1 cell membrane vesicles were selected were recorded as 4T1 lipid vesicles loaded with BLZ-945, and the lipid vesicles loaded with BLZ-945 obtained when 4T1 / SIRPα cell membrane vesicles were selected were recorded as 4T1 / SIRPα lipid vesicles loaded with BLZ-945.

[0060] Taking 4T1 lipid vesicles loaded with BLZ-945 as an example, the particle size, Zeta potential and polydispersity index (PDI) of 4T1 lipid vesicles loaded with BLZ-945 obtained at 7 different mass ratios were measured respectively. The results are shown in Table 1:

[0061] Table 1

[0062]

[0063] The results in Table 1 above show that the 4T1 lipid vesicles loaded with BLZ-945 have a particle size of about 300 nm, a negatively charged surface, and relatively stable nanoparticles can be obtained within a certain mass ratio range, so a wider drug loading range can be obtained. Similarly, the 4T1 / SIRPα lipid vesicles loaded with BLZ-945 prepared in this example also have similar properties to the 4T1 lipid vesicles loaded with BLZ-945, and will not be repeated.

[0064] In addition, this example also takes BLZ-945-loaded 4T1 lipid vesicles and BLZ-945-loaded 4T1 / SIRPα lipid vesicles prepared at a mass ratio of 1:1 as examples, and conducts co-localization experiments and transmission electron microscopy structural studies, both of which present similar results. The following is an example of BLZ-945-loaded 4T1 / SIRPα lipid vesicles prepared at a mass ratio of 1:1.

[0065] The co-localization relationship between the membrane vesicle carrier and rhodamine-labeled DSPE-PEG2000 in the 4T1 / SIRPα lipid vesicles loaded with BLZ-945 prepared under the above mass ratio of 1:1 is shown in the figure. Figure 5 As shown, it proves that the two are well mixed. Figure 5The green channel represents DiO-labeled membrane carriers, the red channel represents RhB, and the orange channel is the colocalization map after fusion, scale bar = 20 μm.

[0066] At the same time, the transmission electron microscopy results of BLZ-945-loaded 4T1 / SIRPα lipid vesicles obtained under the above mass ratio of 1:1 are as follows Figure 6 As shown, it is proved that there are vesicle-like structures in it.

[0067] Example 2

[0068] In this example, the pH response release of the 4T1 lipid vesicles loaded with BLZ-945 and the 4T1 / SIRPα lipid vesicles loaded with BLZ-945 obtained in Example 1 at a mass ratio of 1:1 was studied in two release media at pH = 6.8 and pH = 7.4. Both showed similar pH response phenomena. The following is an example of the release of the drug BLZ-945 in the release media at different pH values. Figure 7 As shown, it can be found that the 4T1 / SIRPα lipid vesicles loaded with BLZ-945 obtained under the above-mentioned mass ratio of 1:1 are released faster and more thoroughly in the release medium with pH=6.8, indicating that it has the ability of pH-responsive drug release and can be used to achieve drug release that responds to the pH of the tumor microenvironment.

[0069] In addition, in this example, the tumor targeting ability of the 4T1 lipid vesicles loaded with BLZ-945 obtained at a mass ratio of 1:1 and the 4T1 / SIRPα lipid vesicles loaded with BLZ-945 obtained at a mass ratio of 1:1 in Example 1 was tested in vivo in mice. At the same time, for comparison, the same in vivo mouse experiment was also carried out using simple 4T1 cell membrane vesicles and 4T1 / SIRPα cell membrane vesicles, and a blank control (CK) was set up. After tail vein injection, the distribution of various components in the mouse body was tracked by fluorescence in each experimental group (respectively referred to as Group A, Group B, Group C, and Group D) and the control group. The final in vivo fluorescence distribution results are shown in the figure. Figure 8 As shown, this proves that whether it is 4T1 lipid vesicles (Group A), 4T1 lipid vesicles loaded with BLZ-945 (Group B), 4T1 / SIRPα cell membrane vesicles (Group C), or 4T1 / SIRPα lipid vesicles loaded with BLZ-945 (Group D), they all have good tumor targeting ability. After tail vein injection, accumulation in the tumor site began to appear at the 12th hour, and strong fluorescence continued until the 48th hour.

[0070] Example 3

[0071] (1) Extraction of macrophage membrane

[0072] M1 macrophages were washed three times with TM buffer at 4°C and then suspended in TM buffer. The cells were then frozen and thawed five times at -80°C and 37°C to obtain cell fragments. A 0.25M sucrose solution equal in volume to the TM buffer used for suspension was added to the cell fragments and centrifuged (4°C, 1000 rpm, 5 min). The supernatant obtained by centrifugation was further subjected to high-speed centrifugation (4°C, 10000 rpm, 30 min). The precipitate obtained by high-speed centrifugation was collected as macrophage membrane vesicles. The macrophage membrane vesicles were resuspended in PBS solution and further homogenized by liposome extrusion. Specifically, the macrophage membrane vesicles were first passed through a 400 nm polycarbonate membrane 15 times with a liposome extruder, and then passed through a 200 nm polycarbonate membrane 15 times with a liposome extruder. The macrophage membrane vesicles after final homogenization were collected and stored at -80°C for later use. The cell membrane protein content was detected using a BCA protein analysis kit to facilitate mixing and proportioning.

[0073] (2) Synthesis of DSPE-PEG2000-CA-CPT

[0074] Weigh 72.1 mg of cis-aconitic anhydride and dissolve it in a reaction bottle containing 6 mL of dry dichloromethane. Place the reaction bottle on ice, slowly add 190 μL of oxalyl chloride while stirring. After 20 minutes of reaction on ice, no bubbles are generated. Then add 60 μL of DMF (catalyst) and place it at room temperature for reaction for 1.5 hours. After the reaction is completed, remove the excess oxalyl chloride by rotary evaporator to obtain the reactant. Then dissolve 42.2 mg of camptothecin CPT in 6 mL of dry dichloromethane, add the solution to the reaction bottle where the above reactant is located, add 12 μL of pyridine, stir magnetically at room temperature overnight, monitor the reaction progress by TLC thin layer chromatography, confirm that the reaction is completed, add 5 mL of saturated potassium chloride solution to terminate the reaction, transfer the solution to a centrifuge tube, extract it with saturated ammonium chloride several times, discard the water layer and spin dry, then separate and collect the product by silica gel column chromatography, and finally spin dry the product to obtain CPT-CA.

[0075] Then weigh 6 mg CPT-CA, 18 mg DSPE-PEG2000-NH2 and 0.8 mg DMAP, add them to the round-bottom flask together with 5 mL dry DMSO, and react overnight in a 36 °C water bath with magnetic stirring under nitrogen protection. Dilute the product and put it into a dialysis bag (MWCO=1000), change the water every 3 hours, and freeze-dry the solution in the bag after dialysis for 48 hours to obtain DSPE-PEG2000-CA-CPT.

[0076] The nuclear magnetic resonance hydrogen spectra of CPT-CA and DSPE-PEG2000-CA-CPT obtained in this step are as follows: Fig. 9 shown.

[0077] (3) Preparation of CPT-loaded macrophage lipid vesicles

[0078] DSPE-PEG2000-CA-CPT and macrophage membrane were mixed in 1 ml of PBS at a mass ratio (calculated as the ratio of the mass of the drug to the mass of the cell membrane protein) of 1:1 and extruded 15 times through a liposome extruder to obtain CPT-loaded macrophage lipid vesicles.

[0079] In this example, a co-localization experiment was performed on the prepared CPT-loaded macrophage lipid vesicles. The results are as follows: Fig.10 As shown, the red fluorescent membrane dye DiD represents the location of the cell membrane, and the blue fluorescence of CPT indicates the location of DSPE-PEG2000-CA-CPT. Fig.10 The results show that the red fluorescence and blue fluorescence completely overlap under the fluorescence microscope, indicating that the membrane vesicles and DSPE-PEG2000-CA-CPT can be evenly and fully mixed.

[0080] Example 4

[0081] In this example, pH-responsive release of CPT-loaded macrophage membrane lipid vesicles prepared in Example 3 was studied in two release media at pH = 6.8 and pH = 7.4. The in vitro release curves of drug CPT in release media at different pH values ​​are shown in Figure 2. Fig.11 As shown, it can be found that the CPT-loaded macrophage membrane lipid vesicles are released faster and more thoroughly in the release medium at pH=6.8, indicating that the release of CPT is significantly increased in an acidic environment, which is mainly because the cis-aconitic anhydride linker has pH responsiveness. Therefore, the CPT-loaded macrophage membrane lipid vesicles prepared by the present invention have the ability to release drugs in response to pH, and can be used to achieve drug release in response to the pH of the tumor microenvironment.

[0082] The above-described embodiments are only some preferred implementations of the present invention, but are not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A pH-sensitive biomimetic drug lipid vesicle, characterized in that: The biomimetic drug lipid vesicle is a drug-loaded lipid vesicle formed by self-assembly after mixing cell membrane vesicles and drug-loaded phospholipids; the drug-loaded phospholipids are obtained by connecting the drug and distearoylphosphatidylethanolamine-polyethylene glycol DSPE-PEG through a pH-sensitive linker aconitic anhydride; when the cell membrane vesicles and the drug-loaded phospholipids are mixed, the cell membrane protein mass: the drug mass is 10:1 to 1:10; The drug is sorastatinib, and the cells from which the cell membrane vesicles are derived are 4T1 cells or 4T1 / SIRPα cells; Alternatively, the drug is camptothecin, and the cells from which the cell membrane vesicles are derived are M1 macrophages.

2. A method for preparing the drug-loaded lipid vesicles as claimed in claim 1, characterized in that: include: S1. After the cells have been proliferated and cultured, they are washed several times with TM buffer solution and then suspended in TM buffer solution. The cells are then lysed by repeated freezing and thawing to obtain cell fragments. Sucrose solution is added to the cell fragments after lysis and the cell membrane fragments and other components are initially separated by centrifugation. The supernatant containing the cell membrane fragments is further centrifuged at high speed. The precipitate obtained by centrifugation is collected and washed and resuspended with PBS solution. The resuspended solution is passed through a polycarbonate membrane several times using a liposome extruder to obtain homogenized cell membrane vesicles. S2, dissolving cis-aconitic anhydride in dry dichloromethane and adding 3 to 6 times the molar equivalent of oxalyl chloride, fully reacting on ice, then adding N,N-dimethylformamide and reacting at room temperature for 1 to 2 h, after the reaction is completed, removing the solvent and excess oxalyl chloride by rotary evaporation, then dissolving the obtained reactant in dry dichloromethane, and adding 0.2 to 0.5 times the molar equivalent of the drug and 0.24 to 1.2 times the molar equivalent of pyridine to react, and after the reaction is completed, separating and purifying to obtain an intermediate product; Then, DSPE-PEG-NH2, 2 to 5 times the molar equivalent of the intermediate product and 1 to 1.5 times the molar equivalent of 4-dimethylaminopyridine are dissolved in dry dimethyl sulfoxide and reacted under the protection of an inert gas. After the reaction is completed, the reaction solution is dialyzed and purified with deionized water and freeze-dried to obtain the drug-loaded phospholipid; S3. According to the ratio of cell membrane protein mass to drug mass of 10:1 to 1:10, the cell membrane vesicles and the drug-loaded phospholipids are mixed, and self-assembled under the assistance of ultrasound or extrusion to obtain drug-loaded lipid vesicles.

3. The preparation method according to claim 2, characterized in that The concentration of the sucrose solution is 0.45-0.55 M, and the amount added is 0.8-1.2 times the volume of the cell disruptor.

4. The preparation method according to claim 2, characterized in that: In S2, cis-aconitic anhydride is dissolved in dry dichloromethane in a reaction container, and then the reaction container is placed on ice and oxalyl chloride is added while stirring. After the reaction is carried out on ice until no bubbles are generated, N,N-dimethylformamide is added to react at room temperature.

5. The preparation method according to claim 2, characterized in that: In S2, the obtained reactant is dissolved in dry dichloromethane, and 0.2 to 0.5 times the molar equivalent of the drug and 0.24 to 1.2 times the molar equivalent of pyridine are added to carry out the reaction. The mixture needs to be stirred at room temperature overnight, and the degree of reaction is monitored by thin layer chromatography. After the reaction is completed, a saturated potassium chloride solution is added to the reaction solution to terminate the reaction, and the organic layer is collected and separated by silica gel column chromatography to obtain the intermediate product.

6. The preparation method according to claim 2, characterized in that: In the S2, DSPE-PEG-NH2, 2 to 5 times the molar equivalent of the intermediate product and 1 to 1.5 times the molar equivalent of 4-dimethylaminopyridine are dissolved in dry dimethyl sulfoxide, and then stirred at room temperature to 36°C for 18 to 24 hours under nitrogen protection. After the reaction, the reaction solution is dialyzed with deionized water for 24 to 48 hours, and the molecular weight cutoff of the dialysis is 950 to 1050. The solution in the dialysis bag is collected and freeze-dried to obtain the drug-loaded phospholipid.

7. Use of the pH-sensitive bionic drug lipid vesicles as claimed in claim 1 in preparing a tumor-targeted drug-carrying nanoplatform.

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