Bionic nano-vesicle expressing CD40L, preparation method and application

Abstract

The invention relates to the technical field of bioengineering, in particular to a bionic nano-vesicle expressing CD40L, a preparation method and application. The method comprises the following steps: coating an oncolytic adenovirus vector with a tumor cell membrane to obtain bionic nano-vesicles; the oncolytic adenovirus vector has a nucleotide sequence capable of expressing CD40L. The preparation for treating malignant tumors, which is prepared from the bionic nano vesicles, is an intraperitoneal injection preparation or an intravenous injection preparation. The bionic nano-vesicles can help adenovirus to escape immune recognition of a body, enter cells in a grid protein dependent form, get rid of CAR dependent transduction of the adenovirus on tumor cells, and achieve the effects of strong targeting and low immunity of the adenovirus in the body; the problems that the immune microenvironment of the body is complex, and the adenovirus is attacked and neutralized can be solved, the tumor cell membrane can well shield the adenovirus, and immune recognition of the body is avoided; the adenovirus can directly reach the tumor part, and the targeting property of the oncolytic virus is improved.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular, to a biomimetic nanovesicle expressing CD40L, a preparation method and an application thereof. Background Art

[0002] Tumor is a disease that seriously threatens human health and is the main obstacle to the extension of human lifespan. Oncolytic virus therapy, as an innovative tumor-targeted gene therapy means, integrates the multiple advantages of targeted drugs, immunotherapy drugs and gene therapy drugs. In 2015, T-VEC, as the world's first and only oncolytic virus proven effective through clinical trials, was approved by the FDA for marketing. Due to its high replication ability, significant killing effect and low side effects, oncolytic virus therapy has quickly become a new focus in the field of tumor treatment research.

[0003] Although it shows great potential, the application of oncolytic adenovirus in tumor treatment still faces some challenges:

[0004] 1) Limited administration route: Oncolytic viruses are mainly administered locally into tumors, which severely limits the clinical application of oncolytic viruses. Most solid tumors are located deep or have multiple metastases, making local administration difficult.

[0005] 2) Insufficient tumor selectivity: Adenovirus infection of tumor cells depends on CAR, but the expression level of CAR on the surface of most cells is low, and the expression levels are uneven. In some tumors, the CAR receptor on the surface often shows downregulation or loss, making it difficult for the virus to infect efficiently, resulting in limited efficacy.

[0006] 3) Pre-existing neutralizing antibodies in patients (such as antibodies against adenovirus type 5) may clear the virus and reduce the efficacy; after the virus is exposed, the body produces neutralizing antibodies, limiting the effect of repeated administration.

[0007] 4) Potential off-target effects: The liver tropism of adenovirus may cause it to infect normal liver cells, triggering inflammatory reactions or organ damage, with poor safety.

[0008] Therefore, an effective oncolytic virus vaccine and its delivery efficiency are the key factors for success. Currently, in the prior art, an oncolytic adenovirus carrying membrane-bound CD40L (OAd5-mCD40L) has been constructed and verified, and the nucleotide sequence of CD40L has been publicly disclosed. This vaccine can activate antigen-presenting cells and improve local tumor immunity in a mouse ovarian cancer model. It can be seen that oncolytic adenovirus has high prospects in tumor treatment, but the above problems still need to be overcome. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a biomimetic nanovesicle expressing CD40L, a preparation method thereof, and an application thereof.

[0010] The technical solution of the present invention for solving the above technical problems is as follows:

[0011] The present invention provides a preparation method of a biomimetic nanovesicle expressing CD40L, which uses a tumor cell membrane to coat an oncolytic adenovirus vector to obtain the biomimetic nanovesicle; the oncolytic adenovirus vector has a nucleotide sequence capable of expressing CD40L.

[0012] On the basis of the above technical solution, the present invention can also be improved as follows.

[0013] Further, the tumor cell membrane is a malignant tumor cell membrane.

[0014] Further, it includes the following steps:

[0015] S1. Extract the tumor cell membrane and perform fragmentation and ultrasonic treatment to obtain tumor cell membrane fragments;

[0016] S2. Obtain the oncolytic adenovirus vector;

[0017] S3. Mix the tumor cell membrane fragments with the oncolytic adenovirus vector to obtain a mixed system;

[0018] S4. Extrude the mixed system and prepare it into the biomimetic nanovesicle.

[0019] Further, in step S1, the time of the ultrasonic treatment is 12 - 25 min, the ultrasonic power is 90 - 120 w, and the ultrasonic frequency is 35 - 45 HZ.

[0020] Further, in step S2, construct the oncolytic adenovirus vector, insert the foreign gene CD40L into the E3 region of the nucleotide sequence of the adenovirus vector type 5 deletion to obtain the oncolytic adenovirus vector.

[0021] Further, in step S3, the mixing ratio of the tumor cell membrane fragments to the oncolytic adenovirus is 90 - 120 μg: 0.5×10 10 -1.5×10 10 vp.

[0022] Further, in step S4, the extrusion method is to perform 10 - 12 extrusion operations on the mixed system using a liposome extruder and a polycarbonate membrane.

[0023] The present invention also provides a biomimetic nanovesicle expressing CD40L, which is prepared by the method as described above. The particle size of the biomimetic nanovesicle is 150 - 220 nm, and the zeta potential of the biomimetic nanovesicle is -12 mV to -5 mV.

[0024] The present invention also provides an application of the biomimetic nanovesicle expressing CD40L as described above, and the biomimetic nanovesicle is used for preparing a preparation for treating ovarian cancer.

[0025] The present invention also provides an oncolytic adenovirus nanoplatform for ovarian cancer, which includes the biomimetic nanovesicle expressing CD40L as described above. The preparation is an intraperitoneal injection preparation or an intravenous injection preparation, and the preparation further includes a pharmaceutically acceptable auxiliary preparation.

[0026] The beneficial effects of the present invention are as follows:

[0027] (1) For the biomimetic nanovesicle expressing CD40L of the present invention, the tumor cell membrane is used to wrap the adenovirus to prepare a virus vesicle at the nanoscale, optimizing the delivery process of the adenovirus. The virus vesicle can help the adenovirus escape the immune recognition of the body, enter the cell in a clathrin-dependent manner, get rid of the CAR-dependent transduction of the adenovirus on tumor cells, and achieve the effects of strong targeting and low immunity of the adenovirus in vivo, providing a new administration strategy for the treatment of adenovirus.

[0028] (2) For the biomimetic nanovesicle expressing CD40L of the present invention, by using the method of biological membrane coating and encapsulation to disguise the adenovirus, problems such as the complex immune microenvironment in ascites and the possible attack and neutralization of the adenovirus can be well solved. The tumor cell membrane can achieve good shielding of the adenovirus and avoid immune recognition of the body.

[0029] (3) For the biomimetic nanovesicle expressing CD40L of the present invention, the homologous targeting ability of the tumor cell membrane can enable the adenovirus to directly reach the tumor site, improving the targeting of the oncolytic virus. Description of the Drawings

[0030] Figure 1 It is the transmission electron microscope image of each experimental group in Example 2 of the biomimetic nanovesicle expressing CD40L of the present invention. Figure 1 In which, a is the naked virus Ad group. Figure 1 In which, b is the cell membrane empty vesicle CM group. Figure 1 In which, c is the virus vesicle Ad@CM group.

[0031] Figure 2 It is the graph of the dynamic light scattering DLS measurement results of the particle size analyzer of each experimental group in Example 2 of the biomimetic nanovesicle expressing CD40L of the present invention. Figure 2 In which, A is the particle size comparison graph. Figure 2In it, B is the dot potential contrast diagram;

[0032] Figure 3 This is the biomimetic nanovesicle expressing CD40L of the present invention. In Example 2, it is the result diagram of the Dot blot hybridization experiment of each experimental group;

[0033] Figure 4 This is the biomimetic nanovesicle expressing CD40L of the present invention. In Example 3, it is the electrophoresis diagram of the evaluation result of the membrane protein characteristics of the nanovesicle Ad@CM with different tumor cell membranes. Figure 4 In it, a is the electrophoresis diagram of the A549 group; Figure 4 In it, b is the electrophoresis diagram of the SKOV3 group; Figure 4 In it, c is the electrophoresis diagram of the ID8 group;

[0034] Figure 5 This is the biomimetic nanovesicle expressing CD40L of the present invention. In Example 4, it is the result diagram of the experiment on the effect of temperature on the endocytosis of virus vesicles. Figure 5 In it, A is the contrast diagram of the +eGFP expression rate in tumor cells infected with naked virus at different temperatures; Figure 5 In it, B is the contrast diagram of the +eGFP expression rate in tumor cells infected with virus vesicles Ad@CM at different temperatures;

[0035] Figure 6 This is the biomimetic nanovesicle expressing CD40L of the present invention. In Example 4, it is the immunofluorescence result diagram after treating cells with clathrin inhibitor. Figure 6 In it, A is the tumor cells infected with naked virus; Figure 6 In it, B is the tumor cells infected with virus vesicles Ad@CM; Figure 6 In it, C is the statistical chart of the eGFP fluorescence result, *** indicates P < 0.001;

[0036] Figure 7 This is the biomimetic nanovesicle expressing CD40L of the present invention. In Example 5, it is the flow cytometry detection result diagram after infecting ID8 cells with different MOIs for 24, 48, and 72 hours;

[0037] Figure 8 This is the biomimetic nanovesicle expressing CD40L of the present invention. In Example 5, it is the contrast diagram of the CD40L expression in each experimental group. Figure 8 In it, A is the experimental result of infecting ID8 cells with Ad-mCD40L@ID8cm; Figure 8 In it, B is the experimental result of infecting 4T1 cells with Ad-mCD40L@4T1 cm; Figure 8 In it, C is the experimental result of infecting B16F10 cells with Ad-mCD40L@B16F10cm. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001;

[0038] Figure 9 For the biomimetic nanovesicles expressing CD40L of the present invention, in Example 6, the result diagram of the in vitro tumor killing effect of viral vesicles, Figure 9 wherein A is the cell survival rate of each experimental group after 24 h of infection of ID8 cells, Figure 9 wherein B is the cell survival rate of each experimental group after 48 h of infection of ID8 cells, Figure 9 wherein C is the cell survival rate of each experimental group after 24 h of infection of 4T1 cells, Figure 9 wherein D is the cell survival rate of each experimental group after 48 h of infection of 4T1 cells;

[0039] Figure 10 For the biomimetic nanovesicles expressing CD40L of the present invention, in Example 7, the fluorescence comparison diagram of HEK293 cells infected with naked virus and viral vesicles after 48 h;

[0040] Figure 11 For the biomimetic nanovesicles expressing CD40L of the present invention, in Example 7, the flow cytometry detection result diagram of HEK293 cells infected with naked virus and viral vesicles;

[0041] Figure 12 For the biomimetic nanovesicles expressing CD40L of the present invention, in Example 8, the evaluation result of the ability of Ad@CM to resist the complex immune microenvironment of ascites, Figure 12 wherein A is the fluorescence diagram, Figure 12 wherein B is the flow cytometry detection result diagram, Figure 12 wherein C is the statistical bar chart of the flow cytometry detection results, * indicates P < 0.05, and * indicates P < 0.01;

[0042] Figure 13 For the biomimetic nanovesicles expressing CD40L of the present invention, in Example 9, the result diagram of the in vitro experiment, Figure 13 wherein A is the comparison diagram of TNF-α secretion amount, Figure 13 wherein B is the comparison diagram of IL-6 secretion amount;

[0043] Figure 14 For the biomimetic nanovesicles expressing CD40L of the present invention, in Example 9, the result diagram of the in vivo experiment, Figure 14 wherein A is the schematic diagram of the experimental process, Figure 14 wherein B is the comparison diagram of TNF-α secretion amount, Figure 14 wherein C is the comparison diagram of IL-6 secretion amount;

[0044] Figure 15The biomimetic nanovesicles expressing CD40L of the present invention. In Example 9, the result graph of the positive rate of HEK293 expressing eGFP detected by flow cytometry, * indicates P < 0.05, and **** indicates P < 0.0001;

[0045] Figure 16 The biomimetic nanovesicles expressing CD40L of the present invention. In Example 10, the distribution of viral vesicles after intraperitoneal administration, Figure 16 In which A is the fluorescence image of the vesicle distribution in mice 48 hours after administration, Figure 16 In which B is the fluorescence image of the vesicle distribution in mice 72 hours after administration, Figure 16 In which C is the comparison graph of fluorescence values of each organ 48 hours after administration, Figure 16 In which D is the comparison graph of fluorescence values of each organ 72 hours after administration, Figure 16 In which E is the fluorescence image of each organ of the mouse;

[0046] Figure 17 The biomimetic nanovesicles expressing CD40L of the present invention. In Example 10, the comparison graph of the content of total viral DNA in the liver and tumor tissues caused by naked virus and viral vesicles at different administration times, Figure 17 In which A is the comparison graph of viral DNA content 48 hours after administration, Figure 17 In which B is the comparison graph of viral DNA content ratio 72 hours after administration, Figure 17 In which C is the comparison graph of the relative content of adenovirus in the liver and tumor, * indicates P < 0.05, *** indicates P < 0.001;

[0047] Figure 18 The biomimetic nanovesicles expressing CD40L of the present invention. In Example 11, the evaluation results of the anti-tumor effect of viral vesicles Ad@CM in vivo, Figure 18 In which A is the administration protocol for the animal experiment, Figure 18 In which B is the comparison graph of small animal imaging analysis of each experimental group, Figure 18 In which C is the fluorescence statistical graph of small animal imaging, Figure 18 In which D is the comparison graph of the ascites condition of the mice, Figure 18 In which E is the statistical graph of the body weight change of each group of mice, Figure 18 In which F is the comparison graph of the ascites volume of each group of mice, *P indicates < 0.05, **P indicates < 0.01, ***P indicates < 0.001. Detailed implementation manners

[0048] The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0049] The preparation method of the biomimetic nanovesicles expressing CD40L of the present invention uses tumor cell membranes to coat oncolytic adenovirus vectors to obtain biomimetic nanovesicles; the oncolytic adenovirus vectors have nucleotide sequences capable of expressing CD40L.

[0050] The preparation method of the biomimetic nanovesicles expressing CD40L of the present invention uses tumor cell membranes to wrap adenovirus (Ad-CD40L) to prepare nanoscale virus vesicles (Ad-CD40L@CM), optimizing the delivery process of Ad-CD40L. The virus vesicles can help Ad-CD40L escape the immune recognition of the body, enter cells in a clathrin-dependent manner, get rid of the CAR-dependent transduction of Ad-CD40L on tumor cells, and achieve the effects of strong targeting and low immunity of Ad-CD40L in vivo, providing a new drug delivery strategy for the treatment of Ad-CD40L.

[0051] Preferably, the tumor cell membrane of the present invention is a malignant tumor cell membrane.

[0052] Specifically, the malignant tumor cell membrane is one of A549 cell membrane, SKOV3 cell membrane, HEK293 cell membrane, ID8 cell membrane, B16F10 cell membrane and 4T1 cell membrane. A549, SKOV3 and HEK293 are human cell lines, and ID8, B16F10 and 4T1 are mouse cell lines.

[0053] Preferably, the preparation method of the present invention includes the following steps:

[0054] S1. Extract the tumor cell membrane and perform fragmentation and ultrasonic treatment to obtain tumor cell membrane fragments.

[0055] Preferably, the time of ultrasonic treatment is 12 - 25 min, the ultrasonic power is 90 - 120 w, and the ultrasonic frequency is 35 - 45 HZ.

[0056] Preferably, the method for extracting the tumor cell membrane is a conventional method. Specifically, collect cells and digest them to obtain a cell suspension, obtain cell precipitates after centrifugation, and repeat grinding and centrifugation multiple times to obtain cell membranes.

[0057] S2. Construct an oncolytic adenovirus vector: Insert the foreign gene CD40L into the E3 region of the adenovirus type 5 vector nucleotide sequence lacking to obtain the oncolytic adenovirus vector Ad-CD40L.

[0058] S3. Mix the tumor cell membrane fragments with the oncolytic adenovirus vector to obtain a mixed system.

[0059] Preferably, the mixing ratio of the tumor cell membrane fragments to the oncolytic adenovirus is 90 - 120 μg: 0.5×10 10 -1.5×10 10 vp.

[0060] S4. Extrude the mixed system and prepare it into biomimetic nanovesicles Ad-CD40L@CM.

[0061] Preferably, the extrusion method is to perform 10 - 12 extrusion operations on the mixed system using a liposome extruder and a polycarbonate membrane.

[0062] The biomimetic nanovesicles expressing CD40L of the present invention are prepared by the method as described above. The particle size of the biomimetic nanovesicles is 150 - 220 nm, and the zeta potential is -12 mV to -5 mV; the particle size and zeta potential are close to those of the empty vesicles, indicating that it has a good encapsulation effect.

[0063] The biomimetic nanovesicles expressing CD40L of the present invention have strong targeting and low immunity effects in vivo.

[0064] The present invention also provides the application of the above biomimetic nanovesicles for preparing a preparation for treating ovarian cancer.

[0065] The oncolytic adenovirus nanoplatform of the present invention includes the biomimetic nanovesicles expressing CD40L as described above. The preparation is an intraperitoneal injection preparation or an intravenous injection preparation, and the preparation also includes a pharmaceutically acceptable auxiliary preparation.

[0066] The present invention uses a biological membrane coating and encapsulation method to disguise the adenovirus, which can well solve problems such as the complex immune microenvironment in ascites and the possible attack and neutralization of adenovirus. The tumor cell membrane can achieve good shielding of the adenovirus and avoid immune recognition by the body. At the same time, the homologous targeting ability of the tumor cell membrane can, to a certain extent, enable the adenovirus to directly reach the tumor site and improve the targeting of the oncolytic virus.

[0067] The present invention is illustrated by the following specific examples. Unless otherwise specified, the experimental methods and materials used in each example are conventional.

[0068] The oncolytic viruses Ad-CD40L, Ad-mCD40L, and Ad-hCD40L used in the following examples were all purchased from Wuhan Kaidejinuo Biotechnology Co., Ltd.

[0069] Example 1 Preparation of Biomimetic Nanovesicles

[0070] The biomimetic nanovesicles in this example are Ad@CM, and the encapsulated oncolytic adenovirus vector is specifically a novel oncolytic adenovirus expressing CD40L, which is constructed by inserting the foreign gene CD40L into the E3 region of the nucleotide sequence of the adenovirus type 5 vector lacking.

[0071] The specific preparation process of this example is as follows:

[0072] (1) Obtain oncolytic virus vector

[0073] The oncolytic virus vector purchased in this example is an oncolytic adenovirus with a deleted adenovirus type 5 as the vector. The 27 bp at positions 920 to 946 in the E1A-CR2 region of its genome is to achieve its oncolytic characteristics, and the E3-ADP region, that is, the adenovirus death protein region, is replaced with an exogenous therapeutic gene fragment carried. This modified oncolytic adenovirus can specifically lyse tumor cells and exempt normal cells, activate the immune response, and exert a bystander effect to achieve tumor elimination.

[0074] For the detection of some verification experiments, in the oncolytic virus vector of this example, the ADP region is replaced by the eGFP DNA fragment. In some experiments, the positive rate of eGFP expression can be used as the basis for the situation of cell uptake of naked virus Ad or virus vesicle Ad@CM.

[0075] In the virus vesicle Ad@CM used in this example, the novel oncolytic adenovirus expressing CD40L is specifically Ad-mCD40L, and mCD40L is murine CD40L. The virus vesicle Ad@CM constructed in this example can also be named Ad-mCD40L@CM.

[0076] The tumor cell membrane specifically used in this example is the ID8 cell membrane, and other tumor cell membranes are also used in subsequent experiments. The specific preparation steps are the same as those in this example.

[0077] (2) Preparation of tumor cell membrane: The extracted tumor cell membrane is subjected to ultrasonic fragmentation treatment, ultrasonicated in an ice bath in an ultrasonic pot for 20 min, the ultrasonic power is 100 w, the ultrasonic frequency is 40 HZ, and it is placed on ice for later use.

[0078] In this example, the specific process of cell membrane extraction is as follows:

[0079] (1) Collect cells: After the cell density in the culture flask reaches 90%, the cells are digested with trypsin into a cell suspension, centrifuged at 1200 rpm × 5 min in a room temperature horizontal centrifuge, and the cells are washed with PBS twice for precipitation, and the centrifugation conditions are the same as above.

[0080] (2) Discard the supernatant, resuspend the cell precipitate with an appropriate amount of membrane extraction reagent A, and lyse on ice for 30 min.

[0081] (3) Use a tissue grinder to grind the cell suspension on ice 40 times in a laminar flow hood, collect the suspension, centrifuge at 3200 rcf × 10 min at 4 °C, and collect the supernatant.

[0082] (4) Transfer the supernatant to a 1.5 ml EP tube, centrifuge at low temperature in a high-speed 4 °C centrifuge, 20000 rcf × 40 min.

[0083] (5) Discard the supernatant, resuspend the cell pellet with 1 ml of PBS to obtain cell membranes, and store them at -80 °C to avoid repeated freezing and thawing.

[0084] (6) CM protein concentration determination: The concentration was determined according to the instruction manual of BCA protein assay kit from Beyotime.

[0085] The following experimental groups were specifically prepared in this example:

[0086] a. Virus vesicles Ad@CM group: The ratio of virus to cell membrane is Ad (1×10 10 vp): 100 μg CM.

[0087] b. Naked virus Ad group: Ad (1×10 10 vp).

[0088] c. Cell membrane empty vesicles CM group: 100 μg CM.

[0089] d. Blank control PBS group: PBS.

[0090] Sample system: Each is 100 μl, and the cells are supplemented with PBS buffer.

[0091] (3) Preparation of biomimetic nanovesicles: Assemble the parts of the liposome extruder, place a 400 nm polycarbonate membrane flat and aligned, and extrude the sample back and forth through the filter membrane 11 times.

[0092] Through the above process, virus vesicles Ad@CM, naked virus Ad, cell membrane empty vesicles CM and blank control PBS group were respectively prepared.

[0093] Example 2 Characterization of Nanovesicles Ad@CM

[0094] In this example, the characterization of the nanovesicles Ad@CM prepared in Example 1 was evaluated. Specifically, transmission electron microscopy was used to characterize the morphological structure of Ad@CM, a particle size analyzer was used for dynamic light scattering (DLS) to measure the particle size and Zeta potential, and the encapsulation of the virus by cancer cell membranes was further detected by Dot blot experiment.

[0095] (1) Morphological Structure of Ad@CM

[0096] The specific experimental process is as follows: Use a copper mesh with a common carbon film, dilute the sample to be tested to an appropriate concentration, take 50 μl of the sample solution to be tested and drop it on the copper mesh. After standing at room temperature for 5 min, drop 50 μl of 1% phosphotungstic acid in the center of the copper mesh for "negative staining", and observe after standing for 5 min and air-drying the sample.

[0097] The test results are as Figure 1 shown. According to Figure 1It can be seen that empty vesicles of different sizes are formed after the extrusion of tumor cell membranes. Viruses can be visually observed to be encapsulated in the tumor cell membrane vesicles in Ad@CM, indicating that the method for synthesizing Ad@CM virus vesicles in Example 1 is feasible.

[0098] (2) Particle size and Zeta potential of Ad@CM

[0099] Specifically, a particle size analyzer was used to measure the particle size and Zeta potential by dynamic light scattering (DLS). As Figure 2 shown, after measurement, the particle size of the empty vesicles of the tumor cell membrane is about 200 nm, and the particle size of the Ad@CM virus vesicles is close to that of the empty vesicles, also about 200 nm. At the same time, in the analysis of the Zeta potential, it was found that the surface potential of adenovirus is negatively charged, and the Zeta potential on the surface of the Ad@CM nanovesicles is very close to that of the empty vesicles, showing a weak negative charge, indicating that the tumor cell membrane has a good encapsulation effect on adenovirus.

[0100] (3) Encapsulation of virus by tumor cell membrane

[0101] The specific process of the Dot blot hybridization experiment is as follows:

[0102] (1) Use a pencil to draw the area to be spotted on the PVDF membrane, and then activate the membrane in methanol.

[0103] (2) Pipette 2 μl of the sample and slowly drop it onto the membrane to reduce the area of solution penetration, and then dry the fiber membrane.

[0104] (3) Immerse the membrane in the blocking solution and incubate at room temperature for 2 h to block non-specific sites, then incubate with the primary antibody (adenovirus Hexon antibody 1:100) in BSA / TBST for 2 h, wash the membrane with TBST for 5 min, and repeat 3 times.

[0105] (4) Then incubate with the secondary antibody for 1 h, wash the membrane with TBST for 5 min, and repeat 3 times.

[0106] (5) Add the chromogenic substrate and react for 5 - 10 min, wash with water to terminate the reaction, and observe the color development.

[0107] As Figure 3 shown, the results of the Ad group showed strong positivity, the CM group showed negative results because it did not contain adenovirus, and the virus vesicles Ad@CM showed weak positivity. The above results indicate that the tumor cell membrane can well shield the surface antigen of adenovirus, and the cancer cell membrane can well encapsulate adenovirus.

[0108] Example 3 Evaluation of the membrane protein characteristics of the nanovesicle Ad@CM

[0109] The purpose of this example is to examine whether the types and contents of membrane surface proteins change after the tumor cell membranes are subjected to ultrasound and extrusion operations. Specifically, polyacrylamide gel electrophoresis (SDS-PAGE) was used to evaluate Ad@CM prepared from A549 cell membranes, SKOV3 cell membranes, and ID8 cell membranes, which were respectively denoted as the A549 group, the SKOV3 group, and the ID8 group. After electrophoresis was completed, the gel was taken out, immersed in Coomassie Brilliant Blue solution for 1 h, and then imaged with a gel imager for observation.

[0110] According to Figure 4 It can be seen that Ad@CM prepared from the three types of tumor cell membranes all contain two components, namely virus and cell membrane, and the related proteins of both are completely retained by the vesicles.

[0111] Example 4 Exploration of the in vitro uptake and endocytic mechanism of the viral vesicle Ad@CM

[0112] Adenovirus infects tumor cells depending on the abundance of coxsackievirus adenovirus receptor (CAR) expressed by tumor cells. However, the expression of CAR in tumor cells is uneven, and there is generally a trend of downregulation of CAR receptors. Therefore, getting rid of CAR-dependent transduction is crucial for improving the adenovirus infection rate to enhance its anti-tumor effect. Therefore, this example explored the endocytic mechanism of the viral vesicle Ad@CM.

[0113] Regarding the effect of temperature on the endocytosis of viral vesicles, in this example, Ad-mCD40L@CM and naked virus were used to infect tumor cells at 37 °C and 4 °C for 2 h respectively, and the uptake of viral vesicles by tumor cells was observed.

[0114] According to Figure 5 A and Figure 5 B in it can be seen that compared with the naked virus, the uptake rate of adenovirus encapsulated by homologous tumor cell membranes by tumor cells is significantly increased, which is mediated by the targeting and adhesion of tumor cell membranes to homologous tumor cells. At the same time, the uptake rate of vesicles by tumor cells increases with the increase of temperature, indicating that the uptake of viral vesicles by cells is through active transport.

[0115] Regarding the dependence on clathrin, in this example, the cells were treated with the clathrin inhibitor chlorpromazine (CPZ) and Pitstop2 respectively, and then the expression of eGFP by the virus was observed under a fluorescence microscope. According to Figure 6 A-C in it can be seen that the endocytic effect of Ad-mCD40L@CM is not affected, while the endocytic effect of viral vesicles is significantly inhibited.

[0116] The above results show that the endocytosis of viral vesicles depends on the clathrin-mediated endocytosis process and does not depend on the CAR receptor.

[0117] Example 5: Tumor cells overexpress CD40L mediated by virus vesicles Ad@CM

[0118] The ability of virus vesicles encapsulating Ad-mCD40L to mediate tumor cells to overexpress CD40L is the basic premise for subsequent experiments. Therefore, in this example, ID8 cells were infected with Ad-mCD40L@ID8cm at different MOIs for 24, 48, and 72 hours, and then flow cytometry was used to detect the expression of CD40L on the tumor cell membrane.

[0119] As Figure 7 and Figure 8 shown, Ad-mCD40L@ID8cm can mediate ID8 cells to successfully overexpress CD40L. At the same time, 4T1 and B16F10 cells were infected with Ad-mCD40L@4T1 cm and Ad-mCD40L@B16F10cm respectively, and the successful expression of CD40L in tumor cells was also observed.

[0120] Example 6: Evaluation of the anti-tumor cell killing effect of virus vesicles Ad@CM in vitro

[0121] The above examples have confirmed that the uptake of adenovirus encapsulated by homologous tumor cell membranes by tumor cells is significantly higher than that of naked virus, increasing the infection rate of adenovirus to tumor cells.

[0122] Based on the experimental results of the above examples, this example continued to evaluate whether the increase in virus infection rate enhanced its anti-tumor effect. In this example, tumor cells were infected with naked virus or virus vesicles at different MOIs, and the survival rate of tumor cells was detected after 24 hours or 48 hours.

[0123] Specifically, this example used M0, Ad-mCD40L, M0@ID8CM, Ad-mCD40L@ID8CM, M0@4T1 CM, and Ad-mCD40L@4T1 CM. M0 is a control virus Ad-eGFP constructed by conventional bioengineering, which does not contain the gene of CD40L.

[0124] As Figure 9 shown, after 24 hours of virus infection of tumor cells, compared with naked virus, virus vesicles have a stronger anti-tumor killing effect, which may be related to the increase in the infection rate of virus vesicles to tumor cells.

[0125] After 8 hours of virus infection of tumor cells, in the murine breast cancer cell line 4T1, virus vesicles did not show a stronger anti-tumor effect. This may be because the virus content in naked virus and virus vesicles is the same, and the long-term continuous infection of virus to tumor cells leads to a smaller difference in the infection rate of the two to tumor cells in the end, so a stronger anti-tumor effect is not shown.

[0126] Example 7: The virus vesicle Ad@CM has the ability to resist neutralizing antibodies in vitro

[0127] Although oncolytic adenovirus vectors have a powerful anti-tumor effect, the neutralizing effect of adenovirus neutralizing antibodies widely present in the human body on adenoviruses greatly reduces their anti-tumor effect.

[0128] The results of the Dot blot experiment have shown that the tumor cell membrane can well shield the adenovirus surface antigen, and the tumor cell membrane forms a natural disguise for adenoviruses. Therefore, in this example, the ability of Ad@CM to resist neutralizing antibodies was evaluated. The virus vesicles were co-incubated with neutralizing antibodies and then used to infect HEK293 cells. After 48 hours, the infection of tumor cells was observed.

[0129] As Figure 10 and Figure 11 shown, compared with naked viruses, the virus vesicles have a stronger ability to resist neutralizing antibodies.

[0130] According to the results of this example, it can be seen that the virus vesicle Ad@CM of the present invention can effectively resist neutralizing antibodies after entering the body, so as to achieve intraperitoneal administration or intravenous administration.

[0131] Example 8: The effect of the virus vesicle Ad@CM on the complex immune microenvironment of ascites

[0132] In this example, it was specifically evaluated whether the virus vesicle Ad@CM has the ability to resist the complex immune environment of ascites.

[0133] The specific test process is as follows:

[0134] (1) Collect mouse ovarian cancer ascites, centrifuge at 1000 rpm for 5 min in a low-temperature 4°C centrifuge, collect the supernatant, and store it in a -80°C refrigerator.

[0135] (2) Digest HEK293 cells into cell suspensions, count 50,000 cells per well with a cell counter, and plate them in a 24-well plate.

[0136] (3) Mix 100 μl of ascites stock solution with virus vesicles (MOI = 10), co-incubate in a 37°C incubator for 30 min, and then add it to the well plate.

[0137] (4) Place the well plate in a 37°C incubator for 24 h, observe with a fluorescence microscope, and then perform flow cytometry.

[0138] In this example, ascites from 5 mice with advanced ovarian cancer was collected and co-incubated with the virus vesicle Ad@CM in vitro and then used to infect HEK293 cells. As Figure 12As shown, ascites did produce a neutralizing effect on adenovirus, and the neutralizing effect was significantly weakened after the adenovirus was coated with tumor cell membrane, which also provided a certain theoretical basis for the intraperitoneal treatment of adenovirus.

[0139] Example 9 Immunogenicity Evaluation of Virus Vesicle Ad@CM

[0140] The clinical application of oncolytic adenovirus has been limited by its strong immune activation effect and the recognition of the body's immune system. Whether the virus vesicle can successfully evade the recognition of the immune system is an important condition for exerting its biological function. Therefore, in this example, the activation effect of the virus vesicle on the immune system was evaluated in in vitro and in vivo experiments.

[0141] In this example, the virus vesicle was co-cultured with mouse immune cells RAW264.7, and the levels of inflammatory cytokines (IL-6, TNF-α) secreted by the immune cells were detected by ELISA. The specific experimental process is as follows:

[0142] Specific details of the experimental groups: PBS blank control group; CM group: 100 μg of CM was diluted to 100 μl and extruded through an extruder; Ad group: 1×1010 vp of the virus was diluted to a final volume of 100 μl and extruded through an extruder; Ad@CM group: 1×1010 vp of the virus and 100 μg of CM were diluted to 100 μl and extruded through an extruder.

[0143] (1) In vitro evaluation: RAW264.7 was digested into a cell suspension, counted at 50,000 per well with a cell counter, and plated into 24-well plates. The next day, virus vesicles or viruses were added to the wells for co-incubation (MOI = 10). After 24 h, the co-culture supernatant was collected, and the secretion of IL-6 and TNF-α in the supernatant was detected according to the instructions of the ELISA kit from Beyotime.

[0144] (2) In vivo experiment: A healthy mouse model was established, and female mice aged 6 - 8 weeks were intraperitoneally injected with each group of drugs on days 0 and 14. On day 21, orbital venous blood of the mice was collected, and then the mice were sacrificed. The schematic diagram of the experimental procedure is as shown in Figure 14 A in

[0145] i. Evaluation of the secretion of inflammatory factors:

[0146] The collected venous blood was centrifuged at 3000 rpm for 15 min at room temperature with a horizontal centrifuge, and the supernatant was collected, which was the serum. The serum was diluted to an appropriate concentration, and the secretion of IL-6 and TNF-α in the serum was detected according to the instructions of the ELISA kit from Beyotime.

[0147] ii. Evaluation of the production of neutralizing antibodies by stimulating the body:

[0148] HEK293 cells were digested into cell suspensions, counted at 50,000 cells per well using a cell counter, and seeded into 24-well plates. After co-incubating mouse serum with virus vesicles M0@CM in vitro at 37 °C for 30 min, HEK293 cells were infected and cultured in an incubator at 37 °C for 24 h. The positive rate of HEK293 expressing eGFP was detected by flow cytometry.

[0149] As Figure 13 The results of in vitro experiments showed that, compared with naked virus, the immunogenicity of virus vesicles was significantly reduced, and the expression of inflammatory cytokines IL-6 and TNF-α was significantly lower than that of naked virus.

[0150] As Figure 14 The results of in vivo experiments showed that virus vesicles could reduce the immune activation effect of the body, and the levels of inflammatory cytokines in mouse serum were significantly lower than those in the naked virus group.

[0151] Figure 15 The experimental results showed the results of the test for stimulating the body to produce neutralizing antibodies. Virus vesicles Ad@CM could stimulate the production of fewer neutralizing antibodies in vivo, which also provided a basis for repeated administration of adenovirus.

[0152] Example 10 Investigation of tumor targeting of virus vesicles Ad@CM

[0153] The ability of adenovirus to target tumor cells is crucial for the therapeutic effect of tumors. The uptake of adenovirus by liver tissue greatly reduces the efficacy of adenovirus application. Therefore, whether virus vesicles Ad@CM can efficiently target tumor cells is an important condition for realizing its biological function.

[0154] In this example, the distribution of virus vesicles after intraperitoneal administration was evaluated in vivo. The specific experimental procedure was as follows:

[0155] (1) Female C57BL / 6 mice, 6 - 8 weeks old, were intraperitoneally inoculated with 1×10 7 ID8-luciferase cells. On the 21st day after tumor inoculation, when obvious ascites appeared in the mice, the experiment began.

[0156] (2) Tumor cell membrane ID8cm was co-incubated with the fluorescent dye DIR in the dark for 30 min.

[0157] (3) Grouping and drug preparation ratio (per portion): Ad group: 1×10 10 vp of virus was diluted to a final volume of 100 μl and extruded through an extruder; Ad@CM: 1×10 10 vp of virus was diluted with 100 μg of CM to 100 μl and extruded through an extruder.

[0158] (4) Intraperitoneally inject 100 μl of naked virus Ad or virus vesicle Ad@CM per mouse, and observe the in vivo distribution of the vesicles by small animal imaging 48 h and 72 h later.

[0159] As Figure 16 shown, 48 h after the treatment with intraperitoneal injection of virus vesicles, the virus vesicles were enriched more in the tumor and liver, and almost no distribution was found in other organs. 72 h after the intraperitoneal injection of virus vesicles, the virus vesicles were mainly targeted at the tumor tissue site, and the DIR fluorescence intensity in the tumor was much higher than that in the liver, indicating that the virus vesicles had stronger preferential targeting to homologous tumors.

[0160] Subsequently, the liver and tumor tissues of the mice in the naked virus Ad group and the virus vesicle Ad@CM group were isolated in this example, the total tissue DNA was extracted, and the content of adenovirus capsid protein fiber in the tissue was detected by qPCR. Using mouse GAPDH as the internal reference gene, the relative content of adenovirus in the liver and tumor was analyzed. As Figure 17 shown, consistent with the small animal imaging results, compared with the naked virus, the virus vesicles showed stronger tumor targeting to homologous tumors and reduced the uptake of the virus by the liver.

[0161] Example 11 Exploration of the in vivo anti-tumor effect of virus vesicle Ad@CM

[0162] Patients with advanced ovarian cancer are often accompanied by extensive peritoneal dissemination. The generation of a large amount of ascites often makes it impossible to directly use a syringe for intratumoral injection treatment. Therefore, for ovarian cancer, a cancer type that is extremely prone to peritoneal dissemination, the common adenovirus administration method is intraperitoneal injection. However, intraperitoneal injection not only dilutes the adenovirus administration concentration to a certain extent, but also the complex immune microenvironment in ascites will affect the efficacy of adenovirus.

[0163] In vitro experiments have demonstrated that virus vesicles can resist the neutralization of adenovirus by complex immune components in mouse ascites, reduce the virus loss rate, and the virus vesicles preferentially target homologous tumors and reduce the uptake by the liver, which provides a basis for the anti-tumor effect of intraperitoneal injection of virus treatment.

[0164] This example continued to explore the anti-tumor effect of virus vesicles in vivo, and the specific experimental process was

[0165] (1) Digest the ID8-luci cells in the logarithmic growth phase into cell suspension, and intraperitoneally inoculate 1×10 7 ID8-luci cells into 6-8-week-old female C57BL / 6 mice, and start the treatment 10 days after the tumor inoculation.

[0166] (2) Grouping and drug preparation ratio (per portion): PBS group: blank control group; CM group: 100 μg of CM was diluted to 100 μl and extruded through an extruder; Ad group: 1×10 10 vp of viruses were diluted to a final volume of 100 μl and extruded through an extruder; Ad@CM group: 1×10 10 vp of viruses and 100 μg of CM were diluted to 100 μl and extruded through an extruder.

[0167] (3) Administer the drug by intraperitoneal injection, 100 μl / animal, four times. On the 18th day of treatment, small animal imaging was performed to observe the treatment effect.

[0168] (4) Measure the tumor volume, body weight of the mice, and the survival status of the mice every day. When the mice have feeding disorders, rapid weight loss, or the tumor volume is too large beyond the ethical range, the mice are euthanized.

[0169] (5) Euthanize the mice on the 25th day after the first drug administration, and draw the ascites volume of the mice for statistical analysis.

[0170] The experimental results of this example are as Figure 18 shown.

[0171] The experimental results show that adenovirus treatment can exert a certain anti-tumor effect. Compared with the PBS and CM groups, the tumor burden of the mice is slightly smaller. After treatment with virus vesicles, we found that the tumor burden and ascites volume of the mice were significantly reduced.

[0172] In the control group, extensive cancer metastases occurred in the abdominal cavity of the mice, and a large amount of bloody ascites was produced. After treatment with virus vesicles, no obvious ascites was found in the abdominal cavity of the mice, which also indicates that virus vesicles play a more powerful tumor inhibitory role in vivo.

[0173] The experimental results of the above examples can show that the present invention coats adenovirus with tumor cell membranes, successfully prepares virus vesicles Ad@CM wrapped with tumor cell membranes, with a diameter of about 200 nm, a weakly negative surface charge, and the virus vesicles completely retain the relevant proteins on the surface of adenovirus and tumor cell membranes. At the same time, the tumor cell membrane has a good shielding effect on the antigen on the surface of adenovirus. This oncolytic adenovirus "disguised" by cancer cell membranes can avoid being neutralized by adenovirus neutralizing antibodies, escape the immune recognition of the body, and play a powerful anti-tumor role. In addition, the results of in vitro and in vivo immunogenicity evaluations also show that virus vesicles can induce the production of fewer inflammatory factors (IL-6, TNF-α), stimulate the body to produce fewer neutralizing antibodies, providing a basis for the repeated administration of adenovirus.

[0174] The infection of target cells by Ad5 is mainly mediated by CAR on the tumor surface. However, most tumor cells have low expression of CAR and there is a downward trend in CAR expression, which limits the infection of the virus. In the present invention, the application of clathrin inhibitors significantly inhibits the endocytosis of virus vesicles, while adenovirus itself enters cells by relying on the binding of the capsid protein fiber to CAR on tumor cells. Therefore, clathrin inhibitors do not affect the infection of adenovirus. Therefore, the strategy of membrane-coated delivery of adenovirus provides a new way for adenovirus to infect tumor cells and increases the infection efficiency of the virus.

[0175] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a biomimetic nanovesicle expressing CD40L, characterized in that: The bionic nanovesicle is obtained by coating an oncolytic adenovirus vector with a tumor cell membrane; the oncolytic adenovirus vector has a nucleotide sequence capable of expressing CD40L.

2. The method for preparing a biomimetic nanovesicle expressing CD40L according to claim 1, characterized in that: The tumor cell membrane is a malignant tumor cell membrane.

3. The method for preparing a biomimetic nanovesicle expressing CD40L according to claim 2, characterized in that: The following steps are involved: S1, extracting the tumor cell membrane, breaking it and performing ultrasonic treatment to obtain tumor cell membrane fragments; S2. obtaining the oncolytic adenoviral vector; S3, mixing the tumor cell membrane fragments with the oncolytic adenovirus vector to obtain a mixed system; S4, extruding the mixed system to prepare the bionic nanovesicles.

4. The method for preparing a biomimetic nanovesicle expressing CD40L according to claim 3, characterized in that: In step S1, the ultrasonic treatment time is 12-25 min, the ultrasonic power is 90-120 w, and the ultrasonic frequency is 35-45 Hz.

5. The method for preparing a biomimetic nanovesicle expressing CD40L according to claim 3, characterized in that: In step S2, the oncolytic adenoviral vector is constructed, and the exogenous gene CD40L is inserted into the E3 region of the nucleotide sequence of the type 5 adenoviral vector to obtain the oncolytic adenoviral vector.

6. The method for preparing a biomimetic nanovesicle expressing CD40L according to claim 3, characterized in that: In step S3, the mixing ratio of the tumor cell membrane fragments and the oncolytic adenovirus is 90-120 μg: 0.5×10 10 -1.5×10 10 vp.

7. The method for preparing a biomimetic nanovesicle expressing CD40L according to claim 3, characterized in that: In step S4, the extrusion method is to use a liposome extruder and a polycarbonate membrane to extrude the mixed system 10-12 times.

8. A biomimetic nanovesicle expressing CD40L, characterized in that: The bionic nanovesicles are prepared by the method according to any one of claims 1 to 7, wherein the particle size of the bionic nanovesicles is 150-220 nm, and the electric potential of the bionic nanovesicles is -12 mv to -5 mv.

9. A use of the biomimetic nanovesicle expressing CD40L as claimed in claim 8, characterized in that: The bionic nanovesicle is used for preparing a preparation for treating ovarian cancer.

10. An oncolytic adenovirus nanoformulation, characterized in that: The biomimetic nanovesicle expressing CD40L as claimed in claim 8, wherein the preparation is an intraperitoneal injection preparation or an intravenous injection preparation, and the preparation further comprises a pharmaceutically acceptable auxiliary preparation.

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