CN115337281A

CN115337281A - Preparation method and application of targeted engineered drug-loaded hybrid cell membrane vesicle

Info

Publication number: CN115337281A
Application number: CN202210827720.XA
Authority: CN
Inventors: 梅林�; 余永康
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2022-07-14
Filing date: 2022-07-14
Publication date: 2022-11-15
Anticipated expiration: 2042-07-14
Also published as: CN115337281B

Abstract

The invention discloses a preparation method and application of a targeted engineered drug-loaded hybrid cell membrane vesicle. The invention firstly prepares the engineering hybrid cell membrane vesicle by preparing a specific stable cell line and a single-component membrane vesicle, and carries out small molecule drug loading to obtain the engineering drug-loaded hybrid cell membrane vesicle. The main components of the engineered drug-loaded hybrid cell membrane vesicle prepared by the invention are derived from organisms, and the engineered drug-loaded hybrid cell membrane vesicle has good biocompatibility, strong targeting property and high bioavailability of drugs; the compound has universal killing property on tumor cells, can obviously inhibit the growth of the tumor cells and cause the immunogenic death of the tumor cells, thereby activating the immune system of an organism; can effectively inhibit the recurrence and metastasis of postoperative tumors, has obvious capacity of targeting postoperative parts, can obviously improve the postoperative life cycle, does not cause tissue damage, and has better in vivo biological safety; chemotherapy and immunotherapy can realize good synergistic effect, and can effectively inhibit the recurrence and metastasis of postoperative tumor.

Description

Preparation method and application of targeted engineered drug-loaded hybrid cell membrane vesicle

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, relates to a preparation method and application of a targeted engineered drug-loaded hybrid cell membrane vesicle.

Background

Cancer is a major killer of human beings, and brings great threat to the psychological and physiological health of people. With respect to cancer treatment, surgical resection remains the current primary method of treating solid tumors. However, recurrence and metastasis of post-operative cancer often results in poor post-operative recovery and low 5-year survival of patients. Therefore, postoperative adjuvant treatment is necessary for postoperative patients. Since recurrence and metastasis usually occur within 5 years during postoperative recovery of cancer, approximately 70% -80% of patients relapse and metastasize between them, and whether tumor recurrence or metastasis, the effect on the patient is significant and may lead to death within a short period of time. Therefore, it is necessary to perform adjuvant treatment during the postoperative recovery period of cancer, and to reduce the incidence of recurrence and metastasis.

At present, the auxiliary treatment aiming at the cancer recovery stage in clinic mainly comprises chemotherapy, radiotherapy, targeted drug therapy, or traditional Chinese medicine therapy, and the like, and can play a role in consolidation, further kill cancer cells remained in a body, and greatly reduce the possibility of relapse. Chemotherapy and immunotherapy are often combined with targeted drugs for treatment in adjuvant therapy, and although the incidence of relapse and metastasis can be reduced to a certain extent, the problems of low combined degree, weak targeting property, low utilization rate and the like of the targeted drugs in chemotherapy and immunotherapy exist, and the effect of combined action needs to be improved; however, there is a certain safety problem in the frequent use of targeted drugs, and therefore, there is a need to research and develop a new drug delivery system or drug delivery system, which provides a new effective strategy for the prevention of recurrence and metastasis after cancer surgery.

The blood platelet can target wounds and Circulating Tumor Cells (CTCs) after operation, effectively improve the bioavailability of the medicament and reduce the side effect of the medicament. Thus, platelets and platelet-derived membrane vesicles are candidates for drug delivery vehicles in post-surgical cancer therapy. Many platelet-based drugs have been developed to explore post-operative tumor treatment. As the prior art discloses a platelet vesicle engineered cell and extracellular vesicle for targeted tissue repair, the use of platelet vesicles to engineer cells and exosomes can treat vascular injury. However, tumor cells have multiple immune escape mechanisms, making it difficult for drugs delivered solely by platelet membrane vesicles to function effectively. For example, oxaliplatin (OXA) is a widely used chemotherapeutic drug in clinical settings, which induces Immunogenic Cell Death (ICD) in tumors, and has a synergistic effect on the immune system. However, the effect of the actual clinical OXA chemotherapy is poor, and the study in the subject group shows that OXA causes the up-regulation of CD155, and the single OXA treatment can further up-regulate the expression of CD155 in tumor cells, so that the tumors are more sensitive to the anti-CD 155 treatment. CD155 is abundantly expressed on the surface of tumor cells, and the combined effect with a co-inhibition receptor TIGIT on the surface of immune cells inhibits the immune system, so that immune escape is realized, and the single drug effect of OXA is poor. While blocking of immunosuppressive signaling pathways restores immunity, such as the CD155/TIGIT pathway. However, the prior art lacks a drug delivery system which has the effects of targeting tumor cells and blocking CD 155/TIGIT.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and shortcomings of the problems and provide a preparation method and application of a targeted engineering drug-loaded hybrid cell membrane vesicle.

The invention aims to provide a preparation method of a targeted engineered drug-loaded hybrid cell membrane vesicle.

The second purpose of the invention is to provide a targeted engineered drug-loaded hybrid cell membrane vesicle.

The third purpose of the invention is to provide the application of the engineered drug-loaded hybrid cell membrane vesicle.

The above purpose of the invention is realized by the following technical scheme:

previous studies in this subject group showed that OXA caused an up-regulation of CD155, and as shown in fig. 1 and 2, treatment with OXA alone further up-regulated CD155 expression in tumor cells, rendering tumors more susceptible to anti-CD 155 treatment. CD155 is expressed on the surface of tumor cells in a large amount, and the combined effect with a co-inhibition receptor TIGIT on the surface of immune cells inhibits an immune system, so that immune escape is realized, the single-effect of OXA is poor, and the blockage of an immunosuppressive signal path can restore immunity.

The invention creatively researches a method for engineering drug-loaded hybrid cell membrane vesicles with targeting tumor cells and CD155/TIGIT blocking effects, firstly constructs a cell line stably expressing TIGIT protein through gene editing and other biotechnology means and physical and chemical methods, then further mixes the cell line with platelet membrane vesicles to prepare hybrid cell membrane vesicles, and finally loads micromolecular chemotherapeutic drugs to obtain the engineering drug-loaded hybrid cell membrane vesicles, thereby providing a new drug system with stronger targeting property and better safety for postoperative recurrence and metastasis of cancers.

The invention provides a preparation method of targeted engineered drug-loaded hybrid cell membrane vesicles, which comprises the following steps:

s1, preparing a stable cell line for over-expressing TIGIT genes;

s2, using the stable cell line of the step S1 as a donor, preparing stable cell line membrane vesicles over-expressing TIGIT genes;

s3, mixing the stable cell system membrane vesicle overexpressing the TIGIT gene with the platelet membrane vesicle, performing ultrasonic treatment, and extruding to obtain an engineered hybrid cell membrane vesicle;

s4, loading the small-molecule chemotherapeutic drugs into the engineered hybrid cell membrane vesicles prepared in the step S3, and mixing to obtain the engineered drug-loaded hybrid cell membrane vesicles.

Preferably, the cell line in step S1 is the HEK-293T cell line.

Preferably, the protein mass ratio of the stable cell-based membrane vesicles to the platelet membrane vesicles used in step S4 is 3 to 5:1.

More preferably, the protein mass ratio of the stable cell-based membrane vesicles to the platelet membrane vesicles is 4:1

In particular, the engineered drug-loaded hybrid cell membrane vesicles prepared by the invention have dual effects of synergetic chemotherapy and immunotherapy. Therefore, as long as small molecular drugs can cause immunogenic death of tumor cells and can cause up-regulation of immune checkpoint proteins such as CD155, PDL1 and the like on the tumor cells, the targeted engineered drug-loaded hybrid cell membrane vesicles obtained by the preparation method can be theoretically used for treating postoperative recurrence and metastasis of cancer.

Preferably, the small molecule drug in step S4 is oxaliplatin, doxorubicin or paclitaxel.

More preferably, the small molecule chemotherapeutic drug is oxaliplatin.

Preferably, the ultrasonic treatment conditions in step S3 are: 3-6 min, temperature-20-5 ℃, and frequency 20-30W.

Preferably, the particle size of the engineered hybrid cell membrane vesicle prepared in step S5 is 100 ± 20nm.

The engineered drug-loaded hybrid cell membrane vesicle prepared by the method retains the targeting protein of platelets and the characteristic protein of a stable cell line on the surface, and the inner cavity is loaded with small-molecule chemotherapeutic drugs. The engineered drug-loaded hybrid cell membrane vesicle prepared by the invention can successfully encapsulate chemotherapeutic drugs; the compound has universal killing property on tumor cells, can obviously inhibit the growth of the tumor cells, and can cause the immunogenic death of the tumor cells so as to activate the immune system of an organism; can effectively inhibit the recurrence and metastasis of postoperative tumors, has obvious capacity of targeting postoperative parts, can obviously improve the survival period after operation, and has better in-vivo treatment effect. Meanwhile, tissue damage cannot be caused, and the results show that the engineered drug-loaded hybrid cell membrane vesicle prepared by the invention has better in-vivo biological safety.

The invention provides an engineered drug-loaded hybrid cell membrane vesicle, which is prepared by the method.

The invention provides application of the engineered drug-loaded hybrid cell membrane vesicle in postoperative recurrence and metastasis of cancer, and application of the engineered drug-loaded hybrid cell membrane vesicle in combined chemotherapy and immunotherapy of postoperative recurrence and metastasis of cancer.

The invention has the following beneficial effects:

the engineered drug-loaded hybrid cell membrane vesicle prepared by the method of the invention retains the targeting protein of platelets and the TIGIT characteristic protein on the surface, has the functions of targeting tumor cells and blocking CD155/TIGIT, and has an inner cavity loaded with micromolecular chemotherapeutic drugs. The main components of the engineered drug-loaded hybrid cell membrane vesicle are from organisms, and the engineered drug-loaded hybrid cell membrane vesicle has good biocompatibility and strong targeting property; the micromolecule chemotherapeutic drug has high bioavailability, universal killing property on tumor cells, can remarkably inhibit the growth of the tumor cells, can cause the immunogenic death of the tumor cells, and thus activates the immune system of an organism; can effectively inhibit the recurrence and metastasis of postoperative tumors, has obvious capacity of targeting postoperative parts, can obviously improve the survival period after operation, has better in-vivo treatment effect, can not cause tissue damage, and has better in-vivo biological safety. The engineered drug-loaded hybrid cell membrane vesicle prepared by the invention can realize good synergistic effect with chemotherapy and immunotherapy drugs, has good effect in targeted therapy of postoperative cancers by combined therapy, and can effectively inhibit relapse and metastasis of postoperative tumors.

Drawings

FIG. 1 is a flow assay (A) and a cellular immunofluorescence assay (B) of mouse breast cancer cells (4T 1 cells) up-regulated CD155 expression following treatment with Oxaliplatin (OXA), scale 50 μm;

FIG. 2 is a graph showing the analysis of the flow assay (A) and the analysis of the cell immunofluorescence assay (B) of human breast cancer cells (MCF-7 cells) up-regulating CD155 expression after treatment with Oxaliplatin (OXA) on a scale of 50 μm;

FIG. 3 is a graph showing the flow analysis of the stable cell line TIGIT cells in example 1;

FIG. 4 is a confocal laser microscopy image of the stable cell line TIGIT cells of example 1 (ruler, 10 μm);

FIG. 5 is a transmission electron microscopy image of engineered hybrid cell membrane vesicles TPNVs of example 4 (ruler, 200 nm);

FIG. 6 is a confocal laser microscopy image (ruler, 10 μm) of the engineered hybrid cell membrane vesicles TPNVs of example 4;

FIG. 7 is a DLS map of the engineered hybrid cell membrane vesicles TPNVs of example 4;

FIG. 8 is an encapsulation efficiency analysis graph of engineered hybrid cell membrane vesicle loaded with oxaliplatin;

FIG. 9 is a graph of the detection of the killing ability of the engineered drug-loaded hybrid cell membrane vesicles O-TPNVs on tumor cells;

FIG. 10 is a graph of the ability of engineered drug-loaded hybrid cell membrane vesicles O-TPNVs to cause immunogenic death of tumor cells (scale, 20 μm);

FIG. 11 is a diagram of in vivo detection of engineered hybrid cell membrane vesicles TPNVs targeting to post-operative sites of tumors (circled portions in the diagram are locations of post-operative wounds);

FIG. 12 is a statistical plot of the volume and size of post-operative relapsed tumors over time for each group of mice;

FIG. 13 is a representative picture of relapsed tumors in various groups of mice;

FIG. 14 is a statistical chart of the weights of relapsed tumors in each group of mice.

FIG. 15 is a graph of survival for groups of mice;

FIG. 16 is a HE slice of lung (scale, 500 μm) of various groups of mice tested for tumor lung metastasis;

FIG. 17 is a statistical graph of the weight of mice in each group over time;

FIG. 18 shows HE sections (scale, 100 μm) of major organs in experimental (O-TPNVs) and control (PBS) groups.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The experimental animals adopted by the invention are approved by animal ethics committee of Zhongshan university (animal ethics number: SYSU-IACUC-2022-000199), and can be used in the invention.

Human embryonic kidney cells HEK-293T were purchased from Chinese academy of sciences and oxaliplatin OXA was purchased from Selleck Chemicals.

Example 1 construction of an overexpression cell line TIGIT cells

Firstly packaging to obtain an expression lentivirus solution specific to a coding TIGIT gene, then infecting HEK-293T cells with the lentivirus solution, and culturing and screening the cells by using a culture medium containing 1-3 mu g/mL puromycin to obtain HEK-293T stable cell line TIGIT cells of over-expression EGFP-TIGIT. After about 2 weeks of screening, gene editing of the cell line TIGIT cells was examined by flow cytometry and confocal microscopy.

The detection result of flow cytometry is shown in fig. 3, compared with the control group, the cell after gene editing presents an obvious EGFP positive peak, and the proportion of the positive cell is up to more than 99%. Meanwhile, the observation result of a confocal microscope is shown in figure 4, which also shows that the EGFP-TIGIT protein is obviously and stably expressed on the HEK-293T cell membrane. The detection results are consistent, and the construction success of the stable cell line TIGIT cells is shown.

Example 2 preparation of engineered drug-loaded hybrid cell membrane vesicles

(1) Preparing TIGIT NVs:

the over-expressed stable cell line TIGIT cells prepared in example 1 were collected, washed with PBS, and then disrupted on ice using a dounce tissue homogenizer. Centrifuging the homogenate for 4-6 min at 4 ℃ under the condition of 2500-3500 g. Collecting supernatant, centrifuging at 15000-25000 g and 4 deg.c for 25-35 min. The supernatant was collected, centrifuged at 80000g to 120000g for 60 to 90min at 4 ℃, removed, and the pellet was washed 2 times with PBS. And (3) carrying out ultrasonic treatment on ice for 3-6 min at the frequency of 20-30W, and then sequentially extruding by extruders at 800nm, 400nm, 200nm and 100nm to obtain the stable cell system membrane vesicle TIGIT NVs.

(2) Preparation of PNVs:

separating the platelets from the blood of the mouse, then repeatedly freezing and thawing the obtained platelets, and then centrifuging the frozen and thawed liquid for 3-5 min at the temperature of 4 ℃ and under the condition of 3500 g-4500 g, and obtaining the precipitate which is the purer platelet membrane. And performing ultrasonic treatment on the obtained platelet membrane on ice for 3-6 min at the frequency of 20-30W, and then sequentially extruding the platelet membrane through extruders at 800nm, 400nm, 200nm and 100nm to obtain the platelet membrane vesicles PNVs.

(3) Synthesizing engineered hybrid cell membrane vesicles TPNVs:

mixing the obtained stable cell system membrane vesicle TIGIT NVs and platelet membrane vesicle PNVs according to the feeding ratio of 3:1 (protein mass), carrying out ultrasonic treatment for 3-6 min, and then extruding through a 100nm extruder to obtain the engineered hybrid cell membrane vesicle TPNVs.

Example 3 preparation of engineered drug-loaded hybrid cell membrane vesicles

(1) Preparing TIGIT NVs:

the over-expressed stable cell line TIGIT cells prepared in example 1 were collected, washed with PBS, and then disrupted on ice using a dounce tissue homogenizer. Centrifuging the homogenate for 4-6 min at 4 ℃ under the condition of 2500-3500 g. Collecting supernatant, centrifuging at 15000-25000 g for 25-35 min at 4 deg.C. The supernatant was collected, centrifuged at 80000g to 120000g for 60 to 90min at 4 ℃, removed, and the pellet was washed 2 times with PBS. And (3) carrying out ultrasonic treatment on ice for 3-6 min at the frequency of 20-30W, and then sequentially extruding by extruders at 800nm, 400nm, 200nm and 100nm to obtain the stable cell system membrane vesicle TIGIT NVs.

(2) Preparation of PNVs:

(3) Synthesizing engineered hybrid cell membrane vesicles TPNVs:

mixing the obtained stable cell system membrane vesicle TIGIT NVs and platelet membrane vesicle PNVs according to the feeding ratio of 4:1 (protein mass), carrying out ultrasonic treatment for 3-6 min, and then extruding through a 100nm extruder to obtain the engineered hybrid cell membrane vesicle TPNVs.

Example 4 preparation of engineered drug-loaded hybrid cell membrane vesicles

(1) Preparing TIGIT NVs:

(2) Preparation of PNVs:

(3) Synthesizing engineered hybrid cell membrane vesicles TPNVs:

mixing the obtained stable cell system membrane vesicle TIGIT NVs and platelet membrane vesicle PNVs according to the feeding ratio of 5:1 (protein mass), carrying out ultrasonic treatment for 3-6 min, and then extruding through a 100nm extruder to obtain the engineered hybrid cell membrane vesicle TPNVs.

In the engineered hybrid cell membrane vesicles prepared in the above examples 2 to 4, when the ratio of TIGIT NVs to PNVs adopted in example 2 is 3:1, the ratio of TIGIT NVs is 3/4, and because TIGIT NVs can play a blocking role, compared with 3 kinds of engineered hybrid cell membrane vesicles with different ratios, the blocking role played by the ratio of 3/4 is relatively weakest among the three. In example 4, when the proportion of TIGIT NVs to PNVs 5:1 is adopted, the proportion of PNVs is lower, 1/6, and in a post-operation targeting drug delivery system, the function of platelets is important, and the targeting property of the platelets can be influenced due to too low proportion. Therefore, combining blocking and targeting considerations, a preferred ratio of TIGIT NVs to PNVs is 4:1. Therefore, the following examples all use the engineered hybrid cell membrane vesicles prepared in example 3 for subsequent experiments.

Example 5 characterization of the Performance of engineered drug-loaded hybrid cell membrane vesicles

The morphology of the engineered hybrid membrane vesicle prepared in example 4 was observed with a transmission electron microscope, and the result is shown in fig. 5, where the hybrid membrane vesicle is spherical or ellipsoidal, and an obvious membrane structure can be observed, with a size of about 100 nm. Then, the membrane fusion state was observed by confocal laser microscopy, and the results are shown in fig. 6, where the cell membranes of two different components had good co-localization, indicating that the membranes were fused together. Then, TPNVs were measured by using a DLS particle size analyzer, and the results are shown in FIG. 7, wherein the particle size of the engineered hybrid cell membrane vesicle is approximately 100nm, and the zeta potential is approximately-20 mV. The detection results of the experiments show that the preparation of the engineered hybrid cell membrane vesicles TPNVs is successful.

Example 6 drug loading of engineered drug-loaded hybrid cell membrane vesicles

100 μ g (protein mass) of the engineered hybrid cell membrane vesicles TPNVs prepared as described in example 4 above and 100 μ g of oxaliplatin OXA were mixed together and sonicated on ice. Followed by centrifugation through an ultracentrifuge or ultrafiltration tube to substantially remove the unloaded oxaliplatin. And detecting the characteristic absorption of the oxaliplatin by using an ultraviolet-visible spectrophotometer or a high performance liquid chromatograph, thereby calculating the entrapment rate and the drug loading rate of the oxaliplatin.

The result is shown in fig. 8, and the encapsulation efficiency of the engineered hybrid cell membrane vesicles TPNVs prepared by the method to the OXA can reach 19.5-27.3%.

Example 7 biological function study of engineered drug-loaded hybrid cell membrane vesicles

1. Ability to inhibit tumor cell growth in vitro

Preparation of different concentrations (0, 0.5, 1, 2.5, 5, 10, 24, 50. Mu.M) of engineered drug-loaded hybrid cell membrane vesicles and co-culture with different types (4T 1, B16F0, MCF-7, heLa) of tumor cells, and 5% CO in a cell culture chamber at 37 ℃ in a manner ₂ Cultured and then tested for its killing of tumor cells by the CCK-8 method (purchased from Biyun Tian).

The results are shown in fig. 9, which shows that the engineered drug-loaded hybrid cell membrane vesicle has universal killing property on 4T1, B16F0, MCF-7 and HeLa tumor cells, and can significantly inhibit the growth of the tumor cells.

2. Ability to cause immunogenic death (ICD) of tumor cells in vitro

In order to explore the killing mechanism of the engineered drug-loaded hybrid cell membrane vesicles on tumor cells, the ability of the engineered drug-loaded hybrid cell membrane vesicles to cause immunogenic death of the tumor cells in vitro was studied. Four groups of experiments are respectively used for research, wherein PBS is a control group, OXA is treated by oxaliplatin, TPNVs is the engineered drug-loaded hybrid cell membrane vesicle obtained by the preparation method of the embodiment 4 of the invention, O-TPNVs is prepared by loading oxaliplatin on the engineered drug-loaded hybrid cell membrane vesicle, the dosage of each group is consistent, 4T1 cells of mouse breast cancer are treated, and then CRT protein and HMGB1 protein of tumor cells are detected.

The results are shown in fig. 10, the obvious eversion of CRT protein and the significant release of HMGB1 protein indicate that the engineered drug-loaded hybrid cell membrane vesicles can cause immunogenic death of tumor cells, thereby activating the body's immune system.

3. In vivo targeting post-operative wound capacity

To study the targeting ability of TPNVs, we constructed a tumor postoperative model of BALB/c mice, and then injected the TPNVs with Cy5.5-NHS fluorescent label into the tail vein of the postoperative mice. After 2h, observation was performed using a small animal in vivo imager.

The results are shown in fig. 11, showing that TPNVs have a clear ability to target post-operative sites.

Example 8 Effect of engineered drug-loaded hybrid cell Membrane vesicles on prevention of postoperative recurrence and metastasis of tumors

In order to research the effect of the prepared engineered drug-loaded hybrid cell membrane vesicles in the postoperative treatment of tumors, firstly, a postoperative tumor model of a BALB/c mouse is constructed and randomly divided into 4 groups, drugs are respectively given, wherein PBS is a control group, OXA is treated by oxaliplatin, TPNVs is the engineered hybrid cell membrane vesicles prepared by the preparation method of the embodiment 4 of the invention, O-TPNVs is prepared by loading oxaliplatin on the engineered drug-loaded hybrid cell membrane vesicles, the dosage of each group is consistent, and then the recurrence condition of the postoperative tumors of each group of mice is monitored. The growth curve of the postoperative recurrent tumor was calculated by measuring the longest diameter a and the shortest diameter b of the tumor by a vernier caliper by the formula tumor volume V = 0.5ab2.

The growth curve of the post-operative recurrent tumor is shown in FIG. 12, and the tumor recurrence in O-TPNVs group mice is the slowest. Representative pictures of each group of relapsed tumors are shown in FIG. 13, and likewise, the minimal volume of relapsed tumors in O-TPNVs mice can be visually observed. The weight of the relapsed tumors in each group is shown in FIG. 14, and the weight of the relapsed tumors in the O-TPNVs group is the smallest. The survival curve of model mice after tumor surgery is shown in FIG. 15, the survival time of O-TPNVs group mice is obviously longest, the survival rate is still as high as 83.3% at 60 days, and other groups of mice die at the time. In addition, in the mouse postoperative metastasis model, HE sections of the lungs of each group of mice are shown in FIG. 16, and no obvious metastasis phenomenon is observed in the lungs of the O-TPNVs group. The experimental results show that the prepared engineered drug-loaded hybrid cell membrane vesicle can effectively inhibit the recurrence and metastasis of postoperative tumors, remarkably improve the survival period of mice after operation, and has better in-vivo treatment effect.

Meanwhile, the in vivo safety of the drug is evaluated, the body weight of each group of mice is recorded before the drug application period and during the treatment period, and the main organs of the mice in the O-TPNVs administration group and the PBS group of the control group are subjected to HE section after the treatment is finished.

Results as shown in fig. 17, there was no significant difference in body weight among the groups of mice, and no sudden loss of a large amount of body weight occurred. Meanwhile, HE sections of the main organs of the experimental and control mice showed no significant tissue damage as shown in fig. 18. The results show that the prepared engineered drug-loaded hybrid cell membrane vesicle has better in-vivo biological safety, and the preliminary verification meets the basic requirements of clinical tests.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.

Claims

1. A preparation method of targeted engineered drug-loaded hybrid cell membrane vesicles is characterized by comprising the following steps:

s1, preparing a stable cell line for over-expressing TIGIT genes;

s2, preparing a stable cell line membrane vesicle for over-expression of the TIGIT gene by using the stable cell line of the step S1 as a donor;

2. The method according to claim 1, wherein the cell line HEK-293T cell line is used in step S1.

3. The method according to claim 1, wherein the protein mass ratio of the stable cell-based membrane vesicles to the platelet membrane vesicles used in step S4 is 3 to 5:1.

4. The method according to claim 3, wherein the protein mass ratio of the stable cell-based membrane vesicles to the platelet membrane vesicles is 4:1.

5. The process of claim 1, wherein the small molecule chemotherapeutic agent in step S4 is oxaliplatin, doxorubicin or paclitaxel.

6. The method according to claim 1, wherein the ultrasonic treatment conditions in step S3 are: 3-6 min, temperature-20-5 ℃, and frequency 20-30W.

7. The preparation method of claim 1, wherein the particle size of the engineered hybrid cell membrane vesicle prepared in step S5 is 100 ± 20nm.

8. An engineered drug-loaded hybrid cell membrane vesicle, prepared by the method of any one of claims 1 to 7.

9. The use of the engineered drug-loaded hybrid cell membrane vesicles of claim 8 for post-operative recurrence and metastasis of cancer.

10. The use of claim 9, wherein the engineered drug-loaded hybrid cell membrane vesicles are used in combination with chemotherapy and immunotherapy for post-operative recurrence and metastasis of cancer.