CN115337281B - Preparation method and application of targeted engineering drug-loaded hybrid cell membrane vesicle - Google Patents
Preparation method and application of targeted engineering drug-loaded hybrid cell membrane vesicle Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a targeted engineering drug-loaded hybrid cell membrane vesicle. The invention firstly prepares the engineering hybridized cell membrane vesicle by preparing a specific stable cell line and a single-component membrane vesicle, and loads small molecular medicines to obtain the engineering medicine-carrying hybridized cell membrane vesicle. The main component of the engineering drug-loaded hybrid cell membrane vesicle prepared by the invention is derived from the organism, and has good biocompatibility, strong targeting property and high bioavailability of the drug; the microbial agent 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 an organism immune system; can effectively inhibit the recurrence and metastasis of postoperative tumor, has obvious capability of targeting postoperative part, can obviously improve the postoperative survival time, can not cause tissue injury, and has better in vivo biosafety; the chemotherapy and the immunotherapy can realize good synergistic effect, and can effectively inhibit the recurrence and metastasis of the tumor after operation.
Description
Technical Field
The invention belongs to the technical field of biological medicine. More particularly, relates to a preparation method and application of a targeted engineering drug-loaded hybrid cell membrane vesicle.
Background
Cancer is a major killer of humans and presents a great threat to the psychological and physiological health of people. With respect to the treatment of cancer, surgical resection remains the primary method of treating solid tumors. However, recurrence and metastasis of postoperative cancer often result in poor patient postoperative recovery and low survival rate for 5 years. Therefore, for postoperative patients, postoperative adjuvant therapy is necessary. Because recurrence and metastasis during postoperative recovery of cancer generally occur within 5 years, about 70% -80% of patients recur and metastasize between them, whether they recur or metastasize, have a great impact on the patient and may cause death to occur in a short period of time. Therefore, it is necessary to perform adjuvant therapy in the postoperative recovery period of cancer, and the occurrence rate of recurrence and metastasis can be reduced.
At present, auxiliary treatment for the convalescence of cancer in clinic mainly comprises chemotherapy, radiotherapy, targeted drug treatment, or traditional Chinese medicine treatment, and the like, which can play a role in consolidation, further kill cancer cells remained in the body and greatly reduce the possibility of recurrence. In the adjuvant therapy, chemotherapy and immunotherapy are often adopted to combine with targeted drugs for treatment, although the occurrence rate of recurrence and metastasis can be reduced to a certain extent, the problems of low combination degree, low targeting, low utilization rate and the like of the targeted drugs in the chemotherapy and immunotherapy exist, and the effect of the combined action is still to be improved; the frequent use of targeted drugs has certain safety problems, so that research and development of a new drug delivery system or drug delivery system is needed, and a new effective strategy is provided for the treatment of cancer postoperative recurrence and metastasis prevention.
Platelets can target wounds and Circulating Tumor Cells (CTCs) after operation, so that the bioavailability of the medicine is effectively improved, and the side effect of the medicine is reduced. Thus, platelets and platelet-derived membrane vesicles are candidates for drug delivery vehicles for post-operative cancer treatment. Many platelet-based drugs have been developed to explore post-operative tumor treatment. Platelet vesicles are used for engineering cells and exosomes to treat vascular injury as the prior art discloses a platelet vesicle engineering cell and exosome for targeted tissue repair. However, tumor cells have multiple immune escape mechanisms, making it difficult for drugs that are simply targeted for delivery with platelet membrane vesicles to function effectively. Oxaliplatin (OXA), for example, is a widely used chemotherapeutic drug in the clinic that induces immunogenic cell death (immunogenic cell death, ICD) of tumors, with a synergistic effect on the immune system. However, the effect of the actual clinical OXA chemotherapy is poor, and previous studies of the subject group show that the OXA can cause up-regulation of CD155, and the single OXA treatment can further up-regulate the expression of CD155 in tumor cells, so that the tumor is more sensitive to the anti-CD 155 treatment. CD155 is expressed on the surface of tumor cells in a large quantity, and the immune system is inhibited by combining with a co-inhibitory receptor TIGIT on the surface of immune cells, so that immune escape is realized, and the effect of the OXA single drug is poor. While blocking of immunosuppressive signaling pathways may restore immunity, such as the CD155/TIGIT pathway. However, there is currently no drug delivery system in the prior art that has both tumor cell targeting and CD155/TIGIT blocking effects.
Disclosure of Invention
The invention aims 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 engineering drug-loaded hybrid cell membrane vesicle.
It is a second object of the present invention to provide a targeted engineered drug loaded hybrid cell membrane vesicle.
A third object of the present invention is to provide the use of engineered drug loaded hybrid cell membrane vesicles.
The above object of the present invention is achieved by the following technical scheme:
previous studies in this group showed that OXA caused up-regulation of CD155, as shown in figures 1 and 2, OXA treatment alone could further up-regulate CD155 expression in tumor cells, rendering the tumor more sensitive to anti-CD 155 treatment. CD155 is expressed on the surface of tumor cells in a large quantity, and is combined with a co-suppression receptor TIGIT on the surface of immune cells to suppress an immune system, so that immune escape is realized, the effect of an OXA single drug is poor, and the blocking of an immune suppression signal channel can restore immunity.
The invention creatively researches a method for preparing the engineering medicine-carrying hybridized cell membrane vesicle with the targeting tumor cells and CD155/TIGIT blocking effect, firstly constructs a cell line for stably expressing the TIGIT protein through biotechnological means such as gene editing and a physicochemical method, then further mixes the cell line with the platelet membrane vesicle to prepare the hybridized cell membrane vesicle, finally loads a micromolecule chemotherapeutic medicine to obtain the engineering medicine-carrying hybridized cell membrane vesicle, and provides a novel medicine system with stronger targeting and better safety for postoperative recurrence and metastasis of cancers.
The invention provides a preparation method of a targeted engineering drug-loaded hybrid cell membrane vesicle, which comprises the following steps:
s1, preparing a stable cell line which overexpresses the TIGIT gene;
s2, preparing a stable cell line membrane vesicle which over-expresses the TIGIT gene by taking the stable cell line of the step S1 as a donor;
s3, mixing a stable cell line membrane vesicle which overexpresses the TIGIT gene with a platelet membrane vesicle, performing ultrasonic treatment, and extruding to obtain an engineering hybrid cell membrane vesicle;
s4, loading small molecular chemotherapeutic drugs into the engineering hybridized cell membrane vesicles prepared in the step S3, and mixing to obtain the engineering drug-loaded hybridized cell membrane vesicles.
Preferably, the cell line in step S1 is a HEK-293T cell line.
Preferably, the protein mass ratio of the stable cell line membrane vesicles to the platelet membrane vesicles used in step S4 is 3-5:1.
More preferably, the protein mass ratio of stable cell line membrane vesicles to platelet membrane vesicles is 4:1
In particular, the engineered drug-loaded hybrid cell membrane vesicles prepared by the invention are dual effects of synergistic chemotherapy and immunotherapy. Therefore, if the small molecular medicine 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 preparation method can be theoretically adopted to obtain the targeted engineering medicine-carrying hybrid cell membrane vesicle 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 agent is oxaliplatin.
Preferably, the ultrasonic treatment conditions in step S3 are: 3-6 min, temperature-20-5 deg.c and frequency 20-30W.
Preferably, the particle size of the engineered hybrid cell membrane vesicles prepared in step S5 is 100±20 nm.
The engineering drug-carrying hybrid cell membrane vesicle prepared by the method has the targeting protein of platelets and the characteristic protein of a stable cell line reserved on the surface, and the inner cavity is loaded with a micromolecular chemotherapeutic drug. The engineering medicine-carrying hybrid cell membrane vesicles prepared by the invention can successfully encapsulate chemotherapeutic medicines; the microbial agent has universal killing property on tumor cells, can obviously inhibit the growth of the tumor cells and can cause immunogenic death of the tumor cells, thereby activating an organism immune system; can effectively inhibit the recurrence and metastasis of postoperative tumor, has obvious capability of targeting postoperative part, can obviously improve the postoperative survival time, and has better in vivo treatment effect. Meanwhile, tissue damage can not be caused, and the results can show that the engineering medicine carrying hybrid cell membrane vesicle prepared by the invention has better in vivo biosafety.
The invention provides an engineering drug-loaded hybrid cell membrane vesicle, which is prepared by the method.
The invention provides application of the engineering drug-loaded hybrid cell membrane vesicle in recurrence and metastasis after cancer operation, and application of the engineering drug-loaded hybrid cell membrane vesicle in combination with chemotherapy and immunotherapy in recurrence and metastasis after cancer operation.
The invention has the following beneficial effects:
the engineering medicine carrying hybridized cell membrane vesicle prepared by the method of the invention has the targeting protein of the platelet and the characteristic protein of the TIGIT reserved on the surface, has the blocking effect of targeting tumor cells and CD155/TIGIT, and the inner cavity of the engineering medicine carrying hybridized cell membrane vesicle is loaded with micromolecule chemotherapeutic medicines. The main component of the engineering drug-loaded hybrid cell membrane vesicle is derived from the organism, and has good biocompatibility and strong targeting property; the small molecule chemotherapeutic medicine has high bioavailability, 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, thereby activating the immune system of the organism; can effectively inhibit the recurrence and metastasis of postoperative tumor, has obvious capability of targeting postoperative part, can obviously improve the lifetime after the operation, has better in vivo treatment effect, can not cause tissue injury, and has better in vivo biosafety. The engineering drug-loaded hybrid cell membrane vesicle prepared by the invention can realize good synergistic effect with chemotherapy and immunotherapy drugs, has good effect in the targeted treatment of postoperative cancers by combined therapy, and can effectively inhibit the recurrence and metastasis of postoperative tumors.
Drawings
FIG. 1 is a flow assay analysis (A) and a cell immunofluorescence assay (B) of up-regulated CD155 expression in mouse breast cancer cells (4T 1 cells) after Oxaliplatin (OXA) treatment, scale 50 μm;
FIG. 2 shows a flow assay (A) and a cell immunofluorescence assay (B) for up-regulating CD155 expression in human breast cancer cells (MCF-7 cells) after Oxaliplatin (OXA) treatment, on a scale of 50 μm;
FIG. 3 is a flow chart showing the results of the stable cell line TIGIT cells assay of example 1;
FIG. 4 is a view of a confocal microscope of the stable cell line TIGIT cells of example 1 (scale, 10 μm);
FIG. 5 is a transmission electron microscopy image of the engineered hybrid cell membrane vesicle TPNVs of example 3 (scale, 200 nm);
FIG. 6 is a laser confocal microscopy image of the engineered hybrid cell membrane vesicle TPNVs of example 3 (scale, 10 μm);
FIG. 7 is a DLS assay of the engineered hybrid cell membrane vesicle TPNVs of example 3;
FIG. 8 is a graph of the encapsulation efficiency analysis of the engineered hybrid cell membrane vesicle loaded oxaliplatin;
FIG. 9 is a graph of the detection of the killing ability of engineered drug-loaded hybrid cell membrane vesicles O-TPNVs against tumor cells;
FIG. 10 is a graph (scale, 20 μm) of the ability of engineered drug loaded hybrid cell membrane vesicles O-TPNVs to cause immunogenic death of tumor cells;
FIG. 11 is a biopsy of the engineered hybrid cell membrane vesicle TPNVs targeted to the post-operative site of a tumor (circled portion of the figure is the post-operative wound site);
FIG. 12 is a statistical plot of recurrent tumor volume size over time following surgery for each group of mice;
fig. 13 is a representative picture of relapsing tumor in each group of mice;
FIG. 14 is a statistical plot of the weight of recurrent tumor in each group of mice;
FIG. 15 is a graph of survival for each group of mice;
FIG. 16 is a graph of HE sections of lung (scale, 500 μm) from groups of mice detecting tumor lung metastasis;
FIG. 17 is a statistical plot of body weight of each group of mice over time;
FIG. 18 shows HE sections (scale, 100 μm) of the major organs of the experimental group (O-TPNVs) and the control group (PBS).
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The experimental animals adopted by the invention are all approved by the ethical committee of Zhongshan university (the ethical number of the animals is SYSU-IACUC-2022-000199), and can be used in the invention.
Human embryonic kidney cells HEK-293T were purchased from the Proc.Natl.Acad., oxaliplatin OXA from Selleck Chemicals.
EXAMPLE 1 construction of an overexpressing cell line TIGIT cells
Firstly packaging to obtain a slow virus solution for expressing the TIGIT gene, then infecting HEK-293T cells with the slow virus solution, and culturing and screening the cells by using a culture medium containing 1-3 mug/mL puromycin to obtain the HEK-293T stable cell line TIGIT cells for over-expressing EGFP-TIGIT. After about 2 weeks of screening, the cell line TIGIT cells were examined for gene editing using flow cytometry and confocal microscopy.
The results of flow cytometry are shown in FIG. 3, and compared with the control group, the cells after gene editing show obvious EGFP positive peaks, and the positive cell proportion is as high as more than 99%. Meanwhile, the confocal microscope observation result is shown in fig. 4, which also shows that EGFP-TIGIT protein is obviously and stably expressed on HEK-293T cell membrane. The detection results are consistent, and the stable cell line TIGIT cells are successfully constructed.
Example 2 preparation of engineered drug loaded hybrid cell membrane vesicles
(1) Preparation of TIGIT NVs:
the overexpressing stable cell line TIGIT cells prepared in example 1 were collected, washed with PBS and then broken up on ice using a dunus tissue homogenizer. Centrifuging the homogenate for 4-6 min at the temperature of 4 ℃ and under the condition of 2500-3500 g. Collecting supernatant, and centrifuging at 4 deg.c and 15000-25000 g for 25-35 min. Collecting the supernatant, centrifuging for 60-90 min at 4 ℃ and 80000-120000 g, removing the supernatant, washing the precipitate for 2 times by using PBS. Ultrasonic treatment is carried out on ice for 3-6 min with the frequency of 20-30W, and then extrusion is carried out through an extruder with the wavelength of 800nm, 400nm, 200nm and 100nm in sequence, thus obtaining the stable cell line membrane vesicle TIGIT NVs.
(2) PNVs were prepared:
separating platelets from the blood of the mice, repeatedly freezing and thawing the platelets, centrifuging the frozen and thawed liquid at the temperature of 4 ℃ and under the condition of 3500-4500 g for 3-5 min, and precipitating to obtain the purer platelet membrane. And (3) carrying out ultrasonic treatment on the obtained platelet membrane ice for 3-6 min at the frequency of 20-30W, and then sequentially extruding the platelet membrane by using an extruder of 800nm, 400nm, 200nm and 100nm to obtain the platelet membrane vesicle PNVs.
(3) Synthetic engineered hybrid cell membrane vesicles TPNVs:
mixing the obtained stable cell line membrane vesicle TIGIT NVs and platelet membrane vesicle PNVs according to a feeding ratio of 3:1 (protein mass), performing ultrasonic treatment for 3-6 min, and extruding through a 100nm extruder to obtain the engineering hybrid cell membrane vesicle TPNVs.
EXAMPLE 3 preparation of engineered drug-loaded hybrid cell membrane vesicles
(1) Preparation of TIGIT NVs:
the overexpressing stable cell line TIGIT cells prepared in example 1 were collected, washed with PBS and then broken up on ice using a dunus tissue homogenizer. Centrifuging the homogenate for 4-6 min at the temperature of 4 ℃ and under the condition of 2500-3500 g. Collecting supernatant, and centrifuging at 4 deg.c and 15000-25000 g for 25-35 min. Collecting the supernatant, centrifuging for 60-90 min at 4 ℃ and 80000-120000 g, removing the supernatant, washing the precipitate for 2 times by using PBS. Ultrasonic treatment is carried out on ice for 3-6 min with the frequency of 20-30W, and then extrusion is carried out through an extruder with the wavelength of 800nm, 400nm, 200nm and 100nm in sequence, thus obtaining the stable cell line membrane vesicle TIGIT NVs.
(2) PNVs were prepared:
separating platelets from the blood of the mice, repeatedly freezing and thawing the platelets, centrifuging the frozen and thawed liquid at the temperature of 4 ℃ and under the condition of 3500-4500 g for 3-5 min, and precipitating to obtain the purer platelet membrane. And (3) carrying out ultrasonic treatment on the obtained platelet membrane ice for 3-6 min at the frequency of 20-30W, and then sequentially extruding the platelet membrane by using an extruder of 800nm, 400nm, 200nm and 100nm to obtain the platelet membrane vesicle PNVs.
(3) Synthetic engineered hybrid cell membrane vesicles TPNVs:
mixing the obtained stable cell line membrane vesicle TIGIT NVs and platelet membrane vesicle PNVs according to a feeding ratio of 4:1 (protein mass), performing ultrasonic treatment for 3-6 min, and extruding through a 100nm extruder to obtain the engineering hybrid cell membrane vesicle TPNVs.
EXAMPLE 4 preparation of engineered drug-loaded hybrid cell membrane vesicles
(1) Preparation of TIGIT NVs:
the overexpressing stable cell line TIGIT cells prepared in example 1 were collected, washed with PBS and then broken up on ice using a dunus tissue homogenizer. Centrifuging the homogenate for 4-6 min at the temperature of 4 ℃ and under the condition of 2500-3500 g. Collecting supernatant, and centrifuging at 4 deg.c and 15000-25000 g for 25-35 min. Collecting the supernatant, centrifuging for 60-90 min at 4 ℃ and 80000-120000 g, removing the supernatant, washing the precipitate for 2 times by using PBS. Ultrasonic treatment is carried out on ice for 3-6 min with the frequency of 20-30W, and then extrusion is carried out through an extruder with the wavelength of 800nm, 400nm, 200nm and 100nm in sequence, thus obtaining the stable cell line membrane vesicle TIGIT NVs.
(2) PNVs were prepared:
separating platelets from the blood of the mice, repeatedly freezing and thawing the platelets, centrifuging the frozen and thawed liquid at the temperature of 4 ℃ and under the condition of 3500-4500 g for 3-5 min, and precipitating to obtain the purer platelet membrane. And (3) carrying out ultrasonic treatment on the obtained platelet membrane ice for 3-6 min at the frequency of 20-30W, and then sequentially extruding the platelet membrane by using an extruder of 800nm, 400nm, 200nm and 100nm to obtain the platelet membrane vesicle PNVs.
(3) Synthetic engineered hybrid cell membrane vesicles TPNVs:
mixing the obtained stable cell line membrane vesicle TIGIT NVs and platelet membrane vesicle PNVs according to a feeding ratio of 5:1 (protein mass), performing ultrasonic treatment for 3-6 min, and extruding through a 100nm extruder to obtain the engineering hybrid cell membrane vesicle TPNVs.
In the engineering hybrid cell membrane vesicles prepared in examples 2 to 4, when the ratio of TIGIT NVs to PNVs is 3:1 in example 2, the ratio of TIGIT NVs is 3/4, and the blocking effect of 3/4 is the weakest in the three compared with the 3 engineering hybrid cell membrane vesicles with different ratios because TIGIT NVs can play a blocking role. When the ratio of TIGIT NVs to PNVs is 5:1 in example 4, the PNVs are present in a lower ratio of 1/6, and in a post-operative targeted drug delivery system, platelet effects are important, and too low a ratio may affect its targeting. Therefore, the ratio of TIGIT NVs to PNVs is preferably 4:1 in consideration of the combined blocking and targeting effects. Thus, the following examples all used 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 cell membrane vesicles prepared in example 3 was observed using a transmission electron microscope, and as shown in fig. 5, the hybrid membrane vesicles were spherical or ellipsoidal, and a distinct membrane structure was observed, with a size of about 100 nm. And then observing the membrane fusion state by using a laser confocal microscope, and the result is shown in fig. 6, the cell membranes of the two different components have good co-localization, and the membranes are fused together. TPNVs were then tested using a DLS particle size analyzer, and the particle size of the engineered hybrid cell membrane vesicles was approximately 100nm and the zeta potential was approximately-20 mV, as shown in FIG. 7. The experimental detection results all show that the engineering hybridized cell membrane vesicle TPNVs are successfully prepared.
EXAMPLE 6 drug loading of engineered drug loaded hybrid cell membrane vesicles
100. Mu.g (protein amount) of the engineered hybrid cell membrane vesicle TPNVs prepared in example 3 above and 100. Mu.g of oxaliplatin OXA were mixed together and sonicated on ice. And then centrifuged through an ultracentrifuge or ultrafiltration tube to sufficiently remove the unloaded oxaliplatin. And detecting the characteristic absorption of the oxaliplatin by using an ultraviolet-visible spectrophotometer or a high performance liquid chromatograph, so as to calculate and obtain the encapsulation rate and the drug loading rate of the oxaliplatin.
As shown in FIG. 8, the encapsulation rate of the engineering hybrid cell membrane vesicle TPNVs prepared by the invention 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
Preparing engineering medicine carrying hybridized cell membrane vesicles with different concentrations (0, 0.5, 1, 2.5, 5, 10, 24 and 50 mu M) and tumor cells of different types (4T 1, B16F0, MCF-7 and HeLa), co-culturing in a cell incubator at 37 ℃ and 5% CO 2 The cells were cultured and tested for their killing by CCK-8 (from Biyun days) method.
The results are shown in fig. 9, which shows that the engineering drug-loaded hybrid cell membrane vesicles have universal killing property on 4T1, B16F0, MCF-7 and HeLa tumor cells, and can remarkably 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 engineering drug-loaded hybrid cell membrane vesicles on tumor cells, we studied their ability to cause immunogenic death of tumor cells in vitro. Four groups of experiments were used to study, wherein PBS was used as a control group, oxA was treated with oxaliplatin, TPNVs were used as engineering drug-loaded hybrid cell membrane vesicles prepared according to the preparation method of example 3 of the invention, O-TPNVs were used to load oxaliplatin with engineering drug-loaded hybrid cell membrane vesicles, the amounts of each group were consistent, 4T1 cells of breast cancer were treated, and then CRT proteins and HMGB1 proteins of tumor cells were detected.
The results are shown in fig. 10, where the apparent eversion of CRT protein and the significant release of HMGB1 protein show that engineered drug loaded hybrid cell membrane vesicles can cause immunogenic death of tumor cells, thereby activating the immune system of the body.
3. In vivo targeting post-operative wound capability
To study the targeting ability of TPNVs, we constructed a tumor post-operation model of BALB/c mice, and then intravenously injected the post-operation mice with Cy5.5-NHS fluorescent-labeled TPNVs. After 2h observations were made using a small animal biopsy imager.
The results are shown in fig. 11, which demonstrates that TPNVs have significant ability to target post-operative sites.
Example 8 effects of engineered drug loaded hybrid cell membrane vesicles on prevention of tumor postoperative recurrence and metastasis
In order to study the effect of the prepared engineering drug-loaded hybrid cell membrane vesicles in postoperative treatment of tumors, a tumor postoperative model of BALB/c mice is firstly constructed, the model is randomly divided into 4 groups and is respectively administered with drugs, wherein PBS is a control group, oxa is treated by oxaliplatin, TPNVs are engineering hybrid cell membrane vesicles obtained by the preparation method of the embodiment 3 of the invention, O-TPNVs are engineering drug-loaded hybrid cell membrane vesicles loaded with oxaliplatin, 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 vernier calipers and calculating the formula tumor volume v=0.5ab2.
The growth curve of postoperative recurrent tumors is shown in FIG. 12, with the slowest recurrence of tumors in the O-TPNVs group of mice. Representative pictures of each group of recurrent tumors are shown in fig. 13, and likewise, it can be intuitively seen that O-TPNVs mice had minimal recurrent tumor volumes. Weight statistics for each group of recurrent tumors as shown in figure 14, the weight of the recurrent tumors in the O-TPNVs group was minimal. The survival graph of model mice after tumor surgery is shown in FIG. 15, the mice in the group of O-TPNVs have the remarkably longest survival time, and the survival rate is still as high as 83.3% at day 60, while the mice in the other groups die. In addition, in the post-operative transfer model of mice, no significant transfer was observed in the lungs of the O-TPNVs group as shown in fig. 16 for HE sections of the lungs of each group. The experimental results show that the prepared engineering drug-loaded hybrid cell membrane vesicles can effectively inhibit the recurrence and metastasis of postoperative tumors, remarkably improve the postoperative survival time of mice, and have better in-vivo treatment effect.
Meanwhile, the in vivo safety of the drug is also evaluated, the body weight of each group of mice is recorded before the administration period and during the treatment period, and main organs of the mice in the experimental group O-TPNVs administration group and the control group PBS group are subjected to HE section after the treatment is finished.
Results As shown in FIG. 17, the weight of each group of mice was not significantly different, and no phenomenon of abrupt loss of a large amount of weight occurred. Meanwhile, HE sections of the main organs of the mice of the experimental group and the control group were shown in fig. 18, and there was no significant tissue damage. These results show that the prepared engineering drug-loaded hybrid cell membrane vesicles have better in vivo biosafety, and the primary verification meets the basic requirements of clinical experiments.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (4)
1. A method for preparing a targeted engineering drug-loaded hybrid cell membrane vesicle, which is characterized by comprising the following steps:
s1, preparing a stable cell line which overexpresses the TIGIT gene;
s2, preparing a stable cell line membrane vesicle which over-expresses the TIGIT gene by taking the stable cell line of the step S1 as a donor;
s3, mixing a stable cell line membrane vesicle which overexpresses the TIGIT gene with a platelet membrane vesicle, performing ultrasonic treatment, and extruding to obtain an engineering hybrid cell membrane vesicle; the protein mass ratio of the stable cell line membrane vesicle to the platelet membrane vesicle is 4:1;
s4, loading small molecule chemotherapeutic drugs into the engineering hybridized cell membrane vesicles prepared in the step S3, and mixing to obtain the engineering drug-loaded hybridized cell membrane vesicles; the small molecule chemotherapeutic drug is oxaliplatin, doxorubicin or paclitaxel;
the ultrasonic treatment conditions in the step S3 are as follows: ultrasonic treatment on ice for 3-6 min and frequency of 20-30W.
2. The method according to claim 1, wherein the cell line HEK-293T is used in step S1.
3. The method of claim 1, wherein the engineered hybrid cell membrane vesicles prepared in step S5 have a particle size of 100±20 nm.
4. An engineered drug-loaded hybrid cell membrane vesicle prepared by the method of any one of claims 1-3.
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