CN114404571A - Chemotherapy drug loaded and TIGIT over-expressed engineered drug loaded cell membrane vesicle, and preparation method and application thereof - Google Patents
Chemotherapy drug loaded and TIGIT over-expressed engineered drug loaded cell membrane vesicle, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method and application of an engineered drug-loaded cell membrane vesicle loaded with small-molecule chemotherapeutic drugs and overexpressed by TIGIT (tungsten inert gas), comprising the following steps of: s1, packaging viruses, and preparing and collecting a specific slow virus solution for encoding a TIGIT gene; s2, infecting a target cell by using the obtained lentivirus solution, and screening by using a resistance medicament, thereby constructing a stable cell line over-expressing TIGIT gene; s3, culturing the obtained stable cell line, extracting cell membranes of the stable cell line, preparing the cell membranes into membrane nano vesicles, and performing centrifugal purification to obtain engineered cell membrane vesicles; s4, loading the obtained cell membrane vesicle with a small-molecule chemotherapeutic drug, and carrying out centrifugal purification to obtain the engineered drug-loaded cell membrane vesicle. The engineered drug-loaded cell membrane vesicle prepared by the invention has good biological safety, shows excellent effect in the immunotherapy of tumors, and has better clinical transformation value and application prospect.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to an engineered drug-loaded cell membrane vesicle loaded with chemotherapeutic drugs and subjected to TIGIT overexpression, and a preparation method and application thereof.
Background
As immunotherapy matures, humans appear to see eosins that overcome cancer. However, despite the unprecedented success of relevant therapies directed against immune checkpoints such as PD-1, CTLA-4, etc., only a fraction of patients have been shown clinically to be in objective remission. Meanwhile, the existing immune check points are difficult to meet the diversity and complexity of clinical tumor patients, and treatment-related toxicity is generated in clinical use, which is called immune-related adverse events. For example, 90% of patients treated with CTLA-4 antibodies and 70% of patients treated with PD-1 antibodies can develop these immune-related adverse events. The development of new immune checkpoints and related treatment protocols is therefore urgently needed to be investigated.
TIGIT is known collectively as the T cell immunoglobulin and immunoreceptor tyrosine inhibitory motif domain (also known as WUCAM, Vstm 3), a member of the CD28 family. TIGIT is mainly expressed in lymphocytes, especially in effector CD8+T cells, natural killer cells, regulatory CD4+T cell and follicular helper CD4+High expression in T cells. The primary ligand of TIGIT in humans and mice is CD155 (also known as PVR), which is barely or weakly expressed in various normal human tissues, but is often overexpressed in human malignanciesAnd (4) expressing. High expression of CD155 promotes tumor cell invasion and migration and is associated with poor tumor progression and prognosis.
The TIGIT/CD155 pathway may specifically suppress anti-tumor immune responses from multiple stages. Given the important role of the TIGIT/CD155 checkpoint pathway in tumor immune escape, drug development for this pathway has also become a research hotspot. However, two independent studies have shown that TIGIT monoclonal antibody is insufficient to inhibit the growth of subcutaneous tumors as a single agent therapy, and thus a combination therapy strategy needs to be developed.
The combination of immunotherapy with each other or with other traditional treatments has been validated in many studies to greatly improve efficacy and reduce toxic side effects. Over the past few decades, increasing preclinical and clinical research evidence suggests that chemotherapy and immunotherapy may be synergistic, and thus their combination is an effective anti-tumor strategy. The IDO inhibitor and the chemotherapeutic drug are combined to be applied to the treatment result of tumor-bearing mice, and the tumors are found to be well controlled. Researchers from the national cancer institute of the united states demonstrated that patients vaccinated with tumor responded better to subsequent chemotherapy than unvaccinated patients. The combination of an effective dose of doxorubicin and tumor immunotherapy successfully activated the anti-tumor response in the CT26 or MCA205 tumor model. Experiments also prove that the nanoparticle co-loaded with the chemotherapeutic drugs paclitaxel and lipopolysaccharide can be more beneficial to tumor regression in animals compared with the treatment of paclitaxel and lipopolysaccharide alone. IL-7 is a cytokine which can regulate the proliferation and development of T cells, and research shows that the in vivo administration of IL-7 and OXA can obviously inhibit the growth of tumors, while the administration of IL-7 alone has little effect.
The main reason for the good efficacy of these studies is that some chemotherapeutic drugs enhance the anti-tumor immune response by inducing Immunogenic Cell Death (ICD) of tumor cells. Tumorigenic ICDs can effectively stimulate the host's immune system primarily from 3 ICD components, which are (1) CRT, a chaperone protein resident in the endoplasmic reticulum cavity that can serve as the "eat me" signal for DCs; (2) HMGB1, a DNA binding protein and TLR-4 mediated DC activator; (3) ATP, is capable of activating P2X7 purinergic receptors on DCs. These three ICD components can facilitate phagocytosis of tumor cells by DCs, thereby facilitating DC processing of tumor-derived antigens and subsequently initiating T lymphocyte-mediated adaptive immune responses. Several chemotherapeutic drugs have been shown to induce ICDs, particularly OXA (Oxaliplatin).
Chemotherapy and immunotherapy present great treatment potential, but only direct injection of these drugs, such as small molecule drugs, monoclonal antibodies, etc., has certain disadvantages, including unstable properties, easy degradation and clearance, short circulation time in vivo, poor targeting ability, etc. With the development and progress of science and technology, the nano delivery system is gradually perfected, and the development of various tumor therapies is greatly promoted. The effective nano delivery system has the capabilities of preventing the medicine from being rapidly degraded, prolonging the detention time of the medicine in vivo, realizing immune escape, releasing the medicine in a targeted manner and carrying the medicine to break through a specific physiological barrier of an organism.
In recent years, biofilm nano-delivery systems have been successfully favored by more and more researchers. The biological membrane derived from cells is a phospholipid bilayer structure, mainly contains lipid and protein as main components, and has important physiological functions. Biological membranes are prepared into nano-sized biological membrane vesicles by various methods, the complexity of the membrane including lipids, proteins and carbohydrates can be preserved, and the further synthetic biological membrane nano-delivery system retains many of the attributes of cell origin. Because the biological membrane is from the cell, the biological membrane nano delivery system constructed based on the method has the following characteristics: (1) the surface characteristics of the source cell membrane are reserved; (2) good biocompatibility and low immunogenicity; (3) avoiding the organism to clear and realizing long-acting circulation; (4) the surface of the membrane can be modified in various ways, so that various additional functions and characteristics can be obtained conveniently; (5) the preparation cost is low, the time consumption is short, and the engineering preparation is facilitated. In the past decade, biofilm-based nano-delivery systems have become a promising strategy for tumor therapy. Various types of cellular biofilms are prepared into nano-delivery systems for tumor therapy, including erythrocytes, tumor cells, leukocytes, platelets and stem cells, which greatly advance the development of tumor therapy.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation method of an engineered drug-loaded cell membrane vesicle which is loaded with chemotherapeutic drugs and over-expressed by TIGIT.
In order to achieve the purpose, the invention adopts the technical scheme that: a preparation method of an engineered drug-loaded cell membrane vesicle loaded with chemotherapeutic drugs and overexpressed in TIGIT (tungsten inert gas) is characterized by comprising the following steps:
s1, packaging lentivirus, culturing HEK-293T cells in a 37 ℃ cell culture box, adding a mixed target plasmid, a packaging plasmid and lipo2000 according to a volume ratio of 1: 1.2-1.5: 2-4, wherein the target plasmid is a plasmid for encoding a TIGIT gene, changing the liquid after 5-9 hours, continuously culturing by using a normal complete culture medium, collecting a culture supernatant, centrifugally removing cells, continuously collecting the supernatant, filtering to remove cell debris in the supernatant, mixing a filtrate and a virus purification reagent according to a volume ratio of 1:4, centrifuging for 20-30 minutes at 3000 g-4000 g after incubation, removing the supernatant, and carrying out heavy suspension precipitation by using BS to obtain a required lentivirus solution; wherein the sequence number of the gene for coding TIGIT is as follows: NM _ 001146325.1. . .
S2, culturing target cells in a cell culture box, wherein the target cells are NIH/3T3 cells, then infecting the NIH/3T3 cells with the obtained lentivirus solution, screening the cells with puromycin after infection, detecting after 1-2 weeks, selecting TIGIT over-expressed cells, and constructing a TIGIT over-expressed stable cell line, namely the TIGIT over-expressed NIH/3T3 cell line.
S3, culturing the obtained stable cell line, collecting and cleaning, crushing to form homogenate, centrifugally extracting cell membranes of the homogenate, preparing the cell membranes into membrane vesicles, and centrifugally purifying to obtain the engineered cell membrane vesicles;
and S4, loading the obtained cell membrane vesicle with a small-molecule chemotherapeutic drug by a co-incubation or ultrasonic method, and centrifugally purifying to obtain the engineered drug-loaded cell membrane vesicle.
Further, the concentration of the puromycin is 2-6 mug/ml. After the slow virus solution infects NIH 3T3 cells, 2-6 mug/mL puromycin is added, and a confocal microscope detection result shows that the cells have obvious target protein TIGIT expression.
Further, the specific step of s 3: collecting a TIGIT over-expressed stable cell line, washing with PBS, crushing with a homogenizer to form homogenate, centrifuging the homogenate at 4 ℃ under the centrifugal force of 1000-1500 g for 6-10 min to remove unbroken cells and large cell fragments, collecting the supernatant, centrifuging at 4 ℃ under the centrifugal force of 3000-4000 g for 6-10 min to remove mitochondria and other organelles, collecting the supernatant, centrifuging at 4 ℃ under 80000-120000 g for 60-90 min to remove the supernatant, washing and precipitating with PBS for 2 times, extruding with extruders with different pore diameters for several times to obtain membrane vesicles, wherein the pore diameter of the extruders is 0.1-1 μm, the pore diameter of the extruders used for several times is reduced in turn, ultracentrifuging the obtained membrane vesicles at 4 ℃ under the centrifugal force of 100000-120000 g for 90-120 min, collecting the precipitate, washing with PBS for 2 times, continuously collecting the precipitate, and (5) resuspending the mixture by using PBS to obtain the engineered cell membrane vesicle. Centrifuging the homogenate for three times, gradually increasing the centrifugal force, gradually removing broken cells and large cell fragments, removing mitochondria and other organelles, and the like, collecting to obtain cell membranes, extruding the membrane vesicles through a plurality of extruders, and gradually reducing the pore diameter of the extruders each time, so that the obtained membrane vesicles have uniform particle size, the collection rate of the membrane vesicles is improved, and the loss is reduced.
Furthermore, the particle size of the engineered cell membrane vesicle is 20-200 nm, and the average particle size is approximately 100 nm. In the structure, the homogenate is centrifuged for three times and extruded for multiple times by an extruder with the aperture of 1-0.1 mu m, and the obtained engineered cell membrane vesicle has uniform particle size and is suitable for drug loading.
Further, the homogenate in S3. is centrifuged for the third time, the supernatant is removed, the precipitate is washed 2 times by PBS, and then the membrane vesicle is obtained by extruding the precipitate through extruders with the aperture of 1 μm, 0.4 μm, 0.2 μm and 0.1 μm in sequence.
Furthermore, the particle size of the engineered cell membrane vesicle is 20-200 nm, and the average particle size is about 100 nm.
Further, in the S4, the oxaliplatin is loaded by a co-incubation or ultrasonic method, wherein the final concentrations of the engineering cell membrane vesicles and the oxaliplatin solution after mixing are 1mg/ml and 1-1.5 mg/ml respectively, the mixture is co-incubated at 37 ℃ or subjected to ultrasonic treatment on ice to obtain a corresponding mixed system, and then the mixed system is centrifuged by an ultracentrifuge or an ultrafiltration tube to obtain the engineering drug-loaded cell membrane vesicles.
Another object of the invention is: the invention also provides an engineered drug-loaded cell membrane vesicle which is loaded with chemotherapeutic drugs and is subjected to TIGIT overexpression, and is characterized by being prepared by the preparation method.
Yet another object of the invention is: the invention also provides an engineered drug-loaded cell membrane vesicle loaded with chemotherapeutic drugs and subjected to TIGIT overexpression, and application of the engineered drug-loaded cell membrane vesicle in preparation of tumor immunotherapy drugs.
Yet another object of the invention is: a drug delivery system characterized by: including engineered drug-loaded cell membrane vesicles loaded with chemotherapeutic drugs and over-expressed in TIGIT as described above.
By adopting the scheme, the invention prepares a stable cell line by infecting target cells with lentiviruses, then extracts cell membranes of the stable cell line, obtains membrane nano vesicles by methods such as extrusion and the like, then loads micromolecular chemotherapeutic drugs, and centrifugally purifies the membrane nano vesicles to obtain the engineered drug-loaded cell membrane vesicles.
The engineering drug-loaded cell membrane vesicle prepared by the method is loaded with oxaliplatin inside and has higher expression of TIGIT protein on the surface, has good biocompatibility, can be applied to preparing tumor immunotherapy drugs, and has better practical value and clinical prospect.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a preparation method of an engineered drug-loaded cell membrane vesicle overexpressed by TIGIT, which is used for preparing a biomembrane system for delivering TIGIT protein firstly. By applying a technical means of gene editing, the TIGIT protein is expressed on a cell membrane, and compared with a TIGIT monoclonal antibody, the stability and the utilization rate of the medicament in vivo are greatly improved.
(2) The engineered drug-loaded cell membrane vesicles or drug delivery systems of the invention perform effective co-administration while delivering oxaliplatin and TIGIT protein. Can realize effective enrichment of the medicine and improve the bioavailability of the medicine. The oxaliplatin kills tumor cells to generate ICD and cause the immune response of an organism, and the inhibition effect of immune cells is relieved by combining with TIGIT, so that the curative effect of 1+1>2 is realized.
(3) The invention uses the engineered cell membrane in a drug delivery system, has high biological safety and better targeting property, can target tumors more effectively, effectively improves the bioavailability of the drug, and has the characteristics of safety, synergy and toxicity reduction.
The invention is further described below with reference to the accompanying drawings.
Drawings
FIG. 1 is a fluorescent graph of HEK-293T cell packaging of engineered cells to produce specific lentiviruses in step one of example 1 of the present invention; 100 μm;
FIG. 2 is a confocal microscope image of the expression of TIGIT protein membrane of stable cell line in step one of example 1 of the present invention; 50 μm;
FIG. 3 is a confocal microscopy image of TIGIT protein expression of stable cell lines of different generations in step one of example 1 of the present invention; 10 mu m;
FIG. 4 is a transmission electron microscopy image of engineered cell membrane vesicles at step one in example 1 of the present invention; 100 nm;
FIG. 5 is a graph of the particle size distribution (A) and Zeta potential detection (B) of engineered membrane vesicles at step one in example 1 of the present invention;
FIG. 6 is a confocal microscope image of engineered cell membrane vesicles at step one in example 1 of the present invention; global plot, 5 μm; top right graph, 1 μm;
FIG. 7 is a statistical chart of Oxaliplatin (OXA) encapsulation efficiency of a small molecule drug in step one of example 1 of the invention;
FIG. 8 is a study of the targeting binding capacity of the engineered cell membrane vesicles and tumor cells in step two of example 1 of the present invention; 10 mu m;
FIG. 9 is a study of the ability of engineered drug-loaded membrane vesicles of different concentrations to inhibit tumor cell growth in vitro in step two of example 1 of the present invention;
FIG. 10 is a study of the immunogenic death capacity of tumor cells caused by in vitro engineering drug-loaded cell membrane vesicles in step two of example 1 of the present invention; 20 μm;
FIG. 11 is a statistical graph of the change in tumor volume size over time for groups of mice after different treatments in step three of example 1 of the present invention;
FIG. 12 is a photograph of the tumor volume of each group of mice treated differently in step three of example 1;
FIG. 13 is a statistical graph of tumor weights of groups of mice treated differently in step three of example 1 according to the present invention;
FIG. 14 is a graph showing survival curves of groups of mice treated differently in step three of example 1;
FIG. 15 is a statistical graph of the weight change over time of various groups of mice treated differently in step three in example 1 of the present invention;
FIG. 16 is HE stained sections of major organs of control (PBS group) and experimental (OXA @ TIGIT MVs) mice in step three of example 1 of the present invention; 200 μm.
Detailed Description
The present invention is further described with reference to the drawings and specific examples, but the present invention is not limited to the following specific embodiments, and those skilled in the art can implement the present invention in other specific embodiments or make simple changes or modifications to the design and concept of the present invention based on the disclosure of the present invention, and fall into the protection scope of the present invention. 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.
Example 1
Preparation of engineered drug-loaded cell membrane vesicles
1. And (4) packaging the lentivirus. HEK-293T cells (purchased externally) are cultured in a 37 ℃ cell culture box, and a target plasmid (a plasmid encoding a TIGIT gene), a packaging plasmid and lipo2000 (liposome 2000) which are mixed well are added according to the proportion of 1: 1.2-1.5: 2-4. And (3) changing the culture solution after 5-9 hours, continuing to culture by using a normal complete culture medium, and observing the transfected HEK-293T cells under a fluorescence microscope at different times. The results are shown in FIG. 1, which shows that HEK-293T cells are efficiently packaging to produce lentiviruses.
The culture supernatant was collected and centrifuged to remove the cells. The supernatant was collected and filtered using a 0.45 μm filter to remove cell debris from the supernatant. And mixing the filtrate with a virus purification reagent (Lenti Concentrator) according to a volume ratio of 1:4, incubating, centrifuging for 20-30min at 3000 g-4000 g, removing supernatant, and carrying out heavy suspension precipitation by using a proper amount of PBS (phosphate buffer solution) to obtain the required lentivirus solution.
2. Constructing a stable cell line overexpressing TIGIT. NIH/3T3 cells (purchased externally) were cultured in a cell incubator, and then an appropriate amount of the above-obtained lentivirus solution was added to infect NIH 3T3 cells. After infection, cells were screened by adding puromycin at 2-6. mu.g/mL for 1-2 weeks. Confocal microscope observation results are shown in FIG. 2, which shows that the EGFP-TIGIT is significantly expressed on NIH/3T3 cells. Fig. 3 shows that TIGIT protein can be expressed by the gene-edited cells for ten generations. The detection results are consistent, and the successful construction of the EGFP-TIGIT stable cell line is shown.
3. Preparing the engineering cell membrane vesicle. Stable cell lines over-expressed with TIGIT in step 2 were collected, washed with PBS and disrupted on ice using a dounce tissue homogenizer. Centrifuging the homogenate at 4 ℃ for 6-10 min at 1000-1500 g to remove unbroken cells and large cell debris. Collecting supernatant, centrifuging at 4 ℃ for 6-10 min by 3000 g-4000 g, and removing mitochondria and other organelles. Collecting supernatant, centrifuging at 4 ℃ for 60-90 min at 80000-120000 g, removing supernatant, and washing the precipitate with PBS for 2 times. Then the mixture is extruded by extruders with the diameters of 1 micron, 0.4 micron, 0.2 micron and 0.1 micron in sequence to obtain uniform membrane vesicles. Then ultracentrifuging for 90-120 min under the conditions of 4 ℃ and 100000-120000 g, collecting the precipitate, and washing for 2 times by PBS. Continuously collecting the precipitate, and resuspending with a proper amount of PBS to obtain the engineered cell membrane vesicle. The morphology was first observed using a transmission electron microscope. The results are shown in fig. 4, which has a typical membrane structure, highly similar to that of natural exosomes in appearance. The particle size distribution of the Brukrains particle size analyzer is detected. As a result, as shown in FIG. 5, the particle size distribution was mainly 20 to 200nm, the average particle size was approximately 100nm, and the Zeta potential was approximately-15 mV to-25 mV. The protein expression was observed by confocal laser microscopy, and the results are shown in fig. 6, indicating that distinct EGFP fluorescent particles were observed. These results indicate that engineered cell membrane vesicles (EGFP-TIGIT MVs) stably expressing a specific protein TIGIT were successfully prepared.
4. Preparing the engineering medicine-carrying cell membrane vesicle. The oxaliplatin loading is realized by a co-incubation and ultrasound method. Firstly, the engineered cell membrane vesicle and the oxaliplatin solution are mixed so that the final concentrations of the two components after mixing are 1mg/ml (protein mass) and 1-1.5 mg/ml respectively. The mixture was then incubated at 37 ℃ or sonicated on ice. The corresponding mixed systems obtained according to the two methods described above are centrifuged using an ultracentrifuge or an ultrafiltration tube to remove substantially the free oxaliplatin. The entrapment rate of oxaliplatin is calculated by detecting the characteristic absorption at 246nm by using an ultraviolet-visible spectrophotometer. The result is shown in figure 7, the encapsulation rate of the ultrasonic method can reach about 25%, the co-incubation encapsulation rate is about 7%, and the effective loading of oxaliplatin is realized.
Second, biological function research of engineering medicine-carrying cell membrane vesicle
1. Study of the ability to target tumor cells in vitro. OFP (orange fluorescent protein) -PVR (poliovirus receptor) plasmids are transfected into B16F10 cells (mouse melanoma cells) at first, and the OFP-PVR B16F10 cells are divided into 2 groups with numbers of (i) and (ii). And secondly, treating the groups with the aPVR antibody for 4-6 hours in advance, and then adding equivalent TIGIT MVs into the two groups respectively for incubation for 2-4 hours. The results of observation using a confocal microscope are shown in FIG. 8 (first group in the upper row and second group in the lower row), which shows that the engineered exosomes prepared can be targeted to tumor cells by the binding force between TIGIT/PVR. This indicates that the engineered exosome has good biological activity in vitro and has the ability of specific targeting binding.
2. The ability to inhibit tumor cell growth in vitro was examined. The CCK-8 method is used for detecting the influence of different concentrations of the engineered drug-loaded cell membrane vesicles on the survival rate of tumor cells (B16F 10 cells and Hela cells). As shown in FIG. 9, the cell line showed significant killing at a concentration of 0 or more for B16F10 cells, and at a concentration of 0.5. mu.g/ml or more for Hela cells. Particularly, when the concentration is 4 mu g/ml, the survival rate of B16F10 cells is about 48 percent, and the survival rate of Hela cells is about 70 percent, and when the concentration is 8 mu g/ml, the survival rate of B16F10 cells is about 41 percent, and the survival rate of Hela cells is about 52 percent, which shows that the engineered drug-loaded cell membrane vesicle can effectively inhibit the growth of tumor cells in vitro.
3. Study of the ability to cause ICD (immunogenic cell death) in tumor cells in vitro. The engineered drug-loaded cell membrane vesicles are co-cultured with tumor cells (B16F 10 cells), and then the eversion of CRT proteins of the tumor cells and the release of nuclear HMGB1 proteins are respectively detected by using a confocal microscope. As shown in FIG. 10, it was observed that tumor cells in the administered group showed more significant CRT eversion (FIG. 10-A) and HMGB1 release (FIG. 10-B). This shows that engineered drug-loaded cell membrane vesicles can effectively cause immunogenic death of tumor cells in vitro, which is the effective basis for combined therapy of oxaliplatin and immunotherapy.
Research on in-vivo tumor inhibition effect of engineered drug-loaded cell membrane vesicle
Experimental animals were used in the relevant experiments, approved by the animal ethics Committee of the university of Zhongshan (approval No. SYSU-IACUC-2020-000430). We constructed a melanoma-bearing model on C57BL/6 mice, and then randomly divided the tumor-bearing mice into 4 groups, and received the following drug treatments: PBS, OXA (oxaliplatin), TIGIT MVs (engineered cell membrane vesicles), OXA @ TIGIT MVs (engineered drug-loaded cell membrane vesicles). The growth of the tumors was then observed and recorded in each group of mice as shown in figure 11. Thereafter, we dissected the tumors of each group of mice, the tumor sizes of which are shown in fig. 12. Tumor weights were weighed and counted, and the results are shown in fig. 13. The survival curves were also recorded and the results are shown in FIG. 14. The results of these experiments all show that the experimental group (OXA @ TIGIT MVs) has a very good in vivo anti-tumor effect.
Meanwhile, in order to verify the in vivo biosafety of the drug, the body weight of each group of mice was recorded, and the results are shown in fig. 15, and each group of mice has no obvious body weight loss. Thereafter, we performed histological examination of the organs. The heart, liver, spleen, lung and kidney of each group of mice were subjected to HE staining, and whether they had tissue damage was observed. The results are shown in figure 16, with no obvious histological lesions, further illustrating the biological safety of the drug.
Claims (10)
1. A preparation method of an engineered drug-loaded cell membrane vesicle loaded with chemotherapeutic drugs and overexpressed in TIGIT (tungsten inert gas) is characterized by comprising the following steps:
s1, packaging lentivirus, culturing HEK-293T cells in a 37 ℃ cell culture box, adding a mixed target plasmid, a packaging plasmid and lipo2000 according to a volume ratio of 1: 1.2-1.5: 2-4, wherein the target plasmid is a plasmid for encoding a TIGIT gene, changing the liquid after 5-9 hours, continuously culturing by using a normal complete culture medium, collecting a culture supernatant, centrifugally removing cells, continuously collecting the supernatant, filtering to remove cell debris in the supernatant, mixing a filtrate and a virus purification reagent according to a volume ratio of 1:4, centrifuging for 20-30 minutes at 3000 g-4000 g after incubation, removing the supernatant, and carrying out heavy suspension precipitation by using BS to obtain a required lentivirus solution; wherein the sequence number of the gene for coding TIGIT is as follows: NM-001146325.1;
s2, culturing target cells in a cell culture box, wherein the target cells are NIH/3T3 cells, then infecting the NIH/3T3 cells with the obtained lentivirus solution, screening the cells with puromycin after infection, detecting after 1-2 weeks, selecting TIGIT over-expressed cells, and constructing a TIGIT over-expressed stable cell line, namely a TIGIT over-expressed NIH/3T3 cell line;
s3, culturing the obtained stable cell line, collecting and cleaning, crushing to form homogenate, centrifugally extracting cell membranes of the homogenate, preparing the cell membranes into membrane vesicles, and centrifugally purifying to obtain the engineered cell membrane vesicles;
and S4, loading the obtained cell membrane vesicle with a small-molecule chemotherapeutic drug by a co-incubation or ultrasonic method, and centrifugally purifying to obtain the engineered drug-loaded cell membrane vesicle.
2. The preparation method of the engineered drug-loaded cell membrane vesicle loaded with chemotherapeutic drugs and overexpressed in TIGIT according to claim 1, wherein the concentration of puromycin is 2-6 μ g/ml.
3. The preparation method of the engineered drug-loaded cell membrane vesicle loaded with chemotherapeutic drugs and overexpressed in TIGIT according to claim 1, wherein the specific steps of S3. are as follows: collecting a TIGIT over-expressed stable cell line, washing with PBS, crushing with a homogenizer to form homogenate, centrifuging the homogenate at 4 ℃ under the centrifugal force of 1000-1500 g for 6-10 min to remove unbroken cells and large cell fragments, collecting the supernatant, centrifuging at 4 ℃ under the centrifugal force of 3000-4000 g for 6-10 min to remove mitochondria and other organelles, collecting the supernatant, centrifuging at 4 ℃ under 80000-120000 g for 60-90 min to remove the supernatant, washing and precipitating with PBS for 2 times, extruding with extruders with different pore diameters for several times to obtain membrane vesicles, wherein the pore diameter of the extruders is 0.1-1 μm, the pore diameter of the extruders used for several times is reduced in turn, ultracentrifuging the obtained membrane vesicles at 4 ℃ under the centrifugal force of 100000-120000 g for 90-120 min, collecting the precipitate, washing with PBS for 2 times, continuously collecting the precipitate, and (5) resuspending the mixture by using PBS to obtain the engineered cell membrane vesicle.
4. The method for preparing a chemotherapeutic drug loaded and TIGIT overexpressed engineered drug loaded cell membrane vesicle according to claim 4, wherein the particle size of the engineered cell membrane vesicle is 20-200 nm, and the average particle size is about 100 nm.
5. The preparation method of the engineered drug-loaded cell membrane vesicle loaded with the chemotherapeutic drug and overexpressed by TIGIT according to claim 4, wherein the homogenate in S3. is centrifuged for the third time, supernatant is removed, and the homogenate is washed and precipitated for 2 times by PBS (phosphate buffer solution), and then extruded by extruders with the pore diameters of 1 μm, 0.4 μm, 0.2 μm and 0.1 μm in sequence to obtain the membrane vesicle.
6. The method for preparing a chemotherapeutic drug-loaded and TIGIT overexpressed engineered drug-loaded cell membrane vesicle according to claim 6, wherein the engineered cell membrane vesicle has a particle size of 20-200 nm and an average particle size of about 100 nm.
7. The preparation method of the engineering drug-loaded cell membrane vesicle loaded with the chemotherapeutic drug and overexpressed by TIGIT according to claim 1, wherein in S4, the oxaliplatin is loaded by a co-incubation or ultrasonic method, wherein the final concentrations of the engineering cell membrane vesicle and the oxaliplatin solution after mixing are 1mg/ml and 1-1.5 mg/ml respectively, the mixture is co-incubated at 37 ℃ or ultrasonically treated on ice to obtain a corresponding mixed system, and then the mixed system is centrifuged by an ultracentrifuge or an ultrafiltration tube to obtain the engineering drug-loaded cell membrane vesicle.
8. An engineered drug-loaded cell membrane vesicle loaded with chemotherapeutic drugs and overexpressed in TIGIT, prepared by the preparation method of any one of claims 1 to 5.
9. An engineered drug-loaded cell membrane vesicle loaded with chemotherapeutic drugs and overexpressed by TIGIT (tungsten inert gas), and application thereof in preparing tumor immunotherapy drugs.
10. A drug delivery system characterized by: comprising the chemotherapeutic drug-loaded and TIGIT over-expressed engineered drug-loaded cell membrane vesicles of any one of claims 1-5, 8.
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