CN113444742A - Preparation method and application of engineered exosome for drug targeted delivery - Google Patents
Preparation method and application of engineered exosome for drug targeted delivery Download PDFInfo
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Abstract
The invention relates to a preparation method and application of an engineered exosome for drug targeted delivery, wherein messenger RNA (mRNA) carrying miRNA or coding toxic protein (such as perforin or granzyme and the like) capable of inducing specific apoptosis is loaded into the exosome. Secreted exosomes loaded with specific nucleic acid cargo were collected and purified. Since a molecular switch ' encryption ' device using miRNA characteristic to the target cell as a trigger signal is pre-installed in the 5 ' -UTR of the target gene, miRNA or mRNA does not function in the host cell. After the engineered exosome is used as a carrier to deliver a carrier-effector (namely miRNA or toxic protein mRNA for inducing apoptosis of target cells) to target cells, specific miRNAs in the cancer cells are used as trigger switches to start the release of the effector (miRNA or toxic protein) and start an apoptosis program, so that the target cancer cells are killed.
Description
Technical Field
The invention relates to a preparation method and application of an engineered exosome for drug targeted delivery.
Background
Exosomes are microvesicles with the size of 40-100 nanometers secreted by various cells, and exosomes, namely insoluble microvesicles, are important pathways for intercellular signal communication discovered in recent years and are important paracrine forms outside soluble cell factors of cells. The properties of exosomes are mainly: (1) the diameter is 40-100 nm; (2) having cytoplasmic and lipocyte membrane components of the source cell; (3) the density is 1.13-1.19 g/ml; (4) contains specific protein of the cell from which the protein is derived and exosome-associated protein such as CD9, CD81, Alix and the like. The Exosome components mainly comprise: the composition of exosome protein is complex as shown in proteomics analysis, and the composition of exosome protein from different cell sources also differs.
In the prior art, targeted drug delivery (for example, targeting cancer) is a key and difficult point in research and development, and a technology for directly targeting cancer cells by engineering exosomes to induce cancer cell apoptosis has not been reported.
Disclosure of Invention
In order to overcome the defects, the invention provides a preparation method and application of an engineered exosome for targeted drug delivery, which is used for triggering cancer cell apoptosis and has an anti-tumor effect.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a preparation method of engineered exosomes for drug targeted delivery is characterized by comprising the following steps of A or B;
a (1) constructing a target gene expression vector; acquiring or constructing an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, a target gene mRNA or miRNA packaging plasmid and a targeting positioning plasmid; the target gene is miRNA or toxic protein gene which promotes cancer cell apoptosis; in the target gene expression vector, a target gene 5' -UTR is inserted or not inserted with an encryption sequence;
(2) co-transfecting HEK293F cells or mesenchymal stem cells by mixed plasmids consisting of a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, an exosome target positioning plasmid and a target gene mRNA or miRNA packaging plasmid and culturing;
(3) isolation of engineered exosomes facilitating targeted drug delivery: collecting the culture solution of the transfected HEK293F cells or mesenchymal stem cells, and filtering to obtain exosomes in the culture solution;
b: (1) constructing a target gene expression vector; obtaining or constructing an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, and a target gene mRNA or miRNA packaging plasmid; the target gene is miRNA or toxic protein gene which promotes cancer cell apoptosis; in the target gene expression vector, a target gene 5' -UTR is inserted with an encryption sequence;
(2) co-transfecting HEK293F cells or mesenchymal stem cells by a mixed plasmid consisting of a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid and a target gene mRNA or miRNA packaging plasmid and culturing;
(3) isolation of engineered exosomes facilitating targeted drug delivery:
and collecting the culture solution of the transfected HEK293F cells or mesenchymal stem cells, and filtering to obtain exosomes in the culture solution.
For ease of description, the plasmids used in the present invention are shown in Table 1 below.
TABLE 1 plasmids used in the present invention
Note: the encrypted sequence is a segment of sequence designed according to miRNA specific to the target cancer cell, so that an effector (miRNA or protein for inducing cancer cell apoptosis) connected behind the encrypted sequence is expressed and acts only in the presence of miRNA characteristic to the target cancer cell.
As a further embodiment, the toxic protein includes, but is not limited to perforin or granzyme.
In the step (1), when a target gene expression vector is constructed, a target gene is directly synthesized, a C/Dbox is arranged at the 3 'end of the target gene, and an encryption sequence is inserted into the 5' -UTR of the target gene and is connected into a pcDNA3.1 vector; the target gene expression vector containing the encrypted sequence is pcDNA3.1-encrypted sequence-miRNA (pGmL3) or toxic protein gene sequence (pGmL1 or 2) -C/Dbox-pAbGH; the exosome cytoplasmic transport helper plasmid is pcDNA3.1-Cx43S368A-pAbGH (pGmL 4); the target gene mRNA or miRNA packaging plasmid is pcDNA3.1-CD63-L7Ae-pAbGH (pGmL 5); the secretion promoting plasmid is pcDNA3.1-STEAP3-p2A-SDC4-p2A-NadB-pAbGH (pGmL 6); the exosome targeting localization plasmid is pcDNA3.1-X-lamp2b-pAbGH (pGmL7), wherein X is a ligand of a characteristic tumor antigen of a target cancer cell surface. The long-term active Cx43S368A mutant is a more potent cytoplasmic delivery aid than wild-type Cx 43. And the pcDNA3.1-STEAP3-p2A-SDC4-p2A-NadB-pAbGH (pGmL6) is provided with 3 genes STEAP3, SDC4 and NadB which are used as a production booster for synthesizing exosomes, and the combined expression of the genes obviously increases the production of exosomes. L7Ae (archaebacteria ribosomal protein large subunit L7Ae) was fused to the C-terminus of CD63, and a C/Dbox was inserted into the 3 '-untranslated region (3' -UTR) of the target gene, and the target nucleic acid was loaded onto the exosome membrane by L7Ae and CDbox recognition. Furthermore, for target cells (e.g., cancer cells expressing PD-L1), ligand X is PD-1. The exosome targeting positioning plasmid is used for positioning exosome on a target cell and acting on the target cell, and because the exosome contains miRNA or toxic protein mRNA which promotes apoptosis, the exosome can mediate apoptosis of the target cell after being positioned and entering the target cell. The miRNA can be miR-1469 (capable of promoting laryngeal cancer cell apoptosis), miR-9-3p (capable of promoting glioma cell apoptosis) and other miRNA capable of promoting cancer cell apoptosis.
As a further technical scheme, the specific process of the step (3) is as follows: collecting culture solution of HEK293F cells or mesenchymal stem cells, centrifuging for 15-20 minutes at 0-4 ℃ under the condition of centrifugal force 600-700g, taking supernatant, centrifuging for 25-30 minutes at 0-4 ℃ under the condition of centrifugal force 11000-12000 g, filtering with a 0.22 mu m filter membrane, taking filtrate, centrifuging for 2-4 hours at 0-4 ℃ under the condition of centrifugal force 150000-160000g, washing and precipitating with PBS solution at 4 ℃ to obtain the engineered exosome for targeted drug delivery. And (3) removing cells and cell fragments by adopting continuously improved centrifugal rotating speed, and finally obtaining the high-concentration and high-purity engineering exosome.
Further, the preservation conditions of the engineered exosome for drug targeted delivery are as follows: the pH value is in the range of 6-8, 0-4 ℃.
In the step A (2), after a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, an exosome target location plasmid and a target gene mRNA or miRNA packaging plasmid are cotransfected with HEK293F cells or mesenchymal stem cells, the cells are cultured at the constant temperature of 37 ℃ and 5 percent of CO is introduced2Obtaining a culture solution of HEK293F cells or mesenchymal stem cells after 48-54 h; during cotransfection, the mass ratio of a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, an exosome targeting positioning plasmid and a target gene mRNA or miRNA packaging plasmid is 1-2.5: 0.5-1: 0.8-1.2: 0.5 to 1; 0.5 to 1;
in the step (2), after a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid and a target gene (mRNA or miRNA) packaging plasmid are cotransfected with HEK293F cells or mesenchymal stem cells, the cells are cultured at the constant temperature of 37 ℃ and 5 percent of CO is introduced2Obtaining a culture solution of HEK293F cells or mesenchymal stem cells after 48-54 h; during cotransfection, the mass ratio of a target gene expression vector to an exosome cytoplasmic transport helper plasmid to an exosome secretion promoting plasmid to a target gene mRNA or miRNA packaging plasmid is 1-2.5: 0.5-1: 0.8-1.2: 0.5-1.
In step (2), the process of co-transfecting HEK293F cells by the mixed plasmid comprises the following steps:
1) cell culture: adding 10-15mL of DMEM medium into a culture dish; seeding number of HEK293F cells was 2 x 106The suspension culture temperature is controlled at 37 deg.C and maintained at 5% CO per ml2When the number of cells reaches 1.2 x 107One/ml for cell transfection;
2) dilution of plasmid DNA: diluting 30. mu.g of the mixed plasmid with 0.5ml of serum-free Opti-MEM medium;
3) to 0.5ml of Opti-MEM dilution containing the above mixed plasmid was added 35 to 50. mu.l of LipofectamineTM LTX with PLUSTMTransfection reagent, mixing plasmid with LipofectamineTMLTX forms a stable complex;
4) in the culture dish, add 0.5ml mixed plasmid-Lipofectamine dropwiseTMShaking the LTX compound;
5) transfected HEK293F cells were placed at 37 ℃ and maintained at 5% CO2Culturing for 48-54h in the incubator.
The invention also provides the engineered exosome for the drug targeted delivery, which is obtained by the preparation method.
The invention also aims to provide application of the engineered exosome in preparing an anti-tumor drug, a drug for apoptosis of cancer cells or a drug targeting delivery carrier.
It is still another object of the present invention to provide the anti-tumor drug of the engineered exosomes as described above.
Compared with the prior art, the invention has the beneficial effects that: the invention loads nucleic acid carrying miRNA triggering cancer cell apoptosis or coding toxic protein (such as perforin and granzyme) triggering apoptosis into exosome after being encrypted (because of the 'encrypted' molecular switch setting, miRNA and coding toxic protein mRNA have no function in host cells), and secreted loading exosome is collected and purified as a carrier to specifically locate the carrier (miRNA or mRNA) to target cells (such as cancer cells), and then uses characteristic miRNAs in the cancer cells as trigger switches to start apoptosis, thereby being used for killing the cancer cells.
Detailed Description
The following describes embodiments of the present invention with reference to examples.
Example 1
(1) Constructing a target gene expression vector; acquiring or constructing an exosome cytoplasmic transportation helper plasmid (pGmL4), an exosome secretion promoting plasmid (pGmL6), an exosome targeting location plasmid (pGmL7) and a target gene mRNA packaging plasmid (pGmL 5); the target gene is toxic protein gene (pGmL1 or pGmL2) (perforin or granzyme); in the target gene expression vector, a target gene 5' -UTR is inserted with an encryption sequence;
(2) co-transfecting HEK293F cells by a mixed plasmid consisting of a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, an exosome target positioning plasmid and a target gene mRNA packaging plasmid, and culturing;
(3) isolation of engineered exosomes facilitating targeted drug delivery:
the culture solution of the transfected HEK293F cells is collected and filtered to obtain exosomes in the culture solution.
When the target gene expression vector is constructed, a target gene is directly synthesized, C/Dbox is arranged on the 3 '-UTR of the target gene, and an encryption sequence is inserted into the 5' end of the target gene and is connected into a pcDNA3.1 vector; in this example, the target gene expression vector containing the encrypted sequence was pGmL1 or pGmL 2; the exosome cytoplasmic transport helper plasmid is pGmL 4; the target gene mRNA packaging plasmid is (pGmL 5); the secretion promoting plasmid of the exosome is pGmL 6; the exosome targeting localization plasmid is pGmL7, wherein X is a ligand of a targeting cancer cell surface antigen (PD-L1), and X is a PD-1 gene.
In the step (2), after a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, an exosome targeting positioning plasmid and a target gene mRNA packaging plasmid are cotransfected with HEK293F cells or mesenchymal stem cells, the cells are cultured at the constant temperature of 37 ℃ and 5 percent of CO is introduced2Obtaining a culture solution of HEK293F cells or mesenchymal stem cells after 48-54 h; during cotransfection, the mass ratio of a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, an exosome targeting positioning plasmid and a target gene mRNA packaging plasmid is 2: 1: 1: 0.5: 0.5;
in step (2), the process of co-transfecting HEK293F cells by the mixed plasmid comprises the following steps:
1) cell culture: adding 15mL of DMEM medium into the culture dish; seeding number of HEK293F cells was 2 x 106Each ml, the suspension culture temperature is controlled at 37 DEG CCO retention 5%2When the number of cells reaches 1.2 x 107One/ml for cell transfection;
2) dilution of plasmid DNA: diluting 30. mu.g of the mixed plasmid with 0.5ml of serum-free Opti-MEM medium;
3) to 0.5ml of Opti-MEM dilution containing the above mixed plasmid was added 50. mu.l of LipofectamineTMLTX with PLUSTMTransfection reagent, mixing plasmid with LipofectamineTMLTX forms a stable complex;
4) in the culture dish, add 0.5ml mixed plasmid-Lipofectamine dropwiseTMShaking the LTX compound;
5) transfected HEK293F cells were placed at 37 ℃ and maintained at 5% CO2The culture was carried out for 48 hours.
The specific process of the step (3) is as follows: HEK293F cells were collected, centrifuged at 700g at 4 ℃ for 15 min, supernatant centrifuged at 12000g at 4 ℃ for 25 min, filtered through a 0.22 μm filter, filtrate centrifuged at 160000g at 4 ℃ for 2h, and the precipitate washed with 4 ℃ PBS solution to give engineered exosome products for targeted drug delivery, including exosome product 1 (containing target genes perforin mRNA, Cx43S368A protein, CD63-L7Ae protein, STEAP3/SDC4/NadB protein and PD-1 protein) and exosome product 2 (containing target genes granzyme mRNA, Cx43S368A protein, CD63-L7Ae protein, STEAP3/SDC4/NadB protein and PD-1 protein) under the following storage conditions: the pH value is 7, 0-4 ℃.
When the tumor cell promoter is used, a target cell of an exosome is a tumor cell, a decryption key (namely a miRNA sequence characteristic of the target cancer cell) is arranged in the tumor cell, after mRNA of toxic protein in the exosome enters the tumor cell, specific miRNA in the target cell is combined with a encryption region of 5' -UTR of the mRNA of the toxic protein, the toxic protein starts to translate, and the cell starts to die. In the case of using the exosome targeting plasmid pGmL7, exosomes would be targeted and enter cancer cells expressing PDL-1 ligand, adding a logical 'and gate' control to the killing of cancer cells.
Example 2
Similar to example 1, the only difference is that in this example no targeting plasmid is required, and expression of the gene of interest (perforin or granzyme) is dependent only on the miRNA characteristic of recognition of the encrypted sequence in the target cancer cells. Exosome product 3 (containing encrypted sequences-target genes perforin mRNA, Cx43S368A protein, CD63-L7Ae protein and STEAP3/SDC4/NadB protein) and exosome product 4 (containing encrypted sequences-target genes granzyme mRNA, Cx43S368A protein, CD63-L7Ae protein and STEAP3/SDC4/NadB protein) were obtained.
Example 3
(1) Constructing a target gene expression vector; acquiring or constructing an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, a target gene miRNA packaging plasmid and an exosome target location plasmid; the target gene is miR-Y (if effector Y is miR-9-3p, glioma cell apoptosis can be promoted, and if Y is miR-1469, laryngeal cancer cell apoptosis can be induced); in the target gene expression vector, a target gene 5' -UTR is inserted with an encryption sequence;
(2) co-transfecting HEK293F cells by a mixed plasmid consisting of a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, a target gene miRNA packaging plasmid and an exosome targeting positioning plasmid, and culturing;
(3) isolation of engineered exosomes facilitating targeted drug delivery:
the culture solution of the transfected HEK293F cells is collected and filtered to obtain exosomes in the culture solution.
When the target gene expression vector is constructed, a target gene is directly synthesized, a C/Dbox is arranged at the 3 'end of the target gene, and an encryption sequence is inserted into the 5' -UTR of the target gene and is connected into a pcDNA3.1 vector; the target gene expression vector containing the encrypted sequence is pcDNA3.1-encrypted sequence-miRNA-C/Dbox-pAbGH (pGmL 3); the exosome cytoplasmic transport helper plasmid is pcDNA3.1-Cx43S368A-pAbGH (pGmL 4); the target miRNA packaging plasmid is pcDNA3.1-CD63-L7Ae-pAbGH (pGmL 5); the secretion promoting plasmid is pcDNA3.1-STEAP3-p2A-SDC4-p2A-NadB-pAbGH (pGmL 6); the exosome targeting localization plasmid is pGmL7, wherein X is a ligand of a targeting cancer cell surface antigen (PD-L1), and X is a PD-1 gene.In the step (2), after CO-transfecting HEK293F cells or mesenchymal stem cells with a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, a target gene miRNA packaging plasmid, a target positioning plasmid and the like, culturing at constant temperature of 37 ℃ and introducing 5% of CO2Obtaining a culture solution of HEK293F cells or mesenchymal stem cells after 48-54 h; during cotransfection, the mass ratio of a target gene expression vector to an exosome cytoplasmic transport helper plasmid to an exosome secretion promoting plasmid to a target gene miRNA packaging plasmid to an exosome targeting positioning plasmid is 2: 1: 1: 0.5: 0.5;
in step (2), the process of co-transfecting HEK293F cells by the mixed plasmid comprises the following steps:
1) cell culture: adding 15mL of DMEM medium into the culture dish; seeding number of HEK293F cells was 2 x 106The suspension culture temperature is controlled at 37 deg.C and maintained at 5% CO per ml2When the number of cells reaches 1.2 x 107One/ml for cell transfection;
2) dilution of plasmid DNA: diluting 30. mu.g of the mixed plasmid with 0.5ml of serum-free Opti-MEM medium;
3) to 0.5ml of Opti-MEM dilution containing the above mixed plasmid was added 50. mu.l of LipofectamineTMLTX with PLUSTMTransfection reagent, mixing plasmid with LipofectamineTMLTX forms a stable complex;
4) in the culture dish, add 0.5ml mixed plasmid-Lipofectamine dropwiseTMShaking the LTX compound;
5) transfected HEK293F cells were placed at 37 ℃ and maintained at 5% CO2The culture was carried out for 48 hours.
The specific process of the step (3) is as follows: collecting HEK293F cells, centrifuging at 4 ℃ for 15 minutes under the condition of 700g of centrifugal force, taking the supernatant, centrifuging at 4 ℃ for 25 minutes under the condition of 12000g of centrifugal force, filtering by using a 0.22 mu m filter membrane, taking the filtrate, centrifuging at 4 ℃ for 2 hours under the condition of 160000g of centrifugal force, washing and precipitating by using a PBS solution at 4 ℃ to obtain an engineered exosome product 5 (containing encrypted sequences-miR-9-3 p, Cx43S368A protein, CD63-L7Ae protein, STEAP3/SDC4/NadB protein and PD-1 protein) and an exosome product 6 (containing encrypted sequences-miR-1469, Cx43S368A protein, CD63-L7Ae protein, STEAP3/SDC4/NadB protein and PD-1 protein) for targeted drug delivery, wherein the conditions are as follows: the pH value is 7, 0-4 ℃.
Example 4
Similar to example 3, the only difference is that no targeting plasmid is required for this example, the objective gene is miR-9-3p which promotes apoptosis in glioma cells, and expresses mirnas which are dependent only on the characteristics of the encrypted sequences recognized in glioma cells, which were designed according to mirnas characteristic of glioma cells, yielding exosome product 7 (containing the encrypted sequences-miR-9-3 p, Cx43S368A protein, CD63-L7Ae protein and STEAP3/SDC4/NadB protein).
Example 5
Similar to example 3, the only difference is that no targeting plasmid is required for this example, the objective gene is that miR-1469, which can induce apoptosis in laryngeal cancer cells, is expressed only depending on miRNA crypt sequences characteristic of recognition of crypt sequences in laryngeal cancer cells, designed according to miRNA characteristic of laryngeal cancer cells, yielding exosome product 8 (containing crypt sequences-miR-1469, Cx43S368A protein, CD63-L7Ae protein and STEAP3/SDC4/NadB protein).
Use of engineered exosomes
The invention loads nucleic acid carrying miRNA triggering cancer cell apoptosis or coding toxic protein (such as perforin and granzyme) triggering apoptosis into exosome after being encrypted (because of the 'encrypted' molecular switch setting, miRNA and coding toxic protein mRNA have no function in host cells), and secreted loading exosome is collected and purified as a carrier to specifically locate the carrier (miRNA or mRNA) to target cells (such as cancer cells), and then uses characteristic miRNAs in the cancer cells as trigger switches to start apoptosis, thereby being used for killing the cancer cells.
Application example 1
The exosome (product 1) containing toxic protein perforin mRNA has killing effect on liver cancer cells HepG 2.
The main materials and sources used in this example are as follows:
cell culture dishes, crystal violet staining solution (fumace, Nanjing), C C C K8 detection kit (Vazyme), 96-well cell culture plates (JET Biofil), annexin V-FITC apoptosis detection kit (fumace, Nanjing), inverted microscope (Nikon, Japan), flow cytometer (Caliburg, USA).
The implementation steps are as follows:
1) digesting the liver cancer cells HepG2, counting the cells after centrifugal precipitation, inoculating 800 liver cancer cells HepG2, adding 2ul of engineered exosome containing toxic protein perforin mRNA after the cells are attached to the wall, and carrying out a plate cloning experiment.
2) Digesting the liver cancer cell HepG2, counting the cells after centrifugal precipitation, inoculating the cells into a 96-well plate, adding 2ul of engineered exosome containing toxic protein perforin mRNA after the cells are attached to the wall, detecting the influence of the exosome on the proliferation capacity of the liver cancer cell HepG2 by using a CCK8 reagent at 0h,12h,24h,36h and 48h respectively, and the results show that the K562 is 100%, 75%, 55%, 36% and 29% of the initial liver cancer cell HepG2 at 0h,12h,24h,36h and 48 h.
3) Digesting the liver cancer cell HepG2, counting the cells after centrifugal precipitation, inoculating the cells into a 6-hole plate, adding 2ul of engineered exosome containing toxic protein perforin mRNA after the cells adhere to the wall, detecting the influence of the exosome on the apoptosis of the liver cancer cell HepG2 by using a flow cytometer, wherein the results show that the apoptosis rates of the cells are 45% and 71% respectively at 24h and 48 h.
Application example 2
Killing effect of miR-9-3 p-containing exosome (product 5) on glioma cells
The main materials and sources used in this example are as follows:
cell culture dishes, crystal violet staining solution (fumace, Nanjing), C C C K8 detection kit (Vazyme), 96-well cell culture plates (JET Biofil), annexin V-FITC apoptosis detection kit (fumace, Nanjing), inverted microscope (Nikon, Japan), flow cytometer (Caliburg, USA).
The implementation steps are as follows:
1) digesting glioma cells, counting the cells after centrifugal precipitation, inoculating 800 glioma cells, adding 1.5ul of engineered exosome containing miR-9-3p after the cells are attached to the wall, and carrying out a plate cloning experiment.
2) Digesting glioma cells, counting the cells after centrifugal precipitation, inoculating the cells into a 96-well plate, adding 1.5ul of engineered exosomes containing miR-9-3p after the cells are attached to the wall, and detecting the influence of the exosomes on the proliferation capacity of the glioma cells by using a CCK8 reagent at 0h,12h,24h,36h and 48h respectively, wherein the results show that the glioma cells are 100%, 77%, 58%, 39% and 30% of the initial glioma cells at 0h,12h,24h,36h and 48 h.
3) Digesting glioma cells, counting the cells after centrifugal precipitation, inoculating the cells into a 6-well plate, adding 1.5ul of engineered exosome containing miR-9-3p after the cells are attached to the wall, detecting the influence of the exosome on the apoptosis of the glioma cells by using a flow cytometer, wherein the results show that the apoptosis rates of the cells are 42% and 70% respectively at 24h and 48 h.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Claims (9)
1. A preparation method of engineered exosomes for drug targeted delivery is characterized by comprising the following steps of A or B;
a (1) constructing a target gene expression vector; acquiring or constructing an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, a target gene mRNA or miRNA packaging plasmid and an exosome target positioning plasmid; the target gene is miRNA or toxic protein gene which promotes cancer cell apoptosis; in the target gene expression vector, a target gene 5' UTR region is inserted or not inserted with an encryption sequence;
(2) co-transfecting HEK293F cells or mesenchymal stem cells by mixed plasmids consisting of a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, an exosome target positioning plasmid and a target gene mRNA or miRNA packaging plasmid and culturing;
(3) facilitating the isolation of engineered exosomes for drug targeted delivery: collecting the culture solution of the transfected HEK293F cells or mesenchymal stem cells, and filtering to obtain exosomes in the culture solution;
b: (1) constructing a target gene expression vector; obtaining or constructing an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, and a target gene mRNA or miRNA packaging plasmid; the target gene is miRNA or toxic protein gene which promotes cancer cell apoptosis; in the target gene expression vector, a target gene 5' -UTR is inserted with an encryption sequence;
(2) co-transfecting HEK293F cells or mesenchymal stem cells by a mixed plasmid consisting of a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid and a target gene mRNA or miRNA packaging plasmid and culturing;
(3) isolation of engineered exosomes facilitating targeted drug delivery: and collecting the culture solution of the transfected HEK293F cells or mesenchymal stem cells, and filtering to obtain exosomes in the culture solution.
2. The method for preparing engineered exosomes for drug targeted delivery according to claim 1, wherein the toxic protein comprises perforin and granzyme.
3. The method for preparing engineered exosome for drug targeting delivery according to claim 1, wherein in the step (1), the target gene expression vector is constructed by directly synthesizing the target gene, arranging C/Dbox at the 3 'end of the target gene, inserting or not inserting an encryption sequence into the 5' -UTR of the target gene and connecting the target gene expression vector to pcDNA3.1 vector; the target gene expression vector containing the encrypted sequence is pcDNA3.1-encrypted sequence (determined according to miRNA characteristic of target cells) -miRNA (or toxic protein gene sequence) -C/Dbox-pAbGH; the target gene expression vector without the encrypted sequence is pcDNA3.1-miRNA (or toxic protein gene sequence) -C/Dbox-pAbGH; the exosome cytoplasmic transport helper plasmid is pcDNA3.1-Cx43S 368A-pAbGH; the target gene mRNA or miRNA packaging plasmid is pcDNA3.1-CD63-L7 Ae-pAbGH; the secretion promoting plasmid is pcDNA3.1-STEAP3-p2A-SDC4-p 2A-NadB-pAbGH; the exosome targeting localization plasmid is pcDNA3.1-X-lamp2b-pAbGH, wherein X is a ligand of a targeting cell.
4. The preparation method of engineered exosomes for drug targeted delivery according to claim 1, wherein the specific process of step (3) is as follows: collecting a culture solution of HEK293F cells or mesenchymal stem cells, centrifuging for 15-20 minutes at 0-4 ℃ under the condition of centrifugal force 600-700g, taking supernatant, centrifuging for 25-30 minutes at 0-4 ℃ under the condition of centrifugal force 11000-12000 g, filtering with a filter membrane of 0.22 mu m, taking filtrate, centrifuging for 2-4 hours at 0-4 ℃ under the condition of centrifugal force 150000-160000g, and washing and precipitating with PBS solution at 4 ℃ to obtain the engineered exosome for targeted drug delivery.
5. The method for preparing engineered exosomes for drug targeting delivery according to claim 1, wherein in the step (2), after a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, an exosome targeting localization plasmid and a target gene mRNA or miRNA packaging plasmid are cotransfected with HEK293F cells or mesenchymal stem cells, the cells are cultured at constant temperature of 37 ℃ and 5% of CO is introduced2Obtaining a culture solution of HEK293F cells or mesenchymal stem cells after 48-54 h; during cotransfection, the mass ratio of a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid, an exosome targeting positioning plasmid and a target gene mRNA or miRNA packaging plasmid is 1-2.5: 0.5-1: 0.8-1.2: 0.5-1: 0.5 to 1;
in the step (2), after a target gene expression vector, an exosome cytoplasmic transport helper plasmid, an exosome secretion promoting plasmid and a target gene mRNA or miRNA packaging plasmid are cotransfected into HEK293F cells or mesenchymal stem cells, the cells are cultured at the constant temperature of 37 ℃ and 5 percent of CO is introduced2Obtaining a culture solution of HEK293F cells or mesenchymal stem cells after 48-54 h; during cotransfection, a target gene expression vector, an exosome cytoplasmic transport helper plasmid and an exosome secretion promoting plasmidAnd the mass ratio of the target gene mRNA or miRNA packaging plasmid is 1-2.5: 0.5-1: 0.8-1.2: 0.5-1.
6. The method for preparing engineered exosomes for drug targeted delivery according to claim 1, wherein in the step (2), the process of mixed plasmid co-transfection of HEK293F cells is as follows:
1) cell culture: adding 10-15mL of DMEM medium into a culture dish; seeding number of HEK293F cells was 2 x 106The suspension culture temperature is controlled at 37 deg.C and maintained at 5% CO per ml2When the number of cells reaches 1.2 x 107One/ml for cell transfection;
2) dilution of plasmid DNA: diluting 30. mu.g of the mixed plasmid with 0.5ml of serum-free Opti-MEM medium;
3) adding 35-50. mu.l of a Lipofectamine LTX with PLUS-transfection reagent to 0.5ml of an Opti-MEM dilution containing the above-described mixed plasmid, so that the mixed plasmid and the Lipofectamine LTX form a stable complex;
4) adding 0.5ml of the complex of the mixed plasmid-Lipofectamine-LTX dropwise into the culture dish, and shaking up;
5) transfected HEK293F cells were placed at 37 ℃ and maintained at 5% CO2Culturing for 48-54h in the incubator.
7. An engineered exosome prepared according to any one of claims 1 to 6 for use in targeted delivery of a drug.
8. The use of the engineered exosome of claim 7 in the preparation of an anti-tumor drug, in the preparation of a drug that causes apoptosis in cancer cells, or in the preparation of a targeted drug delivery vehicle.
9. An antitumor agent comprising the engineered exosome of claim 7.
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