CN115029320B - Engineering exosome for tumor radiotherapy sensitization, preparation method and application - Google Patents
Engineering exosome for tumor radiotherapy sensitization, preparation method and application Download PDFInfo
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- CN115029320B CN115029320B CN202210692258.7A CN202210692258A CN115029320B CN 115029320 B CN115029320 B CN 115029320B CN 202210692258 A CN202210692258 A CN 202210692258A CN 115029320 B CN115029320 B CN 115029320B
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Abstract
The invention discloses an engineering exosome for tumor radiotherapy sensitization, a preparation method and application, wherein the exosome externally modifies tumor targeting peptide, a small interfering RNA nucleic acid drug is wrapped in the exosome, the exosome is an exosome of umbilical cord-derived mesenchymal stem cells, and the exosome modifies tumor targeting polypeptide iRGD through a genetic engineering method. The invention aims to modify tumor targeting peptide iRGD on the surface of umbilical cord-derived mesenchymal stem cell exosomes by genetic engineering means, so that the modified exosomes are combined with tumor cells with high-expression integrins, thereby enhancing the active targeting of the exosomes. By utilizing the engineered exosome, siRNA of STAT3 gene can be efficiently delivered to tumor tissues in a targeting way, so that the STAT3 expression of tumor cells is reduced to inhibit tumor growth, the sensitivity of radiotherapy is enhanced, and a new idea is provided for treating radiotherapy resistance of clinical tumor patients.
Description
Technical Field
The invention belongs to the technical field of biology, in particular to an engineering exosome for tumor radiotherapy sensitization, a preparation method and application.
Background
MSCs are a type of multipotent progenitor cells with self-renewing, multipotent differentiation capability, obtainable from mature tissues, and the major sources include umbilical cord, bone marrow, fat, etc., and because of their self-renewing, multipotent and immunomodulatory properties, MSCs have become a major research focus in the medical field. The mechanism of action of MSCs in vivo is complex, and more researches show that the paracrine mechanism is a main form of MSCs functioning, while the exosomes are main paracrine substances, and the exosomes are expected to be applied to clinical treatment as a cell-free pharmaceutical preparation in the future due to lower toxicity, immunogenicity and adverse reactions.
Exosomes (Exosomes, exo) are nanoscale vesicle-like structures with diameters between 30-150nm, with phospholipid bilayer membranes. The inclusion of large amounts of biologically active substances, such as proteins, mRNA, microRNA, polypeptides, lipids, etc., within the membrane is an important vehicle for intercellular communication and information transfer. Although siRNA is a promising gene targeting drug, siRNA itself has poor stability, and the biggest problem in clinical practice of nucleic acid drugs at present is the lack of better and more targeted in vivo delivery systems. The exosomes serving as transport carriers not only have the advantages of high stability and high selectivity, but also have better affinity with nucleic acid molecules, and can remarkably improve the encapsulation and delivery efficiency. In addition, in the latest research results, exosomes can also act through the blood brain barrier, so that the exosomes have wide application prospects. Improving the targeting property of the medicine is beneficial to improving the curative effect of the medicine, and reasonably modifying the exosomes of the MSCs can lead the exosomes to effectively target into tumor sites, thereby playing a stronger anticancer role.
Signal transduction and transcription activator 3 (signal transducers and activators of transcription, STAT 3) is an important member of the signal transduction and transcription activator family. STAT3 is highly expressed in various tumor tissues and cell lines and is closely related to proliferation differentiation and apoptosis of tumors. If one could try to block expression of STAT3 gene in cancer cells, one would have to greatly attenuate cancer cell proliferation capacity while attenuating apoptosis inhibition. Thus, targeting certain molecules in the blocking signal pathway may be a completely new independently effective approach to tumor therapy
Through the above analysis, the problems and defects existing in the prior art are as follows:
The exosomes in the prior art are not further improved, so that the targeting effect of tumor cells is poor. Moreover, the prior art can not provide theoretical basis for potential drugs for exosomes to inhibit the growth of tumors and sensitize radiotherapy.
Disclosure of Invention
In order to overcome the problems in the related art, the disclosed embodiments of the invention provide an engineering exosome for tumor radiotherapy sensitization, a preparation method and application.
The technical scheme is as follows: an engineering exosome for tumor radiotherapy sensitization, externally modified tumor targeting peptide and internally coated with small interfering RNA nucleic acid medicine; the amino acid sequence of the tumor targeting peptide is c (Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys). The exosome of the umbilical cord-derived mesenchymal stem cells is modified, so that the surface of the exosome membrane is provided with tumor targeting peptide, and the inside of the exosome membrane contains siRNA for radiotherapy sensitization, namely the exosome membrane has new application in the function of the modified exosome.
In one embodiment, the nucleic acid agent is 15-30 nucleotides in length.
In one embodiment, the nucleic acid agent is an siRNA, micro RNA, shRNA or ASO.
In one embodiment, the nucleic acid agent is an siRNA targeting STAT3 gene or an siRNA inhibiting tumor malignancy-associated gene c-myc.
Wherein siSTAT3: GGCGTCCAGTTCACTACTA, sic-myc: CGACGAGACCTTCATCAAA. Purchased from Sharp Biotech Inc. in Guangzhou.
The invention also aims to provide a preparation method of the engineering exosome for tumor radiotherapy sensitization, which comprises the following steps,
Constructing a target gene expression vector GV492-iRGD-Lamp2 b;
the establishment of a mesenchymal stem cell line MSC-GV492-iRGD-Lamp2b for stably expressing a target gene;
Extracting exosomes, namely extracting the exosomes from the supernatant culture solution of the collected MSC-GV492-iRGD-Lamp2b cells to obtain exosomes modified with the iRGD peptide by a genetic engineering method;
Loading of small interfering RNA of exosomes modified with igbd peptide: and (3) transfecting the exosomes modified with the iRGD peptide through a transfection reagent, so as to realize the preparation of the engineering exosomes.
The invention also aims to provide an application of the engineering exosome for tumor radiotherapy sensitization in preparing a drug for enhancing tumor cell targeting by the exosome.
The invention also aims to provide an application of the engineering exosome for tumor radiotherapy sensitization in preparing medicines for inhibiting tumor cell growth and enhancing radiation sensitivity.
The invention also aims to provide an application of the engineering exosome for tumor radiotherapy sensitization in preparing a radiotherapy sensitization tumor targeting drug.
It is another object of the present invention to provide an αvβ3/αvβ5 tumor cell for targeting the pharmacodynamic function of highly expressed engineered exosomes for tumor radiotherapy sensitization.
Another object of the present invention is to provide an animal model construction method for verifying the efficacy of the engineered exosomes for tumor radiotherapy sensitization, the animal model construction method comprising:
Establishing a nude mouse breast cancer model: inoculating 5×10 6 cells under the skin of female Nu/Nu mice, and after 10 days, verifying whether the inoculation is successful; and taking the nude mice after the modeling is successful, and carrying out near infrared living body imaging analysis on the breast cancer model 2, 24 and 48 hours of the administered nude mice for tumor radiotherapy sensitization of the engineering exosomes under the NIR mark.
By combining all the technical schemes, the invention has the advantages and positive effects that:
First, aiming at the technical problems in the prior art and the difficulty in solving the problems, the technical problems solved by the technical proposal of the invention are analyzed in detail and deeply by tightly combining the technical proposal to be protected, the results and data in the research and development process, and the like, and some technical effects brought after the problems are solved have creative technical effects. The specific description is as follows:
The invention successfully expresses the tumor targeting peptide segment iRGD on the surface of the umbilical cord-derived mesenchymal stem cell exosome by a genetic engineering means, so that the modified exosome has the targeting of tumor cells. With this engineered exosome, the present invention delivers siRNA of STAT3 gene into tumor cells, thereby enhancing the radiation sensitivity of tumor cells by reducing STAT3 expression by tumor cells. The invention combines the current advantages and defects of exosomes and RNAi therapy, modifies the exosomes derived from mesenchymal stem cells, improves the tumor targeting capability of the exosomes, loads siRNA into the exosomes, and further improves the delivery efficiency and anti-tumor capability of the siRNA.
Secondly, the technical scheme is regarded as a whole or from the perspective of products, and the technical scheme to be protected has the following technical effects and advantages:
The invention provides an engineering exosome for tumor radiotherapy sensitization, which comprises an exosome, wherein the exosome is an exosome derived from human umbilical mesenchymal stem cells or an exosome derived from placenta mesenchymal stem cells.
The exosome carries the polypeptide iRGD of the active targeting tumor cells, the mesenchymal stem cells of the target genes are established by plasmid construction and slow virus infection methods, then the supernatant culture solution is collected, the exosome with the targeting polypeptide iRGD is obtained by an ultracentrifugation method, and the problem of tumor targeting is solved; the exosomes are loaded with siRNA interfering with tumor malignancy gene STAT3 through transfection experiments, so that the effects of inhibiting the growth of tumors and enhancing the sensitivity of radiotherapy are achieved.
Thirdly, as inventive supplementary evidence of the claims of the present invention, the following important aspects are also presented:
(1) The expected benefits and commercial values after the technical scheme of the invention is converted are as follows: engineered exosomes have strong prospects in targeted drug delivery and cancer treatment, and can be used for clinical transformations.
(2) The technical scheme of the invention fills the technical blank in the domestic and foreign industries: the engineered exosomes are endowed with high-efficiency targeting to the alpha v beta 3/alpha v beta 5 integrin positive tumor cells; and realizes the high-efficiency delivery of the small nucleic acid drug, effectively inhibits the growth of cancer cells and has no obvious toxicity; can be used as potential clinical radiotherapy sensitization medicine.
(3) Whether the technical scheme of the invention solves the technical problems that people want to solve all the time but fail to obtain success all the time is solved: the biggest problem in clinical application of nucleic acid medicaments is the lack of a better and more targeted in-vivo delivery system, so that the nucleic acid medicament is hopeful to become a novel carrier of cancer therapeutic medicaments, and the problems of specificity, effectiveness and safety of the medicaments are solved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the disclosure and together with the description, serve to explain the principles of the disclosure.
FIG. 1 is the construction of the engineered exosomes in example 1: the slow virus successfully infects MSCs, and the detection of MSCs and the expression of the gene fragment of the secretory exosome after the infection;
FIG. 2 is a confocal imaging of the engineered exosomes of example 2, showing successful loading of exosomes siSTAT, green-fluorescently labeled exosomes superimposed with red-fluorescently labeled siSTAT;
FIG. 3 is the result of the exosome nanoparticle transmission electron microscopy analysis of example 2;
FIG. 4 is an in vitro verification of tumor cell targeting (endocytic assay) of exosomes of example 3;
FIG. 5 is an in vivo verification of tumor tissue targeting by exosomes of example 3;
FIG. 6 shows the result of exosomes of example 4 inhibiting tumor cell growth and increasing sensitivity to radiotherapy;
FIG. 7 is a graph showing the result of the exosomes of example 5 further promoting tumor cell apoptosis under irradiation;
FIG. 8 shows the results of exosomes of example 3 inhibiting tumor cell clonogenic capacity and increasing sensitivity to radiotherapy in vitro;
FIG. 9 shows the results of the exosomes of example 3 promoting tumor radiotherapy sensitization in mice.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.
1. Explanation of the examples:
The embodiment of the invention provides an engineering exosome for tumor radiotherapy sensitization, which is externally modified with tumor targeting peptide and internally coated with small interfering RNA nucleic acid medicine; the amino acid sequence of the tumor targeting peptide is c (Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys).
In a preferred embodiment, the nucleic acid agent is 15-30 nucleotides in length.
In a preferred embodiment, the nucleic acid agent is an siRNA, micro RNA, shRNA or ASO.
In a preferred embodiment, the nucleic acid agent is an siRNA targeting STAT3 gene or an siRNA inhibiting tumor malignancy-associated gene c-myc. Wherein siSTAT3: GGCGTCCAGTTCACTACTA, sic-myc: CGACGAGACCTTCATCAAA.
The embodiment of the invention also provides a preparation method of the engineering exosome for tumor radiotherapy sensitization, which comprises the following steps,
Constructing a target gene expression vector GV492-iRGD-Lamp2 b;
the establishment of a mesenchymal stem cell line MSC-GV492-iRGD-Lamp2b for stably expressing a target gene;
extracting exosomes, namely extracting the supernatant culture solution of the collected MSC-GV492-iRGD-Lamp2b cells to obtain exosomes modified with the iRGD peptide by a genetic engineering method;
Loading of small interfering RNA of exosomes modified with igbd peptide: and (3) transfecting the exosomes modified with the iRGD peptide through a transfection reagent, so as to realize the preparation of the engineering exosomes.
The embodiment of the invention also provides an application of the engineering exosome for tumor radiotherapy sensitization in preparing a drug for enhancing tumor cell targeting by the exosome.
The embodiment of the invention also provides an application of the engineering exosome for tumor radiotherapy sensitization in preparing a medicine for inhibiting tumor cell growth and enhancing radiation sensitivity.
The embodiment of the invention also provides an alpha v beta 3/alpha v beta 5 tumor cell, wherein the alpha v beta 3/alpha v beta 5 tumor cell is used for targeting and expressing the drug effect function of the engineering exosome for tumor radiotherapy sensitization.
The embodiment of the invention also provides an animal model construction method for verifying the efficacy of the engineering exosome for tumor radiotherapy sensitization, which comprises the following steps:
Establishing a nude mouse breast cancer model: inoculating 5×10 6 cells under the skin of female Nu/Nu mice, and after 10 days, verifying whether the inoculation is successful; and taking the nude mice after the modeling is successful, and carrying out near infrared living body imaging analysis on the breast cancer model 2, 24 and 48 hours of the administered nude mice for tumor radiotherapy sensitization of the engineering exosomes under the NIR mark.
Example 1: preparation of engineered exosomes with tumor targeting
1) Construction of the Gene expression vector GV492-iRGD-Lamp2b of interest, as shown in FIG. 1, construction of the engineered exosomes in the examples: the lentivirus successfully infects MSCs, and detection of MSCs and expression of the secretory exosome target gene fragment after infection is shown.
The target gene segment iRGD-Lamp2b is inserted into a cloning site BamH I/Age I of a vector GV492, and the targeting peptide can be fused with an N-terminal extracellular domain of the Lamp2b to realize a targeting effect. The target gene expression vector GV492-iRGD-Lamp2b is obtained. The vector was purchased from Sharp Biotechnology Inc. in Guangzhou.
This plasmid was co-transfected with a viral packaging Helper plasmid (Helper 1.0, helper 2.0) into 293T cells. Virus harvesting (i.e. unpurified cell supernatant) is carried out 48-72 hours after transfection is completed, and the corresponding concentration and purification mode is adopted to obtain the lentivirus preservation solution with high titer according to different experimental requirements.
2) Cell line for establishing stable expression target gene by mesenchymal stem cell lentivirus infection
A suspension of cells at a density of 5X 10 4 cells/ml was prepared in DMEM/F12 complete medium and inoculated into six well plates. Culturing at 37 deg.C for 16-24 hr until the cell confluency is 20-30%. After incubation for 24 hours with 1mL of whole medium plus 10 μl of virus solution and 4 μl of virus infection solution P, the virus infection was observed by fluorescence. A cell line MSC-GV492-iRGD-Lamp2b which stably expresses the target gene is established. The vector was purchased from Sharp Biotechnology Inc. in Guangzhou.
3) Exosome extraction
Collecting the culture solution of mesenchymal stem cells successfully infected by slow virus, and centrifuging at the centrifugation speed of 300-500g for 10 minutes at the temperature of 4 ℃ to remove the residual cells in the culture solution; centrifuging 2000-3000g for 30 min to remove cell debris remained in the culture solution, centrifuging for 30 min again, and centrifuging at a speed of 10,000g; finally, the mixture was centrifuged for 70 minutes at a centrifugation speed of 100,000g. The pellet was resuspended in PBS to give the iRGD peptide-modified exosomes.
Exosomes are small vesicles secreted by cells and provided with phospholipid bilayer, wherein the vesicles can be wrapped with drugs, and have no sequence; the tumor targeting peptide iRGD and exosome membrane protein lamp2b are fused and expressed to obtain exosome with the targeting peptide iRGD modified, and the co-expressed fusion protein carrier is ordered from Sharp biological technology Co-Ltd in Guangzhou.
Exosomes are secreted by most cells, and their in vivo composition will vary depending on the cell secreted, but are mostly proteins and RNAs, of many species, requiring a clear sequencing.
4) Exosome siRNA loading
Carrying out siSTAT to load the exosomes obtained in the step 3), and carrying out transfection by using exosome specific transfection reagent Exo-Fect Exosome Transfection Kit. siSTAT3 sequences were purchased from Sharp Biotechnology Inc. in Guangzhou.
A) Transfection system: 10. Mu.L Exo-Fect solution, 2. Mu.L siRNA, 100. Mu.L exosome (50-300. Mu.g), 38. Mu.L PBS, and a total of 150. Mu.L transfection system were mixed by inverting the solution in the transfection system and incubating at 37℃for 10 minutes.
B) Immediately thereafter, the mixture was placed on ice, 30 mu L ExoQuick-TC was added thereto, and the mixture was mixed upside down 6 times, and placed on ice or at 4℃for 30 minutes.
C) Centrifugation was carried out for 3 minutes at a speed of 13,000-14,000rpm, the supernatant was removed and the pellet was resuspended in 300. Mu. LPBS.
For detecting the loading efficiency, siSTAT-cy 3 of the red fluorescent protein is selected, and the transfection efficiency is detected to be 60-85% by a fluorescence spectrophotometer.
Results: as shown in fig. 1, the mesenchymal stem cells expressing green fluorescent protein after successful lentiviral infection. And detecting the expression condition of the target genes of the mesenchymal stem cells and the secretion exosomes thereof by a Real-time PCR method. MSCs and their secreted exosomes after lentivirus infection, the expression of the iRGD-Lamp2b fragment was significantly increased, indicating successful construction of the exosomes expressing the iRGD peptide.
Example 2: fluorescent labeling of engineered exosomes
In order to further visually judge whether the engineered exosomes were successfully prepared, the exosomes and siRNA were respectively fluorescently labeled and imaged. Green Fluorescent labeling of exosomes using kit PKH67 fluorescence CELL LINKER KITS:
1) 10. Mu.L (25. Mu.g) exosomes+0.5ml dilution C, gently mix;
2) 2ul+0.5ml of diluent C, gently mix;
3) Rapidly adding 1 into 2, mixing the samples, and incubating exosome/dye solution in suspension for 15 minutes;
4) Stop staining, add an equal volume (1 mL) of exosome-free serum or 1% bsa;
5) Centrifuging at 100,000g for 70 min, carefully removing the supernatant; 100,000g PBS was washed for 70 minutes, and the supernatant was carefully removed to give stained exosomes.
The results are shown in FIG. 2, where the green-fluorescent labeled exosomes and red-fluorescent labeled siSTAT were superimposed, indicating successful exosome encapsulation of siSTAT. The exosome transmission electron microscope is shown in fig. 3, and the grain size and the morphology of the exosome after engineering are not changed obviously.
Example 3: engineering exosome tumor targeting validation
1) Targeting detection of exosomes to breast cancer cells (in vitro)
Breast cancer MDA-MB-231 cells express αvβ3 in high, MDA-MB-231 cells are inoculated into a six-hole plate according to the density of 2X 10 5 cells/hole, and after the cells are attached, dyeing marked exosomes are added. Exosomes were stained as in example 2, centrifuged and the exosomes were harvested and washed with PBS and finally precipitated and resuspended in 200 μl PBS to give stained exosomes. Adding the stained and incubated exosomes into cells to be observed for co-incubation, and photographing and observing the process of entering the cells by the exosomes with green fluorescent markers at 1 hour and 4 hours after the adding.
As shown in FIG. 4, the exosome Exo-iRGD modified and transformed by iRGD enters MDA-MB-231 cells obviously more than wild exosome Exo, which indicates that the targeting of the modified exosome to tumor cells is enhanced.
2) Detection of targeting of exosomes to breast cancer cells (in vivo)
Establishing a nude mouse breast cancer model: 5X 10 6 cells were inoculated subcutaneously into female Nu/Nu mice, and after 10 days, success was confirmed when tumors grew until large grain tumors appeared. The modeled mouse tail is taken to be injected with 100 mu L of the NIR marked exosome for 2, 24 and 48 hours, and then is placed under near infrared living body imaging for observation.
As shown in FIG. 5, the tumor tissue fluorescence of the tumor-bearing nude mice injected with the iRGD modified exosomes is significantly stronger than that of the wild exosome group.
Example 4: tumor radiation sensitization effect of engineered exosomes (inhibition of proliferation)
MDA-MB-231 cells were seeded into six well plates at a density of 2X 10 5 cells/well, and after the cells had adhered to the walls, the cells were irradiated to 8Gy, gamma rays, according to the groupings. And meanwhile, wild exosomes and engineered exosomes are respectively added into exosome treatment components. After 48h incubation, each group of cells was observed by photographing.
The results are shown in FIG. 6: the growth of breast cancer cells in an engineering exosome Exo-iRGD-siSTAT3 treatment group is obviously inhibited, the cell morphology is worse under the irradiation condition, and after the Exo-iRGD and the Exo-iRGD-siSTAT are treated for 3 days, the cells are obviously reduced and the cell attenuation is prolonged; under the irradiation condition, slag is arranged in the Exo-iRGD-siSTAT group of cells, which shows the worst cell state, so that the engineered exosome can play a very strong role in inhibiting tumor under the irradiation condition, and the radiation sensitization is promoted.
Example 5: tumor radiosensitization of engineered exosomes (promoting apoptosis)
MDA-MB-231 cells were seeded into six well plates at a density of 2X 10 5 cells/well, and after the cells had adhered to the walls, the cells were irradiated to 8Gy, gamma rays, according to the groupings. And meanwhile, wild exosomes and engineered exosomes are respectively added into exosome treatment components. The proportion of apoptotic cells was flow analyzed for each group after 48h incubation.
The results are shown in FIG. 7: exosome-treated groups, increased apoptosis; under the irradiation condition, the Exo-iRGD-siSTAT treatment group has the strongest effect of promoting the apoptosis of breast cancer cells.
Example 6: tumor radiosensitization of engineered exosomes (inhibition of clonogenic potential)
MDA-MB-231 cells are inoculated into a six-hole plate according to the density of 1000 cells/hole, and after the cells are attached, the cells are irradiated with 2,4,6Gy and gamma rays according to groups. And meanwhile, wild exosomes and engineered exosomes are respectively added into exosome treatment components. After 14 days of culture, each group of cells was fixed, and crystal violet staining was performed to observe the clonality of each group of cells.
The results are shown in FIG. 8: exosome treatment group, cell clone number was reduced; under irradiation conditions, exo-iRGD-siSTAT3 treated groups had minimal cell cloning, indicating that the engineered exosomes enhanced the radiation sensitivity of MDA-MB-231 cells.
Example 7: tumor radiation sensitization of engineered exosomes in mice
Establishing a nude mouse breast cancer model: 5X 10 6 MDA-MB-231 cells with the biological luciferase Fluc were inoculated subcutaneously in female Nu/Nu Nu mice, and after 10 days, the success of the inoculation was confirmed when tumors grew to appear as large rice grains. Grouping the nude mice after modeling success, and performing a control group, an Exo-iRGD-siSTAT treatment group, an 8Gy irradiation group and an 8Gy irradiation+Exo-iRGD-siSTAT treatment group; the mice in the irradiation group were locally irradiated with 8Gy tumor, and then, the mice in the exosome treatment group were subjected to 100. Mu.L (100. Mu.g/. Mu.L) of exosome injection by tail vein, once every other day, 3 times of total injection, and then, subjected to bioluminescence imaging observation by in vivo imaging of small animals.
The results are shown in FIG. 9: under the irradiation condition, the biological luminous signals of the Exo-iRGD-siSTAT3 treatment group are weakest, which suggests that the tumor is smallest, and the modified exosome enhances the radiation sensitivity of tumor cells in vivo.
In conclusion, the engineering exosome prepared by the invention is a potential and effective breast cancer cell targeting drug delivery system, and has a good tumor radiation sensitization effect.
In the foregoing embodiments, the descriptions of the embodiments are emphasized, and in part, not described or illustrated in any particular embodiment, reference is made to the related descriptions of other embodiments.
Because the embodiment of the method is based on the same conception, the specific functions and technical effects brought by the embodiment of the method can be seen in the method embodiment section, and the detailed description is omitted.
While the invention has been described with respect to what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (1)
1. The application of an engineering exosome for tumor radiotherapy sensitization in preparing a radiotherapy sensitization tumor targeting drug is characterized in that the engineering exosome for tumor radiotherapy sensitization externally modifies a tumor targeting peptide and internally wraps a small interfering RNA nucleic acid drug; the nucleic acid drug is siRNA targeting STAT3 gene, and the nucleic acid sequence of the siSTAT3 is GGCGTCCAGTTCACTACTA; the tumor is breast cancer;
a method of preparing an engineered exosome for tumor radiotherapy sensitization comprising the steps of:
constructing a target gene expression vector GV492-iRGD-Lamp2b, inserting a target gene fragment iRGD-Lamp2b into a cloning site BamH I/Age I of the vector GV492, and fusing a targeting peptide with an N-terminal extracellular domain of the Lamp2b to realize a targeting effect; obtaining a target gene expression vector GV492-iRGD-Lamp2b; the plasmid and a virus packaging auxiliary plasmid are transfected into 293T cells together, and the virus packaging auxiliary plasmids are Helper 1.0 and Helper 2.0; harvesting the virus at 48-72 h after transfection, and determining to obtain a lentivirus preservation solution with high titer by adopting a corresponding concentration and purification mode according to different experimental requirements;
Establishing a mesenchymal stem cell line MSC-GV492-iRGD-Lamp2b for stably expressing a target gene, preparing a cell suspension with the density of 5X 10 4 cells/ml by using a DMEM/F12 complete culture medium, and inoculating the cell suspension into a six-hole plate; culturing at 37 ℃ to 16-24 h until the cell confluency is 20-30%;
Adding 10 mu L of virus liquid and 4 mu L of virus infection liquid P into 1mL whole culture medium, incubating for 24 hours, and observing the virus infection condition by fluorescence; establishing a cell line MSC-GV492-iRGD-Lamp2b for stably expressing a target gene;
Extracting exosomes, namely extracting the exosomes from the supernatant culture solution of the collected MSC-GV492-iRGD-Lamp2b cells to obtain exosomes modified with the iRGD peptide by a genetic engineering method;
Loading of small interfering RNA of exosomes modified with igbd peptide: and (3) transfecting the exosomes modified with the iRGD peptide through a transfection reagent, so as to realize the preparation of the engineering exosomes.
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