CN114129534A - Engineered exosome and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biological medicines, and discloses an engineered exosome and a preparation method and application thereof. The exosome of the invention is induced by cell THP-1 with (12-) phorbol myristate (-13-) acetate PMA and lipopolysaccharide LPS in turn, and the obtained M1 macrophage is induced by DSPE-PEG2000Culturing in RMPI1640 of Mal, centrifuging the supernatant, ultrafiltering, concentrating, and purifying to obtain purified exosome; loading therapeutic medicine with exosome and modifying with Angiopep-2 or/and CD133aptamer,obtaining the engineered exosome. The engineered exosome obtained by the invention can deliver small-molecule drugs or nucleic acid drugs in a targeted manner through a blood brain barrier, obviously inhibit the growth of glioma cells and glioma stem cells, induce the apoptosis of the glioma cells and the glioma stem cells and prolong the life cycle of tumor-bearing mice, and has no obvious side effect. Therefore, the novel engineered exosomes can be used for effective treatment of brain tumors.
Description
Technical Field
The invention relates to the technical field of biological medicine, and relates to preparation of a novel engineered exosome and application of the novel engineered exosome in treatment of glioma
Background
Glioma is a most threatening tumor in the central nervous system, and the current standard treatment for it is surgical treatment first, combined with radiotherapy and chemotherapy, but the standard treatment is less effective. The 5-year survival of patients is around 5%.
STAT3 is a member of a family of signaling and transcriptional activators that can control transcription of genes downstream of gliomas and promote the development of gliomas, and there is increasing evidence that STAT3 is a promising target for the inhibition of gliomas. Small interfering RNA (siRNA), an artificially synthesized double-stranded RNA that is less toxic and specifically silences intracellular mRNA for a specific protein, has been used to treat a number of refractory tumors, including gliomas. However, the greatest difficulty in treating gliomas using siRNA silencing STAT3 protein is the lack of suitable siRNA carriers. The viral vector has good siRNA delivery capacity, but the immunogenicity and the gene mutagenesis toxicity of the viral vector seriously influence the application of the viral vector. Non-viral vectors, including polymers, inorganic nanoparticles, and liposomes, are also capable of delivering siRNA, but some of the problems of non-viral vectors, such as safety and efficiency of delivery, limit their widespread use. In addition, the application of siRNA for treating glioma also has the problems of potential immunogenicity, poor cellular uptake, short blood circulation, poor plasma stability, incapability of penetrating blood brain barrier and the like.
The current standard treatment regimen for gliomas is surgical treatment followed by radiation therapy and chemical treatment. Temozolomide is a first-line anticancer drug used in standard chemotherapy of glioma. But DNA repair protein O inside glioma cells6Alkylguanine-DNA Alkyltransferase (AGT) can repair temozolomide in guanine O6The induced alkylation injury makes the tumor cells generate drug resistance to temozolomide. Tong (Chinese character of 'tong')By transfer of alkyl groups to Cys145, O of AGT6Benzyl guanine enhances the cytotoxicity of temozolomide, so that the combined use of temozolomide and O6Benzylguanine is a promising therapeutic strategy. Due to O6Benzylguanine has a number of adverse reactions including hepatotoxicity, myelosuppression and pulmonary fibrosis and its inability to cross the blood brain barrier, O6Benzyl guanine cannot be used clinically in large quantities for the treatment of gliomas. Therefore, there is an urgent need to develop a feasible strategy for delivering therapeutic drugs into tumor tissues.
Glioma stem cells are a subset of glioma cells that are strongly resistant to radiation therapy and chemotherapy. The CD133 surface protein is a surface marker of glioma stem cells, so we can target glioma stem cells with CD133 aptamers.
In order to solve the problems, the invention constructs a novel drug carrier; in order to overcome the drug delivery disadvantages of viral and non-viral vectors, the present invention utilizes exosomes as drug targeting delivery vectors. The exosome has many advantages as a natural carrier with huge application potential, such as the capability of penetrating blood brain barrier, good plasma stability, long blood circulation time and low immunogenicity. In view of the tropism of M1 macrophage to tumor cells, the invention utilizes exosome derived from M1 macrophage to carry out engineering transformation, thereby realizing the targeted delivery of the drug. Because the surface of glioma and blood brain barrier endothelial cells over-expresses low-density lipoprotein receptor-related protein 1(LRP-1), An2, which is a ligand of LRP-1, can promote exosome penetration through the blood brain barrier and target glioma.
Disclosure of Invention
In view of some of the problems with current drug delivery for the treatment of gliomas, it is an object of the present invention to provide a novel engineered exosome, as well as methods of preparation and use thereof, in particular for the treatment of gliomas.
The preparation method of the engineered exosome is characterized in that (12-) phorbol myristate (-13-) acetate PMA is used for inducing cell THP-1 patchesInducing adhered THP-1 to form M1 macrophage by LPS, and adding DSPE-PEG2000Culturing M1 macrophage by RMPI1640 of Mal for 2 days, centrifuging culture medium supernatant, ultrafiltering and concentrating the supernatant to obtain crude exosome, and further purifying by using EXOEasy Maxi Kit to obtain purified exosome; loading therapeutic drug into the purified exosomes, then incubating Angiopep-2 or/and CD133aptamer with the exosomes, washing off the unloaded therapeutic drug and the unmodified An2 or/and CD133aptamer by PBS, and obtaining the engineered exosomes, namely the exosomes loaded with the therapeutic drug and modified An2 or/and CD133 aptamer.
Furthermore, the invention also defines a preparation method of the engineered exosome, which is characterized by comprising the following steps:
1) transferring the THP-1 cells onto a culture dish, and incubating the cells with RMPI1640 containing 0.1mg/mL PMA overnight to convert the cells from a suspension state to an adherent state; the cells were then incubated with Opti MEM medium containing 0.4mg/mL LPS, transformed into M1 macrophages, and continued with the solution containing 50. mu.g/mL DSPE-PEG2000-Mal incubation with M1 macrophages;
2) taking the supernatant in the step 1) to carry out centrifugation and filtration in sequence, and centrifuging the filtered solution by using an ultrafiltration tube to obtain a crude exosome extract;
3) purifying the crude extract obtained in the step 2) by using an EXOEasy Maxi Kit to obtain a purified exosome EXO;
4) loading therapeutic drugs and surface modification: loading the therapeutic drug into the purified exosomes obtained in the step 3), adding An2 or/and CD133aptamer for incubation, centrifuging the incubated mixed solution at 4 ℃ by using An ultrafiltration tube, adding PBS for centrifugation, and washing the unloaded therapeutic drug and the unmodified An2 or/and CD133aptamer to obtain the engineered exosomes.
Further, the invention also defines that the supernatant fluid filtration in the step 2) adopts a filter membrane of 0.22 μm for filtration, and the filtrate is centrifuged by an ultrafiltration tube of 100 kDa.
Further, the invention also defines the specific steps of purifying the crude extract by using the EXOEasy Maxi Kit in the step 3) as follows: mixing the crude extract and the XBPBuffer, adding the mixture into a column, placing the column containing the crude extract and the XBPBuffer into a collection tube for centrifugation, removing a filtrate (namely, a liquid flowing into the collection tube), adding the XWPbuffer into the column for centrifugation, removing the filtrate, placing the column into a new collection tube, adding the XE buffer, incubating at room temperature, then centrifuging, taking out the filtrate, adding the filtrate into the column for incubation, and then centrifuging to obtain a purified exosome.
Further, the invention also defines that the therapeutic drug comprises a nucleic acid drug or a small molecule drug; the nucleic acid drug is siRNA, and the small molecule drug is temozolomide or O6-benzylguanine.
Further, the invention also defines targeted therapeutic drug loading using ultrasound, incubation or freeze-thawing, wherein:
the ultrasonic loading process comprises the following steps: adding a targeted therapeutic drug into a purified exosome, performing ultrasound for 30s under the ultrasound power of 25W, stopping the operation for 150s, repeating the operation for 6 times, incubating the exosome subjected to ultrasound at 4 ℃ for 30min, adding An2 or/and a CD133aptamer, incubating for 24h at 4 ℃, centrifuging the incubated mixed solution for 2min at 2000g and 4 ℃ by using a 100kDa ultrafiltration tube, adding PBS (phosphate buffer solution) at 4 ℃ for 2min, and repeating the operation once again to obtain An engineered exosome;
the freeze-thaw loading process comprises the following steps: adding the targeted therapeutic drug into the purified exosome, incubating for 30min at room temperature, -80 ℃ for 10min, incubating for 10min at room temperature, repeating the operation for 3 times, placing the mixture at room temperature for incubating for 30min, adding An2 and/or CD133aptamer, incubating for 24h at 4 ℃, centrifuging the incubated mixed solution for 2min at 4 ℃ by using a 100kDa ultrafiltration tube, adding PBS 2500g, centrifuging for 2min at 4 ℃, and repeating the operation once again to obtain the engineered exosome.
The incubation loading process is as follows: adding a targeted therapeutic drug, An2 or/and CD133aptamer into the purified exosome, incubating for 24h at 4 ℃, centrifuging the incubated mixed solution for 2min at 4 ℃ by using a 100kDa ultrafiltration tube, adding PBS, centrifuging for 2min at 4 ℃, and repeating the operation once again to obtain the engineered exosome.
Furthermore, the invention also defines the engineered exosome obtained by the defining method, wherein the exosome is disc-shaped in morphology, contains CD63 exosome characteristic protein and has the average diameter of 150.8nm, the three engineered exosomes prepared by the invention are: EXO-An2-siRNA (siRNA), EXO-An2-Apt-TMZ (temozolomide) and EXO-An2-Apt-BG (O)6Benzyl guanine), wherein Apt is CD133aptamer (aptamer), EXO-An2-Apt refers to the fact that An2 and Apt are modified on the surface of An exosome, CD133 protein is arranged on the surface of a glioma stem cell, CD133Apt can promote the glioma stem cell to take up the exosome, and therefore the Apt can target the glioma stem cell,
furthermore, the invention also defines the application of the engineered exosome in treating glioma.
Specifically, the invention obtains three engineered exosomes by a defined preparation method, wherein: the prepared EXO-An2-siRNA can knock out mRNA of STAT3 protein, and obviously inhibit expression of STAT3 protein; the prepared EXO-An2-Apt-TMZ can damage glioma cell DNA and induce the apoptosis of glioma; the prepared EXO-An2-Apt-BG can inhibit the activity of AGT drug-resistant enzyme and promote the cytotoxicity of temozolomide.
The invention modifies Angiopep-2(An2) or/and CD133aptamer on the surface of An exosome, and promotes the engineered exosome to penetrate the blood brain barrier and target glioma through the interaction with low-density lipoprotein receptor-related protein 1(LRP-1) or/and CD133 surface protein; the siRNA is delivered through exosomes, and the mRNA of STAT3 protein is knocked out to induce the apoptosis of glioma cells; delivery of temozolomide and O by exosomes6The benzyl guanine induces the apoptosis of glioma cells and glioma stem cells, and provides a feasible idea for developing anti-glioma drugs.
The invention provides a novel engineered exosome with a treatment effect on glioma, and the exosome is loaded with STAT3siRNA, temozolomide or O6-benzylguanine and modification of An2 or/and CD133 aptamer; the CD133aptamer is modified on the surface of the exosome, and the interaction with CD133 surface protein promotes the engineered exosome to target glioma stem cells, so that the engineered exosome obtained by the invention can obviously induce glioma cells and glioma stem cellsAnd (4) apoptosis.
Through tests, the intravenous injection of the exosome provided by the invention can obviously prolong the median survival time of mice, and has no side effect.
In conclusion, the novel engineered exosome provided by the invention has the following beneficial effects:
the invention provides an engineered exosome capable of penetrating a blood brain barrier and targeting glioma, a preparation method thereof and application thereof in treating glioma. The purified exosome is loaded with therapeutic drugs, and An2 or/and CD133aptamer are modified simultaneously to form a final engineered exosome for treating glioma, under the action of An2 and CD133aptamer, the therapeutic drugs can be enriched in the brain and absorbed by glioma and glioma stem cells, and the exosome absorbed by glioma and glioma stem cells releases the therapeutic drugs into cytoplasm and induces apoptosis of glioma and glioma stem cells. The invention improves the treatment effect of the exosome on the glioma by carrying out a series of engineering reconstruction on the exosome.
Drawings
FIG. 1 is a transmission electron micrograph of EXO-An 2;
FIG. 2 is a Western blot gel of EXO-An 2;
FIG. 3 is a Nanoparticle Tracking Analysis (NTA) (particle size and potential) of EXO-An 2;
FIG. 4 is a schematic representation of the flow analysis of U87MG uptake of EXO-An 2;
figure 5 is a schematic of the treatment of glioma cck-8 with EXO-An2-siRNA, representing a significant difference between the two sets of data (p value less than 0.0001);
FIG. 6 is a schematic representation of treatment of glioma cck-8 with EXO-An2-Apt-TMZ and EXO-An 2-Apt-BG;
FIG. 7 is An apoptosis assay for glioma treated with EXO-An 2-siRNA;
FIG. 8 is An in vivo imaging analysis of EXO-An 2;
FIG. 9 is the survival of various groups of tumor-bearing models;
FIG. 10 is a graph of body weight change during treatment for each group of tumor-bearing models;
FIG. 11 is a schematic diagram of exosome preparation.
Detailed Description
In order to better clarify the objects, technical solutions and advantages of the present invention, the present invention will be described in detail with reference to the following examples. The specific embodiments described herein are merely illustrative of the invention and do not limit the practice of the invention.
Example 1 preparation of EXO-An2-siRNA, the preparation process is shown in FIG. 11.
(1) THP-1 was transferred to a 15cm petri dish, cells were incubated overnight with RMPI1640 containing 0.1mg/mL PMA (cells would convert from suspension to adherent), followed by 4h incubation with Opti Men medium containing 0.4mg/mL LPS (THP-1 had converted to M1 macrophages), and continued with DSPE-PEG containing 50. mu.g/mL2000-RMPI 1640 from Mal was incubated with cells for 2 days;
(2) centrifuging the supernatant at 2000g for 15min, filtering the supernatant with 0.22 μm filter membrane, and centrifuging the filtrate with 2200g 100kDa ultrafiltration tube for 20min to obtain crude extract of exosome;
(3) purifying the crude extract by using an EXOEasy Maxi Kit, which comprises the following specific steps of mixing 8mL of the crude extract with 8mL of XBP, adding the mixture into a column, centrifuging for 1min at 500g, removing filtrate, adding 10mLXWP into the column, centrifuging for 5min at 5000g, removing filtrate, placing the column into a new tube, adding 2mL of XE buffer, incubating at room temperature for 1min, centrifuging for 5min at 500g, taking out the filtrate, adding the filtrate into the column, incubating for 1min, and centrifuging for 5min at 5000g to obtain purified exosome EXO;
(4) loading siRNA and surface modification: 0.9nmolsiRNA was added to the purified exosome EXO (100. mu.L, 0.537mg/mL (protein concentration metric)) of step 3) and sonicated as follows: carrying out ultrasonic treatment for 6 times in total with ultrasonic power of 25W, ultrasonic treatment for 30s and stopping for 150s, incubating the ultrasonic exosome EXO-siRNA for 30min at 4 ℃, adding 0.5mg of An2, incubating for 24h at 4 ℃, centrifuging the incubated mixed solution for 2min at 2000g and 4 ℃ by using a 100kDa ultrafiltration tube, adding 150 mu L PBS 2500g, centrifuging for 2min at 4 ℃, and repeating the steps again to obtain the EXO-An 2-siRNA;
(5) or directly adding An2 0.5mg into the exosomal EXO obtained in the step 3), incubating for 24h at 4 ℃, centrifuging the incubated mixed solution for 2min at 2000g and 4 ℃ by using An ultrafiltration tube of 100kDa, adding PBS 2500g at 150 mu L, centrifuging for 2min at 4 ℃, and repeating the steps again to obtain EXO-An2, wherein the appearance of the carrier can be seen through the product.
The transmission electron micrograph of the EXO-An2 obtained in the step (5) of example 1 is shown in fig. 1, the WB glue micrograph of the exosome surface marker protein is shown in fig. 2, the NTA analysis (potential and particle size) is shown in fig. 3, and it can be seen from the graph that the appearance of the anti 2 modified EXO is disc-shaped, contains the exosome characteristic protein CD63, and the particle size and surface potential before and after modification are unchanged, and the average particle size is 150.8 nm;
the invention has three modes of loading nucleic acid drugs (such as STAT3siRNA) or small molecule drugs (such as temozolomide and O6-benzyl guanine) into the purified exosome: sonication, incubation and freeze-thawing, step 4 in example 1 is sonication, and other methods are further described below on the basis of step 3 in example 1 (purified exosomes EXO);
example 1a temozolomide loading and surface modification: (sonication) 3mg of temozolomide was added to purified exosomes (100 μ L, 0.537mg/mL (protein concentration metric)) and sonicated as follows: carrying out ultrasonic power of 25W, carrying out ultrasonic treatment for 30s, stopping ultrasonic treatment for 150s, carrying out ultrasonic treatment for 6 times in total, incubating the ultrasonically treated exosome for 30min at 4 ℃, adding 0.5mg of An2 and 1.8nmol of CD133aptamer, incubating for 24h at 4 ℃, centrifuging the incubated mixed solution for 2min at 2000g and 4 ℃ by using a 100kDa ultrafiltration tube, adding 150 mu L PBS 2500g, centrifuging for 2min at 4 ℃, and repeating the steps again to obtain a novel exosome EXO-An 2-Apt-TMZ;
example 1b temozolomide loading and surface modification: (freezing and thawing method) adding 3mg temozolomide into purified exosome (100 mu L, 0.537mg/mL (protein concentration measurement)) and incubating for 30min at room temperature, incubating for 10min at-80 ℃, then incubating for 10min at room temperature, continuously freezing and thawing for 3 times, placing the mixture at room temperature and incubating for 30min, then adding 1.8nmol of CD133aptamer and incubating for 24h at 4 ℃, centrifuging the incubated mixed solution for 2min at 2000g and 4 ℃ by using a 100kDa ultrafiltration tube, then adding 150 mu L PBS 2500g and centrifuging for 2min at 4 ℃, and repeating once again to obtain novel exosome EXO-Apt-TMZ;
example 1c supporting temozolomide and surface modification: (incubation method) adding 3mg temozolomide and 0.5mg An2 into purified exosome (100 mu L, 0.537mg/mL (protein concentration measurement)) to incubate for 24h at 4 ℃, centrifuging the incubated mixed solution for 2min at 2000g and 4 ℃ by using a 100kDa ultrafiltration tube, adding 150 mu L PBS 2500g to centrifuge for 2min at 4 ℃, and repeating the steps again to obtain the novel exosome EXO-An 2-TM;
example 1d Loading of O6-benzylguanine and surface modification: (ultrasonic method) 0.3mg of O6Benzyl guanine was sonicated in purified exosomes (100 μ L, 0.537mg/mL (protein concentration metric)) as follows: carrying out ultrasonic power of 25W, carrying out ultrasonic treatment for 30s, stopping carrying out ultrasonic treatment for 150s, carrying out ultrasonic treatment for 6 times in total, incubating the ultrasonically treated exosome for 30min at 4 ℃, adding 0.5mg of An2 and 1.8nmol of CD133aptamer, incubating for 24h at 4 ℃, centrifuging the incubated mixed solution for 2min at 2000g and 4 ℃ by using a 100kDa ultrafiltration tube, adding 150 mu L PBS 2500g, centrifuging for 2min at 4 ℃, and repeating once again to obtain a novel exosome EXO-An 2-Apt-BG;
example 1e Loading of O6-benzylguanine and surface modification: (Freeze-thaw method) 0.3mg of O was taken6Adding benzyl guanine into purified exosome (100 mu L, 0.537mg/mL (protein concentration measurement)) and incubating at room temperature for 30min, incubating at-80 ℃ for 10min, incubating at room temperature for 10min, continuously freezing and thawing for 3 times, incubating the mixture at room temperature for 30min, adding 0.5mg of An2 and incubating at 4 ℃ for 24h, centrifuging the incubated mixed solution at 2000g and 4 ℃ for 2min by using a 100kDa ultrafiltration tube, adding 150 mu L PBS 2500g and centrifuging at 4 ℃ for 2min, and repeating the steps again to obtain the novel exosome EXO-An 2-BG;
example 1d Loading of O6-benzylguanine and surface modification: (incubation method) 0.3mg of O was taken6Adding benzyl guanine and 1.8nmol CD133aptamer into a purified exosome (100 mu L, 0.537mg/mL (protein concentration measurement)) to incubate for 24 hours at 4 ℃, centrifuging the incubated mixed solution for 2 minutes at 2000g and 4 ℃ by using a 100kDa ultrafiltration tube, then adding 150 mu L PBS 2500g to centrifuge for 2 minutes at 4 ℃, and repeating the centrifugation for one time to obtain the novel exosome EXO-Apt-B.
Example 2
The EXO-An2 obtained in step (5) of example 1 was stained with DiD and incubated with U87MG, and U87MG was subjected to flow cytometry for uptake of EXO-An2, and U87MG cells were obtained from American Type Culture Collection (ATCC). The method comprises the following specific steps:
0.5mg of An2 was added to exosomes EXO (100. mu.L, 0.537mg/mL), incubated at 4 ℃ for 24h, 0.25. mu.L of DiD dye was added, incubated at 4 ℃ for 30min, centrifuged at 4 ℃ for 2min with 100kDa ultrafiltration tube, 2500g for 2min, 150. mu.L of PBS was added, centrifuged at 4 ℃ for 2min with 100kDa ultrafiltration tube, and repeated once to give EXO-An 2.
EXO-An2 was added to U87MG, incubated for 4h, the supernatant removed, washed with PBS, trypsinized, 800g, centrifuged for 2min, the supernatant removed and suspended in 400 μ L PBS, and the uptake of EXO-An2 by U87MG was analyzed by flow cytometry. The results show that 57.34% of U87MG uptake EXO-An2 relative to exosomes without An2 modification, the results of which are shown in figure 4, and are represented by example 2: an2 can promote the uptake of exosomes of U87MG (glioma).
Example 3
Transfer U87MG to 96-well plates (5X 10)3One/well), adding DMEM medium for incubation for 24h, adding EXO-An2-siRNA (0.09nmol siRNA), EXO-siRNA (0.09nmol siRNA), EXO, siRNA (0.09nmol siRNA), An2(49 μ g) and PBS into the cells for incubation for 48h, removing the supernatant, adding PBS for washing, adding 10 μ L CCK-8 and 100 μ L DMEM for incubation for 2h, and detecting the OD450 value by a microplate reader. As shown in FIG. 5, it is clear that EXO-An2-siRNA can significantly inhibit U87MG activity.
Example 4
Transfer U87MG to 96-well plates (5X 10)3Pieces/well), adding DMEM medium for 24h, adding EXO (0.63mg/mL, 7. mu.L), An2(0.049mg), Apt (0.129nmol), EXO-An2-Apt-TMZ (150. mu.M) and EXO-An2-Apt-BG (30. mu.M) (combination administration), EXO-An2-Apt-TMZ (150. mu.M), EXO-An2-Apt-BG (30. mu.M) and PBS to the cells for 48h, removing supernatant, adding PBS for washing, adding 10. mu.L CCK-8 and 100. mu.L DMEM for 2h, and detecting OD450 value by An enzyme reader. As a result, as shown in FIG. 6, it is clear that EXO-An2-Apt-TMZ can significantly inhibit U87MG activity and that EXO-An2-Apt-TMZ and EXO-An2-Apt-BG are excellent in therapeutic effects when used in combination.
Example 5
Transfer U87MG to 6-well plateMiddle (5X 10)5One/well), adding culture medium, incubating for 24h, adding EXO-An2-siRNA (0.09nmol siRNA), EXO-siRNA (0.09nmol siRNA), EXO, siRNA (0.09nmol siRNA), and PBS into the cells, incubating for 48h, removing supernatant, adding PBS for washing, trypsinizing, centrifuging for 2min at 800g, removing supernatant, adding 195. mu.L Binding Buffer and 5. mu.L Annexin-V-FITC, keeping out of the light, incubating for 15min at room temperature, adding 200. mu.L Binding Buffer for washing cells, centrifuging for 2min at 1000rpm, removing supernatant, suspending the cells in 190. mu.L Binding Buffer, adding 10. mu.L Propidium Iodide, and detecting apoptosis by flow cytometry. The results are shown in fig. 7, and it can be seen from fig. 7 that 94.55% of U87MG underwent apoptosis.
In the embodiment, An in vitro experiment proves that EXO-An2 can target U87MG cells, and simultaneously proves that EXO-An2-siRNA can obviously inhibit the cell activity of U87 MG.
Example 6 (in vivo experiment)
Glioma tumor model construction: BALB/c nude mice (male, 18-20g, 4 weeks) were purchased from Zhejiang medical colleges. mu.L of tumor suspension (10) was injected with a Hamilton syringe5U87MG was suspended in 5. mu.L PBS) and injected into the brain of BALB/c nude mice at specific locations (0.5 mm anterior bregma and 2mm lateral, 3mm deep from dura mater).
In vivo imaging: all tumor-bearing mice were divided into 3 groups of 3 tumor-bearing mice each. At 14 days after tumor inoculation, 100. mu.L of DiD-modified EXO-An2, 100. mu.L of DiD-modified EXO and 100. mu.L of PBS were injected via tail vein into tumor-bearing mice, and 24h later, the mice were treated with the DiD-modified EXO-An2 and PBS
Whole mice were imaged in vivo by Lumina LT Series III. As a result, as shown in fig. 8, it can be seen from fig. 8 that An2 promotes accumulation of exosomes in the brain.
Example 7 (in vivo experiment)
In vivo treatment: all tumor-bearing mice were divided into 4 groups, 5 tumor-bearing mice per group, 100. mu.L of EXO-An2-siRNA (siRNA 0.086 nmol/mouse), 100. mu.L of EXO-An2, 100. mu.L of siRNA (siRNA 0.086 nmol/mouse) and 100. mu.L of PBS were injected into the tumor-bearing mice via tail vein on days 7, 11, 15 and 19 after tumor injection in BALB/c nude mice, and the survival and body weight of the tumor-bearing mice per group were observed. The results are shown in fig. 9 and 10, and it can be confirmed from the results in the figures that: the EXO-An2-siRNA can significantly prolong the survival time of tumor-bearing mice and the weight of the tumor-bearing mice in the group is not changed greatly (less than 20%).
The above description is only for the purpose of general description and implementation of the present invention, and not intended to limit the scope of the invention, but all equivalent modifications made in the light of the present specification and accompanying drawings, or used directly or indirectly to patented other related fields, are deemed to be within the scope of the invention.
Claims (8)
1. A process for preparing the engineered exosome features that the cell THP-1 is sequentially induced by (12-) phorbol myristate (-13-) acetate PMA and lipopolysaccharide LPS to become M1 macrophage, and the obtained M1 macrophage is then induced by DSPE-PEG2000Culturing in RMPI1640 of Mal, centrifuging supernatant of culture medium, ultrafiltering and concentrating the supernatant to obtain crude exosome, and purifying the crude exosome with EXOEasy Maxi Kit to obtain purified exosome; loading the therapeutic drug into the purified exosome, incubating Angiopep-2 or/and CD133aptamer with the exosome, and washing with PBS to obtain the engineered exosome, namely the exosome loaded with the therapeutic drug and modifying An2 or/and CD133 aptamer.
2. The method for preparing an engineered exosome according to claim 1, characterized by comprising the following steps:
1) transferring the THP-1 cells onto a culture dish, and incubating the cells with RMPI1640 containing 0.1mg/mL PMA overnight to convert the cells from a suspension state to an adherent state; the cells were then incubated with Opti MEM medium containing 0.4mg/mL LPS, transformed into M1 macrophages, and continued with the solution containing 50. mu.g/mL DSPE-PEG2000-Mal incubation with M1 macrophages;
2) taking the supernatant in the step 1) to carry out centrifugation and filtration in sequence, and centrifuging the filtered solution by using an ultrafiltration tube to obtain a crude exosome extract;
3) purifying the crude extract obtained in the step 2) by using an EXOEasy Maxi Kit to obtain a purified exosome;
4) loading therapeutic drugs and surface modification: loading the therapeutic drug into the purified exosomes obtained in the step 3), adding An2 or/and CD133aptamer for incubation, centrifuging the incubated mixed solution at 4 ℃ by using An ultrafiltration tube, adding PBS for centrifugation, and washing the unloaded therapeutic drug and the unmodified An2 or/and CD133aptamer to obtain the engineered exosomes.
3. The method for preparing engineered exosome according to claim 2, wherein the supernatant in step 2) is filtered by using 0.22 μm filter membrane, and the filtrate is centrifuged by using 100kDa ultrafiltration tube.
4. The method for preparing engineered exosomes according to claim 2, wherein the specific steps of purifying the crude extract by using the exoasy Maxi Kit in the step 3) are as follows: mixing the crude extract and XBPBbuffer, adding the mixture into a column, placing the column containing the crude extract and XBP buffer into a collection tube, centrifuging, removing the filtrate, adding XWPbuffer into the column, centrifuging, removing the filtrate, placing the column into a new collection tube, adding XE buffer, incubating at room temperature, centrifuging, taking out the filtrate, adding the filtrate into the column, incubating, and centrifuging to obtain the purified exosome.
5. A method of producing an engineered exosome according to claim 1 or 2, in which the therapeutic drug comprises a nucleic acid drug or a small molecule drug; the nucleic acid drug is siRNA, and the small molecule drug is temozolomide or O6-benzylguanine.
6. A method of preparation of engineered exosomes according to claim 1 or 2, characterised in that therapeutic drug loading employs ultrasound, incubation or freeze-thawing.
7. An engineered exosome obtained by the preparation method according to claim 1 or 2, the exosome morphology being disc-shaped, comprising a CD63 exosome characteristic protein.
8. Use of the engineered exosome according to claim 7 in the treatment of glioma.
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