CN117431210A - Extraction method and application for promoting NK cell secretion exosome - Google Patents
Extraction method and application for promoting NK cell secretion exosome Download PDFInfo
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- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
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- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
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- C12N2501/15—Transforming growth factor beta (TGF-β)
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- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
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- C12N2523/00—Culture process characterised by temperature
Abstract
The invention discloses an extraction method for promoting NK cell secretion exosomes and application thereof; the extraction method comprises the following steps: on day 0, NK cells are inoculated in a culture flask for static culture; on days 3 to 19, the cell culture liquid is replaced every few days, and the poured culture waste liquid is collected each time and centrifuged, and the exosomes obtained by each centrifugation are collected independently; on day 20, the culture was stopped, the discharged culture waste liquid was collected again and centrifuged, and the exosomes obtained by centrifugation were collected. In the exosome extraction process, NK cells can be stimulated to achieve the pro-inflammatory effect by intermittently simulating the 38 ℃ temperature of the immune inflammatory reaction in the body, so that the secretion of cytokines (such as TNF-alpha, IL-1 beta and IL-6) is improved in a short period of time by the NK cells, the time for adapting to a fresh culture medium is shortened, and the high-concentration level output of the exosome is maintained.
Description
Technical Field
The invention relates to the field of biological cell preparation, in particular to an extraction method and application for promoting NK cell secretion exosomes.
Background
Targeted drug delivery has proven to be a critical issue in achieving effective cancer treatment. A significant portion of chemotherapeutic agents are less water soluble and therefore require the use of specialized delivery vehicles (e.g., micelles, liposomes, polymeric nanoparticles, or other types of nanoparticles) for parenteral administration. However, these nanosized delivery vehicles are often difficult to manufacture. In addition, traditional drug nano-formulations are typically cleared rapidly from the circulation by the Mononuclear Phagocyte System (MPS), which requires innovative drug delivery system engineering approaches.
Exosomes are a class of natural nanoparticles secreted endogenously by mammalian cells, recently recognized as excellent carriers for drug and gene delivery. They are spherical vesicles of 30-150nm, present in extracellular body fluids and conditioned cell culture media. Exosomes are released by a variety of cell types; when the intracellular multivesicular body fuses with the plasma membrane and releases the intraluminal vesicles into the extracellular microenvironment. The membranphilicity of exosomes suggests that they will be able to effectively interact with target cancer cells as drug carriers and deliver their payload toxicity. Furthermore, alloexosomes have an immune privileged state because their expressed CD47 receptor interacts with signal-modulating protein α, which allows for reduced drug clearance of MPS. These unique features make exosomes an attractive option for use as drug delivery vehicles for cancer therapy.
There are reports of using macrophage-derived exosomes loaded with low molecular chemotherapeutic drugs for primary lung tumor drugs at present, but the drugs have no function of acting on exosomes for preparing metastatic tumors.
Disclosure of Invention
Based on the above problems, the present invention aims to provide an extraction method for promoting secretion of exosomes by NK cells; the exosomes obtained by extraction can be prepared into exosome preparations and exosome solutions for loading medicaments are prepared by using the exosome preparations; the exosome formulation may be used to prepare a medicament for the prevention, treatment, or treatment of a tumor or cancer, while the drug-loaded exosome solution may be used to prepare a medicament for the treatment, prevention, or treatment of a primary or metastatic tumor.
The invention provides an extraction method for promoting NK cell secretion exosomes, which comprises the following steps:
on day 0, 300ml of cell culture medium was added to the flask and inoculated with 5 x 10 in the flask 7 -NK cells; NK cells were then placed in an environment of 37℃at 5v/v% CO 2 Culturing in a static culture box under the condition;
changing liquid once every 3 days from day 3 to day 19, collecting culture recovery liquid poured out every time of changing liquid, then supplementing 500ml of cell culture liquid every time, shaking uniformly, and standing for culture;
stopping NK cell culture on day 20, and collecting culture recovery liquid poured out of the culture flask;
in NK cell culture from day 3 to day 20, the culture recovery liquid collected each time is centrifuged, and the exosomes obtained each time are stored at-20deg.C for use.
The cell culture solution comprises a DMEM or DMEM/F12 basal medium, wherein IL-2 with a final concentration of 1000-2000 IU/ml, IL-15 with a final concentration of 10-50 ng/ml, SCF with a final concentration of 20-40 ng/ml, TGF with a final concentration of 20-40 ng/ml, PDGF with a final concentration of 5-20 ng/ml and StemRegin 1 with a final concentration of 2-5 mu M are added into the basal medium.
In one embodiment, the basal medium is preferably supplemented with IL-2 at a final concentration of 800 to 1500IU/ml, IL-15 at a final concentration of 20 to 40ng/ml, SCF at a final concentration of 25 to 35ng/ml, TGF at a final concentration of 25 to 35ng/ml, PDGF at a final concentration of 8 to 16ng/ml, and StemRegin 1 at a final concentration of 2.5 to 4. Mu.M.
In one embodiment, in the extraction method, in NK cell culture from day 3 to day 19, the stationary culture further comprises the following treatments:
first, the incubator environment was 38℃and 5v/v% CO 2 Standing and culturing for 6 hours under the condition;
next, the incubator environment was 37℃and 5v/v% CO 2 And continuing to perform stationary culture under the condition.
In one embodiment, in the method for extracting, in the cell culture from day 8 to day 20, when the collected culture recovery liquid is subjected to centrifugation, the method further comprises the following steps:
in the cell culture from day 3 to day 20, the culture recovery liquid collected each time is subjected to a first centrifugation treatment according to 3000g and centrifugation for 30min respectively; after each centrifugation, obtaining a first supernatant;
adding a precipitation solution into the first supernatant obtained each time, shaking uniformly, and standing at 2-8 ℃ for 8 hours; and then, respectively centrifuging according to 13000g and carrying out secondary centrifugation for 1h, and removing the secondary supernatant after the centrifugation is finished to obtain the corresponding exosomes.
In one embodiment, in the preparation method, when the precipitating solution is added to the obtained first supernatant each time, the volume ratio of the first supernatant to the precipitating solution is controlled to be 1-3:1; the precipitating solution is 6-12 w/w% PEG6000 solution; preferably, the volume ratio of the first supernatant to the precipitant is 2:1; the precipitation solution is 8w/w% PEG6000 solution.
The invention also provides a preparation method of the NK cell exosome preparation, which comprises the following steps:
firstly, resuspending NK cell exosomes prepared each time by using physiological saline containing 1v/v% human serum albumin to obtain respective corresponding exosome suspensions; wherein, the volume ratio of the physiological saline to the cell culture solution is 1:50;
next, the exosome suspensions were filtered with 0.22 μm filters, respectively, to obtain NK cell exosome preparations.
The invention also provides a preparation method of the exosome solution loaded with the medicine, which comprises the following steps:
taking the NK cell exosome preparation, and adding DSPE-PEG-AA and alkaloid antitumor drugs to obtain exosome mixed solution; wherein, in the exosome mixed solution, the final concentration of DSPE-PEG-AA is 50 mug/ml and the final concentration of alkaloid anti-tumor drug is 100 mug/ml;
treating the exosome mixed solution by adopting ultrasonic waves with ultrasonic frequency of 32 kHz-450 kHz for 10min in an environment of 4 ℃; after the ultrasonic treatment, incubating for 60min at 37 ℃ to obtain the exosome solution loaded with the drug.
The alkaloid antitumor drug is camptothecine antitumor drug (such as irinotecan, topotecan, etc.) or terpenoid antitumor drug (such as paclitaxel).
Preferably, in one embodiment, for the drug-loaded exosome solution prepared above, further purification treatment is required:
removing excessive free DSPE-PEG by size exclusion chromatography with NAP-10 chromatographic column pre-filled with Sephadex G-25, collecting solution to obtain purified drug-loaded exosome solution, and storing at-20deg.C.
The NK cell exosome preparation prepared by the invention can be applied to preparing medicaments for targeting tumors or cancers, for example, the NK cell exosome preparation can be used for preparing medicaments for targeting lung tumors, lung cancers and the like.
The exosome solution loaded with the medicine prepared by the invention can be applied to preparing the medicine for treating the primary or metastatic tumor, for example, the exosome solution loaded with the medicine can be applied to preparing the medicine for treating the primary or metastatic lung tumor.
Compared with the prior art, the invention has the following advantages:
1. in the extraction process of exosomes, the temperature (38 ℃) of immune inflammatory reaction in vivo is intermittently simulated, NK cells can be stimulated to achieve the pro-inflammatory effect, and secretion of cytokines (such as TNF-alpha, IL-1 beta and FasL) is improved in a short period of time by the NK cells, so that the time for adapting to fresh culture medium is shortened, and the high-concentration level output of exosomes is maintained;
2. the cell culture solution can keep stable proliferation of NK cells, prolong the life cycle of the NK cells, and maintain high cell density to achieve the exosome production of high concentration level;
3. the exosomes secreted by NK cells fused by DSPE-PEG-AA membranes have chemotaxis to metastatic tumor cells, and the exosomes are loaded with tumor therapeutic small molecule drugs, so that the exosomes have better targeting property, the ineffective release effect of the drugs is greatly reduced, and the side effect influence of the drugs on organisms is reduced.
Drawings
FIG. 1 is a graph showing NK cell culture growth curves in examples 1 and 2;
FIG. 2 is a bar graph of secreted cytokine concentration during NK cell incubation in example 2;
FIG. 3 is a CD3-CD56+ flowsheet of NK cell culture day 3 of example 1;
FIG. 4 is a CD3-CD56+ flowsheet of NK cell culture day 3 of example 2;
FIG. 5 is a CD3-CD56+ flowsheet of NK cell culture day 20 of example 1;
FIG. 6 is a CD3-CD56+ flowsheet of NK cell culture day 20 of example 2;
FIG. 7 is a graph showing the measurement of the concentration of granules of exosome preparation on day 20 of NK cell culture in example 1;
FIG. 8 is a graph showing the measurement of the concentration of granules of exosome preparation on day 20 of NK cell culture in example 2;
FIG. 9 is a staining microscope image of a tumor chemotactic section of a mouse cell tissue;
FIG. 10 is a graph showing a survival test of mice.
Detailed Description
The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
According to the extraction process for promoting NK cells to secrete exosomes, cell culture solution is firstly required to be prepared, and then exosomes are obtained.
1. Preparation of cell culture solution
The invention takes DMEM or DMEM/F12 as a basic culture medium, and IL-2 with a final concentration of 1000-2000 IU/ml, IL-15 with a final concentration of 10-50 ng/ml, SCF with a final concentration of 20-40 ng/ml, TGF with a final concentration of 20-40 ng/ml, PDGF with a final concentration of 5-20 ng/ml and StemRegin 1 with a final concentration of 2-5 mu M are added into the basic culture medium.
Preferably, in one embodiment, the cell culture solution comprises a DMEM basal medium, and IL-2 with a final concentration of 800-1500 IU/ml, IL-15 with a final concentration of 20-40 ng/ml, SCF with a final concentration of 25-35 ng/ml, TGF with a final concentration of 25-35 ng/ml, PDGF with a final concentration of 8-16 ng/ml and StemRegenin1 with a final concentration of 2.5-4 mu M are added into the basal medium.
Preferably, in another embodiment, the cell culture solution comprises DMEM basal medium to which IL-2 having a final concentration of 1000IU/ml, IL-15 having a final concentration of 10ng/ml, SCF having a final concentration of 20ng/ml, TGF having a final concentration of 40ng/ml, PDGF having a final concentration of 20ng/ml, and StemRegin 1 having a final concentration of 2. Mu.M are added.
Preferably, in one embodiment, the cell culture medium comprises DMEM/F12 basal medium to which IL-2 having a final concentration of 2000IU/ml, IL-15 having a final concentration of 50ng/ml, SCF having a final concentration of 40ng/ml, TGF having a final concentration of 20ng/ml, PDGF having a final concentration of 5ng/ml and StemRegin 1 having a final concentration of 5. Mu.M are added.
Preferably, in one embodiment, the cell culture medium comprises DMEM/F12 basal medium to which IL-2 having a final concentration of 2000IU/ml, IL-15 having a final concentration of 50ng/ml, SCF having a final concentration of 40ng/ml, TGF having a final concentration of 20ng/ml, PDGF having a final concentration of 5ng/ml and StemRegin 1 having a final concentration of 5. Mu.M are added.
Preferably, in one embodiment, the cell culture broth comprises DMEM/F12 basal medium to which IL-2 at a final concentration of 800IU/ml, IL-15 at a final concentration of 40ng/ml, SCF at a final concentration of 25ng/ml, TGF at a final concentration of 35ng/ml, PDGF at a final concentration of 16ng/ml and StemRegin 1 at a final concentration of 4. Mu.M are added.
Preferably, in one embodiment, the cell culture medium comprises DMEM basal medium to which IL-2 having a final concentration of 1500IU/ml, IL-15 having a final concentration of 20ng/ml, SCF having a final concentration of 35ng/ml, TGF having a final concentration of 25ng/ml, PDGF having a final concentration of 8ng/ml, and StemRegin 1 having a final concentration of 2.5. Mu.M are added.
2. Extraction of NK cell exosomes
The extraction method for promoting NK cell secretion exosomes provided by the invention comprises the following steps:
on day 0, 300ml of the above cell culture solution was added to a flask, and the flask was inoculated with a purity of more than 80% and a quantity of 5×10 7 NK cells cultured for less than 14 days; in an incubator at 37℃with 5v/v% CO 2 Standing and culturing under the condition;
the liquid is changed once every 3 days from day 3 to day 19, the culture recovery liquid poured out from the liquid change in each culture bottle is collected, then 500ml of cell culture liquid is added into each culture bottle, and the cells are cultured by standing after shaking;
stopping cell culture on day 20, and collecting all culture recovery liquid poured out of the culture flask;
in the cell culture from day 3 to day 20, the culture recovery liquid collected each time is centrifuged, and the exosomes obtained each time are stored at-20 ℃ respectively for standby.
Preferably, in the above extraction method of exosome secretion, at the time of cell culture from day 3 to day 19, the stationary culture further comprises the following treatments:
first, the temperature of the incubator environment is adjusted to 38 ℃, and the incubator CO is controlled 2 The volume concentration is 5%, and the culture is carried out for 6 hours;
then, the temperature of the incubator environment was adjusted to 37℃to control the incubator CO 2 The volume concentration is 5%, and the stationary culture is continued.
Preferably, when the culture recovery liquid collected each time is subjected to centrifugal treatment in the cell culture from day 3 to day 20 during the extraction of the NK cell exosomes, the method further comprises the following steps:
in the cell culture from day 3 to day 20, the culture recovery liquid collected each time is subjected to a first centrifugation treatment according to 3000g and centrifugation for 30min respectively; after each centrifugation, obtaining a first supernatant;
adding a precipitating solution with the concentration of 6-12 w/w% PEG6000 into the first supernatant obtained each time, wherein the volume ratio of the first supernatant to the precipitating solution is 1-3:1; shaking uniformly, and standing for 8 hours at the temperature of 2-8 ℃; and then, respectively carrying out secondary centrifugation according to 13000g and centrifugation for 1h, and removing second supernatant obtained by each centrifugation after the centrifugation is finished to obtain corresponding exosomes.
The concentration of the PEG6000 precipitation solution can be 6 w/w%, 7 w/w%, 8w/w%, 9 w/w%, 10 w/w% or 12w/w%; the volume ratio of the first supernatant to the precipitation solution is 1:1, 1.5:1, 2:1 or 3:1; the addition of the precipitation solution may be carried out at any temperature of 2-8deg.C, such as 2deg.C, 3deg.C, 5deg.C, 7deg.C or 8deg.C.
In the invention, the exosomes extracted each time can be respectively and independently used for preparing NK cell exosome preparations, and the exosome preparations can be used as biological preparations and can be applied to preparing medicines for preventing and treating tumors or cancers, such as medicines for preparing medicines for treating lung tumors and lung cancers.
3. Preparation of NK cell exosome preparation
The NK cell exosomes obtained by each extraction are respectively prepared into exosome preparations by the following processes:
firstly, resuspending the extracted exosomes with physiological saline containing 1v/v% human serum albumin to obtain exosome suspension; wherein, the dosage of the physiological saline and the cell culture solution is added and prepared according to the volume ratio of 1:50;
next, the exosome suspension was filtered with a 0.22 μm filter to remove cell debris and/or impurities, thereby obtaining NK cell exosome preparation.
4. Preparation of drug-loaded exosome solutions
In the invention, the NK cell exosome preparation prepared from exosome can be used for preparing a drug-loaded exosome solution, and the drug-loaded exosome solution can be used as a biological agent to prepare a drug for treating and preventing primary or metastatic tumors, such as a drug for preparing primary or metastatic lung tumors.
The preparation process of the exosome solution loaded with the medicine comprises the following steps:
taking the prepared NK cell exosome preparation according to the requirement, and adding DSPE-PEG-AA and alkaloid antitumor drugs to obtain exosome mixed solution; wherein, in the exosome mixed solution, the final concentration of DSPE-PEG-AA is 50 mug/ml and the final concentration of alkaloid anti-tumor drug is 100 mug/ml; preferably, the alkaloid antitumor drug is camptothecine antitumor drug or terpenoid antitumor drug; camptothecins antitumor drugs include irinotecan, topotecan, etc.; terpenes antitumor drugs including paclitaxel;
treating the exosome mixed solution by adopting ultrasonic waves with ultrasonic frequency of 32 kHz-450 kHz for 10min in an environment of 4 ℃; after the ultrasonic treatment, incubating for 60min at 37 ℃ to obtain the exosome solution loaded with the drug.
Preferably, the prepared drug-loaded exosome solution is also an initial solution, and excessive DSPE-PEG-AA components are required to be removed, so that the effect of purifying the drug-loaded exosome solution is achieved. Therefore, the purification process of the prepared drug-loaded exosome solution is as follows:
removing excessive free DSPE-PEG-AA by size exclusion chromatography with NAP-10 chromatographic column pre-filled with Sephadex G-25, collecting solution to obtain purified drug-loaded exosome solution, and storing at-20deg.C.
In the preparation process of the drug-loaded exosome solution, DSPE-PEG is a phospholipid-polymer conjugate, and a ligand AA can be accessed to the tail end of the phospholipid-polymer conjugate, so that the ligand AA can target a sigma receptor through high affinity, and the effect of targeting tumor cells is achieved. After being mixed with NK cell exosome preparation, DSPE-PEG-AA is integrated onto the surface of exosome membrane by ultrasonic treatment, and the medicine molecule can permeate into exosome and restore exosome membrane through incubation at 37 deg.c to reach the effect of alkaloid encapsulation inside exosome and prevent DSPE-PEG-AA from permeating out of exosome membrane.
5. Detailed description of the preferred embodiments
The following is a detailed description of several specific embodiments.
Extraction of NK cell exosomes, exosome preparation and preparation of drug-loaded exosome solution
Example 1
1. Acquisition of exosomes
1.1 preparing 5000ml of cell culture solution
A DMEM-based medium was added with IL-2 at a final concentration of 1500IU/ml, IL-15 at a final concentration of 30ng/ml, SCF at a final concentration of 34ng/ml, TGF at a final concentration of 30ng/ml, PDGF at a final concentration of 14ng/ml and StemRegin 1 at a final concentration of 3. Mu.M.
1.2 extraction of NK cell exosomes
On day 0, a 1000ml standard flask was taken, 300ml of cell culture medium was added, and 5×10 cells were inoculated into the flask 7 NK cells cultured for less than 14 days at a purity of greater than 80%; in an incubator at 37℃with 5v/v% CO 2 Standing and culturing for 3 days under the condition;
changing liquid once every 3 days from day 3 to day 19, collecting culture recovery liquid poured out from the liquid change in the culture bottle when changing liquid every time, and then adding 500ml of culture medium into the culture bottle; after shaking NK cells, the cells were first homogenized at 38℃with 5v/v% CO 2 Stationary culturing at 37deg.C for 1 day, followed by 5v/v% CO 2 Continuously standing and culturing for 2 days under the condition; during this period, the culture recovery liquid collected each time is also subjected to the following treatment:
carrying out first centrifugation treatment on the culture recovery liquid collected each time according to 3000g and centrifugation for 30 min; after centrifugation, obtaining a first supernatant;
adding 8w/w% PEG6000 solution into the first supernatant obtained each time according to the volume ratio of the first supernatant to the precipitating solution of 2:1, shaking uniformly, and standing at 5 ℃ for 8 hours; then, respectively carrying out secondary centrifugation according to 13000g and centrifugation for 1h, removing second supernatant obtained by each centrifugation after centrifugation, obtaining corresponding exosomes, and respectively storing at-20 ℃ for later use;
stopping NK cell culture on day 20, collecting the culture recovery liquid completely poured out of the culture flask again, and centrifuging the culture recovery liquid, wherein the operation flow is the same as that of the culture recovery liquid in the period from day 3 to day 19, and the exosomes obtained after centrifugation are also stored at-20 ℃ for standby.
2. Preparation of NK cell exosome preparation
100ml of NK cell exosomes prepared in this example were resuspended in 2ml of physiological saline containing 1% human serum albumin to give an exosome suspension;
filtering the exosome suspension with 0.22 μm filter membrane to remove cell debris and/or impurities to obtain purified NK cell exosome preparation, and collecting for use.
3. Preparation of drug-loaded exosome solutions
Adding DSPE-PEG-AA and irinotecan into the NK cell exosome preparation prepared in the embodiment, and enabling the final concentration of DSPE-PEG-AA to be 50 mug/ml and the final concentration of irinotecan to be 100 mug/ml to obtain exosome mixed solution;
treating the exosome mixed solution by adopting ultrasonic waves with the ultrasonic frequency of 200kHz for 10min in the environment of 4 ℃; after the ultrasonic treatment is finished, incubating for 60min at 37 ℃ to obtain an exosome solution loaded with the drug;
removing excessive free DSPE-PEG-AA by size exclusion chromatography with NAP-10 chromatographic column pre-filled with Sephadex G-25, collecting solution, and storing at-20deg.C.
Example 2
1. Acquisition of exosomes
1.1 preparing 5000ml of cell culture solution
The DMEM/F12 basal medium was used, and IL-2 at a final concentration of 1000IU/ml, IL-15 at a final concentration of 50ng/ml, SCF at a final concentration of 20ng/ml, TGF at a final concentration of 40ng/ml, PDGF at a final concentration of 5ng/ml, and StemRegin 1 at a final concentration of 2. Mu.M were added to the basal medium.
1.2 extraction of NK cell exosomes
On day 0, a 1000ml standard flask was taken, 300ml of cell culture medium was added, and 5×10 cells were inoculated into the flask 7 NK cells cultured for less than 14 days at a purity of greater than 80%; in an incubator at 37℃with 5v/v% CO 2 Standing and culturing for 3 days under the condition;
the liquid is changed every 3 days from day 3 to day 19, the culture recovery liquid poured out from the liquid change in the culture bottle is collected when the liquid is changed every 3 days, 500ml of culture medium is added into the culture bottle, and after shaking up cells, the culture recovery liquid is added into the culture bottle at 37 ℃ and 5v/v percent CO 2 Continuously standing and culturing under the condition; during this period, the culture recovery liquid collected each time is also subjected to the following treatment:
carrying out first centrifugation treatment on the culture recovery liquid collected each time according to 3000g and centrifugation for 30 min; after centrifugation, obtaining a first supernatant;
adding 12w/w% PEG6000 solution into the first supernatant obtained each time according to the volume ratio of the first supernatant to the precipitating solution of 3:1, shaking uniformly, and standing at 5 ℃ for 5h; then, performing secondary centrifugation according to 13000g and centrifugation for 1h, removing second supernatant obtained by centrifugation after centrifugation, obtaining corresponding exosomes, and respectively storing at-20 ℃ for later use;
on day 20, stopping cell culture, collecting the culture recovery liquid completely poured out of the culture flask again, and centrifuging the culture recovery liquid, wherein the operation flow is the same as that of the culture recovery liquid in the period from day 3 to day 19, and the exosomes obtained after centrifugation are also stored at-20 ℃ for standby.
2. Preparation of NK cell exosome preparation
150ml of NK cell exosomes prepared in this example were resuspended in 3ml of physiological saline containing 1% human serum albumin to give an exosome suspension;
filtering the exosome suspension with 0.22 μm filter membrane to remove cell debris and/or impurities to obtain purified NK cell exosome preparation, and collecting for use.
3. Preparation of drug-loaded exosome solution
Adding DSPE-PEG-AA and topotecan into the NK cell exosome preparation prepared in the embodiment, and enabling the final concentration of DSPE-PEG-AA to be 50 mug/ml and the final concentration of topotecan to be 100 mug/ml to obtain exosome mixed solution;
treating the exosome mixed solution by adopting ultrasonic waves with the ultrasonic frequency of 450kHz for 10min in the environment of 4 ℃; after the ultrasonic treatment is finished, incubating for 60min at 37 ℃ to obtain an exosome solution loaded with the drug;
removing excessive free DSPE-PEG-AA by size exclusion chromatography with NAP-10 chromatographic column pre-filled with Sephadex G-25, collecting solution, and storing at-20deg.C.
(II), detection and analysis
1. NK cell culture expansion assay
NK cells in examples 1 and 2 were cultured until day 20, and NK cell expansion growth state was shown in FIG. 1 and Table 1. Table 1 shows the specific data corresponding to fig. 1, which are identical.
TABLE 1 NK cell expansion number
As can be seen from FIG. 1 and Table 1, NK cells were stably and rapidly proliferated during the period from day 3 to day 20 (actual culture time: more than 30 days), indicating that NK cells were high in physiological activity and metabolic secretion level, and the amount of exosomes secreted by NK cells was relatively high at this stage.
2. Cytokine secretion concentration assay
NK cells of example 2 were selected and incubated at 37℃and 38℃respectively, and the results of the assay for the secretion concentration of cytokines such as TNF-. Alpha., IL-1β, fasL were shown in Table 2 and FIG. 2. Table 2 shows the specific data corresponding to fig. 2, which are identical.
TABLE 2 cytokine secretion concentration assay results table
As can be seen from table 2 and fig. 2, in the process of extracting NK cell exosomes, NK cells can be stimulated to achieve pro-inflammatory effect by intermittently simulating the temperature of in vivo immune inflammatory reaction (38 ℃), so that NK cells can increase secretion of cytokines (such as TNF- α, IL-1β, fasL) in a short period of time, so as to shorten the time for adapting to fresh culture medium and achieve the purpose of maintaining the output of exosomes at high concentration level.
3. Assay for NK cell stability
In examples 1 and 2, NK cells were examined for CD3-CD56+ phenotype on days 3 and 20, respectively, using cell flow assays.
The detection results are shown in fig. 3, 4, 5 and 6; wherein FIG. 3 is a CD3-CD56+ flowgram of NK cell culture day 3 of example 1; FIG. 4 is a CD3-CD56+ flowsheet of NK cell culture day 3 of example 2; FIG. 5 is a CD3-CD56+ flowsheet of NK cell culture day 20 of example 1; FIG. 6 is a CD3-CD56+ flow chart of NK cell culture day 20 of example 2.
In FIGS. 3, 4, 5 and 6, the results of the flow assay for CD3-CD56+ phenotypes were 84.58%, 87.35%, 86.96%, 88.25%, respectively; this indicates that NK cells are relatively stable in the culture process, cells are normal in growth and metabolism in the proliferation process, the quantity of secreted exosomes is large, and the activity of exosomes is strong.
4. Concentration detection and analysis of NK cell exosome system granule
In examples 1 and 2, each of which was sampled at day 20, the concentration of NK cell exosome preparations was measured, and the measurement results are shown in Table 3, FIG. 7 and FIG. 8, wherein the data in Table 3 corresponds to the corresponding data in FIGS. 7 and 8, respectively.
TABLE 3 NK extracellular body concentration detection results Table
Exosome preparation concentration was measured using a nanoparticle tracking analyzer (manufacturer: PARTICLE METRIX, germany; model: zeta View PMX-120), and the measurement results are shown in Table 1, FIGS. 6 and 7; in fig. 7 and 8, the corresponding peak diagram is the detection result of the sample in the sample pool, and is derived by the instrument and smoothed; the peak plot corresponding area is expressed as the exosome formulation particle concentration.
The detection parameters of the Zeta View nanoparticle tracking analyzer are as follows:
1) Sample parameters: pH value: 7.0; sample dilution: PBS, wherein: sample temperatures were respectively: example 1 was 27.91 ℃; example 2 was 28.12 ℃;
2) Instrument parameters: laser wavelength: 488nm; filtering wavelength: scattering.
As can be seen from Table 3, FIG. 7 and FIG. 8, the exosome preparation in example 1 had a particle concentration of 8.3X10 11 Particles/mL, exosome Particles concentration 8.6X10 in example 2 11 Particles/mL. This suggests that NK cell secreting exosomes can be produced with a certain particle size, e.g. 115.6nm and 124Exosome preparation at 7 nm.
5. Drug loaded exosome solution targeting assay
As shown in FIG. 9, a staining micrograph of a tumor chemotactic slice taken as an example of example 2 was taken, and examined by a confocal microscope of a lung tissue slice after administration of a mouse (region B and region D respectively represent 3LL-M27 lung tumor cells; region A and region C respectively represent exosomes); the sections were photographed using a fluorescence microscope at 40 x magnification. In fig. 9, letters a to h represent the respective image numbers; wherein:
a-represents a fluorescence microscope image of an exosome in a mouse lung section tissue after administration of an NK cell exosome preparation obtained in example 2 to a model mouse, and a region A is marked;
b-fluorescence microscopy images of 3LL-M27 tumor cells in mouse lung section tissues after administration of NK cell exosome preparation obtained in example 2, labeled region B;
c-represents a fluorescence microscope confocal image of the NK cell exosome preparation obtained in example 2 together with 3LL-M27 tumor cells in the lung section tissue of mice after administration to mice; the image shows that the NK cell exosome preparation and the lung tumor cells are in non-obvious co-localization, and the A+B region is marked;
d-represents fluorescence microscopy images of the drug-loaded exosome solution obtained in example 2 on lung slice tissues of mice after administration to model mice, and identifies region C;
e-represents fluorescence microscopy images of 3LL-M27 tumor cells in mouse lung slice tissues after drug-loaded exosome solution model mice obtained in example 2 were dosed, identifying the D region;
f-represents a fluorescence microscope confocal image of the drug-loaded exosome solution obtained in example 2 together with 3LL-M27 tumor cells in mouse lung slice tissue after administration to a model mouse; the image shows the obvious co-localization of the exosome solution loaded with the drug and the lung tumor cells, and the C+D region is marked;
g-represents that no exosomes were found in the lung section tissue fluorescence microscopy images of mice after administration of NK cell exosome preparations obtained in example 2 to model healthy mice;
h-represents fluorescence microscopy images of lung section tissue of mice after administration of the drug-loaded exosome solution model healthy mice obtained in example 2, no exosomes were found.
Co-localization of exosomes delivered systemically in mice with lung cancer metastasis, NK cell exosomes obtained from exosome extraction process are labeled with DiL dye, e.g., labeled with region a, labeled with region C; 3LL-M27 cells transduced with lentiviral vectors encoding optical reporter GFP fluorescent proteins (e.g., marker B region, marker D region) for intravenous injection into mice; after 4 hours, mice were euthanized, perfused, lung sectioned and stained with DAPI (blue).
Mice were dosed: LLC (Lewis lung carcinoma cells) mouse model construction was performed with 3LL-M27 cells shown by fluorescent proteins (GFP) overexpressing the marker B and D regions (injection 5X 10) 6 Individual cells/100 μl GFP/3 LL-M27), and 21 days later, diL (one of the lipophilic cell membrane dyes) labeled drug-loaded exosome solution was intravenously injected into tumor-bearing mice. After 4 hours, mice were sacrificed and perfused; the lungs were sectioned on a microtome and examined by confocal microscopy.
In fig. 9, the aposome preparation with 20% corresponding image of the identifier a, b, c, g is co-located with lung tumor cells, which indicates that the aposome has targeting effect on lung cancer tumor cells in vivo, but the targeting effect is not strong; the corresponding image of the identifier d, e, f, h is that more than 90% of the exosomes loaded with the drugs are co-located with lung tumor cells, so that the exosomes loaded with the drugs have stronger targeting effect on lung cancer tumor cells in vivo.
6. Mice survival test
Taking example 2 as an example, mice were administered with NK cell exosomes, drug-loaded NK cell exosome solutions and paclitaxel bioalkali solutions, respectively, and their life-time survival was analyzed.
Mice were dosed: 3LL-M27 cells LLC (Lewis lung carcinoma cells) mouse model construction (5X 10 injection) 6 Individual cells/100 μl of 3 LL-M27), and after 2 days, mice were continuously observed for survival after the following operations, respectively.
Experiment group 1: the drug-loaded NK cell exosome solution prepared in example 2 was intravenously injected into tumor-bearing mice (injection 5X 10) 10 100. Mu.L of exosomes);
experiment group 2: the NK cell exosome preparation prepared in example 2 was intravenously injected into tumor-bearing mice (5X 1010/100. Mu.L of exosomes);
experiment group 3: the paclitaxel alkaloid solution was administered alone to tumor-bearing mice intravenously (100. Mu.L of paclitaxel injection, at 10 mg/kg).
As shown in fig. 10, in experimental group 1, when the NK cell exosome preparation was injected into mice with the drug-loaded exosome solution, the survival time of the mice could be greatly prolonged; that is, the NK cell exosome preparation prepared by the method is prepared into an exosome solution loaded with medicines, and has good tumor killing performance.
It is to be understood that the foregoing description of the preferred embodiments of the invention is not to be considered as limiting the scope of the invention, which is defined by the appended claims.
Claims (10)
1. An extraction method for promoting NK cell secretion exosomes is characterized by comprising the following steps:
on day 0, 300ml of cell culture medium was added to the flask and inoculated with 5×10 7 -NK cells; NK cells were then placed in an environment of 37℃at 5v/v% CO 2 Culturing in a static culture box under the condition; the cell culture solution comprises a basal medium, wherein IL-2 with a final concentration of 1000-2000 IU/ml, IL-15 with a final concentration of 10-50 ng/ml, SCF with a final concentration of 20-40 ng/ml, TGF with a final concentration of 20-40 ng/ml, PDGF with a final concentration of 5-20 ng/ml, stemRegenin1 with a final concentration of 2-5 mu M and an Ultroser G serum substitute with a final concentration of 0.1-3% are added into the basal medium;
the liquid is changed once every 3 days from the 3 rd day to the 19 th day,collecting culture recovery liquid poured out after each liquid exchange, then adding 500ml of cell culture liquid each time, shaking uniformly, and standing for culture; in NK cell culture from day 3 to day 19, the culture was performed first in an incubator at 38℃and 5v/v% CO 2 Stationary culturing at 37deg.C under 5v/v% CO for 6 hr 2 Continuously standing and culturing under the condition;
stopping NK cell culture on day 20, and collecting culture recovery liquid poured out of the culture flask;
in NK cell culture from day 3 to day 20, the culture recovery liquid collected each time is centrifuged, and after centrifugation is stopped each time, the obtained exosomes are stored at-20deg.C for use.
2. The method according to claim 1, wherein the basal medium included in the cell culture liquid is a DMEM basal medium, and IL-2 having a final concentration of 1500IU/ml, IL-15 having a final concentration of 30ng/ml, SCF having a final concentration of 34ng/ml, TGF having a final concentration of 30ng/ml, PDGF having a final concentration of 14ng/ml, and StemRegin 1 having a final concentration of 3. Mu.M are added to the basal medium; or alternatively
The basal medium included in the cell culture solution is DMEM/F12 basal medium, and IL-2 with a final concentration of 1000IU/ml, IL-15 with a final concentration of 50ng/ml, SCF with a final concentration of 20ng/ml, TGF with a final concentration of 40ng/ml, PDGF with a final concentration of 5ng/ml and StemRegin 1 with a final concentration of 2 mu M are added into the basal medium.
3. The method according to claim 1, wherein in the NK cell culture on the 3 rd to 20 th days, each time the collected culture recovery liquid is subjected to the centrifugation treatment, the method further comprises the following treatment:
in the cell culture from day 3 to day 20, the culture recovery liquid collected each time is subjected to a first centrifugation treatment according to 3000g and centrifugation for 30min respectively; after each centrifugation, obtaining a first supernatant;
adding a precipitation solution into the first supernatant obtained each time, shaking uniformly, and standing at 2-8 ℃ for 8 hours; and then, respectively carrying out secondary centrifugation according to 13000g and centrifugation for 1h, and removing second supernatant obtained by centrifugation after the centrifugation is finished to obtain corresponding exosomes.
4. The extraction method according to claim 3, wherein the volume ratio of the first supernatant to the precipitating solution is controlled to be 1-3:1 each time the precipitating solution is added to the first supernatant; and the precipitating solution is 6-12 w/w% PEG6000 solution.
5. The extraction method of claim 4, wherein the volume ratio of the first supernatant to the precipitant is 2:1; the precipitation solution is 8w/w% PEG6000 solution.
6. A method for preparing an NK cell exosome preparation, comprising the steps of:
firstly, respectively resuspending NK cell exosomes obtained by the extraction method of any one of claims 1 to 5 with physiological saline containing 1v/v% human serum albumin to obtain respective corresponding exosome suspensions;
next, each of the exosome suspensions was filtered with a 0.22 μm filter membrane, respectively, to obtain respective corresponding NK cell exosome preparations.
7. A method for preparing a drug-loaded exosome solution, comprising the steps of:
taking the NK cell exosome preparation obtained by the preparation method of claim 6, adding DSPE-PEG-AA and alkaloid anti-tumor drugs to obtain exosome mixed solution;
ultrasonic processing the exosome mixed solution for 10min at the ultrasonic frequency of 32 kHz-450 kHz in the environment of 4 ℃; after the ultrasonic treatment, incubating for 60min at 37 ℃ to obtain the exosome solution loaded with the drug.
8. The method of claim 7, wherein the prepared drug-loaded exosome solution is further purified:
removing excessive free DSPE-PEG-AA by using NAP-10 chromatographic column of pre-filled Sephadex G-25 through size exclusion chromatography, collecting solution, and storing at-20deg.C.
9. The use of the NK cell exosome preparation prepared by the preparation method of claim 6 in the preparation of a medicament for preventing and treating lung tumor or lung cancer.
10. Use of the drug-loaded exosome solution prepared by the preparation method of claim 7 or 8 in the preparation of a medicament for preventing and treating primary or metastatic lung tumor.
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