A method for preparing and applying a reovirus complex encapsulated in exosomes derived from mesenchymal stem cells.

Abstract

This invention relates to the field of biomedical technology, specifically a method for preparing and applying a reovirus complex encapsulated in mesenchymal stem cell-derived exosomes. This application uses mesenchymal stem cell-derived exosomes (MSCs). EXO The oncolytic virus carried by the delivery vehicle is reovirus (REO), and MSCs are prepared by extrusion. EXO REO-encapsulated complex (MSC) EXO -REO), confirming MSC EXO It can protect REO from neutralizing antibodies and deliver it to tumor cells to exert an oncolytic effect, and the MSCs prepared in this application... EXO -REO can increase the sensitivity of tumor cells to the killing effect of REO.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a preparation method and application of a mesenchymal stem cell-derived exosome loaded reovirus complex. BACKGROUND

[0002] Oncolytic virus (OVs) has become a new strategy for tumor treatment. Whether it is naturally occurring or genetically engineered OVs, it can specifically infect and kill tumor cells without damaging normal cells. Among the many OVs, reovirus (REO) is special. REO is a naturally occurring non-pathogenic double-stranded RNA (dsRNA) virus that can selectively proliferate in tumor cells and has been shown to have a significant killing effect on tumor cells of various origins. As a leader in the field of OVs research and application, REO has been used as an anti-tumor agent in more than 30 clinical trials worldwide, including a phase III clinical trial for head and neck squamous cell carcinoma. Although REO can be administered intratumorally or intravenously, intratumoral injection is not suitable for hematological tumors, and intravenous administration limits the anti-tumor effect of REO due to neutralizing antibodies in the body.

[0003] Therefore, it is an urgent problem to find a strategy that can enhance the tumor targeting of REO in vivo and help REO escape the blocking effect of neutralizing antibodies on the virus. SUMMARY

[0004] In order to solve the above technical problems existing at present, the present application provides a mesenchymal stem cell-derived exosome loaded reovirus complex and a preparation method and application thereof.

[0005] The mesenchymal stem cell-derived exosome loaded reovirus complex is loaded with reovirus by mesenchymal stem cell exosomes.

[0006] Further, the loading is performed by extrusion.

[0007] The preparation method of the mesenchymal stem cell-derived exosome loaded reovirus complex is prepared by extrusion.

[0008] Further, the extrusion method comprises the following steps:

[0009] The mesenchymal stem cell-derived exosomes and the reovirus are mixed uniformly, and the mixed solution is extruded by an extruder until the loading is completed.

[0010] Further, the extrusion method has the following specific steps: mesenchymal stem cell-derived exosomes and reovirus are mixed in a centrifuge tube at a volume ratio of 1:1, and a pipette is used to fully mix and blow; the extruder (Mini-Extruder set, Avanti, USA) is assembled as required and equipped with a 100 nm membrane, and then a syringe is used to suck a suitable amount of PBS to check for leaks; the syringe is used to suck the mixture of exosomes and virus, and the mixture is extruded back and forth in the extrusion device for 30-50 cycles until the encapsulation is completed.

[0011] Further, the mesenchymal stem cell-derived exosomes are prepared by the following method:

[0012] When the umbilical cord mesenchymal stem cells grow to 60% confluence, they are cultured in a medium containing no exosome serum, and after 48 hours, the supernatant is collected in a centrifuge tube, centrifuged at 300g for 10 minutes, and then the supernatant is centrifuged at 2000g for 20 minutes, followed by 10000g for 30 minutes and filtered through a 0.22μm filter to remove larger size vesicles. Then the supernatant is centrifuged at 100000g for 70 minutes using a high-speed centrifuge, carefully discarded, and then washed once with PBS, followed by 100000g centrifugation for 70 minutes again. All the above centrifugation steps are carried out at 4℃. Finally, the exosome particles are resuspended with PBS and stored at -80℃.

[0013] The mesenchymal stem cell-derived exosome-encapsulated reovirus complex has the application of being used as / preparing a tumor drug.

[0014] Further, the tumor is a solid tumor including colorectal cancer, pancreatic cancer, liver cancer, lung cancer, etc., and a hematological tumor, especially a tumor that is not sensitive to the oncolytic effect of reovirus.

[0015] A pharmaceutical preparation containing the mesenchymal stem cell-derived exosome-encapsulated reovirus complex.

[0016] Further, the pharmaceutical preparation is an antitumor pharmaceutical preparation.

[0017] Since intratumoral injection of reovirus (REO) is not suitable for hematological tumors, and intravenous administration limits the antitumor effect of REO due to factors such as neutralizing antibodies in vivo. The present application uses a delivery carrier to target the delivery of REO to the tumor site, which not only overcomes the hindering effect of neutralizing antibodies in vivo, but also helps REO to be delivered to the tumor site. Currently, the types of carriers used for virus delivery mainly include cell carriers (stem cells, immune cells or inactivated tumor cells), liposomes and exosomes, etc.

[0018] Mesenchymal stem cells (MSCs) are multipotent stromal cells capable of self-renewal and differentiating into multiple cell types. Studies have shown that MSCs can be used as a cell carrier for OVs, and this strategy may be an effective method to improve the efficacy of OVs. Because MSCs have tumor tropism, MSCs loaded with OVs can help deliver viruses to tumor cells, and in addition, MSCs have low immunogenicity, which can protect OVs from neutralizing antibodies and enhance oncolysis.

[0019] Exosomes (EXOs) are nanoscale extracellular vesicles secreted by almost all cell types, have a lipid bilayer structure, and can store nucleic acids, proteins, enzymes and metabolites, etc. They can transfer functional substances to adjacent or distant cells and play a key role in intercellular communication. The biological functions of mesenchymal stem cell-derived exosomes (MSC EXO ) are similar to those of MSCs, and MSC EXO also have the advantages of small size, long circulation half-life, the ability to cross the blood-brain barrier, and ideal biocompatibility, making them ideal drug delivery carriers.

[0020] The present application uses MSC EXO as a delivery tool for REO, compares three different methods for loading REO into MSC EXO , and finally selects the extrusion method to prepare MSC EXO- -REO. EXO The MSC EXO -REO prepared by the extrusion method can maximize the protection of REO from neutralizing antibodies and deliver it to tumor cells to exert oncolytic effects, and the MSC EXO -REO prepared by the present application can improve the sensitivity of tumor cells to REO killing.

[0021] Compared with the prior art, the technical effects created by the present application are embodied in:

[0022] (1) The present application selects MSC EXO as a delivery tool, and MSCs cells are an ideal source for exosome preparation, which can be obtained from various human tissues and can be expanded and prepared into exosomes under GMP conditions.

[0023] (2) The present application compares three different methods for loading REO into MSC EXO , and finally selects the extrusion method to prepare MSC EXO- -REO. EXO ​

[0024] (3) MSC prepared by the application through extrusion method EXO - REO, which has no obvious toxicity to normal cells and can protect REO from the hindering of neutralizing antibodies to deliver REO to tumor cells to exert killing effect.

[0025] (4) MSC prepared by the application EXO - REO can improve the sensitivity of tumor cells to the killing effect of REO, and can significantly increase the killing effect of MSC EXO - REO on tumor cells not sensitive to REO. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a characterization diagram of MSC EXO .

[0027] Figure 2 is a schematic diagram of MSC EXO loading REO.

[0028] Figure 3 is a comparison result diagram of MSC EXO - REO prepared by different methods.

[0029] Figure 4 is a particle size comparison result diagram of MSC EXO , REO and MSC EXO - REO.

[0030] Figure 5 is a comparison diagram of the in vitro killing effect of MSC EXO - REO on tumor cells in the presence of neutralizing antibodies.

[0031] Figure 6 is a comparison diagram of the in vitro effect of MSC EXO - REO on HL60 cells not sensitive to REO and normal peripheral blood mononuclear cells. DETAILED DESCRIPTION

[0032] The technical solutions of the application will be further limited in combination with specific embodiments, but the scope of protection is not limited to the description.

[0033] Example 1: Isolation and identification of MSC EXO

[0034] 1. MSC is extracted by super-speed centrifugation EXO

[0035] ​When the umbilical cord mesenchymal stem cells grow to 60% confluence, replace the culture medium containing exosome-free serum, collect the supernatant into a centrifuge tube after 48 h, centrifuge at 300 g for 10 min, then centrifuge at 2000 g for 20 min, then centrifuge at 10000 g for 30 min and pass through a 0.22 μm filter to remove larger size vesicles. Then use the ultracentrifuge to centrifuge the supernatant at 100000 g for 70 min, carefully discard the supernatant, wash once with PBS, then centrifuge at 100000 g for 70 min again, all the above centrifugation steps are carried out at 4°C. Finally, resuspend the exosome particles with PBS and store at -80°C.

[0036] 2. MSC EXO Identification

[0037] (1) Western blot

[0038] Add protein samples to 5x Loading Buffer, denature in a metal bath, then separate by electrophoresis in a 10% polyacrylamide gel under constant voltage, transfer the proteins to a PVDF membrane by wet transfer under constant current, block with 5% skim milk for 2 h, then wash the membrane with TBST solution 3 times for 10 min each time, after washing, incubate with the corresponding specific primary antibody at 4°C overnight. The next day, wash the membrane with TBST 3 times, then add the appropriate amount of diluted secondary antibody and incubate at room temperature for 1-2 h, after incubation, wash the membrane with TBST 3 times, use ECL ultra-sensitive chemiluminescence reagent to expose and image under a chemiluminescence imaging system and record.

[0039] (2) Detection of exosome particle size

[0040] Use ZetaView instrument to detect exosomes, which tracks and analyzes the Brownian motion of each particle, calculates the hydrodynamic diameter and concentration of nanoparticles using equations. First, calibrate the instrument with standard samples, then dilute the extracted exosomes with PBS to the appropriate concentration for loading, detect the exosome particle size and concentration according to the principle, and generate a report.

[0041] (3) Transmission electron microscopy to identify exosome morphology

[0042] Take 20 μL of exosome sample and drop it on a copper grid, let it stand for 5 min, use filter paper to absorb the excess liquid along the edge, use 1% phosphotungstic acid to stain at room temperature for 1-2 min, use filter paper to absorb the residual stain. After drying, use JEM-1400 FLASH transmission electron microscope to observe and record the imaging.

[0043] (4) Exosome uptake experiment

[0044] PKH67-labeled MSC EXO: Exosome suspension was added to PKH67 dye solution for labeling, then 0.5% BSA solution was added to terminate staining, and then 120000g centrifugation was performed at 4°C for 2h to remove free dye; after centrifugation, the supernatant was discarded, and the cells were resuspended in PBS and mixed thoroughly. THP-1 cells were inoculated into a 24-well plate, and the PKH67-labeled MSC EXO were co-incubated with cells, and PBS labeled with PKH67 was used as a control. After incubation, the cells were stained with DAPI, and the uptake of exosomes by THP-1 cells was observed under a fluorescence microscope.

[0045] Example 2: Different MSC EXO Comparison of REO preparation methods

[0046] (1) Direct mixing method: Equal volumes of MSC EXO and REO were mixed in a plastic centrifuge tube, and a pipette was used to mix thoroughly, and then the mixture was placed on ice for 30min.

[0047] (2) Ultrasonic incubation method: Equal volumes of MSC EXO and REO were mixed and then ultrasonicated in an ice bath. The ultrasonic conditions were as follows: 20% amplitude, 3s on / 3s off, 1min for one cycle, 6 cycles, and 2min interval between each cycle. After ultrasonication, the mixture was allowed to stand for 30min.

[0048] (3) Extrusion method: Equal volumes of MSC EXO and REO were mixed in a plastic centrifuge tube, and a pipette was used to mix thoroughly. The extruder was assembled as required and equipped with a 100nm membrane, and a syringe was used to suck an appropriate amount of PBS for leak detection. Then, the mixture of exosomes and virus was sucked into the syringe and extruded back and forth for 30-50 cycles. After extrusion, the mixture was allowed to stand for 30min, and then it was stored at -80°C.

[0049] Example 3: In vitro killing effect of MSC EXO -REO

[0050] 1. MSC EXO -REO on tumor cells

[0051] (1) MSC EXO -REO in the presence of neutralizing antibodies on THP-1 cells:

[0052] THP-1 cells were resuspended in 1640 medium containing 5% AB serum / FBS, and 10 4 cells / well were inoculated into a 96-well plate, and then REO / MSC EXO -REO were added to the experimental group at an MOI of 1, and PBS / MSC EXOThe 96-well plates were placed in a cell incubator for continuous culture, and after 48 h, the culture plates were taken out and CCK8 reagent was added to each well. After incubation in the incubator for 2 h, the OD value of each well was measured at 450 nm wavelength using an enzyme marker, and the killing rate was calculated according to the formula.

[0053]

[0054] (2) MSC EXO -REO killing effect on HL-60 cells and PBMC:

[0055] To evaluate the MSC EXO -REO killing effect on tumor cells and normal cells in vitro, the present application respectively detected the MSC EXO -REO killing effect on human acute promyelocytic leukemia cell line HL-60 cells (not sensitive to REO) and peripheral blood mononuclear cells (normal cells) in vitro. Well-grown HL-60 / PBMC cells were inoculated into 96-well plates at 10 4 cells / well, and then REO / MSC EXO was added to the experimental group at an infection multiplicity of MOI = 1, while PBS / MSC EXO was set as a control group, and a blank group with culture medium was set, with 3 duplicate wells in each group. The 96-well plates were placed in a cell incubator for continuous culture, and after 48 h, the culture plates were taken out and CCK8 reagent was added to each well. After incubation in the incubator for 2 h, the OD value of each well was measured at 450 nm wavelength using an enzyme marker, and the killing rate was calculated according to the formula.

[0056]

[0057] Results:

[0058] (1) MSC EXO identification: The electron microscope results showed that MSC EXO presented a round membrane vesicle-like structure; the ZetaView results showed that the particle size of MSC EXO was about 123 nm, and Western blot identification showed that MSC EXO expressed TSG101, CD63 and CD81 protein markers, and did not express the negative marker Calnexin; at the same time, the uptake and internalization of MSC EXO by tumor cells (human acute monocytic leukemia cell line THP-1 cells) at 6 h were observed: MSC EXO were observed to enter THP-1 cells at 6 h, as shown in Figure 2 .

[0059] (2) Comparison of different methods of loading reovirus: Comparison of three different methods of loading MSCs with reovirus: direct mixing, sonication incubation and extrusion. EXO Preparation of MSCs with encapsulated REO EXO- REO, the best protocol was selected by testing its killing effect on THP-1 cells in the presence of neutralizing antibodies. Figure A shows MSCs prepared by direct mixing EXO - REO killing effect on THP-1 cells in the presence of neutralizing antibodies; Figure B shows MSCs prepared by sonication incubation EXO - REO killing effect on THP-1 cells in the presence of neutralizing antibodies; Figure C shows MSCs prepared by extrusion EXO - REO killing effect on THP-1 cells in the presence of neutralizing antibodies. Results show that MSCs prepared by extrusion EXO - REO killing effect in the presence of neutralizing antibodies was the most significant, so the subsequent experiments selected the use of MSCs prepared by extrusion EXO - REO. See details in Figure 3 .

[0060] (3) Comparison of MSCs EXO , REO and MSCs EXO - Particle size of MSCs EXO The particle size of MSCs was around 118 nm, the size of REO particles was around 100 nm, and the size of MSCs EXO - REO was close to the size of REO, also around 100 nm. See details in Figure 4 .

[0061] (4) MSCs EXO- - REO killing effect on tumor cells in vitro in the presence of neutralizing antibodies: MSCs EXO - REO killing effect on THP-1 cells in the presence of neutralizing antibodies, the results show that unlike the killing effect of the virus alone group, which was almost completely blocked by the neutralizing antibodies, MSCs EXO - REO killing of THP-1 cells was almost the same as without neutralizing antibodies, which indicates that MSCs EXO - REO protected the killing effect of REO on THP-1 cells in the presence of neutralizing antibodies. See details in Figure 5 .

[0062] (5) MSCs EXO - REO killing effect on human acute promyelocytic leukemia cell line HL-60 cells (not sensitive to REO) and peripheral blood mononuclear cells (normal cells) in vitro: MSCs EXO- The killing effect of REO on HL-60 cells, the results showed that compared with REO, MSC EXO - The killing effect of REO on HL-60 cells was significantly increased, which indicated that MSC EXO - REO can enhance the sensitivity of tumor cells to REO; MSC EXO - The killing effect of REO on PBMC cells, the results showed that compared with REO, MSC EXO - REO had no obvious toxic effect on PBMC, which indicated that MSC EXO - REO had good safety, see Figure 6 .

[0063] Finally, it should be pointed out that the above examples are only more representative examples of the present application. Obviously, the technical solutions of the present application are not limited to the above examples, but can also have many variations. All variations that can be directly derived or inferred from the disclosed content by those of ordinary skill in the art should be considered within the scope of the present application.

Claims

1. A mesenchymal stem cell-derived exosome-encapsulated reovirus complex, characterized in that it is Encapsulating reovirus with umbilical cord mesenchymal stem cell-derived exosomes; The umbilical cord mesenchymal stem cell-derived exosomes express TSG101, CD63 and CD81 protein markers, and do not express negative marker Calnexin. The encapsulation is performed by an extrusion method; the extrusion method uses an extruder equipped with a 100 nm membrane, and the umbilical cord mesenchymal stem cell-derived exosomes and the reovirus are mixed at a volume ratio of 1:1 and extruded back and forth for 30-50 cycles until the encapsulation is completed. The mesenchymal stem cell-derived exosome-encapsulated reovirus complex enhances the sensitivity of tumor cells to the action of reovirus; the tumor is human acute promyelocytic leukemia which is not sensitive to the killing effect of reovirus.

2. A method of preparing the mesenchymal stem cell-derived exosome-encapsulated reovirus complex of claim 1, characterized by, The extrusion method comprises the following steps: The umbilical cord mesenchymal stem cell-derived exosomes and the reovirus are mixed at a volume ratio of 1:1 and uniformly mixed, and the mixed solution is extruded back and forth for 30-50 cycles until the encapsulation is completed.

3. The production method according to claim 2, characterized by, The extrusion method comprises the following steps: the umbilical cord mesenchymal stem cell-derived exosomes and the reovirus are mixed at a volume ratio of 1:1 in a centrifuge tube, and a pipette is used to mix them thoroughly; the extruder is assembled as required and equipped with a 100 nm membrane, and then a syringe is used to suck an appropriate amount of PBS to check for leaks; the syringe sucks the mixture of exosomes and virus and extrudes it back and forth in the extrusion device for 30-50 cycles until the encapsulation is completed.

4. The preparation method according to claim 2, characterized in that, The umbilical cord mesenchymal stem cell-derived exosomes are prepared by the following method: When the umbilical cord mesenchymal stem cells grow to 60% confluence, they are cultured in a medium containing no exosome serum, and the supernatant is collected in a centrifuge tube after 48 hours; the supernatant is centrifuged at 300g for 10 minutes, and then centrifuged at 2000g for 20 minutes; then it is centrifuged at 10000g for 30 minutes and passed through a 0.22μm filter to remove larger-sized vesicles; Then the supernatant is centrifuged at 100000g for 70 minutes using a high-speed centrifuge, and the supernatant is carefully discarded and washed once with PBS, and then centrifuged at 100000g for another 70 minutes; all the above centrifugation steps are carried out at 4℃; finally, the exosome particles are resuspended in PBS and stored at -80℃.

5. Use of the mesenchymal stem cell-derived exosome-encapsulated reovirus complex of claim 1 in the manufacture of a tumor medicament. The tumor is human acute promyelocytic leukemia which is not sensitive to the killing effect of reovirus.

6. A pharmaceutical preparation, characterized by, The mesenchymal stem cell-derived exosome-encapsulated reovirus complex.

7. The pharmaceutical preparation according to claim 6, characterized in that, The pharmaceutical preparation is an antitumor pharmaceutical preparation.

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