Application of HL-60 cell-derived extracellular vesicles as a tumor-targeted drug delivery system

Abstract

The present invention discloses the application of extracellular vesicles derived from HL-60 cells in a drug delivery system for targeting tumors. After LPS and IFN-γ treatment, the expression levels of related pro-inflammatory factors in the extracellular vesicles obtained in neutrophils were significantly upregulated, especially ICAM-1, an important indicator of neutrophil activation, was significantly increased at both mRNA and protein expression levels. Neutrophil-derived EVs with high expression of ICAM-1 are more easily adhered and captured by tumor cells with high expression of integrins. Drug-loaded EVs are prepared by loading Dox with neutrophil-derived EVs, which effectively improves the efficiency of drug-loaded EVs in vivo being taken up by tumor cells and their ability to kill tumor cells.

Description

Technical Field

[0001] The present invention relates to the application of extracellular vesicles derived from HL-60 cells in a drug-carrying system targeting tumors. Background Art

[0002] Chemically synthesized drugs, gene drugs, and especially natural drugs or active ingredients of traditional Chinese medicines have many problems that severely limit their clinical application, such as poor solubility, low stability, and large side effects. Although drug delivery systems that use organic or inorganic materials as synthetic carriers can improve the solubility and bioavailability of drugs, they cannot effectively target the lesion site and may even be regarded as exogenous toxins by the body's immune system and excreted from the body, making them ineffective.

[0003] Extracellular vesicles (EVs) are nanoscale membrane structures produced by cells. They are considered to be an important way to mediate intercellular communication and are involved in a variety of physiological reactions in the body. Due to their endogenous nature and low immunogenicity, EVs can avoid being quickly cleared from the body and will not cause an immune response. Neutrophils have characteristics such as high chemotaxis, but it is not clear whether the EVs from which they are derived can inherit the high chemotaxis of neutrophils. Summary of the invention

[0004] The purpose of the present invention is to provide the use of extracellular vesicles derived from HL-60 cells in a drug delivery system targeting tumors.

[0005] Preferably, the extracellular vesicles are obtained by inducing HL-60 cells to differentiate into neutrophils using all-trans retinoic acid (ATRA), and then stimulating the neutrophils with LPS and IFN-γ.

[0006] Preferably, when the extracellular vesicles are used as a drug delivery system, the drug loaded is a drug with diagnostic or therapeutic activity for medical use.

[0007] Preferably, the drug is one or more of cisplatin, paclitaxel, gemficitabine, vinorelbine, and pemetrexed.

[0008] Beneficial effects: HL-60 cells were induced to differentiate into neutrophils using all-trans retinoic acid (ATRA), and then treated with LPS and IFN-γ. The expression levels of related pro-inflammatory factors in the extracellular vesicles obtained from neutrophils were significantly upregulated, especially the intercellular adhesion molecule 1 (ICAM-1), an important indicator of neutrophil activation, was significantly increased at both mRNA and protein levels.

[0009] Neutrophil-derived EVs that highly express ICAM-1 are more likely to be adhered and captured by tumor cells that highly express integrins. Using neutrophil-derived EVs loaded with Dox to prepare drug-loaded EVs effectively improves the efficiency of drug-loaded EVs being taken up by tumor cells in vivo and their ability to kill tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 , Transmission electron microscopy analysis of extracellular vesicles derived from HL-60 cells.

[0011] Figure 2 , Nanoparticle tracking analysis (NTA) analysis of extracellular vesicles derived from HL-60 cells.

[0012] Figure 3 , in vitro flow cytometry detection of drug uptake efficiency of tumor cells.

[0013] Figure 4 , in vitro immunofluorescence detection of tumor cell drug uptake.

[0014] Figure 5 , detection of the efficiency of drug-loaded vesicles in inducing tumor cell apoptosis.

[0015] Figure 6 , in vivo detection of the therapeutic effect of extracellular vesicles derived from HL-60 cells on tumors.

[0016] Figure 7 , detection of cells' ability to capture vesicles. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0018] ATRA was used to induce HL-60 cells to differentiate into neutrophils. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were used to stimulate neutrophils, and extracellular vesicles were isolated and obtained.

[0019] Steps for inducing HL-60 cells to differentiate into neutrophils:

[0020] (1) Dissolve the purchased ATRA (25 mg) in 10 ml DMSO to make a 25 mg / ml ATRA stock solution.

[0021] (2) Dilute the ATRA stock solution with 1640 complete culture medium, mix gently, and prepare an ATRA mixed solution with a working concentration of 5 μM. Shake the cell culture bottle to suspend the cells, transfer the cell suspension to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and remove the supernatant. Resuspend with the ATRA mixed solution and transfer to a new culture bottle.

[0022] (3) After 3 days of stimulation culture, the supernatant was removed by centrifugation and replaced with fresh 1640 complete culture medium. After 2 days of culture, HL-60 cells differentiated into neutrophils.

[0023] Stimulates neutrophil differentiation to a pro-inflammatory phenotype:

[0024] (1) Dissolve the purchased LPS (10 mg) in sterilized 1xPBS to make a 1 mg / ml LPS stock solution.

[0025] The purchased IFN-γ (50 μg) was dissolved in sterilized ddH2O to prepare a 100 μg / ml IFN-γ stock solution.

[0026] (2) Take the shaken cell culture flask, suspend the cells, transfer the cell suspension to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, and remove the supernatant.

[0027] (3) Take 1~2x10 respectively 7 cells, and resuspended the cells in 1640 culture medium. Add LPS at a working concentration of 1 μg / ml and 20 ng / ml to the dHL-60-N1 group, shake and mix, and stably culture in a cell culture incubator to induce neutrophil differentiation.

[0028] Extraction of extracellular vesicles:

[0029] (1) Preparation of exosome-free serum: Place fetal bovine serum in an ultracentrifuge, centrifuge at 4°C, 160,000 g for 16 h, recover the supernatant to obtain exosome-free serum, and store it at -20°C for later use.

[0030] (2) Cells were cultured in culture medium containing 10% exosome-free serum. After 48 h of culture under different culture conditions, the cell suspension was collected and centrifuged at 300 g for 10 min.

[0031] (3) Collect the supernatant and centrifuge at 2000 g for 10 min to remove cell debris.

[0032] (4) Transfer the supernatant to an ultracentrifuge tube and centrifuge at 160,000 g for 70 min. Remove the supernatant after centrifugation, resuspend the precipitate (vesicles) with 1x PBS, transfer to a 1.5 ml EP tube, and store at -80°C.

[0033] Vesicle drug delivery method:

[0034] (1) Prepare a 1 mg / ml Dox solution using DMSO.

[0035] (2) Add different concentrations of Dox into 1xPBS, detect the absorbance at 480 nm using an enzyme reader, and establish a Dox concentration standard curve.

[0036] (3) The drug was co-incubated with the vesicles by the “direct loading” method. The vesicle protein concentration was standardized to 500 μg / ml, 200 μg / ml Dox was added (ratio 5:2), and after thorough mixing, incubation was carried out at room temperature for 24 h.

[0037] (4) Centrifuge at 300 g for 5 min, transfer the supernatant containing the drug-loaded vesicles to a new EP tube, collect the unloaded Dox precipitate, resuspend it in DMSO, and detect it using an ELISA reader. Calculate the free Dox content based on the standard curve.

[0038] (5) The Dox encapsulation efficiency of vesicles was calculated as follows: encapsulation efficiency (EE%) = 1-[free Dox content (mg) / total amount of Dox added (mg)] x 100%.

[0039] S1. Characterization of the structure of extracellular vesicles derived from HL-60 cells by transmission electron microscopy (TEM) (e.g. Figure 1 ) and nanoparticle tracking analysis (NTA) (e.g. Figure 2 )Analyze the physical properties of drug-loaded vesicles.

[0040] Experimental method: transmission electron microscopy

[0041] (1) Dilute the vesicles with an appropriate amount of 1× PBS, drop the sample on the transmission electron microscope copper grid, let it stand for 1-2 minutes, carefully remove the liquid with filter paper, and let it air dry naturally.

[0042] (2) At room temperature, dye with phosphotungstic acid solution for 3-5 minutes. After dyeing, remove the copper mesh, absorb excess liquid, and let it air dry naturally.

[0043] (3) Observation using a transmission electron microscope.

[0044] S2. To determine whether HL-60 cell-derived extracellular vesicles can improve the drug uptake efficiency of tumor cells, we further studied whether HL-60 cell-derived extracellular vesicles carrying ligands that can selectively interact with tumor cell membrane receptors can enhance the drug delivery potential of nanocarriers. Because anthracycline drugs Dox can spontaneously fluoresce, we co-incubated the drug-loaded vesicles with tumor cells for a short period of time, and then used flow cytometry to detect the intracellular Dox fluorescence intensity to determine the Dox uptake efficiency of tumor cells ( Figure 3 ). At the same time, DiO dye was used to label the drug-loaded vesicles, and the DiO-labeled drug-loaded vesicles were co-incubated with tumor cells for a short period of time, and the fluorescence co-localization in the tumor cells was observed by confocal microscopy. LPS+IFN-γEV-Dox showed strong Dox fluorescence after incubation with LLC cells for 6 hours. In contrast, the Dox fluorescence in tumor cells treated with free Dox and NE-EV-Dox (without inducing pro-inflammatory neutrophils) was weaker. The experimental results show that extracellular vesicles derived from HL-60 cells can effectively improve the drug uptake efficiency of tumor cells as drug delivery carriers and increase the accumulation of drugs in tumor cells ( Figure 4 ).

[0045] Experimental methods Figure 3 ) Flow cytometry for drug uptake in tumor cells

[0046] (1) Lung cancer cells LLC were cultured at 2x10 5 Each well was inoculated into a 24-well plate and cultured overnight.

[0047] (2) Dox and drug-loaded vesicles at specific concentrations were added to the wells, and three parallel wells were set up for each experimental group. The cells were cultured in a cell culture incubator for 4 h.

[0048] (3) Aspirate the culture medium in the wells and wash twice with sterile PBS. Add trypsin to digest the cells. When the cells shrink or slightly fall off, add twice the culture medium to neutralize the trypsin and centrifuge at 800 rpm for 3 min to collect the cells.

[0049] (4) Remove the supernatant, wash the cells once with 1% BSA, and centrifuge at 300 g for 5 min.

[0050] (5) Resuspend the cells with an appropriate amount of 1% BSA and test on an analyzer.

[0051] ( Figure 4 ) Immunofluorescence detection of drug uptake in tumor cells

[0052] (1) Soak the sterilized cell slides in 75% ethanol overnight and wash them three times with sterilized PBS.

[0053] (2) Place the slides in a 24-well plate with approximately 10 5Cells were seeded and cultured overnight.

[0054] (3) Add DiO solution to the vesicles, mix thoroughly, incubate at 37°C for 10 min, and centrifuge to remove free DiO.

[0055] (4) The culture medium in the wells was aspirated and washed once with sterilized PBS. Dox and drug-loaded vesicles were added to the wells according to specific Dox concentrations and treated in a cell culture incubator for 6 h.

[0056] (5) After 4 h, the culture medium was removed and the cells were washed three times with 1x PBS, each time for 5 min.

[0057] (6) Fix with 4% paraformaldehyde at room temperature for 15 min. Wash with 1xPBS three times, 5 min each time.

[0058] (7) Add DAPI stain, incubate at room temperature in the dark for 10 min, and then wash three times with 1x PBS, each time for 5 min.

[0059] (8) Remove the residual liquid, add anti-fluorescence quencher and seal the slide. Use confocal microscopy for examination.

[0060] S3. The experimental results of drug-loaded vesicles inducing tumor cell apoptosis showed that empty vesicles (regardless of whether the ICAM-1 expression level is high or low) themselves have no toxic side effects and no killing ability to tumor cells. After loading with Dox, the drug-loaded vesicles have enhanced cell killing ability compared with free Dox. At the same time, in tumor cells treated with drug-loaded vesicles with high expression of ICAM-1, the ratio of late apoptotic cells and necrotic cells increased significantly, corresponding to the high drug uptake efficiency of tumor cells, indicating that drug-loaded vesicles carrying ICAM-1 promote tumor cells to take up drugs and thus show strong cell killing ability ( Figure 5 ).

[0061] Experimental methods: Cell apoptosis detection

[0062] (1) Lung cancer cells LLC were cultured at 2x10 5 Each well was inoculated into a 24-well plate and cultured overnight.

[0063] (2) Dox and drug-loaded vesicles were added to the wells respectively. Three parallel wells were set up for each experimental group and cultured in a cell culture incubator for 24 h.

[0064] (3) Aspirate the culture medium in the wells and wash twice with sterile PBS. Add EDTA-free trypsin to digest the cells. When the cells shrink or slightly fall off, add twice the culture medium to neutralize the trypsin and centrifuge at 800 rpm for 3 min to collect the cells.

[0065] (4) Remove the supernatant, wash the cells twice with 4°C precooled PBS, use the cell apoptosis kit, resuspend the cells in 250 μl binding buffer, and adjust the cell concentration to 1x10 6 cell / ml.

[0066] (5) Take 100 μl of cell suspension, add 5 μl of Annexin V / PE and 10 μl of 7AAD solution, mix gently, and incubate at room temperature in the dark for 15 min.

[0067] (6) Add 400 μl PBS to wash, centrifuge at 300 g for 5 min, resuspend the cells in an appropriate amount of PBS, and analyze them.

[0068] The two cell proteins were extracted by cell lysate. Figure 6 As shown, the experimental results show that ICAM-1 is mainly expressed in the form of non-secretory protein in pro-inflammatory neutrophils. That is, LPS and IFN-γ can induce the increased expression of adhesion molecule ICAM-1 in neutrophils.

[0069] Experimental method: western blot.

[0070] Vesicles were labeled with DiO dye and incubated with cells for a short period of time. The fluorescence intensity of DiO on cells was detected by flow cytometry to analyze the cells' ability to capture vesicles. The results are shown in Figure 2. Figure 7 As shown in Figure 2, flow cytometry results showed that tumor cells have a good ability to capture neutrophil-derived vesicles, and when the vesicles highly express ICAM-1, the capture efficiency of tumor cells for vesicles is significantly improved. In contrast, fibroblasts with low integrin expression have a lower capture efficiency for vesicles.

[0071] Experimental method: Cellular uptake flow cytometry

[0072] (1) Dissolve the purchased DiO solution in DMSO, store it at -20°C after aliquoting. The working concentration of DiO is 5 μM, Ex / Em=484nm / 501nm.

[0073] (2) LLC cells and NIH 3T3 cells were cultured at 2x10 5 Each well was inoculated into a 24-well plate and cultured overnight.

[0074] (3) Add DiO solution to the vesicles, mix thoroughly, incubate at 37°C for 10 min, and centrifuge to remove free DiO.

[0075] (4) DiO-labeled vesicles were added to the wells respectively. Three parallel wells were set up for each experimental group and cultured in a cell culture incubator for 4 h.

[0076] (5) Aspirate the culture medium in the wells and wash twice with sterile PBS. Add trypsin to digest the cells. When the cells shrink or slightly fall off, add twice the culture medium to neutralize the trypsin and centrifuge at 800 rpm for 3 min to collect the cells.

[0077] (6) Remove the supernatant, wash the cells once with 1% BSA, and centrifuge at 300 g for 5 min.

[0078] (7) Resuspend the cells with an appropriate amount of 1% BSA and test on an analyzer.

Claims

1. Application of extracellular vesicles derived from HL-60 cells in the preparation of a tumor-targeted drug delivery system; the extracellular vesicles are obtained by inducing HL-60 cells to differentiate into neutrophils using ATRA, and then stimulating the neutrophils with LPS and IFN-γ.

2. The use according to claim 1, characterized in that: When the extracellular vesicles are used as a drug-carrying system, the drug loaded is a drug with diagnostic or therapeutic activity for medical use.

3. The use according to claim 2, characterized in that: The drug is one or more of cisplatin, paclitaxel, gemficitabine, vinorelbine, and pemetrexed.

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