Methods for exosome-based targeted therapy

2.5.1 Cell culture and exosome isolation

Murine B cells (M12.4) were used as the source of vehicle exosomes in miRNA inhibitor and miRNA mimic studies. M12.4 cells were cultured in an RPMI medium, in addition to 10 % v/v exosome-depleted FBS (Exo-FBS™, Mountain View, CA, USA), and 1 % w/v penicillin / streptomycin (Gibco®). After 12 h, the cells were stimulated by CD40 (5 μg/ml) (PeproTech, USA) and Interleukin 4 (IL-4) (50 ng/ml) (PeproTech, USA). Three days later, the culture media were collected and exosomes were isolated by Exoquick as described in section 2.2.4. RAW 264.7 macrophages were cultured in Dulbecco’s modified medium (Invitrogen) plus 10 % v/v FBS at seeding density of 8 x 104 _{/well/2 ml medium (passage}

number of 14) in 12-well plates for co-culture experiments. RAW 264.7 macrophages were used for exosome production at seeding density of 3 x 106 /12 ml medium in T-75flasks (passage number of 14).

2.5.2 Optimizing loading conditions of exosomes with miRNA-155

mimic

A PBS suspension of freshly isolated exosomes was diluted in Gene Pulser® (Bio-Rad Laboratories, USA) electroporation buffer at a 1:1 ratio. miRNA-155 mimic or negative miRNA control 1 (Ambion) at a final mass of 150 pmol was added to 0.25 μg/μl, 0.5 μg/μl, 1 μg/μl, and 1.5 μg/μl of exosome sample. Cold (4 oC) electroporation cuvettes (gap width: 0.2 cm) were used and the exosomes were electroporated at various voltages (0.130 kV to 0.200 kV) at a constant capacitance of 100 μF. After optimization of the voltage, the effect of variation in capacitance was assessed. Electroporation was performed using a Gene pulser II System (Bio-Rad Laboratories). One unit of RNase H was administered per exosome suspension for 1 h to eliminate free floating or surface-adsorbed oligonucleotides. Exosomes were next re-isolated using Exoquick-TC™. Synthetic C. elegans (cel)-miRNA-39 (5 µl of a 5 fmol/μL stock tube) was spiked during the total RNA isolation process and used to normalize the qPCR data as an endogenous control The relative amount of encapsulated miRNA-155 was determined using TaqMan® miRNA Assays as described in section 2.1.2.

Next, to determine the most efficient isolation method to re-isolate exosomes after the electroporation procedure, we compared the three methods of ultracentrifugation, CD63 immunomagnetic isolation, and Exoquick-TC™. In the first step, exosomes were isolated using CD63 immunomagnetic beads as delineated in section 2.3.2. 150 pmol of miRNA-155 mimic was electroporated into 50 μg of exosomes. The RNase H was added to the exosome suspension for a total duration of 1 h to eliminate free floating, unloaded miRNA-155 mimic. Exosomes were re-isolated with different isolation methods including ultracentrifugation, immunomagnetic isolation, and Exoquick-TC™. Ultracentrifugation was performed at 100,000 g for 90 min using fixed angle 75 Ti rotor (Beckman Coulter), at 4°C. Exoquick- TC™ and immunomagnetic isolation methods were performed as outlined in section 2.2.4. Cel-miR-39 (150 pmol) was added to the samples before RNA isolation and RNA isolation was done as described in section 2.1.1. The experiments were performed in triplicate and the amount of recovered miRNA-155 was quantified by TaqMan® miRNA Assays as described in section 2.1.2. The cycle number at which the reaction reached an arbitrarily-placed threshold (CT) was determined for each sample and the relative amount of miRNA-155 to

MATERIALS AND METHODS

cel-miR-39 was described using the equation 2−ΔCT where ΔCt = (CtmiRNA-155 – Ctcel-miR-39)

(Schmittgen et al., 2008).

In order to quantify the efficacy of loading, B cell derived exosomes were purified and loaded with miRNA-155 using the optimal conditions (e.g. voltage and concentrations). After loading, exosomes were re-isolated using the Exoquick-TC™ reagent according to the manufacturer’s instructions. To remove aggregates of miRNA-155 mimic outside the exosomes, the exosome pellet was treated with one unit of RNase H. Exogenous cel- miRNA-39 was spiked to the samples before RNA isolation and used an exogenous control to calculate loading efficiency. Subsequently, the total RNA was extracted. The proportion of miRNA-155 mimic that was loaded into the exosomes was calculated using the following formula: 𝑓𝑚𝑖𝑅𝑁𝐴−𝐿𝐸= ( 𝑉_𝑐𝐶_𝑐𝑀𝑊_𝑐 𝑉𝑠𝐶𝑠𝑀𝑊𝑠 ) (𝑅𝐸𝑠 𝑅𝐸𝑐 )

Where fmiRNA-LE (miRNA-LE is the miRNA loading efficiency) is the fraction of miRNA

which was successfully loaded into the exosomes; Vc is the volume of added exogenous

spiked control; Cc is concentration of exogenous control; MWc is molecular weight of the

exogenous control; Vs is the volume of electroporated miRNA sample; Cs is the

concentration of the sample; MWs is the molecular weight of the loaded miRNA-155 mimic; REs is the relative expression of miRNA-155 mimic and REc is relative expression of the

control (spiked cel- miRNA-39). Using this formula, our “optimal” loading protocols led to efficient loading of miRNA-155 mimic into the exosomes (55.06%).

2.5.3 Enzyme-linked immunosorbent assay (ELISA)

TNFα protein levels were measured using a Mouse TNFα ELISA kit (BD Biosciences, USA) as described in section 2.2.10. Supernatants (400 µl) were collected from the cells and centrifuged at 2000 g for 10 min to remove cellular debris and then frozen at −80 °C until use. Protein levels of IL-1β, TNFα, and MCP1 were quantified in the supernatant by ELISA. The quantification of TNFα (BioLegends, USA), MCP1 (BioLegend Inc., USA) and IL-1β (R&D Systems, USA) were carried out based on the manufacturers’ recommendations using a Synergy HTX Multi-Mode ELISA reader.

2.5.4 Lactate Dehydrogenase (LDH) cytotoxicity assay

The lactate dehydrogenase (LDH) level was measured in the supernatant of RAW264.7 macrophages based on the manufacturer's recommendation (Abcam). Released LDH in culture supernatants of RAW macrophages was quantified 36 h after delivery of miRNA- 155 inhibitor via different methods with relevant controls. The percentage of cytotoxicity was calculated by subtracting the LDH content in the supernatant of remaining viable cells from the total LDH in the supernatant of untreated controls. Staurosporine (20 nM) (Abcam) served as a positive control for cell death. The final absorbance was measured at 490 nm with a Synergy™ HTX Multi-Mode Microplate Reader. Results were expressed as the mean of three independent experiments.