Materials

BIS-Tris propane (BTP), glycine (Gly), sodium dodecyl sulfate (SDS), phosphate-buffered saline (PBS), bovine serum albumin (BSA), sodium chloride, Tween 20, and tris (hydroxymethyl) aminomethane (Tris) were obtained from Merck (Darmstadt, Germany). Sodium hydroxide was purchased from Avantor (Gliwice, Poland). All chemicals were of analytical grade. Deionized water was obtained with the Basic 5 water purification system (Hydrolab, Wislina, Poland).

ExpiCHO™ Expression System was obtained from Thermo Fisher Scientific (Waltham, MA, USA).

The 4 mg mL−1 stock solution of 2-nitrophenyl β-d-galactopyranoside (ONPG; Merck) was prepared in 0.1 M phosphate buffer (pH 6.8; Merck). To obtain the ONPG reagent solution, the ONPG stock solution was mixed with 0.1 M MgCl2 (Merck) aqueous solution and 0.1 M phosphate buffer solution in the volumetric ratio of 66:3:201, respectively.

The SDS-PAGE buffer (25 mM Tris, 192 mM glycine, 0.1% m/v SDS), transfer buffer (25 mM Tris, 192 mM glycine), and TBST buffer (Tris Buffered Saline; 20 mM Tris 7.5 pH, 150 mM NaCl, 0.1% v/v Tween 20) were prepared in deionized water and used in western blot experiments.

Expression vector

pCMV Sport-βgal vector encoding β-Gal was purchased from Thermo Fisher Scientific. pTracerBCHE vector expressing the C-terminal truncated form of BChE was constructed by using cloning techniques. An insert encoding BChE was amplified by polymerase chain reaction (PCR) method using template EX-A0103-M51 (GeneCopoeia, Inc) and specific primers: forward NBCHECHO-5′-ATAGATATCAATATGCATAGCAAAGTCACAATC and revers CCHOBCHEHIS-5′-ATAGATATCTTAGTGATGGTGATGGTGATGGACTTTTGGAAAAAATGATGTCCAGAATCG. The insert was cloned into the EcoRV restriction site of the expression vector pTracer-CMV/Bsd (Thermo Fisher Scientific) and introduced into E. coli Stellar™ (Takara Bio, Japan).

Cells culturing and transfection

Chinese hamster ovary (CHO) cells were maintained in ExpiCHO™ Expression Medium (Thermo Fisher Scientific, USA) at 37°C with a humidified atmosphere of 8% v/v CO2 on an orbital shaker at 125 rpm. Cells were transfected with appropriate plasmid DNA after reaching 6 × 106 cells/mL cells’ density. The plasmid DNA and ExpiFectamine™ CHO Reagent were diluted separately with OptiPRO™ medium (Thermo Fisher Scientific, Lithuania) according to the manufacturer’s instructions, connected into one mixture, and incubated for 5 min at room temperature (RT). After this time, ExpiFectamine™ CHO/plasmid complexes were added to the cells. The cells were cultured according to the standard protocol for 8 days. Subsequently, the cultures were centrifuged at 8000 g, 4°C, for 10 min, and the supernatants were kept for the next analyses.

Isolation of vesicles

The culturing media was centrifuged for 30 min at 3000 g at the temperature of 4 °C. 10 mL of collected supernatant was ultrafiltrated down to 0.5 mL with Vivaspin 20 concentrator (100 kDa MWCO, PES, Sartorius) according to the vendor’s recommendations. The retentate was fractionated into 25 fractions, 0.5 mL each, with qEVoriginal 35 nm Gen 2 (IZON) SEC column. The elution was conducted with PBS solution. The obtained fractions were stored at 4 °C.

Protein assay

Total protein content assay was conducted with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to vendors’ recommendations. Bovine serum albumin (BSA) was used for calibration. The samples and standards were mixed with 6% m/m SDS solution in a 9 to 1 volumetric ratio. The measurements were performed in 96-well plates using an Infinite M200 plate reader (Tecan, Mannedorf, Switzerland).

Nanoparticles tracking analysis (NTA)

NTA analysis was performed with the Nanosight NS300 instrument (Malvern Instruments, UK) and controlled with the NTA software (version 3.2 Dev Build 3.2.16, Malvern Instruments, UK). Samples were diluted in PBS to obtain around 40–100 particles per frame. A 305-nm laser and an sCMOS camera (camera level = 15–16; detection threshold = 5; focus = 180–220; flow rate = 100) were used to track particles. Each sample was analyzed twice. The single analysis included 5 films (60 s each).

Capillary electrophoresis (CE)

The CE experiments were performed with PACE MDQ plus system (Sciex, Framingham, MA, USA) equipped with a photodiode array detector and controlled with 32 Karat software (version 10.2, Sciex). Separation was conducted in uncoated silica capillaries (50 μm i.d. × 30.2 cm of total length; Polymicro Technologies, West Yorkshire, UK). The background electrolyte was composed of 50 mM BTP and 75 mM Gly (pH 9.5). The sample was injected hydrodynamically (5 s, 3.45 kPa). The electrophoresis was carried out at 10 kV (25 °C) and the separation process was monitored at 200 and 230 nm. Capillary conditioning was described elsewhere [13].

Transmission electron microscopy (TEM)

TEM analysis was performed with Tecnai G2 T12 Spirit BioTwin microscope (FEI Company, Hillsboro, OR, USA). Before the analysis, the isolates (5 μL) were deposed on the formvar support on a copper mesh (200 mesh, Agar Scientific, Stansted, UK), contrasted with a 1% m/v uranyl acetate, and left for drying.

β-Galactosidase activity assay

270 μL of ONPG reagent solution was mixed with 30 μL of a sample. The absorbance was measured with an Infinite M200 plate reader (Tecan) at 420 nm wavelength in a time interval of 1 min. The β-galactosidase activity was determined as an increment of absorbance in time.

Measurement of BChE activity by Ellman’s assay

BChE activity of each SEC fraction was determined spectrophotometrically by modified Ellman’s method [14] using BTC (S-butyrylthiocholine iodide) as a substrate. The assay was performed in 96-well microtiter plates in a final reaction volume of 200 μL of 100 mM phosphate buffer (pH 7.4) with a final concentration of 0.5 mM DTNB (5,5′-Dithiobis(2-nitrobenzoic acid) and 5 mM BTC as described previously [15]. The absorbance was monitored at 412 nm by repeated measurements at 1-min intervals for 10 min by a microplate reader spectrophotometer (Tecan Infinite M200 Pro, Tecan Group Ltd., Männedorf, Switzerland) at 25 °C.

Western blot

The SEC fractions were concentrated with Amicon ® Ultra 10K (Merck) concentrator. The total protein concentration of samples was measured with a DC protein assay (Bio-Rad, Hercules, CA, USA). On each lane of the 12% m/v SDS-PAGE gel, 30 mg of total protein amount from certain fractions was loaded. The proteins were separated at 100V for 90 min. Next, the proteins were blotted onto a PVDF membrane by using a wet transfer system at 100 V for 1 h. The membrane was blocked with TBST buffer with 3% m/v skim milk for 1 h at 4°C. The proteins were detected using the primary rabbit monoclonal antibodies: anti-Hsp70, anti-TSG101, anti-CD63 (Abcam, Trumpington, UK), and the secondary antibodies anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad, USA). The signals were measured in the ChemiDoc Touch Imaging system (Bio-Rad, USA) with a Clarity Western ECL Substrate chemiluminescence kit (Bio-Rad, USA).