Qnano gold

Particles in the nanometer range (150–900 nm) were determined with TRPS using the qNano Gold (IZON, Nottingham, UK). For analysis, pore NP 300 (A57745, IZON) was chosen, a stretch of 47.01 mm and a pressure of around 15 mbar were applied. All samples, buffers, and the calibration particles CPC 400 (mean diameter 350 nm, 7.56 × 10⁸ mg/mL; IZON) were spiked with a small amount of NaCl solution up to a final concentration of 140 mM of sodium chloride.
The lower fluid chamber was loaded with 80 µL of the respective buffer and the voltage was set to 0.34 V for the analysis of the Avastin vial and to 0.38 V for all remaining samples.
Twenty-five microliters of spiked sample or buffer were pipetted into the upper cell and measured in triplicates for 10 minutes. Results were calculated using the included software IZON CONTROL SUITE (IZON), with the focus on particle sizes of 150 and 300 nm.
To ensure no aggregation occurred due to spiking the samples, size and polydispersity (PDI) were counterchecked with dynamic light scattering for all samples used for TRPS measurements.

Hemolysis of serum samples was evaluated through spectrophotometric analysis. The absorbance of a representative 100 µL aliquot from each sample was measured at two wavelengths (λ = 541 nm and 576 nm) using the Synergy 2-BioTek plate reader (Agilent Technologies, Inc., Santa Clara, CA, USA): only samples with an absorbance value <0.2 were considered non-hemolyzed and were used for further processing [76 (link)]. Briefly, a 1 mL aliquot from non-hemolyzed sera was thawed at 4 °C and centrifuged at 1500× g, 10′, 4 °C; the supernatant was centrifuged again at 8000× g, 10′, 4 °C to remove potential debris: supernatants from the second centrifugation were used for sEV isolation. sEVs were isolated from 500 uL of serum by using SEC, through qEV original/70nm chromatography columns (IZON Science, Christchurch, New Zealand). Sterile PBS, filtered through 0.2 µm filters, was used as eluent and pooled fractions 7–10 were collected in a final volume of 2 mL and stored at −80 °C until further processing, according to the manufacturer’s instructions. For three randomly chosen GBM and control samples, an aliquot of 100 µL was assayed by TRPS through qNano Gold (IZON Science) to determine the size and the concentration of isolated EVs. TRPS data analysis was performed through qNANO Control Suite Software v. 3.4 (IZON Science).

Following isolation, ADNVs in PBS were quantified through tunable resistive pulse sensing (qNANO Gold, Izon Science Ltd., Cambridge, MA, USA). The analysis also provided data on size distribution of particles in the ADNV fraction. The nanopore (NP150, Izon Science Ltd., Cambridge, MA, USA) was stretched 49 mm wide using a digital caliber. The preparation of the instrument was performed with reagents provided by the manufacturer following the manufacturer’s instructions. Each sample was analyzed at two pressure points, 10 atm and 20 atm. During each measurement, the particle rate was maintained above 200 particles/min, and the total particle count surpassed 500 particles. Calibration particle (CPC200, Izon Science Ltd., Cambridge, MA, USA) measurements were taken at both 10 atm and 20 atm and were used for calibrating each sample measurement during data analysis. The protein content was also established through Bradford assay with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. Absorbance was measured at 570 nm using multilabel plate reader Victor 3 (Perkin Elmer, Milano, Italy).

The qNano Gold instrument (Izon Science, Christchurch, New Zeeland) was employed to measure the size distribution and concentration of the isolated NPs using the tunable resistive pulse sensing (TRPS) principle as already reported [53 (link),54 (link)]. Briefly, 35 μL of purified particles were analyzed with a qNano Gold instrument using a NP200 Nanopore (Izon Science) and applying 49 mm stretch, 0.1 V, and 20 mBar parametric conditions. The calibration particles (CPC100, Izon Science) were assayed before the experimental samples under identical conditions. Size and concentrations (2000 events each) were finally determined using the qNano software provided by Izon Science (Izon Control Suite version 3.1).
Zeta potential was evaluated by a Litesizer 500 Particle Analyzer (Anton Paar, Turin, Italy) in aqueous suspension.
DSC and FT-IR analyses on BBR-NPs were performed with the same experimental conditions used to characterize the BBR salts (see Section 2.2).
TEM ultrastructural analysis of NPs was carried out using a Zeiss EM900 electron microscope (Zeiss) operating at 80 kV.

Rat LMVECs were transiently transfected, using Polyjet reagent, with plasmid cDNA encoding GFP-tagged constructs: wild-type (p18^wt) and non-endosomal binding (p18^{N39 (link)}) p18, dominant active (Rab4^Q67L), and non-endosomal binding (Rab4^{S22 (link)N}) Rab4, or GFP vector control. Alternatively LMVECs were transiently transfected with p18 or scrambled control siRNA (300 nM) using DharmaFect4. Overexpression of constructs was assessed by GFP fluorescence quantified using the Victor fluorometer (Perkin Elmer). At 42 h post-transfection, cells were exposed to LPS (1 µg/ml) for a further six hours. Media from cells was then removed and centrifuged at 300 × g, 4℃ for 10 min twice, with pellet discarded each time. Supernatant was then centrifuged at 100,000 × g, 4℃ for one hour. The pellet, comprising of EDEVs, was resuspended in sterile Dulbecco's PBS (DPBS) for permeability assays and quantification using qNano Gold (Izon Science) at 0.5 V, 45 mm stretch, 15 mbar pressure, using the NP200 pore and normalized to media as a control, RNAlater for miRNA analysis, or lysed for protein concentration analysis.^{23 (link),24 (link)}