Ns500

Nanoparticle tracking analyses were performed using a NanoSight NS500 instrument (NanoSight NTA 2.3 nanoparticle tracking and analysis release version build 0033; Malvern Instruments, Malvern, United Kingdom) following the manufacturer’s instructions. Samples were diluted 1:1000 with Dulbecco’s PBS to obtain 10:100 particles per image (optimal ∼50 particles per image). The NS500 instrument measured the rate of Brownian motion of nanoparticles in a light-scattering system that provides a reproducible platform for specific and general nanoparticle characterization (NanoSight, Amesbury, United Kingdom). Suspended nanoparticles were passed through the laser beam that traversed the sample chamber and scattered the light, discerning the nanoparticles through a microscope. The samples were mixed before introducing them into the chamber (temperature, 25°C), and the camera level was set to obtain an image that had sufficient contrast to clearly identify particles while minimizing background noise (camera level, 10; capture duration, 30 seconds). The captured videos (two videos per sample) were then processed and analyzed. Each video file was processed and analyzed to give the mean and mode of particle sizes, along with the concentration and the number of particles. An Excel spreadsheet was automatically generated and data were imported into GraphPad Prism 7 (La Jolla, CA).

FBH were bought from the feed market in the governorate of Kafr El-Sheikh, Egypt. FBH was converted to nanoparticle size using the method of Khataee et al. [22 (link)] with a slight modification. To create the FBH-NPs, the FBH was ground to a micro-particle size range of 100–150 µm using a Black & Decker grinder (Model BD54958, Black & Decker Inc, Minneapolis, MN USA) and crushed using a high-energy planetary ball-mill (Model PM2400, Kian Madan Pars Co, Tehran, Iran) at a speed of rotation of 320 rpm for 2 h. The ball-milling procedure was carried out at atmospheric conditions (26 °C at 1 atm) with a 10:1 ball-to-powder mass ratio. Finally, the average size of the homogeneous FBH-NPs was determined using a zetasizer (NS500, Malvern Panalytical, Worcestershire, UK). The obtained FBH-NPs (with an average size of 81 ± 4 nm) were then packed in a dark glass vial without oxygen and used in the tests immediately. To limit antioxidant loss, all operations used to make FBH-NPs were carried out under controlled settings.

The size distribution was checked by the nanoparticle tracking analysis (NTA) method using a NanoSight NS500 (Malvern, Grovewood road, UK) by using minor modification of manufacturer’s methods. Samples were diluted sufficiently for the contrast and minimal background level. The quick measurement mode was performed to find the optimal condition. Then, total 5 numbers of particle motion video were recorded automatically using standard measurement mode (temperature: 20.3 or 20.4 °C and viscosity: 0.99 to 1.0 cP). All other conditions were constant.

The size and number of B. cepacia OMVs were measured using a NanoSight NS500 instrument with a 488 nm laser module and sCMOS camera module (Malvern Instruments, Worcestershire,UK) [39 (link)]. The purified OMVs were diluted in MilliQ water to a concentration of approximately 8–9 × 10⁸ particles/mL for 50–100 particles per frame in the NTA measurement. OMV samples were loaded in the sample chamber and then videos were recorded for 30s three times. The captured data were analyzed using NTA 3.1 software build 3.1.46. All measurements were performedin triplicate at room temperature.

The size of the peptide/SSO complex was measured by Nanoparticle Tracking Analysis (NTA). The complexes were formulated as described above and measured on the NS500 instrument (Malvern Instruments, Malvern, Worcestershire, UK). The movement of nanoparticles for each sample was recorded five times (30 s each video). In order to process the videos, the camera gain and minimum detection threshold was set to 14 and 7, respectively.