Qnano

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the International Animal Care and Use Committee of the University of North Carolina at Greensboro (Protocol Number: 11–02). All techniques were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.
A panel of fullerene derivatives was synthesized at Luna Innovations and characterized for particle size using dynamic light scattering (Malvern Instruments, Zetasizer Nano ZS, Westborough, Massachusetts, USA), qNano (Izon Science, qNano, Cambridge, Massachusetts, USA) and nano particle tracking analysis, (Malvern Instruments, Nanosight LM10, Westborough, Massachusetts, USA), zeta potential (Malvern Instruments, Zetasizer Nano ZSP, Westborough, Massachusetts, USA), NMR (Agilent Technologies, 400 Mhz NMR Spectrometer, Santa Clara, California, USA), and FT-IR (Agilent Technologies, Varian 670 FT-IR, Santa Clara, California, USA). A representative physiochemical characterization schematic for the two fullerene derivatives used for the in vivo studies (ALM, a liposome encapsulated C₇₀ fullerene and TGA, a water-soluble C₇₀ fullerene conjugated with four glycolic acids) is shown in [27 (link),31 (link)].

Tunable resistive pulse sensing (TRPS) was performed using a qNano (Izon, Cambridge, MA, USA) according to the manufacturer’s instructions. Samples were diluted 50-fold with PBS (-) and were measured using TRPS. The measured value was multiplied by the dilution factor to calculate the concentration of nanoparticles in the sample.

Extracted exosome samples were examined for particle size using TRPS (qNano; Izon Science Ltd., New Zealand) according to the manufacturer’s instructions, and the data were analyzed with Izon Control Suite v.3.3.2.2000 (Izon Science Ltd.).

Relative surface charge and size distribution analysis of WT, and mutants chiA and chiB was performed using a qNano (Izon Science). The qNano utilizes Tunable Resistive Pulse Sensing technology to allow for a high-throughput, particle-by-particle, analysis of particle size, surface charge, and electrophoretic mobility [20] (link), [43] (link). All qNano experiments were performed using the manufacturer's established protocols [19] (link)–[21] , [43] (link). Briefly, overnight cultures of WT, chiA, and chiB were pelleted (five minutes at 4,500×g) and washed three times with sterile PBS. For each measurement, 40 μl of the washed bacterial suspension was added to the top fluid cell and a minimum of 1,000 blockade events were recorded. Measurements were taken at 48.49 mm of applied stretch with an applied voltage of 0.10 V. An applied pressure of 5 cm H₂O was applied to the top fluid cell using the Izon Science variable pressure module. The size distribution and relative surface charge analysis was performed using IZON proprietary software V2.2.