Poster N16 - An in-vitro macrophage toxicity study of engineered high-energy silver nanoparticles

Introduction: EVQ-218 is a novel allotrope of silver nanoparticles developed via a patented nanosecond pulsed
laser-based, high-pressure manufacturing process, that uses secondary laser systems to modify
nanoparticles in the ejecta plume during particle formation. The surface properties of EVQ-218
show unique stability and antimicrobial properties without any ROS generation for cytotoxicity.
Measuring up to the NIST standard, EVQ-218 has shown strong antibiofilm and antibacterial
efficacy towards healthcare-associated infections (HAI) that can potentially benefit in mitigating
medical device-related infections. These unique properties of EVQ-218 led to our current study to
check the interaction of EVQ-218 with phagocytic cells like macrophages. In this study, EVQ-218
was evaluated for toxicity, uptake, and phagocytic activity against three different macrophage cell
lines: RAW 264.7 murine macrophages, MH-S murine alveolar macrophages, and THP-1 human
monocyte-like cells.

Materials and

Methods: EVQ-218 was manufactured using ultra-pure 18 MΏ water and metallic silver rods from ESPI
Metals, Oregon, USA, via a patented method [1]. The nanoparticles were characterized using
Dynamic Light Scattering (DLS) and Scanning Transmission Electron Microscopy (STEM) for
size, polydispersity (PDI), and morphology. RAW 264.7, MH-S, and THP-1 cells were cultured
following standardized cell culture protocols. THP-1 cells were differentiated into macrophages
using Phorbol 12-myristate 13-acetate (PMA). In-vitro CCK-8 assays were performed to
determine the dose-dependent toxicity of EVQ-218. The cellular uptake of the particles for
nontoxic doses was confirmed via Scanning Transmission Electron Microscopy (STEM) with dual
Energy Dispersive Spectroscopy (EDS) detectors. Phagocytosis assay was performed by analyzing
the uptake of IgG-FITC coated latex beads upon EVQ-218 nontoxic dose treatment.

Results, Conclusions, and Discussions: Particle size and PDI of EVQ-218 from DLS measurement was around 13 nm with a PDI of 0.2.
STEM demonstrated a similar size distribution of the particles (Fig 1). IC50 value of EVQ-218
against RAW 264.7 and MH-S cells was around 8 µg/ml, and THP-1 showed an IC50 of around 17
µg/ml (Fig. 2). STEM-EDS data demonstrated the presence of Ag inside the cells without any
significant or visible change to the cells’ morphology. The phagocytic assay showed a change in
phagocytosis upon EVQ-218 treatment in a dose-dependent manner (Fig. 3).

The cytotoxicity towards the cells is mostly attributed to cell-nanoparticle interaction since EVQ218 did not show Ag nanoparticle release that can cause cell cytotoxicity [2]. The STEM data for cellular uptake did not show any presence of trace materials, except for sulfur, which is due to the
presence of mercaptoethanol added to cell culture media. The phagocytosis assay for THP-1 cells
showed a decrease in phagocytosis with increased doses, and it was statistically significant only
for a 15 µg/ml dose. The phagocytic activity for RAW 264.7 and MH-S did not change at lower
doses, and only a significant increase was observed at 6 µg/ml for RAW 264.7 cells. The change
in phagocytosis depends on cellular division. Since THP-1 cells do not divide, their phagocytic
uptake of IgG-FITC-coated latex beads gets lower in higher nanoparticle concentrations, whereas
RAW 264.7 and MH-S cells are proliferative cell lines that divide with time, and the nanoparticle
load gets diluted with each cellular division.

This study creates a baseline for EVQ-218 nanoparticles and their interaction with the first line of
immune cell macrophages. Three different macrophage cell lines showed dose-dependent toxicity,
whereas human THP-1 cells showed more tolerance than murine cells. At nontoxic doses, the
internalization of particles was observed with no visible toxicity to the cells. The phagocytosis
assay showed cell line-dependent phagocytic activity.

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