An in vitro study on macrophage toxicity of allotrope silver nanoparticles

Abstract

Recent studies have established EVQ-218 as a novel type of allotrope silver nanoparticle (AgNP). The patented laser-based method of manufacture imparts EVQ-218 with unique surface properties that enhance particle stability without capping agents and other stabilizing chemistry. In this study, EVQ-218 was screened against RAW 264.7 murine macrophages, MH-S murine alveolar macrophages, and THP-1 human monocyte cell lines. IC₅₀ values were obtained, and cellular uptake and phagocytosis assays were conducted. Results of this study indicate that the intracellular uptake of EVQ-218 did not make any apparent physiologically significant change in cell morphology and phagocytic activity of macrophages at nontoxic doses.

Introduction

Silver (Ag) and silver-based nanoparticles (AgNPs) have gained enormous interest because of their unique electrical and optical properties, catalytic activity, surface-enhanced Raman scattering, surface-modifying ability, and chemical stability [1, 2]. The antimicrobial properties of AgNPs make them one of the most commonly used nanoparticles in biomedical applications that include the coating of implants, wound dressings, and prostheses [3–7]. AgNPs are also used in consumer products, including in food and cosmetic industries [8, 9]. With the increased use and availability of AgNPs, the interaction of these nanoparticles with the human body is increasing to a higher extent, which leads to the necessity of understanding the short, mid, and long-term effects of silver nanoparticles on human health [10].

Silver possesses a strong affinity towards the protective and redox-reactive -SH group, and it can also mediate oxidative stress to cells, which can pose a risk when using AgNPs in different applications [11]. Studies have shown that inhaled AgNPs in rats reached the liver and brain, and subcutaneously injected AgNPs have been found in the kidney, lung, brain, liver, and spleen [12, 13]. Previous studies have also shown the adverse effects of AgNPs on different cell lines, including glioblastoma cells, THP-1 monocyte-derived macrophages, and rat alveolar macrophages [14–16]. The reported adverse in vitro toxic effects of AgNPs on cells include the reactive oxygen species (ROS) generation, mitochondrial membrane interaction, and leaching of Ag⁺ ions from AgNPs [17, 18]. Also, AgNPs have been found to alter cell morphology and induce genotoxicity in both normal and cancer cell lines [15].

It is known that physicochemical properties such as morphology, size, shape, surface charge, and surface chemistry impact the toxicity of the particles towards cells, tissues, and organs [14, 19–21]. In addition, it is known that cytotoxicity towards nanoparticles is cell-dependent [22].

Here, we synthesized unique AgNPs named EVQ-218, using high energy and pressure akin to diamond-like bond formations, resulting in an allotrope of silver, which are 10 nm, spherical, non-emissive particles and do not show a zone of inhibition in bacterial disk diffusion studies [23]. EVQ-218 shows nearly equivalent properties in terms of morphology and particle uniformity with National Institute of Standards and Technology (NIST) standard materials, as well as a longer shelf life [23]. Dissolution and surface chemistry studies also showed EVQ-218 as a clean, stable, and non-emissive alternative to traditional silver nanoparticles [23]. Treatment of polymers and textiles with EVQ-218 creates a bacteriostatic array preventing bacterial colonization. EVQ-218 also did not show enough generation of ROS for cytotoxicity in previous studies [24]. These unique properties of EVQ-218 led us to explore the toxic aspects of these particles in vitro, specifically to macrophages.

Considering the first line of defense, macrophages are a part of the mononuclear phagocytic system and are responsible for clearing foreign and potentially harmful particles. They are also involved in different immunological and inflammatory processes [16]. Inhalation, ingestion, and dermal contact can cause cell-nanoparticle interaction, and macrophages are one of the first cells likely to interact with the nanoparticles [16, 21]. Macrophages can recognize pathogens, cellular debris, and foreign particles and are responsible for their clearance through phagocytosis [25].

Despite the benefits of AgNPs, including antimicrobial, antibacterial, and therapeutic applications, their acute, sub-chronic, and chronic toxicological effects on macrophages are largely unknown [26, 27]. A study using AgNPs with different sizes reported a decrease in cell viability and increased cytokine and chemokine levels with increasing AgNP concentration [16]. Changes in Nitric Oxide (NO) production have also been reported in a study where murine peritoneal macrophages were exposed to AgNPs [28]. It is imperative to study how EVQ-218 interacts with macrophages, which could potentially benefit the further translation of these particles for future biomedical applications.

This study aims to understand the interaction of EVQ-218 with macrophages regarding toxicity, uptake, and phagocytic activity. To have a comparative understanding of the effect of EVQ-218 with murine and human macrophage cell lines, RAW 264.7 murine macrophages, which is a model macrophage cell line, murine alveolar macrophages MH-S, and human monocyte cells THP-1 (which can be differentiated into macrophages) are used throughout this study. The cytotoxicity, phagocytic activity, and cellular uptake of the nanoparticles are reported herein.

Materials

Gibco^™ RPMI 1640 cell culture media and Fetal Bovine Serum (FBS) were purchased from ThermoFisher Scientific (Waltham, Massachusetts, USA). Phorbol 12-myristate 13-acetate (PMA) was purchased from Invivogen (San Diego, California, USA) and Dimethyl Sulfoxide (DMSO) was obtained from ATCC (Manassas, Virginia, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Phagocytosis Assay Kit (IgG-FITC) was purchased from Cayman Chemicals (Ann Arbor, Michigan, USA).

Methods

Particle synthesis and characterization

EVQ-218 was produced via patented methods [29], using 18 mΩ water and 99.999% silver rod (ESPI Metals, Oregon, USA). After initial particle characterization, a bulk sample was concentrated with Tween-80 (targeting 800 μg/mL EVQ-218, 1% Tween 80). The resulting sample of EVQ-218 contained 870 μg/mL Ag and maintained original particle characteristics upon reconstitution. The nanoparticles were characterized using Dynamic Light Scattering (DLS) and Scanning Transmission Electron Microscopy (STEM) for size, polydispersity (PDI), and morphology.

Cell culture

The in vitro studies of EVQ-218 were evaluated on three different cell lines: RAW 264.7 murine macrophages, MH-S murine alveolar macrophage cells, and THP-1 human monocytes. The cells were obtained from ATCC (Manassas, Virginia, USA). All the cell lines were grown in Gibco^™ RPMI 1640 media supplemented with 10% Fetal Bovine Serum (FBS) in a 37° C incubator with 5% CO₂.

In vitro cytotoxicity

For the CCK-8 cytotoxicity assay, 15,000, 25,000, and 40,000 cells were seeded in each well of a 96-well plate for RAW 264.7, MH-S cells, and THP-1 cells, respectively. RAW 264.7 and MH-S cells were seeded in a 96-well plate for 24 h in regular cell culture media. THP-1 cells were seeded in media supplemented with Phorbol 12-myristate 13-acetate (PMA) at a 50 ng/ml concentration for differentiation into macrophages. The PMA-contained media was replaced with fresh THP-1 cell culture media. The cells were allowed to rest for an additional 24 h before performing the assay. Twenty-four hours after seeding the cells, the cells were dosed with different EVQ-218 doses (6 μg/ml to 26 μg/ml for RAW 264.7 cells, 2 μg/ml to 26 μg/ml for MH-S cells, and 4 μg/ml to 50 μg/ml for THP-1 cells). After 24 h, the media was aspirated, and 10% CCK-8 reagent was added following the manufacturer’s protocol. The cells were incubated for an hour before the absorbance values were measured at 450 nm using the Spectramax M2 plate reader (Molecular Devices, USA). Cells treated with only media were considered negative controls, and cells treated with 5% (v/v) DMSO were considered positive controls. Each experiment was performed in replicates of 6 (n = 6).

Phagocytosis

In order to check the effect of EVQ-218 on the phagocytic activity of the macrophage cell lines, a phagocytosis assay was performed according to the manufacturer’s protocol (Cayman Chemicals, Ann Arbor, Michigan, USA). In brief, the cells were seeded on a 6/48-well plate for 24 h, and the nontoxic doses of EVQ-218 from the CCK-8 study (2–6 μg/ml for RAW 264.7 and MH-S cells and 5–15 μg/ml for THP-1 cells) were used to dose the cells. After 24 h, the media was replaced with the latex beads fluorescently coated with Rabbit IgG and kept for 2 h. After that, the cells were washed with PBS, trypsinized, collected, and analyzed with flow cytometry (Cytoflex S Cytometer, Beckman Coulter, USA) for phagocytic activity. The data was collected through CytExpert (version 2.4.0.28 Beckman Coulter) and post-analyzed using FlowJo. The phagocytic activity was measured via the geometric mean fluorescent intensity present in the cells and normalized to the control cells without exposure to nanoparticles.

Cellular uptake

Cellular uptake of EVQ-218 was visualized by transmission electron microscopy (TEM). Briefly, the cells were seeded, grown overnight in a 6-well plate, treated with a 6 μg/ml dose of the nanoparticles, and incubated for 24 h. Then, the cells were washed, fixed using a fixative solution, harvested, and refrigerated overnight. The cell pellets were embedded in resin and cut into ultrathin sections for imaging using a JEOL JEM 2800 microscope with a dark field camera and dual high-resolution EDS detectors. For the images of healthy cell morphology in the supplementary information, a similar procedure were followed, and the images were taken using a JEOL JEM 1400 microscope.

Statistical analysis

The data were presented as mean ±standard deviation. Each experimental group was replicated in at least n= 3, and the difference between each group was analyzed by the student’s t-test. The IC₅₀ values were determined using log(inhibitor) vs. response using GraphPad Prism (Version 10.1,2). P value <0.05 between different groups was considered as statistically significant.

Results

Particles demonstrate uniform size with low polydispersity

The STEM images in Fig. 1 show the spherical morphology and uniform particle distribution of EVQ-218. The average hydrodynamic diameter and the PDI of the particles determined via DLS were 13.5 nm and 0.2, respectively.

Fig. 1.

EVQ-218 characterization. (Left) STEM images of EVQ-218 suitable for particle size analysis. Note the uniform, spherical particle nature. (Right) Particle size distribution of EVQ-218 obtained from STEM image analysis

EVQ-218 demonstrates a dose-dependent toxicity to macrophages

For MH-S and RAW cell lines, more than 90% cell viability was observed at 6 μg/ml nanoparticle concentration. THP-1 cell line showed more than 90% cell viability at 10 μg/ml. The IC₅₀ values for the RAW 264.7 and MH-S cell lines were 8.7 μg/ml and 8.6 μg/ml, whereas the value for the IC₅₀ of THP-1 cells was found to be 17.9 μg/ml (Fig. 2).

Fig. 2.

Viability of macrophages exposed to EVQ-218 silver nanoparticles. A RAW 264.7 macrophages, (B) MH-S macrophages, (C) THP-1 macrophages. Data represents Mean ± SD. Red line depicting the non-linear curve fit, whereas the blue line showing the average viability values with error bars

Phagocytic activity is macrophage-type dependent

The phagocytic activity for the THP-1 cells decreased by about 25% with increased dosage, where the 15 μg/ml dose was found statistically significant. The phagocytic activity of MH-S and RAW 264.7 upon EVQ-218 dosage did not show any significant change, except the 6 μg/ml dose in RAW cells showed a significant increase (Fig. 3).

Fig. 3.

Phagocytic activity of macrophages upon EVQ-218 silver nanoparticle treatment with (A) RAW 264.7 cells (B) MH-S cells (C) THP-1 cells. The Mean Fluorescence Intensity (MFI) for the treated groups is presented in % of non-treated controls. Data represent Mean ± SD, *P < 0.05

Cellular uptake of EVQ-218 does not alter macrophage morphology

The cellular uptake of EVQ-218 is presented in Fig. 4 for RAW 264.7, MH-S, and THP-1 cells. The STEM images showed the presence of EVQ-218 inside the cells. The cellular size and morphology also indicated healthy cell morphology. Here, we define “healthy cell morphology” as no visible changes or damage in the cell membranes, nucleus, and organelles. Elemental analysis showed the presence of Ag in the dispersive X-ray spectra, which confirmed the cellular uptake and internalization of EVQ-218 (Fig. 5,6,7 in the supplementary information). Healthy and unhealthy RAW 264.7 cell morphology is provided in Fig. 8 in supplementary information.

Fig. 4.

STEM images of EVQ-218 treated macrophages showing particle internalization (in the yellow arrows) and healthy cell morphology. a RAW 264.7 cells ((d) and (e) are zoom in regions); (b) MH-S cells ((f) and (g) are zoom in regions); (c) THP-1 cells ((h) and (i) are zoom in regions)

Discussion

In this work, we aim to evaluate the interaction of our unique AgNP EVQ-218 with macrophage cell lines. Macrophage cell models have been chosen since systemic exposure to silver nanoparticles will be mostly encountered by macrophages and will be cleared by them. In order to achieve that, we performed cytotoxicity, cellular uptake, and phagocytic activity of the particles with the different cell lines.

The CCK-8 assay-based cytotoxicity study showed that the viability of all three cell lines decreased in a dose-dependent manner. Previous studies also suggested the size and dose-dependent toxicity of AgNPs with different cell lines [30]. The IC₅₀ value of the murine macrophages was nearly half that of human macrophages, indicating that murine macrophages are more sensitive to EVQ-218 than human macrophages, which had also been reported before [31]. The potential reason behind this variability might include differences in cellular interactions and uptake of nanoparticles between cell lines. It has previously been established that the cytotoxicity caused by AgNP is generally attributed to multiple factors, including reactive oxygen species (ROS) generation, cell-nanoparticle interaction, DNA damage, upregulation of inflammatory cytokines, genotoxicity, and the release of Ag⁺ ions from the nanoparticles [1, 11, 32]. Studies have also shown that upon cellular uptake, smaller-sized nanoparticles (< 10nm) release more Ag⁺ ions than larger nanoparticles due to the increased surface area of the particles [21]. As such, cytotoxicity from smaller NPs would be due to ion emission, whereas larger nanoparticles primarily induce toxicity due to the cell-nanoparticle interaction [30, 33]. Since EVQ-218 has a diameter of approximately 10–13 nm and does not release Ag⁺ ions [23], we hypothesize that the direct cell surface-nanoparticle interaction predominantly controls its cytotoxicity [33]. However, particles’other physicochemical properties, such as shape, surface charge, coating, capping agent, and geometry can also mediate different mechanisms of toxicity [1]. One predominant mechanism for AgNP-induced toxicity can be the reactive oxygen species (ROS) generation by the particles and the oxidative stress on the macrophages; however, previous studies have shown that EVQ-218 does not show enough generation of ROS for cytotoxicity [24]. To further investigate the mechanisms of toxicity, additional molecular mechanistic studies are required in the future.

The phagocytosis assay with EVQ-218 to macrophages showed a dose-dependent decrease in the clearance of phagocytic beads compared to the control for the THP-1 cell line. At the highest dose, the decrease was significant compared to non-treated control cells; however, the changes were not physiologically significant since the fold change was less than 2 [34]. MH-S cells did not show any significant difference in phagocytic activity. Phagocytic activity for RAW 264.7 cells showed no significant change for 2 and 4 μg/ml doses. However, at 6 μg/ml dose, the phagocytic activity for RAW 264.7 was higher than in non-treated control cells.

The difference in the phagocytic activity of the cell lines might be attributed to the cell lines from different species and the proliferation capacity of the cell lines since functional activities can vary among macrophages from different species, even for macrophages within the same species [35]. THP-1 differentiated macrophages do not possess cell division capability. We hypothesize that with increased AgNP doses, more internalization of Ag nanoparticles might reduce the internalization of phagocytic beads, which resulted in a decreased phagocytosis with higher dose concentrations. In contrast, MH-S and RAW 264.7 cells being proliferative cell lines, their newborn daughter cells might have reduced the nanoparticle load, and the phagocytic capacity did not show any significant change compared to the non-treated control cells. For both MH-S and RAW 264.7 cells, the highest dose showed increased phagocytic activity with RAW 264.7 cells showing significant changes. This phenomenon can be explained by triggering macrophage activation when exposed to the particles, which results in rapid proliferation and division of the macrophages, leading to a higher phagocytic uptake of the beads.

Decreased phagocytosis of bacteria and pathogens may reduce immune response during infections. However, the in vitro study of macrophage phagocytic ability in THP-1, MH-S, and RAW 264.7 cells showed that in vitro phagocytic activity is cell-line dependent. The variation in cytotoxicity and phagocytic activity from one cell line to another was also observed in previous studies [30].

Conclusion

The in vitro studies with EVQ-218 on three different macrophage cell lines showed the nontoxic dose ranges and IC₅₀ values for the macrophages, whereas the phagocytosis activity assay showed mostly no significant change for MH-S and RAW 264.7 cell lines and a dose-dependent decrease in the THP-1 cell line. The STEM images showed healthy morphology upon EVQ-218 uptake, and the EDS spectra confirmed the presence of silver inside all the cell lines. These studies provide a baseline for the in vitro macrophage interaction for EVQ-218 that can be expanded for more specific and mechanistic studies in the future, targeting different applications. Further, in vivo studies are needed to establish a safe dose for the utility of EVQ-218 in specific biological applications.

Supplementary Material

Supplementary Information The online version contains supplementary material available at 10.1007/s11051-025-06333-y.

Data availability

No datasets were generated or analysed during the current study.