Differences in uptake of 14 nm PEG-liganded gold nanoparticles into BEAS-2B cells is dependent on their functional groups.

Physico-chemical characteristics of nanoparticles have been shown to alter the uptake and toxicity of nanoparticles. This study investigated the uptake of six gold nanoparticles (AuNPs) into the human bronchial epithelial cell line BEAS-2B. The AuNPs studied included colloidal citrate-stabilised AuNPs of 14 nm in diameter; and 14 nm AuNPs conjugated to functional groups via polyethylene glycol (PEG), namely hydroxyl-PEG (POH), carboxyl-PEG (PCOOH), biotin-PEG (PBtn), nitrilotriacetic acid-PEG (PNTA), and azide-PEG (PAZ). Uptake was visualised by dark field microscopy using the CytoViva Hyperspectral Imaging system and after a 2 hour incubation at 37 °C, uptake was observed in cells treated with the citrate-stabilised and PCOOH AuNPs. However, no uptake was observed for the POH, PBtn, PNTA, or PAZ AuNPs, even after 24 h of incubation. An investigation into the energy dependence of uptake of the citrate-stabilised and PCOOH AuNPs showed that uptake was an active process. Cells pre-treated with either chlorpromazine or genistein as endocytosis inhibitors for clathrin- and caveolae-mediated pathways respectively, prior to addition of AuNPs, suggested a caveolae-dependent mechanism of endocytosis. These results further support recent findings on the mechanism of intracellular uptake and localisation and the subsequent toxicity of nanoparticles.

Cytotoxicity, intracellular localization and exocytosis of citrate capped and PEG functionalized gold nanoparticles in human hepatocyte and kidney cells.

Surface-modified gold nanoparticles (AuNPs) are nanomaterials that hold promise in drug delivery applications. In this study, the cytotoxicity, uptake, intracellular localization, and the exocytosis of citrate-stabilized (Cit-AuNP) and polyethylene glycol (PEG)-modified gold nanoparticles with the carboxyl (COOH) terminal functional group were assessed in human embryonic kidney (HEK 293) and the human caucasian hepatocytes carcinoma (Hep G2) cell systems, representing two major accumulation sites for AuNPs. The zeta (ζ)-potential measurements confirmed the negative surface charge of the AuNPs in water and in cell growth medium. The transmission electron microscopy confirmed the size and morphology of the AuNPs. Both types of AuNPs were shown to induce cytotoxic effects in cells. The Hep G2 cells were more sensitive cell type, with the COOH-PEG-AuNPs inducing the highest toxicity at higher concentrations. Dark field microscopy and TEM images revealed that the AuNPs were internalized in cells, mostly as agglomerates. TEM micrographs further revealed that the AuNPs were confined as agglomerates inside vesicle-like compartments, likely to be endosomal and lysosomal structures as well as in the cytosol, mostly as individual particles. The AuNPs were shown to remain in cellular compartments for up to 3 weeks, but thereafter, clearance of the gold nanoparticles from the cells by exocytosis was evident. The results presented in this study may therefore give an indication on the fate of AuNPs on long-term exposure to cells and may also assist in safety evaluation of AuNPs.

Challenges facing sterilization and depyrogenation of nanoparticles: effects on structural stability and biomedical applications.

This review outlines and compares techniques that are currently available for the sterilization of nanoparticles and addresses the topic of endotoxin contamination. Several techniques are available for the removal of microbial contamination from nanoparticles developed for use in nanomedicine applications. These techniques include filtration, autoclaving and irradiation, as well as formaldehyde, ethylene oxide and gas plasma treatments. Of these sterilization methodologies, filtration may potentially remove microbial contamination without altering the physicochemical properties of the carrier nanoparticles, nor affecting their toxicity and functionality. However, no single process may be applied to all nanoparticle preparations and, therefore, it is recommended that each nanoparticle-drug system be validated on a case-by-case basis. From the clinical editor: This comprehensive review covers the currently available methods for removal of microbial contaminations from nanoparticles for nanomedicine applications. The review highlights the pros and cons of each available method. Authors conclude that there is no single best method and recommend a customized approach for each nanoparticle system.

Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines.

BACKGROUND: Reliable in vitro toxicity testing is needed prior to the commencement of in vivo testing necessary for hazard identification and risk assessment of nanoparticles. In this study, the cytotoxicity and uptake of 14 nm and 20 nm citrate stabilised gold nanoparticles (AuNPs) in the bronchial epithelial cell line BEAS-2B, the Chinese hamster ovary cell line CHO, and the human embryonic kidney cell line HEK 293 were investigated. METHODS: Cytotoxicity of the AuNPs was assessed via traditional XTT-, LDH-, and ATP-based assays, followed by cell impedance studies. Dark-field imaging and hyperspectral imaging were used to confirm the uptake of AuNPs into the cells. RESULTS: Interference of the AuNPs with the XTT- and ATP-based assays was overcome through the use of cell impedance technology. AuNPs were shown to be relatively non-toxic using this methodology; nevertheless CHO cells were the most sensitive cell type with 20 nm AuNPs having the highest toxicity. Uptake of both 14 nm and 20 nm AuNPs was observed in all cell lines in a time- and cell type-dependent manner. CONCLUSIONS: Using the cell impedance and dark-field hyperspectral imaging technologies, it was possible to study the toxicity of AuNPs in different cell lines and show that these cells could internalize AuNPs with their subsequent intracellular aggregation. It was also possible to show that this toxicity would not correlate with the level of uptake but it would correlate with cell-type and the size of the AuNPs. Therefore, these two label-free methodologies used in this study are suitable for in vitro studies on the effects of AuNPs, and could present themselves as appropriate and valuable methodologies for future nanoparticle toxicity and uptake studies.

Mechanisms facilitating the uptake of carboxyl-polythene glycol-functionalized gold nanoparticles into multicellular spheroids.

OBJECTIVES: Nanomedicines represent theragnostic alternatives to traditional candidate drugs, with increased targeting and delivery potential due to their size and functional tailorability. Biological activity typically relies on nanomaterials permeating into the intracellular environment, necessitating characterization of uptake and intracellular trafficking pathways. Spheroids' three-dimensional architecture and heterogenous cellular distribution offer an in-vivo-representative platform to assess the biological activity of nanoparticles (NPs). This study aimed to develop an A549 alveolar carcinoma spheroid model as a NP uptake assessment platform for carboxyl-polythene glycol-functionalized gold NPs affording further biological characterization opportunities in nanomedicine. METHODS: A549 spheroids were generated via the liquid overlay method, and their morphology and viability were assessed for 21 days. Cytotoxicity was assessed via lactate dehydrogenase release. NP uptake was elucidated using uptake pathway inhibition, combined with CytoViva hyperspectral imaging of sectioned spheroids to count internalized NPs. KEY FINDINGS: Cytotoxicity was absent for all exposure groups. Clathrin-mediated endocytosis was the primary endocytic mechanism (33.5-54.8% of uptake), which may precede lysosomal degradation. Lysosomal membrane permeabilization appears to be a potential downstream application. Low penetration into spheroids (4.5 µm) suggests the failure of NPs to traverse cellular layers in the spheroid. CONCLUSIONS: Although poor uptake was observed, a multicellular spheroid model of A549 alveolar carcinoma cells was established, allowing for similar future uptake assessment of various NPs.

COVID-19 cases among medical laboratory services staff in South Africa, 2020-2021: A cohort study.

Medical laboratory workers may have an increased risk of COVID-19 due to their interaction with biological samples received for testing and contamination of documents. Records of COVID-19 laboratory-confirmed positive cases within the medical laboratory service were routinely collected in the company's Occupational Health and Safety Information System (OHASIS). Surveillance data from the OHASIS system were extracted from 1 April 2020 to 31 March 2021. An epidemic curve was plotted and compared to that for the country, along with prevalence proportions and incidence rates. The odds of COVID-19 infection were categorised by job and compared to the US Occupational Risk Scores. A logistic regression model assessed the risk of COVID-19 infection per occupational group. A total of 2091 (26% of staff) COVID-19 positive cases were reported. The number of COVID-19 cases was higher in the first wave at 46% (967/2091) of cases, than in the second wave 40% (846/2091) of cases. There was no significant difference in COVID-19 prevalence between male and female employees. The job categories with the most increased risk were laboratory managers [AOR 3.2 (95%CI 1.9-5.1)] and laboratory support clerks [AOR 3.2 (95%CI 1.9-5.2)]. Our study confirms that some categories of medical laboratory staff are at increased risk for COVID-19; this is a complex interaction between workplace risk factors, community interaction, socioeconomic status, personal habits, and behaviour. Targeted interventions are recommended for high-risk groups. OHASIS has the potential to generate data for surveillance of health care workers and contribute towards a South African risk profile.

From the Cover: An Investigation of the Genotoxicity and Interference of Gold Nanoparticles in Commonly Used In Vitro Mutagenicity and Genotoxicity Assays.

The suitability of 4 in vitro assays, commonly used for mutagenicity and genotoxicity assessment, was investigated in relation to treatment with 14 nm citrate-stabilized gold nanoparticles (AuNPs). Specifically, the Ames test was conducted without metabolic activation, where no mutagenic effects were observed. High resolution transmission electron microscopy and Cytoviva dark-field image analysis showed that AuNPs did not enter the bacterial cells, thus confirming the unreliability of the Ames test for nanoparticle mutagenicity studies. In addition, the Chinese hamster ovary (CHO) cell line was used for Comet, Chromosome aberration and Micronucleus assays. CHO cells were treated with AuNPs for 20 h at 37 °C. Cytotoxicity was not detected by cell impedance studies even though AuNP uptake was confirmed using Cytoviva image analysis. The DNA damage was statistically significant in treated cells when assessed by the Comet assay. However, minimal and nonstatistically significant chromosomal DNA damage was observed using the chromosome aberration and micronucleus assays. In this study, we showed that false positive results obtained with Comet assay may have been due to the possibility of direct contact between the residual, intracellular AuNPs and DNA during the assay procedure. Therefore, the chromosome aberration and micronucleus assays are better suited to assess the genotoxic effects of nanoparticles due to low probability of such direct contact occurring. Genotoxic effect of 14 and 20 nm citrate-stabilized, as well as, 14 nm PCOOH AuNPs were also investigated using chromosome aberration and micronucleus assays. Based on our acceptance criteria for a positive genotoxic response, none of the AuNPs were found to be genotoxic in either of these assays.

Gold nanoparticle interference study during the isolation, quantification, purity and integrity analysis of RNA.

Investigations have been conducted regarding the interference of nanoparticles (NPs) with different toxicological assay systems, but there is a lack of validation when conducting routine tests for nucleic acid isolation, quantification, integrity, and purity analyses. The interference of citrate-capped gold nanoparticles (AuNPs) was investigated herein. The AuNPs were added to either BEAS-2B bronchial human cells for 24 h, the isolated pure RNA, or added during the isolation procedure, and the resultant interaction was assessed. Total RNA that was isolated from untreated BEAS-2B cells was spiked with various concentrations (v/v%) of AuNPs and quantified. A decrease in the absorbance spectrum (220-340 nm) was observed in a concentration-dependent manner. The 260 and 280 nm absorbance ratios that traditionally infer RNA purity were also altered. Electrophoresis was performed to determine RNA integrity, but could not differentiate between AuNP-exposed samples. However, the spiked post-isolation samples did produce differences in spectra (190-220 nm), where shifts were observed at a shorter wavelength. These shifts could be due to alterations to chromophores found in nucleic acids. The co-isolation samples, spiked with 100 µL AuNP during the isolation procedure, displayed a peak shift to a longer wavelength and were similar to the results obtained from a 24 h AuNP treatment, under non-cytotoxic test conditions. Moreover, hyperspectral imaging using CytoViva dark field microscopy did not detect AuNP spectral signatures in the RNA isolated from treated cells. However, despite the lack of AuNPs in the final RNA product, structural changes in RNA could still be observed between 190-220 nm. Consequently, full spectral analyses should replace the traditional ratios based on readings at 230, 260, and 280 nm. These are critical points of analyses, validation, and optimization for RNA-based techniques used to assess AuNPs effects.