Engineered nanoparticles (ENPs) (e.g. zinc oxide (nZnO) and iron oxide (nFeOx) and organic pollutants (e.g. triclosan (TCS)) are among emerging contaminants (ECs) of environmental concern. However, to date there is limited knowledge on their fate and potential deleterious effects in the ecological systems. Following simultaneous and/or sequential release of ECs in the ecological systems; they co-exist as mixtures defined by complex permutations. As such, toxicological outcomes of individual ECs may be altered as those of mixtures formed may exhibit synergistic, antagonistic or additive effects. Influenced by water physicochemical parameters, chemical interactions between multiple contaminants and the unique properties of ENPs like photoactivity, adsorption capacity and dissolution may alter the toxic outcomes to bacteria. Yet, currently, the environmental fate and toxicity ENPs as mixtures, particularly in natural water are limited. In this work, Bacillus subtilis was used as a model organism to assess the toxicity of nZnO and maghemite iron oxide (γ-nFe2O3) as individual ENPs, binary mixtures, and ternary mixtures with TCS. Natural water from two river sources, the Elands River (ER) and the Bloubank River (BR) were used to generate environmentally relevant data; and four endpoints were used to evaluate toxicological outcomes (cell viability, cell membrane integrity, ATP production, oxidative stress from reactive oxygen species (ROS). Aggregation of the two ENPs were significantly different between the two river water matrixes with higher aggregates observed in BR water. nZnO induced significant reduction in cell viability and membrane integrity at higher tested concentrations in ER; but none in BR under visible light. A higher decrease in ATP levels was observed in ER than in BR, and ROS production was negligible irrespective of the ENP type and exposure media under visible light. Conversely, γ-nFe2O3 induced no significant effects on B. subtilis on all tested endpoints. nZnO induced concentration-dependent effects on the cell membrane integrity of B. subtilis in both river water samples under solar irradiation. For binary mixtures of ENPs under solar irradiation, nZnO toxicity was found to be concentration-dependent, with more pronounced effects in ER than BR water due to water chemistry. However, toxic effects were mitigated by γ-nFe2O3 in the binary mixtures, linked to heteroaggregation between the ENPs. Solar irradiation induced ROS had minimal effect on the toxicity of ENPs. For ternary mixtures, toxicity of TCS was more pronounced at the highest concentration. However, the effects were not water chemistry dependent, compared to the observed effects from nZnO. In addition, more distinctive mitigating effects were observed in ternary mixtures, where nZnO dissolution was significantly lower in the presence of TCS. These findings demonstrated that observed differences in the effects of nZnO towards B. subtilis, either in binary or ternary systems were influenced by the nature of interactions (TCS and γ-nFe2O3) as well as water chemistry of natural water in focus. Therefore, the unique physicochemical properties of natural aqueous media were established to be the key determinant attributes in enhancing or inhibiting the effects of ENPs on bacteria, and, the co-existence of ECs of different types and classes may lead to reduction of toxic effects of individual contaminants. / Thesis (PhD)--University of Pretoria, 2020. / Chemical Engineering / PhD / Unrestricted

The physical characteristics (i.e., nanostructure and stiffness) of the extracellular matrix where cells reside have been shown to profoundly affect numerous cellular events in vivo and also been employed to modulate cell behavior in vitro, yet how these physical cues regulate cell behavior is still elusive. Therefore, we engineered a variety of nanotopographies with different shapes and dimensions, and investigated how the nanotopographical cue, through focal adhesions-cytoskeleton-nucleus pathway, affected cell phenotype and function. We further designed and fabricated well-defined substrates which had either identical biochemical cue (adhesive ligand presentation) but different nanotopographical cues or identical nanotopography but different biochemical cues, and dissected the roles of these cues in cell modulation. In addition, we revealed that the human mesenchymal stem cells (hMSCs) could obtain nanotopographical memory from the past culture environment, and the nanotopographical memory influenced the future fate decision of the hMSCs. Moreover, we evaluated the effects of substrate nanotopographical and stiffness cues on the fibrogenesis of human lung fibroblasts in response to carbon nanotubes and highlighted the significance of these physical cues in the development of physiologically relevant in vitro models for nanotoxicological study. The mechanistic understanding of the physical regulation of cell behaviors will provide important insight into the advancement of cell culture technologies and the recreation of biomimetic in vitro tissue/organ models.

Primarily concerned with manipulation and study of matter at the nanoscale, the concept of nanoscience encompasses ideas such as nanomaterial synthesis, characterization, and applications to modern scientific and societal problems. These problems encompass a broad range of issues such as energy storage and conversion, medical diagnostics and treatment, environmental remediation and detection, carbon economy and as well as many others. Silica nanoparticles of porous morphology have broad application to many of these issues. In particular, the utility of silica nanoparticles is facilitated by their large intrinsic surface area, tunable surface chemistry, and synthetic variability in both their size and morphology. This facilitates applications to these problems. However, extensive characterization and deeper understanding is needed before full implementation in key applications can be realized. The work described in this thesis aims to explore fundamental and applied characterization of silica nanoparticles that might be used in biomedical and environmental applications. Fundamental studies of functionalized nanomaterials using NMR spectroscopy reveal complex, dynamic phenomena related to-and ultimately deriving from-the intrinsic and/or modified surface chemistry. Applied studies of nanomaterial-biological interfaces demonstrate free radical chemistry as dominating the toxic response of the materials when exposed to biological systems of interest. Characterization of protein adsorbed on the interface reinforces the ubiquitous nature of protein adsorption on nanomaterial surface in biological and environmental media. Overall, this work illuminates and highlights complex changes that take place in aqueous solution for silica nanoparticles of varied morphology and surface chemistry.

Gold nanoparticles (GNPs) are currently being intensely investigated for their potential use in biomedical applications. Nanotoxicity studies are urgently needed to validate their safety in clinical practice. The objective of this research was to assess the acute, subacute, and chronic effects of a single intravenous exposure to commercially available GNPs in two in vivo models, mice and rats. Gold nanoparticles were purchased and independently characterized. Animals were exposed to either 1000 mg GNPs/kg body weight (GNP group) or an equivalent volume of phosphate buffered saline (PBS group) intravenously via the tail vein. Subsets of animals were euthanized 1, 7, 14, 21, 28 days (female BALB/c mice and female F344 rats) or 20 weeks (female and male C57BL/6 mice) post-exposure and samples were collected for biochemistry, histopathology, electron microscopy, and atomic absorption spectrometry analysis. Independent characterization demonstrated that the physicochemical properties of the purchased GNPs were in good agreement with the information provided by the supplier. Important differences in GNP-induced immune responses were identified when comparing mice and rats 1 to 28 days post-exposure. Gold nanoparticles stimulated the formation of liver microgranulomas in mice, along with transiently increased serum levels of the proinflammatory cytokine interleukin-18. No such alterations were found in rats. Species differences in GNP biodistribution and excretion were also detected, with higher relative accumulation of GNPs in spleen and longer fecal excretion in rats. In the long-term (20 weeks after dosing), exposure to GNPs incited chronic inflammation in mice, characterized by the persistence of microgranulomas in liver, spleen, and lymph nodes, as well as further increased serum levels of interleukin-18. Impairment of body weight gain was also observed in the GNP-exposed group. No sex differences were detected. In conclusion, GNPs are not innocuous and have the ability to incite a robust macrophage response in mice. However, considering the mildness of the toxic effects identified despite the high dose selected for the study, GNPs continue to have great potential for biomedical uses. Further studies are needed in order to determine specific mechanisms of toxicity and the role of chronic inflammation in the development of adverse effects after co- or post-exposures. / Ph. D.

No description available.

Advancing applications of engineered nanomaterials (ENM) in various fields create the opportunity for intended (e.g. drug and gene delivery) or unintended (e.g. occupational and environmental) exposure to ENM. However, the knowledge of ENM-toxicity is lagging behind their application development. Understanding the ENM hazard can help us to avoid potential human health problems associated with ENM applications as well as to increase their public acceptance. Ceria (cerium [Ce] oxide) ENM have many current and potential commercial applications. Beyond the traditional use of ceria as an abrasive, the scope of ceria ENM applications now extends into fuel cell manufacturing, diesel fuel additives and for therapeutic intervention as a putative antioxidant. However, the biological effects of ceria ENM exposure have yet to be fully defined. Both pro-and anti-oxidative effects of ceria ENM exposure are repeatedly reported in literature. EPA, NIEHS and OECD organizations have nominated ceria for its toxicological evaluation. All these together gave us the impetus to examine the oxidative stress effects of ceria ENM after systemic administration. Induction of oxidative stress is one of the primary mechanisms of ENM toxicity. Oxidative stress plays an important role in maintaining the redox homeostasis in the biological system. Increased oxidative stress, due to depletion of antioxidant enzymes or molecules and / or due to increased production of reactive oxygen (ROS) or nitrogen (RNS) species may lead to protein oxidation, lipid peroxidation and/or DNA damage. Increased protein oxidation or lipid peroxidation together with antioxidant protein levels and activity can serve as markers of oxidative stress. To investigate the oxidative stress effects and the mechanisms of ceria-ENM toxicity, fully characterized ceria ENM of different sizes (~ 5nm, 15nm, 30nm, 55nm and nanorods) were systematically injected into rats intravenously in separate experiments. Three brain regions (hippocampus, cortex and cerebellum) were harvested from control and ceria treated rats after various exposure periods for oxidative stress assessment. The levels of oxidative stress markers viz. protein carbonyl (PC), 3-nitrotyrosine (3NT), and protein bound 4-hydroxy-2-trans-nonenal (HNE) were evaluated for each treatment in each control and treated rat organ. Further, the levels and activities of antioxidant proteins, such as catalase, glutathione peroxidase (GPx), glutathione reductase (GR), super oxide dismutase (SOD), were measured together with levels of heat shock proteins heme oxygenase -1 and 70 (HO-1 and Hsp-70). In addition, the levels of pro-inflammatory cytokines IL-1β, TNF-α, pro-caspase-3, and autophagy marker LC-3A/B were measured by Western blot technique. In agreement with the literature-proposed model of oxidative stress hierarchy mechanism of ENM-toxicity, the statistical analysis of all the results revealed that the ceria ENM-induced oxidative stress mediated biological response strongly depends on the exposure period and to some extent on the size of ceria ENM. More specifically, a single intravenous injection of ceria ENM induced tier-1 (phase-II antioxidant) response after shorter exposure periods (1 h and 20 h) in rat brain. Upon failure of tier-1 response after longer exposure periods (1 d to 30 d), escalated oxidative stress consequently induced tier-2 and tier-3 oxidative stress responses. Based on our observations made at chronic exposure period (90 d) after the single i.v. injection of ceria ENM, we could extend the model of oxidative stress hierarchy mechanisms for ceria-ENM-induced toxicity. Considering the evaluation of all the oxidative stress indices measured in 3-brain regions, oxidative stress effects were more prominent in hippocampus and the least in cerebellum, but no specific pattern or any significant difference was deduced.

Esta tese de doutoramento relata estudos sobre a interação de nanotubos de carbono com nanomateriais, biomoléculas e células, com o propósito de obter informações relevantes para o desenvolvimento de biossensores e para o campo da nanotoxicologia. No primeiro estudo, foram produzidos e caracterizados três tipos de eletrodos modificados com filmes multicamadas obtidos através da técnica de automontagem. Estes filmes continham nanotubos de carbono de parede simples (SWNT), ftalocianina tetrasulfonada de níquel (NiTsPc) e o dendrímero poli(amidoamina) de geração 2 (PAMAM G2), e estes polieletrólitos foram organizados nos seguintes sistemas: (PAMAM G2/NiTsPc), (PAMAM G2/SWNT) e (nanocompósito SWNT/PAMAM G2 / NiTsPc). Medidas de voltametria cíclica com a sonda ferrocianeto de potássio revelaram que os três sistemas podem ser aplicados como eletrodos descartáveis por serem instáveis e que os dois sistemas com NiTsPc apresentam um amplo intervalo de potencial para detecção de analitos sem a interferência dos picos redox da Pc. Também foram conduzidos ensaios de citometria de fluxo para avaliação da toxicidade de nanocompósitos contendo nanotubos de carbono e poli(amidoamina) de gerações 2, 4 e 6 em células F C3H, correspondentes a fibroblastos saudáveis de fígado humano. Os resultados mostraram que o contato com os nanomateriais provoca uma queda significativa na viabilidade deste tipo de célula, e apontam para a necessidade de aprofundar a investigação sobre os efeitos biológicos deste nanocompósito para que ele seja aplicado com segurança como um vetor de drogas e material genético. A última parte da tese é dedicada a explorar ferramentas computacionais para elucidar os mecanismos de formação do nanocompósito SWNT/PAMAM G2 e sua interação com modelos de membrana celular. Simulações por dinâmica molecular revelaram que a estabilidade do nanocompósito é mantida por interações entre as paredes apolares dos nanotubos e as cadeias internas não polares do dendrímero. O estudo envolvendo bicamadas lipídicas sugeriu que a presença de espécies aniônicas, como as fosfatidilserinas, é crucial para iniciar a ligação desta nanopartícula à membrana celular. O contato do nanocompósito com a bicamada resultou na extração destrutiva de lipídeos da membrana, um efeito deletério que pode causar danos às células. / This thesis describes the interaction between carbon nanotubes and nanomaterials, biomolecules and cells, to obtain relevant information for the development of biosensors and the progress of the nanotoxicology field. In a first study, we produced and characterized three types of modified electrodes made from layer-by-layer films. These nanostructures had single-walled carbon nanotubes (SWNT), nickel tetrasulfonated phthalocyanine (NiTsPc) and poly(amidoamine) dendrimer, generation 2 (PAMAM G2). These polyelectrolytes were organized in the following multilayers: (PAMAM G2/NiTsPc), (PAMAM G2/SWNT) and (SWNT/PAMAM G2 nanocomposite / NiTsPc). Cyclic voltammetry measurements with a potassium ferrocyanide probe revealed that the three systems can be used as disposable electrodes for being unstable and that the two systems with NiTsPc exhibit a wide useful potential interval for detection without the interference of the Pc redox peaks. We also performed flow cytometry experiments to evaluate the toxicity of nanocomposites containing carbon nanotubes and poly(amidoamine) generations 2, 4 and 6 in F C3H cells, derived from healthy human liver fibroblasts. The results showed that the contact with these nanomaterials decreases the viability of this type of cell and point to the need to further determine the biological effects of this nanocomposite before it can be safely applied as a vector for drugs and genetic material. The last part of the thesis explores computational tools to unravel the mechanisms behind the formation of the nanocomposite SWNT/PAMAM G2 and its interaction with cell membrane models. Molecular dynamics simulations revealed that the stability of the nanocomposite is kept mainly by interactions between the apolar nanotube walls and the inner non polar chains of the dendrimer. The study involving lipid bilayers suggested that the presence of anionic species, such as phosphatidylserines, is crucial to trigger the binding of this nanoparticle to the cell membrane. The contact of the nanocomposite with the bilayer resulted in the destructive extraction of lipids from the membrane, an effect that ultimately causes cell damage.

Esta tese de doutoramento relata estudos sobre a interação de nanotubos de carbono com nanomateriais, biomoléculas e células, com o propósito de obter informações relevantes para o desenvolvimento de biossensores e para o campo da nanotoxicologia. No primeiro estudo, foram produzidos e caracterizados três tipos de eletrodos modificados com filmes multicamadas obtidos através da técnica de automontagem. Estes filmes continham nanotubos de carbono de parede simples (SWNT), ftalocianina tetrasulfonada de níquel (NiTsPc) e o dendrímero poli(amidoamina) de geração 2 (PAMAM G2), e estes polieletrólitos foram organizados nos seguintes sistemas: (PAMAM G2/NiTsPc), (PAMAM G2/SWNT) e (nanocompósito SWNT/PAMAM G2 / NiTsPc). Medidas de voltametria cíclica com a sonda ferrocianeto de potássio revelaram que os três sistemas podem ser aplicados como eletrodos descartáveis por serem instáveis e que os dois sistemas com NiTsPc apresentam um amplo intervalo de potencial para detecção de analitos sem a interferência dos picos redox da Pc. Também foram conduzidos ensaios de citometria de fluxo para avaliação da toxicidade de nanocompósitos contendo nanotubos de carbono e poli(amidoamina) de gerações 2, 4 e 6 em células F C3H, correspondentes a fibroblastos saudáveis de fígado humano. Os resultados mostraram que o contato com os nanomateriais provoca uma queda significativa na viabilidade deste tipo de célula, e apontam para a necessidade de aprofundar a investigação sobre os efeitos biológicos deste nanocompósito para que ele seja aplicado com segurança como um vetor de drogas e material genético. A última parte da tese é dedicada a explorar ferramentas computacionais para elucidar os mecanismos de formação do nanocompósito SWNT/PAMAM G2 e sua interação com modelos de membrana celular. Simulações por dinâmica molecular revelaram que a estabilidade do nanocompósito é mantida por interações entre as paredes apolares dos nanotubos e as cadeias internas não polares do dendrímero. O estudo envolvendo bicamadas lipídicas sugeriu que a presença de espécies aniônicas, como as fosfatidilserinas, é crucial para iniciar a ligação desta nanopartícula à membrana celular. O contato do nanocompósito com a bicamada resultou na extração destrutiva de lipídeos da membrana, um efeito deletério que pode causar danos às células. / This thesis describes the interaction between carbon nanotubes and nanomaterials, biomolecules and cells, to obtain relevant information for the development of biosensors and the progress of the nanotoxicology field. In a first study, we produced and characterized three types of modified electrodes made from layer-by-layer films. These nanostructures had single-walled carbon nanotubes (SWNT), nickel tetrasulfonated phthalocyanine (NiTsPc) and poly(amidoamine) dendrimer, generation 2 (PAMAM G2). These polyelectrolytes were organized in the following multilayers: (PAMAM G2/NiTsPc), (PAMAM G2/SWNT) and (SWNT/PAMAM G2 nanocomposite / NiTsPc). Cyclic voltammetry measurements with a potassium ferrocyanide probe revealed that the three systems can be used as disposable electrodes for being unstable and that the two systems with NiTsPc exhibit a wide useful potential interval for detection without the interference of the Pc redox peaks. We also performed flow cytometry experiments to evaluate the toxicity of nanocomposites containing carbon nanotubes and poly(amidoamine) generations 2, 4 and 6 in F C3H cells, derived from healthy human liver fibroblasts. The results showed that the contact with these nanomaterials decreases the viability of this type of cell and point to the need to further determine the biological effects of this nanocomposite before it can be safely applied as a vector for drugs and genetic material. The last part of the thesis explores computational tools to unravel the mechanisms behind the formation of the nanocomposite SWNT/PAMAM G2 and its interaction with cell membrane models. Molecular dynamics simulations revealed that the stability of the nanocomposite is kept mainly by interactions between the apolar nanotube walls and the inner non polar chains of the dendrimer. The study involving lipid bilayers suggested that the presence of anionic species, such as phosphatidylserines, is crucial to trigger the binding of this nanoparticle to the cell membrane. The contact of the nanocomposite with the bilayer resulted in the destructive extraction of lipids from the membrane, an effect that ultimately causes cell damage.

Knowledge of cell electronics has led to their integration to medicine either by physically interfacing electronic devices with biological systems or by using electronics for both detection and characterization of biological materials. In this dissertation, an electrical impedance sensor (EIS) was used to measure the electrode surface impedance changes from cell samples of human and environmental toxicity of nanoscale materials in 2D and 3D cell culture models. The impedimetric response of human lung fibroblasts and rainbow trout gill epithelial cells when exposed to various nanomaterials was tested to determine their kinetic effects towards the cells and to demonstrate the biosensor’s ability to monitor nanotoxicity in real-time. Further, the EIS allowed rapid, real-time and multi-sample analysis creating a versatile, noninvasive tool that is able to provide quantitative information with respect to alteration in cellular function. We then extended the application of the unique capabilities of the EIS to do real-time analysis of cancer cell response to externally applied alternating electric fields at different intermediate frequencies and low-intensity. Decreases in the growth profiles of the ovarian and breast cancer cells were observed with the application of 200 and 100 kHz, respectively, indicating specific inhibitory effects on dividing cells in culture in contrast to the non-cancerous HUVECs and mammary epithelial cells. We then sought to enhance the effects of the electric field by altering the cancer cell’s electronegative membrane properties with HER2 antibody functionalized nanoparticles. An Annexin V/EthD-III assay and zeta potential were performed to determine the cell death mechanism indicating apoptosis and a decrease in zeta potential with the incorporation of the nanoparticles. With more negatively charged HER2-AuNPs attached to the cancer cell membrane, the decrease in membrane potential would thus leave the cells more vulnerable to the detrimental effects of the applied electric field due to the decrease in surface charge. Therefore, by altering the cell membrane potential, one could possibly control the fate of the cell. This whole cell-based biosensor will enhance our understanding of the responsiveness of cancer cells to electric field therapy and demonstrate potential therapeutic opportunities for electric field therapy in the treatment of cancer.

Cerium oxide nanoparticles (nanoceria) have shown great potential as antioxidant and radioprotective agents for applications in cancer therapy. Recently, various polymer-coated nanoceria preparations have been developed to improve their aqueous solubility and allow for surface functionalization of these nanoparticles. However, the interaction of polymer-coated nanoceria with cells, their uptake mechanism, and subcellular localization are poorly understood. Herein, we engineered polymer-coated cerium oxide nanoparticles with different surface charges (positive, negative, and neutral) and studied their internalization and toxicity in normal and cancer cell lines. The results showed that nanoceria with a positive or neutral charge enters most of the cell lines studied, while nanoceria with a negative charge internalizes mostly in the cancer cell lines. Moreover, upon entry into the cells, nanoceria is localized to different cell compartments (e.g., cytoplasm and lysosomes) depending on the nanoparticles surface charge. The internalization and subcellular localization of nanoceria plays a key role in the nanoparticles cytotoxicity profile, exhibiting significant toxicity when they localize in the lysosomes of the cancer cells. In contrast, minimal toxicity is observed when they localize into the cytoplasm or do not enter the cells. Taken together, these results indicate that the differential surface-charge-dependent localization of nanoceria in normal and cancer cells plays a critical role in the nanoparticles toxicity profile.