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Scholarly Works (9 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2022)

The ascension of nanomaterials in the past decade for sensing, imaging, and delivery platforms in the biomedical fields is attributable to their unique physiochemical properties. It is understood that upon incubation with biological media, pristine nanomaterials will inherit a biological identity through the adsorption of biomolecules onto the material surfaces. The attached biomolecules, also known as the biocorona, can govern the extent of toxicity of the xenobiotic nanomaterial; in some cases, the biocorona can impede toxicity, while in other cases, it can exacerbate nanomaterial lethality. Yet, there is still an enormous knowledge gap regarding how nano-sized materials interact with biomolecules and impact biological systems.

In this dissertation, the toxicological profile of 40 engineered nanomaterials were assessed in mammary epithelial cells, as detailed in Chapter 1. From this collection, a subset were chosen for further investigation which are detailed in the following chapters. Tungsten disulfide nanosheets were further evaluated in Chapter 2 for their toxicity in lung epithelial cells. Additionally, the mechanism of cell death and impact on the mitochondria were assessed following cellular exposure to tungsten disulfide nanosheets. Since this material was passivated with a surfactant there was no formation of biocorona and thus, toxicity observed was due to the material itself. In Chapter 3, there is a transition into evaluating the toxicity of reduced graphene oxide nanosheets by modulating their surface chemistry with the formation of protein corona. Specifically, reducing the surfactant concentration on reduced graphene oxide nanosheets permitted the adsorption of larger quantities of proteins. Moreover, characterization of the protein corona correlated with the measured biological impacts. In Chapter 4, the lipid and protein corona, also known as the biocorona, were extracted from digested food-grade titanium dioxide nanomaterials for characterization. The bioinformatic profile corroborates previous findings our collaborators observed in a tri-culture small intestinal epithelial model. Lastly, the biocorona from digested polyethylene-based nanoplastics were extracted and characterized in Chapter 5. The lipid and protein profile from the nanoplastics’ biocorona also correlated with the biological impacts observed in the tri-culture model of gastrointestinal cells. This work utilizes analytical chemistry and molecular biology techniques to gauge nanomaterial toxicity, isolate and characterize biocoronas, and identify correlations between biocoronas and biological impacts observed in lung, gastrointestinal and mammary cells. The approach taken in this work adds fundamental knowledge in understanding the impact nanomaterials have on different biological systems as well as the complexities of biocorona formation for future developments of safer nanotechnologies for biological applications.

    Cover page: On Nano-Bio Interactions: Investigation of Biological Impacts Induced by Nanomaterials & BiocoronasCreative Commons 'BY' version 4.0 license
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2020)
    Increasing applications of nanomaterials in consumer goods, industrial products, medical practices, etc., calls for the development of tools for rapid separation, quantification, and sizing of nanoparticles to ensure their safe and sustainable employment. While many techniques are available for characterization of pure, homogeneous nanomaterial preparations, particle sizing and counting remains difficult for heterogeneous mixtures that resulted from imperfect synthesis conditions, aggregation from product instability, or degradation during storage. Herein, nanoparticle tracking analysis (NTA) was coupled to asymmetrical flow field flow fraction (AF4) using a splitter manifold to enable online particle separation and counting. The high pressure and flow rate in AF4 were reduced to the levels compatible with NTA by the proper flow splitting design, and a syringe pump was employed to withdraw fluid through the exit port of the NTA and maintain consistent flow rates entering NTA for proper particle sizing. Successful AF4-NTA coupling was demonstrated by analyzing a mixture of polystyrene particles with the average diameters of ∼50, 100, and 200 nm. Good correlation was observed between the amount of each type of particle injected to and measured by the hyphenated system. The particle concentrations acquired using online and offline coupling of AF4-NTA also agreed well with each other. The nonspherical nanoparticles like gold nanorods and hexagonal boron nitride nanosheets were also analyzed to demonstrate the versatile applicability of this system. Our work has proved that AF4-NTA can achieve accurate online particle counting on different populations of the nanomaterials in a mixture, which cannot be done by either AF4 or NTA alone. It will be a valuable tool for rapid characterization of heterogeneous nanomaterial solutions without purification to fulfill the regulation requirement on the nanomaterial-containing products.
      Cover page: Asymmetrical Flow Field Flow Fractionation Coupled to Nanoparticle Tracking Analysis for Rapid Online Characterization of Nanomaterials.
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2023)
      Escalating the production and application of tungsten disulfide (WS2) nanosheets inevitably increases environmental human exposure and warrants the necessity of studies to elucidate their biological impacts. Herein, we assessed the toxicity of WS2 nanosheets and focused on the impacts of low doses (≤10 μg/mL) on normal (BEAS-2B) and tumorigenic (A549) lung epithelial cells. The low doses, which approximate real-world exposures, were found to induce cell apoptosis, while doses ≥ 50 μg/mL cause necrosis. Focused studies on low-dose exposure to WS2 nanosheets revealed more details of the impacts on both cell lines, including reduction of cell metabolic activity, induction of lipid peroxidation in cell membranes, and uncoupling of mitochondrial oxidative phosphorylation that led to the loss of ATP production. These phenomena, along with the expression situations of a few key proteins involved in apoptosis, point toward the occurrence of mitochondria-dependent apoptotic signaling in exposed cells. Substantial differences in responses to WS2 exposure between normal and tumorigenic lung epithelial cells were noticed as well. Specifically, BEAS-2B cells experienced more adverse effects and took up more nanosheets than A549 cells. Our results highlight the importance of dose and cell model selection in the assessment of nanotoxicity. By using doses consistent with real-world exposures and comparing normal and diseased cells, we can gain knowledge to guide the development of safety precautions for mitigating the adverse impacts of nanomaterial exposure on human health.
        Cover page: Low-Dose Exposure of WS2 Nanosheets Induces Differential Apoptosis in Lung Epithelial CellsCreative Commons 'BY-NC-ND' version 4.0 license
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2019)
        The molecular structure of a protein could be altered when it is attached to nanoparticles (NPs), affecting the performance of NPs present in biological systems. Limited proteolysis coupled with LC-MS/MS could reveal the changes in protein structure when it binds to a variety of entities, including macro-molecules and small drugs, but it has not yet been applied to study protein-NP interaction. Herein, adsorption of proteins, transferrin, and catalase on the polystyrene (PS) or iron oxide (IO) NPs was analyzed with this method. Both increased and decreased proteolytic efficiency in certain regions on the proteins were observed. Identification of the peptides affected by protein-NP interaction led to proper prediction of alterations to protein function as well as to colloidal stability of NPs. Overall, the present work has demonstrated the utility of limited proteolysis in helping to elucidate the potential biological outcomes of the protein-NP conjugate, obtaining knowledge to guide improvement of the rational design of the protein-conjugated NPs for biomedical applications and to understand the biological behaviors of the engineered NPs.
          Cover page: Mapping Molecular Structure of Protein Locating on Nanoparticles with Limited Proteolysis
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2020)
          In the presence of biological matrices, engineered nanomaterials, such as TiO2, develop a biomolecular corona composed of lipids, proteins, etc. In this study, we analyzed the biocorona formed on the food grade TiO2 (E171) going through an in vitro simulated gastrointestinal digestion system in either a fasting food model (FFM), a standardized food model (SFM), or a high fat food model (HFFM). Lipids and proteins were extracted from the biocorona and underwent untargeted lipidomic and label-free shotgun proteomic analyses. Our results showed that the biocorona composition was different before and after food digestion. After digestion, more diverse lipids were adsorbed compared to proteins, most of which were the enzymes added to the simulated digestion system. The corona lipid profile was distinct from the digested food model they presented in, although similarity in the lipid profiles between the corona and the food matrix increased with the fat content in the food model. The corona formed in the two low-fat environments of FFM and SFM shared a higher degree of similarity while very different from their corresponding matrix, with some lipid species adsorbed with high enrichment factors, indicating specific interaction with the TiO2 surface outperforming lipid matrix concentration in determination of corona formation. Formation of the biocorona may have contributed to the reduced oxidative stress as well as toxicological impacts observed in cellular studies. The present work is the first to confirm persistent adsorption of biomolecules could occur on ingested nanomaterials in food digestae. More future studies are needed to study the in vivo impacts of the biocorona, and shed lights on how the biocorona affects the biotransformations and fate of the ingested nanomaterials, which may impose impacts on human health.
            Cover page: Lipid and protein corona of food-grade TiO2 nanoparticles in simulated gastrointestinal digestion
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2022)
            Despite mounting evidence of micro-nanoplastics (MNPs) in food and drinking water, little is known of the potential health risks of ingested MNPs, and nothing is known of their potential impact on nutrient digestion and absorption. We assessed the effects of environmentally relevant secondary MNPs generated by incineration of polyethylene (PE-I), on digestion and absorption of fat in a high fat food model using a 3-phase in vitro simulated digestion coupled with a tri-culture small intestinal epithelium model. The presence of 400 μg/mL PE-I increased fat digestion by 33% and increased fat absorption by 147 and 145% 1 and 2 h after exposure. Analysis of the PE-I lipid corona during digestion revealed predominantly triacylglycerols with enrichment of fatty acids in the small intestinal phase. Protein corona analysis showed enrichment of triacylglycerol lipase and depletion of β-casein in the small intestinal phase. These findings suggest digestion of triacylglycerol by lipase on the surface of lipid-coated MNPs as a potential mechanism. Further studies are needed to investigate the mechanisms underlying the greater observed increase in fat absorption, to verify these results in an animal model, and to determine the MNP properties governing their effects on lipid digestion and absorption.
              Cover page: Incineration-Generated Polyethylene Micro-Nanoplastics Increase Triglyceride Lipolysis and Absorption in an In Vitro Small Intestinal Epithelium Model
              • Article
              • Peer Reviewed
              UC Riverside Previously Published Works (2020)
              Effective in silico methods to predict protein corona compositions on engineered nanomaterials (ENMs) could help elucidate the biological outcomes of ENMs in biosystems without the need for conducting lengthy experiments for corona characterization. However, the physicochemical properties of ENMs, used as the descriptors in current modeling methods, are insufficient to represent the complex interactions between ENMs and proteins. Herein, we utilized the fluorescence change (FC) from fluorescamine labeling on a protein, with or without the presence of the ENM, as a novel descriptor of the ENM to build machine learning models for corona formation. FCs were significantly correlated with the abundance of the corresponding proteins in the corona on diverse classes of ENMs, including metal and metal oxides, nanocellulose, and 2D ENMs. Prediction models established by the random forest algorithm using FCs as the ENM descriptors showed better performance than the conventional descriptors, such as ENM size and surface charge, in the prediction of corona formation. Moreover, they were able to predict protein corona formation on ENMs with very heterogeneous properties. We believe this novel descriptor can improve in silico studies of corona formation, leading to a better understanding on the protein adsorption behaviors of diverse ENMs in different biological matrices. Such information is essential for gaining a comprehensive view of how ENMs interact with biological systems in ENM safety and sustainability assessments.
                Cover page: Prediction of protein corona on nanomaterials by machine learning using novel descriptors
                • Article
                • Peer Reviewed
                UC Riverside Previously Published Works (2022)
                Applications of reduced graphene oxide (rGO) in many different areas have been gradually increasing owing to its unique physicochemical characteristics, demanding more understanding of their biological impacts. Herein, we assessed the toxicological effects of rGO in mammary epithelial cells. Because the as-synthesized rGO was dissolved in sodium cholate to maintain a stable aqueous dispersion, we hypothesize that changing the cholate concentration in the dispersion may alter the surface property of rGO and subsequently affect its cellular toxicity. Thus, four types of rGO were prepared and compared: rGO dispersed in 4 and 2 mg/mL sodium cholate, labeled as rGO and concentrated-rGO (c-rGO), respectively, and rGO and c-rGO coated with a protein corona through 1 h incubation in culture media, correspondingly named pro-rGO and pro-c-rGO. Notably, c-rGO and pro-c-rGO exhibited higher toxicity than rGO and pro-rGO and also caused higher reactive oxygen species production, more lipid membrane peroxidation, and more significant disruption of mitochondrial-based ATP synthesis. In all toxicological assessments, pro-c-rGO induced more severe adverse impacts than c-rGO. Further examination of the material surface, protein adsorption, and cellular uptake showed that the surface of c-rGO was coated with a lower content of surfactant and adsorbed more proteins, which may result in the higher cellular uptake observed with pro-c-rGO than pro-rGO. Several proteins involved in cellular redox mediation were also more enriched in pro-c-rGO. These results support the strong correlation between dispersant coating and corona formation and their subsequent cellular impacts. Future studies in this direction could reveal a deeper understanding of the correlation and the specific cellular pathways involved and help gain knowledge on how the toxicity of rGO could be modulated through surface modification, guiding the sustainable applications of rGO.
                  Cover page: Biological Impacts of Reduced Graphene Oxide Affected by Protein Corona Formation
                  • Article
                  • Peer Reviewed
                  UC Riverside Previously Published Works (2021)
                  Extracellular vesicles (EVs) are essential intercellular communicators that are of increasing interest as diagnostic biomarkers. Exploring their biological functions and clinical values, however, remains challenging due to their small sizes and high heterogeneity. Herein, we report an ultrasensitive method that employs target-initiated construction of DNA nanostructure to detect single EVs with an input as low as 100 vesicles/μL. Taking advantage of both DNA nanostructure labeling and EV membrane staining, the method can also permit calibration-free analysis of the protein profiles among different EV samples, leading to clear EV differentiation by their cell of origin. Moreover, this method allows co-localization of dual protein markers on the same EV, and the increased number of EVs carrying dual tumor proteins present in human serum could differentiate cancer patients at the early developmental stage from healthy controls. Our results demonstrate the great potential of this single-EV visualization method in non-invasive detection of the EV-based protein biomarkers for cancer diagnosis and treatment monitoring.
                    Cover page: Calibration-free analysis of surface proteins on single extracellular vesicles enabled by DNA nanostructure
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