Physiological and Environmental Impacts of Nanomaterials-Biomolecular Coronae

Engineered nanomaterials (ENMs) exhibit unique electronic and optical properties, which are suitable for the broad spectrum of applications including biomedical, aerospace, textiles, agriculture. Such unique properties of ENMs also raise significant concerns over their adverse ecological and physiological effects. In the past two decades, ENMs were found to exhibit complex interactions in biological milieu. It is expected that physicochemical properties of ENMs and their surrounding environment influence their environmental and physiological fate and transport. Indeed, a comprehensive understanding of interactions at the nano-bio interface is critical to designing environmentally and physiologically benign ENMs pivotal to the future growth of nanomedicine and its applications. To this end, the present work experimentally investigates nanomaterial-biomolecular corona (or biocorona) using spectroscopic techniques and biological assays. Specifically, the influence of defects, surface functionalization, and physiological environment on the formation of biocorona and ensuing biological responses is presented. The first two chapters (Chapters 1 and 2) provide a succinct introduction to nanotoxicity with focus on biocorona formation and various spectroscopic characterization techniques utilized. In Chapter 3, the variation in biocorona formation due to defects in the structure of single-walled and multi-walled carbon nanotubes (SWCNTs and MWCNTs) is presented. Briefly, it was found that defects result in significant changes in the nature and amount of proteins adsorbed on SWCNTs due to differences in their charge distribution. In case of MWCNTs, carboxylated MWCNTs with higher density of defects resulted in a stronger binding and efficient delivery of antigens. In Chapter 4, a unique study exploring the effect of diseased (hyperlipidemic) physiological state on the biocorona composition (instead of the normal healthy state) is presented. Our findings suggest that disease-induced variations in the physiological environment have a significant impact on nanomaterial-biomolecular corona, cell response, and cellular association. In Chapter 5 provides new spectroscopic insights into charge transfer interactions between biomolecules (aromatic amino acid complexes) and emerging two-dimensional (2D) ENMs like graphene, graphene oxide, and BN. The adsorption of amino acids on 2D materials was observed to considerably alter their biological response in terms of generation of reactive oxygen species. Also, the influence of different surface functional groups on Ag nanoparticles on conformational changes in apolipoprotein biocorona and ensuing biological is explored. Finally, Chapter 6 explores environmental effects of graphene, BN upon their interaction with natural organic matter (NOM). The results show that the delocalized π-electron cloud in graphene facilitated a significant charge transfer and chemisorption of NOM unlike BN. Also, BN was found to result in algal cell rupture and reduction in photosynthetic activity unlike graphene

Ultrasensitive Immunosensing Platform Based on Analyte Induced Disruption of Luminescence Quenching (AIDLuQ)

In this study, we design an extremely fast and sensitive immunosensing platform using graphene as the sensing platform. A solution containing a mixture of graphene nanoplatelets and gold nanoparticles was coated on to a copier paper using a spray gun to form a uniform coating. Fluorescent quantum dots (QDs) functionalized with antibodies (Ab) were drop casted on to this platform, whose fluorescence was quenched by the graphene on the graphene/gold paper. With the addition of the antigen to this graphene/gold-QD-Ab complex, a disruption of quenching was observed, and the fluorescence intensity increased with increasing concentration of the antigen. A detection limit of as low as 10 fM was obtained for the detection of human Immunoglobulin G (IgG)

Spectroscopic Insights into the Nano-Bio Interface

Engineered nanomaterials (ENMs) strongly interact with biomolecules due to their unique physicochemical properties. From the standpoint of nanotoxicity, it is imperative to achieve a comprehensive understanding of various nano-bio interactions to ultimately design benign ENMs that do not elicit adverse physiological responses. Spectroscopic tools are ideal for elucidating the underlying biophysical mechanisms of nano-bio interactions. In this chapter, we review spectroscopy techniques, such as Raman, infrared, circular dichroism (CD), and hyperspectral imaging, to illuminate the nano-bio interface. Particularly, we discuss the role of spectroscopic tools in gaining a fundamental understanding of the formation and influence of protein corona on ENM physiological responses

Variations in biocorona formation related to defects in the structure of single walled carbon nanotubes and the hyperlipidemic disease state

Ball-milling utilizes mechanical stress to modify properties of carbon nanotubes (CNTs) including size, capping, and functionalization. Ball-milling, however, may introduce structural defects resulting in altered CNT-biomolecule interactions. Nanomaterial-biomolecule interactions result in the formation of the biocorona (BC), which alters nanomaterial properties, function, and biological responses. The formation of the BC is governed by the nanomaterial physicochemical properties and the physiological environment. Underlying disease states such as cardiovascular disease can alter the biological milieu possibly leading to unique BC identities. In this ex vivo study, we evaluated variations in the formation of the BC on single-walled CNTs (SWCNTs) due to physicochemical alterations in structure resulting from ball-milling and variations in the environment due to the high-cholesterol disease state. Increased ball-milling time of SWCNTs resulted in enhanced structural defects. Following incubation in normal mouse serum, label-free quantitative proteomics identified differences in the biomolecular content of the BC due to the ball-milling process. Further, incubation in cholesterol-rich mouse serum resulted in the formation of unique BCs compared to SWCNTs incubated in normal serum. Our study demonstrates that the BC is modified due to physicochemical modifications such as defects induced by ball-milling and physiological disease conditions, which may result in variable biological responses

MEMO: Dataset and Methods for Robust Multimodal Retinal Image Registration with Large or Small Vessel Density Differences

The measurement of retinal blood flow (RBF) in capillaries can provide a powerful biomarker for the early diagnosis and treatment of ocular diseases. However, no single modality can determine capillary flowrates with high precision. Combining erythrocyte-mediated angiography (EMA) with optical coherence tomography angiography (OCTA) has the potential to achieve this goal, as EMA can measure the absolute 2D RBF of retinal microvasculature and OCTA can provide the 3D structural images of capillaries. However, multimodal retinal image registration between these two modalities remains largely unexplored. To fill this gap, we establish MEMO, the first public multimodal EMA and OCTA retinal image dataset. A unique challenge in multimodal retinal image registration between these modalities is the relatively large difference in vessel density (VD). To address this challenge, we propose a segmentation-based deep-learning framework (VDD-Reg) and a new evaluation metric (MSD), which provide robust results despite differences in vessel density. VDD-Reg consists of a vessel segmentation module and a registration module. To train the vessel segmentation module, we further designed a two-stage semi-supervised learning framework (LVD-Seg) combining supervised and unsupervised losses. We demonstrate that VDD-Reg outperforms baseline methods quantitatively and qualitatively for cases of both small VD differences (using the CF-FA dataset) and large VD differences (using our MEMO dataset). Moreover, VDD-Reg requires as few as three annotated vessel segmentation masks to maintain its accuracy, demonstrating its feasibility.Comment: Submitted to IEEE JBH

Charge-transfer interactions induce surface dependent conformational changes in apolipoprotein biocorona

Upon introduction into a biological system, engineered nanomaterials (ENMs) rapidly associate with a variety of biomolecules such as proteins and lipids to form a biocorona. The presence of “biocorona” influences nano–bio interactions considerably, and could ultimately result in altered biological responses. Apolipoprotein A-I (ApoA-I), the major constituent of high-density lipoprotein (HDL), is one of the most prevalent proteins found in ENM-biocorona irrespective of ENM nature, size, and shape. Given the importance of ApoA-I in HDL and cholesterol transport, it is necessary to understand the mechanisms of ApoA-I adsorption and the associated structural changes for assessing consequences of ENM exposure. Here, the authors used a comprehensive array of microscopic and spectroscopic tools to elucidate the interactions between ApoA-I and 100 nm Ag nanoparticles (AgNPs) with four different surface functional groups. The authors found that the protein adsorption and secondary structural changes are highly dependent on the surface functionality. Our electrochemical studies provided new evidence for charge transfer interactions that influence ApoA-I unfolding. While the unfolding of ApoA-I on AgNPs did not significantly change their uptake and short-term cytotoxicity, the authors observed that it strongly altered the ability of only some AgNPs to generate of reactive oxygen species. Our results shed new light on the importance of surface functionality and charge transfer interactions in biocorona formation

Defect density in multiwalled carbon nanotubes influences ovalbumin adsorption and promotes macrophage activation and CD4(+) T-cell proliferation

Carbon nanotubes (CNTs) are of great interest for the development of drugs and vaccines due to their unique physicochemical properties. The high surface area to volume ratio and delocalized pi-electron cloud of CNTs promote binding of proteins to the surface forming a protein corona. This unique feature of CNTs has been recognized for potential delivery of antigens for strong and long-lasting antigen-specific immune responses. Based on an earlier study that demonstrated increased protein binding, we propose that carboxylated multiwalled CNTs (MWCNTs) can function as an improved carrier to deliver antigens such as ovalbumin (OVA). To test this hypothesis, we coated carboxylated MWCNTs with OVA and measured uptake and activation of antigen-presenting cells (macrophages) and their ability to stimulate CD4+ T-cell proliferation. We employed two types of carboxylated MWCNTs with different surface areas and defects (MWCNT-2 and MWCNT-30). MWCNT-2 and MWCNT-30 have surface areas of ~215 m2/g and 94 m2/g, respectively. The ratios of D- to G-band areas (ID/IG) were 0.97 and 1.37 for MWCNT-2 and MWCNT-30, respectively, samples showing that MWCNT-30 contained more defects. The increase in defects in MWCNT-30 led to increased binding of OVA as compared to MWCNT-2 (1,066±182 µg/mL vs 582±41 µg/mL, respectively). Both types of MWCNTs, along with MWCNT–OVA complexes, showed no observable toxicity to bone-marrow-derived macrophages up to 5 days. Surprisingly, we found that MWCNT–OVA complex significantly increased the expression of major histocompatibility complex class II on macrophages and production of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin 6), while MWCNTs without OVA protein corona did not. The coculture of MWCNT–OVA-complex-treated macrophages and OVA-specific CD4+ T-cells isolated from OT-II mice demonstrated robust proliferation of CD4+ T-cells. This study provides strong evidence for a role for defects in carboxylated MWCNTs and their use in the efficient delivery of antigens for the development of next-generation vaccines

Contribution of engineered nanomaterials physicochemical properties to mast cell degranulation

The rapid development of engineered nanomaterials (ENMs) has grown dramatically in the last decade, with increased use in consumer products, industrial materials, and nanomedicines. However, due to increased manufacturing, there is concern that human and environmental exposures may lead to adverse immune outcomes. Mast cells, central to the innate immune response, are one of the earliest sensors of environmental insult and have been shown to play a role in ENM-mediated immune responses. Our laboratory previously determined that mast cells are activated via a non-FcεRI mediated response following silver nanoparticle (Ag NP) exposure, which was dependent upon key physicochemical properties. Using bone marrow-derived mast cells (BMMCs), we tested the hypothesis that ENM physicochemical properties influence mast cell degranulation. Exposure to 13 physicochemically distinct ENMs caused a range of mast degranulation responses, with smaller sized Ag NPs (5 nm and 20 nm) causing the most dramatic response. Mast cell responses were dependent on ENMs physicochemical properties such as size, apparent surface area, and zeta potential. Surprisingly, minimal ENM cellular association by mast cells was not correlated with mast cell degranulation. This study suggests that a subset of ENMs may elicit an allergic response and contribute to the exacerbation of allergic diseases

Charge-transfer interactions induce surface dependent conformational changes in apolipoprotein biocorona

Upon introduction into a biological system, engineered nanomaterials (ENMs) rapidly associate with a variety of biomolecules such as proteins and lipids to form a biocorona. The presence of “biocorona” influences nano–bio interactions considerably, and could ultimately result in altered biological responses. Apolipoprotein A-I (ApoA-I), the major constituent of high-density lipoprotein (HDL), is one of the most prevalent proteins found in ENM-biocorona irrespective of ENM nature, size, and shape. Given the importance of ApoA-I in HDL and cholesterol transport, it is necessary to understand the mechanisms of ApoA-I adsorption and the associated structural changes for assessing consequences of ENM exposure. Here, the authors used a comprehensive array of microscopic and spectroscopic tools to elucidate the interactions between ApoA-I and 100 nm Ag nanoparticles (AgNPs) with four different surface functional groups. The authors found that the protein adsorption and secondary structural changes are highly dependent on the surface functionality. Our electrochemical studies provided new evidence for charge transfer interactions that influence ApoA-I unfolding. While the unfolding of ApoA-I on AgNPs did not significantly change their uptake and short-term cytotoxicity, the authors observed that it strongly altered the ability of only some AgNPs to generate of reactive oxygen species. Our results shed new light on the importance of surface functionality and charge transfer interactions in biocorona formation