High Aspect Ratio Nanomaterials Research Articles

Modular design of cyclic peptide – polymer conjugate nanotubes for delivery and tunable release of anti-cancer drug compounds

High aspect-ratio nanomaterials have recently emerged as promising drug delivery vehicles due to evidence of strong cellular association and prolonged in vivo circulation times. Cyclic peptide - polymer conjugate nanotubes are excellent candidates due to their elongated morphology, their supramolecular composition and high degree of pliability due to the versatility in manipulating amino acid sequence and polymer type. In this work, we explore the use of a nanotube structure on which a potent anti-cancer drug, camptothecin, is attached alongside hydrophilic or amphiphilic RAFT polymers, which shield the cargo. We show that subtle modifications to the cleavable linker type and polymer architecture have a dramatic influence over the rate of drug release in biological conditions. In vitro studies revealed that multiple cancer cell lines in 2D and 3D models responded effectively to the nanotube treatment, and analogous fluorescently labelled materials revealed key mechanistic information regarding the degree of cellular uptake and intracellular fate. Importantly, the ability to instruct specific drug release profiles indicates a potential for these nanomaterials as vectors which can provide sustained drug concentrations for a maximal therapeutic effect.

Pulmonary Toxicity of Boron Nitride Nanomaterials Is Aspect Ratio Dependent.

Boron nitride (BN) nanomaterials have drawn a lot of interest in the material science community. However, extensive research is still needed to thoroughly analyze their safety profiles. Herein, we investigated the pulmonary impact and clearance of two-dimensional hexagonal boron nitride (h-BN) nanosheets and boron nitride nanotubes (BNNTs) in mice. Animals were exposed by single oropharyngeal aspiration to h-BN or BNNTs. On days 1, 7, and 28, bronchoalveolar lavage (BAL) fluids and lungs were collected. On one hand, adverse effects on lungs were evaluated using various approaches (e.g., immune response, histopathology, tissue remodeling, and genotoxicity). On the other hand, material deposition and clearance from the lungs were assessed. Two-dimensional h-BN did not cause any significant immune response or lung damage, although the presence of materials was confirmed by Raman spectroscopy. In addition, the low aspect ratio h-BN nanosheets were internalized rapidly by phagocytic cells present in alveoli, resulting in efficient clearance from the lungs. In contrast, high aspect ratio BNNTs caused a strong and long-lasting inflammatory response, characterized by sustained inflammation up to 28 days after exposure and the activation of both innate and adaptive immunity. Moreover, the presence of granulomatous structures and an indication of ongoing fibrosis as well as DNA damage in the lung parenchyma were evidenced with these materials. Concurrently, BNNTs were identified in lung sections for up to 28 days, suggesting long-term biopersistence, as previously demonstrated for other high aspect ratio nanomaterials with poor lung clearance such as multiwalled carbon nanotubes (MWCNTs). Overall, we reveal the safer toxicological profile of BN-based two-dimensional nanosheets in comparison to their nanotube counterparts. We also report strong similarities between BNNTs and MWCNTs in lung response, emphasizing their high aspect ratio as a major driver of their toxicity.

36 An Investigation into Mechanisms Responsible for (geno)Toxicity Induced by Exposure to Industrially-Relevant Multi-Component Nanomaterials and high Aspect Ratio Nanomaterials

Abstract Production and application of multi-component nanomaterials (MCNMs) and high aspect ratio nanomaterials (HARN) has raised questions as to their inhalation risk. A PRISMA-orientated systematic literature review indicated a key knowledge gap related to the (geno)toxicity of metal oxide MCNMs and HARN. The aim of this study was to conduct a mechanistic-based toxicology assessment of a number of industrially-relevant MCNMs and HARN materials, in order to fill this knowledge gap and provide vital hazard assessment towards these materials’ safe and sustainable design. A549 monocultures (2.5x105), and A549/dTHP-1 co-cultures (1:10) were seeded in 12-well transwell-membranes at the air-liquid interface. Cell cultures were exposed, via aerosol, to Printex90, LaCo0.5Ni0.05O3, LaCo0.475Ni0.475Pt0.05O3, LaCo0.475Ni0.475Pd0.05O3, LaCo0.25Ni0.75O3, LaNiO3, NiZnFe4O8, ZnFe2O4, and NiFe2O4 to human exposure-relevant concentrations of 390, 780, 3100 ng/cm2 (24hrs). Aerosolised MCNM and HARN were characterised following acellular deposition via electron microscopy. Cellular cytotoxicity, barrier integrity, (pro)inflammatory/fibrotic response, oxidative stress and mutagenicity/DNA damage was assessed. Results indicate barrier integrity and IL-8 production were unaffected by Printex90, LaCo0.5Ni0.05O3, and ZnFe2O4 exposure. A dose-dependent increase in micronucleus frequency was observed for these four materials. Investigations are continuing to determine the mechanisms associated with this noted genotoxic effect, as well as upon further MCNMs/HARN (i.e. Fe2CoO4(Fe1/Co3), Silica-Ref-Std). Emphasis is being placed upon elucidating specific structure-activity relationships for each MCNM/HARN by testing specific key events of inhalation-based adverse outcome pathways relevant to the in vitro models used. This project was funded by EU H2020 research and innovation programme, grant agreement No. 953183 (HARMLESS).

Carbon nanotube pathogenicity conforms to a unified theory for mesothelioma causation by elongate materials and fibers

The purpose of this review is to elucidate how dimensional and durability characteristics of high aspect ratio nanomaterials (HARN), including carbon nanotubes (CNT) and metal nanowires (MeNW), contribute to understanding the fiber pathogenicity paradigm (FPP), including by explaining the structure-activity relationships (SAR) of a diverse range of natural and synthetic elongate materials that may or may not contribute to mesothelioma development in the lung. While the FPP was originally developed to explain the critical importance of asbestos and synthetic vitreous fiber length, width, aspect ratio and biopersistence in mesothelioma development, there are a vast number of additional inhalable materials that need to be considered in terms of pathogenic features that may contribute to mesothelioma or lack thereof. Not only does the ability to exert more exact control over the length and biopersistence of HARNs confirm the tenets of the FPP, but could be studied by implementating more appropriate toxicological tools for SAR analysis. This includes experimentation with carefully assembled libraries of CNTs and MeNWs, helping to establish more precise dimensional features for interfering in lymphatic drainage from the parietal pleura, triggering of lysosomal damage, frustrated phagocytosis and generation of chronic inflammation. The evidence includes data that long and rigid, but not short and flexible multi-wall CNTs are capable of generating mesotheliomas in rodents based on an adverse outcome pathway requiring access to pleural cavity, obstruction of pleural stomata, chronic inflammation and transformation of mesothelial cells. In addition to durability and dimensional characteristics, bending stiffness of CNTs is a critical factor in determining the shape and rigidity of pathogenic MWCNTs. While no evidence has been obtained in humans that CNT exposure leads to a mesothelioma outcome, it is important to monitor exposure levels and health effect impacts in workers to prevent adverse health outcomes in humans.

High aspect ratio nanomaterial-induced macrophage polarization is mediated by changes in miRNA levels.

Inhalation of nanomaterials may induce inflammation in the lung which if left unresolved can manifest in pulmonary fibrosis. In these processes, alveolar macrophages have an essential role and timely modulation of the macrophage phenotype is imperative in the onset and resolution of inflammatory responses. This study aimed to investigate, the immunomodulating properties of two industrially relevant high aspect ratio nanomaterials, namely nanocellulose and multiwalled carbon nanotubes (MWCNT), in an alveolar macrophage model. MH-S alveolar macrophages were exposed at air-liquid interface to cellulose nanocrystals (CNC), cellulose nanofibers (CNF) and two MWCNT (NM-400 and NM-401). Following exposure, changes in macrophage polarization markers and secretion of inflammatory cytokines were analyzed. Furthermore, the potential contribution of epigenetic regulation in nanomaterial-induced macrophage polarization was investigated by assessing changes in epigenetic regulatory enzymes, miRNAs, and rRNA modifications. Our data illustrate that the investigated nanomaterials trigger phenotypic changes in alveolar macrophages, where CNF exposure leads to enhanced M1 phenotype and MWCNT promotes M2 phenotype. Furthermore, MWCNT exposure induced more prominent epigenetic regulatory events with changes in the expression of histone modification and DNA methylation enzymes as well as in miRNA transcript levels. MWCNT-enhanced changes in the macrophage phenotype were correlated with prominent downregulation of the histone methyltransferases Kmt2a and Smyd5 and histone deacetylases Hdac4, Hdac9 and Sirt1 indicating that both histone methylation and acetylation events may be critical in the Th2 responses to MWCNT. Furthermore, MWCNT as well as CNF exposure led to altered miRNA levels, where miR-155-5p, miR-16-1-3p, miR-25-3p, and miR-27a-5p were significantly regulated by both materials. PANTHER pathway analysis of the identified miRNA targets showed that both materials affected growth factor (PDGF, EGF and FGF), Ras/MAPKs, CCKR, GnRH-R, integrin, and endothelin signaling pathways. These pathways are important in inflammation or in the activation, polarization, migration, and regulation of phagocytic capacity of macrophages. In addition, pathways involved in interleukin, WNT and TGFB signaling were highly enriched following MWCNT exposure. Together, these data support the importance of macrophage phenotypic changes in the onset and resolution of inflammation and identify epigenetic patterns in macrophages which may be critical in nanomaterial-induced inflammation and fibrosis.

Direct Piercing of Ag Nanowires into Living Macrophages: Implications for Nanotoxicity

High aspect ratio nanomaterials, such as nanowires, are known to exhibit unique properties and have a wide range of applications owing to their geometry and small diameters. Prior work has indicated that silver nanowires (AgNWs, more than 15 μm long) could lead to frustrated phagocytosis in lung macrophages, whose characteristics differ from those among the micro- and macro-wires with the same chemical compositions. This work unveils whether other pathways occur upon exposure of living cells to AgNWs. Using high-resolution laser scanning confocal microscopy, the precise locations of AgNWs with respect to cells can be visualized upon exposure of a macrophage cell line, NR8383 cells, to AgNWs. Cellular membranes and intracellular components, such as actin, are also tagged to allow direct and detailed visualization of cellular components with respect to AgNWs during exposures. Direct piercing of AgNWs into cells was captured, where a portion of wires was poked into a cell without significant structural perturbation of its local surroundings. While AgNWs were predicted to be able to poke through the cell membrane from computation studies, direct piercing has not been experimentally observed until the present work. Other scenarios include frustrated phagocytosis and failed attempts by AgNWs to pierce due to the membrane wrapping of the invading portion of the AgNWs resisting their entry. The comprehensive studies of AgNW–cell interactions are of fundamental importance in understanding nanocytotoxicity and nanotoxicity. The knowledge gained shall facilitate the development of preventive measures for the potential adversarial impacts of nanowires on the environment and human health, thus enabling broader applications of nanomaterials.

DNA delivery by high aspect ratio nanomaterials to algal chloroplasts

Single walled carbon nanotubes (SWCNTs) are coated with polyethylenimine (PEI) of different molecular weight for DNA delivery into algal chloroplasts without the use of mechanical aid for understanding their impact on cell viability and function.

Grouping MWCNTs based on their similar potential to cause pulmonary hazard after inhalation: a case-study

BackgroundThe EU-project GRACIOUS developed an Integrated Approach to Testing and Assessment (IATA) to support grouping high aspect ratio nanomaterials (HARNs) presenting a similar inhalation hazard. Application of grouping reduces the need to assess toxicity on a case-by-case basis and supports read-across of hazard data from substances that have the data required for risk assessment (source) to those that lack such data (target). The HARN IATA, based on the fibre paradigm for pathogenic fibres, facilitates structured data gathering to propose groups of similar HARN and to support read-across by prompting users to address relevant questions regarding HARN morphology, biopersistence and inflammatory potential. The IATA is structured in tiers, allowing grouping decisions to be made using simple in vitro or in silico methods in Tier1 progressing to in vivo approaches at the highest Tier3. Here we present a case-study testing the applicability of GRACIOUS IATA to form an evidence-based group of multiwalled carbon nanotubes (MWCNT) posing a similar predicted fibre-hazard, to support read-across and reduce the burden of toxicity testing.ResultsThe case-study uses data on 15 different MWCNT, obtained from the published literature. By following the IATA, a group of 2 MWCNT was identified (NRCWE006 and NM-401) based on a high degree of similarity. A pairwise similarity assessment was subsequently conducted between the grouped MWCNT to evaluate the potential to conduct read-across and fill data gaps required for regulatory hazard assessment. The similarity assessment, based on expert judgement of Tier 1 assay results, predicts both MWCNT are likely to cause a similar acute in vivo hazard. This result supports the possibility for read-across of sub-chronic and chronic hazard endpoint data for lung fibrosis and carcinogenicity between the 2 grouped MWCNT. The implications of accepting the similarity assessment based on expert judgement of the MWCNT group are considered to stimulate future discussion on the level of similarity between group members considered sufficient to allow regulatory acceptance of a read-across argument.ConclusionThis proof-of-concept case-study demonstrates how a grouping hypothesis and IATA may be used to support a nuanced and evidence-based grouping of ‘similar’ MWCNT and the subsequent interpolation of data between group members to streamline the hazard assessment process.

(Invited) Carbon Nanotubes for Plant Genetic Engineering

Plants will benefit from genetic engineering to feed our growing global population in the midst of climate change, to provide our most clinically-relevant drugs, and for our environmental remediation efforts. New generation genome engineering technologies, such as CRISPR/Cas, hold the potential to alter plant genes in a precise and targeted manner. The first step of any plant genetic engineering application is to deliver important biomolecular cargoes into the desired subcellular locations of plant cells. However, the limitations of traditional plant gene delivery methods, i.e. Agrobacterium and gene gun, prevent plant genetic engineering from reaching its full potential.Given the lack of efficient biomolecule delivery vehicles to plants and potential impact of plant biotechnology on food security and sustainability, we developed nanoparticle-based platforms to efficiently deliver small interfering RNA1,2 and plasmid DNA3,4 cargoes into plant cells in a species-independent manner, which eliminates the use of gene gun and addresses the main delivery bottlenecks of plant genetic engineering. We show that pristine single walled carbon nanotubes (SWCNTs) can adsorb small interfering RNA molecules through pi-pi stacking for plant delivery, whereas a covalently modified and positively charged SWCNT platform is more efficient for large double-stranded DNA delivery to mature plant leaf cells.This talk will first briefly summarize the development, characterization and biological verification of each nanoparticle platform for plant delivery applications. Next, I will present our recent results of CRISPR/Cas gene editing in plant leaves using the SWCNT DNA delivery platform5. The talk will be concluded by discussing the several assays performed to demonstrate the effect of nanoparticles on plant health, including an RNA-sequencing analysis of nanoparticle-treated plant leaves6. We believe the SWCNT delivery technologies that are discussed in this talk will have an immense impact on food security and sustainability by enabling the generation of improved plants that can rapidly reach the market in a cost-effective manner. Demirer G.S.*, Zhang H.* et al., DNA Nanostructures Coordinate Gene Silencing in Mature Plants. PNAS (2019). Demirer G.S. et al., Carbon Nanocarriers Deliver siRNA to Intact Plant Cells for Efficient Gene Knockdown. Science Advances (2020). Demirer G.S. et al., High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material Without Transgenic DNA Integration in Mature Plants. Nature Nanotechnology (2019). Demirer G.S. et al., Carbon nanotube–mediated DNA delivery without transgene integration in intact plants. Nature Protocols (2019). Demirer G.S. et al., Nanotechnology to advance CRISPR–Cas genetic engineering of plants. Nature Nanotechnology (2021).González-Grandío E., Demirer, G.S. et al. Carbon nanotube biocompatibility in plants is determined by their surface chemistry. bioRxiv (2021).

High-Speed Imaging of the Ultrasonic Deagglomeration of Carbon Nanotubes in Water

Ultrasonic treatment is effective in deagglomerating and dispersing nanoparticles in various liquids. However, the exact deagglomeration mechanisms vary for different nanoparticle clusters, owing to different particle geometries and inter-particle adhesion forces. Here, the deagglomeration mechanisms and the influence of sonotrode amplitude during ultrasonication of multiwall carbon nanotubes in de-ionized water were studied by a combination of high-speed imaging and numerical modeling. Particle image velocimetry was applied to images with a higher field of view to calculate the average streaming speeds distribution. These data allowed direct comparison with modeling results. For images captured at higher frame rates and magnification, different patterns of deagglomeration were identified and categorized based on different stages of cavitation zone development and for regions inside or outside the cavitation zone. The results obtained and discussed in this paper can also be relevant to a wide range of carbonaceous and other high aspect ratio nanomaterials.

Immobilization of FeFe-hydrogenase on black TiO2 nanotubes as biocathodes for the hydrogen evolution reaction

Hydrogenases are attractive biocatalysts for utilization in electrochemical devices as potential replacement for Pt in hydrogen evolving electrodes. In this work, we investigate the immobilization of ferredoxin tagged FeFe-hydrogenase (Fd-HydA1) on black TiO2 nanotubes (bTNTs), with uniform nanotube opening diameters of 140 nm. By utilizing an immunogold labelling method, we show that the enzymes attach on the top surface of the bTNTs film rather than on the inner nanotube walls, reflecting the difficulty to insert enzymes into high aspect-ratio nanomaterials for O2-shielding. Nevertheless, cyclic voltammetry demonstrates direct electron transfer between Fd-HydA1 and bTNTs for the hydrogen evolution reaction (HER) in neutral media. This work provides new insight towards design of new nanostructured electrodes for enzyme immobilization.

(Nanocarbons Division SES Young Investigator Award) Nanomaterials Engineering to Probe and Control Living Systems

Unique physical, chemical, and optical phenomena arise when materials are confined to the nano-scale. We are accustomed to making observations and predictions for the behavior of living systems on a macroscopic scale that is intuitive for the time and size scales of our day-to-day lives. However, the building blocks of life: proteins, nucleic acids, and cells, occupy different spatiotemporal scales. Our lab focuses on understanding and exploiting tunable optical and mechanical properties of nanomaterials to access information about biological systems stored at the nano-scale. In the context of leveraging nanomaterial optical properties, we present recent work on developing and implementing dopamine nanosensors to image dopamine volume transmission in the extracellular space of the brain striatum. We validate our dopamine nanosensor in acute striatal slices with electrical and optogenetic stimulation of dopamine release, and show disrupted dopamine release or reuptake kinetics when brain tissue is exposed to dopamine agonist or antagonist drugs [1,2]. In the context of leveraging nanomaterial chemical properties, we also discuss how high aspect ratio nanomaterials can be synthesized to carry biomolecular cargo to living systems. In particular, genetic engineering of plants is at the core of environmental sustainability efforts, but the physical barrier presented by the cell wall has limited the ease and throughput with which exogenous biomolecules can be delivered to plants. We will describe how nanomaterials engineering principles can be leveraged to manipulate living plants [3, 4], in efforts to reconcile the benefits of crop genetic engineering with the demand for non-GMO foods [5]. Our work in the agricultural space provides a promising tool for species-independent, targeted, and passive delivery of genetic material, without transgene integration, into plant cells for rapid and parallelizable testing of plant genotype-phenotype relationships.1. Beyene, A.B., McFarlane, I.R., Pinals, R.L, Landry, M.P.‡ Stochastic Simulation of Dopamine Neuromodulation for Implementation of Fluorescent Neurochemical Probes in the Striatal Extracellular Space. ACS Chemical Neuroscience 8 (10), 2275-2289 (2017).2. Beyene, A. G., Delevich, K., O’Donnell, J.T.D., Piekarski, D.J., Lin, W.C., Thomas, A.W., Yang, S.J., Kosillo, P., Yang, D., Wilbrecht, L., Landry, M.P.‡ Imaging Striatal Dopamine Release Using a Non-Genetically Encoded Near-Infrared Fluorescent Catecholamine Nanosensor. Science Advances 5 (7), 1-11 (2019)3. Demirer, G.S., Zhang, H., Matos, J., Goh, N., Cunningham, F.J., Sung, Y., Chang, R., Aditham, A.J., , Chio, L., Cho, M.J., Staskawicz, B., Landry, M.P.‡ High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material Without DNA Integration in Mature Plants. Nature Nanotechnology (2019). DOI: 10.1038/s41565-019-0382-5NNANO-180816844. Demirer, G.S., Zhang, H., Goh, N.S., Grandio, E.G., Landry, M.P.‡ Carbon nanotube-mediated DNA delivery without transgene integration in intact plants. Nature Protocols (2019). DOI: 0.1038/s41596-019-0208-95. Landry, M.P.‡, Mitter, N.‡ How nanocarriers delivering cargoes in plants can change the GMO landscape. Nature Nanotechnology 2019, 14; 512–514

A Protein Corona-Based Design Strategy for Carbon Nanotube Sensors

Unpredictable protein adsorption on nanoparticles remains a considerable challenge towards effectively applying nanotechnologies in biological environments. However, if well understood and controlled, proteins adsorbed on nanoparticles can be exploited to develop protein-based nanosensors. Herein, we employ our knowledge of spontaneous “protein corona” formation on single-walled carbon nanotubes (SWCNTs) in biofluids1 towards rational nanosensor design based on protein-SWCNT complexation.2 SWCNTs are implemented as the sensor platform due to their desirable optical and physical characteristics, including photostable fluorescence in the near-infrared tissue transparency window3 and high aspect ratio that confers delivery capabilities.4 We functionalize carbon nanotubes with proteins that, in turn, bind to their natural ligand partners. The adsorbed protein thus acts as the recognition moiety that interacts with the environment and detects the analyte of interest, tethered to the SWCNT that serves as the signal transducer. This nanosensor platform combines the sensitive and selective binding capabilities of proteins for their ligand partners with the near-infrared fluorescence readout of SWCNTs that is modulated upon protein-ligand binding. In this manner, we demonstrate that although formation of the nanoparticle protein corona can impede the intended nanobiotechnology application, it also presents an opportunity to create improved protein-nanoparticle architectures by manipulating selective protein adsorption on the nanoparticle surface to generate nanosensors for detection of SARS-CoV 2 infections.5 References Pinals, R.L., et al., Quantitative Protein Corona Composition and Dynamics on Carbon Nanotubes in Biological Environments. Angew Chem Int Ed Engl, 2020.doi:10.1101/2020.01.13.905356.Pinals, R. L., Yang, D., Lui, A., Cao, W. & Landry, M. P. Corona Exchange Dynamics on Carbon Nanotubes by Multiplexed Fluorescence Monitoring. J. Am. Chem. Soc. 142, 1254–1264 (2020).Boghossian, A. A. et al. Near-Infrared Fluorescent Sensors based on Single-Walled Carbon Nanotubes for Life Sciences Applications. ChemSusChem 4, 848–863 (2011).Demirer, G. S. et al. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat. Nanotechnol. 14, 456–464 (2019).Pinals, R., Ledesma, F., Yang, D., Jeong, S., Navarro, N., Pak, J.E., Landry, M.P. Rapid SARS-CoV-2 Detection by Carbon Nanotube-Based Near-Infrared Nanosensors. (2020) DOI: 10.1101/2020.11.02.20223404

An integrated approach to testing and assessment of high aspect ratio nanomaterials and its application for grouping based on a common mesothelioma hazard

Here we describe the development of an Integrated Approach to Testing and Assessment (IATA) to support the grouping of different types (nanoforms; NFs) of High Aspect Ratio Nanomaterials (HARNs), based on their potential to cause mesothelioma. Hazards posed by the inhalation of HARNs are of particular concern as they exhibit physical characteristics similar to pathogenic asbestos fibres. The approach for grouping HARNs presented here is part of a framework to provide guidance and tools to group similar NFs and aims to reduce the need to assess toxicity on a case-by-case basis. The approach to grouping is hypothesis-driven, in which the hypothesis is based on scientific evidence linking critical physicochemical descriptors for NFs to defined fate/toxicokinetic and hazard outcomes. The HARN IATA prompts users to address relevant questions (at decision nodes; DNs) regarding the morphology, biopersistence and inflammatory potential of the HARNs under investigation to provide the necessary evidence to accept or reject the grouping hypothesis. Each DN in the IATA is addressed in a tiered manner, using data from simple in vitro or in silico methods in the lowest tier or from in vivo approaches in the highest tier. For these proposed methods we provide justification for the critical descriptors and thresholds that allow grouping decisions to be made. Application of the IATA allows the user to selectively identify HARNs which may pose a mesothelioma hazard, as demonstrated through a literature-based case study. By promoting the use of alternative, non-rodent approaches such as in silico modelling, in vitro and cell-free tests in the initial tiers, the IATA testing strategy streamlines information gathering at all stages of innovation through to regulatory risk assessment while reducing the ethical, time and economic burden of testing.

Nanoporous multimetallic Ir alloys as efficient and stable electrocatalysts for acidic oxygen evolution reactions

The dearth of appropriate electrocatalysts for stable anodic water splitting, oxygen evolution reaction (OER), in acid has given rise to concerted efforts toward making iridium-based high aspect ratio nanomaterials, as iridium and its higher valent oxides have been shown time and again to exhibit the most optimal balance between activity and durability. Here, we show a dealloying strategy to synthesize free-standing 3D, oxide skinned nanoporous Ir electrocatalysts (np-Ir) with demonstrated enhanced activity and durability in comparison to more traditional IrOx nanoparticulate catalysts. The metallic core and absence of any binder/support result in low electrode and charge transfer resistance, ultimately giving rise to lower OER overpotentials and improved activity.

(Invited) A Protein Corona-Based Design Strategy for Carbon Nanotube Sensors

Unpredictable protein adsorption on nanoparticles remains a considerable challenge towards effectively applying nanotechnologies in biological environments.1 However, if well understood and controlled, proteins adsorbed on nanoparticles can be exploited to develop protein-based nanosensors.Herein, we employ our knowledge of spontaneous “protein corona” formation on single-walled carbon nanotubes (SWCNTs) in biofluids towards rational nanosensor design based on protein-SWCNT complexation.2 SWCNTs are implemented as the sensor platform due to their desirable optical and physical characteristics, including photostable fluorescence in the near-infrared tissue transparency window3 and high aspect ratio that confers delivery capabilities.4 We functionalize carbon nanotubes with proteins that, in turn, bind to their natural ligand partners. The adsorbed protein thus acts as the recognition moiety that interacts with the environment and detects the analyte of interest, tethered to the SWCNT that serves as the signal transducer. This nanosensor platform combines the sensitive and selective binding capabilities of proteins for their ligand partners with the near-infrared fluorescence readout of SWCNTs that is modulated upon protein-ligand binding. In this manner, we demonstrate that although formation of the nanoparticle protein corona can impede the intended nanobiotechnology application, it also presents an opportunity to create improved protein-nanoparticle architectures by manipulating selective protein adsorption on the nanoparticle surface. References Pinals, R. L. et al. Protein Corona Composition and Dynamics on Carbon Nanotubes in Blood Plasma and Cerebrospinal Fluid. bioRxiv 2020.01.13.905356 (2020) doi:10.1101/2020.01.13.905356.Pinals, R. L., Yang, D., Lui, A., Cao, W. & Landry, M. P. Corona Exchange Dynamics on Carbon Nanotubes by Multiplexed Fluorescence Monitoring. J. Am. Chem. Soc. 142, 1254–1264 (2020).Boghossian, A. A. et al. Near-Infrared Fluorescent Sensors based on Single-Walled Carbon Nanotubes for Life Sciences Applications. ChemSusChem 4, 848–863 (2011).Demirer, G. S. et al. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat. Nanotechnol. 14, 456–464 (2019).

Biomolecule Exchange Dynamics on Carbon Nanotubes Via Multiplexed Fluorophore Quenching

Adsorption of biomolecules on single-walled carbon nanotubes (SWCNTs) has enabled developments in molecular sensing, in vivo imaging, and gene delivery applications.1,2 This noncovalent functionalization of SWCNTs preserves the pristine SWCNT atomic structure, thus retaining the intrinsic near-infrared fluorescence for sensing or imaging functions, and offers a reversible binding mode for cargo delivery. However, biomolecule adsorption is an inherently dynamic process, where exchange occurs between molecules in the bulk solution and molecules on the nanoparticle surface, into what is known as the corona phase. The nature, strength, and kinetics of both the biomolecule binding and unbinding processes on SWCNTs are important contributors to the success of SWCNT-based technologies. Understanding this binding process is especially important for intended uses of functionalized SWCNTs in biological environments, where native biomolecules such as proteins compete with the original biomolecule to occupy the SWCNT surface. Such biofouling of the SWCNT surface disrupts the intended functionality of the nanoparticle and can further lead to adverse biocompatibility outcomes.We present a generalizable method to study the corona exchange dynamics between solution-phase and corona-phase biomolecules on SWCNTs in real-time.3 This approach exploits the quenching property of fluorophores proximal to the SWCNT surface to monitor ligand binding and unbinding events. Real-time tracking of adsorption and desorption events on the SWCNT surface are conducted with systematic variation of the molecular entities and solution conditions, with a specific focus on DNA (as the sensing moiety) and protein (as the biofouling agent). Binding profiles are extracted from the experimental assay and used to inform a kinetic model of the system. The work presented herein develops an understanding of the fundamental corona exchange mechanism and provides insight into performance of these designed SWCNT-based systems in biologically relevant, protein-rich conditions. References Demirer, G. S. et al. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nature Nanotechnology 1 (2019) doi:10.1038/s41565-019-0382-5.Beyene, A. G. et al. Imaging striatal dopamine release using a nongenetically encoded near infrared fluorescent catecholamine nanosensor. Science Advances 5, eaaw3108 (2019).Pinals, R. L., Yang, D., Lui, A., Cao, W. & Landry, M. P. Corona Exchange Dynamics on Carbon Nanotubes by Multiplexed Fluorescence Monitoring. J. Am. Chem. Soc. (2019) doi:10.1021/jacs.9b09617. Figure 1

Novel insights into biodegradation, interaction, internalization and impacts of high-aspect-ratio TiO2 nanomaterials: A systematic in vivo study using Drosophila melanogaster

The elongated nature of the high-aspect-ratio nanomaterials (NMs) can help us to obtain valuable information on its biodegradation, physical interaction with target-cells, and internalization. Three different length nano-titanium have been studied using Drosophila, TEM, and different biological markers. Nano-titanium, regardless of its shape, was eroded and degraded just entering the gut lumen of the larvae. Results showed that the distinguished shape of nanowires helps to understand the interactions of NMs with the intestinal barrier. The peritrophic membrane, as the first defense line of the intestinal barrier, succeeded in the reservation of NMs, though the perpendicular particles of nanowires stabbing it, making pores, and permitting their translocation into intestinal cells. On the other side, the exposure to TiO2NPs did not decrease egg-to-adult viability, but all its different shapes, especially nanowires, mediated a wide molecular response including changes of expression in genes involved in stress, antioxidant, repair, and physical interaction responses. All these changes concerning their ability to elevate ROS levels ultimately led to potential genotoxicity. So, the high aspect ratio NMs are efficient in understanding the outstanding issues of NMs exposure, but at the same time could induce genotoxic impact rather than the low aspect ones.

Abstract 3815: Cellular oxidative stress and immunological effects of carbon-based nanomaterials towards lung carcinogenicity

Abstract Lung cancer accounts for most number of cancer related death, reporting over 26.5% of all cancer deaths in United States alone. Among the risk factors, along with smoking, occupational as well as environmental exposure of carcinogens is considered as major driver of lung cancer in most cases. In that context, high aspect ratio nanomaterials (HARNs) with low density, such as carbon nanotubes (CNTs), remain efficient air-borne particulate matters during every step of their life cycle, from production to usage to disposal. Consequently, inhalation and potentially carcinogenic nanomaterial exposure to pulmonary tissues are anticipated, which may considerably facilitate lung cancer occurrence and mortality. Apart from pulmonary interaction, nanomaterial exposure via skin or oral route may play a significant role in adverse human health effects, especially when the use of nanomaterials in food and cosmetic industries are growing exponentially. CNTs are made of flat sheets of sp2 hybridized carbon atoms layered in hexagonal geometry that are rolled into unified cylindrical structures. CNTs are subdivided into single-walled (SWCNT), double-walled (DWCNT), or multi-walled carbon nanotube (MWCNT) based on the number of carbon layers in nanotubes. Size of CNTs can range from one to several nanometers in diameter and up to several micrometers in length, exhibiting a fibrous-like length-to-width (aspect) ratio similar to asbestos fibers and potentially harbor asbestos-like pulmonary fibrosis and lung cancer risks associated with their long-term exposure. Considerable efforts has been made to study Nano-toxicity but how cellular toxicological and immunological responses are affected by nanomaterial exposure is largely unknown. The present study investigated the effects of different CNT exposure on cellular proliferation, uptake, oxidative stress, DNA damage, Stem cell-like properties along with activation of cellular innate immune responses of human lung epithelial cells and immune cells to determine the underlying cross-talks. Using a physiologically relevant long-term-low-dose exposure model, we found that both single-walled and multi-walled carbon nanotubes, induced particle-specific cellular proliferation, tumor sphere formation, reactive oxygen species (ROS) and super oxide (SO), DNA-strand breaks, mitochondrial depolarization and reduced apoptosis, indicating the genotoxicity and carcinogenic potential of CNTs. We also determined distinct immune activation signature in these cells upon CNTs exposure that closely resembles “immune mediated” carcinogenesis signature as reported in “Inflammatory Cancer (C3)” subtypes. Finally, through in silico analyses and validation we have determined potential CNT exposure induced immune effectors to devise better targeted therapeutic intervention against nanomaterial exposure induced carcinogenesis. Citation Format: Rajib Ghosh, Liying W. Rojanasakul, Yon Rojanasakul. Cellular oxidative stress and immunological effects of carbon-based nanomaterials towards lung carcinogenicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3815.

(Nanocarbons Division SES Young Investigator Award Address) Nanomaterials Engineering to Probe and Control Living Systems

Unique physical, chemical, and optical phenomena arise when materials are confined to the nano-scale. We are accustomed to making observations and predictions for the behavior of living systems on a macroscopic scale that is intuitive for the time and size scales of our day-to-day lives. However, the building blocks of life: proteins, nucleic acids, and cells, occupy different spatiotemporal scales. Our lab focuses on understanding and exploiting tunable optical and mechanical properties of nanomaterials to access information about biological systems stored at the nano-scale. In the context of leveraging nanomaterial optical properties, we present recent work on developing and implementing dopamine nanosensors to image dopamine volume transmission in the extracellular space of the brain striatum. We validate our dopamine nanosensor in acute striatal slices with electrical and optogenetic stimulation of dopamine release, and show disrupted dopamine release or reuptake kinetics when brain tissue is exposed to dopamine agonist or antagonist drugs [1,2]. In the context of leveraging nanomaterial chemical properties, we also discuss how high aspect ratio nanomaterials can be synthesized to carry biomolecular cargo to living systems. In particular, genetic engineering of plants is at the core of environmental sustainability efforts, but the physical barrier presented by the cell wall has limited the ease and throughput with which exogenous biomolecules can be delivered to plants. We will describe how nanomaterials engineering principles can be leveraged to manipulate living plants [3, 4], in efforts to reconcile the benefits of crop genetic engineering with the demand for non-GMO foods [5]. Our work in the agricultural space provides a promising tool for species-independent, targeted, and passive delivery of genetic material, without transgene integration, into plant cells for rapid and parallelizable testing of plant genotype-phenotype relationships. 1. Beyene, A.B., McFarlane, I.R., Pinals, R.L, Landry, M.P.‡ Stochastic Simulation of Dopamine Neuromodulation for Implementation of Fluorescent Neurochemical Probes in the Striatal Extracellular Space. ACS Chemical Neuroscience 8 (10), 2275-2289 (2017).2. Beyene, A. G., Delevich, K., O’Donnell, J.T.D., Piekarski, D.J., Lin, W.C., Thomas, A.W., Yang, S.J., Kosillo, P., Yang, D., Wilbrecht, L., Landry, M.P. ‡ Imaging Striatal Dopamine Release Using a Non-Genetically Encoded Near-Infrared Fluorescent Catecholamine Nanosensor. Science Advances 5 (7), 1-11 (2019)3. Demirer, G.S., Zhang, H., Matos, J., Goh, N., Cunningham, F.J., Sung, Y., Chang, R., Aditham, A.J., , Chio, L., Cho, M.J., Staskawicz, B., Landry, M.P. ‡ High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material Without DNA Integration in Mature Plants. Nature Nanotechnology (2019). DOI: 10.1038/s41565-019-0382-5NNANO-180816844. Demirer, G.S., Zhang, H., Goh, N.S., Grandio, E.G., Landry, M.P. ‡ Carbon nanotube-mediated DNA delivery without transgene integration in intact plants. Nature Protocols (2019). DOI: 0.1038/s41596-019-0208-95. Landry, M.P. ‡, Mitter, N.‡ How nanocarriers delivering cargoes in plants can change the GMO landscape. Nature Nanotechnology 2019, 14; 512–514