Investigating the impact of gold nanoparticles on cells: from transcription to behavior

Gold nanoparticles (Au NPs) are being research extensively for various biomedical applications; their applicability arises from a unique set of optical and physical properties brought on by their nanoscale dimensions.1 Furthermore, facile and scalable synthetic and surface functionalization strategies for Au NPs make these properties highly tunable.1 These areas of research are still relatively new and the number of publications per year referring to gold nanomaterials has skyrocketed from 11 in 1990, to 673 in 2000, to more than 31,400 in 2015 (Web of Science database, topic search = “gold nano*”, accessed March 2, 2016). The potential application of Au NPs for disease detection, diagnosis and therapy has motivated numerous analyses of their interactions with biomolecules, cells, animals, humans and the environment.1,2 A vast majority of studies aimed at gaining a better understanding of how cells interact with and are influenced by Au NPs have focused mainly on measuring cytotoxicity and simple cell processes like proliferation or NP uptake. While Au NPs larger than 4-5 nm in diameter (with appropriate, non-toxic surface coatings) have been shown to be largely non-cytotoxic, there can be subtle non-toxic effects induced by Au NPs.3 The adsorption of soluble proteins onto NP surfaces (the protein corona) is highly studied, but little attention has been paid to how those interactions perturb gene expression of cells or to understanding NP interactions with other types of biomolecules. This thesis aims to look deeper into how molecular level effects of NPs in cells and cellular environments can lead to down-stream changes to cell gene expression and behavior. Firstly, the impact of Au nanorods (Au NRs) on 3D cancer cell migration via interactions between Au NRs and extracellular matrix (ECM) structural proteins was examined.4 While experiments on the influence of NPs on cell behaviors exist, nearly all of these studies neglect the impact of the ECM. In vivo cells exist in complex, fibrous 3D environments and series of intricate biochemical, cell-cell and cell-ECM interactions govern behaviors such as migration. Cancer cell migration allows tumor cells to spread and metastasize to new areas of the body, but little is known about how Au NR interaction with the ECM after injection and targeting to tumors may affect this process. The inevitable contact of in vivo Au NRs with the ECM presents a possible source of unintended side effects. In order to study how gold nanoparticles can influence ECM properties and cell-ECM interactions, we have created a nested-gel type I collagen matrix for measuring whether Au NRs alter the migration of MDA-MB-231 human breast cancer cells in 3D collagen environments. In contrast to the few studies of Au NR-induced migration changes in 2D environments, our results show that Au NRs in a model ECM increase the frequency of spontaneous cellular migration. The presence of negatively-charged polyelectrolyte-coated Au NRs during the collagen self-assembly process was shown to induce mechanical and structural changes, to alter molecular diffusion, and to affect cellular adhesion, morphology, locomotion strategy and protease expression. The results demonstrate the indirect impact nanoparticles can exert on cell behaviors within three-dimensional ECMs. The shape and surface chemistry of Au NPs was also investigated in terms of the role of these factors in cellular transcriptomic (gene expression) responses after both short- and long-term exposures.5 Respectively, human dermal fibroblasts (HDF) and prostate cancer (PC3) cells were exposed to 0.1 nM (24 hours) and 1.0 nM (48 hours) concentrations of Au NPs of four different, but related surface chemistries. A combination of microarray gene expression analysis techniques and typical cellular characterization was used to learn more about how the nature of the Au NP surface coating influences cells on a molecular level. Identity, charge and lability of surface coatings (and cell type and dosing parameters) were all implicated as important factors to consider in order to predict gene expression responses. In a separate study, HDF cells were exposed to 0.1 nM (low-dose) Au NPs of different shapes and surface coatings for 20 weeks. The long-term effects of acute (24 hour) and chronic (20 weeks) exposure were measured by viability, proliferation, NP uptake, and morphology studies combined with gene expression analysis of genes related to stress and toxicity pathways. It is rare to find chronic exposure studies, especially with Au NPs, and these experiments showed that acute, sub-cytotoxic doses of NPs may induce long-term stress on cells. These cells were found to react very differently to acute versus chronic doses of the same NPs after 20 weeks. Additionally, surface coating was shown to have a much larger impact on determining NP-cell interactions than shape of Au NPs. In all, we have expanded the collective understanding of the molecular interactions Au NPs experience inside cells based on surface chemistry, shape, dosage and exposure time and parameters. References 1. Dreaden, E.C.; Alkilany, A.M.; Huang, X.; Murphy, C.J.; El-Sayed, M.A. The Golden Age: Gold Nanoparticles for Biomedicine. Chem. Soc. Rev. 2012, 41, 2740–2779. 2. Yang, X.; Yang, M.; Pang, B.; Vara, M.; Xia, Y. Gold Nanomaterials at Work in Biomedicine. Chem. Rev. 2015, 115, 10410–10488. 3. Alkilany, A.M.; Lohse, S.E.; Murphy, C.J. The Gold Standard: Gold Nanoparticle Libraries to Understand the Nano-Bio Interface. Acc. Chem. Res. 2013, 46, 650–661. 4. Grzincic, E.M.; Murphy, C.J. Gold Nanorods Indirectly Promote Migration of Metastatic Human Breast Cancer Cells in Three-Dimensional Cultures. ACS Nano 2015, 9, 6801–6816. 5. Grzincic, E.M.; Yang, J.A.; Drnevich, J.; Falagan-Lotsch, P.; Murphy, C.J. Global Transcriptomic Analysis of Model Human Cell Lines Exposed to Surface-Modified Gold Nanoparticles: The Effect of Surface Chemistry. Nanoscale 2015, 7, 1349–1362

Discovery of Stable and Selective Antibody Mimetics from Combinatorial Libraries of Polyvalent, Loop-Functionalized Peptoid Nanosheets.

The ability of antibodies to bind a wide variety of analytes with high specificity and high affinity makes them ideal candidates for therapeutic and diagnostic applications. However, the poor stability and high production cost of antibodies have prompted exploration of a variety of synthetic materials capable of specific molecular recognition. Unfortunately, it remains a fundamental challenge to create a chemically diverse population of protein-like, folded synthetic nanostructures with defined molecular conformations in water. Here we report the synthesis and screening of combinatorial libraries of sequence-defined peptoid polymers engineered to fold into ordered, supramolecular nanosheets displaying a high spatial density of diverse, conformationally constrained peptoid loops on their surface. These polyvalent, loop-functionalized nanosheets were screened using a homogeneous Förster resonance energy transfer (FRET) assay for binding to a variety of protein targets. Peptoid sequences were identified that bound to the heptameric protein, anthrax protective antigen, with high avidity and selectivity. These nanosheets were shown to be resistant to proteolytic degradation, and the binding was shown to be dependent on the loop display density. This work demonstrates that key aspects of antibody structure and function-the creation of multivalent, combinatorial chemical diversity within a well-defined folded structure-can be realized with completely synthetic materials. This approach enables the rapid discovery of biomimetic affinity reagents that combine the durability of synthetic materials with the specificity of biomolecular materials

Gold Nanorods Indirectly Promote Migration of Metastatic Human Breast Cancer Cells in Three-Dimensional Cultures

Gold nanomaterials are intensively studied for applications in disease detection, diagnosis and therapeutics, and this has motivated considerable research to determine their interaction with biomolecules, cells and cell behaviors. However, few studies look at how nanomaterials alter the extracellular matrix (ECM) and cell–ECM interactions. Nanomaterials in the body would interact with the entire cellular environment, and it is imperative to account for this when studying the impact of nanomaterials on living systems. Furthermore, recent evidence finds that migration rates of cells in 2D can be affected by nanomaterials, and uptake of the nanomaterials is not necessary to exert an effect. In this study, three-dimensional nested type I collagen matrices were utilized as a model ECM to study how gold nanorods affect the migration of MDA-MB-231 human breast cancer cells. Spontaneous cell migration through collagen containing gold nanorods was found to increase with increasing concentrations of gold nanorods, independent of intracellular uptake of the nanorods. Gold nanorods in the collagen matrix were found to alter collagen mechanical properties and structure, molecular diffusion, cellular adhesion, cell morphology, mode of migration and protease expression. Correlation between decreased cellular adhesion and rounded cell morphology and locomotion in nanorod-containing collagen suggests the induction of an amoeboid-like migratory phenotype

Lipid-anchor display on peptoid nanosheets <i>via</i> co-assembly for multivalent pathogen recognition

A facile route to a diversity of functionalized two-dimensional bionanomaterials was developed based on the aqueous co-assembly of lipidated small molecules and nanosheet-forming peptoids.</p

Uniform, Large-Area, Highly Ordered Peptoid Monolayer and Bilayer Films for Sensing Applications.

The production of atomically defined, uniform, large-area 2D materials remains as a challenge in materials chemistry. Many methods to produce 2D nanomaterials suffer from limited lateral film dimensions, lack of film uniformity, or limited chemical diversity. These issues have hindered the application of these materials to sensing applications, which require large-area uniform films to achieve reliable and consistent signals. Furthermore, the development of a 2D material system that is biocompatible and readily chemically tunable has been a fundamental challenge. Here, we report a simple, robust method for the production of large-area, uniform, and highly tunable monolayer and bilayer films, from sequence-defined peptoid polymers, and their application as highly selective molecular recognition elements in sensor production. Monolayers and bilayer films were produced on the centimeter scale using Langmuir-Blodgett methods and exhibited a high degree of uniformity and ordering as evidenced by atomic force microscopy, electron diffraction, and grazing incidence X-ray scattering. We further demonstrated the utility of these films in sensing applications by employing the biolayer interferometry technique to detect the specific binding of the pathogen derived proteins, shiga toxin and anthrax protective antigen, to peptoid-coated sensors

Uniform, Large-Area, Highly Ordered Peptoid Monolayer and Bilayer Films for Sensing Applications

The production of atomically defined, uniform, large-area 2D materials remains as a challenge in materials chemistry. Many methods to produce 2D nanomaterials suffer from limited lateral film dimensions, lack of film uniformity, or limited chemical diversity. These issues have hindered the application of these materials to sensing applications, which require large-area uniform films to achieve reliable and consistent signals. Furthermore, the development of a 2D material system that is biocompatible and readily chemically tunable has been a fundamental challenge. Here, we report a simple, robust method for the production of large-area, uniform, and highly tunable monolayer and bilayer films, from sequence-defined peptoid polymers, and their application as highly selective molecular recognition elements in sensor production. Monolayers and bilayer films were produced on the centimeter scale using Langmuir–Blodgett methods and exhibited a high degree of uniformity and ordering as evidenced by atomic force microscopy, electron diffraction, and grazing incidence X-ray scattering. We further demonstrated the utility of these films in sensing applications by employing the biolayer interferometry technique to detect the specific binding of the pathogen derived proteins, shiga toxin and anthrax protective antigen, to peptoid-coated sensors