Fabrication of Single-Nanocrystal Arrays.

To realize the full potential of nanocrystals in nanotechnology, it is necessary to integrate single nanocrystals into addressable structures; for example, arrays and periodic lattices. The current methods for achieving this are reviewed. It is shown that a combination of top-down lithography techniques with directed assembly offers a platform for attaining this goal. The most promising of these directed assembly methods are reviewed: capillary force assembly, electrostatic assembly, optical printing, DNA-based assembly, and electrophoretic deposition. The last of these appears to offer a generic approach to fabrication of single-nanocrystal arrays.

Particokinetics and in vitro dose of high aspect ratio nanoparticles.

Computational particokinetics models become essential in the design and interpretation of in vitro nanoparticle toxicology assays involving submerged adherent cell cultures. Yet, none of the current models offers the possibility of addressing the delivered dose of high-aspect ratio nanoparticles, including nanorods, nanotubes, and nanofibers. Here we present a straightforward method that lends this ability to any of the models used currently.

Beyond Global Charge: Role of Amine Bulkiness and Protein Fingerprint on Nanoparticle-Cell Interaction.

Amino groups presented on the surface of nanoparticles are well-known to be a predominant factor in the formation of the protein corona and subsequent cellular uptake. However, the molecular mechanism underpinning this relationship is poorly defined. This study investigates how amine type and density affect the protein corona and cellular association of gold nanoparticles with cells in vitro. Four specific poly(vinyl alcohol-co-N-vinylamine) copolymers are synthesized containing primary, secondary, or tertiary amines. Particle cellular association (i.e., cellular uptake and surface adsorption), as well as protein corona composition, are then investigated. It is found that the protein corona (as a consequence of "amine bulkiness") and amine density are both important in dictating cellular association. By evaluating the nanoparticle surface chemistry and the protein fingerprint, proteins that are significant in mediating particle-cell association are identified. In particular, primary amines, when exposed on the polymer side chain, are strongly correlated with the presence of alpha-2-HS-glycoprotein, and promote nanoparticle cellular association.

Direct Assembly of Large Area Nanoparticle Arrays.

A major goal of nanotechnology is the assembly of nanoscale building blocks into functional optical, electrical, or chemical devices. Many of these applications depend on an ability to optically or electrically address single nanoparticles. However, positioning large numbers of single nanocrystals with nanometer precision on a substrate for integration into solid-state devices remains a fundamental roadblock. Here, we report fast, scalable assembly of thousands of single nanoparticles using electrophoretic deposition. We demonstrate that gold nanospheres down to 30 nm in size and gold nanorods <100 nm in length can be assembled into predefined patterns on transparent conductive substrates within a few seconds. We find that rod orientation can be preserved during deposition. As proof of high fidelity scale-up, we have created centimeter scale patterns comprising more than 1 million gold nanorods.

Directed Chemical Assembly of Single and Clustered Nanoparticles with Silanized Templates.

The assembly of nanoscale materials into arbitrary, organized structures remains a major challenge in nanotechnology. Herein, we report a general method for creating 2D structures by combining top-down lithography with bottom-up chemical assembly. Under optimal conditions, the assembly of gold nanoparticles was achieved in less than 30 min. Single gold nanoparticles, from 10 to 100 nm, can be placed in predetermined patterns with high fidelity, and higher-order structures can be generated consisting of dimers or trimers. It is shown that the nanoparticle arrays can be transferred to, and embedded within, polymer films. This provides a new method for the large-scale fabrication of nanoparticle arrays onto diverse substrates using wet chemistry.

Form Follows Function: Nanoparticle Shape and Its Implications for Nanomedicine.

This review is a comprehensive description of the past decade of research into understanding how the geometry and size of nanoparticles affect their interaction with biological systems: from single cells to whole organisms. Recently, there has been a great deal of effort to use both the shape and the size of nanoparticles to target specific cellular uptake mechanisms, biodistribution patterns, and pharmacokinetics. While the successes of spherical lipid-based nanoparticles have heralded marked changes in chemotherapy worldwide, the history of asbestos-induced lung disease casts a long shadow over fibrous materials to date. The impact of particle morphology is known to be intertwined with many physicochemical parameters, namely, size, elasticity, surface chemistry, and biopersistence. In this review, we first highlight some of the morphologies observed in nature as well as shapes available to us through synthetic strategies. Following this we discuss attempts to understand the cellular uptake of nanoparticles through various theoretical models before comparing this with observations from in vitro and in vivo experiments. In addition, we consider the impact of nanoparticle shape at different size regimes on targeting, cytotoxicity, and cellular mechanics.

Assumption-free morphological quantification of single anisotropic nanoparticles and aggregates.

Characterizing the morphometric parameters of noble metal nanoparticles for sensing and catalysis is a persistent challenge due to their small size and complex shape. Herein, we present an approach to determine the volume, surface area, and curvature of non-symmetric anisotropic nanoparticles using electron tomography and design-based stereology without the use of segmentation tools or modeling of the particles. Finally, we apply these tools to aggregates to estimate their fractal dimension.

A new angle on dynamic depolarized light scattering: number-averaged size distribution of nanoparticles in focus.

Size polydispersity is a common phenomenon that strongly influences the physicochemical properties of nanoparticles (NPs). We present an analytical approach that is universally applicable to characterizing optically anisotropic round NPs and determines directly the number-averaged size distribution and polydispersity via depolarized dynamic light scattering (DDLS). To demonstrate, we use aqueous suspensions of Au NPs of different sizes and surface functionalization.

Plasmonic nanoparticles and their characterization in physiological fluids.

Nanoparticles possess unique properties beyond that of classical materials, and while these properties can be used for designing a dedicated functionality, they may also pose a problem to living organisms, to human health and the environment. The specific primary routes by which nanoparticles may interact with the human body include inhalation, injection, ingestion and application to the skin. Independent of the entry route, the particles inevitably encounter a complex physiological fluid populated with e.g. proteins, vitamins, lipids and salts/ions. Different consequences of such an encounter may include formation of a surface-bound protein layer, particle dissolution or aggregation, which are expected to have a crucial impact on cellular interaction. Understanding cellular responses to nanoparticle interactions starts with understanding particle behavior in physiological fluids. Nanoparticles are now available in practically any size, shape and functionalization, to promote distinct optical, magnetic, and physico-chemical properties, making the prediction of their behavior, in physiological fluids, not a trivial task. Characterization has therefore become of paramount importance. In this review, we give an overview about the diversity of physiological fluids as well as present an inventory of the most relevant experimental techniques used to study plasmonic nanoparticles.

Ultrathin Ceramic Membranes as Scaffolds for Functional Cell Coculture Models on a Biomimetic Scale.

Epithelial tissue serves as an interface between biological compartments. Many in vitro epithelial cell models have been developed as an alternative to animal experiments to answer a range of research questions. These in vitro models are grown on permeable two-chamber systems; however, commercially available, polymer-based cell culture inserts are around 10 µm thick. Since the basement membrane found in biological systems is usually less than 1 µm thick, the 10-fold thickness of cell culture inserts is a major limitation in the establishment of realistic models. In this work, an alternative insert, accommodating an ultrathin ceramic membrane with a thickness of only 500 nm (i.e., the Silicon nitride Microporous Permeable Insert [SIMPLI]-well), was produced and used to refine an established human alveolar barrier coculture model by both replacing the conventional inserts with the SIMPLI-well and completing it with endothelial cells. The structural-functional relationship of the model was evaluated, including the translocation of gold nanoparticles across the barrier, revealing a higher translocation if compared to corresponding polyethylene terephthalate (PET) membranes. This study demonstrates the power of the SIMPLI-well system as a scaffold for epithelial tissue cell models on a truly biomimetic scale, allowing construction of more functionally accurate models of human biological barriers.

Fate of cellulose nanocrystal aerosols deposited on the lung cell surface in vitro.

When considering the inhalation of high-aspect ratio nanoparticles (HARN), the characterization of their specific interaction with lung cells is of fundamental importance to help categorize their potential hazard. The aim of the present study was to assess the interaction of cellulose nanocrystals (CNCs) with a multicellular in vitro model of the epithelial airway barrier following realistic aerosol exposure. Rhodamine-labeled CNCs isolated from cotton (c-CNCs, 237 ± 118 × 29 ± 13 nm) and tunicate (t-CNCs, 2244 ± 1687 × 30 ± 8 nm) were found to display different uptake behaviors due to their length, although also dependent upon the applied concentration, when visualized by laser scanning microscopy. Interestingly, the longer t-CNCs were found to exhibit a lower clearance by the lung cell model compared to the shorter c-CNCs. This difference can be attributed to stronger fiber-fiber interactions between the t-CNCs. In conclusion, nanofiber length and concentration has a significant influence on their interaction with lung cells in vitro.

Thermally reversible self-assembly of nanoparticles via polymer crystallization.

The directed self-assembly of gold nanoparticles through the crystallization of surface-grafted polyethylene oxide (PEO) in ethanol-water mixtures is described. This process is fully reversible and tunable through either the size of the core or the polymeric coating. Characterization by X-ray scattering and electron microscopy of the self-assembled structures reveals order at the nanoscale, typically not the case for thermoresponsive gold nanoparticles coated with lower or upper critical solution temperature polymers. A further novelty is the result of selective binding of calcium ions to the PEO in the fluid state: a reversible thermoresponsive transition become irreversible.

An in vitro testing strategy towards mimicking the inhalation of high aspect ratio nanoparticles.

BACKGROUND: The challenge remains to reliably mimic human exposure to high aspect ratio nanoparticles (HARN) via inhalation. Sophisticated, multi-cellular in vitro models are a particular advantageous solution to this issue, especially when considering the need to provide realistic and efficient alternatives to invasive animal experimentation for HARN hazard assessment. By incorporating a systematic test-bed of material characterisation techniques, a specific air-liquid cell exposure system with real-time monitoring of the cell-delivered HARN dose in addition to key biochemical endpoints, here we demonstrate a successful approach towards investigation of the hazard of HARN aerosols in vitro. METHODS: Cellulose nanocrystals (CNCs) derived from cotton and tunicates, with differing aspect ratios (~9 and ~80), were employed as model HARN samples. Specifically, well-dispersed and characterised CNC suspensions were aerosolised using an "Air Liquid Interface Cell Exposure System" (ALICE) at realistic, cell-delivered concentrations ranging from 0.14 to 1.57 µg/cm2. The biological impact (cytotoxicity, oxidative stress levels and pro-inflammatory effects) of each HARN sample was then assessed using a 3D multi-cellular in vitro model of the human epithelial airway barrier at the air liquid interface (ALI) 24 hours post-exposure. Additionally, the testing strategy was validated using both crystalline quartz (DQ12) as a positive particulate control in the ALICE system and long fibre amosite asbestos (LFA) to confirm the susceptibility of the in vitro model to a fibrous insult. RESULTS: A rapid (≤ 4 min), controlled nebulisation of CNC suspensions enabled a dose-controlled and spatially homogeneous CNC deposition onto cells cultured under ALI conditions. Real-time monitoring of the cell-delivered CNC dose with a quartz crystal microbalance was accomplished. Independent of CNC aspect ratio, no significant cytotoxicity (p>0.05), induction of oxidative stress, or (pro)-inflammatory responses were observed up to the highest concentration of 1.57 µg/cm2. Both DQ12 and LFA elicited a significant (p<0.05) pro-inflammatory response at sub-lethal concentrations in vitro. CONCLUSION: In summary, whilst the present study highlights the benign nature of CNCs, it is the advanced technological and mechanistic approach presented that allows for a state of the art testing strategy to realistically and efficiently determine the in vitro hazard concerning inhalation exposure of HARN.

Polyvinyl alcohol as a biocompatible alternative for the passivation of gold nanorods.

The functionalization of gold nanorods (GNRs) with polymers is essential for both their colloidal stability and biocompatibility. However, a bilayer of the toxic cationic surfactant cetyl trimethylammonium bromide (CTAB) adsorbed on the nanorods complicates this process. Herein, we report on a strategy for the biocompatible functionalization of GNRs with a hydrophobic polymeric precursor, polyvinyl acetate, which is then transformed into its hydrophilic analogue, polyvinyl alcohol. This polymer was chosen due to its well-established biocompatibility, tunable "stealth" properties, tunable hydrophobicity, and high degree of functionality. The biocompatibility of the functionalized GNRs was tested by exposing them to primary human blood monocyte derived macrophages; the advantages of tunable hydrophobicity were demonstrated with the long-term stable encapsulation of a model hydrophobic drug molecule.

Surface charge of polymer coated SPIONs influences the serum protein adsorption, colloidal stability and subsequent cell interaction in vitro.

It is known that the nanoparticle-cell interaction strongly depends on the physicochemical properties of the investigated particles. In addition, medium density and viscosity influence the colloidal behaviour of nanoparticles. Here, we show how nanoparticle-protein interactions are related to the particular physicochemical characteristics of the particles, such as their colloidal stability, and how this significantly influences the subsequent nanoparticle-cell interaction in vitro. Therefore, different surface charged superparamagnetic iron oxide nanoparticles were synthesized and characterized. Similar adsorbed protein profiles were identified following incubation in supplemented cell culture media, although cellular uptake varied significantly between the different particles. However, positively charged nanoparticles displayed a significantly lower colloidal stability than neutral and negatively charged particles while showing higher non-sedimentation driven cell-internalization in vitro without any significant cytotoxic effects. The results of this study strongly indicate therefore that an understanding of the aggregation state of NPs in biological fluids is crucial in regards to their biological interaction(s).