Leveraging proteomics to compare submerged versus air-liquid interface carbon nanotube exposure to a 3D lung cell model.

With the emerging concern over the potential toxicity associated with carbon nanotube inhalation exposure, several in vitro methods have been developed to evaluate cellular responses. Since the major concern for adverse effects by carbon nanotubes is inhalation, various lung cell culture models have been established for toxicity testing, thus creating a wide variation of methodology. Limited studies have conducted side-by-side comparisons of common methods used for carbon nanotube hazard testing. The aim of this work was to use proteomics to evaluate global cellular response, including pro-inflammatory and pro-fibrotic mediators, of a 3D lung model composed of macrophages, epithelial cells, and fibroblasts which mimics the human alveolar epithelial tissue barrier. The cells were exposed to Mitsui 7 (M-7) multi-walled carbon nanotubes (MWCNT) under submerged and air-liquid interface (ALI) conditions and discovery proteomics identified 3500 proteins. The M-7 ALI exposure compared to control was found to increase expression in proteins related to oxidative stress that were not found to be enriched in submerged exposure. Comparison of MWCNT exposure methods, M-7 ALI exposure versus M-7 submerged exposure, yielded protein enrichment in pathways known to be associated with carbon nanotube exposure stress response, such as acute phase response signaling and NRF2-mediated oxidative stress response. This study demonstrates a comparison of commonly deployed carbon nanotube exposure methods. These data should be considered by the nanotoxicology community when interpreting or cross comparing in vitro exposure results.

A novel sample holder for 4D live cell imaging to study cellular dynamics in complex 3D tissue cultures.

Three dimensional (3D) co-cultures to mimic cellular dynamics have brought significant impacts in tissue engineering approaches for biomedical research. Herein, we present a novel sample holder combined with time-lapse fluorescence imaging technique, referred as 4D live cell imaging, allowing direct visualization of various cells up to 24 hours. We further extended our approach to monitor kinetics and dynamics of particle uptake by cells and translocation across tissue membranes.

A critical review of the current knowledge regarding the biological impact of nanocellulose.

Several forms of nanocellulose, notably cellulose nanocrystals and nanofibrillated cellulose, exhibit attractive property matrices and are potentially useful for a large number of industrial applications. These include the paper and cardboard industry, use as reinforcing filler in polymer composites, basis for low-density foams, additive in adhesives and paints, as well as a wide variety of food, hygiene, cosmetic, and medical products. Although the commercial exploitation of nanocellulose has already commenced, little is known as to the potential biological impact of nanocellulose, particularly in its raw form. This review provides a comprehensive and critical review of the current state of knowledge of nanocellulose in this format. Overall, the data seems to suggest that when investigated under realistic doses and exposure scenarios, nanocellulose has a limited associated toxic potential, albeit certain forms of nanocellulose can be associated with more hazardous biological behavior due to their specific physical characteristics.

Decoupling the shape parameter to assess gold nanorod uptake by mammalian cells.

The impact of nanoparticles (NPs) upon biological systems can be fundamentally associated with their physicochemical parameters. A further often-stated tenet is the importance of NP shape on rates of endocytosis. However, given the convoluted parameters concerning the NP-cell interaction, it is experimentally challenging to attribute any findings to shape alone. Herein we demonstrate that shape, below a certain limit, which is specific to nanomedicine, is not important for the endocytosis of spherocylinders by either epithelial or macrophage cells in vitro. Through a systematic approach, we reshaped a single batch of gold nanorods into different aspect ratios resulting in near-spheres and studied their cytotoxicity, (pro-)inflammatory status, and endocytosis/exocytosis. It was found that on a length scale of ∼10-90 nm and at aspect ratios less than 5, NP shape has little impact upon their entry into either macrophages or epithelial cells. Conversely, nanorods with an aspect ratio above 5 were preferentially endocytosed by epithelial cells, whereas there was a lack of shape dependent uptake following exposure to macrophages in vitro. These findings have implications both in the understanding of nanoparticle reshaping mechanisms, as well as in the future rational design of nanomaterials for biomedical applications.

A lock-in-based method to examine the thermal signatures of magnetic nanoparticles in the liquid, solid and aggregated states.

We propose a new methodology based on lock-in thermography to study and quantify the heating power of magnetic nanoparticles. Superparamagnetic iron oxide nanoparticles exposed to a modulated alternating magnetic field were used as model materials to demonstrate the potency of the system. Both quantitative and qualitative information on their respective heating power was extracted at high thermal resolutions under increasingly complex conditions, including nanoparticles in the liquid, solid and aggregated states. Compared to conventional techniques, this approach offers a fast, sensitive and non-intrusive alternative to investigate multiple and dilute specimens simultaneously, which is essential for optimizing and accelerating screening procedures and comparative studies.

Distribution of Silica-Coated Silver/Gold Nanostars in Soft- and Hardwood Applying SERS-Based Imaging.

Nanoparticles (NPs) in aqueous suspension have just begun to be exploited for the preservative treatment of wood. However, at present, there is very little information available on the distribution of NPs in wood after impregnation, due to associated analytical challenges. In this study, we present the detection of model NPs in softwood and hardwood by surface-enhanced Raman spectroscopy (SERS). SERS is a highly sensitive analytical method requiring no fluorescent labeling. The NP distribution after impregnation is evaluated with one representative species of the two wood types. To show the feasibility of the method, we prepared SERS-active Au/Ag nanostars coated with silica to act as a model NP system. We show herein that NPs can be imaged in very low quantities in both wood types without any matrix interactions. The presence of the NPs in the wood was confirmed by scanning electron microscopy (SEM) imaging and energy dispersive X-ray analysis (EDX). The fast detection of NPs in a complex matrix, without complicated sample preparation, marks a huge step forward in the development and application of nanotechnology for wood preservation and the quest to optimize the properties of one of the world's most important raw materials.

Synthesis, characterization, antibacterial activity and cytotoxicity of hollow TiO2-coated CeO2 nanocontainers encapsulating silver nanoparticles for controlled silver release.

Biomaterials as implants are being applied more extensively in medicine due to their on-going development and associated improvements, and the increase in human life expectancy. Nonetheless, biomaterial-related infections, as well as propagating bacterial resistance, remain significant issues. Therefore, there is a growing interest for silver-based drugs because of their efficient and broad-range antimicrobial activity and low toxicity to humans. Most newly-developed silver-based drugs have an extremely fast silver-ion release, increasing adverse biological impact to the surrounding tissue and achieving only short-term antimicrobial activity. Nanoencapsulation of these drugs is hypothesized as beneficial for controlling silver release, and thus is the aim of the present study. Initially, an amorphous or crystalline (anatase) titania (TiO2) coating was synthesized around silver nanoparticle-containing (AgNP) ceria (CeO2) nanocontainers using a sonication method forming AgNP/CeO2/TiO2 nanocontainers. These nanocontainers were characterized by high-resolution transmission electron microscopy, scanning electron microscopy, powder X-ray diffraction, gas sorption experiments and energy-dispersive X-ray spectroscopy. Silver release, monitored by using inductively coupled plasma optical emission spectroscopy, showed that these containers prevented silver release in water at neutral pH, and released the silver in concentrated nitric acid solution (pH = 1.1). The AgNP/CeO2/TiO2 nanocontainers showed an antibacterial activity against E. coli, however a concentration-dependent cytotoxicity towards a model epithelial barrier cell type (A549 cells) was observed. These nanocontainers offer the concept of potentially controlling silver delivery for the prevention of implant-associated infections.

Characterizing nanoparticles in complex biological media and physiological fluids with depolarized dynamic light scattering.

Light scattering is one of the few techniques available to adequately characterize suspended nanoparticles (NPs) in real time and in situ. However, when it comes to NPs in multicomponent and optically complex aqueous matrices - such as biological media and physiological fluids - light scattering suffers from lack of selectivity, as distinguishing the relevant optical signals from the irrelevant ones is very challenging. We meet this challenge by building on depolarized scattering: Unwanted signals from the matrix are completely suppressed. This approach yields information with an unprecedented signal-to-noise ratio in favour of the NPs and NP-biomolecule corona complexes, which in turn opens the frontier to scattering-based studies addressing the behaviour of NPs in complex physiological/biological fluids.

Integrating silver compounds and nanoparticles into ceria nanocontainers for antimicrobial applications.

Silver compounds and nanoparticles (NPs) are gaining increasing interest in medical applications, specifically in the treatment and prevention of biomaterial-related infections. However, the silver release from these materials, resulting in a limited antimicrobial activity, is often difficult to control. In this paper, ceria nanocontainers were synthesized by a template-assisted method and were then used to encapsulate silver nitrate (AgNO3/CeO2 nanocontainers). Over the first 30 days, a significant level of silver was released, as determined using inductively coupled plasma optical emission spectroscopy (ICP-OES). A novel type of ceria container containing silver NPs (AgNP/CeO2 containers) was also developed using two different template removal methods. The presence of AgNPs was confirmed both on the surface and in the interior of the ceria containers by X-ray diffraction (XRD), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Upon removal of the template by calcination, the silver was released over a period exceeding three months (>90 days). However, when the template was removed by dissolution, the silver release was shortened to ≤14 days. The antimicrobial activity of the silver-containing CeO2 containers was observed and the minimum bactericidal concentration (MBC) was determined using the broth dilution method. Investigation on human cells, using a model epithelial barrier cell type (A549 cells), highlighted that all three samples induced a heightened cytotoxicity leading to cell death when exposed to all containers in their raw form. This was attributed to the surface roughness of the CeO2 nanocontainers and the kinetics of the silver release from the AgNO3/CeO2 and AgNP/CeO2 nanocontainers. In conclusion, despite the need for further emphasis on their biocompatibility, the concept of the AgNP/CeO2 nanocontainers offers a potentially alternative long-term antibactericidal strategy for implant materials.

In vitro dosimetry of agglomerates.

Agglomeration of nanoparticles in biological fluids is a pervasive phenomenon that leads to difficulty in the interpretation of results from in vitro exposure, primarily due to differing particokinetics of agglomerates to nanoparticles. Therefore, well-defined small agglomerates were designed that possessed different particokinetic profiles, and their cellular uptake was compared to a computational model of dosimetry. The approach used here paves the way for a better understanding of the impact of agglomeration on the nanoparticle-cell interaction.

Magnetic microreactors for efficient and reliable magnetic nanoparticle surface functionalization.

Microreactors have attracted wide attention in the nano- and biotechnology fields because they offer many advantages over standard liquid phase reactions. We report the development of a magnetic microreactor for reliable, fast and efficient surface functionalization of superparamagnetic iron oxide nanoparticles (SPIONs). A comprehensive study of the development process in terms of setup, loading capacity and efficiency is described. We performed experimental and computational studies in order to evaluate the trapping efficiencies, maximum loading capacity and magnetic alignment of the nanoparticles. The results showed that capacity and trapping efficiencies are directly related to the flow rate, elution time and reactor type. Based on our results and the developed magnetic microreactor, we describe a model multistep surface derivatization procedure of SPIONs.

Improved dynamic response assessment for intra-articular injected iron oxide nanoparticles.

The emerging importance of nanoparticle technology, including iron oxide nanoparticles for monitoring development, progression, and treatment of inflammatory diseases such as arthritis, drives development of imaging techniques. Studies require an imaging protocol that is sensitive and quantifiable for the detection of iron oxide over a wide range of concentrations. Conventional signal loss measurements of iron oxide nanoparticle containing tissues saturate at medium concentrations and show a nonlinear/nonproportional intensity to concentration profile due to the competing effects of T1 and T2 relaxation. A concentration calibration phantom and an in vivo study of intra-articular injection in a rat knee of known concentrations of iron oxide were assessed using the difference-ultrashort echo time sequence giving a positive, quantifiable, unambiguous iron signal and monotonic, increasing concentration response over a wide concentration range in the phantom with limited susceptibility artifacts and high contrast in vivo to all other tissues. This improved dynamic response to concentration opens possibilities for quantification due to its linear nature at physiologically relevant concentrations.

Superparamagnetic iron oxide binding and uptake as imaged by magnetic resonance is mediated by the integrin receptor Mac-1 (CD11b/CD18): implications on imaging of atherosclerotic plaques.

INTRODUCTION: Superparamagnetic iron oxide nanoparticles (SPIONs) have been successfully used for magnetic resonance imaging (MRI) of atherosclerotic plaques. Endocytosis into monocytes/macrophages has been proposed as the mechanism for SPION uptake, but a specific receptor has not been identified yet. A potential candidate is the versatile integrin Mac-1 (CD11b/CD18, alphaMbeta2), which is involved in leukocyte adhesion, complement activation and phagocytosis. METHODS AND RESULTS: Intracellular SPION-accumulation was confirmed in cultured human monocytes using immunohistochemistry and iron staining. Recombinant cells expressing Mac-1 in different activation states as well as human monocytes with or without PMA stimulation were incubated either with an unspecific IgG or a CD11b-blocking antibody. Thereafter, cells were incubated with FITC-labeled amino-covered SPIONs or ferumoxtran-10 SPIONs and signal intensity was quantified by flow cytometry. Depending on the activation status of Mac-1, a significant increase in SPION binding/uptake was observed, independent on surface coating. Furthermore, SPION binding/uptake was significantly reduced after CD11b blockade. Results were confirmed in recombinant cells incubated with amino-PVA SPIONs and ferumoxtran-10, using T2(*)-weighted 3T MRI. CONCLUSION: The integrin Mac-1 is directly involved in SPION binding/uptake. Thus, monocytes abundantly expressing Mac-1 and especially activated monocytes expressing activated Mac-1 may be useful vehicles for high resolution MRI labeling of atherosclerotic plaques.

Development of functionalized superparamagnetic iron oxide nanoparticles for interaction with human cancer cells.

Our goal is to develop, characterize and optimize functionalized super paramagnetic iron oxide nanoparticles (SPION) demonstrating the capacity to be internalized by human cancer cells. SPION (mean diameter 9nm) were coated with various ratios to iron oxide of either polyvinyl alcohol (PVA), carboxylate-functionalized PVA, thiol-functionalized PVA and amino-functionalized PVA (amino-PVA). The interaction with cells and cytotoxicity of the SPION preparations were determined using human melanoma cells. From the four functionalized SPION preparations, only the amino-PVA SPION demonstrated the capacity to interact with, and were not cytotoxic to, human melanoma cells. This interaction with melanoma cells was dependent on the amino-PVA to iron oxide ratio, was an active and saturable mechanism displayed by all cells in a culture. These functionalized SPION were characterized by transmission electron microscopy and electrophoretic mobility. The physical comportment of SPION changed at specific PVAs to iron oxide ratios, and this ratio corresponded to the ratio of optimal interaction with cells. In conclusion, the successful development of functionalized SPION displaying potential cellular uptake by human cancer cells depends both on the presence of amino groups on the coating shell of the nanoparticles and of its ratio to the amount of iron oxide.