Calum Kinnear

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).

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

BackgroundThe 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.MethodsCellulose 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.ResultsA 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.ConclusionIn 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.

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.

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.

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.

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.

Ultrathin ceramic membranes as scaffolds for functional cell coculture models on a biomimetic scale

Abstract 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.

High-content analysis of factors affecting gold nanoparticle uptake by neuronal and microglial cells in culture

Owing to their ubiquitous distribution, expected beneficial effects and suspected adverse effects, nanoparticles are viewed as a double-edged sword, necessitating a better understanding of their interactions with tissues and organisms. Thus, the goals of the present study were to develop and present a method to generate quantitative data on nanoparticle entry into cells in culture and to exemplarily demonstrate the usefulness of this approach by analyzing the impact of size, charge and various proteinaceous coatings on particle internalization. N9 microglial cells and both undifferentiated and differentiated SH-SY5Y neuroblastoma cells were exposed to customized gold nanoparticles. After silver enhancement, the particles were visualized by epipolarization microscopy and analysed by high-content analysis. The value of this approach was substantiated by assessing the impact of various parameters on nanoparticle uptake. Uptake was higher in microglial cells than in neuronal cells. Only microglial cells showed a distinct size preference, preferring particles with a diameter of 80 nm. Positive surface charge had the greatest impact on particle uptake. Coating with bovine serum albumin, fetuin or protein G significantly increased particle internalization in microglial cells but not in neuronal cells. Coating with wheat germ agglutinin increased particle uptake in both N9 and differentiated SH-SY5Y cells but not in undifferentiated SH-SY5Y cells. Furthermore, internalization was shown to be an active process and indicators of caspase-dependent apoptosis revealed that gold nanoparticles did not have any cytotoxic effects. The present study thus demonstrates the suitability of gold nanoparticles and high-content analysis for assessing numerous variables in a stringently quantitative and statistically significant manner. Furthermore, the results presented herein showcase the feasibility of specifically targeting nanoparticles to distinct cell types.

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.

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.