Probing nano-scale viscoelastic response in air and in liquid with dynamic atomic force microscopy

We perform a comparative study of dynamic force measurements using an Atomic Force Microscope (AFM) on the same soft polymer blend samples in both air and liquid environments. Our quantitative analysis starts with calibration of the same cantilever in both environments. Intermodulation AFM (ImAFM) is used to measure dynamic force quadratures on the same sample. We validate the accuracy of the reconstructed dynamic force quadratures by numerical simulation of a realistic model of the cantilever in liquid. In spite of the very low quality factor of this resonance, we find excellent agreement between experiment and simulation. A recently developed moving surface model explains the measured force quadrature curves on the soft polymer, in both air and liquid

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

Biological response of an in vitro human 3D lung cell model exposed to brake wear debris varies based on brake pad formulation

Wear particles from automotive friction brake pads of various sizes, morphology, and chemical composition are significant contributors towards particulate matter. Knowledge concerning the potential adverse effects following inhalation exposure to brake wear debris is limited. Our aim was, therefore, to generate brake wear particles released from commercial low-metallic and non-asbestos organic automotive brake pads used in mid-size passenger cars by a full-scale brake dynamometer with an environmental chamber simulating urban driving and to deduce their potential hazard in vitro. The collected fractions were analysed using scanning electron microscopy via energy-dispersive X-ray spectroscopy (SEM-EDS) and Raman microspectroscopy. The biological impact of the samples was investigated using a human 3D multicellular model consisting of human epithelial cells (A549) and human primary immune cells (macrophages and dendritic cells) mimicking the human epithelial tissue barrier. The viability, morphology, oxidative stress, and (pro-)inflammatory response of the cells were assessed following 24 h exposure to similar to 12, similar to 24, and similar to 48 A mu g/cm(2) of non-airborne samples and to similar to 3.7 A mu g/cm(2) of different brake wear size fractions (2-4, 1-2, and 0.25-1 A mu m) applying a pseudo-air-liquid interface approach. Brake wear debris with low-metallic formula does not induce any adverse biological effects to the in vitro lung multicellular model. Brake wear particles from non-asbestos organic formulated pads, however, induced increased (pro-)inflammatory mediator release from the same in vitro system. The latter finding can be attributed to the different particle compositions, specifically the presence of anatase.Web of Science9272351233

Collective and single cell behavior in epithelial contact inhibition

Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as "contact inhibition". The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured MDCK cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition

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

Formation of drug nanocrystals under nanoconfinement afforded by liposomes

Nanocrystals of drug substances have important therapeutic applications, but their preparation is often difficult due to size control in bottom up approaches, or energetic milling and surface activation in top down processing. In this study, confinement within liposome nanocompartments is demonstrated to enable drug crystallization with a high aspect ratio but limited growth resulting in nanocrystals, using a simple freeze–thaw process which is anticipated to be amenable to large scale preparation. After the freeze–thaw, cryo-transmission electron microscopy (cryoTEM) imaging and cryo-electron tomography revealed that the majority of the liposomes contained a single drug nanocrystal, observed to physically stretch but not burst the liposomes, and the composition of the freeze–thaw medium altered the aspect ratio of the drug nanocrystals. Small angle X-ray scattering and dynamic depolarized light scattering were used to confirm the asymmetric nature of particles in suspension to exclude the cryoTEM preparation process as a contributor to the particle morphology. In assessing potential use in controlled release drug delivery, the in vitro release rate of ciprofloxacin from liposomes containing the nanocrystals revealed that the rate of dissolution of the nanocrystals became the rate controlling step, in contrast to the lipid bilayer rate controlling function prior to the formation of the crystals. The ability to modulate the release rate of the active ingredient in a complex formulation using simple physical means (e.g., freeze/thaw) is an attractive possibility, especially in highly regulated industries such as pharmaceuticals where qualitative and quantitative changes of composition would require extensive safety evaluations

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing