Toxicity Assessments of Multisized Gold and Silver Nanoparticles in Zebrafish Embryos

The potential toxicity of nanoparticles is addressed by utilizing a putative attractive model in developmental biology and genetics: the zebrafish (Danio rerio). Transparent zebrafish embryos, possessing a high degree of homology to the human genome, offer an economically feasible, medium‐througput screening platform for noninvasive real‐time assessments of toxicity. Using colloidal silver (cAg) and gold nanoparticles (cAu) in a panoply of sizes (3, 10, 50, and 100 nm) and a semiquantitative scoring system, it is found that cAg produces almost 100% mortality at 120 h post‐fertilization, while cAu produces less than 3% mortality at the same time point. Furthermore, while cAu induces minimal sublethal toxic effects, cAg treatments generate a variety of embryonic morphological malformations. Both cAg and cAu are taken up by the embryos and control experiments, suggesting that cAg toxicity is caused by the nanoparticles themselves or Ag⁺ that is formed during in vivo nanoparticle destabilization. Although cAg toxicity is slightly size dependent at certain concentrations and time points, the most striking result is that parallel sizes of cAg and cAu induce significantly different toxic profiles, with the former being toxic and the latter being inert in all exposed sizes. Therefore, it is proposed that nanoparticle chemistry is as, if not more, important than specific nanosizes at inducing toxicity in vivo. Ultimately such assessments using the zebrafish embryo model should lead to the identification of nanomaterial characteristics that afford minimal or no toxicity and guide more rational designs of materials on the nanoscale.     

Real‐Time Label‐Free Monitoring of Nanoparticle Cell Uptake

The surface plasmon resonance technique in combination with whole cell sensing is used for the first time for real‐time label‐free monitoring of nanoparticle cell uptake. The uptake kinetics of several types of nanoparticles relevant to drug delivery applications into HeLa cells is determined. The cell uptake of the nanoparticles is confirmed by confocal microscopy. The cell uptake of silica nanoparticles and polyethylenimine–plasmid DNA polyplexes is studied as a function of temperature, and the uptake energies are determined by Arrhenius plots. The phase transition temperature of the HeLa cell membrane is detected when monitoring cell uptake of silica nanoparticles at different temperatures. The HeLa cell uptake of the mesoporous silica nanoparticles is energy‐independent at temperatures slightly higher than the phase transition temperature of the HeLa cell membrane, while the uptake of polyethylenimine–DNA polyplexes is energy‐dependent and linear as a function of temperature with an activation energy of Ea = 62 ± 7 kJ mol^?1 = 15 ± 2 kcal mol^?1. The HeLa cell uptake of red blood cell derived extracellular vesicles is also studied as a function of the extracellular vesicle concentration. The results show a concentration dependent behavior reaching a saturation level of the extracellular vesicle uptake by HeLa cells.     

Real‐Time Tracking of Superparamagnetic Nanoparticle Self‐Assembly

The spontaneous self‐assembly process of superparamagnetic nanoparticles in a fast‐drying colloidal drop is observed in real time. The grazing‐incidence small‐angle X‐ray scattering (GISAXS) technique is employed for an in situ tracking of the reciprocal space, with a 3 ms delay time between subsequent frames delivered by a new generation of X‐ray cameras. A focused synchrotron beam and sophisticated sample oscillations make it possible to relate the dynamic reciprocal to direct space features and to localize the self‐assembly. In particular, no nanoparticle ordering is found inside the evaporating drop and near‐surface region down to a drop thickness of 90 µm. Scanning through the shrinking drop‐contact line indicates the start of self‐assembly near the drop three‐phase interface, in accord with theoretical predictions. The results obtained have direct implications for establishing the self‐assembly process as a routine technological step in the preparation of new nanostructures.     

Modulation of Electron Tunneling in a Nanoparticle Array by Sound Waves: An Avenue to High‐Speed,High‐Sensitivity Sensors

Using an electrostatic self‐assembly process, metal nanoparticles are deposited on polyelectrolyte fibers such that the interparticle distance between the nanoparticles is comparable to the polyelectrolyte's molecular width. By modulating the dielectric properties of the interparticle polymer layer, a highly sensitive, reversible humidity sensor with an ultrafast response time of ≈3 ms is demonstrated. The higher sensitivity at low humidity shows a conductivity increase by over two orders of magnitude in response to a change in relative humidity from 21 to 1%.     

In vitro Toxicity Testing of Nanoparticles in 3D Cell Culture

Common 2D cell cultures do not adequately represent the functions of 3D tissues that have extensive cell–cell and cell–matrix interactions, as well as markedly different diffusion/transport conditions. Hence, testing cytotoxicity in 2D cultures may not accurately reflect the actual toxicity of nanoparticles (NPs) and other nanostructures in the body. To obtain more adequate and detailed information about NP–tissue interactions, we here introduce a 3D‐spheroid‐culture‐based NP toxicology testing system. Hydrogel inverted colloidal crystal (ICC) scaffolds are used to create a physiologically relevant and standardized 3D liver tissue spheroid model for in vitro assay application. Toxicity of CdTe and Au NPs are tested in both 2D and 3D spheroid cultures. The results reveal that NP toxic effects are significantly reduced in the spheroid culture when compared to the 2D culture data. Tissue‐like morphology and phenotypic change are identified to be the major factors in diminishing toxicity. Acting as an intermediate stage bridging in vitro 2D and in vivo, our in vitro 3D cell‐culture model would extend current cellular level cytotoxicity to the tissue level, thereby improving the predictive power of in vitro NP toxicology.     

Zebrafish Embryos Allow Prediction of Nanoparticle Circulation Times in Mice and Facilitate Quantification of Nanoparticle–Cell Interactions

The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real‐time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse.     

Real‐Time Analysis of Magnetic Hyperthermia Experiments on Living Cells under a Confocal Microscope

Combining high‐frequency alternating magnetic fields (AMF) and magnetic nanoparticles (MNPs) is an efficient way to induce biological responses through several approaches: magnetic hyperthermia, drug release, controls of gene expression and neurons, or activation of chemical reactions. So far, these experiments cannot be analyzed in real‐time during the AMF application. A miniaturized electromagnet fitting under a confocal microscope is built, which produces an AMF of frequency and amplitude similar to the ones used in magnetic hyperthermia. AMF application induces massive damages to tumoral cells having incorporated nanoparticles into their lysosomes without affecting the others. Using this setup, real‐time analyses of molecular events occurring during AMF application are performed. Lysosome membrane permeabilization and reactive oxygen species production are detected after only 30 min of AMF application, demonstrating they occur at an early stage in the cascade of events leading eventually to cell death. Additionally, lysosomes self‐assembling into needle‐shaped organization under the influence of AMF is observed in real‐time. This experimental approach will permit to get a deeper insight into the physical, molecular, and biological process occurring in several innovative techniques used in nanomedecine based on the combined use of MNPs and high‐frequency magnetic fields.     

Biomimicry 3D Gastrointestinal Spheroid Platform for the Assessment of Toxicity and Inflammatory Effects of Zinc Oxide Nanoparticles

Our current mechanistic understanding on the effects of engineered nanoparticles (NPs) on cellular physiology is derived mainly from 2D cell culture studies. However, conventional monolayer cell culture may not accurately model the mass transfer gradient that is expected in 3D tissue physiology and thus may lead to artifactual experimental conclusions. Herein, using a micropatterned agarose hydrogel platform, the effects of ZnO NPs (25 nm) on 3D colon cell spheroids of well‐defined sizes are examined. The findings show that cell dimensionality plays a critical role in governing the spatiotemporal cellular outcomes like inflammatory response and cytotoxicity in response to ZnO NPs treatment. More importantly, ZnO NPs can induce different modes of cell death in 2D and 3D cell culture systems. Interestingly, the outer few layers of cells in 3D model could only protect the inner core of cells for a limited time and periodically slough off from the spheroids surface. These findings suggest that toxicological conclusions made from 2D cell models might overestimate the toxicity of ZnO NPs. This 3D cell spheroid model can serve as a reproducible platform to better reflect the actual cell response to NPs and to study a more realistic mechanism of nanoparticle‐induced toxicity.     

Inertial Microfluidic Cell Stretcher (iMCS): Fully Automated,High‐Throughput,and Near Real‐Time Cell Mechanotyping

Mechanical biomarkers associated with cytoskeletal structures have been reported as powerful label‐free cell state identifiers. In order to measure cell mechanical properties, traditional biophysical (e.g., atomic force microscopy, micropipette aspiration, optical stretchers) and microfluidic approaches were mainly employed; however, they critically suffer from low‐throughput, low‐sensitivity, and/or time‐consuming and labor‐intensive processes, not allowing techniques to be practically used for cell biology research applications. Here, a novel inertial microfluidic cell stretcher (iMCS) capable of characterizing large populations of single‐cell deformability near real‐time is presented. The platform inertially controls cell positions in microchannels and deforms cells upon collision at a T‐junction with large strain. The cell elongation motions are recorded, and thousands of cell deformability information is visualized near real‐time similar to traditional flow cytometry. With a full automation, the entire cell mechanotyping process runs without any human intervention, realizing a user friendly and robust operation. Through iMCS, distinct cell stiffness changes in breast cancer progression and epithelial mesenchymal transition are reported, and the use of the platform for rapid cancer drug discovery is shown as well. The platform returns large populations of single‐cell quantitative mechanical properties (e.g., shear modulus) on‐the‐fly with high statistical significances, enabling actual usages in clinical and biophysical studies.     

Temperature‐Controlled High‐Speed AFM: Real‐Time Observation of Ripple Phase Transitions

With nanometer lateral and Angstrom vertical resolution, atomic force microscopy (AFM) has contributed unique data improving the understanding of lipid bilayers. Lipid bilayers are found in several different temperature‐dependent states, termed phases; the main phases are solid and fluid phases. The transition temperature between solid and fluid phases is lipid composition specific. Under certain conditions some lipid bilayers adopt a so‐called ripple phase, a structure where solid and fluid phase domains alternate with constant periodicity. Because of its narrow regime of existence and heterogeneity ripple phase and its transition dynamics remain poorly understood. Here, a temperature control device to high‐speed atomic force microscopy (HS‐AFM) to observe dynamics of phase transition from ripple phase to fluid phase reversibly in real time is developed and integrated. Based on HS‐AFM imaging, the phase transition processes from ripple phase to fluid phase and from ripple phase to metastable ripple phase to fluid phase could be reversibly, phenomenologically, and quantitatively studied. The results here show phase transition hysteresis in fast cooling and heating processes, while both melting and condensation occur at 24.15 °C in quasi‐steady state situation. A second metastable ripple phase with larger periodicity is formed at the ripple phase to fluid phase transition when the buffer contains Ca²⁺. The presented temperature‐controlled HS‐AFM is a new unique experimental system to observe dynamics of temperature‐sensitive processes at the nanoscopic level.     

Simple and Rapid High‐Yield Synthesis and Size Sorting of Multibranched Hollow Gold Nanoparticles with Highly Tunable NIR Plasmon Resonances

Branched gold nanoparticles with sharp tips are considered excellent candidates for sensing and field enhancement applications. Here, a rapid and simple synthesis strategy is presented that generates highly branched gold nanoparticles with hollow cores and a ca.100% yield through a simple one‐pot seedless reaction at room temperature in the presence of Triton X‐100. It is shown that multibranched hollow gold nanoparticles of tunable dimensions, branch density and branch length can be obtained by adjusting the concentrations of the reactants. Insights into the formation mechanism point toward an aggregative type of growth involving hollow core formation first, and branching thereafter. The pronounced near‐infrared (NIR) plasmon band of the nanoparticles is due to the combined contribution from hollowness and branching, and can be tuned over a wide range (≈700–2000 nm). It is also demonstrated that the high environmental sensitivity of colloidal dispersions based on multibranched hollow gold nanoparticles can be boosted even further by separating the nanoparticles into fractions of given sizes and improved monodispersity by means of a glycerol density gradient. The possibility to obtain highly monodisperse multibranched hollow gold nanoparticles with predictable dimensions (50–300 nm) and branching and, therefore, tailored NIR plasmonic properties, highlights their potential for theranostic applications.     

Optofluidic Platform for Real‐Time Monitoring of Live Cell Secretory Activities Using Fano Resonance in Gold Nanoslits

An optofluidic platform for real‐time monitoring of live cell secretory activities is constructed via Fano resonance in a gold nanoslit array. Large‐area and highly sensitive gold nanoslits with a period of 500 nm are fabricated on polycarbonate films using the thermal‐annealed template‐stripping method. The coupling between gap plasmon resonance in the slits and surface plasmon polariton Bloch waves forms a sharp Fano resonance with intensity sensitivity greater than 11 000% per refractive index unit. The nanoslit array is integrated with a cell‐trapping microfluidic device to monitor dynamic secretion of matrix metalloproteinase 9 (MMP‐9) from human acute monocytic leukemia cells in situ. Upon continuous lipopolysaccharide (LPS) stimulation, MMP‐9 secretion is detected within 2 h due to ultrahigh surface sensitivity and close proximity of the sensor to the target cells. In addition to the advantage of detecting early cell responses, the sensor also allows interrogation of cell secretion dynamics. Furthermore, the average secretion per cell measured using our system well matches previous reports while it requires orders of magnitude less cells. The optofluidic platform may find applications in fundamental studies of cell functions and diagnostics based on secretion signals.