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Jacopo M Fontana,1 Huijuan Yin,1 Yun Chen,2 Ricardo Florez,1 Hjalmar Brismar,1 Ying Fu1 1Section of Cellular Biophysics, Department of Applied Physics, Royal Institute of Technology, Science for Life Laboratory, Solna, 2Department of Molecular and Clinical Medicine/Clinical Physiology, The Sahlgrenska Academy and University Hospital, University of Gothenburg, Gothenburg, Sweden Abstract: Colloidal semiconductor quantum dots (QDs) have been extensively researched and developed for biomedical applications, including drug delivery and biosensing assays. Hence, it is pivotal to understand their behavior in terms of intracellular transport and toxicological effects. In this study, we focused on 3-mercaptopropionic acid-coated CdSe-CdS/ZnS core-multishell quantum dots (3MPA-QDs) converted from the as-grown octadecylamine-coated quantum dots (ODA-QDs) and their direct and dynamic interactions with human umbilical vein endothelial cells (HUVECs). Live cell imaging using confocal fluorescence microscopy showed that 3MPA-QDs first attached to and subsequently aggregated on HUVEC plasma membrane ~25 min after QD deposition. The aggregated QDs started being internalized at ~2 h and reached their highest internalization degree at ~24 h. They were released from HUVECs after ~48 h. During the 48 h period, the HUVECs responded normally to external stimulations, grew, proliferated and wound healed without any perceptible apoptosis. Furthermore, 1) 3MPA-QDs were internalized in newly formed LysoTracker-stained early endosomes; 2) adenosine 5'-triphosphate-induced [Ca2+]i modulation caused a transient decrease in the fluorescence of 3MPA-QDs that were attached to the plasma membrane but a transient increase in the internalized 3MPA-QDs; and 3) fluorescence signal modulations of co-stained LysoTracker and QDs induced by the lysosomotropic agent Gly-Phe-β-naphthylamide were spatially co-localized and temporally synchronized. Our findings suggest that 3MPA-QDs converted from ODA-QDs are a potential nontoxic fluorescent probe for future use in clinical applications. Moreover, the photophysical strategy and techniques reported in this work are easily applicable to study of direct interactions between other nanoparticles and live cells; contributing to awareness and implementation of the safe applications of nanoparticles. Keywords: colloidal semiconductor quantum dots, human umbilical vein endothelial cell, intracellular labeling, nanotoxicity, adenosine 5'-triphosphate, endosome

Huijuan Yin,1 Jacopo M Fontana,1 Johan Solandt,1,2 Johnny Israelsson Jussi,1 Hao Xu,1 Hjalmar Brismar,1 Ying Fu1 1Section of Cellular Biophysics, Department of Applied Physics, Royal Institute of Technology (KTH), Science for Life Laboratory, Solna, 2AstraZeneca R&D, Mölndal, Sweden Abstract: While adverse effects of nanoparticles on lung health have previously been proposed, few studies have addressed the direct effects of nanoparticle exposure on the airway epithelium. In this work, we examine the response of the pulmonary airway to nanoparticles by measuring intracellular Ca2+ concentration ([Ca2+]i) in the Calu-3 epithelial layer stimulated by 3-mercaptopropionic-acid (3MPA) coated CdSe-CdS/ZnS core-multishell quantum dots (QDs). Simultaneous transient transepithelial electrical resistance (TEER) decrease and global [Ca2+]i increase in Calu-3 epithelial layer, accompanied by cell displacements, contraction, and expansion, were observed under QD deposition. This suggests that a QD-induced global [Ca2+]i increase in the Calu-3 epithelial layer caused the transient TEER decrease. The [Ca2+]i increase was marked and rapid in the apical region, while [Ca2+]i decreased in the basolateral region of the epithelial layer. TEER transient response and extracellular Ca2+ entry induced by QD deposition were completely inhibited in cells treated with stretched-activated (SA) inhibitor GdCl3 and store-operated calcium entry (SOCE) inhibitor BTP2 and in cells immersed in Ca2+-free medium. The voltage-gated calcium channel (VGCC) inhibitor nifedipine decreased, stabilized, and suppressed the TEER response, but did not affect the [Ca2+]i increase, due to QD deposition. This demonstrates that the Ca2+ influx activated by QDs’ mechanical stretch occurs through activation of both SA and SOCE channels. QD-induced [Ca2+]i increase occurred in the Calu-3 epithelial layer after culturing for 15 days, while significant TEER drop only occurred after 23 days. This work provides a new perspective from which to study direct interactions between airway epithelium and nanoparticles and may help to reveal the pathologies of pulmonary disease. Keywords: Calu-3 epithelial layer, quantum dot, intracellular Ca2+ concentration [Ca2+]i, transepithelial electrical resistance, cell movement

We demonstrate a laboratory method for imaging small blood vessels using x-ray propagation-based phase-contrast imaging and carbon dioxide (CO2) gas as a contrast agent. The limited radiation dose in combination with CO2 being clinically acceptable makes the method promising for small-diameter vascular visualization. We investigate the possibilities and limitations of the method for small-animal angiography and compare it with conventional absorptionbased x-ray angiography. Photon noise in absorption-contrast imaging prevents visualization of blood vessels narrower than 50 &mgr;m at the highest radiation doses compatible with living animals, whereas our simulations and experiments indicate the possibility of visualizing 20 &mgr;m vessels at radiation doses as low as 100 mGy. Experimental computed tomography of excised rat kidney shows blood vessels of diameters down to 60 &mgr;m with improved image quality compared to absorption-based methods. With our present prototype x-ray source, the acquisition time for a tomographic dataset is approximately 1 h, which is long compared to the 1-20 min common for absorption-contrast micro-CT systems. Further development of the liquid-metal-jet microfocus x-ray sources used here and high-resolution x-ray detectors shows promise to reduce exposure times and make this high-resolution method practical for imaging of living animals. [ABSTRACT FROM AUTHOR]

Abstract: The interaction between the dopaminergic and glutamatergic systems governs normal behavior and is perturbed in many psychiatric disorders including schizophrenia. Hypofunction of the D1 family of receptors, to which the D1 and D5 subtypes belong, is a typical feature of schizophrenia. Here we have used confocal live cell imaging of neurons to examine the distinct roles of the D1 and D5 receptors in the intra-neuronal interaction with the glutamatergic system. Using fluorescently tagged D1 or D5 expressed in cultured striatal neurons, we show that both receptor subtypes are primarily transported via lateral diffusion in the dendritic tree. D1 is to a much larger extent than D5 expressed in spines. D1 is primarily expressed in the head whereas D5 is largely localized to the neck of the spine. Activation of N-methyl-d-aspartic acid (NMDA) receptors slowed the diffusion rate and increased the number of D1 positive spines, while no effect on D5 diffusion or spine localization could be observed. The observed differences between D1 and D5 can be attributed to structural differences in the C-terminus and its capacity to interact with NMDA receptors and PSD-95. Identification of a unique role of D1 for the intra-neuronal interaction between the dopaminergic and glutamatergic systems will have implications for the development of more specific treatments in many neuropsychiatric disorders. [Copyright &y& Elsevier]