Drug Evaluation Based on a Multi-Channel Cell Chip with a Horizontal Co-Culture.

We developed a multi-channel cell chip containing a three-dimensional (3D) scaffold for horizontal co-culture and drug toxicity screening in multi-organ culture (human glioblastoma, cervical cancer, normal liver cells, and normal lung cells). The polydimethylsiloxane (PDMS) multi-channel cell chip (PMCCC) was based on fused deposition modeling (FDM) technology. The architecture of the PMCCC was an open-type cell chip and did not require a pump or syringe. We investigated cell proliferation and cytotoxicity by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-dphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and analysis of oleanolic acid (OA)-treated multi-channel cell chips. The results of the MTT and LDH assays showed that OA treatment in the multi-channel cell chip of four cell lines enhanced chemoresistance of cells compared with that in the 2D culture. Furthermore, we demonstrated the feasibility of the application of our multi-channel cell chip in various analysis methods through Annexin V-fluorescein isothiocyanate/propidium iodide staining, which is not used for conventional cell chips. Taken together, the results demonstrated that the PMCCC may be used as a new 3D platform because it enables simultaneous drug screening in multiple cells by single point injection and allows analysis of various biological processes.

Tumor Homing Reactive Oxygen Species Nanoparticle for Enhanced Cancer Therapy.

Multifunctional nanoparticles that carry chemotherapeutic agents can be innovative anticancer therapeutic options owing to their tumor-targeting ability and high drug-loading capacity. However, the nonspecific release of toxic DNA-intercalating anticancer drugs from the nanoparticles has significant side effects on healthy cells surrounding the tumors. Herein, we report a tumor homing reactive oxygen species nanoparticle (THoR-NP) platform that is highly effective and selective for ablating malignant tumors. Sodium nitroprusside (SNP) and diethyldithiocarbamate (DDC) were selected as an exogenous reactive oxygen species (ROS) generator and a superoxide dismutase 1 inhibitor, respectively. DDC-loaded THoR-NP, in combination with SNP treatment, eliminated multiple cancer cell lines effectively by the generation of peroxynitrite in the cells (>95% cell death), as compared to control drug treatments of the same concentration of DDC or SNP alone (0% cell death). Moreover, the magnetic core (ZnFe2O4) of the THoR-NP can specifically ablate tumor cells (breast cancer cells) via magnetic hyperthermia, in conjunction with DDC, even in the absence of any exogenous RS supplements. Finally, by incorporating iRGD peptide moieties in the THoR-NP, integrin-enriched cancer cells (malignant tumors, MDA-MB-231) were effectively and selectively killed, as opposed to nonmetastatic tumors (MCF-7), as confirmed in a mouse xenograft model. Hence, our strategy of using nanoparticles embedded with ROS-scavenger-inhibitor with an exogenous ROS supplement is highly selective and effective cancer therapy.

Bifunctional Au@Bi2 Se3 Core-Shell Nanoparticle for Synergetic Therapy by SERS-Traceable AntagomiR Delivery and Photothermal Treatment.

For the first time, topological insulator bismuth selenide nanoparticles (Bi2 Se3 NP) are core-shelled with gold (Au@Bi2 Se3 ) i) to represent considerably small-sized (11 nm) plasmonic nanoparticles, enabling accurate bioimaging in the near-infrared region; ii) to substantially improve Bi2 Se3 biocompatibility, iii) water dispersibility, and iv) surface functionalization capability through straightforward gold-thiol interaction. The Au@Bi2 Se3 is subsequently functionalized for v) effective targeting of SH-SY5Y cancer cells, vi) disrupting the endosome/lysosome membrane, vii) traceable delivery of antagomiR-152 and further synergetic oncomiR knockdown and photothermal therapy (PTT). Unprecedentedly, it is observed that the Au shell thickness has a significant impact on evoking the exotic plasmonic features of Bi2 Se3 . The Au@Bi2 Se3 possesses a high photothermal conversion efficiency (35.5%) and a remarkable surface plasmonic effect (both properties are approximately twofold higher than those of 50 nm Au nanoparticles). In contrast to the siRNA/miRNA delivery methods, the antagomiR delivery is based on strand displacement, in which the antagomiR-152 is displaced by oncomiR-152 followed by a surface-enhanced Raman spectroscopy signal drop. This enables both cancer cell diagnosis and in vitro real-time monitoring of the antagomiR release. This selective PTT nanoparticle can also efficiently target solid tumors and undergo in vivo PTT, indicating its potential clinical applications.

Effect and possible mechanisms of melatonin treatment on the quality and developmental potential of aged bovine oocytes.

After reaching the metaphase II (MII) stage, unfertilised oocytes undergo a time-dependent process of quality deterioration referred to as oocyte aging. The associated morphological and cellular changes lead to decreased oocyte developmental potential. This study investigated the effect of exogenous melatonin supplementation on in vitro aged bovine oocytes and explored its underlying mechanisms. The levels of cytoplasmic reactive oxygen species and DNA damage response in bovine oocytes increased during in vitro aging. Meanwhile, maturation promoting factor activity significantly decreased and the proportion of morphologically abnormal oocytes significantly increased. Melatonin supplementation significantly decreased quality deterioration in aged bovine MII oocytes (P<0.05). Additionally, it decreased the frequency of aberrant spindle organisation and cortical granule release during oocyte aging (P<0.05). In the melatonin-supplemented group, mitochondrial membrane potential and ATP production were significantly increased compared with control. Furthermore, melatonin treatment significantly increased the speed of development of bovine oocytes to the blastocyst stage after in vitro fertilisation and significantly decreased the apoptotic rate in the blastocysts (P<0.05). The expression of Bax and Casp3 in the blastocysts was significantly reduced after treatment with melatonin, whereas expression of Bcl2 significantly increased (P<0.05). In conclusion, these findings suggest that supplementation of aged bovine oocytes with exogenous melatonin improves oocyte quality, thereby enhancing the developmental capacity of early embryos.

Difficulty-related changes in inter-regional neural synchrony are dissociated between target and non-target processing.

The major purpose of this study was to explore the changes in the local/global gamma-band neural synchronies during target/non-target processing due to task difficulty under an auditory three-stimulus oddball paradigm. Multichannel event-related potentials (ERPs) were recorded from fifteen healthy participants during the oddball task. In addition to the conventional ERP analysis, we investigated the modulations in gamma-band activity (GBA) and inter-regional gamma-band phase synchrony (GBPS) for infrequent target and non-target processing due to task difficulty. The most notable finding was that the difficulty-related changes in inter-regional GBPS (33-35 Hz) at P300 epoch (350-600 ms) completely differed for target and non-target processing. As task difficulty increased, the GBPS significantly reduced for target processing but increased for non-target processing. This result contrasts with the local neural synchrony in gamma-bands, which was not affected by task difficulty. Another major finding was that the spatial patterns of functional connectivity were dissociated for target and non-target processing with regard to the difficult task. The spatial pattern for target processing was compatible with the top-down attention network, whereas that for the non-target corresponded to the bottom-up attention network. Overall, we found that the inter-regional gamma-band neural synchronies during target/non-target processing change significantly with task difficulty and that this change is dissociated between target and non-target processing. Our results indicate that large-scale neural synchrony is more relevant for the difference in information processing between target and non-target stimuli.

Electrochemical Detection of Human Mesenchymal Stem Cell Differentiation on Fabricated Gold Nano-Dot Cell Chips.

Human mesenchymal stem cells (MSCs) have the capacity for self-renewal and maintain pluripotency, which is defined by their ability to differentiate into cells such as osteoblasts, neurons, and glial cells. In this study, we report a method for defining the status of human MSCs based on electrochemical detection systems. Gold nano-dot structures were fabricated using a nanoporous alumina mask, and the structural formations were confirmed by scanning electron microscopy (SEM). Human MSCs were allowed to attach to RGD (Arg-Gly-Asp) peptide nanopatterned surfaces, and electrochemical tools were applied to the MSCs attached on the chip surface. The cultured MSCs were shown to differentiate into neural cell types, as indicated by immunocytochemical staining for tyrosine hydroxylase and beta tubulin III. Following treatment with basic fibroblast growth factor (bFGF) for 14 days, most of the B10 cells exhibited bipolar or multipolar morphology with branched processes, and the proportion of B10 cells expressing neuronal cell markers considerably increased. Electrophysiological recordings from MSCs treated with bFGF for 5-14 days were examined with cyclic voltammetry, and the electrochemical signals were shown to increase during differentiation from MSCs to neuronal cells. This human MSC cell line is a useful tool for studying organogenesis, specifically neurogenesis, and in addition, the cell line provides a valuable source of cells for cell therapy. The electrochemical measurement system proposed here could be utilized in electrical cell chips for numerous applications, including cell differentiation, disease diagnosis, drug detection, and on-site monitoring.

Microfluidic chip for the detection of biological toxic effects of polychlorinated biphenyls on neuronal cells.

The aim of this study was to develop a microfluidic neuronal cell chip device to monitor the toxic effects of polychlorinated biphenyls (PCBs) on PC-12 neuronal cells. PCBs adversely influence the activities of neuronal cells in the nervous system. In PC-12 cells, the production and secretion of dopamine decreases in response to PCB exposure. The microfluidic device that we developed to measure the amount of dopamine by cyclic voltammetry is composed of a control layer, a fluidic layer, and a gold electrode-patterned glass wafer. The control channel in the control layer functions as a microvalve to control the flow of the fluidic channel in the fluidic layer. The fluidic layer consists of 3 reaction chambers as well as fluidic channels. Three electrodes, including the working electrode, counter electrodes, and a reference electrode, are placed in a fluidic chamber. The electrochemical signals of dopamine, either from a standard dopamine solution or from the culture supernatant from cultured PC-12 cells, were obtained using a fabricated microfluidic neuronal cell chip by cyclic voltammetry. When PCBs were added to cultures of PC-12 cells, the amount of dopamine secreted from the PC-12 cells decreased due to the reduced activity of PC-12 cells. The fabricated neuronal cell chip was capable of detecting the toxic effect of dopamine on neuronal cells at concentrations of 10 microg/L and over. The practicality of the developed microfluidic neuronal cell chip was validated using river water spiked with PCBs.

Fronto-temporal interactions in the theta-band during auditory deviant processing.

Mismatch negativity (MMN) is a negative component of event-related potential (ERP) that reflects auditory deviant detection. Previous studies repeatedly suggested that MMN is generated by the fronto-temporal network. However, it is still unclear how the frontal and temporal areas interact. To verify this, we observed the spatiotemporal pattern of inter-regional functional connectivity using phase synchrony and Granger causality, by analyzing event-related electroencephalograms (EEGs) elicited by standard and deviant tones in an oddball paradigm. Strong theta-band phase synchrony and bidirectional Granger causality were observed between the frontal and temporal areas during the processing of auditory deviants, especially at the temporal interval of MMN. Our results support the hypothesis that fronto-temporal interactions are crucial to the generation of MMN during auditory deviant processing.

Cell chip based monitoring of toxic effects on dopaminergic cell.

Oxidative stress has been implicated in pesticide-induced neurotoxicity, base on its role in cascade of biochemical changes that lead to dopaminergic neuronal cell death. The present study examined the role of oxidative stress and the electrochemical detection by polychlorinated biphenyls (PCBs)-induced toxicant in SH-SY5Y cell. The cells were seed in the RED (Arg-Gly-Asp) nanopatterned coating gold substrate and treated with different concentration of PCBs for 24 h in culture, which induced the change of the cyclic voltammetry (CV) current peak. The CV results showed that PCB significantly decreased the current peaks in dose and time-dependent manner. After antioxidant treatment, the CV of the PCB-treated cell chip increased the current peak. Especially, gluthaione and catalase prevent PCB-induced decrease of CV current peak in the cell. The results demonstrated that the current peak decreased by the PCB and recovered by the antioxidant enzyme. In conclusion, results suggest that the electrochemical-based chip provide crucial information to improvement toward a cell chip system for drug screening application.

Fabrication of gold nanodot arrays on a transparent substrate as a nanobioplatform for label-free visualization of living cells.

Two-dimensional gold (Au) nanodot arrays on a transparent substrate were fabricated for imaging of living cells. A nanoporous alumina mask with large-area coverage capability was prepared by a two-step chemical wet etching process after a second anodization. Highly ordered Au nanodot arrays were formed on indium-tin-oxide (ITO) glass using very thin nanoporous alumina of approximately 200 nm thickness as an evaporation mask. The large-area Au nanodot arrays on ITO glass were modified with RGD peptide (arginine; glycine; aspartic acid) containing a cysteine (Cys) residue and then used to immobilize human cancer HeLa cells, the morphology of which was observed by confocal microscopy. The confocal micrographs of living HeLa cells on Au nanodot arrays revealed enhanced contrast and resolution, which enabled discernment of cytoplasmic organelles more clearly. These results suggest that two-dimensional Au nanodot arrays modified with RGD peptide on ITO glass have potential as a biocompatible nanobioplatform for the label-free visualization and adhesion of living cells.

Thalamic T-type Ca²+ channels mediate frontal lobe dysfunctions caused by a hypoxia-like damage in the prefrontal cortex.

Hypoxic damage to the prefrontal cortex (PFC) has been implicated in the frontal lobe dysfunction found in various neuropsychiatric disorders. The underlying subcortical mechanisms, however, have not been well explored. In this study, we induced a PFC-specific hypoxia-like damage by cobalt-wire implantation to demonstrate that the role of the mediodorsal thalamus (MD) is critical for the development of frontal lobe dysfunction, including frontal lobe-specific seizures and abnormal hyperactivity. Before the onset of these abnormalities, the cross talk between the MD and PFC nuclei at theta frequencies was enhanced. During the theta frequency interactions, burst spikes, known to depend on T-type Ca(2+) channels, were increased in MD neurons. In vivo knockout or knockdown of the T-type Ca(2+) channel gene (Ca(V)3.1) in the MD substantially reduced the theta frequency MD-PFC cross talk, frontal lobe-specific seizures, and locomotor hyperactivity in this model. These results suggest a two-step model of prefrontal dysfunction in which the response to a hypoxic lesion in the PFC results in abnormal thalamocortical feedback driven by thalamic T-type Ca(2+) channels, which, in turn, leads to the onset of neurological and behavioral abnormalities. This study provides valuable insights into preventing the development of neuropsychiatric disorders arising from irreversible PFC damage.

Analysis of effect of nanoporous alumina substrate coated with polypyrrole nanowire on cell morphology based on AFM topography.

In this study, in situ electrochemical synthesis of polypyrrole nanowires with nanoporous alumina template was described. The formation of highly ordered porous alumina substrate was demonstrated with Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). In addition, Fourier transform infrared analysis confirmed that polypyrrole (PP) nanowires were synthesized by direct electrochemical oxidation of pyrrole. HeLa cancer cells and HMCF normal cells were immobilized on the polypyrrole nanowires/nanoporous alumina substrates to determine the effects of the substrate on the cell morphology, adhesion and proliferation as well as the biocompatibility of the substrate. Cell adhesion and proliferation were characterized using a standard MTT assay. The effects of the polypyrrole nanowires/nanoporous alumina substrate on the cell morphology were studied by AFM. The nanoporous alumina coated with polypyrrole nanowires was found to exhibit better cell adhesion and proliferation than polystyrene petridish, aluminum foil, 1st anodized and uncoated 2nd anodized alumina substrate. This study showed the potential of the polypyrrole nanowires/nanoporous alumina substrate as biocompatibility electroactive polymer substrate for both healthy and cancer cell cultures applications.

Cell-based chip for the detection of anticancer effect on HeLa cells using cyclic voltammetry.

HeLa cells directly immobilized on gold-patterned silicon substrate were used to assess the biological toxicity of anticancer drugs (hydroxyurea and cyclophosphamide). Immobilization of HeLa cells was confirmed by optical microscopy, and cell growth, viability and drug-related toxicity were examined by cyclic voltammetry and potentiometric stripping analysis. The voltammetric behaviors of HeLa cells displayed a quasi-reversible pattern with the peak current exhibiting a linear relationship with cell number. The attached living cells were exposed to different concentrations of hydroxyurea and cyclophosphamide as anticancer drugs, which induced the change of cyclic voltammetry current peak. As the exposed concentration of anticancer drugs was increased, the change of current peak was increased, which indicates the decrease of cell viability. Trypan Blue dyeing was performed to confirm the results of the effect of anticancer drugs on the cell viability which was obtained from cyclic voltammetry assay. The proposed direct cell immobilization method technique can be applied to the fabrication of cell chip for diagnosis, drug detection, and on-site monitoring.