Inverted Fluorescence Microscope Research Articles

Developing a fluorescence immunosensor for detection of HER2-positive breast cancer based on graphene and magnetic nanoparticles

Breast cancer is regarded as the most common cause of death after lung cancer among women. Using graphene quantum nanoparticles (GQNPs) to construct a biosensor increases the sensitivity, rapidness, and flexibility of biological tests to detect cancer cells. In this study, we developed a fluorescence immunosensor based on GQNPs and magnetic nanoparticles (MNPs) for selective detection of HER2-positive breast cancer. The GQNPs and MNPs were conjugated with Herceptin individually which was confirmed by physicochemical studies. Then, the conjugates were exposed to the SK-BR3 breast cancer cells to form a sandwich structure of MNP-Herceptin-SK-BR3 cell-Herceptin-GQNP. Then, an inverted fluorescence microscope was used to detect breast cancer cells after isolating them using a magnet. The results showed a high sensitivity (even one cell ml−1) and specificity of the designed biosensor for the detection of SK-BR3 cells within 30 min.

The effect of varying radius of curvature on mixing in elliptical spiral microchannels

Effective mixing is essential for a wide range of microfluidics applications. Passive micromixers have attracted much attention during recent years due to their low-cost and simple fabrication procedures with less power input in their implementation to high-throughput microfluidics platforms. Increasing the efficiency of micromixers is possible with an optimum geometry of inertial microfluidic channels, which utilize Dean vortices and Dean flows for the enhancement of mixing. Recently, micromixers with curved microchannels have been introduced to the literature. Yet, the enormous potential of elliptical spiral microchannels has not been adequately revealed. This study aims to assess the mixing performance of five-loop spiral polydimethylsiloxane micromixers with spiral structures and elliptic structures, which have different initial aspect ratios with varying radii of curvature along the channel. The performances of these micromixers were evaluated by comparing the mixing indices and intensity profiles obtained from inverted fluorescence microscopy images over a broad range of Reynolds numbers between 10 and 100. Elliptical spiral microchannels were able to provide mixing indices up to 98% at a Reynolds number of 20. Overall, the utilization of ellipse-shaped microchannels appear to provide a passive control over the mixing performance at several locations through the microchannel.

Force-Controlled Mechanical Stimulation and Single-Neuron Fluorescence Imaging of Drosophila Larvae

Studying the neural response of a Drosophila larva to touch stimulation could decipher neural basis of the creature's danger-escaping behaviors. This letter reports force-controlled robotic mechanical stimulation and single-neuron fluorescence imaging of Drosophila larvae. A force control architecture based on a model compensation-prediction scheme and a switched fuzzy-PID controller was used for regulating the touch force applied to a larva at the micronewton level. The developed force control system demonstrates a settling time of 0.15 s, zero overshoot and a resolution of <; 50 μN. Established based on a high-resolution inverted fluorescence microscope, our robotic system is capable of simultaneously applying a controlled touch force to a larva and quantifying the fluorescence signal transmission inside a single neuron responsive to body touch stimulation. Using this system, we examined, for the first time, the quantitative relationship between the applied force level (range: 0.25-2 mN) and the change in transmission signal of the class III ddaA neuron. The touch force threshold at which the neuron starts to get activated was determined to be in the range of 0.25-0.5 mN. This work may contribute to new studies on sensory mechanotransduction in Drosophila larvae.

3D-printed microplate inserts for long term high-resolution imaging of live brain organoids

BackgroundOrganoids are a reliable model used in the study of human brain development and under pathological conditions. However, current methods for brain organoid culture generate tissues that range from 0.5 to 2 mm of size, which need to be constantly agitated to allow proper oxygenation. The culture conditions are, therefore, not suitable for whole-brain organoid live imaging, required to study developmental processes and disease progression within physiologically relevant time frames (i.e. days, weeks, months).ResultsHere we designed 3D-printed microplate inserts adaptable to standard 24 multi-well plates, which allow the growth of multiple organoids in pre-defined and fixed XYZ coordinates. This innovation facilitates high-resolution imaging of whole-cerebral organoids, allowing precise assessment of organoid growth and morphology, as well as cell tracking within the organoids, over long periods. We applied this technology to track neocortex development through neuronal progenitors in brain organoids, as well as the movement of patient-derived glioblastoma stem cells within healthy brain organoids.ConclusionsThis new bioengineering platform constitutes a significant advance that permits long term detailed analysis of whole-brain organoids using multimodal inverted fluorescence microscopy.

An Optically Reconfigurable Förster Resonance Energy Transfer Process for Broadband Switchable Organic Single-Mode Microlasers

An Optically Reconfigurable Förster Resonance Energy Transfer Process for Broadband Switchable Organic Single-Mode Microlasers

Coaxial electrospun PVA/PCL nanofibers with dual release of tea polyphenols and ε-poly (L-lysine) as antioxidant and antibacterial wound dressing materials

Preparing wound dressing with dual-delivery of antioxidant and antibacterial agents is highly desirable in clinical wound treatment. Herein, a series of coaxial nanofiber membranes loaded with antioxidant tea polyphenols (TP) in the core and antibacterial ε-poly (L-lysine) (ε-PL) in the shell layer were successfully fabricated by coaxial electrospinning. The physicochemical characterizations by transmission electron microscopy, inverted fluorescence microscopy and fourier transform infrared spectroscopy confirmed the formation of core-shell structure. The results of in vitro drug release indicated that ε-PL exhibited a fast release profile while TP released in a sustained manner, which is favorable to the achievement of quick bacteria inhibition in the initial phase as well as long-term antioxidant activity during wound healing. The antioxidant activity of coaxial nanofibers was found to be increased with the increment of TP content and incubation time. The antibacterial assays against Escherichia coli and Staphylococcus aureus demonstrated that the incorporation of ε-PL in the coaxial nanofibers led to strong antibacterial activity. Additionally, all the coaxial nanofibers possessed good cytocompatibility. Therefore, the prepared coaxial nanofibers simultaneously incorporated with ε-PL and TP are promising as potential wound dressing materials.

Crystal structure, DNA binding, cytotoxicity and anticancer ability of Zn(II) complex constructed by 2-(1,2,4)triazol-1-yl-isonicotinic acid

A novel Zn(II) complex was designed and synthesized under hydrothermal condition based on the ligand 2-(1,2,4)triazol-1-yl-isonicotinic acid. The crystal data were collected by X-ray single crystal diffractometer, and the purity of the complex was verified using elemental analysis and powder X-ray diffractometer. UV–Vis spectroscopy analyzed the binding mode of the complex and CT-DNA and calculated the binding constant Kb value of 3.36 × 103 M−1. Fluorescence spectroscopy further studied the interaction mode of the complex with CT-DNA, and the quenching constant Ksv value was 0.06. Gel electrophoresis was used to study the ability of the complex to cut pBR322 plasmid DNA. Flow cytometry detected the apoptosis ratio of the complex to HeLa cells, and the total apoptosis rate reached 71.4%. The fluorescent inverted microscope was used to observe the morphological changes of the cells by double staining, and detect the apoptotic effect on HeLa cells.

Realization of flexible and parallel laser direct writing by multifocal spot modulation.

In this investigation, we propose a strip segmentation phase (SSP) method for a spatial light modulator (SLM) to generate independent multifocal spots when the beam passes through a high numerical aperture (NA) lens. With the SSP method, multifocal spots can be generated with each spot independently, flexibly and uniformly distributed. The performance of the SSP method is first validated with numerical simulation. Then, by applying the modulation method with SLM and importing the beams into an inverted fluorescence microscopy system with a high-NA lens, the spot distribution and their shapes can be observed by fluorescent image. The fluorescent image exhibits high uniformity and high consistency with the aforementioned numerical simulations. Finally, we dynamically load a series of phase maps on SLM to realize continuous and independent spot movement in a multifocal array. By laser direct writing on photoresist, a complex NWU-shape structure can be realized flexibly with multi-task fabrication capability. The SSP method can significantly improve the efficiency and flexibility of laser direct writing. It is also compatible with most recent techniques, e.g., multiphoton absorption, stimulated emission depletion and photo-induced depolymerization etc., to realize parallel super-resolution imaging and fabrications.

A9 DORSAL ROOT GANGLIA NEURONAL RESPONSES AND SUBSTANCE P PRODUCTION ARE HIGHER IN MALE MICE

Abstract Background Abdominal pain is a common complaint in patients with chronic gastrointestinal disorders. Accumulating evidence suggests that gut microbiota is an important determinant of gut function, including visceral sensitivity. Germ-free (GF) mice have been shown to display visceral hypersensitivity, which normalizes after colonization. Sex also appears to play a key role in visceral sensitivity, as women report more abdominal pain than men. Thus, both gut bacteria and sex are important in the regulation of gut nociception, but the underlying mechanisms remain poorly understood. Aims To investigate the role of gut microbiota and sex in abdominal pain. Methods We used primary cultures of sensory neurons from dorsal root ganglia (DRG) of female and male conventionally raised (SPF) or germ-free (GF) mice (7–18 weeks old). To study the visceral afferent activity in vitro, calcium mobilization in DRG sensory neurons was measured by inverted fluorescence microscope using a fluorescent calcium probe Fluo-4 (1mM). Two parameters were considered i) the percentage of responding neurons ii) the intensity of the neuronal response. First, DRG sensory neurons were stimulated by a TRPV1 agonist capsaicin (12.5nM, 125nM and 1.25µM) or by a mixture of G-protein coupled receptors agonist (GPCR: bradykinin, histamine and serotonin; 1µM, 10µM and 100µM). We next measured the neuronal production of substance P and calcitonin gene-related peptide (CGRP), two neuropeptides associated with nociception, in response to capsaicin (1.25µM) or GPCR agonists (100µM) by ELISA and EIA, respectively. Results The percentage of neurons responding to capsaicin and GPCR agonists was similar in male and female SPF and GF mice. However, the intensity of the neuronal response was higher in SPF male compared to SPF female in response to capsaicin (125nM: p=0.0336; 1.25µM: p=0.033) but not to GPCR agonists. Neuronal activation was similar in GF and SPF mice of both sexes after administration of capsaicin or GPCR agonists. Furthermore, substance P and CGRP production by sensory neurons induced by capsaicin or GPCR agonists was similar in SPF and GF mice, regardless of sex. However, while the response to capsaicin was similar, the GPCR agonists-induced production of substance P was higher in SPF male mice compared to SPF females (p=0.003). The GPCR agonists-induced production of CGRP was similar in SPF male and female mice. Conclusions Our data suggest that at the level of DRG neurons, the absence of gut microbiota does not predispose to visceral hypersensitivity. The intensity of DRG neuronal responses to capsaicin and the GPCR agonists-induced production of substance P are higher in male compared to female mice, in contrast to previously published studies in various models of acute and chronic pain. Further studies are thus needed to investigate the role of sex in visceral sensitivity. Funding Agencies CIHR

Inhibition of glioma using a novel non-neurotoxic vesicular stomatitis virus.

The aim of this study was to demonstrate the in vivo safety and antitumor effect of a novel recombinant vesicular stomatitis virus (VSV): G protein less (GLESS)-fusion-associated small transmembrane (FAST)-VSV. Viral infection efficiency and cell proliferation were detected using an inverted fluorescence microscope and alarmaBlue assay, respectively. To evaluate the safety of the virus, different doses of GLESS-FAST-VSV and a positive control virus (VSV∆M51) were injected into normal F344 rats and C57BL/6 mice, and each animal's weight, survival time, and pathological changes were examined on the following day. To evaluate the efficacy of the virus, RG2 and GL261 cells were used to construct rat and mouse glioma models, respectively, via a stereotactic method. After multiple intratumoral injections of the virus, tumor growth (size) and the survival time of the animals were observed. In vitro experiments showed that GLESS-FAST-VSV could infect and kill brain tumor cells and had less toxic effects on normal cells. After direct injection of GLESS-FAST-VSV into the animal brains, all animals tolerated the virus well, and no animal death, encephalitis, or ventriculitis was observed. In contrast, all animals that received brain injections of VSV∆M51 in the brain died. Moreover, multiple injections of GLESS-FAST-VSV in brain tumors significantly prolonged the survival of normal-immunity animals harboring brain tumors. GLESS-FAST-VSV exhibited little neurotoxicity and could be injected directly into the tumor to effectively inhibit tumor growth and prolong the survival of normal-immunity animals, laying a theoretical foundation for the early application of such viruses in clinical trials.

Near-Infrared Light-Triggered Thermosensitive Liposomes Modified with Membrane Peptides for the Local Chemo/Photothermal Therapy of Melanoma.

PurposeA near-infrared (NIR)-triggered trans-activating transcriptional activator (TAT)-based targeted drug delivery system for the combined chemo/photothermal therapy of melanoma, namely, TAT-TSL-TMZ (temozolomide)/IR820, was developed for the first time.MethodsTAT-TSL-TMZ/IR820 liposomes were synthesized via thin-film dispersion and sonication. IR820 and TMZ were encased in the inner layer and lipid bilayer of the liposomes, respectively.ResultsDynamic light scattering results showed that the liposomes had an average hydrodynamic size of 166.9 nm and a zeta potential of −2.55 mV. The encapsulation rates of TMZ and IR820 were 35.4% and 28.6%, respectively. The heating curve obtained under near-infrared (NIR) laser irradiation showed that TAT-TSL-TMZ/IR820 liposomes had good photothermal conversion efficiency. The in vitro drug release curve revealed that NIR laser irradiation could accelerate drug release from TAT-TSL-TMZ/IR820 liposomes. The results of inverted fluorescence microscopy and flow cytometry proved that the uptake of TAT-TSL-TMZ/IR820 liposomes by human melanoma cells (MV3 cells) was concentration-dependent and that the liposomes modified with membrane peptides were more likely to be ingested by cells than unmodified liposomes. Confocal laser scanning microscopy indicated that TAT-TSL-TMZ/IR820 liposomes entered MV3 cells via endocytosis and was stored in lysosomes. In addition, TAT-TSL-TMZ/IR820 liposomes exposed to NIR laser showed 89.73% reduction in cell viability.ConclusionThis study investigated the photothermal conversion, cell uptake, colocation and chemo/photothermal effect of TAT-TSL-TMZ/IR820 liposomes.

Glucose absorption regulation and mechanism of the compounds in Lilium lancifolium Thunb on Caco-2 cells

In this paper, the Caco-2 cell was used to study the glucose absorption regulation and mechanism of kaempferol, caffeic acid and quercetin-3-O-β-D-galactoside in Lilium lancifolium Thunb in vitro. Glucose oxidase-peroxidase (GOD-POD) method was used to measure glucose consumption in supernatant. The fluorescent D-glucose analog (2-NBDG) was used as a tracer probe to study the changes in the fluorescence intensity of 2-NBDG uptake by Caco-2 cells with an inverted fluorescence microscope. Western blotting and quantitative real-time PCR were used to detect the protein expression and mRNA transcription of SGLT1 and GLUT2. The results showed that caffeic acid and quercetin-3-O-β-D-galactoside could significantly promote the absorption of glucose by normal Caco-2 cells compared with the control group (P < 0.001). Both caffeic acid and quercetin-3-O-β-D-galactoside could significantly promote the uptake of glucose tracer 2-NBDG on Caco-2 cells. Caffeic acid and quercetin-3-O-β-D-galactoside could significantly promote SGLT1 and GLUT2 protein expression levels and mRNA transcription (P < 0.001, P < 0.01, P < 0.05). The mechanism might be related to the promotion of SGLT1 and GLUT2 protein expression levels and mRNA transcription.

The rigidizable behavior of the deployable hindwings of the Asian ladybeetle during flight

Deployable hindwings support the excellent flight performance of Coleoptera during flapping flight, exhibiting rigidizable behavior without refolding during flight. This paper investigates the inner structure of the veins of the Asian ladybird beetle (Harmonia axyridis) hindwing by histological sections and inverted fluorescence microscopy. Considering the hollow and solid structures of the veins and the wing membrane, two three-dimensional (3-D) models of the right hindwings are established for comparison: Model I with a solid C + ScA vein and Model II with a hollow C + ScA vein, and the other veins are solid. The relationships among the attack angles (− 60°, − 30°, 0°, 30° and 60°), locations at which the pressure is set and bending deformation tendencies of the two models are discussed. These related data are obtained by simulation. The results demonstrate that the hollow structures of the veins have a higher flexibility and self-adaption deformation than those of the solid veins, and sinusoidal relationships are observed between the resulting deformations and attack angles. The relationship between the rigidizable behavior of deployable hindwings and vein structure is discussed.

Fabrication Protocol for Thermoplastic Microfluidic Devices: Nanoliter Volume Bioreactors for Cell Culturing.

Microfluidic devices consist of microchannels etched or embossed into substrates made of polymer, glass or silicon. Intricate connections of the microchannels to reactors with some smart mechanical structures such as traps or curvatures fulfil the desired functionalities such as mixing, separation, flow control or setting the environment for biochemical reactions. Here, we describe the fabrication methods of a thermoplastic microbioreactor step by step. First, material selection is made, then, production methods are determined with the equipment that can be easily procured in a laboratory. COP with its outstanding characteristics among many polymers was chosen. Two types of microbioreactors, with and without electrodes, are designed with AutoCAD and L-edit softwares. Photolithography and electrochemical wet etching are used for master mold preparation. Thermal evaporator is employed for pure chromium and gold deposition on COP substrate and etchants are used to form the interdigitated electrodes. Once the master mold produced, hot embossing is used to obtain the designed shape on drilled and planarized COP. Cover COP, with or without electrodes, is bonded to the hot embossed COP via thermo-compression and thermoplastic microfluidic device is realized. Tubings are connected to the device and a bridge between the macro and micro world is established. Yeast or mammalian cells labeled or tagged with GFP/RFP on specific gene products are loaded into the microfluidic device, and real time data on cell dimensions and fluorescence intensity are collected using inverted fluorescence microscope, and finally image processing is used to analyze the acquired data.

The gastroprotective effect of alpinia officinarum extract on indomethacin-induced topical injuries in RGM-1 Cells: Involvement of H+/K+-ATPase- and mitochondrial-mediated apoptosis

Background: Topical effects are essential mechanisms of nonsteroidal anti-inflammatory drugs (NSAIDs)-induced gastric damage. Our previous study showed that the extract of Alpinia officinarum (AOE) may have some protective effects against the topical effects of indomethacin (INDO). The aim of this study was to elucidate the protective effects and mechanisms of A. officinarum against INDO-induced topical injuries to gastric mucosa. Materials and Methods: 0.5 mM INDO was used to cause topical effects to rat gastric epithelial cells (RGM-1). Meanwhile, AOE (2.5 μg/mL) and galangin (GAL) (0.05 mM) were added, respectively, to explore their protective effects. The cell proliferation, mitochondrial viability, and mitochondrial-mediated apoptosis were assessed by flow cytometry, inverted fluorescence microscope, or microplate reader. Pro- and cleaved-caspase-3 were detected by Western blot method. Results: AOE and GAL could significantly protect INDO-damaged RGM-1 cells by promoting cell proliferation, upregulating mitochondrial viability, inhibiting mitochondrial cytochrome c release into cytoplasm, inhibiting lipid peroxidation and caspase-3 activity, and suppressing H+/K+-ATPase activity. Conclusion: The gastroprotective effects of AOE and GAL were closely associated with suppressing the gastric acid secretion and restraining mitochondrial-mediated apoptosis. These data provided new perceptions into interpreting the underlying mechanisms of gastroprotective effects of A. officinarum and showed a promising clinical use in treating gastric mucosal injury induced by NSAIDs.

Life-Inspired Endogenous Dynamic Behavior of Lipid Droplet-like Microcompartments in Artificial Adipocyte-like Structures

Chemical systems that can replicate cellular behaviors are gaining increasing attention and are being used to study various biological processes. Here, a protein- or amylose-based assembly at an oi...

PH-sensitive β-cyclodextrin derivatives for the controlled release of Podophyllotoxin

An effective tumor targeting drug delivery systems was designed and synthesized by conjugating pH-sensitive maleamide derivatives to Mono-(6-deoxy-6-amino)-β-CD. Their characteristics and inclusion behaviors with insoluble anticancer drug PPT were investigated in both solution and solid state by means of 1H NMR and 2D-ROESY, XRD, DSC and SEM, which reveal PPT is successfully encapsulated in the cavity of CD derivatives with different stability constants (Ks). Water solubility of PPT are significantly increased to 60.35 and 22.89 mg·mL−1 after formation of inclusion complexes with host-1 and host-2, compared with free PPT (0.12 mg·mL−1). Their acid-controlled release has been studied in vitro by 1H NMR and UV-Vis spectra, living cells incubated with host 1-2 were observed by Inverted fluorescence microscope to confirm pH-response releasing. Moreover, host-1/PPT and host-2/PPT maintain effective cell proliferation inhibition to human cancer, while their cytotoxicity to normal cell is significantly reduced. Our work shows inspiring potential in tumor-targeted delivery and acid-controlled release of PPT both in vitro.

CD44-Targeting Oxygen Self-Sufficient Nanoparticles for Enhanced Photodynamic Therapy Against Malignant Melanoma.

ObjectiveNanotechnology-based photodynamic therapy (PDT) is a relatively new anti-tumor strategy. However, its efficacy is limited by the hypoxic state in the tumor microenvironment. In the present study, a poly(lactic-co-glycolic acid) (PLGA) nanoparticle that encapsulated both IR820 and catalase (CAT) was developed to enhance anti-tumor therapy.Materials and MethodsHA-PLGA-CAT-IR820 nanoparticles (HCINPs) were fabricated via a double emulsion solvent evaporation method. Dynamic light scattering (DLS), transmission electron microscopy (TEM), laser scanning confocal microscopy, and an ultraviolet spectrophotometer were used to identify and characterize the nanoparticles. The stability of the nanoparticle was investigated by DLS via monitoring the sizes and polydispersity indexes (PDIs) in water, PBS, DMEM, and DMEM+10%FBS. Oxygen generation measurement was carried out via visualizing the oxygen bubbles with ultrasound imaging system and an optical microscope. Inverted fluorescence microscopy and flow cytometry were used to measure the uptake and targeting effect of the fluorescent-labeled nanoparticles. The live-dead method and tumor-bearing mouse models were applied to study the HCINP-induced enhanced PDT effect.ResultsThe results showed that the HCINPs could selectively target melanoma cells with high expression of CD44, and generated oxygen by catalyzing H2O2, which increased the amount of singlet oxygen, ultimately inhibiting tumor growth significantly.ConclusionThe present study presents a novel nanoplatform for melanoma treatment.

Effect of Pulse Interval on Promoting Secretion of Neurite-Inducing Factor from Glioma Cells in the Application of Nanosecond High Electric Field Pulses

1. Purpose We previously discovered and reported that the application of nanosecond high electric field pulses transiently increased intracellular Ca2+ concentration in glioma cells to promote the secretion of neurite-inducing factor. Moreover, it has been clarified that the electric field strength, the number of pulse application, and the pulse width have a strong effect on the secretory ability as a condition for promoting the secretion of these neurite-inducing factors. In this presentation, we further report that even nanosecond pulses with a weak electric field strength which does not cause the increase of intracellular Ca2+ concentration with a single pulse can induce the intracellular Ca2+ increase and neurite-inducing factor secretion from glioma cells by applying multiple pulses with short intervals. The considered mechanism also will be discussed in this presentation. さらに表示 2. Experimental method Rat glioma C6 cells were seeded on an ITO electrode and cultured overnight to adhere. To monitor intracellular Ca2+ concentration the cells were treated with a medium containing Fura-2 AM for 30 minutes to introduce the Ca2+ indicator into the cells. After washing the cells on the electrode, Hanks Balanced Salt Solution (HBSS) was put in the chamber, and an aluminum plate electrode serving as a counter electrode was faced to ITO electrode with a spacer in thickness of 30 µm, and set on the stage of an inverted fluorescence microscope. After two minutes from the start of measurement, nanosecond electric field pulse was applied in various voltage and pulse interval, and changes in intracellular Ca2+ concentration were observed. Furthermore, thapsigargin and 2-aminoethoxydiphenylborane (2-APB) were used to confirm that the increase in intracellular Ca2+ concentration may be due to Ca2+ release from the endoplasmic reticulum via IP3 receptors. In addition, after applying a nanosecond high electric field pulse to C6 cells in the same manner in a clean bench, the HBSS was immediately replaced with DMEM medium containing 10% FBS, and the cells were cultured for 1 day or 2 days. The culture supernatant (Conditioned Medium: CM) of the C6 cells was then collected, and exchanged completely with the medium of PC12 cells, which is a neural progenitor cell model. The morphological change of PC12 cells was observed and recorded for 6 days. The PC12 cells which extended the neurites more than twice as the cell body were recognized as neuronal differentiated cells. By calculating the differentiation rate of PC12 cells, it was evaluated how the secretion of the neurite-inducing factor from C6 cells was promoted or not. 3. Results and discussion Three pulses with a pulse width of 140 ns and an electric field strength of 23 kV / cm were applied at intervals of 2 seconds, 30 seconds, and 1 minute to C6 cells and intracellular Ca2+ concentration changes and neurite-inducing factor secretion was examined. It was demonstrated that intracellular Ca2+ gradually increased at shorter intervals of 2 seconds and 30 seconds, but not increased at intervals of 1 minute. In addition, intracellular Ca2+ concentration increase was observed even when pulse application was done in the absence of extracellular Ca2+. It indicated that this increase in intracellular Ca2+ concentration is due to Ca2+ release from organelles. Furthermore, intracellular Ca2+ concentration elevation was not induced by the same pulse application after treatment with thapsigargin or 2-APB. Therefore, it was considered that Ca2+ was released from the endoplasmic reticulum via IP3 receptors by the pulse application in this condition. Figure 1 (a) and (b) show images of PC12 cells cultivated in the CM of C6 cells which were exposed to three pulses with 1 minute or 2 seconds intervals, respectively. From these results, it was demonstrated that the secretion of the neurite-inducing factor was promoted by the multi-pulse application with short intervals. Furthermore, it was found that the Ca2 + release via IP3 receptors of the endoplasmic reticulum and following promotion of the secretion of neurite-inducing factor could be induced by shortening the nanosecond multi-pulse interval in the application of weak electric field. Now, we are planning to clarify what kind of gene expression in C6 cells is induced by pulse application to identify and quantify the secreted neurite-inducing factor. Figure 1

Synthesis of boron carbonitride nanosheets using for delivering paclitaxel and their antitumor activity

As a structural analog of graphene and boron nitride, hexagonal boron carbonitride nanosheets (BCNNSs) are supposed to be a potential drug deliverer. In the present work, an improved solid-state reaction method combined with ultrasonic exfoliating was reported for preparing BCNNSs. Vapor-solid (VS) mechanism was proposed to be responsible for the formation of BCNNSs. The BCNNSs were further modified by DSPE-mPEG-5000 to improve their dispersion in aqueous solution. It was found that the BCNNSs-PEG nanocomplex could be efficiently taken in by MDA-MB-231 breast cancer cells evidenced by inverted fluorescence microscopy. The PEGylated BCNNSs showed an outstanding ability to load paclitaxel through π-π interaction and hydrophobic interaction, and BCNNSs-PEG-loaded paclitaxel presented higher cytotoxicity in comparison with free paclitaxel. BCNNSs may become a promising candidate for delivering paclitaxel and other hydrophobic drugs.