Takashi Jin

Fluorescent Platinum Nanoclusters: Synthesis, Purification, Characterization, and Application to Bioimaging†

Noble-metal nanoclusters consisting of several atoms have been gaining much attention as novel fluorescent markers owing to their optical properties, which include size-dependent emission wavelength and discrete electronic state, features that are similar to semiconductor quantum dots. However, their smaller size and lower cytotoxicity in some ways make metal nanoclusters superior. Dickson and coworkers reported the synthesis and characterization of gold and silver 7] nanoclusters and successful bioimaging of actin filament labeled with these nanoclusters, while Lin et al. used peptide-conjugated gold nanoclusters as nuclear targeting and intracellular imaging probes. However, among the noble-metal clusters, platinum clusters have not yet been used as bioimaging probes except as a reducing catalyst. Herein, we describe water-soluble platinum nanoclusters that are less cytotoxic and emit a brighter fluorescence at 470 nm than other fluorescent nanomaterials, such as gold clusters. The synthesis of such platinum nanoclusters is achieved by a simple chemical reduction; they are purified by size-exclusion high performance liquid chromatography (HPLC). The size of the noble-metal nanoclusters is crucial because it affects the photoluminescence wavelength. 2, 3a, 4a,5a] This parameter can be regulated by a macromolecule-based template such as a dendrimer. One example is the fourth-generation polyamidoamine dendrimer (PAMAM (G4-OH)), which has been used as a molecular template in the synthesis of fluorescent gold nanoclusters. 8a,b] This dendrimer has nanometer-scale size and a uniform structure comprising an internal core and an external shell. Since the internal core contains tertiary amines that can form coordination bonds with the metallic ions, PAMAM dendrimers can trap these metal ions to form metal nanoclusters. Herein, we apply PAMAM (G4-OH) to synthesize Pt nanoclusters. Electronspray ionization (ESI) mass spectrometry confirmed that the synthesized nanoclusters were atomically monodispersed Pt5. Platinum nanoclusters were prepared by reducing H2PtCl6 (6.0 mL, 0.5m) with NaBH4 (3.0 mmol) in the presence of PAMAM (G4-OH) (0.5 mmol, 71.4 mg; Scheme 1). NaBH4

Dose-dependent in-vivo toxicity assessment of silver nanoparticle in Wistar rats.

This study aims to suggest the limits of silver nanoparticle (AgNP) uses for medicinal purpose and was performed to explore the effect of various doses of silver nanoparticle in rats. Four different doses of AgNP (4, 10, 20, and 40 mg/kg) were injected intravenously. For safety evaluation of injected AgNP, body weight, organ coefficient, whole blood count, and biochemistry panel assay for liver function enzyme (AST, ALT, ALP, and GGTP), comet assay, ROS, and histological parameter were performed; 10–12 week old animals were randomly divided into groups of six individuals each for control, and doses of 40, 20, 10, and 4 mg/kg AgNP injected. Significant changes were observed (p < 0.01) in hematological parameters (WBC count, platelets counts, haemoglobin, and RBC count) in the 40 and 20 mg/kg groups. The changes were non-significant in the other groups (4 and 10 mg/kg group). In the 40 mg/kg group, a significant increase was also found in liver function enzymes like ALT and AST (p < 0.01), ALP (p < 0.01), GGTP (p < 0.01), and bilirubin (p < 0.01). ROS in blood serum increased in the high dose group. Tail migration in single cell gel electrophoresis in the 40, 20, 10, 4 mg/kg, and control groups was 34.9, 29.5, 17.8, 5.8, and 0.0 µm, respectively, which indicated damage in the DNA strand in the high dose group. EDXRF showed a ∼ 10-times increase in silver concentration in the 40 mg/kg group and TEM image also showed particle deposition in the 40 mg/kg group. This study indicates that the AgNP in doses (< 10 mg/kg) is safe for biomedical application and has no side-effects, but its high dose (> 20 mg/kg) is toxic.

Preparation and characterization of highly fluorescent, glutathione-coated near infrared quantum dots for in vivo fluorescence imaging.

Fluorescent probes that emit in the near-infrared (NIR, 700–1,300 nm) region are suitable as optical contrast agents for in vivo fluorescence imaging because of low scattering and absorption of the NIR light in tissues. Recently, NIR quantum dots (QDs) have become a new class of fluorescent materials that can be used for in vivo imaging. Compared with traditional organic fluorescent dyes, QDs have several unique advantages such as size- and composition-tunable emission, high brightness, narrow emission bands, large Stokes shifts, and high resistance to photobleaching. In this paper, we report a facile method for the preparation of highly fluorescent, water-soluble glutathione (GSH)-coated NIR QDs for in vivo imaging. GSH-coated NIR QDs (GSH-QDs) were prepared by surface modification of hydrophobic CdSeTe/CdS (core/shell) QDs. The hydrophobic surface of the CdSeTe/CdS QDs was exchanged with GSH in tetrahydrofuran-water. The resulting GSH-QDs were monodisperse particles and stable in PBS (phosphate buffered saline, pH = 7.4). The GSH-QDs (800 nm emission) were highly fluorescent in aqueous solutions (quantum yield = 22% in PBS buffer), and their hydrodynamic diameter was less than 10 nm, which is comparable to the size of proteins. The cellular uptake and viability for the GSH-QDs were examined using HeLa and HEK 293 cells. When the cells were incubated with aqueous solutions of the GSH-QDs (10 nM), the QDs were taken into the cells and distributed in the perinuclear region of both cells. After 12 hrs incubation of 4 nM of GSH-QDs, the viabilities of HeLa and HEK 293 cells were ca. 80 and 50%, respectively. As a biomedical utility of the GSH-QDs, in vivo NIR-fluorescence imaging of a lymph node in a mouse is presented.

Multilayered, core/shell nanoprobes based on magnetic ferric oxide particles and quantum dots for multimodality imaging of breast cancer tumors

Multilayered, core/shell nanoprobes (MQQ-probe) based on magnetic nanoparticles (MNPs) and quantum dots (QDs) have been successfully developed for multimodality tumor imaging. This MQQ-probe contains Fe(3)O(4) MNPs, visible-fluorescent QDs (600 nm emission) and near infrared-fluorescent QDs (780 nm emission) in multiple silica layers. The fabrication of the MQQ-probe involves the synthesis of a primer Fe(3)O(4) MNPs/SiO(2) core by a reverse microemulsion method. The MQQ-probe can be used both as a fluorescent probe and a contrast reagent of magnetic resonance imaging. For breast cancer tumor imaging, anti-HER2 (human epidermal growth factor receptor 2) antibody was conjugated to the surface of the MQQ-probe. The specific binding of the antibody conjugated MQQ-probe to the surface of human breast cancer cells (KPL-4) was confirmed by fluorescence microscopy and fluorescence-activated cell sorting analysis in vitro. Due to the high tissue permeability of near-infrared (NIR) light, NIR fluorescence imaging of the tumor mice (KPL-4 cells transplanted) was conducted by using the anti-HER2 antibody conjugated MQQ-probe. In vivo multimodality images of breast tumors were successfully taken by NIR fluorescence and T(2)-weighted magnetic resonance. Antibody conjugated MQQ-probes have great potential to use for multimodality imaging of cancer tumors in vitro and in vivo.

Bio-distribution and toxicity assessment of intravenously injected anti-HER2 antibody conjugated CdSe/ZnS quantum dots in Wistar rats

Anti-HER2 antibody conjugated with quantum dots (anti-HER2ab-QDs) is a very recent fluorescent nanoprobe for HER2+ve breast cancer imaging. In this study we investigated in-vivo toxicity of anti-HER2ab conjugated CdSe/ZnS QDs in Wistar rats. For toxicity evaluation of injected QDs sample, body weight, organ coefficient, complete blood count (CBC), biochemistry panel assay (AST, ALT, ALP, and GGTP), comet assay, reactive oxygen species, histology, and apoptosis were determined. Wistar rat (8–10 weeks old) were randomly divided into 4 treatment groups (n = 6). CBC and biochemistry panel assay showed nonsignificant changes in the anti-HER2ab-QDs treated group but these changes were significant (P < 0.05) in QDs treated group. No tissue damage, inflammation, lesions, and QDs deposition were found in histology and TEM images of the anti-HER2ab-QDs treated group. Apoptosis in liver and kidney was not found in the anti-HER2ab-QDs treated group. Animals treated with nonconjugated QDs showed comet formation and apoptosis. Cadmium deposition was confirmed in the QDs treated group compared with the anti-HER2ab-QDs treated group. The QDs concentration (500 nM) used for this study is suitable for in-vivo imaging. The combine data of this study support the biocompatibility of anti-HER2ab-QDs for breast cancer imaging, suggesting that the antibody coating assists in controlling any possible adverse effect of quantum dots.

Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells

The early detection of HER2 (human epidermal growth factor receptor 2) status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs) have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs) using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC). As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs) with fluorescence quantum yields of 0.23∼0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line) was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission) on the fluorescence image of KPL-4 cells.

Expanded palette of Nano-lanterns for real-time multicolor luminescence imaging

Significance The application of luminescence imaging has been limited mainly by the two drawbacks of luciferases: low brightness and poor color variants. Here, we report the development of cyan and orange luminescent proteins approximately 20 times brighter than the wild-type Renilla luciferase. The color change and enhancement of brightness were both achieved by exploring bioluminescence resonance energy transfer (BRET) from enhanced Renilla luciferase to a fluorescent protein, a technology that we previously reported for the development of the bright yellowish-green luminescent protein Nano-lantern. These cyan and orange Nano-lanterns along with the original yellow Nano-lantern enable monitoring of multiple cellular events, including dynamics of subcellular structures, gene expressions, and functional status, such as intracellular Ca2+ change. Fluorescence live imaging has become an essential methodology in modern cell biology. However, fluorescence requires excitation light, which can sometimes cause potential problems, such as autofluorescence, phototoxicity, and photobleaching. Furthermore, combined with recent optogenetic tools, the light illumination can trigger their unintended activation. Because luminescence imaging does not require excitation light, it is a good candidate as an alternative imaging modality to circumvent these problems. The application of luminescence imaging, however, has been limited by the two drawbacks of existing luminescent protein probes, such as luciferases: namely, low brightness and poor color variants. Here, we report the development of bright cyan and orange luminescent proteins by extending our previous development of the bright yellowish-green luminescent protein Nano-lantern. The color change and the enhancement of brightness were both achieved by bioluminescence resonance energy transfer (BRET) from enhanced Renilla luciferase to a fluorescent protein. The brightness of these cyan and orange Nano-lanterns was ∼20 times brighter than wild-type Renilla luciferase, which allowed us to perform multicolor live imaging of intracellular submicron structures. The rapid dynamics of endosomes and peroxisomes were visualized at around 1-s temporal resolution, and the slow dynamics of focal adhesions were continuously imaged for longer than a few hours without photobleaching or photodamage. In addition, we extended the application of these multicolor Nano-lanterns to simultaneous monitoring of multiple gene expression or Ca2+ dynamics in different cellular compartments in a single cell.

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V

Single molecule fluorescence polarization techniques have been used for three-dimensional (3D) orientation measurements to observe the dynamic properties of single molecules. However, only few techniques can simultaneously measure 3D orientation and position. Furthermore, these techniques often require complex equipment and cumbersome analysis. We have developed a microscopy system and synthesized highly fluorescent, rod-like shaped quantum dots (Q rods), which have linear polarizations, to simultaneously measure the position and 3D orientation of a single fluorescent probe. The optics splits the fluorescence from the probe into four different spots depending on the polarization angle and projects them onto a CCD camera. These spots are used to determine the 2D position and 3D orientation. Q rod orientations could be determined with better than 10° accuracy at 33 ms time resolution. We applied our microscopy and Q rods to simultaneously measure myosin V movement along an actin filament and rotation around its own axis, finding that myosin V rotates 90° for each step. From this result, we suggest that in the two-headed bound state, myosin V necks are perpendicular to one another, while in the one-headed bound state the detached trailing myosin V head is biased forward in part by rotating its lever arm about its own axis. This microscopy system should be applicable to a wide range of dynamic biological processes that depend on single molecule orientation dynamics.

Real-Time Nanoscopy by Using Blinking Enhanced Quantum Dots

Superresolution optical microscopy (nanoscopy) is of current interest in many biological fields. Superresolution optical fluctuation imaging, which utilizes higher-order cumulant of fluorescence temporal fluctuations, is an excellent method for nanoscopy, as it requires neither complicated optics nor illuminations. However, it does need an impractical number of images for real-time observation. Here, we achieved real-time nanoscopy by modifying superresolution optical fluctuation imaging and enhancing the fluctuation of quantum dots. Our developed quantum dots have higher blinking than commercially available ones. The fluctuation of the blinking improved the resolution when using a variance calculation for each pixel instead of a cumulant calculation. This enabled us to obtain microscopic images with 90-nm and 80-ms spatial-temporal resolution by using a conventional fluorescence microscope without any optics or devices.

Synthesis and optical properties of emission-tunable PbS/CdS core–shell quantum dots for in vivo fluorescence imaging in the second near-infrared window

Near-infrared (NIR) fluorescence imaging at wavelengths from 1000 to 1500 nm (2nd-NIR window) is a promising modality for in vivo fluorescence imaging because of the deeper tissue penetration with lower tissue scattering of the 2nd-NIR light. For such in vivo fluorescence imaging, highly fluorescent probes in the 2nd-NIR wavelength region are needed. Although single-walled carbon nanotubes and Ag2S quantum dots (QDs) have recently appeared as 2nd-NIR fluorescent probes, their fluorescence brightness is relatively low (quantum yields <6%). In this study, we developed a synthetic method for preparing highly fluorescent PbS/CdS core–shell QDs (quantum yields, 17% in water) with narrow band widths (<200 nm) that emit in the 2nd-NIR region. By overcoating of a CdS shell onto a PbS QD core, we could easily control the emission wavelengths of the PbS/CdS QDs at 1000 to 1500 nm. To use the QDs for in vivo imaging, we investigated the optical properties of QDs (penetration depth and blurring of fluorescence images in slices of skin, brain, and heart in mice) in the 2nd-NIR region. We found that the 2nd-NIR fluorescence imaging at ca.1300 nm using the PbS/CdS QDs results in the highest signal to background ratio with a low blurring for in vivo imaging. To confirm the capabilities of the PbS/CdS QDs for in vivo imaging, we conducted fluorescence angiography imaging of a mouse head.