Narufumi Kitamura

Preparation for highly sensitive MRI contrast agents using core/shell type nanoparticles consisting of multiple SPIO cores with thin silica coating.

We describe here the facile and robust preparation methods for the multiple-SPIO-containing silica-coated core/shell type nanoparticles which can serve as a highly sensitive MRI contrast agent. The imidazolium-tethered core/shell type particles were synthesized, and the centrifugal selection for the multiple-SPIO-containing particles and the etching process to fabricate thin silica layers were carried out to improve the proton relaxivity of water tissue. We found that the synthetic particles can provide approximately 7-fold clearer contrasts than that of the particles before treatments. In addition, the particles can show good dispersibility at least for 1 week in aqueous media.

Bimodal quantitative monitoring for enzymatic activity with simultaneous signal increases in 19F NMR and fluorescence using silica nanoparticle-based molecular probes.

We describe the bimodal quantitative assay for enzymatic activity in (19)F NMR spectroscopy and fluorescence spectroscopy using a nanoparticle-based molecular probe. Perfluorinated dendrimers were tethered on silica nanoparticles with a phosphate-caged fluorescein as a linker. Before enzymatic reaction, the molecular rotation of the perfluorinated dendrimers should be highly restricted, and the (19)F NMR signals from the perfluorinated dendrimers were too broad to be detected relative to the noise level. Fluorescence signals of fluorescein were suppressed by the presence of the diphosphate groups. Following the enzymatic reaction with an alkaline phosphatase, perfluorinated dendrimers and fluorescein were released, and the NMR signals of perfluorinated dendrimers and strong fluorescence from fluorescein were correspondingly observed. The enzymatic activity and reaction rates of the hydrolysis of alkaline phosphatase were detected from the increases of fluorescence and (19)F NMR signals. Finally, the feasibility of the probe in the presence of miscellaneous molecules under biomimetic conditions was demonstrated by determining of the enzymatic activity in cell lysate. Quantitative analysis using both (19)F NMR spectroscopy and fluorescence spectroscopy can be accomplished.

Reversible signal regulation system of 19F NMR by redox reactions using a metal complex as a switching module

We present here the reversible signal regulation of (19)F NMR using the transition metal complex as a switching module. Water-soluble fluorinated dendrimers containing ferrocene were synthesized as a probe, and the signal intensities of (19)F NMR of the probes were monitored by changing the valence state of the ferrocene moiety. By oxidation of the ferrocene moiety, the relaxation of the nearby fluorine atoms was accelerated via the paramagnetic relaxation enhancement, as a result the (19)F NMR signal from the probe was significantly decreased. The reduction of the ferrocenium cation of the probe recovered the signal intensity. Finally, in combination with the difference of the fluorescence quenching ability between ferrocene and ferrocenium cation, we constructed the multimodal (19)F NMR/fluorescence probe based on the redox switching system.

Heavy metal-free 19F NMR probes for quantitative measurements of glutathione reductase activity using silica nanoparticles as a signal quencher

For the quantitative assessment of the glutathione reductase (GR) activity with a (19)F NMR spectroscopy, we developed the heavy metal-free probes based on silica nanoparticles modified with water-soluble perfluorinated dendrimers via the disulfide linkers. Before enzymatic reaction, the molecular rotation of the perfluorinated dendrimers is highly restricted, and the magnitude of (19)F NMR signals from the perfluorinated dendrimers can be suppressed. By the reductive cleavage of the disulfide linkers with the reduced glutathione-mediated enzymatic reaction of GR, perfluorinated dendrimers can be released from the surfaces of the nanoparticles. Consequently, the (19)F NMR signals of perfluorinated dendrimers were recovered. The enzymatic activity of GR was determined from the increase of the magnitude of (19)F NMR signals. Finally, to demonstrate the feasibility of the probe in the presence of miscellaneous molecules under bio-mimetic conditions, the comparison study was executed with the cancer cell lysate. The value determined from our method showed a good agreement with that from the conventional method.

Assembly system of direct modified superparamagnetic iron oxide nanoparticles for target-specific MRI contrast agents.

We report the direct modification of SPIOs with a biomolecule and the target-specific assembly system of these modified SPIOs for using MRI contrast agents. The transverse relaxation rate of the aqueous solutions containing the modified SPIOs was altered by the dispersion state.

Reduced glutathione-resisting 19F NMR sensors for detecting HNO

The (19)F NMR probes for the HNO detection are reported. We synthesized the probe molecules with the paramagnetic Cu(II) complex and fluorine atoms using a cubic silsesquioxane. By using the magnetism changes of the Cu(II) to Cu(I) in the complex by the reduction with HNO, the (19)F NMR signal intensities of the probe increased. Noteworthily, our probes have superior resistance to reduced glutathione which is the major intracellular molecule to maintain the reductive environment and the competitor in the reduction of Cu(II) against HNO.

Properties of Superparamagnetic Iron Oxide Nanoparticles Assembled on Nucleic Acids

We report the direct modification of SPIOs with a biomolecule, and the sequence-specific assembly of the modified SPIOs was achieved with the aptamer-small molecule interaction. In addition, the transverse relaxation rate of the aqueous solutions containing the modified SPIOs was altered by the dispersion state.

Heat-initiated detection for reduced glutathione with 19F NMR probes based on modified gold nanoparticles

For detecting reduced glutathione (GSH) with a (19)F NMR spectroscopy with time-specificity, we developed the probes based on gold nanoparticles modified with the fluorinated groups via the thermally-cleavable linkers. Before the heating treatment with the probe, the maleimide moiety as a binding site with GSH in the probe is inactivated by cycloaddition of furan. At this silent state, the magnitude of (19)F NMR signals from the fluorinated groups was suppressed. By heating for the activation of the probe, the maleimide moiety was produced via retro Diels-Alder reaction, and (19)F NMR signals were observed. From this moment, GSH started the reaction with the probe via Michael addition to the maleimide moiety, leading to the observation of the new peak in (19)F NMR spectra. Finally, the amounts of GSH were determined from the increase of the magnitude of (19)F NMR signals.

X-ray computed tomography imaging of a tumor with high sensitivity using gold nanoparticles conjugated to a cancer-specific antibody via polyethylene glycol chains on their surface

Abstract Contrast agents are often used to enhance the contrast of X-ray computed tomography (CT) imaging of tumors to improve diagnostic accuracy. However, because the iodine-based contrast agents currently used in hospitals are of low molecular weight, the agent is rapidly excreted from the kidney or moves to extravascular tissues through the capillary vessels, depending on its concentration gradient. This leads to nonspecific enhancement of contrast images for tissues. Here, we created gold (Au) nanoparticles as a new contrast agent to specifically image tumors with CT using an enhanced permeability and retention (EPR) effect. Au has a higher X-ray absorption coefficient than does iodine. Au nanoparticles were supported with polyethylene glycol (PEG) chains on their surface to increase the blood retention and were conjugated with a cancer-specific antibody via terminal PEG chains. The developed Au nanoparticles were injected into tumor-bearing mice, and the distribution of Au was examined with CT imaging, transmission electron microscopy, and elemental analysis using inductively coupled plasma optical emission spectrometry. The results show that specific localization of the developed Au nanoparticles in the tumor is affected by a slight difference in particle size and enhanced by the conjugation of a specific antibody against the tumor.

Quantitative diagnosis of HER2 protein expressing breast cancer by single-particle quantum dot imaging.

Overexpression of HER2 is one of the major causes of breast cancer, and therefore precise diagnosis of its protein expression level is important. However, current methods estimating the HER2‐expression level are insufficient due to problem with the lack of quantification. This might result in a gap between diagnostics and therapeutics targeting HER2. Therefore, a new effective diagnostic method is needed. We developed a new immunohistochemical (IHC) technique with quantum dots (QD)‐conjugated trastuzumab using single‐particle imaging to quantitatively measure the HER2 expression level. Tissues from 37 breast cancer patients with available detailed clinical information were tested by IHC with QDs (IHC‐QD) and the correlation with IHC with 3,3′‐diaminobenzidine (DAB), fluorescence in situ hybridization (FISH), and IHC‐QD was examined. The number of QD‐conjugated trastuzumab particles binding specifically to a cancer cell was precisely calculated as the IHC‐QD score. The IHC‐QD score in 37 cases was correlated proportionally with the score of HER2 gene copy number as assessed by FISH (R = 0.83). When HER2 positivity was judged to be positive, the IHC‐QD score with our cut‐off level was exactly concordant with the FISH score with a cut‐off value of 2.0. Furthermore, IHC‐QDs score and time to progression (TTP) of trastuzumab therapy were well correlated in HER2‐positive cases (R = 0.69). Conversely, the correlation between FISH score and TTP was not observed. We developed a precisely quantitative IHC method using trastuzumab‐conjugated QDs and single‐particle imaging analysis and propose the possibility of using IHC‐QDs score as a predictive factor for trastuzumab therapy.