Kazunori Ishimura

Superparamagnetic Nanoparticle Clusters for Cancer Theranostics Combining Magnetic Resonance Imaging and Hyperthermia Treatment

Superparamagnetic nanoparticles (SPIONs) could enable cancer theranostics if magnetic resonance imaging (MRI) and magnetic hyperthermia treatment (MHT) were combined. However, the particle size of SPIONs is smaller than the pores of fenestrated capillaries in normal tissues because superparamagnetism is expressed only at a particle size <10 nm. Therefore, SPIONs leak from the capillaries of normal tissues, resulting in low accumulation in tumors. Furthermore, MHT studies have been conducted in an impractical way: direct injection of magnetic materials into tumor and application of hazardous alternating current (AC) magnetic fields. To accomplish effective enhancement of MRI contrast agents in tumors and inhibition of tumor growth by MHT with intravenous injection and a safe AC magnetic field, we clustered SPIONs not only to prevent their leakage from fenestrated capillaries in normal tissues, but also for increasing their relaxivity and the specific absorption rate. We modified the clusters with folic acid (FA) and polyethylene glycol (PEG) to promote their accumulation in tumors. SPION clustering and cluster modification with FA and PEG were achieved simultaneously via the thiol-ene click reaction. Twenty-four hours after intravenous injection of FA- and PEG-modified SPION nanoclusters (FA-PEG-SPION NCs), they accumulated locally in cancer (not necrotic) tissues within the tumor and enhanced the MRI contrast. Furthermore, 24 h after intravenous injection of the NCs, the mice were placed in an AC magnetic field with H = 8 kA/m and f = 230 kHz (Hf = 1.8×109 A/m∙s) for 20 min. The tumors of the mice underwent local heating by application of an AC magnetic field. The temperature of the tumor was higher than the surrounding tissues by ≈6°C at 20 min after treatment. Thirty-five days after treatment, the tumor volume of treated mice was one-tenth that of the control mice. Furthermore, the treated mice were alive after 12 weeks; control mice died up to 8 weeks after treatment.

Magnetically responsive smart nanoparticles for cancer treatment with a combination of magnetic hyperthermia and remote-control drug release.

We report the synthesis of smart nanoparticles (NPs) that generate heat in response to an alternating current magnetic field (ACMF) and that sequentially release an anticancer drug (doxorubicin, DOX). We further study the in vivo therapeutic efficacy of the combination of magnetic hyperthermia (MHT) and chemotherapy using the smart NPs for the treatment of multiple myeloma. The smart NPs are composed of a polymer with a glass-transition temperature (Tg) of 44°C, which contains clustered Fe3O4 NPs and DOX. The clustered Fe3O4 NPs produce heat when the ACMF is applied and rise above 44°C, which softens the polymer phase and leads to the release of DOX. The combination of MHT and chemotherapy using the smart NPs destroys cancer cells in the entire tumor and achieves a complete cure in one treatment without the recurrence of malignancy. Furthermore, the smart NPs have no significant toxicity.

Light and electron microscopic immunohistochemistry of the localization of adrenal steroidogenic enzymes.

Recent immunohistochemical studies have revealed the precise localization of the enzymes involved in adrenal steroidogenesis. Light microscopical investigations showed that cytochromes P450 of cholesterol side‐chain cleavage enzyme (P450scc) and of 11β‐hydroxylase (P45011β), 3β‐hydroxysteroid dehydrogenase/Δ5‐4 isomerase (3βHSD), and 21‐hydroxylase (P450C21) are localized in all the adrenocortical cells, especially in those of the zona fasciculata‐reticularis. 17α‐Hydroxylase/C17‐20 lyase (P45017α, lyase) is present in the zona fasciculata‐reticularis cells of human, bovine, pig, and guinea‐pig adrenals, but absent in the adrenals of some rodents such as rat, hamster, and mouse. Aldosterone synthase (P450aldo) is contained only in the zona glomerulosa cells. In the rat adrenal, P45011β, which catalyzes the conversion of deoxycorticosterone to corticosterone, is localized in the zona fasciculata‐reticularis cells. Electron microscopic investigations demonstrated that P450scc and P45011β are colocalized in the matrix side of inner mitochondrial membrane including cristae, while 3βHSD, P450C21, and P45017α, lyase are present in the membranes of smooth endoplasmic reticulum (SER). These results clearly indicate that aldosterone, the most potent mineralocorticoid, is synthesized in the zona glomerulosa cells, and glucocorticoids, such as corticosterone and cortisol, are produced in the zona fasciculata‐reticularis cells. The conversion of cholesterol to pregnenolone and the final steps of corticosteroid synthesis occur in the mitochondria, while the intermediate steps, leading to the synthesis of deoxycorticosterone or deoxycortisol from pregnenolone, take place in the SER membranes. Microsc. Res. Tech. 36:445–453, 1997.

One-Pot Synthesis and Characterization of Three Kinds of Thiol−Organosilica Nanoparticles

Using a one-pot synthesis, thiol-organosilica nanoparticles (NPs) made from (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)triethoxysilane, and (3-mercaptopropyl)methyldimethoxysilane have been successfully prepared. We compared the synthesis processes of thiol-organosilica NPs made of these three kinds of organosilicates, as well as particles made from tetraethoxysilicate (TEOS), at concentrations varying between 6.25 and 200 mM. We examined three types of synthetic conditions: the Stöber method, in which particles are prepared in 65% ethanol, and two entirely aqueous solvent syntheses, containing either 2% or 27% ammonium hydroxide. The synthetic mixtures were examined using transmission electron microscopy (TEM) to evaluate the as-prepared NPs. The formation trends and rates for these organosilica NPs vary with differing organosilicate precursors, concentrations, and synthetic conditions. The Stöber method is not suitable for formation of thiol-organosilica NPs as compared with the case of TEOS, but the conditions without ethanol and with 27% ammonium hydroxide are suitable for the formation of thiol-organosilica NPs. The size distributions of the formed NPs were evaluated using TEM and dynamic light scattering. The mean diameters of NPs increase with increasing concentrations of silicate, but the size distributions of NPs prepared under various conditions also differ between silicate sources. Thiol-organosilica NPs internally functionalized with fluorescent dye were also prepared using a one-pot synthesis and were characterized using fluorescence microscopy. The thiol-organosilica NPs retain fluorescent dye maleimide very well. In addition, rhodamine B-doped thiol-organosilica NPs show higher fluorescence than thiol-organosilica NPs prepared with rhodamine red maleimide. The surface of thiol-organosilica NPs contains exposed thiol residues, allowing the covalent attachment of fluorescent dye maleimide and protein maleimide. This is the first report describing the synthesis of thiol-organosilica NPs made of three kinds of thiol-organosilicates, differences in nanoparticle formation due to the kinds and concentrations of thiol-organosilicate and due to synthetic conditions, and the advantages of thiol-organosilica NPs due to the existence of both interior and exterior thiol residues.

Developmental Changes in Long-Form Leptin Receptor Expression and Localization in Rat Brain

The expression and localization of long-form leptin receptor (OB-Rb) were studied immunocytochemically in the brain of fetal and adult rats using a polyclonal antibody that specifically recognized OB-Rb. At 14 days of gestation, immunoreactive cells were observed in the ventricular layer, which contains premature neuronal cells. At 18 days of gestation, they were weakly stained but obvious in the paraventricular nucleus (PVN), and ependymal cells also showed immunoreactivity. At birth, the immunoreactivity of OB-Rb in the PVN seemed to be much lower than that in adult rats and remained low during the suckling period. Considering the presence of neuroendocrine and structural neuronal abnormalities in Lepob/Lepob mice with genetic leptin deficiency, these results suggest that the expression of OB-Rb in premature neuronal cells may have some function, and that the regulation of energy balance by leptin through hypothalamic regions, such as PVN, may not yet be developed in the perinatal period.

Immunocytochemical localization of aromatase-containing neurons in the rat brain during pre- and postnatal development

Abstractthe present immunohistochemical study demonstrates the ontogenetic appearance of aromatase-immunoreactive neurons in several discrete regions of the hypothalamus and limbic system in the rat brain, using a purified antibody against human placental aromatase cytochrome P450. Immunoreactive cells were first detected in the preoptic area on the 13th day of embryonic life (E 13), and additionally in the bed nucleus of the stria terminalis on E 15. Labeled cells were also found in the medial amygdaloid nucleus and the ventromedial nucleus on E 16, and some were detected in the arcuate nucleus on E 19. As gestation progressed, the number and the immunoreactivity of these cells gradually increased and peaked within definite periods of perinatal life and there-after declined or disappeared. The immunoreactive cells were also found in the central amygdaloid nucleus and the lateral septal nucleus, and in the ventral pallidum, after the 14th day of postnatal life (P 14) and 30th day (P 30), respectively. The distribution of aromatase-immunoreactive neurons was similar between the sexes, while the immunoreactivity was higher in males than in females after late gestational days. No immunoreaction was detectable in other regions of the telencephalon or midbrain at any time periods studied. The aromatase-immunoreactive neurons in the specific regions may be involved in the sexual differentiation of the brain.

Imaging of size-dependent uptake and identification of novel pathways in mouse Peyer's patches using fluorescent organosilica particles

UNLABELLED We investigated size-dependent uptake of fluorescent thiol-organosilica particles by Peyers patches (PPs). We performed an oral single-particle administration (95, 130, 200, 340, 695 and 1050 nm) and a simultaneous dual-particle administration using 2 kinds of particles. Histological imaging and quantitative analysis revealed that particles taken up by the PP subepithelial dome were size dependent, and there was an optimal size range for higher uptake. Quantitative analysis of simultaneous dual-particle administration revealed that the percentage of fluorescence areas for 95, 130, 200, 340, 695 and 1050 nm with respect to 110 nm area was 124.0, 89.1, 73.8, 20.2, 9.2 and 0.5%, respectively. Additionally, imaging using fluorescent thiol-organosilica particles could detect 2 novel pathways through mouse PP epithelium: the transcellular pathway and the paracellular pathway. The uptake of nanoparticles based on an optimal size range and 2 novel pathways could indicate a new approach for vaccine delivery and nanomedicine development. FROM THE CLINICAL EDITOR Studying various sizes of fluorescent organosilica particles and their uptake in Peyers patches, this team of authors determined the optimal size range of administration. Two novel pathways through mouse Peyers patch epithelium were detected, i.e., the transcellular pathway and the paracellular pathway. This observation may have important applications in future vaccine delivery and nano-drug delivery.

Arachidonate 12-lipoxygenase of rat pineal glands : catalytic properties and primary structure deduced from its cDNA

When a crude extract of rat pineal glands (the 1000 x g supernatant of a homogenate) was incubated with arachidonic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid was found as a major product. The 12-lipoxygenase of rat pineal gland also reacted with linoleic and alpha-linolenic acids at 35% and 101% the rate of arachidonate 12-oxygenation, respectively. Upon Western blot analysis using polyclonal antibody against porcine leukocyte 12-lipoxygenase, the cytosol fraction of rat pineal gland showed a positive band with a molecular weight of approx. 74 kDa. A full-length cDNA for this enzyme was cloned from a cDNA library of rat pineal gland and the identity of the 12-lipoxygenase cDNA was confirmed by its expression in E. coli. The amino acid sequence of the enzyme was deduced from the nucleotide sequence of the cDNA, encoding 663 amino acids with a calculated molecular weight of 75,305. The enzyme showed 72% identity of amino acid sequence with porcine leukocyte 12-lipoxygenase and 73% with bovine tracheal 12-lipoxygenase, but only 59% with human platelet 12-lipoxygenase. Taken together, the high reactivity with C-18 fatty acids, the immunoreactivity and the amino acid homology data indicate that the rat pineal 12-lipoxygenase is more closely related to leukocyte 12-lipoxygenase than to platelet 12-lipoxygenase. Upon RNA blot analysis, by far the highest content of 12-lipoxygenase mRNA was observed in the pineal gland and negligible amounts of mRNA were detected in other parts of the brain. The predominant presence of 12-lipoxygenase mRNA in pineal gland was confirmed by in situ hybridization of rat brain. Significant amounts of 12-lipoxygenase mRNA were also detected in rat spleen, aorta, lung and leukocytes.

Presence of estrogen receptors in aromatase-immunoreactive neurons in the mouse brain

The present study was undertaken to demonstrate estrogen receptors in aromatase-immunoreactive neurons showed the immunoreactivity for estrogen receptors in the cell nuclei in the medial subdivision of the bed nucleus of the stria terminalis and the posterodorsal division of the medial amygdaloid nucleus, but a few did in the medial preoptic area. These results suggest that aromatase-immunoreactive neurons in the former two areas are regulated by steroids through estrogen receptors in their cell nuclei.

Immunohistochemical localization of estrogen receptors within aromatase-immunoreactive neurons in the fetal and neonatal rat brain

We elucidated the anatomical relationship between estrogen receptors and aromatase, the enzyme converting androgens to estrogens, in the fetal and neonatal rat brain by means of double immunohistochemical labeling, using antibodies against rat estrogen receptors and human placental aromatase cytochrome P450. Numerous aromatase-immunoreactive neurons were found in the medial preoptic area, the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus. Estrogen receptors were also abundant in these areas. Most of the aromatase-immunoreactive neurons showed immunoreactivity for estrogen receptors in the medial subdivision of the bed nucleus of the stria terminalis and in the posterodorsal division of the medial amygdaloid nucleus. There were also many double-labeled cells in the ventromedial nucleus. However, in the medial preoptic area the localization of aromatase-immunoreactive neurons was distinct from that of neurons containing estrogen receptors. These results suggested that estrogens, which are converted from androgens in aromatase-containing neurons, are involved in the sexual differentiation of the brain through estrogen receptors within aromatase-immunoreactive neurons in the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus, but through estrogen receptors in aromatase-immunonegative neurons in the medial preoptic area.