Noriyoshi Manabe

Use of fluorescent quantum dot bioconjugates for cellular imaging of immune cells, cell organelle labeling, and nanomedicine: surface modification regulates biological function, including cytotoxicity.

With the development of nanotechnology, nanoscale products that are smaller than several hundred nanometers have been applied to all areas of science and technology. Nanoscale products, including carbon nanotubes, fullerene derivatives, and nanocrystal quantum dots (QDs), are wide spread as novel tools in various fields, not only in materials engineering, electronics, plastics, and the automobile and aerospace industries, but also in molecular biology and medicine. At present, QDs have been widely used in biological and medical studies because of their superior photoemission and photostability. Although the physical and chemical properties of QDs have been circumstantially investigated, little is known about any harmful effects of QDs on human health. Here we report on the toxicity and biological behavior of QDs in vitro and in vivo. The toxicity of the core constituent chemicals such as cadmium and selenium has been identified. Recently, the surface molecules surrounding QDs have been intensively investigated. Accumulating evidence that toxic surface-covering molecules showed their cytotoxicity and biomolecules conjugated with QDs maintained their biological effects indicates that at least the biological properties of QDs are attributable to the QD-capping material rather than to the core metalloid complex itself.

Quantum Dot as a Drug Tracer In Vivo

Quantum dots (QDs) have been applied to a wide range of biological studies by taking advantage of their fluorescence properties. There is almost no method to trace small molecules including medicine. Here, we used QDs for fluorescent tracers for medicine and analyzed their kinetics and dynamics. We conjugated QDs with captopril, anti-hypertensive medicine, by an exchange reaction while retaining the medicinal properties. We investigated the medicinal effect of QD-conjugated captopril (QD-cap) in vitro and in vivo. We also evaluated the concentration and the distribution of the QD-cap in the blood and the organs with their fluorescence. We demonstrate that the QD-cap inhibits the activity of ACE in vitro. The QD-cap reduced the blood pressure of hypertensive model rats. The concentration of the QD-cap in the blood was measured by using the standard curve of the fluorescence intensity. The blood concentration of the QD-cap decrease exponentially and QD-cap has approximately the same half-life as that of captopril. In addition, the fluorescence of the QDs revealed that QD-cap accumulates in the liver, lungs, and spleen. We succeeded in analyzing the dynamics and kinetics of small molecules using fluorescence of QDs

Simultaneous multicolor detection system of the single-molecular microbial antigen with total internal reflection fluorescence microscopy.

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystal QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD‐conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD‐conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high‐sensitivity detection systems.

Organ distribution of quantum dots after intraperitoneal administration, with special reference to area-specific distribution in the brain

Quantum dots (QDs) are well known for their potential application in biosensing, ex vivo live-cell imaging and in vivo animal targeting. The brain is a challenging organ for drug delivery, because the blood brain barrier (BBB) functions as a gatekeeper guarding the body from exogenous substances. Here, we evaluated the distribution of bioconjugated QDs, i.e., captopril-conjugated QDs (QDs-cap) following intraperitoneal injection into male ICR mice as a model system for determining the tissue localization of QDs, employing ICP-MS and confocal microscopy coupled with spectrometric analysis. We have demonstrated that intraperitoneally administered QDs-cap were delivered via systemic blood circulation into liver, spleen, kidney and brain at 6 h after injection. QDs-cap were located predominantly inside the blood vessels in the liver, kidney and brain, but a few were distributed in the parenchyma, especially noteworthy in the brain. Careful studies on acute as well as chronic toxicity of QDs in the brain are required prior to clinical application to humans.

Immune Response Induced by Fluorescent Nanocrystal Quantum Dots In Vitro and In Vivo

Fluorescent nanocrystal quantum dots (QDs) are widely used as novel tools in various biological fields including cellular biology, molecular biology, and even in basic and clinical medical fields, due to their far brighter photoemission and photostability. Although many amounts of biological studies, including in vivo experiments, were circumstantially investigated, there is no informative report that investigates whether the QDs affect the mammalian immune system. This study investigated the immune response and biological behavior of QDs in vitro and in vivo. The immune response to QDs by both lymphocytes and kinds of macrophages in vitro and in vivo was investigated. Co-culture of QDs with immune cells showed that apparently normal production of cytokines and chemokines in both mouse CD4+ lymphocytes and peritoneal F4/80+ macrophages (PM phi). In addition, the bionanocomplex of QDs with enhanced-green-fluorescent-protein (eGFP)-encoding nucleotides successfully induced the expression of eGFP protein by PMphi. However, direct injection of QD+nucleotides bionanocomplex aqueous solution into the peritoneal cavity of mice resulted in the inflammation with the infiltration of inflammatory cells into the peritoneal cavity. Furthermore, QD+nucleotides bionanocomplex (but not QD bionanocomplex without nucleotides), induced the production of both proinflammatory cytokines and chemokines by PM phi in vitro. These results indicated that QDs covered with nucleotides caused the peritoneal inflammation in vivo via activation of PMphi and probably nonimmune cells. Taken together, these data indicated that QDs affect the proliferation of immune cells, but not in immune response including cytokine production. We propose here that all nanotechnology researchers should confirm the biological responses of their nanoscale products, because the biological response against nanoscale products can be occurred by not only in immune cells but also other nonimmune cells.

Visualizing Vitreous Using Quantum Dots as Imaging Agents

Vitreous is transparent tissue located between the lens and the retina of the eye, thus, difficult to look at by even ophthalmological microscope. But vitreous is connected with some sight-threatening eye diseases, for example, retinal detachment, macular hole, epi-retinal membrane, and so forth. Quantum dots (QDs) have been applied to a wide range of biological studies by taking advantage of their fluorescence properties. We established a novel technique of aqueous colloidal QD (ACQD) as a vitreous lesion detector. When compared with some conventional dyes used for clinical situation, i.e. fluorescein, indocyanine green, and triamcinolone acetonide, ACQD exerted a higher performance to detect a Weiss Ring. Furthermore ACQD is also effective to perform vitrectomy, an eye surgery to cut and eliminate vitreous. Some functional structures in vitreous are detected clearly when ACQD was injected into an enucleated porcine eye. We demonstrated that ACQD enabled any ophthalmic surgeon to perform vitrectomy reliably, easily, and more safely. Taken together, the ACQD-oriented vitreous staining system will promote ophthalmological science, and it will raise the cure rate of eye diseases

Roles of Intramolecular and Intermolecular Hydrogen Bonding in a Three-Water-Assisted Mechanism of Succinimide Formation from Aspartic Acid Residues

Aspartic acid (Asp) residues in peptides and proteins are prone to isomerization to the β-form and racemization via a five-membered succinimide intermediate. These nonenzymatic reactions have relevance to aging and age-related diseases. In this paper, we report a three water molecule-assisted, six-step mechanism for the formation of succinimide from Asp residues found by density functional theory calculations. The first two steps constitute a stepwise iminolization of the C-terminal amide group. This iminolization involves a quintuple proton transfer along intramolecular and intermolecular hydrogen bonds formed by the C-terminal amide group, the side-chain carboxyl group, and the three water molecules. After a conformational change (which breaks the intramolecular hydrogen bond involving the iminol nitrogen) and a reorganization of water molecules, the iminol nitrogen nucleophilically attacks the carboxyl carbon of the Asp side chain to form a five-membered ring. This cyclization is accompanied by a triple proton transfer involving two water molecules, so that a gem-diol tetrahedral intermediate is formed. The last step is dehydration of the gem-diol group catalyzed by one water molecule, and this is the rate-determining step. The calculated overall activation barrier (26.7 kcal mol−1) agrees well with an experimental activation energy.

GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

Evaluation of Anti-Inflammatory Drug-Conjugated Silicon Quantum Dots: Their Cytotoxicity and Biological Effect

Silicon quantum dots (Si-QDs) have great potential for biomedical applications, including their use as biological fluorescent markers and carriers for drug delivery systems. Biologically inert Si-QDs are less toxic than conventional cadmium-based QDs, and can modify the surface of the Si-QD with covalent bond. We synthesized water-soluble alminoprofen-conjugated Si-QDs (Ap-Si). Alminoprofen is a non-steroid anti-inflammatory drug (NSAID) used as an analgesic for rheumatism. Our results showed that the “silicon drug” is less toxic than the control Si-QD and the original drug. These phenomena indicate that the condensed surface integration of ligand/receptor-type drugs might reduce the adverse interaction between the cells and drug molecules. In addition, the medicinal effect of the Si-QDs (i.e., the inhibition of COX-2 enzyme) was maintained compared to that of the original drug. The same drug effect is related to the integration ratio of original drugs, which might control the binding interaction between COX-2 and the silicon drug. We conclude that drug conjugation with biocompatible Si-QDs is a potential method for functional pharmaceutical drug development.

Conjugation of Quantum Dots and JT95 IgM Monoclonal Antibody for Thyroid Carcinoma Without Abolishing the Specificity and Activity of the Antibody

Among the immunoglobulins, IgM class-antibodies are now considered to be potent immunological reagents for anticancer remedies. However, only a few reports are available about the effective labeling of IgM with enzymes, fluorescence, or other bioreactive reagents. Here, we report an effective application of luminescent semiconductive nanoparticles, quantum dots (QDs), as a labeling material of the IgM antibody. The CdSe carboxyl QDs were reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfo- succinimide in 2-(morpholino) ethanesulfonic acid. The reacted QDs were then coupled to JT95 IgM antibody, which recognizes thyroid carcinoma associated antigen. The specificity and activity of the conjugates were tested by immunoblot, immunoquantitive assay and immunohistological imaging. The QDs were firmly conjugated with JT95 IgM monoclonal antibody. In immunoblot assay, QD-JT95 conjugates directly detected the target molecules without obstructing the binding site. In immunoquantitive assay, the conjugates could quantify the antigen in the range of 1.56-100 μg/mL. Also, QDs-labeled antibody detected the antigen on plasma membrane. Our results demonstrate that labeling of JT95 and other IgM class antibodies with QDs is feasible. This approach may be an important method for the medical application of IgM in the diagnosis and treatment of cancers.