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.
Fluorescent nanoparticles, such as nanocrystal quantum dots (QDs), have potential to be applied to molecular biology
and bioimaging, since some nanocrystals emit higher and longer lasting fluorescence than conventional organic probes
do. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully
introduced the 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/geneconstruct
was added to the culture media. The gene-expression efficiency varied depending on multiple factors around
QDs, such as 1) the reading direction of gene fragments, 2) the quantity of gene fragments attached on the surface of
QD-constructs, 3) the surface electronic charges varied according to the structure of 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/geneconstruct
system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs
was disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without
using either viral vectors or specific liposomes.
These results suggest that inappropriate treatment and disposal of QDs may still have risks to the environmental
pollution including human health under certain conditions. Here we propose the further research for the immune and
physiological responses in not only immune cells but also other cells, in order to clear the effect of all other nanoscale
products as well as nanocrystal QDs.
Carbon group quantum dots (QDs) such as carbon, silicon and germanium, have potential for biomedical applications
such as bio-imaging markers and drug delivery systems and are expected to demonstrate several advantages over
conventional fluorescent QDs such as CdSe, especially in biocompatibility. We assessed biocompatibility of newly
manufactured silicon QDs (Si-QDs), by means of both MTT assay and LDH assay for HeLa cells in culture and thereby detected the cellular toxicity by administration of high concentration of Si-QD (>1000 μg/mL), while we detected the high toxicity by administration of over 100 μg/mL of CdSe-QDs. As a hypothesis for the cause of the cellular toxicity, we measured oxy-radical generation from the QDs by means of luminol reaction method. We detected generation of oxy-radicals from the Si-QDs and those were decreased by radical scavenger such as superoxide dismutase (SOD) and N-acetyl cysteine (NAC). We concluded that the Si-QD application to cultured cells in high concentration led cell membrane damage by oxy-radicals and combination usage with radical scavenger is one of the answers.
Quantum dots (QDs) have brighter and longer fluorescence than organic dyes. Therefore, QDs can be applied to
biotechnology, and have capability to be applied to clinical technology. Currently, among the several types of QDs,
CdSe with a ZnS shell is one of the most popular QDs to be used in biological experiments. However, when the CdSe-QDs were applied to clinical technology, potential toxicological problems of CdSe core should be considered. To overcome the problem, silicon nanocrystals, which have the potential of biocompatibility, could be a candidate of alternate probes. Silicon nanoparticles have been synthesized using several techniques. Recently, novel silicon nanoparticles were reported to be synthesized with the combination methods, radio frequency sputtering method and hydrofluoric-etching method In order to assess the biocompatibility of the Silicon nanoparticles, we performed two different cytotoxicity assays, cell iability/proliferation assay using the mitochondrial activity assay and cell membrane damage assay using the lactate dehydrogenase assay. At the 112 μg/mL of silicon nanoparticles (the maximum concentration in this study), we could detected the cell membrane damage of HeLa cells and the decrease of hepatocytes viability. We concluded that we could use the silicon nanoparticles as bioimaging marker but the attention should be given when Silicon nanoparticles were applied to cells in high concentration.
Quantum dots (QDs) have brighter and longer fluorescence than organic dyes. Therefore, QDs can be applied to biotechnology, and have capability to be applied to medical technology. Currently, among the several types of QDs, CdSe with a ZnS shell is one of the most popular QDs to be used in biological experiments. However, when the CdSe QDs were applied to clinical technology, potential toxicological problems due to CdSe core should be considered. To eliminate the problem, silicon nanocrystals, which have the potential of biocompatibility, could be a candidate of alternate probes.
Silicon nanocrystals have been synthesized using several techniques such as aerosol, electrochemical etching, laser pyrolysis, plasma deposition, and colloids. Recently, the silicon nanocrystals were reported to be synthesized in inverse micelles and also stabilized with 1-heptene or allylamine capping. Blue fluorescence of the nanocrystals was observed when excited with a UV light. The nanocrystals covered with 1-heptene are hydrophobic, whereas the ones covered with allylamine are hydrophilic. To test the stability in cytosol, the water-soluble nanocrystals covered with allylamine were examined with a Hela cell incorporation experiment. Bright blue fluorescence of the nanocrystals was detected in the cytosol when excited with a UV light, implying that the nanocrystals were able to be applied to biological imaging.
In order to expand the application range, we synthesized and compared a series of silicon nanocrystals, which have variable surface modification, such as alkyl group, alcohol group, and odorant molecules. This study will provide a wider range of optoelectronic applications and bioimaging technology.
Fluorescent nanoparticles, such as nanocrystal quantum dots (QDs), have potential to be applied to molecular biology and bioimaging, since some nanocrystals emit higher and longer lasting fluorescence than conventional organic probes do. Here we report an example of labeling immune cells by QDs. We collected splenic CD4<sup>+</sup> T-lymphocyte and peritoneal macrophages from mice. Then cells were labeled with QDs. QDs are incorporated into the T-lymphocyte and macrophages immediately after addition and located in the cytoplasm via endocytosis pathway. The fluorescence of QDs held in the endosomes was easily detected for more than a week. In addition, T-lymphocytes labeled with QDs were stable and cell proliferation or cytokine production including IL-2 and IFN-γ was not affected. When QD-labeled T-lymphocytes were adoptively transferred intravenously to mice, they remained in the peripheral blood and spleen up to a week. Using QD-labeled peritoneal macrophages, we studied cell traffic during inflammation on viscera in peritoneum cavity. QD-labeled macrophages were transplanted into the peritoneum of the mouse, and colitis was induced by intracolonic injection of a hapten, trinitrobenzensulfonic acid. With the aid of stong signals of QDs, we found that macrophage accumuled on the inflammation site of the colon. These results suggested that fluorescent probes of QDs might be useful as bioimaging tools for tracing target cells in vivo.
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 nanocrystals 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 <i>in situ</i> 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.
Fluorescent nanoparticles, such as nanocrystal quantum dots (QDs), novel nanometer-size probes and have the potential to be used as easy imaging tool for molecular biology and bioimaging including medical applications, since some nanocrystals emit higher and far longer fluorescence than conventional organic probes. QDs are now becoming widely used in biotechnology and medical applications. QDs have several advantages over organic fluorophores with regard to high luminescence, stability against photobleaching, and a range of fluorescence wavelengths from blue to infrared depending on the particle size. In this review, we reported labeling of some kinds of immune cells and biomolecules with several QDs coated with hydrophilic carboxyl/amine groups, and reported that we could image the circulation of mouse lymphocytes <i>in vivo</i> by QDs. In addition, we also reported here about the cytotoxicity of these nanocrystals.