Hydrophilic Hyperbranched Polymer-Coated siRNA/Polyamidoamine Dendron-Bearing Lipid Complexes Preparation for High Colloidal Stability and Efficient RNAi.

RNA interference (RNAi) using siRNA has gained much attention for use in therapies for cancer and genetic disorders. To establish RNAi-based therapeutics, the development of efficient siRNA nanocarriers is desired. Earlier, we developed polyamidoamine dendron-bearing lipids able to form complexes with nucleic acids as gene vectors. Especially, dendron lipids with unsaturated alkyl chains (DL-G1-U2) induced efficient endosomal escape by membrane fusion, leading to efficient transfection in vitro. For this study, dendron lipids having oleyl/linoleyl groups (DL-G1-U3) were designed to increase membrane fusogenic activity further. Indeed, DL-G1-U3/siRNA complexes achieved higher membrane fusogenic activity and knockdown of the target gene more efficiently than conventional DL-G1-U2/siRNA complexes did. A hydrophilic polymer, hyperbranched polyglycidol lauryl ester (HPG-Lau), was modified further on the surface of DL-G1-U3/siRNA complexes to provide colloidal stability. Surface modification of HPG-Lau increased the colloidal stability in a physiological condition more than complexes without HPG-Lau. Importantly, HPG-Lau-coated DL/siRNA complexes showed identical RNAi effects to those of parental DL/siRNA complexes, whereas the RNAi activity of poly(ethylene glycol)-bearing lipid (PEG-PE)-modified DL/siRNA complexes was hindered completely. Introduction of unsaturated bonds into dendron lipids and selection of suitable hydrophilic polymers for nanocarrier modification are important for obtaining efficient siRNA vectors toward in vivo siRNA delivery.

D/H exchange in C-H bonds of fatty acids: implication for geographical discrimination of food materials.

The stable isotope analysis of non-exchangeable hydrogen in food materials has been believed as a powerful tool for tracing geographical origins and delivery of the materials. However, little information is available for the D/H exchanges even in hydrophobic molecules, which leads to potential uncertainty on the observed results. To further evaluate the reliability of hydrogen isotopes of organic molecules, we examined the isotope exchangeability in fatty acids within phospholipid bilayers exposed to D(2)O for 12h. The results indicate that the isotope ratio is clearly increased during all examines (e.g., 93-328 ‰ in 50% D(2)O), in which its magnitude depends on type of fatty acids and state of bilayers. However, these results also indicate very clearly that the observed exchange rate is negligible if samples are exposed to natural abundance of deuterium (0.0156%) in natural and laboratory environments before/during analysis.

Characterization of Japanese polished rice by stable hydrogen isotope analysis of total fatty acids for tracing regional origin.

We determined the δD values of the total fatty acids of Japanese rice to test the hypothesis that there is a wide variation in the hydrogen isotopic composition (δD) of the total fatty acids of Japanese rice sourced from different growth areas in Japan and to distinguish the minor differences among these growth areas. The δD values showed a wide variation ranging from -216‰ (Hokkaido) to -183‰ (Okinawa), indicating a good correlation with the corresponding variations for ambient water (r = 0.63) and mean temperature (r = 0.84). These results suggest that the δD values for the total fatty acids can be representative of the growth environments in Japan.

Statistical analysis of rice samples for compositions of multiple light elements (H, C, N, and O) and their stable isotopes.

Stable isotopic compositions and elemental contents of the H, C, N, and O in 163 rice samples were analyzed. The samples were taken from three different farming countries; Japan (n = 103), United States of America (n = 30), and Australia (n = 21), in addition of Asian rice samples from Thailand (n = 2), Vietnam (n = 1), and China (n = 6) as comparison. They were mostly short grain samples known as "Koshihikari," with several samples of middle and long grains included. All samples were grown in the presence of either natural manure or artificial fertilizer. The climate of the rice farming environment was diverse, from arid to humid. Excluding deltaD data showing large uncertainty, according to the statistical analysis of the principal components based on the stable isotopic compositions such as delta(13)C, delta(15)N, and delta(18)O of rice samples, the Japanese rice samples were clearly distinctive from the Australian and the American rice samples. This fact may be explained by the regional differences in isotopic signatures of the climate, utilized nutrition, and/or quality of irrigation water among the farming countries. This statistical distinction could be one of the useful tools to extract the rice samples grown in Japan from those grown in the other countries.

MALDI mass spectrometry using 2,4,6-trihydroxyacetophenone and 2,4-dihydroxyacetophenone with cyclodextrins: suppression of matrix-related ions in low-molecular-weight region.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of a model peptide, Substance P (SubP), was carried out using 2,4,6-trihydroxyacetophenone (THAP) and 2,4-dihydroxyacetophenone (DHAP) with cyclodextrins (cyclodextrin-supported matrix). It was found that the use of a cyclodextrin-supported matrix simplified the mass spectrum in the low-molecular-weight region. The interaction between THAP/DHAP and cyclodextrin (CD) was studied by UV-vis absorption spectroscopy and the incorporation of matrix molecules into the cyclodextrin cavity was confirmed by (1)H-NMR spectroscopy. DHAP showed tight incorporation with betaCD (betaCD(DHAP)) rather than THAP and it was found that the matrix-related peaks could be weakened by less than one third of the peak intensity of a protonated analyte. The betaCD(DHAP) matrix was applied to the measurements of two low-molecular-weight compounds; adenosine and adrenaline. It became clear that the cyclic structure of the CD and the host-guest interaction between betaCD and the matrix molecule were important to reduce the matrix-related peaks of THAP and DHAP.

Direct observation of dispersion and mixing processes in microfluidic systems.

The diffusion phenomena, dispersion and mixing processes of the sample solute (Basic Blue 3 dye and KMnO4 aqueous solutions) were directly observed in laminar flow in glass microchannels. Quasi steady-state UV-visible absorption spectrometry was carried out using CCD camera images of the colored sample dispersion and mixing processes, and the absorbance change (DeltaAbs) was discussed based on the dimensionless parameter, tau which represents the flow time renormalized to the diffusion coefficient and the channel cross section. It was found that DeltaAbs showed almost the same tau dependence, even though the solutions and the microchannel sizes differed in laminar flow, if the microchannel fabrication method was the same. On the basis of this fundamental result, the total microchannel length required for the reaction of 2,3-diaminonaphthalene (DAN) and NO2- at a flow rate of 2 microL min(-1) was calculated, and the obtained value ( approximately 100 mm) showed very good agreement with our previous microchip research. It was concluded that both results were useful for designing the microchannel width, depth and length to control the chemical reaction time in recent microfluidic systems.

Suppression of matrix-related ions using cyclodextrin in MALDI mass spectrometry.

An effective technique for the suppression of matrix-related ions in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have been developed. Using typical organic matrices such as THAP (2,4,6-trihydroxyacetophenone) and CHCA (alpha-cyano-4-hydroxycinnamic acid) in a cyclodextrin cavity, we successfully measured the mass peaks of only protonated matrix ions and significantly suppressed their intensities and fragmentation. In addition, it became possible to analyze the mass peak of the analyte molecules (substance P and adenosine) without any interference from the matrix. We believe that this technique could be a powerful tool for MALDI mass spectrometry, particularly for low-molecular-weight compounds.

Stable carbon, nitrogen, and oxygen isotope analysis as a potential tool for verifying geographical origin of beef.

Stable isotope analysis of organic elements such as carbon and nitrogen has been employed as a powerful tool for provenance determination of food materials, because isotopic compositions of the materials reflect many factors in natural environment. In this study, we examined carbon, nitrogen, and oxygen isotope signatures of beef from Australia, Japan, and USA, in order to confirm the method as a potential tool for verifying geographical origin of beef commercially distributed in Japan. Defatted dry matter of beef from USA was characterized by higher carbon isotopic composition (-13.6 per thousand to -11.1 per thousand) than that from Japan (-19.6 per thousand to -17.0 per thousand) and Australia (-23.6 per thousand to -18.7 per thousand). That from Australia was characterized by higher oxygen isotopic composition (+15.0 per thousand to +19.4 per thousand) than that from Japan (+7.3 per thousand to +13.6 per thousand) and USA (+9.5 per thousand to +11.7 per thousand). The oxygen isotopic composition in Japanese beef showed a positive correlation with the isotopic composition of cattle drinking water, the difference in which is clearly latitude dependent. These results suggest that a comparison of carbon, nitrogen, and oxygen isotopic compositions is applicable as a potential tool to discriminate the provenance of beef not only between different countries (i.e. Australia, Japan, and USA) but also among different regions within Japan.

Geographical origin of polished rice based on multiple element and stable isotope analyses.

We determined carbon and nitrogen contents (C and N contents) and stable carbon, nitrogen, and oxygen isotopic compositions (δ(13)C, δ(15)N, and δ(18)O) of polished rice in order to develop a simple method to discriminate its geographical origin. As a first attempt, we examined a single cultivar, Koshihikari rice, from 14 different cultivation areas including Australia (n=1), Japan (n=12), and USA (n=1). For all rice samples, C and N contents and the isotopic compositions are consistent with those of general plant materials, being 37.2-40.0% (C content), 0.8-1.4% (N content), -27.1 to -25.4% (δ(13)C), +0.4 to +9.0% (δ(15)N), and +18.8 to +22.9% (δ(18)O). However, its cultivated area is clearly distinguished by a pentagonal radar plot based on the elemental and isotopic compositions. Thus, the comparison of C and N contents and δ(13)C, δ(15)N, and δ(18)O values would potentially be useful for rapid and routine discrimination of geographical origin of the polished rice.

Determination of oxygenated volatile organic compounds in ambient air using canister collection-gas chromatography/mass spectrometry.

This research attempts to establish a method to measure 11 kinds of oxygenated volatile organic compound (OVOC) in ambient air by using the canister collection-gas chromatography/mass spectrometry (GC/MS) method. Since several compounds such as acetone exhibited high blank concentrations due to their laboratory use, stringent quality control was conducted for the VOC-free added water and the VOC-free nitrogen gas. In order to prevent the decline of recovery rates due to lack of sufficient relative humidity, it is necessary to add VOC-free water when pressurizing and diluting the air samples. Thus, all the target compounds in ambient air were obtained from the canisters at high recovery rates without significant contamination. Furthermore, the canister collection-GC/MS method makes it possible to apply simultaneous air monitoring of OVOCs as well as volatile hazardous air pollutants without additional sampling.

Development of microchip for rapid pretreatment of trichloroethylene and tetrachloroethylene volatilized from polluted soil.

In the field of environmental analysis, microchip technology has many attractive features and is expected to realize the miniaturization of analytical instruments allowing on-site and multipoint monitoring using this technology. We propose a new soil analytical system for detecting trichloroethylene (TCE) and tetrachloroethylene (PCE) using a microchip. The experimental conditions for the pretreatment microchip were optimized. Under the optimized conditions, the detection limits of TCE and PCE were 15 and 9 ppbv, and the determination limits of TCE and PCE were 62 and 72 ppbv, respectively.

Desorption/ionization on carbon mass spectrometry: study of amorphous-carbon property.

In this study, an amorphous carbon was proposed as a new ionization substrate for laser desorption/ionization (LDI) and its efficiency for soft laser ionization without any matrix molecules was focused on the soft laser ionization effect of the amorphous carbon substrate was compared with that of a stainless substrate by LDI and matrix-assisted laser desorption/ionization (MALDI) using a 266-nm irradiation laser. The result of the amorphous carbon substrate was indicated to have a soft ionization effect similar to the result of MALDI. In addition, from the experimental result achieved by soft laser ionization using not only 266-nm-wavelength but also 355-nm-wave length irradiation lasers, the amorphous carbon substrate had a high degree of generality for selecting a sample similarly to soft ionization. The same experimental condition was carried out for other amorphous carbon substrates with different surface roughnesses in order to clarify the ionization mechanism on the amorphous carbon substrates. From the results, it was suggested that factors besides the difference in surface roughness exist for laser ionization efficiency.

Femtosecond/picosecond time-resolved fluorescence study of hydrophilic polymer fine particles.

Femtosecond/picosecond time-resolved fluorescence study of hydrophilic polymer fine particles (polyacrylamide, PAAm) was reported. Ultrafast fluorescence dynamics of polymer/water solution was monitored using a fluorescent probe molecule (C153). In the femtosecond time-resolved fluorescence measurement at 480 nm, slowly decay components having lifetimes of tau(1) approximately 53 ps and tau(2) approximately 5 ns were observed in addition to rapid fluorescence decay. Picosecond time-resolved fluorescence spectra of C153/PAAm/H2O solution were also measured. In the time-resolved fluorescence spectra of C153/PAAm/H2O, a peak shift from 490 to 515 nm was measured, which can be assigned to the solvation dynamics of polymer fine particles. The fluorescence peak shift was related to the solvation response function and two time constants were determined (tau(3) approximately 50 ps and tau(4) approximately 467 ps). Therefore, the tau(1) component observed in the femtosecond time-resolved fluorescence measurement was assigned to the solvation dynamics that was observed only in the presence of polymer fine particles. Rotational diffusion measurements were also carried out on the basis of the picosecond time-resolved fluorescence spectra. In the C153/PAAm/H2O solution, anisotropy decay having two different time constants was also derived (tau(6) approximately 76 ps and tau(7) approximately 676 ps), indicating the presence of two different microscopic molecular environments around the polymer surface. Using the Stokes-Einstein-Debye (SED) equation, microscopic viscosity around the polymer surface was evaluated. For the area that gave a rotational diffusion time of tau(6) approximately 76 ps, the calculated viscosity is approximately 1.1 cP and for tau(7) approximately 676 ps, it is approximately 10 cP. The calculated viscosity values clearly revealed that there are two different molecular environments around the polyacrylamide fine particles.

Inhibitory analysis of the effect of polycyclic aromatic hydrocarbons on the activity of chitinase by means of liquid chromatography-mass spectrometry of chitin oligosaccharides.

The analytical method of determining enzyme activity by liquid chromatography-mass spectrometry (LC/MS) was developed and applied for investigation of the effect of polycyclic aromatic hydrocarbons (PAHs) on the enzyme activity of chitinase. The measurement of chitinase activity by LC/MS is useful in order to use the nonderivatized substrate, which can show in vivo chitinase activity. Substrate consumption and product formation were monitored in order to determine chitinase activity. It was shown that, for the first time, in vitro addition of PAHs inhibited the activity of chitinase in a noncompetitive manner. The IC(50) value of benzo[a]pyrene was 1.4 microM, and PAHs containing four or more aromatic rings showed the same or higher inhibitory effect, whereas PAHs with a lower number of aromatic rings showed lower inhibition of the chitinase activity than benzo[a]pyrene.

Novel method to identify illegal diesel fuel, I: use of adamantane or [1, 2-D2] n-tetradecane as chemical marker.

In this study, we demonstrated a new identification method of illegal diesel fuel using adamantane (tricyclo[3,3,1,1,(3,7)]decane) or [1, 2-D(2)] n-tetradecane as a potential chemical marker instead of coumarin. In a mixing system of diesel fuel with marker-labeled kerosene, the concentration of kerosene in the mixed oil is quantitatively determined using specific ion fragments of the markers using GC/MS analysis, in which the detection limit is 12.5 ng/microl for adamantane or 7.1 ng/microl for [1, 2-D(2)] n-tetradecane. Coumarin, the currently used chemical marker, can be easily removed by sulfuric acid treatment, which is a primal cause of environmental pollution due to the illicit disposal of the strong acidic by-product. However, adamantane and [1, 2-D(2)] n-tetradecane cannot be removed by sulfuric acid treatment. Thus, although the detection limit of adamantane and [1, 2-D(2)] n-tetradecane is higher by approximately one or two orders of magnitude than that of coumarin, they cannot be removed by sulfuric acid treatment. Therefore, adamantane and [1, 2-D(2)] n-tetradecane are strong candidates as potential markers instead of coumarin, leading to the prevention of the production of illegal diesel fuel and a reduction in environmental pollution.

A simple fluorometric method for the determination of sulfur dioxide in ambient air with a passive sampler.

A simple and rapid fluorescence detection method was used for the determination of sulfur dioxide (SO(2)) in ambient air collected by a passive sampler. Gaseous SO(2) was absorbed as sulfite ions into an absorbing solution of triethanolamine (TEA) contained in an adsorbent sheet and reacted with N-(9-acridinyl)maleimide (NAM), which was used as a fluorescent reagent. TEA was also used as a buffer solution in this study. The calibration curve of SO(2) showed a linear correlation R(2)=0.992, and the relative standard deviation (RSD) was 2.4% for 50 ppb SO(2) with six repeated measurements. It was satisfactory to apply this method to determine the SO(2) level for simultaneous multipoint monitoring.

Effect of aspect ratio on chemical reactions on microchip.

Parallel two-phase laminar flow, which is formed when two solutions flow in microchannels, has been developed and has advanced unique research in the area of microchip analysis. In two-phase laminar flow, channel size has a significant effect on the efficiency of chemical reactions. However, the sizes of microchannels vary greatly in many studies. In this paper, we report on the effect of microchannel size on chemical reactions on a microchip. Aspect ratio is defined as the ratio of depth to width of a microchannel. Five microchips with different aspect ratios (from 0.50 to 2.00) were fabricated by mechanical machining. The reaction of nitrous acid and Saltzman reagent was carried out on these microchips and the absorbance was measured on-line in a capillary tube, which was attached to the outlet on the microchip. The results showed that the color reaction occurred more efficiently as the aspect ratio increased. This result is expected to be useful when determining the size of microchannels.

Characterization of hydrophilic polymer fine particles by steady-state fluorescence spectroscopy.

We synthesized hydrophilic polymer particles based on acrylamide, and their chemical properties are investigated by fluorescence spectroscopy. The morphology of the synthesized polymers was monitored by scanning electron microscopy (SEM) and a dynamic light-scattering analyzer, and it was observed that the synthesized polymers are spherical with a median diameter of approximately 500 nm. A fluorescent probe molecule (C153) was introduced into the polymer/water solution, and the steady-state fluorescence spectrum was observed. In the C153/polymer/water solution, strong fluorescence inHtensity from the C153 molecules was observed with a maximum intensity at 515 nm, whereas the C153/water solution only gave very weak fluorescence with a maximum at 540 nm. Since C153 is hardly soluble in water, it was concluded that the C153 molecules existed selectively around the particle surfaces. Because of the difference between the fluorescence spectra, it was found that the chemical properties around the polymer surface were very different from that of the bulk water.

Application of a monolithic silica capillary adsorbent for the preconcentration of airborne trichloroethylene and tetrachloroethylene.

Monolithic silica with a bimodal pore structure was prepared in a fused silica capillary by the sol-gel process and investigated as an adsorbent for the preconcentration of airborne trichloroethylene and tetrachloroethylene. The airborne trichloroethylene and tetrachloroethylene are adsorbed when they flow through the silica capillary column and can be desorbed by liquid hexane followed by GC/MS analysis. The monolithic silica adsorbent is mechanically stable and no frits or other special structures are needed to retain them in place. In addition, owing to the smaller size of the capillary column, only a small volume of desorption solvent is needed. The present investigation demonstrates the satisfactory applicability of monolithic silica as a capillary adsorbent.