Enhancement of gene transfer activity mediated by mannosylated dendrimer/alpha-cyclodextrin conjugate (generation 3, G3).

To enhance gene transfer activity of dendrimers, we prepared its conjugate (generation 3, G3) with alpha-cyclodextrin bearing mannose (Man-alpha-CDE conjugates) with various degrees of substitution of the mannose moiety (DSM5, 10, 13, 20) and compared their cytotoxicity and gene transfer activity, and elucidated the enhancing mechanism for the activity. Of the various carriers used here, Man-alpha-CDE conjugate (G3, DSM10) provided the highest gene transfer activity in NR8383, A549, NIH3T3 and HepG2 cells, being independent of the expression of mannose receptors. Gene transfer activity of Man-alpha-CDE conjugate (G3, DSM10) was not decreased by the addition of 10% serum in A549 cells. Cytotoxicity of the polyplex with Man-alpha-CDE conjugates (G3, DSM10) was not observed in A549 and NIH3T3 cells up to the charge ratio of 200/1 (carrier/pDNA). The gel mobility and particle size of polyplex with Man-alpha-CDE conjugate (G3, DSM10) were relevant to those with alpha-CDE conjugate (G3), but zeta-potential, DNase I stability, pDNA condensation of the former polyplex were somewhat different from those of the latter one. Cellular association of polyplex with Man-alpha-CDE conjugate (G3, DSM10) was almost comparable to that with dendrimer (G3) complex and alpha-CDE conjugate (G3). The addition of mannan and mannose attenuated gene transfer activity of Man-alpha-CDE conjugate (G3, DSM10) in A549 cells. Alexa-pDNA complex with TRITC-Man-alpha-CDE conjugate (G3, DSM10), but not the complex with TRITC-alpha-CDE conjugate (G3), was found to translocate to nucleus at 24 h after incubation in A549 cells. HVJ-E vector including mannan, but neither the vector alone nor the vector including dextran, suppressed the nuclear localization of TRITC-Man-alpha-CDE conjugate (G3, DSM10) to a striking degree after 24 h incubation in A549 cells. These results suggest that Man-alpha-CDE conjugate (G3, DSM10) has less cytotoxicity and prominent gene transfer activity through not only its serum resistant and endosome-escaping abilities but also nuclear localization ability.

Composition of chemical species of selenium contained in selenium-enriched shiitake mushroom and vegetables determined by high performance liquid chromatography with inductively coupled plasma mass spectrometry.

Selenium (Se) species in Se-enriched shiitake mushroom (Lentinula edodes) were identified and quantified by high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICPMS). Two types of Se-enriched shiitake obtained from selenite- or selenate-fertilized mushroom beds were used. More than 80% of Se in both shiitake samples could not be extracted with 0.2 M HCl. Protease digestion released a large amount of selenomethionine from the shiitake enriched with selenite. However, most of the Se in the shiitake enriched with selenate was not released by protease but was released by a cell wall digestive enzyme and most of the Se released was identified as selenate. These results indicate that the main Se species in the shiitake enriched with selenite or selenate is selenomethionine bound to protein or selenate bound to polysaccharides in the cell wall, respectively. Several Se-enriched vegetables grown on a soil fertilized with selenate were also analyzed by HPLC-ICPMS. Four Se species, selenate, Se-methylselenocysteine, selenomethionine, gamma-glutamyl-Se-methylselenocysteine, and an unknown Se compound were detected in the vegetables. The composition of Se species varied with the kinds or parts of vegetables. The main Se species in bulbs, leaves or flowers of the Se-enriched garlic, onions, cabbage and ashitaba were selenate, Se-methylselenocysteine or gamma-glutamyl-Se-methylselenocysteine, while those in fruit bodies of the peppers and pumpkin were selenomethionine bound to protein. Bioavailabilities of Se in the shiitake mushroom enriched with selenite and the vegetables enriched with selenate are expected to be high, but that in shiitake enriched with selenate may be low.

Improvement of gene delivery mediated by mannosylated dendrimer/alpha-cyclodextrin conjugates.

The purpose of this study is to evaluate in vitro and in vivo gene delivery efficiency of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)) bearing mannose (Man-alpha-CDE conjugates) with the various degrees of substitution of the mannose moiety (DSM) as a novel non-viral vector in a variety of cells. Man-alpha-CDE conjugates (DSM 3.3 and 4.9) were found to have much higher gene transfer activity than dendrimer, alpha-CDE conjugate and Man-alpha-CDE conjugates (DSM 1.1 and 8.3) in various cells, which are independent of the expression of cell surface mannose receptors. Cellular association of pDNA complexes with dendrimer, alpha-CDE conjugate and Man-alpha-CDE conjugate (DSM 3.3) and their cytotoxic effects differed only very slightly. Surface plasmon resonance study demonstrated that the specific binding activity of Man-alpha-CDE conjugates to concanavalin A was not very strong. Much more conjugation of the mannose moiety to alpha-CDE conjugates provided unfavorable physicochemical properties of pDNA complexes for gene transfer, e.g. the low interaction with pDNA, the low enzymatic stability of pDNA and the lack of pDNA compaction. Man-alpha-CDE conjugate (DSM 3.3) provided gene transfer activity higher than dendrimer and alpha-CDE conjugate in kidney 12 h after intravenous injection in mice. These results suggest the potential use of Man-alpha-CDE conjugate (DSM 3.3) as a non-viral vector.

Preparation of selenium-enriched sprouts and identification of their selenium species by high-performance liquid chromatography-inductively coupled plasma mass spectrometry.

Sprouts of several plants (10 families and 28 species) were cultivated in a high selenium environment, and the chemical species of selenium in these selenium-enriched sprouts were identified by using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). Cultivation of sprouts of kaiware daikon (type of radish) with 5.0 microg/ml or 10.0 microg/ml of selenium as selenite inhibited the growth. However, no abnormalities in the shape or color were apparent even in the sprouts exposed to 10.0 microg/ml of selenium. The selenium concentration in the sprouts of most plants examined was higher than that from environmental exposure. Among the types of selenium that were accumulated, a large part (69-98%) was extractable in 0.2 M HCl. Chemical analysis of selenium in the HCl extract showed that the main selenium species in all the sprouts examined was Se-methylselenocysteine. In addition to Se-methylselenocysteine, selenomethionine, non-metabolized selenite, gamma-glutamyl-Se-methylselenocysteine and an unknown selenium compound were also detected in several high-selenium sprouts. Since higher anticarcinogenic activities of these monomethylated selenoamino acids have been observed, it is anticipated that such selenium-enriched sprouts will be used as a foodstuff for cancer prevention.