Biomedical subjects
Publications and source records attributed to Hideyoshi Harashima.
The effects of cationic liposomes complexed with plasmid DNA on the process of transcription was examined using a recently developed rapid cell free translation system. The findings indicate that the liposome itself inhibited the process when the ratio of DNA/liposome typically used in transfection studies was used.
The optimization of drug disposition in the body leads to an increase in its therapeutic effect and to a decrease in adverse effects. Liposomes can serve as a potential drug carrier for achieving this. However, the behavior of a drug carrier system under in vivo conditions is complex. Therefore, a more complete understanding of the pharmacokinetics of liposomes themselves, as well as that of the encapsulated drug, is required. The optimization of the pharmacokinetics of liposomes can be performed by linking a pharmacodynamic model of the free drugs that are encapsulated into liposomes. Sensitivity analysis was applied to optimize the delivery system to maximize the antitumor effect of liposomal doxorubicin (DOX). Advanced technology for ligand-mediated selective targeting and intracellular targeting is also introduced for antitumor agents and for gene delivery systems.
Efficient nuclear entry of exogenous DNA is one of the key factors toward gene therapy success with nonviral vectors. To re-address the effects of a nuclear localization signal (NLS) peptide attached directly to DNA, we prepared three dumbbell-shaped, green fluorescent protein (GFP)-encoding DNAs containing one or two NLS peptides. The peptide was conjugated to the loop-forming oligodeoxyribonucleotides by cross-linking reactions between the peptide and a modified uracil base with a dioxaoctylamino linker, and the oligonucleotides were then ligated to the DNA molecules. The NLS-conjugated DNA dumbbells were microinjected into the cytosols and nuclei of simian COS-7 cells. In addition, unconjugated DNA dumbbells, with or without a modified uracil base, were also examined for comparison. The GFP gene was expressed with efficiencies in the order of the unmodified DNA >or= the NLS-conjugated DNA > the unconjugated DNA with the base modification, with both cytosolic and intranuclear microinjections. Thus, we concluded that (i) one or two NLS peptide(s) did not dramatically improve the nuclear entry of DNA and that (ii) chemical modification of DNA reduced the transcription efficiency or stability in the nucleus.
In order to identify factors affecting cationic liposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining lipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.