| 研究実績の概要 |
The basic purpose of this project was to visualize with the electron microscope (the EM) the mechanism of entry of the important cell-penetrating therapeutic agents available today, with the hypothesis that this entry is via endosome-rupture after cells have “endocytosed” the agents.
The agents we obtained from different laboratories all totally failed to provide any tools that would be visible by electron microscopy, or potent enough to penetrate cells and thus be used for drug delivery.
As a last resort, we finally fell back on simply trying to achieve just one of the desired characteristics of drug delivery agents. Among the different endosome-disrupting peptides we sought to characterize, we chose the HA2 peptide, derived from Influenza virus, and the GALA peptide, a de novo-designed peptide. However, such endosome disrupting peptides are of small molecular weight, and thus are totally invisible in the electron microscope. Therefore, in order to study their mechanism of action, we had to combine the use of these peptides with probes that we could actually see by electron microscopy, and then follow the uptake of these 'markers' at different stages of the internalization-mechanism.
We did all of this by intensive use of classical thin section electron microscopy, as well as by our own very special and unique procedures for quick-freeze, freeze-etch electron microscopy. Sadly, we utterly failed to achieve any success in these efforts, and learned absolutely nothing about how or why or when any of these cell-penetrating peptides ultimately reach the cytoplasm.
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