2013 Fiscal Year Annual Research Report
フリーズエッチ電顕法による薬剤・遺伝子導入用ナノ粒子の細胞内取込み機構の解明
| Project/Area Number |
25253004
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (A)
|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
HEUSER John 京都大学, 物質-細胞統合システム拠点, 教授 (40571815)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
諸根 信弘 京都大学, 物質-細胞統合システム拠点, 講師 (50399680)
村上 達也 京都大学, 物質ー細胞統合システム拠点, 助教 (90410737)
|
|---|
| Project Period (FY) |
2013-05-31 – 2016-03-31
|
| Keywords | electron microscopy / drug delivery / endocytosis |
|---|
| Research Abstract |
The basic purpose of this project is to visualize with the electron microscope (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. We believe that visualizing endosome rupture is terribly important for understanding why these agents are effective and for pointing the way for the development of future improved therapeutics. This is a vital medical goal because it represents the bottleneck between further industrial production of more modern therapeutics, and future clinical application of these new agents.
Maximizing this effectiveness can only be achieved by understanding their cellular mechanisms of uptake, which can only be achieved by directly observing the uptake in the EM. The work in this laboratory is therefore dedicated to this fundamental purpose.
The goal of the initial grant period was to establish the methodology that would be used to observe therapeutic endocytosis in the EM. This task was made more difficult because we could not get any agents that were both (1) visible in the EM and (2) potent enough to enter cells. Moreover, all the agents were either overtly toxic to cells or else they entered the cells very poorly. This failure involved our having to test a wide range of potential therapeutics -everything from metal zeolites to organic proteolipid complexes- and each test required its own EM.
|
| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The agents obtained from different laboratories failed to provide a tool visible by electron microscopy or potent enough to penetrate cells and thus be used for drug delivery. Therefore, we set out on our own to develop our own uptake complex, one that we knew would be visible in the electron microscope and that seemed likely it would serve as a good platform for the same sorts of derivatization that have been used for the standard therapeutics of today. Again, we came up short, because despite its proving to be readily visible in the electron microscope, it proved to be exceedingly difficult to derivatize without running into all sorts of problems like simple old precipitation and loss of the material.
Short of our goal of finding a good therapeutic to study, and hamstrung in our development of our own endocytic uptake particle, we turned back to basics to ask whether the sort of changes in endosomes that people predicted to occur when these agents were applied could even be seen properly in the electron microscope, at all.
The classical example is the polycationic polymer polyethyleneimine -PEI-, which is supposed to break endosomes via what it is called the “proton sponge” mechanism.
This mechanism is based on the supposition that agents like PEI must absorb protons when they find themselves in the endosome acidic environment. This absorption causes them to swell; swelling that ultimately causes the endosome to rupture.
We have unfortunately proven that when these agents are protonated they do not swell and therefore this mechanism must be totally impossible.
|
| Strategy for Future Research Activity |
Haven failed to obtain a drug delivery material good enough to enter cells and be seen by electron microscopy from collaborators, we will struggle to produce this material by ourselves, as well as characterize the mechanism of cellular internalization by electron microscopy.
In order to achieve one of the desired characteristics of drug delivery agents, being able to reach the cytoplasm, we will start working with endosome disrupting peptides. These kinds of peptides are well known for inducing endosome damage and endosome release. At neutral pH these peptides are in a random coil structure, but upon acidification in the endosome they adopt an α-helix structure that is able to insert in the endosome membrane and damage it enough to produce the desired endosomal escape.
Among the different endosome disrupting peptides we are planning to use HA2 peptide, derived from Influenza virus, and GALA peptide, a de novo designed peptide.
However, endosome disrupting peptides are of small molecular weight and thus invisible in electron microscopy. Therefore, we will combine the use of these peptides with probes that can be easily seen by electron microscopy (hemocyanin protein, quantum dots, gold nanoparticles), and follow the uptake at different stages of the internalization mechanism, to ultimately elucidate when and how do they reach the cytoplasm. This will be done by the classical thin section electron microscopy technique combined with freeze fracture electron microscopy.
|
