Site-specific cell perforation technique

Details Site-specific cell perforation technique Site-specific cell perforation technique

2000-12-28

2004-06-22

Leffers, Gerry (Department: 1636)

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

Cell membrane or cell surface is target

C435S173100, C435S325000, C435S449000, C435S455000, C435S460000

Reexamination Certificate

active

06753171

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a method for perforating a membrane by partially treating the membrane (cell membrane, etc.) with a membrane-denaturing agent, etc. It also relates to a membrane-disrupting material having a membrane-denaturing effect.
BACKGROUND ART
In gene therapy and artificial substance production systems using living organisms, means of introducing a nucleic acid, a protein, and such, into the interior of a cell are extremely important. On the other hand, techniques for extracting structures such as the nucleus of a cell, are also gaining wide attention. In other words, it can be said that injecting and extracting substances into/from cells, the basic unit constituting organisms, is a fundamental technique of bioengineering.
Conventional substance introduction techniques can be roughly categorized as follows:
a) Introduction techniques targeting non-specific cell groups
b) Introduction techniques targeting a specific cell
Examples of technique a) are, those using viral vectors (retroviral vectors, etc.), non-viral vectors (lipofectin, etc.), electroporation, calcium phosphate method, particle-gun method, etc. Technique b) could be exemplified by the microinjection method (“Fundamental techniques of gene therapy”, Youdosha (1995)).
Generally, technique b) is used against large cells, such as egg cells. One reason for this is that the microinjection method uses shear force of a capillary glass tube to disrupt the cell membrane, and therefore, a technical limit probably arises due to the cell size. Also, this method requires skilled experience on the part of the handler, and therefore, automation is difficult. Furthermore, in many cases the pipette cannot be inserted due to the flexibility of the cell membrane of normal cells, except egg cells.
In technique a), a non-specific cell group is randomly treated, and it is hoped that the objective substance will be introduced to a part of the cell group. Therefore, very rarely does the objective substance get introduced into all cells. Also, it is generally difficult to separate only cells into which the objective substance has been introduced.
Moreover, a sophisticated micromanipulator is required to extract structures within the cell without causing them any damage, having drawbacks similar to the above microinjection method.
Thus, at a time when cell treatment has become a routine technique in medicine/engineering, the enhancement of reproducibility/precision of cell treatments is a vital issue. For example, when treating reproductive cells, considering that only an extremely small amount of egg cells can be obtained from each individual animal compared to somatic cells, an egg cell is a valuable genetic resource. Therefore, the fact that the success rate of their treatment largely depends on the technical experience of the technician is a grave problem from an engineering point-of-view.
Furthermore, all the above treatments were limited to single cells, migratory cells, or cells such as cancer cells that could be isolated/re-introduced from/into the body. Therefore, it was extremely difficult to modify treatments so that they could be used for cells that are inseparable from the body, such as nerve cells.
Other than the above examples, the possibility of technological developments that owe to cell modifications is on the increase. A few typical examples are,
1) To produce cloned animals, it is necessary to inject the nucleus as or chromosomal genes via the egg cell membrane, but the success rate of this procedure, is extremely low.
2) It a specific cell into which a magnetic structure has been incorporated can be made, it will be possible to magnetically control the location of the cell. The method of introducing magnetic bacteria-derived magnetosome-formation gene is generally used, and although there are successful examples, in most cases it is suitable to insert artificial magnetic structures when using cells, and such, for medical purposes.
3) When preparing micromachines such as micro surgical instruments, it is easily postulated that a cutting output sufficient for dissecting the cell membrane would not be obtained by mere physical means. Also, membrane disruption by mere chemical reactions would have problems regarding the regulation of the disruption.
4) In measuring action potential/electric stimulation of nerve cells, the measurement/stimulation were both done extracellularly by practical electrodes, except in fundamental research. This was the causative problem impeding the enhancement of precision, since it weakened the detection signal and increased stimulation input compared to the potential threshold value involved in original neuro activities. If it is possible to implant an electrode within nerve cells, not only would a measurement/stimulation equivalent to the original potential threshold value of nerves become possible, it will also enable information exchange between the electrode and nerve with a one-to-one precision.
5) In the field of energy conversion engineering, studies on placing micro photoelectric converting elements comprising photo potential onto artificial membranes are being conducted aiming at artificial photosynthesis. This approach aims to use the electromotive force given by photoelectric converting elements to generate transmembrane potential. If it becomes possible to place such micro photoelectric converting elements within cell membranes/mitochondrial membranes, it will enable the use of light to supply the energy needed for cellular metabolisms. Namely, it might be possible supplement various cells the ability to use photo energy just as the plants do.
DISCLOSURE OF THE INVENTION
A fundamental problem underlying these cell treatments is the lack of techniques that could control the disruption of the cell membrane. Various toxins have been examined for long for the purpose of just disrupting the cell. However, they could not respond to the demands of cytoengineering, which is to partially, and temporarily disrupt the cell membrane without causing cell death. Also there were limitations to the method utilizing physical shear force by using micro glass tubes, etc. The present invention attempts to resolve the issue of developing a technique by which a biomembrane could be perforated using a method other than physical shear force while regulating the disruption of the membrane, namely developing a membrane disruption regulating technique.
Though there are various membrane disruption techniques as mentioned above, those that could site-specifically disrupt a membrane were limited. Microinjectors and micromanipulators cause partial membrane disruption, however, they relied on physical shear force to disrupt and perforate the membrane. Namely, another issue that the present invention attempts to resolve is to develop a component for perforating a biomembrane while regulating the disruption using a method other than physical shear force.
What is needed for perforating a biomembrane while regulating the disruption of a biomembrane, is the regulation of the site and degree of disruption. Therefore, the inventors conducted zealous investigations on which methods would enable the denaturation and perforation of a membrane while regulating the membrane disruption activity.
Enzymatic disruption using lipases and proteases, and methods using &bgr;-rays and laser-beams could be exemplified as methods that partially and temporarily disrupt a membrane. However, the inventors focused their attention on the phospholipid radical sequential peroxidation reaction.
Active oxygen such as singlet oxygen, and superoxide radicals peroxidize unsaturated phospholipids of the cell membrane by sequential reactions. As countermeasures, cells have radical scavengers such as &agr;-tocopherol (vitamin E), and L-ascorbic acid (a water soluble anti-oxidant; vitamin C), superoxide dismutase (SOD) and such oxidation defense mechanisms, to resist oxidation (“Free Radicals in Biology and Medicine”, Oxford university Press (1985)).
When such sequential oxidations exceed the oxidation defense cap

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