U.S. patent application number 10/836773 was filed with the patent office on 2005-01-27 for site-specific cell perforation technique.
Invention is credited to Karube, Isao, Saitoh, Takashi.
Application Number | 20050019922 10/836773 |
Document ID | / |
Family ID | 13711081 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050019922 |
Kind Code |
A1 |
Karube, Isao ; et
al. |
January 27, 2005 |
Site-specific cell perforation technique
Abstract
A technique for controlling membrane denaturation reactions
other than physical shear force was developed. For example, the
present invention provides, a method for causing membrane
disruption at a specific site by reacting a stimulus such as light
with a compound that is activated by the stimulus, where the
reaction occurs on a membrane such as a biomembrane. It also
provides a membrane structure such as cells in which a specific
site has been disrupted, which are obtained by the present method.
Introduction of substances such as genes also became possible by
using this membrane structure. Further provided is a
membrane-destroying member for disrupting a membrane at a specific
site. Thus, the present invention enabled, for example, easy
membrane penetration using components constituting microelectrodes,
micromanipulators, and microinjectors, which were conventionally
hardly usable in penetrating cell membranes.
Inventors: |
Karube, Isao; (Kanagawa,
JP) ; Saitoh, Takashi; (Tokyo, JP) |
Correspondence
Address: |
Michael L. Goldman
Nixon Peabody LLP
Clinton Square
P.O. Box 31051
Rochester
NY
14603-1051
US
|
Family ID: |
13711081 |
Appl. No.: |
10/836773 |
Filed: |
April 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10836773 |
Apr 30, 2004 |
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09623970 |
Dec 28, 2000 |
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6753171 |
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09623970 |
Dec 28, 2000 |
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PCT/JP99/01223 |
Mar 12, 1999 |
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Current U.S.
Class: |
435/446 ;
435/488 |
Current CPC
Class: |
C12N 13/00 20130101;
C12N 15/89 20130101; C12M 35/00 20130101 |
Class at
Publication: |
435/446 ;
435/488 |
International
Class: |
C12N 015/01; C12N
015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 1998 |
JP |
10/80177 |
Claims
What is claimed is:
1. A method of denaturing or perforating a specific site of a
membrane, the method comprising contacting the whole or part of the
membrane with a reagent containing a specific compound that induces
a membrane-denaturing reaction by a specific stimulation, and
giving said stimulation.
2. The method of claim 1, wherein the membrane is a cell membrane,
a cell wall, a biomembrane, or an artificial membrane.
3. The method of claim 1 or 2, wherein the region stimulated is,
included in the region contacted with the reagent.
4. The method of claim 1 or 2, wherein the region contacted with
the reagent is included in the region stimulated.
5. The method of claim 4, wherein the reagent is contacted using a
support.
6. A method of any one of claim 1 to 5, wherein the specific
stimulation is light, and the compound is a photosensitizing
compound.
7. A membrane obtained by the method of any one of claims 1 to 6,
wherein the specific site has been perforated or denatured, or a
membrane structure containing said membrane.
8. The membrane or membrane structure containing said membrane of
claim 7, wherein the membrane is a cell membrane, a biomembrane, or
an artificial membrane.
9. The membrane structure of claim 7 or 8, which is a cell, a
micelle, or a liposome.
10. A method of injecting a compound into a structure, the method
comprising mixing the structure of any one of claims 7 to 9 with a
complex comprising a compound to be injected and a carrier.
11. The method of 10, wherein the carrier is a liquid or solid.
12. A method of claim 10 or 11, wherein the substance is a nucleic
acid or protein.
13. A membrane-destroying member for denaturing or perforating a
specific site of a membrane, which comprises a support, and a
membrane denaturation promoting portion comprising a membrane
denaturation force other than physical shear force, which was
formed on at least one site on the surface of the support.
14. The membrane-destroying member of claim 13, wherein the support
is rod-shaped, tube-shaped, needle-shaped, or spherical.
15. The membrane-destroying member of claim 13 or 14, wherein the
membrane denaturation promoting portion is coated or fixed with a
compound that generates the membrane denaturation reaction at the
membrane denaturation promoting portion.
16. The membrane-destroying member of claim 15, which utilizes a
sequential peroxidation reaction of the membrane components, in
which the membrane denaturation reaction is started by a
direct/indirect production reaction of reactive oxygen species.
17. The membrane-destroying member of claim 15 or 16, wherein the
membrane denaturation reaction is induced by a specific stimulation
and reaction precursor, and includes the reaction that denatures or
perforates the membrane.
18. The membrane-destroying member of claim 17, wherein the
specific stimulation is light, and the reaction precursor is a
photosensitizing compound.
19. The membrane-destroying member of any one of claims 13 to 18,
wherein the support penetrates the membrane following membrane
denaturation or disruption, and said penetrated membrane closely
contacts with said support.
20. The membrane-destroying member of any one of claims 13 to 19,
wherein the membrane is a cell membrane, a biomembrane or an
artificial membrane.
Description
TECHNICAL FIELD
[0001] 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
[0002] 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.
[0003] Conventional substance introduction techniques can be
roughly categorized as follows:
[0004] a) Introduction techniques targeting non-specific cell
groups
[0005] b) Introduction techniques targeting a specific cell
[0006] 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)).
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] Other than the above examples, the possibility of
technological developments that owe to cell modifications is on the
increase. A few typical examples are,
[0013] 1) To produce cloned animals, it is necessary to inject the
nucleus or chromosomal genes via the egg cell membrane, but the
success rate of this procedure, is extremely low.
[0014] 2) If 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.
[0015] 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.
[0016] 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.
[0017] 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
[0018] 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.
[0019] 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.
[0020] 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.
[0021] Enzymatic disruption using lipases and proteases, and
methods using .beta.-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.
[0022] 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 .alpha.-tocopherql (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)).
[0023] When such sequential oxidations exceed the oxidation defense
capacity, phospholipid membrane disruption rapid progresses in an
exponential manner, and the cell becomes unable to maintain
metabolism as the membrane loses its permeation inhibiting
capacity. If these sequential reactions progress further, the cell
will ultimately perish.
[0024] Photosensitizers (PS) are molecules that trigger such lipid
sequential peroxidation reactions by producing active oxygen using
light. Rose Bengal, porphyrin, and such can be given as
photosensitizers in general use.
[0025] By utilizing such photosensitizers as membrane-denaturing
agents, when denaturing the membrane, it will be sufficient to
conduct sequential peroxidation reactions partially on the minimum
objective cell surface, for a short period of time. Furthermore,
the membrane damaged by the sequential peroxidation reactions at
the time of membrane perforation, is expected to be repaired after
the perforation by the fluidity of the membrane itself, or by the
aforementioned anti-oxidation systems.
[0026] The inventors coated terthiophene
(5'5"-bis(aminomethyl)-2,2':5',2"- -terthiophene dihydrochloride),
a type of photosensitizer, on the surface membrane of the nerve
cell line PC12. Photosensitizers are membrane-denaturing agents
that could be controlled by light exposure. By measuring membrane
resistance, the inventors revealed that membrane resistance,
namely, the ion permeability of the membrane increases due to the
action of the photosensitizer, which is activated by exposing the
whole cell to laser beams. Furthermore, by controlling the amount
of beams and amount of photosensitizer, the change in membrane
resistance caused by laser beam exposure could be regulated in at
least three ways
[0027] 1) No change in membrane resistance
[0028] 2). The resistance decreases and then recovers
[0029] 3) The resistance is lost
[0030] Noteworthy is the fact that the inventors found that the
duration it takes for the membrane permeability to recover to the
state prior to disruption, was around 30 sec at suitable
conditions.
[0031] Also, a similar change in membrane resistance was observed
when only the axon of the cell was exposed to laser beams.
[0032] To examine whether membrane denaturation by photosensitizers
could be applied for introducing substances into cells, the
inventors attempted the adaptation into microinjection treatment.
At the time of microinjection treatment, an injection solution
containing Lucifer Yellow CH (LY), a water-soluble fluorescent dye,
was prepared, and the ability to inject LY into PC12 cells was used
as the determining indicator of the success of the injection. The
injection was also automated by using an electric manipulator to
exclude, as much as possible, man-induced influences on the
evaluation of the success rate.
[0033] Using such an injection treatment system, the inventors
evaluated how the success rate of the injection varied according to
the presence or absence of the photosensitizer, terthiophene
(5'5"-bis(aminomethyl)-2,2':- 5',2"-terthiophene dihydrochloride,
100 .mu.M) within the injection solution, and according to the
presence or absence of a two-minute exposure treatment by a 100 W
mercury lamp was done, was measured.
[0034] As a result, when light exposure was done using a
photosensitizer-containing injection solution, the success rate of
the injection turned out to be 80%. Other controls resulted in an
approximately 0 to 10% success rate. Therefore, by utilizing
membrane denaturation, a significant improvement in the success
rate of the injection was accomplished.
[0035] Furthermore the inventors compared the cell survival rate
between photosensitizer-treatment and normal treatments taking the
LY retention rate of cells following injection treatment as the
indicator of cell survival. 90% of the photosensitizer-treated
cells survived for three to six days, whereas normally treated
cells had a survival rate of only 10%, the survival rate of
photosensitizer-treated cells being significantly high.
[0036] These results show that compared to injection techniques
using physical shear force, those using membrane denaturation are
clearly superior as means that suppress damage to cells.
[0037] The above results showed that suitable membrane perforation
is possible by combining a photosensitizer and light. Namely,
conditions in which the cell membrane is repaired without the cell
being perished can be easily discovered. Naturally, it goes without
saying that by preparing a membrane-destroying member by coating a
membrane-disrupting material such as a photosensitizer on a
support, the membrane could be contacted with this
membrane-destroying member easily.
[0038] For example, this membrane-destroying member could be
contacted with cells by utilizing a floating membrane-destroying
member, for example microbeads coated with a membrane-denaturing
agent, and contacting this membrane-destroying member using laser
tweezers. Under such contact conditions, it is also possible to
start the membrane denaturation reaction and then introduce this
membrane-destroying member into cells. The membrane-denaturing
agent itself may be a membrane structure, such as a micelle.
[0039] Furthermore, the inventors converted the scanning probe of
the atomic force microscope, into an electrode, and coated the
probe tip with the photosensitizer
5'5"-bis(aminomethyl)-2,2':5',2"-terthiophene dihydrochloride to
create a novel component. When this iphotosensitizer-coated
component is inserted into the cell membrane, resistancecaused by
the cell membrane could be observed between the electrode within
and without the membrane. Since the physical shear force of the
electrodes of the atomic force microscope is not intense enough to
cause cell perforation, the newly prepared component showed that it
could be used as a component that is equipped with both the
electrode function of the atomic force microscope and the
controllable membrane-disrupting function. Also, by coating or
fixing photosensitizing compounds onto already existing numerous
components, it is possible to give these components the
controllable membrane-disrupting function in addition to their
original functions.
[0040] Namely, the present invention features:
[0041] (1) a method of denaturing or perforating a specific site of
a membrane the method comprising contacting the whole or part of
the membrane with a reagent containing a specific compound that
induces a membrane-denaturing reaction by a specific stimulation,
and giving said stimulation;
[0042] (2) the method of (1), wherein the membrane is a cell
membrane, a cell wall, a biomembrane, or an artificial
membrane;
[0043] (3) the method of (1) or (2), wherein the region stimulated
is included in the region contacted with the reagent;
[0044] (4) the method of (1) or (2), wherein the region contacted
with the reagent is included in the region stimulated;
[0045] (5) the method of (4), wherein the reagent is contacted
using a support;
[0046] (6) a method of any one of (1) to (5), wherein the specific
stimulation is light, and the compound is a photosensitizing
compound;
[0047] (7) a membrane obtained by the method of any one of (1) to
(6), wherein the specific site has been perforated or denatured, or
a membrane structure containing said membrane;
[0048] (8) the membrane or membrane structure containing said
membrane of, (7), wherein the membrane is a cell membrane, a
biomembrane, or an artificial membrane;
[0049] (9) the membrane structure of (7) or (8), which is a cell, a
micelle, or a liposome;
[0050] (10) a, method of injecting a compound into a structure, the
method comprising mixing the structure of any one of (7) to (9)
with a complex comprising a compound to be injected and a
carrier;
[0051] (11) the method of (10), wherein the carrier is a liquid or
solid;
[0052] (12) a method of (10) or (11), wherein the substance is a
nucleic acid or protein;
[0053] (13) a membrane-destroying member for denaturing or
perforating a specific site of a membrane, which comprises a
support, and a membrane denaturation promoting portion comprising a
membrane denaturation force other than physical shear force, which
was formed on at least one site on the surface of the support;
[0054] (14) the membrane-destroying member of (13), wherein the
support is rod-shaped, tube-shaped, needle-shaped, or
spherical;
[0055] (15) the membrane-destroying member of (13) or (14), wherein
the membrane denaturation promoting portion is coated or fixed with
a compound that generates the membrane denaturation reaction at the
membrane denaturation promoting portion;
[0056] (16) the membrane-destroying member of (15), which utilizes
a sequential peroxidation reaction of the membrane components, in
which the membrane denaturation reaction is started by a
direct/indirect production reaction of reactive oxygen species;
[0057] (17) the membrane-destroying member of (15) or (16), wherein
the membrane denaturation reaction is induced by a specific
stimulation and reaction precursor, and includes the reaction which
denatures or perforates the membrane;
[0058] (18) the membrane-destroying member of (17), wherein the
specific stimulation is light, and the reaction precursor is a
photosensitizing compound;
[0059] (19) the membrane-destroying member of any one of (13) to
(18), wherein the support penetrates the membrane following
membrane denaturation or disruption, and said penetrated membrane
closely contacts with said support;
[0060] (20) the membrane-destroying member of any one of (13) to
(19), wherein the membrane is a cell membrane, a biomembrane or an
artificial membrane.
[0061] As to the combination of the compound and stimulation used
for denaturing or perforating the membrane, any combination may be
used, as long as it can perforate the membrane in a controllable
manner, without completely destroying the membrane. As to the
stimulations used, electromagnetic waves including light, particle
rays including radiation, heat, cooling, electricity, magnetism,
oscillations including ultrasonic waves, physical contact, chemical
substances, as well as, living beings in general including cells,
viruses, and such can be given as examples. These stimulations may
be used alone or together with others.
[0062] As compounds that are used to denature and perforate the
membrane, enzymes involved in membrane denaturation and disruption,
antibody molecules, membrane bound proteins, glycoproteins, lipids,
and such may be used. Photosensitizers such as porphyrin, rose
bengal, methylene blue, acid red, alpha-terthienyl, etc., or their
derivatives may also be used. Oxidants such as reactive oxygen
species, reductants, explosive compounds such as
nitro-glycerin/picric acid, magnetic particulates/magnetic fluids,
metal particles/conductor particles/insulator
particles/photoelectric converting elements/piezoelectric elements,
and such-may also be suitably used. These compounds may be used
alone or together with others.
[0063] The membrane to be denatured or perforated, may be a
membrane containing photoelectric converting elements and
piezoelectric elements, or may be cell membrane or cell wall of
animals/plants, biomembranes, or artificial membranes. As a
biomembrane, a cellular coat including cell wall, cellular inner
membrane including cell membrane, nucleus membrane, viral membrane,
cytoplasmic micro tubule, microsome membrane, golgi apparatus
membrane, lysosome membrane, endoplasmicreticulum membrane,
tonoplastmembrane, plastidmembrane, peroxysome membrane, ribosome
membrane, mitochondrial membrane, and such can be given. These may
be combined to make a reconstituted membrane. Examples of
artificial membranes are, protein membranes, lipid membranes, high
molecular (collagen, etc.) membranes, metal membranes, conductor
membranes, mitochondrial membranes, insulator membranes, electric
conductive high molecular (such as polyacetylene, polythiophene)
membranes, etc.
[0064] As membrane disruption methods the following are
provided.
[0065] First is the method, wherein a compound is contacted with
the cell membrane, and a part of the contact region is stimulated
to denature or perforate only a range smaller than the region where
the compound and the membrane contacted. For example, by exposing
light through a micro alit against cells treated with a
water-soluble) photosensitizing compound solution, only the cell
membrane portion that was exposed can be denatured, or
perforated.
[0066] Second is the method, wherein a compound is contacted with a
part of the cell membrane, and a region larger than the contact
region that is stimulated to denature or perforate only the region
where the compound and the membrane contacted. For example, by
coating a part of a micro support obtained by processing silicon
crystals, contacting the region coated with the photosensitizing
compound with the cell surface under a microscope, and stimulating
with light, will cause membrane disruption only to the region
contacted with the support.
[0067] Examples of supports having membrane-destroying member as
constitutive elements are, crystals, macro compounds such as
C.sub.60, micro pipettes, glass micro electrodes, patch electrodes,
metal micro electrodes, wires, whisker, living organisms including
cells, magnetic particulates/magnetic fluids, metal
particles/conductor particles/insulator particles/photoelectric
converting elements/piezoelectric elements, micro structures such
as micromachines, as well as objects in which these are
conjugated.
[0068] Light is well used as the specific stimulation,
corresponding compounds being photosensitizing compounds. Dyes can
be generally used as photosensitizing compounds. Among dyes,
porphyrin, rose bengal, methylene blue, acid red, alpha-terthienyl,
etc., or their derivatives are well used.
[0069] By suitably using the above methods, a membrane in which a
portion is denatured or perforated can be provided. Also, as a
membrane structure containing a membrane that has been denatured or
perforated, a membrane which could be fixed on crystal oscillators
and electrode basel plates, and in which the resonance
frequency/fluidity/adsorption and such physical features could be
modified, or a membrane in which the permeability/permeable site of
a substance could be regulated by contacting with a gas/liquid, and
such membranes can be given. Also, it may be a membrane in which
the cell membrane or cell wall of animals/plants, biomembranes, or
artificial membranes has been membrane denatured/perforated. As a
biomembrane, a cellular coat including cell wall, cellular inner
membrane including cell membrane, nucleus membrane, viral membrane,
cytoplasmic micro tubule, microsome membrane, golgi apparatus
membrane, lysosome membrane, endoplasmic reticulum membrane,
tonoplast membrane, peroxysome membrane, plastid membrane, ribosome
membrane, mitochondrial membrane, and such can be given. These may
be combined to make a reconstituted membrane. Examples of
artificial membranes are, membranes containing a magnetic structure
at a high density, protein membranes, lipid membranes, high
molecular (such as collagen) membranes, metal membranes, conductor
membranes, insulator membranes, electric conductive high molecular
(polyacetylene, polythiophene, etc.) membranes, etc. As structures
containing membranes subjected to denaturation and perforation,
cells and micelles having a specific number of holes can be given.
As cells, animal cells, plant cells, microbial cells, reproductive
cells, somatic cells, and such can be given.
[0070] Compounds that are to be injected can be substances that
permeate the membrane easily by normal diffusion, or substances
that are to be passed through the membrane artificially in large
amounts, specifically, nucleic acids, proteins, lipids, membrane
structures etc.
[0071] Carriers are gases, liquids, or solids that can be used to
dissolve, or suspend the substance to be injected, examples being
buffers in which nucleic acids are dissolved, etc.
[0072] The form of membrane-destroying member may be any used
according to the objective, as long as it can carry out
controllable disruption of the membrane. The membrane-destroying
member contains a support and a membrane denaturation-accelerating
site, and this membrane denaturation-accelerating site may be on
the support surface according to the purpose, or may be a part of
the surface. It may be kenzan (needlepoint flower holder)-shaped,
spherical, needle-shaped, rod-shaped, tube-shaped, or may be
provided in a combined shape of these. Examples of tube-shaped
supports are, specifically, pipettes, tubes, injection needles,
etc. Spherical supports could be beads that could be handled by the
laser tweezer technique.
[0073] When coating or fixing a compound that generates a membrane
denaturation reaction in a support constituting the
membrane-destroying member, the methods that could be used are,
solvent evaporation drying, spattering, vacuum deposition, plasma
polymerization, chemisorption, physisorption, radical
polymerization, ion polymerization, etc. The support and the
compound may indeed be indirectly bound via a mediator.
[0074] When the membrane denaturation reaction utilizes a
sequential peroxidation reaction of the membrane components that is
started by a direct/indirect production reaction of reactive oxygen
species, it is possible to initiate the production reaction by
suitably supplying light energy, electrical energy, chemical
energy, etc. Specifically, light is electromagnetic waves from the
deep ultra-violet region to the deep infrared region with a
wavelength of about 180 nm. Light due to laser oscillations may
also be used. The above-described photosensitizing compounds may be
used in this case as well.
[0075] When membrane denaturation or disruption is generated using
membrane-denaturing component, there are cases where close contact
of the penetrated membrane and the membrane-denaturing component or
a support constituting the membrane-denaturing component is
suitable. Namely, in the case of a membrane-denaturing component
that is tube-shaped and is connected to a pump, and to which
manipulations such as microinjection and micromanipulation could be
done, it is suitable that the penetrated membrane and the
membrane-denaturing component or a support constituting the
membrane-denaturing component are in close contact at the time of
substance transport into/out of the cell.
[0076] Membrane-denaturing component controllable by location
controlling apparatus are also well used in cell treatments, etc.
Examples of location controlling apparatus are, scanning probe
microscopes of atomic force microscopes and such, laser tweezers,
micromanipulators, etc. Specifically, as a support constituting the
membrane-denaturing component, atomic force microscope's scanning
probe, or the proximal light scanning microscope's scanning probe,
and such could be used.
[0077] The present invention can be utilized as a technology in
various fields by suitable simple-and-clear applications carried
out by one skilled in the art. One example is indicated below.
[0078] First, as the membrane manipulation, membrane
disruption/denaturation and other manipulations could be carried
out on an objective membrane by ligating or using together with the
membrane-denaturing or disrupting agent, a manipulator comprising
the function of manipulating an object having a membrane such as a
cells/viruses, and a binder having the ability to bind/contact the
target membrane. The manipulator, binder, and the membrane
denaturing or disrupting agent may be used alone, or one of these
could serve as the other two. In this case, the target membrane is
a liposome/cell membrane/intracellular organelle membrane/viral
membrane, and such, and the binder is a polyclonal
antibody/monoclonal antibody/metal beads/plastic
beads/virus/cell/living organism, etc. Utilized environment may be
within the atmosphere, within a liquid, within the body, etc.
Examples of cell/virus manipulations are,
denaturation/disruption/growth acceleration or
suppression/transformation/cell death induction/division or fusion
acceleration/agglutination or dissociation acceleration/substance
uptake or excretion acceleration, etc.
[0079] The invention may also be applied as substance
introduction/excretion techniques due to the temporary perforation
of the cell membrane. For example, in the creation of clone
animals, and gene therapy, the introduction of genes into cells is
an important manipulation.
[0080] The invention may also be applied for cell fusion. It could
be done by using the membrane denaturation reaction of the present
invention, which utilizes chemical substances such as polyethylene
glycol, and viruses such as Sendai virus, for the cell fusion
treatment.
[0081] Furthermore, it is possible to create a battery using the
cell membrane potential caused by electrodes inserted into the
cell. A major problem carried by micromachines and internal
therapeutic instruments, is the securing of a power source for
operation. By using membrane-disrupting material and electrodes,
the potential that is within and without the cell membrane could be
used as the power supply, and it will be possible to construct a
system having suitable cells as batteries. The present invention
could also be applied to create ultra microsurgery tools for
cellular levels, such as cell scalpels. Though there are various
dissecting instruments such as various types of scalpels, even if
they are very small instruments, they cannot go beyond dissecting
tissues. Dissecting instruments usable beyond the level of cells
did not exist until now, but the membrane disruption technique of
the present invention could be applied for dissecting cell
membranes and intracellular membranes such as nucleus membrane.
[0082] The present invention could also be provided as a disruption
technique that could be used site-specifically for
micelles/liposomes containing medications for drug delivery systems
(DDS) used in gene therapy. Namely, DDS research for using the
system intensively around the target affected area, while
suppressing side effects of the medications are being carried out,
and by applying the present invention for disrupting microcapsules
containing medications, an effective DDS could be accomplished.
[0083] The present invention could also be applied in manipulations
of cellular organelles. Namely, the above-described
micromanipulators and microinjectors could be used to easily
manipulate cellular organelles such as lysosomes and the nucleus,
to enable effective cell treatments. Specifically, egg cell
manipulations and such (clone creation, etc.) in reproductive
engineering could be given as an example.
[0084] The present invention could also be used for novel function
expression and arrangement of functional molecules targeting
flat/spherical membranes. In biomembranes, membrane proteins having
various functions drift within the membrane while exerting their
diverse functions, such as metabolism of chemical
substances/transmission of electrons within the membrane surface
and within and without the membrane, carrying out the functions
alone or in suitable fissions-fusions. Such functional expressions
by combinations of migratory functional units within the membrane
surface and outside the membrane, are exactly what could be called
micro chemical plants. Using such functional expressions of
biomembranes--the scene of reaction--as a model, if biological
functional molecules, and artificial objects such as piezoelectric
elements, photoelectric converting elements, and memory elements
would also become incorporatable as membrane devices by controlling
the intake of various functional units into artificial membranes
and biomembranes using the membrane disruption/denaturation
technique, it will be possible to construct reaction systems with
an extremely high degree of freedom.
[0085] Furthermore, cellular functions could be expanded by
membrane manipulations and addition of devices. Namely, by using
the membrane perforation technique of the present invention, novel
functions could be given to already existing cells, by fusing a
cell and an artificial functioning body. For example, a leukocyte
could be magnetically guided to an affected area by creating a
leukocyte in which a micro magnetosome particle deriving from
magnetic bacteria has been inserted. This is a novel technique that
could be called, not only a drug delivery system, but also a cell
delivery system.
[0086] Also, by incorporating a photoelectric converting element
into a membrane, it is possible to furnish cells with a function
that is specific to plant cells, which is the ability to convert
the energy of light into a chemical energy.
[0087] Furthermore, by applying a photoelectric converting element
incorporated in the cell membrane for the input/output of nerve
cell signals, it is possible to connect nerve cells directly with a
photo information-processing computer.
[0088] Use of micromachines for therapeutic purposes is drawing
great interest. Since such micromachines need to be small enough to
enter even blood vessels, the energy source becomes restricted.
Namely, energy supply via wires is difficult, and on the other
hand, energy that could be mounted on the micromachine is highly
trivial. Therefore, it is clear that an output provided by physical
means by a built-in energy source would be overwhelmingly
insufficient to cause influences such as cell disruption and cell
denaturation when using micromachines. It could be said that
membrane-disrupting agents and activation of these are
indispensable in cell treatment. As an example, by using a
photosensitizer that is activated by infrared light such as
porphyrin as the membrane disruptor, the energy needed for membrane
disruption could be supplied from outside the body by infrared
lasers, etc.
[0089] By connecting an electrode to each nerve, the present
invention could also be applied to create a nerve interface that
exchanges information between nerves and electronic information
instruments. Neural information is normally transmitted by the
alterations in the cell membrane potential, namely, by
action-potential. To generate and measure this action potential,
various analyses are being carried out regarding nerve-electronic
instrument interfaces that input/output neural information, termed
neural interfaces, however the distance between the electrodes and
cells, and the degree of accumulation have become drawbacks.
Although glass microelectrodes used for fundamental research
purposes can be inserted into the cell membrane, or adsorped onto
it to enable direct measurement/manipulation of cell membrane
potential, high density accumulation was impossible since these
electrodes are made by heat-processing glass tubes with diameters
of only a few millimeters. On the other hand, metal microelectrodes
that could be easily accumulated by semiconductor processing
techniques, carry problems in cell membrane penetration ability,
and stimulation/measurement have been done extracellularly, which
is inefficient and also carries problems in site-specificity. By
combining the membrane perforation technique of the present
invention and metal electrode technique, it is possible to
perforate the cell membrane, and set up micro metal electrodes
within the cell. This will create an ideal, neural interface where
a cell and electrode could be connected one-to-one, or where
several electrodes could be connected to a single cell.
[0090] The present invention could also be applied for the
elucidation of cellular functions of the body such as for
fundamental research of the brain. Namely, for brain function
analysis, it is vital to analyze reciprocal information exchange
between nerve cells. What is done at present is loading membrane
potential sensitive dyes into nerves, and simultaneously measuring
multi points of neural activity by optical measurement of the
alteration of membrane potential as alterations in
absorbance/fluorescence. However, in this case, it is impossible to
optically carry out inputs into nerves, and to provide
inputs/outputs to nerves, there is no alternative but to rely on
electrical means, namely, electrodes. Analysis of the neural
information processing mechanism by culturing nerve cells upon
basal plate electrodes to form an artificial network, and research
of applying nerve cells themselves as computing elements are also
being carried out. Even in this case, signal inputting into nerves
has become the stumbling block. Electrodes upon basal plates are
extracellular electrodes, and in order to give nerve cells a
stimulation to reach action potential generating threshold, the
situation was such that an action potential could be finally
generated as a total sum of stimulations received by each nerve
following stimulation of nerve cell population upon the basel
plate. The keys in assembling electrodes for exchanging information
with each nerve are, downsizing electrodes into the cellular level,
and means for connecting electrodes to each cell. This objective
could be fulfilled by inserting intracellular electrodes obtained
by combining micro metal electrodes downsized by semiconductor
processing techniques, and the membrane perforation technique.
[0091] The present invention could also be utilized for functional
electrical stimulation, and such, and for enhancing the
accumulation/precision of stress-measuring therapeutic electrodes.
As a part of rehabilitation medicine, a method called functional
electrical stimulation is being used, in which metal electrodes are
inserted into nerve bundles and electrical stimulation is given to
improve nerve/muscle functions. At present, the method has not gone
beyond stopped at setting up electrodes in a few places within the
nerve bundle, and is insufficient in the aspects of site
specificity/precision of the nerve stimulation. By conjugating the
membrane perforation technique and already existing electrode
accumulation technique, one-to-one joining of a nerve and electrode
will become possible, and thereby, stimulating only the nerve that
needs functional improvement could be accomplished.
[0092] The present invention could also be applied as a technique
for transmitting signals from nerves to various artificial organs.
Organs embedded within the body are controlled, not by direct
neural information of the body, but strictly by indirect control.
One example is artificial urethral valve control. These valves that
are made by shape-memory metals, open by heating and close at
normal body temperature. This problem is, that the closing and
opening is controlled by an external heating device switch and
cannot be directly controlled by the will of the patient. If
stabilization/enhancement of precision of neural information
becomes possible by neural interfaces using membrane-penetrating
electrodes, these valves could be controlled as if they were a part
of the body of the user. It is true that artificial urethral valves
are used only a few times throughout the day, and the handling, the
opening/closing, is also easy, and therefore, not much
inconveniences are caused by switch manipulations outside the body
in daily life. However, to control artificial organs that carry out
more complicated acts in place of visceral functions, it is
indispensable to make the control signal source be from autonomous
nerves.
[0093] The present invention could also be used for
connecting/controlling prosthetic hand/legs equipped with sensory
organs and joints that are controllable similarly to the human
body. At present, there is a remarkable enhancement in performance
of externally powered prosthetic hands/legs for functional
assistance following dismembering. However, the controlling
information source utilizes myoelectric power left in the wearer in
most cases, and the power is overwhelmingly short compared to the
information amount originally needed to control the limbs. Also,
the transmitting of sensation through the prosthetic hand/legs to
the wearer is limited to just physical contact information via the
area connected to prosthetic limb. Therefore, even if the handling
of the prosthetic hands/legs is learned by training, the reality is
that the limbs are being used while enduring major inconveniences.
One of the reasons impeding the enhancement of features of
prosthetic limbs, is the fact that the information exchange
pathways between the wearer and the prosthetic limb are poor. If
stabile/highly precise measurement of each neural information
becomes possible by neural interfaces using membrane penetrating
electrodes, by joining the interface to the nerve that was
connected to the wearer's amputated limb, this nerve can be used as
the controlling information source, and sensory signal input
terminal. Namely, it will be possible to control a prosthetic limb
equipped with a movement performance/sensory organ similar to the
body.
[0094] The present invention could also be utilized for controlling
an artificial sensory organ (visual/auditory) and connecting it to
the body. Artificial inner ears for repairing auditory functions
already exist. This substitutes the functions of the ear drum-inner
ear through a microphone and signal converting circuit, and several
tens' of electrodes are set up in the cochlear organs, auditory
nerves are stimulated electrically, and the auditory information is
sent to the brain. The biggest reason why this instrument
established itself as a therapeutic instrument is in the cochlear
organs, in which auditory nerves are aligned according to frequency
bands. Being a special case, information can be easily sent to the
nerves from the electrodes in this instrument, but it is impossible
to apply this electrode technique of the artificial inner ear to
other organs. One of the technical targets is an electrode system
sufficient to connect to the optic nerve, which contains one
million neural axons per a 1 cm diameter, as the optic nerve is
indispensable in repairing functions of the sense of sight, which
among sensory organs is the most important information source of
the body. Although such a highly dense, accumulative nerve
electrode does not exist at present, the neural interface using
membrane-penetrating electrodes will enable a highly dense
accumulation able to respond to the optical nerve. Already, optical
products using the charge-coupled device (CCD) has enabled one
million picture elements at a low price, and using such photo
information inputting instruments as functional artificial eyes, it
will be possible to provide the human body with a sense of sight
via the neural interface.
[0095] The present invention may also be used to expand brain
functions. Although the brain, is energy-saving, has advantages
such as parallel processing, and such, as an information processing
device compared to semiconductor micro processors, it also carries
weaknesses such as, the imprecision of information retention,
difficulty of learning, etc. In order to overcome these weaknesses
while taking use of the advantages of the brain, it is also
possible to use together the brain and already existing
semiconductor devices via the neural interface.
BRIEF DESCRIPTION OF THE DRAWINGS
[0096] FIG. 1 shows the structural formula of the
5'5"-bis(aminomethyl)-2,- 2':5',2"-terthiophene (BAT)
dihydrochloride.
[0097] FIG. 2 shows the flow chart of membrane perforation
technique.
[0098] FIG. 3 shows an example of the relationship between a
membrane-destroying member having a pipe-shaped support, and a
membrane structure that is treated by the membrane-destroying
member.
[0099] FIG. 4 shows an example of the relationship between a
membrane-destroying member having a spherical or bead-shaped
support and a membrane structure that is treated by the
membrane-destroying member.
[0100] FIG. 5 shows an example of the relationship between a
membrane-destroying member having a rod-shaped support, and a
membrane structure that is treated by the membrane-destroying
member.
[0101] FIG. 6 shows an example of the relationship between a
pipe-shaped membrane-destroying member having a support maintaining
a liquid containing a membrane denaturation promoter, and a
membrane structure that is treated by the membrane-destroying
member.
[0102] FIG. 7 shows an example of the relationship between a
membrane-destroying member having an atomic force microscope
probe-shaped support, and a membrane structure that is treated by
the membrane-destroying member.
[0103] FIG. 8 is a schematic diagram showing an instrument for
measuring intracellular potential and membrane resistance using a
patch electrode.
[0104] FIG. 9 indicates the relationship between the intensity of
the light stimulation and alteration in cell membrane potential or
alteration in membrane resistance.
[0105] FIG. 10 shows the flow chart of an example of the
photosensitizing injection.
[0106] FIG. 11 shows a schematic diagram of an example of the
photosensitizing injection.
[0107] FIG. 12 shows a comparison of the success rate of the
injection by a normal physical injection treatment, and the novel
photosensitizing injection treatment.
[0108] FIG. 13 shows a comparison of the alteration in cell
survival rate by normal physical injection treatment, and the novel
photosensitizing injection treatment.
MODE FOR CARRYING OUT THE INVENTION
[0109] The invention shall be described with reference to examples
below, but it is not to be construed as being limited thereto.
EXAMPLE 1
Culturing Nerve Cell Line PC-12
[0110] Cells of the established nerve cell line PC 12 used as a
model of the central nervous system, are ganglia-like cells of
adrenal medulla origin. These PC 12 cells were cultured using a
NeuroBasal Medium (GIBCO BRL) (pH 7.3) containing 10%
heat-inactivated horse serum, 5% bovine fetal serum, 7.35 mg/l
L-glutamic acid, and 2 mM L-glutamine, under 95% CO.sub.2.
[0111] Passaging was done by detaching cells from the walls of the
culture flask by spraying culture medium onto cells, collecting
cells by centrifuging at 300 g, for 5 min, inoculating 1 to
3.times.10.sup.4 cells/cm per 1 ml into a culture flask (IWAKI
Glass) with a bottom surface area of 25 cm.sup.2, and changing the
medium every two to three days.
[0112] When differentiating PC 12 cells into nerve-like cells, 2.5S
of the mouse nerve growth factor (NGFY was added to the medium to a
final concentration of 50 ng/ml. The method of preparing the NGF
(Murine, 2.5S) dispersed solution for adding into the medium is as
follows.
[0113] 1) Phosphate buffered saline (PBS; composition being,
KH.sub.2PO.sub.4 2.10 g/l, NaCl 90.00 g/l, NaHPO.sub.4.7H.sub.2O
7.26 g/l, pH adjusted to 7.4 by adding 1N NaOH) was prepared.
[0114] 2) 2 mg of Bovine Serum Albumin (BSA) was dispersed in 1000
.mu.l of the above-described PBS (pH 7.4), and the dispersed
solution was sterilized by filtering through a 0.22 .mu.m
pore-sized filter.
[0115] 3) The total volume was made into 200 .mu.l by adding 100
.mu.l of this sterilized solution to 100 .mu.l NGF solution (GIBCO
BRL), 8 .mu.l of this solution was divided into mini-tubes, and
cryopreserved at -20.degree. C.
[0116] PC 12 cells were differentiated by adding the divided NGF
solutions so as to dilute the solution 1000 times.
[0117] PC12 adhered weakly onto the walls of the plastic bottle,
and grew while forming small clusters. Collagen dishes (IWAKI
Glass) were used to culture the neuralyzed cells.
[0118] For the electrophysiological experiment described below,
cells differentiated into nerve-like cells 6 days before were
used.
EXAMPLE 2
Synthesis of 5'5"-bis(aminomethyl)-2,2':5',2"-terthiophene
[0119] The photosensitizer used was
5'5"-bis(aminomethyl)-2,2':5',2"-terth- iophene (BAT), a derivative
of .alpha.-terthienyl. This compound was synthesized according to
"Muguruma et al., J. Heterocyclic Chem., 33, 1-6 (1996)", and was
provided in the state of BAT dihydrochloride. The structure of BAT
dihydrochloride is shown in FIG. 1.
[0120] The thiophene oligomer having amino-methyl residue at the
terminal, has a high solubility compared with other derivatives of
the same type because of this amino-methyl residue. Solubility
changes with the dissociation state of this amino-methyl residue.
On the other hand, in the case of the BAT of the present invention,
it has a bivalent positive charge within acidic aqueous solutions
and dissolves easily. In aqueous solutions near a pH region
suitable for the body (around 7.4), it has a feature of having both
positively-charged monovalent BAT maintaining a high solubility,
and non-charged BAT that agglutinates easily into a colloid state.
Under this pH condition, by perfusing cells within a BAT-dispersed
solution, this molecule can easily attach onto the cell surface.
The hydrophilicity of BAT molecule is a novel feature that is not
seen in either modified thiophene oligomers starting with
.alpha.-terthienyl, or in other molecules designed as conductive
high molecular monomers.
EXAMPLE 3
The Measurement of Membrane Resistance and Membrane Potential
Following Light Exposure
[0121] Since there is a need to monitor cellular level micro
membrane damages, including the recovering process within seconds
to a few minutes, the intermembrane potential of the cell membrane,
or the ionic current passing through the cell membrane was measured
using the patch clamp method, which is an electrophysiology
experiment technique.
[0122] The photosensitizer BAT was dispersed in HEPES (25 mM,
pH7.4) buffer. The BAT dispersed solution to be added topically
near the cells by a micropipette had a BAT concentration of 2 mM,
and the dispersed solution to be added throughout the perfusate had
a BAT concentration of 0.2 mM.
[0123] Cells were incubated at room temperature within an
electrophysiology experimental culture medium.
[0124] The culture medium used for this experiment had a
composition of NaCl, 124 mM; KCl, 5 mM; CaCl.sub.2.2H.sub.2O, 2.4
mM; MgSO.sub.4.7H.sub.2O, 1.3 mM; glucose 10 mM, in which the pH
was adjusted to 1.4 by finally adding NaOH. To prevent any
influence due to evaporation, the electrophysiology experiment
culture medium was changed every 40 min at the latest by a
pipette.
[0125] BAT was added to a final concentration of 49 .mu.M. Exposed
light quantity was in three stages, being 0.47 J/cm.sup.2, 0.94
J/cm.sup.2, and 1.57 J/cm.sup.2.
[0126] The solution within the patch electrodes had the composition
of KCl 132 mm, NaCl, 0.8 mM, MgCl.sub.2, 2 mM, HEPES 30 mM,
Na.sub.2ATP 4 mM, GTP 0.3 mM, and EGTA 6.5 mM, in which the pH was
adjusted to 7.3 by finally, adding NaOH.
[0127] As the excitation power source, 50 mw, 363 nm argon ion
laser standardly equipped within a confocal laser scanning
microscope (CLSM) MRC-1000 UV (BIO-RAD laboratories) was used.
{fraction (1/16)}.sup.th (1/4.sup.th to the direction of the X axis
and Y axis, 117 .mu.m.times.170 .mu.m) of the full screen of the
microscope is scanned with the above-described laser beams.
Adjustments were done so that the whole image of the patched target
cell came into this area. 50 mW laser beam is a 100% output.
According to the scanning speed, the exposed duration was selected
from {fraction (1/16)}, 1/4, {fraction (1/32)} sec. The light
reduction by the lens was also utilized. When the zoom function was
used, the light is scanned on a concentrated area smaller than the
normal area, and therefore, the light quantity per unit area
increases in proportion to the square of the zoom magnification.
Since the excitation light has to pass through the plastic of the
collagen-coated dish before reaching the cells, there is a
necessity to consider light reduction.
[0128] Also, in the excitation light exposure, a TTL signal coupled
with an electrophysiological recording is suitably transmitted to
the excitation light source, to synchronize the exposure and
electrical measurement.
[0129] At the start of the electrdphysiological experiment, the
cell membrane potential was maintained between -80 to -60 mV. The
resistance of the patch electrode was 3.about.4 M.OMEGA., and the
above described solution was filled into electrodes, and used.
[0130] As for the amplifier for cell membrane potential
measurement, Axopatch 1-D (Axon Instruments) was used. The membrane
resistance was calculated by the change in membrane potential when
a 1 Hz rectangular hyperpolarizing current was passed through for
350 mili sec. The amount of current passed through (0.1 or 00.15
nA) was selected so that the change in membrane potential due to
the passing of current did not exceed 30 mV. Under these experiment
conditions, PC12 cells did not generate action potential. The
measured potential/current value was analyzed by Axoscope ver.1.1
software (Axon Instruments). The result is shown in FIG. 9.
[0131] In FIG. 9, the horizontal axis shows time lapse (unit;sec).
In the vertical axis, the top box shows the cell membrane potential
(unit; %) when normalization was done taking the value prior to
exposure as 100%, and the bottom box shows cell membrane potential
(unit; MV). Membrane resistance reflects membrane permeation
inhibiting capacity, and membrane potential reflects the active ion
transport capacity due to the functions of various intermembrane
ion transportation systems and ion permeation inhibiting
capacity.
[0132] For the light quantity of 1.57 J/cm.sup.2, A was the weakest
(0.47 J/cm.sup.2), B was the second (0.94 J/cm.sup.2), and C was
the strongest (1.57 J/cm.sup.2). In A, although cell membrane
resistance/membrane potential changes a little following light
exposure, a big change was not seen. In B, a decrease in cell
membrane resistance/depolarization of the membrane potential
occurred after a lag period of a few seconds. Also, under these
conditions, it was seen that the resistance/potential recovered to
the levels prior to light exposure after 30 sec. This is thought to
be due to the membrane being repaired by vital reactions following
a temporary disruption. In C, cell membrane resistance/membrane
potential both disappeared in 8 sec after a few second lag period
following light exposure, and was constant thereafter. This is
thought to be due to a lack of a repairing reaction following a
temporary disruption of the membrane, namely due to an irreparable
membrane disruption.
EXAMPLE 4
The Treatment of Spraying BAT into Target Cells by a Micro Glass
Pipette
[0133] A microinjector was connected to a micro glass pipette held
by a micromanipulator, and BAT dispersed solution (BAT
concentration was 2 mM, aqueous solvent) was filled into this micro
glass pipette. The tip of this glass pipette was connected to a
patch electrode and setup within 200 .mu.m proximity of the cells
in which the membrane potential/membrane resistance was to be
measured. BAT dispersed solution was released by compressing the
micro glass pipette to attach BAT onto the cell membrane of target
cells. When laser beams were applied similar to Example 3,
depolarization of the cell membrane potential was observed.
EXAMPLE 5
Microinjection Treatment Using Site-Specific Membrane
Disruption
[0134] Introduction of substances using membrane disruption was
applied for microinjection treatment.
[0135] To determine the success or failure of microinjection
treatment, Lucifer Yellow CH(LY), a water-soluble fluorescent dye,
was added to the injection solution. After injection treatment, if
a yellow fluorescence of LY origin was observed within the cell by
a fluorescent microscope, then, the injection treatment was judged
to have been successful. The variation of the success rate of cell
injection was evaluated by the presence or absence of the
photosensitizer BAT within the injection solution, and the presence
or absence of a BAT excitation light exposure.
[0136] Since LY is a low-toxic fluorescent dye used in
microinjections, it has a feature of transmigrating into daughter
cells at the time of cell division (Cell & Tissue Res., 234,
309-318 (1983)). Also, since LY is highly soluble in water, and is
superior in diffusion, it is also used as a fluorescent labeling
agent for nerve cells (Cell & Tissue Res., 254, 561-571(1988)).
It also has a feature of rapidly (in a matter of minutes)
transmigrating between cells ligated by a gap junction, an
intercellular liquid-liquid junction (SHIN-SEIRIGAKU TAIKEI 7
development/differentiati- on physiology, chapter 4, intercellular
ligations I. Electrical bonds, IGAKU SHOIN, 1991). It was
reportedly used as a similar gap junction formation marker in the
PC12 cells utilized in the present invention (J. Neurosci., 14,
3945-3957 (1994)). Although LY-injected cells undergo cell death
against an excessive LY excitation light exposure (Science, 206,
702-704 (1979)), the variation of the success rate of cell
injection was evaluated by the presence or absence of BAT within
the injection solution, and the presence or absence of a. BAT
excitation light exposure.
[0137] Normally, to make microinjection successful, a glass
capillary having an opening with a diameter of a few hundred
nanometers should be contacted with cells at high speed, to
instantly penetrate/perforate the cell membrane or the nucleus
membrane physically. For the experiments, a program operative
electric micromanipulator (Eppendorf, Micromanipulator 5171) and
electric injector (Eppendorf, Transjector 5246) were used, and it
was possible to set the capillary contact speed to an arbitrary
value. For the injection capillary, a mass-produced commercially
available product made for the apparatus was used (Eppendorf,
FemtoTips). Since the injection was automated by the electric
manipulator, which resulted in a good reproducibility, and since a
commercially available capillary with a highly conformed shape was
used, compared to self-made capillaries, statistical processing of
the success and efficiency of the injection could be easily done.
The micromanipulator was attached to a fluorescence microscope
(OLYMPUS OPTICAL, IX70 fluorescence microscope spec). Light of a
100 W argon lamp, which is the epi-illumination fluorescence light
source equipped in the microscope, was treated by transmitting
through an ultraviolet ray excitation filter set (OLYMPUS OPTICAL,
U-MWU mirror unit), and the resulting ultraviolet light was used as
the excitation source of the photosensitizer. The light exposing
area was set to an approximately 100 .mu.m diameter, according to
the aperture of the fluorescence optical system of the microscope.
Light of the 100 W argon lamp was treated by transmitting through
an ultraviolet ray excitation filter set (OLYMPUS OPTICAL, U-MWU
mirror unit), and the resulting ultraviolet light was used also as
the LY excitation source for determining that LY has been injected
into cells.
[0138] In such a system, the photosensitizer BAT is concentrated in
the 0.5 .mu.m diameter region in contact with the capillary, and as
for the other areas, BAT was diluted rapidly by diffusion.
Therefore, in the capillary contact region, when considering the
BAT photosensitizing action produced by 100 .mu.M BAT in the
injection solution, BAT concentration at the areas other than the
capillary contact region is thought to be negligible.
[0139] At the time of injection, after setting the operation range
of the micromanipulator so as to allow the capillary to pierce
cells at a contact speed 1000 .mu.m s.sup.-1, the capillary is
operated at a low speed of 7 .mu.m s.sup.-1, and contacted with
cells in such a manner that cell membrane is not perforated
physically. Under these conditions, the change in the success rate
of the cell injection by the photosensitizing action of BAT was
compared.
[0140] The photosensitizer BAT used was that synthesized/provided
by Muguruma et al. Commercially available products were used for
the following reagents.,
[0141] Sodium chloride (NaCl, Kishida Kagaku), potassium chloride
(KCl, Kishida Kagaku), disodium hydrogenphosphate
(Na.sub.2HPO.sub.4, Wako Pure Chemicals), potassium
dihydrogenphosphate (KH.sub.2PO.sub.4, Wako Pure Chemicals),
fluorescent marker Lucifer Yellow CH, Lithium salt (LY, ex.428 nm,
em.536 nm. Molecular Probes).
[0142] The injection solution was prepared by dissolving the
following compositions in purified water to the indicated final
concentration (photosensitizer BAT 100 .mu.M, HCl 50 .mu.M,
fluorescent marker LY 2 mM, NaCl 8 g/l, KCl 0.2 g/l,
Na.sub.2HPO.sub.4 1.15 g/l, KH.sub.2PO.sub.4 0.2 g/l.
[0143] A control injection solution not containing BAT was also
prepared.
[0144] As the target cells for the injection, established nerve
cell line PC12 was used. This cell line was obtained from the
Institute of Physical and Chemical Research (RIKEN) Cell Bank and
was cultured according to Example 1. Cells to be injection treated
were passaged to a .phi.35 mm collagen coated dish (Iwaki Glass) at
a cell density of 90000 cells/dish. The cells were incubated in a
CO.sub.2 incubator (Form a Scientific), at 37.degree. C., 5% CO,
95% air, and 100% humidity.
[0145] At the time of injection treatment, a Hib-A culture medium
made by supplementing Hibernate A Media (Hib-A, Gibco BRL)
[NeurOReport, 7, 1509-1512 (1996)] with 10% horse serum (Gibco
BRL), 5% bovine fetal serum (Mitsubishi Chemicals, Dialyzed Fetal
Calf Serum obtained from Nakashibetsu Calf), 7.35 mg/l L-glutamic
acid (Gibco BRL), and 2 mM L-glutamine,) was used. The horse serum
used had been heat-inactivated at 56.degree. C. for 30 min.
[0146] For the above solution preparation, water purified by the
water purification apparatus Biocel A10/Elix 10 (MILLIPORE) was
used.
[0147] FIG. 10 shows the flow chart of injection'treatment
steps.
[0148] Below is the relevant supplementary information.
[0149] 1) At the time of injection treatment, all 3 ml of the
NeuroBasal medium was removed from dish in which the cells were
cultured, and the cells on the dish were washed by running 1 ml of
phosphate buffered saline (PBS, 7.4, containing neither Ca nor Mg)
(GIBCO BRL) over them. Then, all the PBS was removed, washed again
with 1 ml of fresh PBS, then, all of this PBS was removed, and
finally, the dish was filled with 2 ml of Hib-A culture medium for
injection treatment. The cells were maintained in this Hib-A
culture medium.
[0150] 2) As for the micromanipulator used, in order to enable the
capillary tip to pierce cells at the time of injection treatment,
it was necessary to set the threshold position (Z limit) where the
capillary tip nears most to the face of the dish. The manipulator
was set so that this Z limit position coincided with the position
of the nucleus of cells on the dish.
[0151] 3) The capillary position was altered to 30 .mu.m above Z
limit. The approach speed input value was altered to 700 .mu.m
s.sup.-1 (effective value 1000 .mu.m s.sup.-1), and injection
duration to 1.1 s (effective value 1.0 s) for other conditions as
well. Under these conditions, the Z limit position was adjusted to
enable a physical microinjection treatment with an 80% or more
success rate against 10 or more cells. When the success rate fell,
the Z limit was re-set.
[0152] 4) The capillary position was altered to 10 .mu.m above Z
limit. The approach speed input value was set to 5 .mu.m s.sup.-1
and injection duration was changed to 124 s (effective value 120
s).
[0153] 5) The epi-illumination fluorescence light source of the
microscope was switched to violet excitation (U-MWBV mirror-unit)
filter set suitable for observing LY fluorescence, and the release
of LY from the capillary was observed by the Clear function
(injection solution within the capillary is compressed with a
pressure of 7000 hPa to removeclogging in the capillary). If the
capillary is clogged, a new one was used.
[0154] 6) The injection pressure was set to a pressure sufficient
enough for a slow-release of LY from the capillary tip. The
pressure sufficient enough for a slow-release of LY from the
capillary tip had a wide range of variation from 10 hPa to 1000 hPa
depending on the condition of the capillary tip, and therefore,
suitable pressure corrections were done. The reason for this
variation was the adherence of micro debris such as cell membrane
fragments onto the capillary tip. When such debris adhered onto the
capillary tip, the amount of the effective injection solution
released varied significantly even when an equal pressure was
applied, and hence, corrections had to be done.
[0155] 7) In the visual field of the microscope, the cell position
and capillary position were adjusted so that the capillary tip came
onto the center of the cell. BAT was excited by applying
ultraviolet rays, by switching the epi-illumination fluorescence
light source to ultraviolet excitation (U-MWBV mirror-unit) filter
set. To suppress the influence by light other than ultraviolet rays
for excitation, the permeation light source for cell observation
was shut-off.
[0156] 8) Injection treatment Switch of the manipulator was turned
on. Capillary neared the cells, and after releasing the injection
solution at that location for the set-duration, at the
set-pressure, the capillary moved back to its original position.
This whole process was carried out automatically.
[0157] 9) After completing the injection treatment, the filter of
the epi-illumination fluorescence light source of the microscope
was changed from that for ultra violet rays to that for violet rays
(U-MWBV mirror unit), LY excitation light was applied, and it was
verified that cells had been LY-stained. When injection is done to
dead cells, LY rapidly leaks out of the cell membrane and loses its
fluorescence, and therefore, such cells are excluded from the
data.
[0158] 10) The permeation light source for cell observation is
opened again, and while observing the cells, the capillary tip is
positioned on the next cell.
[0159] 11) Injection treatment is repeated from 5).
[0160] After injection treatment, Hib-A culture medium for
injection treatment is removed from the dish, the dish is washed
twice with 1 ml of PBS, and the culture medium is changed to 3 ml
of NeuroBasal culture medium. Thereafter, the culture medium is
changed according to normal procedure.
[0161] Penicillin/streptomycin mixture was added to the NeuroBasal
culture medium used after the injection treatment to prevent
contamination by fungi and bacteria.
[0162] When evaluating the success rate of microinjection due to
the photosensitizing mechanism, it was necessary to carry out the
injection under conditions in which the physical shear force of the
pipette tip is not involved. To carry out a suitable injection, it
is important to adjust the threshold limit to which the capillary
can reach (Z limit). Therefore, the approach speed was lowered to 7
.mu.m s.sup.-1 after verifying that an injection with a success
rate of 80% or more was possible at the set Z limit, under the
normal approach speed of 1000 .mu.m s.sup.-1. At this approach
speed, although the capillary reaches the same location as that
when the injection succeeded, it hardly penetrates the cell
membrane. Under such approach conditions where membrane perforation
by physical force is difficult, the success rate of injection using
the photosensitizing mechanism was compared. The results are shown
in FIG. 12.
[0163] In FIG. 12, the horizontal axis indicates the injection
treatment conditions, and the vertical axis shows the success rate
of the injection (unit; %).
[0164] When an injection solution using BAT was used, the inventors
succeeded in obtaining an 80% success rate after a 2 min exposure
of ultraviolet rays (succeeded in 25 of the 30 cells, 83%).
[0165] If the exposure was not done when the capillary was in
contact with the cell, there was hardly any diffusion of LY into
the cell (n=30, succeeded in 4 cells, 13%). Also, when an injection
solution not containing BAT was used, the injection rate dropped to
0 to 10% (capillary approach speed 7 .mu.m s.sup.-1) irrespective
of the presence or absence of an UV exposure (under UV exposure,
succeeded in 0 out of 30 cells, 0%) (under no UV exposure,
succeeded in 3 out of 30 cells, 10%)
[0166] The survival rate of cells to which microinjection treatment
by a physical shear force was given, and that for photosensitizing
microinjection treated cells were compared.
[0167] LY was injected into cells by microinjection. In dead cells
in which the cell membrane had collapsed, LY rapidly diffuses, and
staining is lost (Cell, 14, 741-759 (1978)). Therefore, LY
maintaining rate of injection treated cells was used as an
indicator of cell survival rate, and the survival rate after 3 to 6
days following injection treatment was compared. The results are
shown in FIG. 13.
[0168] In FIG. 13, the horizontal axis indicates the number of days
following injection treatment (unit, day), and the vertical axis
shows the survival rate (unit; %).
[0169] Compared with the control where normal LY injection was
done, the survival rate on the 3.sup.rd day following LY-BAT
treatment was 30% or lower (LY:17%, BAT+LY:30%). On the other hand,
in the photosensitizing, injection-treated group, 90% of cells
survived even after three to six days following injection treatment
(IBAT+LY+UV: 91%). For all these three injection conditions, LY
retention rate did not change during the 3.sup.rd to 6.sup.th
days.
[0170] In the present injection treatment, when injection solution
not-containing BAT was used, and when light exposure was not done,
only a 10% of the cells were LY stained. Compared to such control
treatments, when injection solution containing BAT was used, and
light exposure was done, the success rate of LY staining was
significantly high being approximately 80%. This shows that BAT
contributed to cell perforation following light exposure.
[0171] After injection treatment, if it was a normal membrane
shearing injection, the cell survival rate was 20 to 30%. Since
cells treated by a photosensitizing injection had a survival rate
of approximately 90%, it shows how little the cells are damaged by
the photosensitizing injection.
[0172] LY injected into cells rapidly disperses into the cytoplasm
and stains cells. However, in dead cells, in which the ion barrier
ability of the cell membrane is lost, LY diffuse into the exterior
of the cells in one to two seconds following injection. This rapid
dispersion of LY has been reported to occur also through the gap
junction, an intercellular liquid-liquid connection pathway (Cell
& Tissue Res., 234, 0.309-318 (1983)) (J. Neurosci., 14,
3945-3957 (1994)). When considering these reports of rapid LY
dispersion, in cells retaining LY after injection treatment, it is
thought that perforated cell membrane has closed following
injection treatment. Such a rapid membrane repair is also supported
by the recovery of membrane potential/membrane resistance within a
few minutes following light exposure, as shown in Example 3.
[0173] Two types of mechanisms are thought to be involved in this
closing mechanism. One is due to the anti-oxidation mechanism of
cells, by a metabolical repair of the oxidized membrane (J.
Neurochem., 68, 1904-1910 (1997)). The other is not a physiological
repair but the closing mechanism of the damaged site by the:
fluidity of membrane lipids (Proc. Natl. Acad. Sci., 69, 2056-2060
(1972)) (J. Am. Chem. Soc., 94, 4475-4481 (1972)).
[0174] In the present injection treatment system, when the
capillary is parted from the cell, the perforated site of the
membrane is thought to close due to the flowing of cell membrane
components that are unaffected by the photosensitizer. The fact
that a similar cell membrane closing occurs even after a mere
physical cell membrane perforation, can be given as a reason for
the above assumption. Earlier, a physical cell membrane perforation
by micro glass pipette and patch pipette was described. After such
a physical perforation, inmost cases, the perforated part of the
cell membrane closes after the pipette is removed, and such a rapid
re-closure is thought to be due to the fluidity/self-organizability
of membrane lipids (Biomembranes and Bioenergy, Third edition, 7.
The reconstitution of the biomembrane, Tokyo Daigaku Shuppan
(1985)).
[0175] Membrane recovery due to biochemical metabolisms cannot
result in membrane closing in a matter of seconds. Indeed,
biochemical membrane repair may occur, however, such mechanisms may
take a few hours to a few days to normalize an oxidized cell
membrane.
[0176] When considering electrode connection techniques, the fact
that LY had been injected into cells by a photosensitizing
injection, is extremely significant. As mentioned earlier, LY is
able to penetrate and stain not only cells into which LY is
directly injected, but also adjoining cells connected via gap
junctions (Cell & Tissue Res., 234, 309-318 (1983))[J.
Neurosci., 14, 3945-3957 (1994)].
[0177] Intercellular electrical connection is one of the functions
of the gap junction. (SHIN-SEIRIGAKU TAIKEI 7
development/differentiation physiology, chapter 4, intercellular
ligations I. Electrical bonds, IGAKU SHOIN, 1991) in such cells
like cardiomuscular cells, a large number of muscle cells are
electrically connected via gap junctions, and generate a
synchronized contraction of the whole muscular tissue against an
electrical stimulation.
[0178] Since cell-staining by LY was possible by the present BAT
photosensitizing mechanism injection, it is thought that an
electrical connection, at least to the extent of a gap junction,
has been attained between the solution within the capillary and
cytoplasm. Namely, this result suggests that an electrical
connection was: accomplished not by a physical perforation, but by
light exposure.
[0179] By adding the photosensitizer BAT to the injection solution,
and carrying out injection treatment, the possibility of a
light-regulated perforation technique that does not rely on
physical shear force was shown. Furthermore, this technique was
superior to normal microinjections that rely on physical shear
force as it suppresses cell damage given by such injections.
EXAMPLE 6
The Creation of a Membrane-Destroying Member Using the Atomic Force
Microscope Scanning Probe
[0180] The probe-tip (the surface used for detection) of the
commercially available, silicon single crystal scanning probe
processed by etching (Nanosensors, silicon beam silicon single
crystal scanning probe, cantilever 130 .mu.m) was plated with a 220
nm layer of gold (Au) by spattering (Shibaura Seisakujo, spattering
pressure 0.3 Pa, output 100 W). The areas other than the metal
terminal at the detecting side, and the metal terminal at the
instrument connecting side were insulated with a 100 nm layer of
silicon dioxide. This scanning probe was equipped to the atomic
force microscope (Nanoscope III, Digital Instruments), and the
metal terminal at the instrument connecting side was connected to
the minus electrode of an electroporation apparatus (Gene Pulser,
BIO-RAD laboratories). The plus electrode of the electroporation
apparatus was connected to the metal basel plate (copper, platinum,
etc.) on the AFM sample plate, and prior to breakdown, namely, the
probe-tip insulated by silicon dioxide, was connected to the basel
plate. Also, a 3 M.OMEGA. resistance was serially connected between
the scanning probe basel plate and electroporation apparatus, to
prevent destruction of the scanning, probe due to the excessive
current that result after the breakdown of the probe. The electric
capacity of the electroporation apparatus was set to 0.25 .mu.F,
the voltage to 50 V, and current was momentarily passed between the
basel plate and probe-tip of the scanning probe.
[0181] By this electrification, the insulation at the tip of the
probe was destructed to expose the metal at the tip of the probe.
Thus, a micro metal electrode was completed, in which only the
probe-tip of the scanning probe was exposed.
[0182] This probe-tip of the micro metal electrode was immersed in
a BAT 2 mm acidic solution (pH 3.0). By this manipulation, BAT was
adhered onto the electrode. Finally, the electrode was washed with
purified water to remove excessive BAT.
[0183] By the above process, a membrane-destroying member having
both the function of a scanning probe of an atomic force
microscope, and the function of an electrode was created.
[0184] These results showed the possibility of controlling
perforation by light exposure by pinpoint use of photosensitizers
in the connecting/embedding of various devices in small cells.
INDUSTRIAL APPLICABILITY
[0185] A technique controlling membrane denaturation reaction other
than physical shear force was developed. Thereby, it was possible
to conduct membrane denaturation and membrane perforation more
easily than by conventional techniques. For example, easy membrane
penetration became possible where it was conventionally difficult
with micro components constituting microelectrodes,
micromanipulators, microinjectors, etc. Effective introduction of
genes and such into cells also became possible.
* * * * *