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Claims  |
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We claim:
1. A process for preparing a mass of loaded cells suspended in a solution,
which cells by their loading are provided with material intended for
chemical or physical interaction with substances present outside the
cells, comprising the steps of suspending living animal cells selected
from the group consisting of erythrocytes, lymphocytes, thrombocytes and
leucocytes, and having cell membranes, in a cell-compatible solution,
increasing the permeability of the cell membranes by the effect of osmotic
pressure, or by the effect of an electric field, or both, incorporating
loading material selected from the group consisting of medicaments and
radionuclides into the cells by passage of said material from a
cell-compatible solution through the membranes of increased permeability,
restoring the original permeability of the membranes by healing up the
membranes by regeneration effect, then separating the cells from the
solution in which they were suspended and putting them for preservation in
suspension in a physiological solution of the same osmolarity as the
loaded cell content, said process incorporating the improvement consisting
in that:
in the step of incorporating loading material into the cells, a medicament
material having a capability of reacting chemically or physically with
substances in physiological solution outside the cell, is incorporated
which is of a kind which when so incorporated without the incorporation of
any other material would prematurely destroy the cell membranes, and at
least one additional material selected from the group consisting of
blood-compatible sugars and proteins which are capable of providing
hydrogen-bridge-bonding with said medicament material or of entering into
covalent bonds therewith is incorporated along with said medicament
material into said cells to such an extent that the reaction of said
medicament material with the cell membranes is inhibited while its
intended capability for interaction with substances external to the loaded
cells is not thereby impaired, said additional material being added to
said cell-compatible solution used in said incorporating step in such
dosing that after incorporation of said medicament and said additional
material in the loaded cells the interaction of said first material with
the cell membrane is inhibited for a predetermined time.
2. A process for preparing a mass of loaded cells suspended in a solution,
which cells by their loading are provided with material intended for
chemical or physical interaction with substances present outside the
cells, comprising the steps of suspending in a cell-compatible solution
living animal cells selected from the group consisting of erythrocytes,
lymphocytes, thrombocytes and leucocytes, and having cell membranes,
increasing the permeability of the cell membranes by the effect of osmotic
pressure, or by the effect of an electric field, or both, incorporating
loading a medicament material into the cells by passage of said medicament
material from a cell-compatible solution through the membranes of
increased permeability, restoring the original permeability of the
membranes by healing up the membranes by regeneration effect, then
separating the cells from the solution in which they were suspended and
putting them for preservation in suspension in a physiological solution of
the same osmolarity as the loaded cell content, said process incorporating
the improvement consisting in that:
in the step of incorporating material into the cells, a first material is
incorporated which is of a kind which is compatible with the cell
membranes and therefore produces no destruction of the cell membranes,
which first material is selected from the group consisting of medicaments
having a capability of reacting chemically or physically with substances
in physiological solution outside the cell, and there is also incorporated
into the loaded cells, at the time their cell membranes have an increased
permeability, a second material added to the cell-compatible solution
utilized in said step of incorporating material, said second material
being selected from the group consisting of enzymes capable of decomposing
proteins and enzymes capable of decomposing lipids, and having a gradual
destructive effect on the cell membranes and being supplied with such
dosing in said cell-compatible solution, that after the healing up of the
cell membranes and the separation of the loaded cells from the solution
containing materials to be incorporated in the cells, the cell membranes
will be destroyed after a predetermined period of time.
3. A process as defined in claim 1, in which in the step of incorporating
loading material into the cells, a second additional material is provided
in the cell-compatible solution used in said step and is thereby
incorporated in the loaded cells, which second additional material is a
material selected from the group consisting of enzymes capable of
decomposing proteins and enzymes capable of decomposing lipids, and having
a gradual destructive effect on the cell membrane, said additional
material being provided in said physiological cell-compatible solution in
such dosing, that after the healing up of the cell membrane and the
separation of the loaded cells from the solution used in the
material-incorporating step, the cell membranes of the loaded cells will
be destroyed in a predetermined period of time.
4. A mass of loaded cells suspended in a physiological solution containing
a first material extraneous to the original cell content which is a
medicament capable of chemical or physical interaction with substances
contained in physiological solution external to the cells and which has a
destructive effect on the membrane of the cells, and containing also a
second material selected from the group consisting of blood-compatible
sugars and proteins which are capable of forming hydrogen bonds or
covalent bonds with said first material and thereby inhibiting said
destructive effect on the cell membranes for at least a predetermined
period of time.
5. A mass of loaded cells suspended in a physiological solution in which
the cells contain a first material selected from the group consisting of
medicaments having a capability of reacting chemically or physically with
substances in physiological solution external to the cells and also a
second material selected from the group consisting of enzymes capable of
decomposing proteins and enzymes capable of decomposing lipids, and having
a gradual destructive effect on the cell membranes, said second material
being present in such relative quantity that the cell membranes are not
destroyed thereby until a predetermined time after the preparation of the
said suspension of loaded cells in said physiological solution.
6. A mass of loaded cells suspended in a physiological solution as defined
in claim 5, in which said loaded cells contain a third material selected
from the group consisting of blood-compatible sugars and proteins which
are capable of forming hydrogen-bridge-bonds or covalent bonds with said
second material and thereby inhibiting the destructive effect of said
second material on the membranes of the cells for a predetermined period
of time. |
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Claims |
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Description |
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This invention relates to a process of preparation of a physiological
solution containing a mass of loaded cells in suspension, such cells
having a concentration of material loaded therein designed for chemical or
physical interaction with substances located outside of the cell membrane.
In such a process, the permeability of living animal cells of the kind
having a cell membrane suspended in a cell-compatible solution is
increased by the effect of osmotic pressure or by the effect of an
electric field. In such a process, also, the interaction material or
materials are drawn out of the surrounding cell-compatible solution by
permeation through the cell membranes of which the permeability has been
increased, by simultaneous exchange with the cell content in the interior
of the cells to be loaded, and then the material or materials in question
are locked in the cells by regeneration of the cells that heals the
changes produced in the cell membrane by the effect of osmotic pressure or
by the effect of an electric field, after which the loaded cells are
separated from the cell-compatible solution containing the loading
material and are suspended for preservation and storage in a physiological
solution having an osmolarity that corresponds to the osmolarity of the
content of the loaded cells. The term "osmolarity" refers to a
concentration of particles of molecular size in terms of the osmotic
pressure, so that the preceding statement refers to a solution in which
osmosis will not occur during storage.
Processes of the above kind for preparation of a suspension of loaded cells
in a physiological solution are known from German Pat. No. 23 26 244 and
from German published patent applications (OS) 23 26 161 and 24 95 119.
The patent just mentioned relates to processes for incorporating of
complex-forming materials in loaded cells obtained from living cells of
living organisms. The processes disclosed in German (OS) No. 23 26 161
concern the incorporation of catalytically active materials such as
enzymes or pharmaceutics in loaded cells. Both of the processes just
mentioned seek to increase the permeability of the cell membrane by the
action of osmotic pressure on the membrane. In the process disclosed in
German (OS) 24 05 119, on the other hand, the increase of the permeability
is obtained by the effect of an electric field.
In addition to the designation "loaded cells" which is intended to express
that the cells are "loaded" with materials that are distinct from the
normal cell content, other terms have been used for the products of the
known processes of the above-described kind, such as "membrane vesicles",
"ghost cells" and "membrane envelopes".
As cells for the known processes for preparing a mass of suspended loaded
cells, there are used both cells that occur as individual cells in a
physiological solution, as for example erythrocytes, lymphocytes,
thrombocytes or leukocytes and also cells, such as for example liver
cells, that are organized in tissues as associations of cells clinging one
to another. The cell binding of a tissue is releasable by biochemical or
biophysical procedures, so that in this fashion also a suspension of cells
in a solution can be obtained.
In the performance of the known processes, particularly for incorporation
of extraneous materials in the loaded cells, a specific property of the
membrane of living cells is used, namely that a permeability increase
produced within certain limits can be reduced back to normal healing
through regeneration of the cells. The healed membrane of the loaded cells
thus regains the semipermeable properties of the membrane of the original
cells. Apart from the cases in which materials are used which destroy the
membrane after their incorporation in the loaded cells and are thereby set
free, it is then possible to bring the material incorporated in the loaded
cells into interaction with substances present in a physiolgical solution
outside of the loaded cells without the material incorporated and locked
in the loaded cells getting into the physiological solution. This takes
place when the loaded cells are immersed into the physiological solution
containing the substances in question and the substance, by permeation,
gets through the semipermeable membranes of the loaded cells. It is thus,
for example, possible, with the enzyme invertase locked in loaded cells,
to convert cane sugar (sucrose) into glucose and fructose, since cane
sugar as well as glucose and fructose get through the membrane, while the
enzyme invertase remains locked in the loaded cells. It is also possible,
for example, to load cells with urease and then to inject the loaded cells
thus produced into the blood vessels of a human body without releasing the
urease out of the loaded cells into the blood, and thereby to break down
urea contained in the blood which penetrates into the loaded cells.
It has been found, however, that a great many of the materials incorporated
and locked in loaded cells enter into interaction with the cell membrane
and this interaction has the result of destroying the cell membrane. The
loaded cells produced according to the known methods make no provision for
any restraint or other influence on the progress of the destruction of the
cell membrane in the manner just mentioned and for that reason, loaded
cells prepared according to the known processes, in the cases in which the
cell membrane is attacked, are either used only for a very limited time
that can be very short according to the nature of the interaction with the
cell membrane, or else such loaded cells are regarded as not useable at
all.
THE PRESENT INVENTION
It is an object of the present invention to provide a process for preparing
a mass of loaded cells suspended in a physiological solution of the
above-described type with which it is possible to lock in the loading
material in the loaded cells for a considerable time exceeding a
predetermined minimum for a predetermined time.
Briefly, when the loading material, if loaded alone in the loaded cells,
would lead to premature destruction of the cell membrane, additional
material for loading in the interior of the cells having raised cell
membrane permeability is provided in the solution of said loading
material, which additional materials form hydrogen bonds or covalent bonds
with the loading material that is provided for interaction with substances
that may be present outside the loaded cells. The result is that the
loading materials are unable to react with the membrane, while their
intended effect is nevertheless not impaired. The dosing in the
physiological solution provided for material exchange is such that after
the incorporation of the main loading material and the additional material
in the loaded cells, the interaction of the materials with the cell
membrane is hindered for a predetermined time.
In application of the process of the invention, to give an example, the
material methotrexate, that is used for treating tumors but nevertheless
attacks the cell membrane, is incorporated in loaded cells together with a
protein, for example albumin, or with a sugar, for example sucrose or a
similar polysaccharide, with the result that the membranes of the loaded
cells remain stable about twice as long as would be the case without the
addition of protein or sugar. By use of the process according to the
invention, therefore, the range of application of the known processes are
extended in a highly advantageous manner.
In the case of loading materials that are compatible with the cell membrane
and therefore cause no destruction of the cell membrane, by another aspect
of the invention dealing with the same problem of providing the
preservability of loaded cells for a predetermined period, there is
loaded, through the cell membranes when they have increased permeability,
along with the aforesaid loading material, a material that does have a
destructive effect on the cell membrane which is introduced in the
physiological solution provided for the material exchange in such dosing
that, after the healing of the cell membrane and the separation of the
loaded cells from the solution containing the loading materials, the cell
membranes of the loaded cells will be destroyed after a predetermined
time.
If for example a mass of membrane vesicles is produced with erythrocytes as
a starting cell material, in accordance with the invention, in which the
enzyme arginase that is used in enzyme deficiency diseases, together with
a predetermined dose of a material having a destructive effect on the cell
membranes, as for example proteolytic enzymes and substances producing the
breakdown of lipids (pronase, phospholiphase, trypsin), then after
injecting the cells so loaded into the blood circulation system of an
animal body, the medicament is set free in the body after a predetermined
time and thus brought into action. It is of course possible to inject into
a blood vessel a mixture of loaded cells with different doses of the cell
membrane destroying agent and in that way to control in a predetermined
manner the progress of the liberation of the medicament in the body.
The applicability of a mass of loaded cells prepared by the process of the
present invention extends thus far beyond the field of application of the
loaded cells prepared according to the heretofore known processes. The
loaded cells produced by the processes according to the invention can,
according to their particular purpose, be introduced either as
semipermeable holders for materials designed for interaction with
substances present outside the cell membrane or, on the other hand, as
carriers for materials that will be set free from their contained
condition after a predetermined time. A fully new field is thus opened up,
particularly for medical application.
It has been found particularly useful to introduce a cell-membrane
destroying agent into the cell-compatible solution provided for material
exchange through the membrane in such dosing, that after the healing of
the cell membranes and the separation of the loaded cells, the cell
membranes of the loaded cells are destroyed after a predetermined time,
taking account of the addition to the loading materials of materials that
inhibit the destruction of the cell membranes by formation of hydrogen
bonds (sometimes referred to as "hydrogen bridges") or by formation of
covalent bonds, and not relying on the loading materials designed to
interact with substances in the solution outside the cell membranes for
the membrane-destructive effect. In that way it is possible to reduce to a
negligible contribution the undesired effect of a loading material
provided merely for the interaction with substances that may be present
outside the cell membranes, for example the agent 6-fluoro-uracil
sometimes used for chemotherapy against cancer, and to provide the
intended destruction of the cell membranes solely by the effect of the
agent provided particularly for that purpose, whereby a better time
control of the desired destruction of the cell membranes is brought about.
In the performance of the process according to the invention, the
permeability increase of the cell membranes can be produced either by the
effect of osmotic pressure or by the effect of an electric field, as may
be desired according to the requirements of the particular case.
For the case that the teaching provided in the above-cited German patent
and German OS No. 23 26 191 regarding permeability increase by the
operation of osmotic pressure, the process of the present invention can be
carried out as follows:
The cells provided for the preparation of the mass of loaded cells are
first put into a cell-compatible solution that, for example, can be an
aqueous solution containing at least 0.5 mM per liter of magnesium and/or
calcium ions as well as potassium ions, the solution having an osmolarity
that is so low compared with the osmolarity of the cell content that, as
the result of the osmotic pressure thereby produced in the cells, the
permeability of the cell membranes is increased--without however
destroying the membranes.
Erythrocytes are used for preparation of the loaded cells. The osmolarity
difference to be provided amounts approximately to a factor of 15. If the
cell-compatible solution does not already contain the materials to be
loaded into the cells, this material should then at this point be added.
Furthermore, the materials to be included in the cells in accordance with
the present invention are also introduced into the cell-compatible
solution in the appropriate dosing. After the material exchange between
the materials present in the cell-compatible solution and the cell
contents through the cell membranes now having an increased permeability,
and the content of the thus produced loaded cells practically corresponds
to that of the cell-compatible solution, as a next step, the osmolarity of
the cell-compatible solution is increased to that of the original cell
content by the addition of the osmotically active materials, such as
calcium, potassium and sodium ions. By osmotically active materials there
are here understood materials that have a reflection coefficient of about
0.8, but, however, because they are in general contained in a
cell-compatible solution, build up a sufficiently high osmotic pressure.
After a dwell time, during which the cell membranes heal up, the loaded
cells so formed are separated from the cell-compatible solution and the
mass of loaded cells thus produced is poured into an isotonic
physiological liquid. When erythrocytes are used, it is practical, for
healing away the changes of the cell membranes produced by permeability
increase, to let the cells stand for about five minutes at 0.degree. C.
and then to warm them up to body temperature for about 30 to 60 minutes.
For the case in which the teaching of German OS No. 24 05 119 regarding
permeability increase by the effect of an electric field is to be used,
the performance of the method of the present invention is carried out as
follows:
The cells provided for the preparation of the mass of loaded cells are put
into an electrically conducting liquid forming a cell-compatible
electrolyte solution which is preferably at a temperature lying between
0.degree. C. and 25.degree. C. As a next step, the electrolyte solution
containing the cells is subjected to an electric field having a strength
from 10.sup.3 to 10.sup.5 V/cm until the permeability of the cell
membranes is increased to such an extent that molecules with a radius of
at least 0.5 nm can pass through the cell membranes. For this purpose, it
is convenient and practical to pass the electrolyte solution through a
focus of an electric field. The resulting permeability increase can be
recognized, for example in the application of the process to erythrocytes,
by the discoloration of the electrolyte liquid as the result of the
hemoglobin going out of the cell interiors and by the decoloration of the
erythrocytes. In the case in which the materials and substances that are
to be incorporated in the loaded cells are already in the cell-compatible
electrolyte solution, the material exchange takes place right after the
permeability increase. It is however also possible, after the permeability
increase and still before the performance of the healing of the cell
membranes, to put the cells into a cell-compatible solution of which the
osmolarity corresponds to the osmolarity of the cell content of the
original cells. In this cell-compatible solution, in which are contained
the materials to be loaded into the cells, the material exchange between
these materials and the cell content then takes place. After a dwell time
in which the cell membranes heal, the loaded cells thus formed are
separated from the cell-compatible solution and the mass of loaded cells
thus prepared is then poured into an isotonic physiological solution for
preservation and storage. When erythrocytes are used, it is practical to
prepare the loaded cells in a potassium chloride solution and then to
transfer the loaded cells into an isotonic sodium chloride solution that
corresponds to blood serum in its ion concentration and osmolarity.
EXAMPLE I
Erythrocytes, obtained from citrated blood by new stages of centrifuging
are suspended in a solution in the ratio of one part by volume of
erythrocytes to ten parts by volume of the solution, the solution
containing:
105 mM KCl; 20 mM NaCl; 4 mM MgCl.sub.2 ; 7,6 mM Na.sub.2 HPO.sub.4 ; 2.4
mM NaH.sub.2 PO.sub.4 and 10 mM glucose.
The pH value of the solution was 7.2
10 ml of the suspension so produced was exposed to an electrical field
strength of 12 kV/cm at 0.degree. C. in an apparatus suitable for the
purpose for 40usec. About one minute after the application of the
electrical field that was followed with hemolysis, 5 mM per liter of
methotrexate that had been marked with tritium and 0.1% by volume of
albumin was added to the solution. After the hemolysis, that lasted about
five minutes, the solution was held for another five minutes at 0.degree.
C. in order that an equilibrium could be reached between the cell
interiors and the external solution that contained the methotrexate. As a
next step, the temperature of the solution was raised to 37.degree. C., in
order to accelerate the healing-up of the changes produced by the electric
field in the membranes. The healing-up process was terminated after about
twenty minutes. The loaded cells were then centrifuged out for ten minutes
under an accelerative force 10,000 times the value of the acceleration of
gravity, and the sediment of loaded cells thus obtained was suspended in a
physiological solution that has the following composition:
138.6 mM NaCl; 12.3 mM Na.sub.2 HPO.sub.4 ; 2.7 mM NaH.sub.2 PO.sub.4.
The pH value of the solution was 7.4 and the suspension concentration of
the solution 6%. In order to determine the effect of the locked-in
albumin, after twenty hours, the loaded cells were centrifuged out of the
solution and the radioactivity in the solution and in the still intact
loaded cells was measured. The same measurements were carried out on
loaded cells that were prepared in the same manner as described above, but
without inclusion of albumin. A comparison of the measured values showed
that after twenty hours 33% more intact cells with locked-in albumin were
present than there were without locked-in albumin.
EXAMPLE II
The loaded cells were produced in the manner described in Example I, but
instead of the addition of methotrexate and albumin into the solution
containing the erythrocytes, sucrose that had been marked with the
radio-nuclide C 14 and also pronase P were added to the solution
containing the erythrocytes, still before the application of the electric
field. The sucrose content in the solution was 10 mM and that of pronase P
was 0.01 mg per 100 ml.
The effect of the pronase P locked in the loaded cells was, as described in
Example I, determined by measurement of the radioactivity in the loaded
cells and by comparison with loaded cells in which sucrose, but no pronase
P had been locked in. After twenty hours, the quantity of intact cells
containing pronase P was only 11% of the quantity of intact loaded cells
in which no pronase P had been locked in.
EXAMPLE III
The loaded cells were produced as described in Example I, except that
instead of the addition of methotrexate and albumin to the solution
containing erythrocytes, methotrexate that had been marked with tritium,
albumin and phospholiphase C were added to the solution, still before the
application of the electric field. The content of methotrexate was 5 mM,
the content of albumin 0.1% by volume and the content of phospholiphase C
0.01 mg per 100 ml.
As shown by a comparative measurement for a case in which no phospholiphase
C had been incorporated, after twenty hours only 17% of the intact loaded
cells were present compared to the number of them found in the comparison
measurement.
EXAMPLE IV
For preparation of loaded cells by the effect of osmotic pressure,
erythrocytes were suspended in a volume ratio of 1:1 in an isotonic,
phosphate-buffered NaCl solution of the following composition:
138.6 mM NaCl; 12.3 mM Na.sub.2 HPO.sub.4 ; 2.7 mM NaH.sub.2 PO.sub.4.
The pH value of the solution was 7.4.
1 ml of the suspension so produced was added, for increasing the
permeability of the membranes of the cells, with stirring, to 10 ml of a
solution that contained 5 mM of methotrexate that had been marked with
tritium, 4 mM of MgSo.sub.4 and 50 mM of sucrose. This solution was
allowed to stand five minutes at 0.degree. C. As the next step, the
osmolarity of the original solution was restored by adding a corresponding
quantity of a 2 molar KCl solution. The solution was then allowed to stand
another five minutes at 0.degree. C. and immediately thereafter the
temperature was raised for twenty minutes to 37.degree. C. in order to
accelerate the healing of the membranes. The loaded cells so produced were
centrifuged out of the solution, after which the cells were incubated in
an isotonic, phosphate-buffered sodium chloride solution of the
above-given composition. The loaded cells so produced contained
practically the same concentration of methotrexate as the external medium,
namely 98%.
A comparative measurement for checking the holding capability of the loaded
cells produced showed the same results as in Example I.
Although the process of the present invention has been illustrated with
reference to particular examples, it will be understood that variations
and modifications are possible within the inventive concept.
Just as the suspensions of loaded cells in physiological solutions prepared
according to previously known methods, the suspension of loaded cells in
physiological solutions prepared according to the present invention are
usable both for the treatment of animals and for the treatment of human
beings. In the case of treatment of human beings, it is of course
desirable to use human cells for the starting material in the preparation
of the loaded cell suspension: erythrocytes from human blood, for example.
The present invention widens the applicability for medical and other
physiological treatment of loaded cell suspension which, as prepared
according to previously known methods, have already established a certain
acceptability for use in medical and physiological treatment. It should be
mentioned that the additives introduced in the cell-loading step in
accordance with the present invention for inhibiting the destruction of
cell membranes by other loading materials by the formation of hydrogen
bonds or covalent bonds are classes of materials, represented by proteins
and sugars, regarding which there is a great deal of information available
regarding the compatibility of such materials with the blood of human
patients.
The terms "animal cells" and "animal body" as used herein, therefore, are
to be understood as including human cells and a human body, respectively.
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