DE2405119C3 - Method and device for increasing the permeability of the skin of cells of living beings - Google Patents

Description

40

The invention relates to a method for increasing the permeability of the skin of cells Living beings and a device for carrying out the method.

According to a fact that was not previously published, cells of living beings are introduced into a solution containing complexing agents or catalytically active substances with a lower osmolarity than the cell content. As a result of the increased permeability of the cell membrane, there is an exchange of substances between the solution contained in the cell interior and the solution containing the complexing agents or the catalytically active substances. The osmolarity of the solution containing the cells is then increased by adding osmotically active substances such as calcium, potassium and sodium ions to the osmolarity of the cell content of the originally introduced cells, whereby the cell membrane loses its permeability for the complexing agents or catalytically active substances contained in the cell interior which it thus includes. Cells containing complexing agents treated in this way are used to separate ionized substances having chemical or physical properties from an aqueous solution. The cells containing the complexing agents are inserted into the aqueous solution containing the ionized substances, so that the ionized substances migrate through the cell membrane acting as a membrane and are converted by the complexing agents into difficultly dissociable or difficultly soluble complexes. By separating the cells from the aqueous solution, the ionized substances bound in the cells are also separated from the aqueous solution.

Cells containing catalytically active substances are used to build up or break down through The chemical properties of excellent substances contained in an aqueous solution are used Cells are used in the aqueous solution until the to be built up or to be broken down in the aqueous solution contained substances due to the permeability of the skin of the cells in the interior of the Cells migrated, the build-up or breakdown of the substances has ended, and the substances pass through the skin of the Cells have migrated into the aqueous solution, whereupon the substances built up or broken down are known per se Way to be separated from the aqueous solution.

The process step causing the increase in permeability, in which the cells are in a solution with osmolarity lower than the osmolarity of the cell content is introduced, however, is temporal expensive, since the increase in permeability takes place only slowly and, moreover, several for the process step important variables must be observed. In addition, bacterial cells must be used as cells can be used and it is necessary to remove the cell wall, an additional one in itself known process step can be used to detach the cell wall.

It is the object of the invention to provide a method for increasing the permeability of the skin of cells To create living beings is easy, quick and therefore in is economically feasible, with an increase in permeability is achieved, the one As far as possible exchange of inside the cell and in a solution in which the cells are introduced macromolecules having a radius of at least 5 Å have been made possible. Included it should be possible to heal the increase in permeability achieved in a simple process step. It is Another object of the invention is to create a device for carrying out the method.

This object is achieved with a method of the kind referred to according to the invention in that the cells having electrical current conductive in a lying between 0 ⁰ C and 25 ° C temperature, a physiological electrolyte solution forming liquid to be entered as a suspension, whereupon the physiological electrolyte solution thus formed, containing the cells, ^{is exposed to an electric field with a field strength of IO 3} to ICV / cm until a radius of at least 5 A showing macromolecules through the membrane acting as a membrane between the solution contained in the cell interior and the physiological electrolyte solution can be exchanged.

The process according to the invention can be carried out batchwise or continuously. To carry out the discontinuous procedure is a container in which two electrodes are arranged with filled with a physiological electrolyte solution containing cells of living beings as a suspension and on the Electrolyte solution given an electrical impulse. For the continuous implementation of the process according to the invention; becomes in one with physiological

3 4

Container filled with electrolyte solution, on whose electro-organic or inorganic complexing agent

which a constant electric field is applied, which is reversible. In doing so, the cells are transformed into one that

A solution containing cells as a suspension containing physiological complexing agents, their osmolarity Electrolyte solution passed through the electric field within the limits of the osmolaticity of the cell contents This is best done by the fact that the container 5 original cells and the osmolarity of the

continuously fresh, the cells differing as a suspension aqueous solution, used until

containing physiological electrolyte solution supplied as a result of the exchange of substances through the membrane

and at the same time from the container the cell membrane acting on the electrical power in a state of equilibrium between

Electrolyte solution containing cells exposed to the field , the solution contained in the interior of the cell and the the

is sucked off. At the same time, the solution contained in the complexing agent becomes the cell content

Electrolyte solution in the electric field heat generated practically dissipated from the solution containing the complexing agents. A very convenient practice exists
in that the containing the cells as a suspension
physiological electrolyte solution through the focus of a
focused electric field is conducted. Here- 15
through being better utilization of the electrical
Field and at the same time ensures that the
cells carried along by the electric field all one

are exposed to almost the same field strength. unm «v ··» ·· »·» ... «...« ... "

As has been shown, the is used to increase the 20 cells in the aqueous solution, bi

Permeability of the cell membrane, required residence time of the aqueous solution to be separated ionisi

_ .. · _ jj: "Increase ^J ~" ° u: i: "a · j .._" uj: "" 1 ". Vi.mkran iuirlri"nH>"7f" llhaiit

is equivalent to. For annealing of the permeability increase it, the solution containing the cells after heating is maintained for about 1 to 2 hours at a temperature in the range of 15 to 4O ⁰ C. Subsequently, the cells containing the complexing agent are separated from the solution containing the complexing agents. To enrich the ionized substances contained in the aqueous solution, the

- ·· · ·· -η- · - ■ ": —— ° tzt until the from the

„„ _W1 , _6V .. _β ionized substances

through the cell membrane, which acts as a membrane, into the interior of the cells migrated and by the complexing agents in difficult to dissociate or difficult to dissolve complexes

Cells in the electric field causing the increase in permeability are very low, so that

Cells with increased permeability of the cell membrane simply «...» ,, .. ν ^ ν ..... ·

_{and i 5} which can be produced quickly and also with high yield have been transferred. In a further process stage which is known _{per se, the cells}

The method according to the invention is advantageously separated from the aqueous solution, ter way by means of a device of the initially Die according to the method according to the invention

designated type feasible, which from a cell produced by living beings are also formed in a manner formed from electrically non-conductive material, 30 favorable in processes for the construction and dismantling of a passage with a diameter of little - due to chemical properties ^of 8 ", jn least 20 μηι having partition in two
Chambers divided container consists, in which
Chambers each have an electrode and
wherein on the container wall enclosing the chambers, line connections for the supply of
physiological electrolyte solution are provided and
one of the chambers being the container wall

penetrating, directed towards the passage _aH .u .. ₆ ..w, .... —..-. · —- ■ --- - ■. "__ _t , _{t hi (} . The feed nozzle for the physio-aqueous solution containing the cells differs as long as the electrolyte solution is used, and the electrolyte solution passes through the other chamber as a result of the increased permeability of the Zeilhau

^y Exchange of substances in the solution inside the cell

and the cell contents of the solution containing the catalytically active substances practically correspond to the solution containing the catalytically active substances. Then, the solution containing the cells after heating is maintained for about 1 to 2 hours at a temperature in the range of 15 to 4O ⁰ C. Subsequently, the catalytically active substances are contained-

VUIl UUIV-Il v.UCllll3t.llt! -. IgWiJi-IiUi ₁ - .. ~ - ~ _o

at least 0.5 mM magnesium and / or calcium as well as aqueous solution containing potassium ions, solutes by means of the build-up or the breakdown promoting catalytically active substances can be used. The cells become catalytically active Solution containing substances, the osmolarity of which is within the limits of the osmolarity of the cell content of the original cells and from the osmolarity of the

The suction line for the cells that penetrates the container wall and is also directed towards the passage containing physiological electrolyte solution are designed protruding.

As has been shown, the increase in permeability of the skin of the cells is due to the heating of the cells containing solution to a temperature between 15 ° C and 40 ° C lying temperature can be healed after 1 to 2 hours.

45

lying temperature after 1 dis / atunuen ausncnudi. wciucn uv uiv ^ _ul - _v ..- ~. . . ,

S use of bacterial cells, the cells 50 are the cells of the catalytic acting,

expediently these heated to a temperature of 20 ⁰ C and with the use of erythrocytes to a temperature of about 37 ° C. Because the increased permeability of the tent skin can be healed, the cells can be used to take up macromolecules of the most varied of types and thus for various purposes. According to the method according to the invention

The cells produced by living beings are therefore also in »hui ··> uiv. "U ^ .. ₆ ~ ^. _D "

Favorable way in process for the separation of by 60 or degraded substances are then in per se chemical or physical properties separated from the aqueous solution in a known manner, Drew, ionized substances, such as heavy metal ions. In the drawing are two exemplary embodiments of the

or the like of one presented in an at least 0.5 mM device according to the invention. Magnesium and / or calcium and potassium ions
containing aqueous solution, such as seawater, 65
Fresh water, sewage or the like, dissolved substance mixture by means of the separation promoting, with the
substances to be separated establish a connection in more detail

containing solution separated. For carrying out processes for the creation or degradation of substances the cells are then placed in the aqueous solution e ^ until the to be built up or to be broken down in the aqueous solution contained substances by the than Membrane; , doing acting of the cells in the interior of the skin Cells migrated, the build-up or breakdown of the substances has ended, and the substances pass through the skin of the Cells have migrated into the aqueous solution. The on-

^{1 ;} - to " ^: " ^u

It shows

Fig. 1 shows one of a through a partition in two-chamber divided container device according to the invention,

F i g. 2 shows an embodiment of the device according to

of the invention with a container-shaped partition wall adapted to the shape of the container.

As can be seen from the drawing, the container 1 is divided into the chambers 4 and 5 by an intermediate wall 2 which has a passage 3. An electrode 6 is arranged in each of the chambers 4 and 5. Line connections 7 are provided on the container wall for the separate passage of physiological electrolyte solution into the chambers 4 and 5. To carry out the method according to the invention, the physiological electrolyte solution containing cells is fed from the outside through the feed nozzle 8 of the chamber 4 and sucked out of the container 1 again through the passage 3, which surrounds the focus of the electric field, via the suction tube 9 and in a in the drawing, not shown, collected the suction pump upstream, cooled collecting vessel. The loss of physiological electrolyte solution is compensated for via the supply lines 7.

In Fig. 2 is a variant of the device according to FIG Invention shown in which the partition 2 designed in the form of a container and concentric to the Container 1 and the suction pipe 9 is arranged. In the suction pipe 9 is also to control the temperature To exclude excessive heating and thus damage to the cells, a thermocouple 10 is provided. In the embodiment of the device according to the invention shown in FIG. 2 there is also a Another line connection It is arranged on the container t, but only for venting the apparatus serves.

Embodiment:

About 100 ml of fresh bovine blood was collected in an isotonic sodium citrate solution and the solution thus formed was centrifuged off in a centrifuge at 1200 g. Then about 30 ml of the concentrated erythrocytes centrifuged in 100 ml of a buffer solution containing 150 mM NaCl, 16 mM KCl, 4 mM MgCl ₂ , 2 mM CaCl ₂ and 5 mM Tris per liter and their pH by adding Hydrochloric acid had been adjusted to 7.4, washed twice with each centrifugation. The erythrocyte concentrate was then diluted in a ratio of 10: 3 with buffer solution to which 1 mM adenosine triphosphate had been added per liter.

Subsequently, the solution containing the erythrocytes was passed through the feed nozzle 8 of a solution shown in FIG. 2, which was simultaneously ^{fed to the buffer solution cooled to O 0} C and serving as a physiological electrolyte solution. The diameter and the length of the passage 3 provided in the partition 2 and the distance between the tip of the feed nozzle 8 and the passage 3 were 0.45 mm. A voltage of 350 V was applied to the electrodes. The throughput of erythrocytes through the device was such that the amount of erythrocytes used had passed the device in about 30 minutes. The erythrocytes collected in the collecting vessel were ^{centrifuged off at 0 °} C. and 13,000 g for about 15 minutes.

0.5 ml of the centrifuged erythrocytes were then suspended in a solution of 5 ml of buffer solution and 0.2 ml of an iodine ³¹ -albumin solution, the specific activity of which was 0.1 mCi / ml, and reduced to 0 ^{0 for about one hour} C held. The solution was then warmed and held at 37 ° C. for about two hours. The erythrocytes were then centrifuged for 15 minutes at 13,000 g and the centrifuged erythrocytes were washed twice in buffer solution containing 0.1% albumin as a carrier, each time being centrifuged off. The activity of the iodine ³¹ remaining in the erythrocytes was measured in a TriCarb liquid scintillator after the erythrocytes had been disrupted. The activity measured corresponded to a 31% uptake of iodine ¹³¹ by the erythrocytes from the solution containing ^{iodine 13l-albumin.}

For this purpose 2 sheets of drawings

Claims (2)

Patent claims: 1. A method for increasing the permeability of the skin of cells of living beings, characterized in that the cells are entered as a suspension in a liquid which conducts electrical current and forms a physiological electrolyte solution, at a temperature ^{between 0 ° and 25 0 C,} whereupon the physiological electrolyte solution thus formed, containing the cells, ^{is exposed to an electric field with a field strength of 10 3} to ^{10 5} V / cm until macromolecules with a radius of at least 5 Λ pass through the membrane acting as a membrane between those contained in the cell interior Solution and the physiological electrolyte solution are exchanged. 2. Apparatus for performing the method according to claim 1, characterized by a through one formed from electrically non-conductive material, a passage (3) with a diameter at least 20 μιη having intermediate wall (2) in two chambers (4, 5) divided container (1), wherein an electrode (6) is arranged in each of the chambers (4, 5) and where the The container wall surrounding the chambers, line connections (7) for the supply of physiological Electrolyte solution are provided and in which one chamber (4) is a container wall penetrating feeding nozzle (8) directed towards the passage (3) for the one containing the cells physiologically 3 electrolyte solution and into the other chamber (5; une the container wall penetrating, suction line (9) for the lines also directed towards the passage (3) physiological electrolyte solution are designed to protrude.