DE102005035456A1 - A method of electromanipulating cells to alter the membrane permeability of these cells using lipophilic ions as a medium supplement - Google Patents

Abstract

The invention relates to methods for treating cells with electric fields to change the membrane permeability of these cells using lipophilic ions as a medium additive. More particularly, the invention relates to methods of electroporation and electrofusion of cells using lipophilic ions as a medium additive.

Description

The The present invention relates to methods of treating cells with electric fields to change the membrane permeability of these cells using lipophilic ions as a medium supplement. More particularly, the invention relates to methods of electroporation and electrofusion of cells using lipophilic ions as a medium supplement. By this addition of lipophilic ions is the efficiency and, above all, the reproducibility elevated as well as the otherwise relatively high variation of electroporation or electrofusion reduced.

electrotransfection and electrofusion of cells are routinely used in many areas Biotechnology and biomedicine for used the manipulation of the genome and the cytosol. Both methods are based on the application of high intensity and very intense electric field pulses short duration, leading to a reversible electrical membrane breakthrough (so-called electroporation) lead. A wide range of molecules, including hormones, Proteins, RNA and DNA, can be transformed into living cells by electroporation be introduced. In dielectrophoretically contacted cells leads the Membrane breakthrough in the contact zone to fuse the cells, what kind of the production of hybrid cells (electrofusion) can be used can.

As a rule, field strengths of a few kV / cm and a pulse duration of a few 10 μs (to ms) are used for the electroporation / fusion of mammalian cells. The applied field leads to an exponential membrane charge, which can be indicated by the induced transmissive voltage V _g : V G (t) = 1.5aE 0 cosθ (1-exp (-t / τ m )) (1), where a is the cell radius, θ the polar angle to the direction of the applied field E ₀ and τ _m the time constant of the membrane charging. The membrane breakthrough occurs when V _g exceeds the critical value V _c = 1 volt.

wobei Cm [μF/cm²] die flächenspezifische Membrankapazität ist und σ_i und σ_e für die spezifischen Leitfähigkeiten [mS/cm] des Zytosols und des äußeren Porations- bzw. Fusionsmediums stehen.The time constant τ _{m of} the membrane charging is calculated using the following equation:

where Cm [μF / cm ² ] is the area-specific membrane capacity and σ _i and σ _{e are} the specific conductivities [mS / cm] of the cytosol and the outer poration or fusion medium, respectively.

If the cell radius is given a typical value of 7 μm in Equation 1, the minimum critical field strength for the breakdown in the membrane regions facing the electrodes (θ = 0) can be calculated (provided that the pulse duration is much longer than the relaxation time of the membrane is t >> τ _m ): E _crit = V _c / ₍ 1.5 × a) ≈ 1 kV / cm. For larger cells, according to Eq. 1 much smaller field strengths needed. The time constant τ _m in mammalian cells is in the range of 0.1-1 μs (equation 2: Cm 1 μF / cm ² , σ _i 5 mS / cm, σ _e = 0.1-10 mS / cm, see below). Therefore, the used pulse durations of 10-100 μs are much longer than τ _m, so that Eq. 1 can be simplified: V _g = 1.5aE ₀ cosθ.

in the It is known in the prior art that the efficiency of electrotransfection and -Electrofusionsverfahren by the use of weak-conductive, hypotonic media during the pulse application can be significantly increased [Zimmermann & Neil, 1996; Sukhorukov et al. 1998]. Such media contain as the main osmoticum different Sugar and sugar derivatives. Among other things, sugars become monomeric Carbohydrates, such as glucose, sorbitol, inositol or mannitol, and disaccharides, including Sucrose or trehalose, commonly used [Zimmermann & Neil, 1996].

• 1) Das hypotone Anschwellen von Zellen fördert den Membrandurchbruch, weil die induzierte Membranspannung linear vom Zellradius abhängt (s.o. Gl. 1).
• 2) Das Verringern der äußeren Leitfähigkeit verbessert stark die Elektroinjektion aufgrund der transienten Elektrodeformationskraft F_DEF auf die Zellmembran:
  
  Wenn σ_i = σ_e, bleibt die Deformationskraft aus: F_D = 0. Unter der Bedingung σ_i < σ_e wird auf die Zellmembran eine Elongationskraft F_D in Feldrichtung ausgeübt, deren Ausmaß mit sinkender σ_e zunimmt (Gl. 3). Gleichzeitig führt die Senkung der σ_e zu einer starken Zunahme der Mengen an elektroinjizierten Molekülen [Sukhorukov et al., 1998].
• 3/4) Bei der Elektrofusion sind niedrig leitende Medien obligatorisch. Analog zur Deformationskraft nehmen sowohl die elektrische Polarisation der Zelle bzw. der induzierte Zelldipol μ_c (Gl. 4) als auch die positive dielectrophoretische Kraft im MHz-Bereich (1-10 MHz) (Gl. 5) mit sinkender σ_e zu [Jones, 1995]:
  
  wobei ε_eε₀ = 80 × 8.8510^-12 F/m die Permittivität des wässrigen Außenmediums ist. Der Term ∇E₀ ² spiegelt die Inhomogenität des Feldes wider. Die Verminderung der σ_e führt außerdem zu einer Erhöhung der Dipol-Dipol-Wechselwirkungen, was die gegenseitige Anziehung der Fusionspartner begünstigt.
• 5) Zusätzlich zu den oben beschriebenen physikalischen Effekten erleichtert das hypotone Anschwellen die Elektrofusion durch die Auflösung des Zytoskeletts, das Glätten der Zellmembranen (Retraktion der Mikrovilli) und die Erhöhung der Mobilität der Membranbestandteile.

The reduction of osmotic pressure and conductivity serves several purposes in electroporation and fusion:

• 1) The hypotonic swelling of cells promotes membrane breakthrough because the induced membrane voltage depends linearly on the cell radius (see Eq. 1).
• 2) Reducing the outer conductivity greatly improves the electroinjection due to the transient electrodefractive force F _DEF on the cell membrane:
  
  If σ _i = σ _e , the deformation force remains: F _D = 0. Under the condition σ _i <σ _e , an elongation force F _D in the field direction is exerted on the cell membrane, the extent of which increases with decreasing σ _e (equation 3). At the same time, the decrease in σ _e leads to a large increase in the amounts of electroinjected molecules [Sukhorukov et al., 1998].
• 3/4) In electrofusion, low-conductivity media are mandatory. Analogous to the deformation force, both the electrical polarization of the cell or the induced cell dipole μ _c (equation 4) and the positive dielectrophoretic force in the MHz range (1-10 MHz) (equation 5) increase with decreasing σ _e to [Jones , 1995]:
  
  where ε _e ε ₀ = 80 × 8.8510 ^-12 F / m is the permittivity of the aqueous external medium. The term ∇E ₀ ² reflects the inhomogeneity of the field. The decrease in σ _{e also} leads to an increase in the dipole-dipole interactions, which favors the mutual attraction of the fusion partners.
• 5) In addition to the physical effects described above, hypotonic swelling facilitates electrofusion by dissolving the cytoskeleton, smoothing the cell membranes (retraction of the microvilli), and increasing the mobility of the membrane constituents.

• (1) niedrige Transfektions- und Fusionsraten bei besonders „Feld-resistenten" Zelltypen,
• (2) hohe Sterblichkeitsraten von Zellen nach der Elektroporation/fusion,
• (3) starke „batch-to-batch" Schwankungen der Transfektions- und Fusionsraten sowie
• (4) sehr oft schwankende Reproduzierbarkeit in der Elektroinjektions- bzw. Fusionsausbeute.

However, despite widespread use of electrotransfection / electroporation and electrofusion processes, there are still a number of unresolved problems in the current methods, such as:

• (1) low transfection and fusion rates in particularly "field resistant" cell types,
• (2) high mortality rates of cells after electroporation / fusion,
• (3) strong batch-to-batch variations in transfection and fusion rates as well
• (4) very often fluctuating reproducibility in the electroinjection or fusion yield.

Task of invention

A Object of the present invention is therefore in the provision new improved cell electrification techniques with increased efficiency and reproducibility. A so in Related task is the provision of Means for improving conventional electromanipulation methods, in particular electroporation and electrofusion processes.

incoming Investigations of the inventors led to the surprising Result that by adding lipophilic ions to the treatment medium, in particular a hypotonic treatment medium, the efficiency and Reproducibility of electromanipulation procedures for cells, such as. Electroporation and electrofusion process, greatly improved and in particular the yield and viability of the electromanipulated Cells increased significantly can be.

Accordingly The above-mentioned objects are achieved by the invention the provision of a method of treating cells with electric fields according to claim 1, wherein the treatment medium contains lipophilic ions, and the use of these lipophilic ions according to claim 23 and preferred embodiments The invention is the subject of the dependent claims.

description the invention

starting point for the present invention have been experimental findings that many cells, especially mammalian cells, especially under hypotonic conditions as typically while electroporation / electrofusion, to a complex Volume regulation are able.

Recent investigations [Reuss et al., 2004] have shown that after rapid initial swelling of the cells in hypotonic sugar media, depending on the one used Sugar, both a slower secondary volume increase as well Shrinkage of the cells can occur. Both can be fast Outflow of the electrolyte by so-called. Volume-sensitive channels from the Lead cytosol.

In hypotonic poration and fusion media containing an oligosaccharide (e.g., trehalose, sucrose or raffinose) a regulatory volume decrease (so-called "regulatory volume decrease" = RVD) in all So far, human and mammalian cells have been studied. This means, After a short period of swelling (1-3 min) cells in hypotonic Medium her original Volume (despite persistent hypotensive stress) within 15-20 can regulate min.

Electrophysiological studies by other authors suggest that initial cell swelling may activate the volume-sensitive chloride channels, resulting in chloride outflow from the cell and cell membrane depolarization. The membrane depolarization causes activation of the voltage-dependent K ⁺ channels and a K ⁺ outflow. The net KCl outflow along with the osmotic water outflow is expected to restore normal (ie, isotonic) cell volume [Fürst et al., 2002; Lang, 1998].

in the Unlike oligosaccharides found in hypotonic solutions monomeric Sugar derivatives (for example sorbitol, inositol or glucose) no RVD instead [Reuss et al., 2004]. In some cell types, you even see one secondary slower swelling of the cells. The difference between oligo- and monosaccharides could by the size selectivity of the volume-sensitive channels ("volume-sensitive channels "= VSC) explained become. Such channels are ubiquitous in the membranes of mammalian cells present [Kirk, 1997; Strange, 1994].

However, even in media without RVD, eg in inositol or sorbitol-substituted media, hypotonic stress leads to considerable electrolyte losses and a reduction in cytosolic conductivity (eg by means of electro-rotation spectra ( 7A occupied).

The ion losses from the cytosol reduce the cytosolic σ _i conductivity, which greatly reduces the porosity and fusion relevant deformation force (equation 3), cell polarization μ _c (equation 4) and dielectrophoretic force (equation 5). In addition, the ion outflow from the cytosol results in significant loss of vitality in cells.

These Effects can the positive effects of hypotonic media in electroporation / electrofusion completely or partially ruined.

To now, in literature, the complex volume response of cells, especially mammalian cells, in hypotonic sugar media and the associated ion fluxes, however not yet relevant to their relevance to electroporation and fusion discussed.

The present invention is now based u. a. on the surprising experimental Found that the addition of lipophilic ions, especially anions, Among other things, this volume regulation to prevent the treatment medium and thus also the efficiency of electroporation or electrofusion can increase.

The Adsorption or the incorporation of lipophilic anions into the cell membrane possibly leads to inhibit the ion channels involved in volume regulation. alternative It may be that the lipophilic ions in a direct or indirect way the probability of occurrence of the channels or regulate their other transport properties. From both Model ideas can be derived that when using lipophilic Ionic ions lost from the cytosol during hypotonic stress be greatly reduced. On the one hand, this is the cell volume stabilized, on the other hand, the ion content and the conductivity of the cytosol are kept at high levels.

Without to commit to a theory of the mechanism of action to want to become, is present assumed that this inhibition of certain ion channels a Consequence of the change, especially increase, the membrane capacity the cell, in dependence from their membrane voltage, by adsorption and / or incorporation the lipophilic ions is in the membrane.

Extensive studies by the inventors have revealed that the lipophilic ion-induced Changing the membrane capacitance as a function of the membrane voltage usually follows a certain relationship and preferably the behavior of a symmetrical or asymmetric bell curve with a voltage V _max , at which the increase in capacitance C (V _max ) is a maximum.

The Hang the parameters of the bell curve thereby of the chemical nature of the lipophilic ions and of the Nature of the membrane (or cell type) from. Based on the voltage dependence The bell curve of a lipophilic ion can draw conclusions about the distribution of the lipophilic ions are pulled in the membrane.

• 1) die drastische Erniedrigung des Oberflächenpotentials führt zu einem so steilen Potentialverlauf über die Membran, daß lokale Durchbrüche erzeugt werden bzw. lokale Durchbrüche durch überlagerte externe Felder erleichtert werden (Elektroporation, Elektrofusion).
• 2) der veränderte Potentialverlauf führt zur Beeinflussung von spannungsabhängigen Transportsystemen in der Membran (Volumenregulation).
• 3) Neben der „Membran-Asymmetrie" wird eine „Hemisphären-Asymmetrie" der Zelle als ganzes auftreten. Diese erleichtert den elektrischen Durchbruch auf zwei Arten. Auf der einen Seite der Zelle ist das Potential zum Durchbruch bereits nahezu erreicht und die extern applizierte Durchbruchsspannung kann geringer als normal gewählt werden. Auf der anderen Seite der Zelle ist der anliegende Potentialgradient „ungünstig" für die extern angelegte Durchbruchsspannung. Diese Zellseite ist also durch die lipophilen Ionen „geschützt". Alternativ kann man dies im Rahmen der externen Feldamplitude bzw. in der Länge/Kürze des applizierten Feldpulses nutzen. Beide Hemisphären werden unterschiedliche Aufladungszeiten aufweisen. Dies hat zur Folge, daß dieser „asymmetrische" Durchbruch für die Zelle schonender ist als der normale Durchbruch.

An equal distribution of the lipophilic ions on both sides of the membrane should be present at the membrane voltage, at which the maximum capacity increase is detected. By contrast, the most extreme unequal distribution of the lipophilic ions (either completely on the cytosolic or extracellular membrane side) is present if no increase in capacity by the lipophilic ions can be measured. An uneven distribution of the lipophilic ions should lead to a strong lowering of the surface potential (up to ΔV ~ -400 mV) on one side of the membrane. This will usually lead to a drastic change in the potential profile across the membrane. This "membrane asymmetry" effect may facilitate electromanipulations of treated cells in several ways:

• 1) the drastic reduction of the surface potential leads to such a steep potential profile across the membrane that local breakthroughs are generated or local breakthroughs are facilitated by superimposed external fields (electroporation, electrofusion).
• 2) The altered potential course leads to the influence of voltage-dependent transport systems in the membrane (volume regulation).
• 3) In addition to the "membrane asymmetry", a "hemispheric asymmetry" of the cell as a whole will occur. This facilitates the electrical breakdown in two ways. On one side of the cell, the potential for breakdown is already almost reached and the externally applied breakdown voltage can be chosen to be lower than normal. On the other side of the cell, the applied potential gradient is "unfavorable" for the externally applied breakdown voltage, so this cell side is "protected" by the lipophilic ions. Alternatively, one can use this in the context of the external field amplitude or in the length / shortness of the applied field pulse. Both hemispheres will have different charging times. As a result, this "asymmetric" breakthrough is gentler on the cell than the normal breakthrough.

• – Messung der elektrischen Membrankapazität der biologischen Zelle in Abhängigkeit von einer bestimmten Membranspannung, wobei die Spannungsabhängigkeit der durch lipophile Ionen induzierten Membrankapazität mit mehreren verschiedenen lipophilen Ionen im Behandlungsmedium gemessen wird, und
• – Feststellung und Auswahl der lipophilen Ionen, für welche die durch lipophile Ionen induzierte Membrankapazität der Zelle ein vorbestimmtes Auswahlkriterium erfüllt. Dieses Auswahlkriterium kann beispielsweise die Form oder Position der Glockenkurve bei bestimmten Membranspannungen sein.

This finding allows the selection of particularly suitable lipophilic ions by determining their effects on changing the membrane capacity under given conditions. Such a selection process will typically include the following steps:

• Measuring the electrical membrane capacity of the biological cell as a function of a specific membrane voltage, the voltage dependence of the lipophilic ion-induced membrane capacity being measured with a plurality of different lipophilic ions in the treatment medium, and
• Detection and selection of the lipophilic ions for which the lipophilic ion-induced membrane capacity of the cell meets a predetermined selection criterion. This selection criterion can be, for example, the shape or position of the bell curve at certain membrane stresses.

ever according to the desired Results and applications can e.g. those lipophilic ions are selected for which the membrane capacity at the natural Membrane voltage of the cell is at a minimum (i.e. or hyperpolarized flank of the bell curve), or those for which the membrane capacity at the natural Membrane voltage of the cell has a maximum.

In an embodiment In the invention, those lipophilic ions are selected for which the membrane capacity at the natural Membrane voltage of the cell a little or no increased value has, i. those lipophilic ions, their characteristic bell curve looks like that at the natural Membrane voltage has a falling edge. In this case For example, the lipophilic ions are expected to be at natural membrane tension uneven about the Be distributed membrane and cause the above usable asymmetry effect. This concerns e.g. hyperpolarized cells that are extremely weak Are exposed to fusion medium.

lipophilic Ions, for which the membrane capacity at the natural Membrane voltage of the cell has a maximum, for applications be used in which a high membrane capacity or a Uniform distribution of lipophilic ions is useful. An example therefor are cells with inhomogeneous membrane structure, which in this way a more homogeneous membrane charging, e.g. most mammalian cells, in which the negatively charged phosphatidylserine mainly on the cytosolic side of the plasma membrane is located.

After this The natural Diaphragm voltage (i.e., no applied field) of ion gradients permeable ions between the outer bath medium (Treatment medium) and the interior of the cell depends these are adjusted by adding salts to the treatment medium become. Suitable soluble Salts can For example, sodium, potassium or barium ions include but not limited to this.

After this the change the membrane capacity on the one hand by the choice of lipophilic ions and on the other hand by change the natural one Membrane voltage can be influenced, it means that a desired Distribution of lipophilic ions either unchanged Membrane tension by the choice of a lipophilic ion with the desired Shape (e.g., flank or maximum) of the bell curve at this voltage or at a certain ion by corresponding displacement of the natural Diaphragm voltage up to the voltage corresponding to the corresponding bell curve section corresponds, can be obtained.

One further advantage in setting cells to a specific one Membrane potential (e.g., using cell cycle synchronized Cells, adjustment of the membrane potential by suitable salt conditions) lies in the better reproducibility of the results.

As already mentioned, the lipophilic ion-induced change in membrane capacity can be used to completely or partially eliminate the volume regulation of cells in hypotonic media. As discussed above, this leads to significantly reduced electrolyte losses and thus to an improvement of the basic conditions for electromanipulation processes such as electroporation or electrofusion. Losses from the cytosol reduce the cytosolic σ _i , which greatly reduces the porosity and fusion relevant deformation force (equation 3), cell polarization μ _c (equation 4), and dielectrophoretic force (equation 5). In addition, the ion outflow from the cytosol results in significant loss of vitality in cells.

By the elimination of these negative effects can increase the efficiency and Reproducibility of Elektromanipulationsverfahren considerably increased become.

As from the above can be seen, the presence of lipophilic ions or by the induced change the membrane capacity but also under non-hypotonic, e.g. isotonic, conditions too lead a membrane charging, those desired or required in the case of electronic manipulation methods Membrane breakthrough much easier. Thus, the inventive use of lipophilic ions also perform easier and more efficient Electromanipulation methods such as electroporation or electrofusion in non-hypotonic media. Avoidance of hypotonic stress for the cells would be from great Advantage and in any case affects the survival rates of the manipulated Cells cheap out.

suitable Cells for the application of the method according to the invention or the use according to the invention of lipophilic ions are basically all cells natural or of synthetic origin. For example, synthetic cells can be used artificial membrane- Vesicles, liposomes or micelles. Natural cells include prokaryotic Cells, e.g. Bacteria or yeasts, or eukaryotic cells, e.g. animal or plant cells. Preferred animal cells are Mammalian cells, especially human cells. In a preferred embodiment the cells are living cells.

The used according to the invention lipophilic ions can Cations, anions or a mixture of cations and anions.

cations are specially for preferred for use in plant cells, while anions in animal Cells, in particular mammalian cells, are particularly preferred.

The cations used in the invention may consist of one species or comprise several different cations. Typically, the lipophilic cations used will contain at least one of the group of cations comprising cations of lipophilic metal complexes, lipophilic organometallic compounds, and lipophilic organic compounds having at least one positively charged functional group. Specific examples are rhenium hexacarbonyl (Re (CO) ₆ ) ⁺ , tetraphenylphosphonium or tetraphenylarsenium compounds. Other suitable compounds will be readily apparent to one skilled in the art through routine experimentation.

The anions used in the invention may consist of one species or ver several include different anions. Typically, the lipophilic anions used will contain at least one of the group of anions comprising anions of lipophilic metal complexes, lipophilic organometallic compounds and lipophilic organic compounds having at least one deprotonatable functional group.

Specific, non-limiting examples of lipophilic ions which can be used according to the invention, in particular lipophilic metal complexes, are the following:

Preferably contain the lipophilic anions at least one substance from the Group of anions, the anions of lipophilic tungsten complexes (LTC), in particular lipophilic tungsten carbonyl complexes, dipicrylamine (DPA) and derivatives thereof, anions of lipophilic organic compounds, the at least one carboxylate, sulfonate, sulfate, thiosulfate or thiocyanate group contain, in particular arachidonic acid, or anions of an optionally substituted tetraphenylborate or phenyldicarbaundecane-decane (PCB) or a tetracyano borate or tetratrifluoromethyl borate.

ausgewählt,
wobei: (1) = [C₁₂H₄N₇O₁₂]^-, 2,2',4,4',6,6'-Hexanitrodiphenylamid (Dipikrylamin = DPA); (2) = das Anion von Et₄N[W(CO)₅(SCNOC₆H₄)], Tetraethylammoniumbenzoxazolidin-2-thion-1-yl-pentacarbonylwolframat, MW 604 (abgekürzt WO). (3) das Anion von Et₄N[W₂(CO)10(μ-S₂CH)], Tetraethylammoniumdecacarbonyl-μ-dithioformiato-diwolframat, MW 725 (hier im folgenden als LTC^- ("Lipophilic Tungsten Complex") oder WW abgekürzt); (4) Das Anion von Na[B(C₆H₅)₄], Natriumtetraphenylborat, MW 342.Particularly preferred are the lipophilic anions from the group of the following anions

selected,
wherein: (1) = [C ₁₂ H ₄ N ₇ O _12] ^-, 2,2 ', 4,4', 6,6'-Hexanitrodiphenylamid (hexanitrodiphenylamine = DPA); (2) = the anion of Et ₄ N [W (CO) ₅ (SCNOC ₆ H ₄ )], tetraethylammoniumbenzoxazolidine-2-thion-1-yl-pentacarbonyl-tungstate, MW 604 (abbreviated to WO). (3) the anion of Et ₄ N [W ₂ (CO) 10 (μ-S ₂ CH)] Tetraethylammoniumdecacarbonyl-μ-dithioformiato-diwolframat, MW 725 (hereinafter referred to as LTC ^- ( "Lipophilic Tungsten Complex") or WW abbreviated); (4) The anion of Na [B (C ₆ H ₅ ) ₄ ], sodium tetraphenylborate, MW 342.

By the use of several lipophilic ions of different nature in the same or different concentrations to the same Time or in time, an "overlap" of the individual characteristics of the lipophilic ions are achieved. This can be done for different purposes, e.g. to guarantee more robust, i. reproducible and less error prone procedures of Be an advantage.

The usable according to the invention Lipophilic ions are well-known compounds in the art and commercially available or can be produced by known routine methods.

Basically suitable for all ions that are capable are, the membrane capacity to increase that the increase the capacity a symmetrical or asymmetric bell curve in dependence from the trans-membrane voltage. Exemplary this is for some Ions (tungsten compounds, DPA, etc.).

After this a major application of the present invention is the electromanipulation live cells, the lipophilic ions should be in this Case for live cells are physiologically compatible. For some applications it is particularly preferred that the lipophilic ions used are degradable by UV light and physiological for living cells compatible Degradation products result. Both conditions are e.g. in the lipophilic anionic tungsten complex of the above formula 3 in an ideal manner Fulfills. For the It will be apparent to those skilled in the art that similar compounds are readily available Efficacy, tolerability and the like Degradation behavior can be produced.

• – Bereitstellung von mindestens einer Zelle (1) in einem Behandlungsmedium (2), und
• – Beaufschlagung der Zelle (1) mit mindestens einem elektrischen Spannungspuls, gekennzeichnet durch den Schritt:
• – Zusatz von lipophilen Ionen zu dem Behandlungsmedium (2).

According to the invention, the lipophilic ions are typically used in a method for treating cells with electric fields for changing the membrane permeability of these cells, which comprises at least the following steps:

• - provision of at least one cell ( 1 ) in a treatment medium ( 2 ), and
• - loading the cell ( 1 ) with at least one electrical voltage pulse, characterized by the step:
• Addition of lipophilic ions to the treatment medium ( 2 ).

Such a method generally comprises the various known or conceivable electromani Pulsion method for changing the membrane permeability of cells in which the cells are exposed to an electrical voltage pulse, and in particular methods for electrotransfection / electroporation or electrofusion.

The lipophilic ions are added to the treatment medium ( 2 ) is generally added in a concentration in the range from 1 nM to 100 μM, preferably 0.1 μM to 60 μM, particularly preferably 1 μM to 50 μM.

In a specific embodiment of the method according to the invention, the treatment medium ( 2 ) also an osmotically active substance for generating a hypotonic stress for the at least one biological cell ( 1 ) was added. Such substances include all substances known for this purpose, preferably mono- and / or disaccharides, eg sucrose, trehalose, glucose, mannitol, inositol, sorbitol etc.

Preferably, the application of the biological cell ( 1 ) with the electrical voltage pulse with a delay time after the addition of the osmotically active substance, wherein the delay time is preferably selected in the range of 15 s to 10 min.

investigations the inventors have shown that in electrotransfection / poration and electrofusion procedures predict the duration of hypotonic stress the field pulse application has a significant impact on efficiency this method has.

Based on biophysical and volumetric experiments, the Duration of hypotonic treatment in the electromanipulation protocols be optimized significantly. In these experiments were hypotonic Poration and fusion media used, whose composition in Tables 1 and 2 is given. For washing the cells were also used isotonic media.

a) Electrofusion:

Essential higher Yields of living hybrid cells could be improved by optimization the timing of the pulse application can be achieved. The hybrid yields strongly depend on the duration of the hypotonic stress before the pulse application from. A short hypotonic treatment of 2 min, the end of the rapid swelling phase corresponded, gave the best results. Longer incubation times (e.g. 20 and 40 min, respectively) reduced the fusion yields (data not shown).

b) Electro-transfection with the GFP plasmid:

Analogous for electrofusion revealed a brief hypotonic treatment (i.e., 2 min before the pulse application) usually much higher yields on GFP-transfected cells as a longer incubation of 10 min (Table 4). In general, the incubation time-dependent transfection yields correlated (TY) very well with the transient changes in cell size (see Eq. 1) in different cell types.

Longer incubation led always to a decrease in cell proliferation (PF, e.g., from 47 to 33% in HEK cells in inositol 150), indicating a decreased viability of the cells (due to electrolyte loss).

These strong dependence electroporation / transfection or electrofusion efficiency from the duration of the hypotonic stress can lead to a further improvement the above-described Elektromanipulationsverfahren using of lipophilic ions by optimizing the periods of hypotonic stress be used. It should be noted that through this temporal optimization in some cases Even without the use of lipophilic ions already very high efficiencies achieved with lipophilic ions only slightly increased could become. However, the use of lipophilic ions brings in any case, the additional Advantage of a more robust protocol, i. H. of a protocol that not on the precise Setting optimal time values to achieve high efficiencies instructed. This is just in the commercial application this process a big one practical advantage.

Preferably, the addition of the lipophilic ions to the treatment medium ( 2 ) in a sufficient concentration and for a sufficient period of time before the voltage pulse that a doping of the membranes of the cells to be treated with these lipophilic ions takes place. The term "doping" in this context includes the adsorption of the ions on the membrane and / or the incorporation of the ions into the membrane Suitable concentrations and time periods (usually of the order of minutes) With the knowledge of the present disclosure, those skilled in the art will readily appreciate in routine experimentation.

figure description

1 Schematic representation of the method according to the invention

2 Schematic representation of a test arrangement for carrying out the method according to the invention

3 Schematic representation of the method for selecting suitable lipophilic ions

4 Representation of "bell curves" showing the capacity change of the cell membrane of oocytes as a function of the voltage applied to the membrane for three different lipophilic anions.

A: DPA (compound 1)

The lower two curves represent the results of the control measurements in the absence of the lipophilic ion DPA at the beginning (squares) and at the end (triangle with the top down) of the measurement series. The top curve (filled circles) shows the results in the presence of extracellular 50 μM DPA and the underlying curve (triangle topped) in the presence of extracellular 50 μM DPA and 2.8 mM BaCl ₂ .

B: WO (compound 2)

The lower two curves represent the results of the control measurements in the absence of the lipophilic ion WO at the beginning (squares) and at the end (triangle with the top down) of the measurement series. The top curve (triangle with the tip up) shows the results in the presence of extracellular 10 μM WO and 2.8 mM BaCl ₂ and the underlying curve (solid circles) in the presence of extracellular 10 μM WO.

C: WW (connection 3, LTC)

The lower two curves represent the results of the control measurements in the absence of the lipophilic ion WW at the beginning (squares) and at the end (triangle with the tip down) of the measurement series. The top curve (solid circles) shows the results in the presence of extracellular 10 μM WW and the underlying curve (triangle with the tip up) in the presence of extracellular 10 μM WW and 2.8 mM BaCl ₂ .

5 Time courses of relative volumes of activated B lymphocytes (A) and Jurkat cells (B and C) in highly hypotonic fusion media (75 and 100 mOsm, respectively) containing either sorbitol or trehalose as the major osmotic agent. In hypotonic trehalose medium (filled circles), both cell types showed RVD after the first rapid swelling. In contrast, hypotonic sorbitol eliminated RVD in Jurkat cells (data not shown) or even induced a secondary swelling (A, open circles). The tungsten complex LTC partially or completely inhibited RVD in B lymphocytes in Jurkat cells (squares in A and B, respectively). Arachidonic acid picked up the RVD in Jurkat cells (C, squares).

6 Complete or partial inhibition of RVD in mammalian cells by various structurally unrelated lipophilic anions (compounds (1) (DPA), (2) (WO) and (4) TPB) (AC). In contrast, the lipophilic cation tetraphenylphosphonium (TPP, 10-50 μM) did not affect the RVD (C, squares).

• A: H7-Zellen wurden 15-20 min in isotonen oder hypotonen Sorbitmedien (ausgefüllte bzw. leere Kreise) derselben Leitfähigkeit von 120 μS/cm inkubiert. Im Vergleich zu isotonen Bedingungen führte hypotoner Stress zu einer deutlichen Verschiebung des zytosolischen Peaks f_c2 zu einer niedrigeren Frequenz, was eine signifikante Verringerung der zytosolischen Leitfähigkeit σ_i anzeigt.
• B: Im Vergleich zur unbehandelten Kontrolle (offene Kreise) verursachte 10 μM LTC einen zusätzliche Antifeld-Peak (f_LTC) und verschob auch die f_c2 zu einer höheren Frequenz. Der letztere Effekt ist auf verringerten Elektrolytverlust aus dem Zytosol zurückzuführen.

7 Typical rotational spectra of H7 and Jurkat cells (A and B, respectively).

• A: H7 cells were incubated for 15-20 min in isotonic or hypotonic sorbitol media (filled or empty circles) of the same conductivity of 120 μS / cm. Compared to isotonic conditions, hypotension resulted in a significant shift of the cytosolic peak f _c2 to a lower frequency, indicating a significant reduction in cytosolic σ _i .
• B: In comparison to the untreated control (open circles), 10 μM LTC caused an additional anti-field peak (f _LTC ) and also shifted the f _c2 to a higher frequency. The latter effect is due to decreased electrolyte loss from the cytosol.

Tabelle 2. Zusammensetzung von Medien, die in dem verbesserten Elektrofusionsprotokoll verwendet wurden

Tabelle 3. Elektrische Eigenschaften von H7- and Jurkat-Zellen gemäß ROT-Spektren, die in isotonen und hypotonen Medien unterschiedlicher Zuckerzusammensetzung gemessen wurden

• ^aDie mittleren C_m-, σ_i- and ε_i Werte (±SA) wurden durch Anpassung des Einschalenmodells an die ROT-Spektren der in 6 gezeigten Zellen (nicht mit LTC^- behandelt) erhalten. Hinsichtlich weiterer Details siehe Legende zu 6.
• ^bDie ν-Werte wurden durch Volumenmessungen erhalten und repräsentieren das relative Zellvolumen, gemessen 20-25 min nach dem hypotonen Schock.

8th Effect of LTC on the Electrojection of Propidium Iodide Dependence of the electrically induced PI uptake on the applied field strength E ₀ with a pulse duration of 40 μs. The data points are the mean PI acquisition values obtained from three independent flow cytometric determinations. Within the range of field strength examined, PI uptake in cells treated with 10 μM LTC (solid circles) was greater than control (open circles). Table 1. Composition of washing and pulse solutions used for electrotransfection

Table 2. Composition of media used in the improved electrofusion protocol

Table 3. Electrical properties of H7 and Jurkat cells according to ROT spectra measured in isotonic and hypotonic media of different sugar composition

• ^a The mean C _m , σ _i and ε _i values (± SA) were obtained by fitting the shell model to the ROT spectra of the in 6 cells shown (not with LTC ^- treated) was obtained. For more details see legend 6 ,
• ^b The ν values were obtained by volume measurements and represent the relative cell volume, measured 20-25 min after the hypotonic shock.

Legend to Table 4:

• ^a T is the duration of the hypotonic treatment before the first electropulse; ν (T) is the relative cell volume at time T of the pulse application after the hypotonic shock.
• ^b The TA and GE values represent the percentage or geometric mean fluorescence intensity of GFP positive cells, as determined from GF histograms. GE geometric mean values of non-transfected cells (ie, autofluorescence) were 3 ± 0.3 au. The data represent the mean ± SA of 3-9 experiments.
• ^c PF is the number of viable cells 48 h after electro-transfection, expressed as a percentage of the original number of cells.
• ^d The transfection license TE is defined as TA × PF in percent. TE is proportional to the number of live cells expressing the reporter gene.

The explain the following examples special embodiments However, without limiting it to the present invention.

EXAMPLE 1

/ Transfection electroporation of cells whose plasma membrane has been doped with lipophilic anions

For electrotransfection, Jurkat T lymphocyte cells, HEK 293 cells and mouse fibroblast L929 cells were incubated in complete Complete Growth Medium (abbreviated CGM) supplemented with 10% (v / v). fetal calf serum, cultured at 37 ° C under 5% CO _2. Before electro-transfection, the adherent cells (ie, HEK 293 and L929 cells) were detached with 0.5 mg / ml trypsin without EDTA to prevent contamination of the pulse medium with this chelator The enzyme was removed by washing with CGM.

• 1. Waschen der Zellen mit einer isotonen Waschlösung (IWS-P, siehe Tabelle 1);
• 2. Suspension von Zellen (10⁶ Zellen/ml) in einer hypotonen Pulslösung (HPS, Tabelle 1), enthaltend 10 μg/ml pEGFP-C1 (Clontech, Heidelberg, Deutschland), kodierend für ein grünes Fluoreszenzprotein (GFP);
• 3. Inkubation der Zellen für 2 oder 10 min. in HPS;
• 4. Applikation eines einzigen exponentiell abnehmenden Pulses bei den Zellproben (~800 μl) bei Raumtemperatur (RT etwa 22-24°C) unter Verwendung des Multiporators (Eppendorf Hamburg, Deutschland) und steriler Küvetten mit planaren Aluminiumelektroden in einem Abstand von 4 mm;
• 5. Nachinkubation der Zellen in Pulsmedium für 10 min bei RT;
• 6. Sanfte Überführung der Zellen in 5 ml vorgewärmtes CGM und 2tägige Kultivierung, um die maximale GFP-Epression erzielen;
• 7. Analyse mittels Durchflußzytometrie und elektronischer Zellzählung.

The electrotransfection / poration protocols included the following steps:

• 1. washing the cells with an isotonic wash solution (IWS-P, see Table 1);
• 2. suspension of cells (10 ⁶ cells / ml) in a hypotonic pulse solution (HPS, Table 1) containing 10 μg / ml pEGFP-C1 (Clontech, Heidelberg, Germany) encoding a green fluorescent protein (GFP);
• 3. Incubate the cells for 2 or 10 min. in HPS;
• 4. Application of a single exponentially decreasing pulse to the cell samples (~ 800 μl) at room temperature (RT about 22-24 ° C) using the Multiporator (Eppendorf Hamburg, Germany) and sterile cuvettes with planar aluminum electrodes at a distance of 4 mm;
• 5. re-incubation of the cells in pulse medium for 10 min at RT;
• 6. Gentle transfer of the cells to 5 ml prewarmed CGM and 2 days of culture to achieve maximal GFP expression;
• 7. Analysis by flow cytometry and electronic cell counting.

L929 cells were given a pulse of 3 kV / cm field strength and 100 μs duration in 100 mOsm pulse media (HPS, Table 1) electrotransfected. Jurkat cells were at 1.2 kV / cm and 40 μs transfected in 100 mOsm HPS. HEK cells were injected at 1.5 kV / cm and 70 μs in Treated 150 mOsm HPS pulse media.

to Investigation of the effect of a lipophilic anion herichtlich the Avoidance or minimization of unwanted electrolyte losses and the stabilization of cell volume became different poration media with 10 μM the lipophilic tungsten complex of the above formula 3, abbreviated LTC ( "Lipophilic tungsten complex ") added.

table Figure 4 shows the results obtained with different medium compositions and time conditions for the hypotonic shock was received.

The Adding the LTC resulted in especially with the longer ones Incubation times in hypotonic media, to a significant increase the transfection efficiency.

The improved transfection yields could also be confirmed in additional experiments with electroinjection of the marker propidium iodide ( 8th ). The pulsed media contained 25 μg / ml propidium iodide (PI), a cationic, normally non-membrane permeable dye, which was named Bin on nucleic acids shows a strong fluorescence. PI fulfilled two tasks, first as a dye that allows live cells to be distinguished from dead (short term vitality test) and secondly as an indicator of transient electrically induced membrane permeability. The PI staining of the cells was analyzed by flow cytometry within 10-15 minutes after pulse application.

EXAMPLE 2

Electrofusion of cells

For the electrofusion, human B lymphocytes were isolated from the peripheral blood of healthy donors and activated with 2.4 μg / ml phytohemagglutinin (PHA-L) as known. The activated cells were cultured at 37 ° C in a 5% CO ₂ enriched atmosphere. When the cell density exceeded ~ 5 x 10 ⁶ cells / ml, the cells were diluted to a density of 1 x 10 ⁶ cells / ml. The activated B lymphocytes were fused to the human mouse heteromyeloma cell line H73C11 (hereinafter referred to as H7 cells) grown under standard conditions.

For electrofusion, B lymphocytes and H7 cells were mixed in CGM in a 1: 1 ratio, pelleted by centrifugation at 200 x g for 10 min, and in a hypotonic, sugar-supplemented (HFS) osmolality solution of 75 or 100 mOsm (see Tab. 2) resuspended. The cells were then washed twice with HFS (recentrifugation at 200 x g for 10 min and resuspension). 200 μl of the cell suspension in HFS were pipetted into a helical chamber consisting of a Perspex tube around which two platinum wires were wound at a distance of 200 μm. The final cell density was 3 × 10 ⁵ cells / ml. According to this standard protocol, the cells were exposed to hypotonic stress for at least twenty minutes during the washing steps before the first fusion pulse was applied. In the improved protocols, the duration of hypotensive stress was significantly reduced by using isotonic wash solutions (IWS-F, Table 2).

The Eppendorf Multiporator was used for electrofusion. The cells were first approximated dielectrophoretically by an alternating field of 45 V _PP amplitude and 2 MHz frequency for 30 s. Thereafter, the RF field was turned off and fusion initiated by three rectangular DC pulses of 30 V amplitude and 15 μs duration. Then again a 2 MHz field of 5 V _PP amplitude was applied to hold the cells in place during the ensuing fusion process. The helical chamber was then held at RT without disturbance for 10 min. The chamber was then rinsed with 1 ml of isotonic CGM and cells were placed in 4 wells of a 24-well plate filled with 1 ml each of isotonic CGM. After 24 hours, the selection HAT medium in which only the hybridoma cells proliferated was added to the wells. Hybridoma colonies were counted 1-3 weeks after electrofusion.

The Presence of lipophilic anions, e.g. WW, increased the efficiency and reproducibility electrofusion, especially at prolonged duration of the hypotonic Stresses, clearly. In particular, a reproducible high Yield of viable Obtained hybrid cells.

EXAMPLE 3

determination the lipophilic anion-induced membrane capacitance course in oocytes depending on the membrane voltage

Measurements were made on Xenopus laevis oocytes using the two-electrode voltage clamp technique in ND96 solution. To calculate the membrane capacity Cm (U _H ) in the presence and absence of the lipophilic anions, voltage ramp protocols were programmed with the Clampex 9.2 ^® software. In these, the potential was first clamped to a certain value and voltage ramps (dU / dt) were driven to defined voltages. (Measurements were also made on the natural membrane voltage without voltage clamp).

The ND96 solution contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl ₂ , 1 mM MgCl ₂ , 5 mM Hepes, and was adjusted to pH 7.4 with NaOH. For ND96 + 2.8 mM BaCl ₂ , 1.8 mM CaCl ₂ and 1 mM MgCl _{2 were} replaced by 2.8 mM BaCl ₂ .

For the measurements Collagenase-treated stage IV-VI oocytes were used.

The results of the measurements with three different lipophilic anions (compounds 1 to 3) are in 4 shown.

The Determination of the voltage dependence the capacity increase ("bell curve") for the ions 1 to 3 shows that DPA and WO have nearly identical bell curves. By contrast, points WW (LTC) a strongly shifted bell curve, with a maximum from -130 mV (compared to -70 mV and -80 mV at DPA and WO, respectively).

It is therefore to be assumed that at normal cell potentials in the case of WO and DPA there is a distribution of the lipophilic ions (V _M = -30-70 mV), which is close to the uniform distribution. On the other hand, under normal cell conditions, LTC (WW) should be unevenly accumulated on the side of the cytosol.

Quoted literature

• Prince, J., Gschwentner, M., Ritter, M., Botta, G., Jakab, M., Mayer, M., Garavaglia, L., Bazzini, C., Rodighiero, S., Meyer, G., Eichmüller, S., Woell, E., Paulmichl, M. 2002. Molecular and functional aspects of anionic channels activated during regulatory volume decrease in mammalian cells. plowman Arch. -Eur. J. Physiol. 444: 1-25
• Jones, T.B. 1995. Electromechanics of Particles. Cambridge University Press, New York
• Kirk, K. 1997. Swelling-activated organic osmolyte channels. J. Membr. Biol. 158: 1-16
• Lang, F. 1998. Cell Volume Regulation, Karger, Basle
• Miller, K.J., Sukhorukov, V.L., Zimmermann U. 2001. Reversible electropermeabilization of mammalian cells by high-intensity, ultra-short pulses of submicrosecond duration. J. Membr. Biol. 184: 161-170
• Reuss, R., Ludwig, J., Shirakashi, R., Ehrhart, F., Zimmermann, H., Schneider, S., Weber, M. M., Zimmermann, U., Schneider, H., Sukhorukov, V. L. 2004. Intracellular delivery of carbohydrates into mammalian cells through swelling-activated pathways. J. Membr. Biol. 200: 67-81
• Strange, K., 1994. Cellular and Molecular Physiology of Cell Volume Regulation, CRC, Boca Raton, FL
• Sukhorukov, V.L., Mussauer, H., Zimmermann, U. 1998. The effect of electrical deformation forces on the electropermeabilization of erythrocyte membranes in low-and high-conductivity media. J. Membrane Biol. 163: 235-245
• Zimmermann, U., Friedrich, U., Mussauer, H., Gessner, P., Hämel, K., Sukhorukov, V.L. 2000. Electromanipulation of mammalian cells: fundamental and application. IEEE Trans. Plasma Sci. 28: 72-82
• Zimmermann, U., Neil, G.A. 1996. Electromanipulation of Cells, CRC, Boca Raton, FL

Claims (33)

Method of treating cells with electric fields to modify the membrane permeability of these cells, comprising the steps of: - providing at least one cell ( 1 ) in a treatment medium ( 2 ), and - loading the cell ( 1 ) with at least one electrical voltage pulse, characterized by the step: - addition of lipophilic ions to the treatment medium ( 2 ). Method according to claim 1, which is an electromanipulation, in particular electroporation or electrofusion, which represents cell (s) or includes. The method of claim 1 or 2, wherein the cells prokaryotic or eukaryotic cells. The method of claim 3, wherein the cells are live Cells are. The method of claim 1 or 2, wherein the cells natural or artificial membrane- Vesicles, liposomes or micelles are. Process according to at least one of Claims 1 to 5, in which the lipophilic ions are added to the treatment medium ( 2 ) in a concentration ranging from 1 nM to 100 μM. Process according to at least one of Claims 1 to 6, in which the addition of the lipophilic ions to the treatment medium ( 2 ) leads to a doping of the membranes of the cells to be treated with these lipophilic ions. Method according to at least one of claims 1 to 7, wherein the lipophilic ions are cations, anions or a mixture made of cations and anions. Method according to at least one of claims 1 to 8, in which the lipophilic ions are anions. The method of claim 9, wherein the lipophilic Anions contain at least one substance from the group of anions, the anions of lipophilic metal complexes, lipophilic organometallic Compounds and lipophilic organic compounds with at least a deprotonatable functional group. The method of claim 10, wherein the lipophilic anions contain at least one of anionic group, the anion of lipophilic tungsten complexes (LTC), especially lipophilic tungsten carbonyl complexes, dipicrylamine (DPA) and derivatives thereof, derivatives of lipophilic organic compounds containing at least a carboxylate, sulfonate, sulfate, thiosulfate or thiocyanate group, in particular arachidonic acid, or anions of an optionally substituted tetraphenylborate or Phenyldicarbaundecandecaborans (PCB) or a Tetracyanoborats [B (CN) ₄ ] ^- or Tetratrifluormethylborats [B (CF ₃ ) ₄ ] ^- includes. The method of claim 11, wherein the lipophilic anions are selected from the group of the following anions

are selected. Method according to at least one of the preceding Claims, in which the lipophilic ions for living cells are physiologically compatible. Method according to at least one of the preceding Claims, in which the lipophilic ions are degradable by UV light and for living Cells physiologically acceptable Degradation products result. Method according to at least one of claims 1 to 14, in which the lipophilic ions have the membrane capacity C of the Cell in dependence increase from the membrane voltage of the cell. Method according to Claim 15, in which the dependence on the membrane voltage has the behavior of a symmetrical or asymmetrical bell curve with a voltage V _max at which the capacitance increase C (V _max ) is maximal. Method according to at least one of the preceding claims, in which a selection of the lipophilic ions is provided in a preliminary test, comprising the steps of: measuring the electrical membrane capacity of the biological cell as a function of an applied membrane voltage, wherein the voltage dependence of the membrane capacity induced by lipophilic ions several different lipophilic ions in the treatment medium ( 2 ) and determination and selection of the lipophilic ions for which the lipophilic ion-induced membrane capacity of the cell ( 1 ) fulfills a predetermined selection criterion. Process according to Claim 17, in which the lipophilic ions are selected for which the membrane capacity at the natural membrane voltage of the cell ( 1 ) is minimal or at one of the falling edges of the bell curve defined in claim 16. Process according to Claim 17, in which the lipophilic ions are selected for which the membrane capacity at the natural membrane voltage of the cell ( 1 ) has a maximum. Method according to at least one of claims 17 to 19, with the step: - adding a salt into the treatment medium ( 2 ) for adjusting the natural membrane voltage of the cell ( 1 ). Method according to at least one of the preceding claims, with the step: - Addition of an osmotically active substance into the treatment medium ( 2 ) for generating hypotonic stress for the at least one biological cell ( 1 ). Process according to Claim 21, in which the application of the biological cell ( 1 ) with the electrical voltage pulse with a delay time after the addition of the osmotically active substance, wherein the delay time in the range of 15 s to 10 min is selected. Use of lipophilic ions for addition into a treatment medium ( 2 ) in which cells are subjected to treatment with electric fields involving an electrical voltage pulse. Use according to claim 23, wherein the cells prokaryotic or eukaryotic cells. Use according to claim 24, wherein the cells are living cells. Use according to claim 25, wherein the cells natural or artificial membrane- Vesicles, liposomes or micelles are. Use according to at least one of claims 23 to 26, in which treatment with electric fields electroporation or electrofusion. Use according to at least one of claims 23 to 27, where the lipophilic ions are anions. Use according to claim 28, wherein the lipophilic Anions contain at least one substance from the group of anions, the anions of lipophilic metal complexes, lipophilic organometallic compounds and lipophilic organic compounds having at least one deprotonatable includes functional group. Use according to claim 29, wherein the lipophilic anions comprise at least one of anionic groups, the anions of lipophilic tungsten complexes (LTC), in particular lipophilic tungsten coxbonyl complexes, dipicrylamine (DPA) and derivatives thereof, derivatives of lipophilic organic compounds containing at least a carboxylate, sulfonate, sulfate, thiosulfate or thiocyanate group, in particular arachidonic acid, or anions of an optionally substituted tetraphenylborate or Phenyldicarbaundecandecaborans (PCB) or a Tetracyanoborats [B (CN) ₄ ] ^- or Tetratrifluormethylborats [B (CF ₃ ) ₄ ] ^- includes. Use according to claim 30, wherein the lipophilic anions are selected from the group consisting of the following anions

are selected. Method according to at least one of the preceding Claims, in which several lipophilic ions of different nature in the same or different concentrations at the same time or in time sequence are used. Lipophilic metal complexes selected from the following group: