WO1999010337A1 - Tetraether lipid derivatives and liposomes and lipid agglomerates containing tetraetherlipid derivatives, and use thereof - Google Patents

Abstract

Conventional liposomes which are used for transporting pharmaceutical active agents in eukaryotic cells or for lipofection can only be preserved for a limited period, are not acid-stable and require a number of set parameters in order to achieve satisfactory results. Less sensitive liposomes are therefore highly desirable. The inventive tetraetherlipid derivatives are very stable and are well suited to lipofection.

Description

 Liposomes and lipid agglomerates containing tetraether lipid derivatives and tetraether lipid derivatives and their use

The present invention relates to tetraether lipid derivatives, the liposomes and lipid agglomerates containing the tetraether lipid derivatives according to the invention and their use.

Scientific research has a great need for methods for the transfection of cells in cell culture and multicellular organisms with nucleic acids. The conventional methods such as electroporation, DEAE and "calcium phosphate"-assisted transfection, microinjection or ballistic methods have the disadvantage that they often only achieve low transfection efficiencies, the cell survival rates are very low and / or that they cannot be carried out on multicellular cells. Viral and retroviral transfection systems are more efficient, but involve their own risks, such as an increased immune response or uncontrolled integration into the target genome. The transfection of non-viral nucleic acids with the help of liposomes, also called lipofection, therefore represents a successful and already frequently used alternative to the methods described above.

Liposomes are artificially produced uni- or multilamellar lipid vesicles that enclose an aqueous interior. They are generally similar to biological membranes and are therefore often easily integrated into the membrane structure after being attached to them. With this membrane fusion, the contents of the liposome interior are discharged into the lumen enclosed by the biological membrane. Alternatively, the liposomes are brought into the cytosol of the cell to be transfected by endocytotic processes; they are then either destroyed in the cytosol or, as such, interact with the core membrane. In the latter case, the compounds contained in the aqueous interior of the liposome are largely protected against proteolytic or nucleolytic attacks.

Liposomes can therefore be used as transport vehicles for substances such as nucleic acids and pharmaceuticals. For example, the cosmetics industry produces liposome-containing skin creams that target active ingredients into the epidermis and deeper cell layers. For the production of liposomes, mainly natural Before soybean or egg yolk lecithins or defined natural or artificial phospholipids such as cardiolipin, sphingomyelin, lysolecithin and others are used. By varying the polar head groups (choline, ethanolamine, serine, glycerol, inositol), the length and the degree of saturation of the hydrocarbon chains, size, stability and ability to take up and release the associated molecules are influenced.

One of the main disadvantages of the liposomes known to date is their poor stability. Liposomes formed from normal double-layer forming phopholipids can only be kept for a short time even in the cooled state. Their storage stability can be e.g. by including phosphatidic acid, but the stability thus improved is still insufficient for many purposes. In addition, conventional liposomes are not acid-stable and are therefore unsuitable for the transport of active pharmaceutical ingredients that pass through the stomach after oral administration, nor for liposome-assisted DNA transfection under slightly acidic pH conditions.

For scientific and medical lipofections in mammalian cells, liposome-forming lipid mixtures, e.g. Lipofectamin®, Lipofectin® or DOTAP®, often used. In addition to the disadvantages already mentioned, their use also means the need to precisely determine a large number of parameters (e.g. cell density, nucleic acid amount, proportion of lipids added, volume of liposome preparation, etc.) because there is only a very narrow parameter optimum with sufficient Transfektioπseffizienzen can be achieved. This makes transfections using commercial lipofection reagents very complex and cost-intensive. Furthermore, large variations between the individual batches can be observed with the above-mentioned products, which makes them less reliable in practice.

It is therefore an object of the present invention to produce lipofection agents with greater mechanical and chemical stability and thus an extended shelf life, which enable simple and reliable use.

This object is achieved according to the invention by providing tetraether lipid derivatives (hereinafter also abbreviated as "TEL derivatives") with the general formula (I): S ¹

where S ¹ and S ^{2 may be the} same or different and each has the following meaning:

and

Y can mean -NR ^ R ^J or -N ^ R 4 ₀ T5rR-> 6 ";

X ¹ and X ² may be the same or different and are each independently selected from the group comprising a branched or unbranched alkylene or alkenylene having 2 to 20 carbon atoms;

R ¹ to R ^δ may be the same or different and are each independently selected from the group consisting of: hydrogen, branched or unbranched alkyl, alkenyl, aralkyl or aryl groups having 1 to 12 carbon atoms, where in each case one of the radicals R ² to R ^{6 can} further comprise an antibody against cell surface molecules or a ligand for cell surface receptors; and

π can be an integer between 0 and 10,

and modifications thereof formed by the formation of pentacycles in the basic tetraether structure.

The lipid structure of the tetraether lipid derivatives according to the invention consists of a 72-membered macrotetraether cycle, the basic structure of which is a dibiphytanyl-diethyl-tetraether heterocycle. In this, the ra carbon atoms of the phytanyl chain of 2 diether molecules are covalently linked to one another. Tetraether lipids are already known and have so far only been found in archaebacteria. Depending on e.g. from the growth temperature, pentacycles can form within the dibiphytanyl chains, which give the lipid a specific physico-chemical character. With every pentacyclization, the basic structure loses two hydrogen atoms. A summary of all previously known basic structures of archaebacterial lipids is contained in Langworthy and Pond (System Appl. Microbiol. 7, 253-257, 1986).

The naturally occurring lipid structure has now been derivatized according to the invention in order to be suitable for incorporation into liposomes or lipid agglomerates intended for transfection. For this purpose, side chains are introduced which are positively charged either per se through the formation of quaternary ammonium salts or under physiological conditions, ie at a pH of 7.35 to 7.45. Lipids derivatized in this way are particularly suitable for contacting negatively charged molecules, for example nucleic acid molecules, and for example enclosing them in liposomes. Since a possible use of the lipids according to the invention is in the formation of liposomes or lipid agglomerates for therapeutic applications, the lipids according to the invention can additionally be coupled with molecules which enable the lipids to be specifically docked onto special cells. Examples are antibodies against cell surface antigens, especially those that are selectively expressed on the target cells. Also possible are ligands for receptors that can be found selectively on the surface of certain cells, and biologically active peptides that enable organ or cell-specific targeting in vivo (Ruoslati, Science, 1997). The latter were also referred to as ligands for the purposes of this application.

The particular advantage of the tetraether lipid derivatives according to the invention is that their lipid structure contains no double bonds and is therefore insensitive to oxidation. Furthermore, instead of the lipid ester bonds contained in lipids from eubacteria and eukaryotes, it only contains lipid ether bonds which are also present at high proton concentrations, as e.g. occur in the stomach, not be attacked.

In preferred embodiments, the substituents π S ¹ and S ² are the same at both ends of the tetraether lipid backbone. Based on natural tetraetheriipids, this enables synthesis without the interim use of protective groups. The identity of the substituents S ¹ and S ² is particularly preferred in those cases in which none of the radicals R ¹ to R ^{6 is} an antibody or ligand for a cell surface receptor.

In a preferred embodiment of the tetraether lipid derivative according to the invention, the group X ^{1 in} both S ¹ and S ^{2 is} an alkylene or alkylene having 2 to 10, preferably 3 to 6, carbon atoms. In very particularly preferred embodiments, X ¹ is Propylene.

The group X ² is also preferably an alkylene or alkenylene with 2 to 10, preferably with 3 to 6, carbon atoms. Propylene radicals are also particularly preferred for X ² .

n can mean 0 to 10. In preferred embodiments, n is 0 to 3, very particularly preferably 0.

The tetraether lipid derivatives according to the invention are preferably produced from natural tetraether lipids which can be isolated, for example, from archaebacteria. As already mentioned above, pentacycles within the dibiphytanyl chains occur to a certain extent in the tetraetheriipids isolated from natural sources. The extent of Pentacyclization can be influenced by the growth temperature. There are normally 0 to 8 pentacycles per tetraether backbone, with most lipid molecules having between 1 and 5 pentacycles at a cultivation temperature of 39 °, while predominantly 3 to 6 pentacycles are observed at a cultivation temperature of 59 °. The following table provides information on the distribution of the pentacyclization at a cultivation temperature of 39 ° C or 59 ° C:

TABLE

In further embodiments of the tetraether lipid derivative according to the invention, Y means both in S ¹ and in S ² -NR ² R ³ . R ² and R ^{3 are} preferably hydrogen, branched or unbranched alkyl, alkenyl, araikyl or aryl groups, particularly preferably hydrogen, methyl, ethyl or propyl groups. In a further preferred embodiment, Y in both S and S ^{2 is} a quaternary ammonium salt, the radicals R ⁴ , R ⁵ and R ^{6 of which are} preferably also hydrogen, branched or unbranched alkyl, alkenyl, araikyl or aryl groups with 1 to 12 carbon atoms, particularly preferably hydrogen, methyl, ethyl or propylene. In each case one of the radicals R ² to R ⁶ can comprise an antibody against cell surface molecules or a ligand for cell surface receptors.

In particularly preferred embodiments, the tetraether lipid derivative according to the invention has the general formula (I) with the substituents S ¹ and S ² given below: Connection A:

S ¹ and S ² : -CO-NH- (CH ₂ ) ₃ -NH ₂ ;

Compound B:

S ¹ and S ² : -CO-NH- (CH ₂ ) ₃ -N (CH ₃ ) ₂ :

Compound C:

S ¹ and S ² : -CO-NH- (CH ₂ ) ₃ -N ^Θ (CH ₃ ) ₃

The tetraether lipid derivatives according to the invention can be obtained, for example, from the total lipid extract of archaebacteria, for example the total lipid extract of the archaebacterium Thermoplasmic acidophilum. Thermoplasmas grow between pH 1 and 4 and at temperatures from 33 to 67 ° C. Thermoplasma acidophilum can, as shown in Example 1, be cultivated. Usually a culture of the microorganism grown up to an OD ₅₇ a of about 0.6 is harvested and the harvested microorganisms are either used directly for lipid production or freeze-dried and stored. To obtain the tetraether lipids from the total lipid extract of Thermoplasma acidophilum, the latter is subjected to acid hydrolysis in order to produce tetraethers (Example 2 and Fig. 7). Oxidation of the free hydroxyl groups on the tetraether to dicarboxylic acid compounds, reaction of these dicarboxylic acid compounds with SOCI ₂ and reaction of the carboxylic acid chlorides formed with Amineπ results in the formation of various tetraether lipid derivatives (Example 3 and Fig. 7).

The TEL derivatives according to the invention can be used to produce lipofection agents. Lipofection agents are, for example, liposomes or lipid agglomerates. Methods of making liposomes are well known. In general, the lipid intended for the preparation of the liposomes is first dissolved in an organic solvent and a lipid film is formed by evaporation. The lipid film is dried well to remove all solvent residues. Then the Lipids of this film are resuspended in a suitable buffer system. For medical-pharmaceutical purposes, for example, physiological saline, pH 7.4, is suitable, but other buffer systems (for example Mcllvaine buffer) or unbuffered solutions, such as, for example, unbuffered potassium or sodium chloride solutions, can also be used. The amount of buffer should be calculated so that it later results in a liposome dispersion with a maximum of 15-20 mg lipid / ml buffer. By shaking by hand, large multilamellar vesicles with a size distribution in the μm range can first be formed. The formation of these vesicles can be facilitated by using two glass spheres and / or an ultrasound bath with low sound intensity. The following methods were tested for the actual preparation of unilameilar liposomes of a defined size and were found to be suitable for the production of liposomes from tetraether lipid derivatives according to the invention:

(a) Ultrasound treatment: The suspension of multiiamellar liposomes is 5 at 20 kHz

Minutes sonicated (Braπson Sonifer B 15). Liposomes with a diameter of around 500 nm are formed.

(b) Extrusion through a French pressure cell: The suspension of multiiamellar liposomes is at 16000 p.s.i. extruded four times in the French pressure cell (SLM-Aminco Inc., Urbana IL, USA). Liposomes with a diameter of around 120 nm are formed.

(c) Extrusion through polycarbonate filters: The suspension of multiiamellar liposomes is extruded through polycarbonate filters of a defined pore size (LiposoFasf ¹ , Avestin, Ottawa, Canada). When using filters with a pore size of 100 or 200 nm, liposomes are formed which have a size distribution slightly above the pore size. Extrusion through filters of this small pore size can simultaneously serve as sterile filtration, provided all other requirements for sterile filtration (for example, exact separation of the sterile and sterile sides of the filtration apparatus) are met. A few preparations can be made to facilitate the actual filtration:

(ca) The suspension of multiiamellar liposomes is sonicated at 20 kHz for between 1 and 5 minutes (see (a)) (cb) The suspension of multiiamellar liposomes is frozen 1-3 times and thawed again.

(cc) The suspension of multiiamellar liposomes according to (ca) or (cb) is prefiltered through a polycarbonate filter with a pore size of 600 to 800 nm.

Following the ultrasound treatment or extrusion, the liposomes can be centrifuged in a 3200 Eppendorf centrifuge for 10 minutes in order to remove non-liposomal material. Intact, closed vesicles remain in the supernatant.

Liposomes can be further produced by detergent solubilization with subsequent detergent dialysis. For this purpose, a lipid film is first formed as described above. This is suspended in a detergent-containing buffer system (examples of dialysable detergents: octyl-β-D-glucopyranoside or octyl-β-D-thioglucopyranoside). The molar ratio (TEL derivative: detergent) for the detergents mentioned should be between 0.05 and 0.3 and the amount of buffer should be calculated in such a way that a liposome dispersion with a maximum of 15-20 mg lipid per ml buffer results later. Mixing micelles from detergent and TEL derivative is formed by shaking by hand.

The suspension of the mixed micelles is now in dialysis tubes, e.g. transferred into a Lipoprep® dialysis cell or into a Mini-Lipoprep® dialysis cell (Diachema AG, Langnau, Switzerland) and dialyzed at RT for 24 hours. When the detergent is removed by dialysis, the mixed micelles form liposomes with a diameter of approximately 400 nm.

The liposome preparation can be centrifuged in a 3200 Eppendorf centrifuge for 10 minutes to remove non-liposomal material. Intact, closed vesicles remain in the supernatant.

The lipid agglomerates according to the invention consist of one or more layers of the TEL derivatives according to the invention. Negatively charged molecules, for example nucleic acid molecules, can be sandwiched between two such layers. Lipofection agents, which consist 100% of TEL derivatives according to the invention, have proven to be exceptionally stable, storable almost indefinitely and permeable to protons. For many applications, including the formulation of pharmaceuticals with acid-labile active ingredients and the transfection of eukaryotic cells, pure TEL-derivative lipofection agents are therefore the means of choice.

It has also proven to be advantageous to prepare lipofection agents which, in addition to a proportion of TEL derivative, contain conventional double-layer-forming phospholipids, this percentage by weight being based on the total lipid (mixed liposomes or mixed lipid agglomerates). The production of mixed lipofection agents is carried out analogously to the production of pure TEL derivative lipofection agents.

These double-layer-forming phospholipids can be, for example, sphingomyeline, cephaline, lecithin and cardioiipin. The addition of cationic lipids such as DOTMA (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride) (Life Technologies, Gaithersberg, USA) or DOTAP (N- [1- (2,3-Dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride) (Boehringer Mannheim, FRG) or DOSPER (Boehringer Mannheim). To support the transfection efficiency, neutral lipids such as e.g. DOPE (dioleoylphosphatidylethanolamine) (Sigma) can be included in the preparation of the TEL derivative liposomes (Example 5). In a further preferred embodiment, cholesterol and / or its derivatives are incorporated into the membrane. In addition, any other lipid suitable for the formation of liposomes or lipid agglomerates can be used. The particular advantage of the mixed lipofection agents according to the invention is their improved (increased) stability due to the TEL derivative component, compared e.g. with liposomes without TEL derivative content.

In the mixed liposuction agents according to the invention, the ratio of tetraether lipid derivative to further lipids is preferably 5: 1 to 1: 5. In particularly preferred embodiments, the ratio is 1: 2 to 1: 0.5, particularly preferably 1.1. All information relates to weight ratios.

In the following, the terms "TEL derivative liposome", "Liposome from pure TEL derivative" etc. refer to liposomes whose lipid content is 100% from the invention TEL derivatives, preferably TEL consists of Thermoplasma acidophilum. The same applies to the terms "tetraether lipid derivative agglomerate" and related terms.

"Mixed liposomes" and "mixed lipid agglomerates" additionally include conventional phospholipids, and the terms "liposome" and "lipid agglomerates" include both liposomes and lipid agglomerates from pure TEL derivatives as well as mixed liposomes and mixed lipid agglomerates.

The liposomes according to the invention including the mixed liposomes and the lipid agglomerates including the mixed agglomerates can serve as transport vehicles for nucleic acids and / or cosmetic, pharmaceutical active substances. The liposomes and lipid agglomerates according to the invention also enable targeted gene transfer. For this, nucleic acids, e.g. DNA or RNA sequences which contain genes or gene fragments and are present in linear form or in the form of closed circular vectors which may contain further genetic material, packed in pure TEL liposomes or mixed liposomes, pure lipid agglomerates and mixed agglomerates and the ones to be transfected Cells added in vitro or in vivo. If the liposome membrane or aggiomerate surface also contains antibodies for which corresponding proteins or peptides are present on the cells to be reached by gene therapy, the contact between antigen on the target cell and antibodies in the membrane of the liposome according to the invention or Aggiomerate surface promoted contact between the liposome or aggiomerate surface and the target cell.

The invention further relates to a pharmaceutical composition which contains the liposomes or lipid agglomerates according to the invention.

The liposomes and / or lipid agglomerates according to the invention are preferably used as transport vehicles in the case of active substances in which oral administration is not possible because these active substances would be inactivated in the acidic gastric fluid or would be broken down in the small intestine by lipases or peptidases. However, the liposomes and lipid agglomerates according to the invention are acid-stable and can therefore pass freely through the stomach, for example into the small intestine, and included active substances which normally do not enter the bloodstream after oral administration can bring about resorption there. The active substances can be contained in the aqueous lumen of the liposome, be present between the layers of the lipid agglomerates or be integrated into them. If the active substance to be transported is nucleic acids, it is usually protected in the liposome or between the agglomerate layers. In a further embodiment, the active ingredient is covalently bound to the TEL contained in the liposome. Preferred active ingredients are, for example, cytostatics, immunosuppressants, immunostimulants or vaccines and hormones. The localization of the active substance in the lumen or in the membrane depends on its water or lipid solubility and is determined by the oil / water distribution coefficient. Many active substances have an intermediate distribution, ie both a certain water and lipid solubility, according to which they are distributed within a liposome on the lumen and membrane. Further examples of active ingredients dissolved in the lumen are vaccines, hormones and water-soluble components or derivatives of daunorubicin and doxorubicin or of methylprednisolone. A preferred methylprednisolone derivative is the sodium salt of methylprednisolone hydrogen succinate. In contrast, preferred covalently bound active ingredients are, for example, antibodies, hormones, lectins and interleukins or active fragments of this group of substances.

It could be shown that the liposomes or lipid agglomerates according to the invention can interact particularly well with cell membranes. E.g. the uptake of tetraetheriipid derivatives coupled with the lipophilic fluorescent dye rhodamine through the cell membrane of Baby Hamster Kidney (BHK) cells (see Fig. 1 and Example 5.2).

With the liposomes or lipid agglomerates according to the invention, tumors could therefore also be targeted, for example by tumor-specific antibodies or antibody fragments are built into the liposome membrane, which guide the liposomes of the lipid agglomerates filled with active substances into tumor cells in a targeted manner. Chemotherapy with targeted chemotherapy helps to drastically reduce unwanted side effects.

In a further embodiment according to the invention, the TEL derivatives according to the invention serve in pure form or as a constituent of pure or mixed liposomes or lipid agglomerates as the basis for the manufacture of medical ointments or skin creams.

Another possible area of application for the TEL derivatives according to the invention, for liposomes or lipid agglomerates from pure TEL derivatives from Thermoplasma acidophilum or the mixed liposomes or mixed lipid agglomerates containing these TEL derivatives is the use of these substances in medical diagnostics. Here, e.g. Microtiter plates are coated with TEL derivatives, TEL derivative liposomes or TEL derivative mixed liposomes, which have been obtained by conjugation with specific antibodies or antigens. Such coated substrates can e.g. be used for the immunometric determination of proteins, peptides or metabolic products.

The following figures and examples illustrate the invention.

Description of the pictures

Figure 1: shows microscopic images of BHK cells that have been pre-incubated with rhodamine-coupled tetraether lipid derivative B (10 μg / ml) for 70 min.

Figure 1A is a phase contrast image,

Figure 1 B shows the fluorescence, recorded with red emission and green extinction filters.

Figure 2 shows the ethidium bromide fluorescence of DNA tetraether lipid derivative complexes with different DNA: lipid ratios. The experiment on which the figure is based is described in Example 4.2.2.

Figure 3 shows an agarose gel (1%) stained with ethidium bromide, on which DNA / tetraether lipid derivative complexes with different molar ratios of DNA to tetraether lipid have been electrophoresed. The DNA applied is pSV-lacZ, the TEL derivative used was Compound B. Similar results were obtained with Compounds A and C.

The traces show in detail:

Track 1: size marker (λHindlll: 23130, 9416, 6557, 4361, 2322, 2027, 5634 and 125 bp)

Lane 2: molar DNA lipid ratio of 1 0 Lane 3: molar DNA lipid ratio of 1 0.7 Lane 4: molar DNA lipid ratio of 1 1, 4 Lane 5: molar DNA lipid ratio of 1 2.1 Lane 6: molar DNA lipid ratio of 1 2.8.

Figure 4: shows the efficiency of transfection of BHK cells with the tetraetheriipid derivative B at a constant DNA concentration of 1.4 μg / ml, depending on the lipid concentration in the medium. The same results have been obtained with the compounds A and C.

Figure 5: shows the time course of the expression of β-galactosidase after incubation of BHK cells with tetraether lipid derivative complexes which contained the plasmid pSV-lacZ (Promega) coding for β-galactosidase. 1.2 μg of DNA and 5 μg of tetraetheriipid derivative B were used per transfection approach. The same results have been obtained with the tetraetheriipid derivatives A and C.

Figure 6: shows the transfection efficiency of pure tetraether lipid derivative

Liposomes compared to mixed liposomes with DOPE (molar ratio 1: 1), PC (molar ratio 1: 1) and MPL (molar ratio tetraetheriipid derivative B to MPL as 1: 2) (MPL: "main phospholipid" from Thermoplasma acidophilum, total phospholipid fraction, see PCT / EP97 / 01011). The results with derivatives A and B are comparable. Figure 7: shows the reaction scheme for the synthesis of various TEL derivatives. The tetraether macro cycle is represented by the rectangle central to each formula. In detail shows:

Fig. 7a the implementation of a natural tetraether lipid, e.g. isolated from Thermoplasma acidophilum, with 1 molar hydrochloric acid in methanol to remove the sugar residues for dihydroxyl compound (2). Compound (2) is then converted to dicarboxylic acid (3).

Fig. 7b shows the conversion of the dicarboxylic acid (3) to the corresponding dicarboxylic acid chloride (4). The dicarboxylic acid chloride serves as a starting material for the reaction with amines. According to reaction 4a, 1,3-diaminopropane is reacted to give the tetraetheriipid derivative (A). According to reaction no. 4b, the dicarboxylic acid chloride is reacted with 3-dimethylaminopropylamine to give the tetraetheriipid derivative B.

Fig. 7c The tetraetheriipid derivative B reacts with dimethyl sulfate to give the tetraetheriipid derivative C.

example 1

Cultivation and conservation of Thermoplasma acidophilum

1.1 medium

The culture medium for Thermoplasma acidophilum is composed of Freundt's medium (Christiansen et al., (1975)), a 20% (w / v) glucose solution and a 20% (w / v) yeast extract solution (Difco). Freundt's medium is sterilized in situ at 120 ° C, the 10 l fermenter 25 min and the 50 l fermenter 45 min. The glucose and yeast solutions are sterilized separately at 110 ° C for 10 min and are only added to the medium immediately before the inoculation. 1.1.1 For the inoculation with freezer cells, 94 vol.% Freundt's medium, 5 vol.% Glucose solution and 1 vol.% Yeast extract solution are added.

1.1.2 For the inoculation with a suspension culture 84 vol .-% Freundt's medium, 5% glucose solution and 1% difol solution are combined as above.

1.2 inoculation

2.1 In the case of culture from freezer cells, 1 ml / l freezer cells are added to the medium described in 1.1. The lag phase is 2 to 3 days.

2.2 For cultures with suspension culture inoculum, 10 vol.% Suspension culture are added to the medium described in 1.2. The lag phase in this case is only a few hours.

1.3 Cultivation of Thermoplasma acidophilum

Thermoplasma acidophilum is preferably grown in 10 l or 50 l fermenters. All Fermeπterteile must be made of sulfuric acid-resistant material, e.g. Braun Biostat S (10 I, glass body), Braun Biostat 50 D (50 I, stainless steel body).

Starting from freezer cells, which have been preserved as described below, a 10 l fermenter can be inoculated directly without prior bottle cultivation. The normal conditions for fermenter cultivation are 59 ° C and pH 2. After reaching an optical density of 0.4 (at 578 nm), 1% by volume of yeast extract solution is added for the first time, and again after another 8 hours. The addition of yeast extract causes the pH in the medium to rise and is compensated for by adding appropriate amounts of 1 M sulfuric acid. After reaching an optical density (578 nm) of 0.6 to 0.7, 5 l of the 10 l fermenter culture are removed as inoculum for a 50 l fermenter. This is cultivated as usual, with 1 vol.% Yeast extract being added after 20 and 28 hours. After reaching the stationary phase, 1 liter is used as an inoculum for a further 10 l fermenter. Thermoplasma acidophilum grows aerobically, but high oxygen concentrations are not beneficial for growth. The oxygen regulation is set to 0.02 to 0.03 vvm (volume of aeration per volume of medium per minute) in the 10 l fermenter and to 0.04 vvm in the 50 l fermenter. During growth, the oxygen content of the medium decreases constantly and rapidly below the limit of measurability, but does not have to be counter-regulated. The pH is continuously measured during cultivation and regulated when there are changes in pH.

1.4 Cell harvest

The growth of Thermoplasma acidophilum reaches the stationary phase after approx. 40 hours (OD ₅₇ 8 of approx. 0.6). The harvest should therefore take place after approx. 40 hours with an OD ₅ 78 of 0.6, maximum 0.7.

The cell culture from a 10 l fermenter is centrifuged in a Heraeus stock centrifuge at 3000 x g for 15 minutes, the supernatant is discarded, the precipitate is resuspended in Freundt's solution. The suspension is centrifuged for 10 minutes in a Christian centrifuge at maximum speed (4500 rpm, corresponding to about 3000 xg), the process is repeated at least twice, with Freuπdt's solution being replaced by sulfuric acid aqua bidest, pH 2, until the Pellet is white. Finally, the white wet cell mass is bidistilled in aqua. suspended, frozen in methanol / dry ice and freeze-dried to constant weight.

The cell culture of the 50 l fermenter is concentrated to a volume of 2 l using a Pelicon tangential flow filter system. This concentrate is washed with sulfuric acid distilled water, pH 2, until the filtrate is clear and undyed. Now the concentrated cell suspension is centrifuged (see above), the pellet in double-distilled water. resuspended, recentrifuged, resuspended, frozen and freeze-dried to constant weight.

1.5 Optical control of the purity of the Thermoplasma acidophilum culture

Thermoplasma acidophilum appears in the light microscope after the above culture with a cell density of 10 ⁷ cells / ml in mostly isodiametric form with a diameter between see 1 and 3 μm, some cells have pleio orphes appearance. According to laser light scatter measurements in the Malvern particle sizer, the maximum size distribution is 2.3 μm. The cells usually appear round by freeze-fracture electron microscopy, pleio-morphic cells have longitudinal axes between 1, 1 and 2.7 μm and transverse axes around 0.6 μm. The cells show a typical fracture behavior that distinguishes them from most other cells: they break perpendicular (transverse) to the membrane plane, along the hydrocarbon chains and not along the inner interface of the double layer (tangential).

Given the growth conditions (59 ° C, pH 2), only a few organisms can grow into the culture, e.g. Bacillus acidocaldarius. Cultivation at pH 1.5 also prevents this, but is associated with a loss of yield with regard to the desired lipid component. Thus, the optimum breeding is at pH 2 while observing as sterile ("germ-reduced") conditions as possible.

1.6 Preservation

Thermoplasma acidophilum is preserved at -80 ° C or in liquid N ₂ . For preservation, an active culture (8-10 l) is adjusted to pH 5.0-5.5 by adding calcium carbonate. After sedimentation of calcium sulfate and excess calcium carbonate (let it stand still for at least 30 min), the supernatant is removed and centrifuged under sterile conditions for 15 min at 3000 xg. The supernatant is discarded and the Thermoplasma acidophilum cells of the pellet are resuspended in freshly prepared 10 mM sodium citrate buffer, pH 5.5. The suspension is portioned on ice in cryocups to 0.5 to 3.0 ml, the cryocups are frozen in liquid nitrogen for one hour and then stored therein or at -80.degree.

For cultivation, the cryocups are thawed with the cells in a water bath at 37 ° C.

It is imperative that the preservation steps are carried out sterile, since the pH of 1-2, which prevents the growth of most microorganisms during cultivation, is not maintained. Example 2

Extraction and purification of the tetraether lipids from Thermoplasma acidophilum

2.1 Extraction of total lipid

Total lipid extraction is performed with freeze-dried material. For this purpose, 8 to 10 g of freeze-dried cell mass are extracted with 300 ml of a petroleum ether (fraction 60 to 80 ° C.) / 2-propanol mixture (77:23 v / v) in a Soxhlet apparatus (recurrent, closed system) under reflux for 40 hours . The extraction described is almost quantitative and leads to approx. 1 g of pure total lipid fraction.

750 mg of the total lipid fraction from Thermoplasma acidophilum are refluxed in 800 ml of a 1 M HCl solution in methanol for 10 h. After evaporation of the solvent, the residue is dissolved in 100 ml of CHCl ₃ and washed with H ₂ O (5 x 100 ml). The CHCI ₃ fraction is evaporated and the residue is separated chromatographically on silica gel (silica gel, Merck; eluent CHCI ₃ ). The fractions are collected and analyzed on TLC plates (eluent: CHCIs / methanol 9: 1), with isolated tetraethers serving as the standard (Freisleben et al., 1993, Appl. Microbiol. Biotechnol., 40, 745-52). After evaporation of the eluent, the yield is approximately 390 mg of tetraether lipids.

Example 3

Preparation of tetraether lipid derivatives A, B and C

3.1 Manufacture of dicarboxylic acid compounds

80 mg of tetraether lipids are dissolved with stirring in a solution of 1 mg of 4-methoxy-TEMPO radical in 2 ml of CH ₂ Cl ₂ at 4 ° C. (J. Org. Chem., 1985, 50, p. 1332). 5 ml of sodium hypochlorite (0.35 M, pH 8.6) are added to the solution and the mixture is stirred vigorously at 4 ° C. for 5 minutes. After adding 30 ml of CHCI ₃ , the organic phase Washed 5 times with 30 ml 0.25 M HCI. The organic phase is evaporated and the residue is chromatographed on silica gel (eluent: CHCIs / methanol / acetic acid 100: 5: 0.1). The fractions are collected and analyzed on TLC plate (mobile solvent: CHCIs methanol / acetic acid 100: 5: 01). After evaporation of the eluent, approx. 47 mg dicarboxylic acid compound are obtained from the corresponding fractions (approx. 50% yield).

3.2 Manufacture of dicarboxylic acid chloride

45 mg of the dicarboxylic acid compound are dissolved in a solution of thionyl chloride (100 μl) in 5 ml of dry CH ₂ CI ₂ and refluxed. After about 6 hours the solvent has evaporated. The resulting dicarboxylic acid chloride can be used without further purification steps.

3.3. Manufacture of TEL derivatives

3.2.1 Tetraetheriipid derivative B

50 μl of 3-dimethylaminopropylamine are added to a solution of approximately 45 mg of dicarboxylic acid chloride in 5 ml of dry CH ₂ Cl. After 5 minutes, the mixture is washed five times with 30 ml of water, the solvent is evaporated and the residue is chromatographically separated on silica gel (eluent: CHCls / methanol / acetic acid, 80: 20: 0.5). The fractions are collected and analyzed on TLC plates with the same eluent as mentioned above. The solvent is evaporated from the fractions in which the tetraetheriipid derivative A is found. The process leads to approx. 35 mg of derivative B.

3.2.2 Tetraetheriipid derivative A

Derivative A is obtained by a similar process, in which 100 μl of 1,3-diaminopropane are used instead of 100 μl of 3-dimethylaminopropylamine.

3.2.3 Tetraetheriipid derivative C Derivative C is obtained by dissolving derivative B in a solution of 10 μl dimethyl sulfate in CH ₂ CI ₂ (5 ml). After 20 h the solvent is evaporated, the residue is dissolved in 10 ml of CHCl ₃ and washed three times in 20 ml of 0.1 M HCl. After evaporation of the organic solution, about 10 mg of derivative C are obtained (yield about 95%).

3.4 Preparation of a fluorescence-labeled tetraether lipid derivative

To clarify the question of whether the positively charged tetraether lipid derivatives according to the invention can penetrate cells, a new fluorescent tetraether lipid derivative has been synthesized.

Fluorescence-labeled tetraetheriipid derivative is obtained by adding 2 mg rhodamine isothiocyanate and 5 μl triethylamine to a solution of 2 mg derivative A in 2 ml dry CH ₂ CI ₂ . After 15 h, the solution is washed five times with 30 ml of water, the solvent is evaporated and the residue is chromatographically separated on silica gel (eluent: CHCl / methanol / acetic acid, 80: 20: 0.5). The fractions are collected and samples thereof are analyzed on TLC plates. The fractions containing the fluorescent lipid are combined and the solvent is evaporated. The process leads to approximately 2 mg of tetraether lipid rhodamine.

Example 4

Manufacture of lipofection agents

4.1 Empty lipofection agent

The tetraether lipid derivatives A, B or C are used for the production of lipofection agents. To form lipofection agents, the tetraetheriipid derivative is dissolved in chloroform / methanol (1/1, v / v) (2 mg / ml). Evaporation of the solvent creates a lipid film. The lipid film is hydrated in buffer A (150 mM NaCl, 50 mM Hepes, pH 7.4) at room temperature for 48 h (final concentration 0.5-2 mg lipid / 500 μl buffer A), then in an ultrasonic bath (Branson 1210) Sonicated for 15 min and finally 10 min at room temperature with an ultrasound tip (Branson B15, "cycle mode ", position 40). The lipid solution in buffer A appears cloudy without aggregates or recognizable precipitates.

4.2 DNA lipofection agent complexes

4.2.1 Procedure

To prepare DNA-lipofection agent complexes, 3-5 μl of lipids (1 mg / ml) suspended in buffer A are dissolved in 100 μl of serum-free medium. 1-2 μg DNA (1 mg / ml) are diluted in 100 μl serum-free medium. The two solutions are mixed gently and incubated for 15 min at room temperature to form DNA-lipid complexes. Typically, no aggregates occur during complex formation. 800 μl of serum-free medium are added before each transfection, so that a final volume of 1 ml is reached.

4.2.2 Evidence of the formation of the DNA lipofection agent complexes

The formation of the DNA lipofection agent complexes was examined fluorometrically. It is known that the formation of complexes from DNA and cationic lipids prevents the binding of ethidium bromide to DNA (Gershon et al., Biochemistry 32, 7143-7151, 1993). Since the fluorescence of ethidium bromide-DNA complexes is proportional to the amount of free DNA in solution, this method can be used to quantify DNA-lipid complexes. For this purpose, DNA-lipid complexes of tetraetheriipid derivative B and pSV-lacZ with different tetraetheriipid derivative: DNA ratios in 1 ml of 150 mM NaCl, 50 mM Hepes, pH 7.4 were pre-formed. Ethidium bromide was then added to a final concentration of 10 ^'7 M. The fluorescence of ethidium bromide-DNA complexes was monitored by excitation at 518 nm and measurement of the emission at 605 nm. The results are shown in Figure 2 and indicate that the formation of DNA tetraether lipid derivative complexes occurs at a molar ratio of 1: 1.

The formation of the complexes was also demonstrated using 1% agarose gelπ. In this independent test, the formation of DNA Tetraether lipid derivative complexes made visible by the disappearance of free DNA (Figure 3, lane 4).

These results, which were obtained in two independent experiments, indicate that the formation of tetraether lipid derivative-DNA complexes occurs at a molar ratio of 1: 1.

Example 5

Transfection with tetraether lipid derivative-containing lipofection agent

5.1 Transfection with pSV-lacZ

Transfections are carried out with a pSV-lacZ plasmid (Promega) as a reporter gene construct. 1 x 10 ⁵ BHK (Baby Hamster Kidney) cells are plated on 6-well plates. After 24 h incubation at 37 ° C in a CO ₂ -gassed atmosphere, the cells reach a density of 25-50% and can be used for transfection. The cells are washed with 2 ml of serum-free medium immediately before the transfection. 1 ml of the solution containing the DNA lipid complex (see Example 4.2) is added. After 5 h the medium containing the DNA is replaced by normal growth medium containing 10% serum. Shorter incubation times (2-4 h) also lead to successful transfections.

19 h after the transfection, the cells are washed once in PBS, fixed with ice-cold methanol (-20 ° C.), washed three times with PBS and for 16 h in a solution of 4 mM Ferrycyanid (Sigma), 1 mM MgCl ₂ , 0, 1% X-gal (Roth) incubated in PBS, pH 7.4 to detect the expression of β-galactosidase.

The transformation efficiency of the tetraether lipid derivatives A, B and C was compared with the commercially available transfection agents Lipofectin® and Lipofectamin® (Gibco / BRL). The transfection efficiency was given as a percentage of blue cells, based on the total number of cells. The comparison showed that the transforma- efficiency for all compounds according to the invention was in the same order of magnitude as for the commercial agents.

The optimal ratio of DNA to lipid was also determined using the plasmid pSV-lacZ. The best transfection results were obtained with a molar ratio of 1: 1 DNA / üpid (Figure 4). The maximum of the β-galactosidase expression was observed 19 h after the start of the common incubation of the cells with the DNA-lipid complex. As shown in Figure 5, the trafection efficiency under these conditions was 12%, based on the total number of cells used.

5.2 Transfection with fluorescent tetraetheriipid derivatives

A fluorescence-labeled lipofection agent suspension is prepared by sonicating 1 mg of a mixture of tetraetheriipid derivative A, B or C and tetraether lipid-rhodamine (see Example 3.3) (100: 1 molar ratio) in 1 ml of a buffer made of 150 mM NaCl, 50 mM Hepes, pH 7.4. The lipid concentration of rhodamine-labeled liposomes or DNA-lipid complexes ranges from 10-100 μg / ml. The cells are incubated for 70 min at 38 ° C. with the rhodamine-labeled lipofection agent, washed three times with PBS and then analyzed in a “reverse fluorescent microscope”.

The analysis showed a diffuse fluorescence that was observed in the cytoplasm of the cells (Figure 1). This result allows the conclusion that the tetraether lipid derivatives according to the invention cross the cell membrane.

6. Transfection efficiency of mixed lipofection agents

It has been shown in the literature (Feigner et al., Proc. Natl. Acad. Sci. 84, 7413-7417, 1987) that mixing Lipofectin® with DOPE (Sigma) (1: 1, w / w) increased Transfection rates can be achieved. Therefore, the influence of the addition of various lipids to the tetraether lipid derivatives according to the invention on the efficiency of cell transfection was examined. The following was added: MPL (total phospholipid fraction from Thermoplasma acidophilum), PC (phosphatidylcholine) and DOPE (dioleylphosphatidylethanolamine). It was observed that mixing the tetraether lipid derivatives A, B or C according to the invention with MPL in a ratio of 1: 1 (w / w) does not impair the transfection efficiency, while mixing the tetraether lipid derivatives according to the invention with MPL at a mixing ratio of 1: 2 (molar ratio of tetraether lipid derivative / MPL) severely affects the transfection efficiency. A mixture of tetraetheriipid derivatives according to the invention with DOPE (molar ratio of 1: 1) increases the transfection efficiency, while a mixture of tetraetheriipid derivative with PC (molar ratio of 1: 1) lowers it.

These experiments show that DOPE is a promising addition to the tetraetheriipid derivative A. The results are shown graphically in Figure 6.

Claims

claims Tetraetheriipid derivative of the general formula I:

where S ¹ and S ^{2 may be the} same or different and each has the following meaning:

Y can mean -NR R ^J or -N * R ⁴ DR5 ³ ΓR ^Ö ; X ¹ and X ² may be the same or different and are each independently selected from the group comprising a branched or unbranched alkylene or alkenylene having 2 to 20 carbon atoms; R ¹ to R ⁶ can be the same or different and are each independently selected from the group comprising: hydrogen, branched or unbranched alkyl, alkenyl, aralkyl or aryl groups having 1 to 12 carbon atoms, where in each case one of the radicals R ² to R ^{6 can} further comprise an antibody against cell surface molecules or a ligand for cell surface receptors; and n can be an integer between 0 and 10, and modifications thereof formed by the formation of pentacycles in the basic tetraether structure. 2. Tetraetheriipid derivative according to claim 1, wherein S ¹ and S ^{2 are the} same. 3. Tetraetheriipidderivat according to claim 1 or 2, wherein X ^{1 is} an alkylene or alkenylene with 2 to 10, preferably with 3 to 6 carbon atoms. 4. tetraetheriipid derivative according to any one of claims 1 to 3, wherein X ^{2 is} an alkylene or alkenylene having 2 to 10, preferably having 3 to 6 carbon atoms. 5. tetraetheriipid derivative according to any one of claims 1 to 4, wherein n is 0 to 3, preferably 0. 6. A tetraetheriipid derivative according to any one of claims 1 to 5, wherein each Y is -NR ² R ³ and wherein R ² and R ³ are selected from the group comprising: -H, -CH ₃ , -C ₂ H ₅ and -C ₃ H ₇ . 7. tetraetheriipid derivative according to any one of claims 1 to 5, wherein Y is each -N ^Θ R ⁴ R ⁵ R ⁶ and wherein R ⁴ , R ⁵ and R ^{s may be the} same or different and are selected from the group comprising: -H, -CH ₃ , -C ₂ H ₅ and -C ₃ H ₇ . 8. Tetraetheriipid derivative according to claim 1 with the formula A:

where S? 1 ¹ - = S θ2: is and means O C -N - (CH ₂ ), -NH (A) H 9. tetraetheriipid derivative according to claim 1 with the formula B:

where S ¹ = S and means O N - (CH ₂ ) ₃ - N (CH ₃ ) ₂ (B) H 10. tetraetheriipid derivative according to claim 1 with the formula C:

where S = S and means O C - N - (CH ₂ ) ₃ - N © (, CH ₃ ) ₃ (C) H 11. Liposome containing one or more tetraether lipid derivatives according to one of claims 1 to 10. 12. Liposome according to claim 11, containing further double-layer-forming cationic or neutral lipids. 13. Liposome according to claim 12, containing the cationic lipid DOTMA and / or DOTAP and / or DOSPER. 14. Liposome according to one of claims 11 to 13, containing the neutral lipid DOPE and / or cholesterol. 15. lipid agglomerate containing one or more layers of one or more tetraether lipid derivatives according to any one of claims 1 to 10. 16. Lipid agglomerate according to claim 15, containing further double-layer-forming cationic or neutral lipids. 17. Lipid agglomerate according to claim 16, containing the cationic lipid DOTMA and / or DOTAP and / or DOSPER. 18. Lipid agglomerate according to claim 16, containing the neutral lipid DOPE and / or cholesterol. 19. Liposome according to one of claims 11 to 14 or lipid agglomerate according to one of claims 15 to 18, wherein the weight ratio of tetraetheriipid derivative to further lipids is 5: 1 to 1: 5. 20. The liposome or lipid agglomerate according to claim 19, wherein the weight ratio of tetraetheriipid derivative to further lipid is between 1: 2 and 1: 0.5, preferably 1: 1. 21. Liposome according to one of claims 11 to 14 or lipid agglomerate according to one of claims 15 to 18, further comprising nucleic acid molecules. 22. Pharmaceutical composition containing liposomes according to one of claims 11 to 14 or lipid agglomerates according to one of claims 15 to 18 and a physiologically acceptable diluent. 23. In vitro transfection kit. containing liposomes according to one of claims 11 to 14 and / or lipid agglomerates according to one of claims 15 to 18 and suitable buffers. 24. Use of liposomes according to one of claims 11 to 14 or of lipid agglomerates according to one of claims 15 to 18 for the manufacture of a medicament for gene therapy in mammals. 25. Use of liposomes according to one of claims 11 to 14 or a lipid agglomerate according to one of claims 15 to 18 for the transfection of eukaryotic cells in vitro.