WO1999010337A1 - Derives de tetraetherlipides, liposomes contenant des derives de tetraetherlipides et agglomerats de lipidiques ainsi que leur utilisation - Google Patents

Derives de tetraetherlipides, liposomes contenant des derives de tetraetherlipides et agglomerats de lipidiques ainsi que leur utilisation Download PDF

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Publication number
WO1999010337A1
WO1999010337A1 PCT/EP1998/005264 EP9805264W WO9910337A1 WO 1999010337 A1 WO1999010337 A1 WO 1999010337A1 EP 9805264 W EP9805264 W EP 9805264W WO 9910337 A1 WO9910337 A1 WO 9910337A1
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Prior art keywords
lipid
derivative
tetraetheriipid
liposomes
tetraether
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PCT/EP1998/005264
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German (de)
English (en)
Inventor
H.-J. Freisleben
Emmanouil Antonopoulos
Maxim Balakirev
Larissa Balakirev
Klaus Hartmann
Felix Gropp
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Bernina Biosystems Gmbh
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Priority to AU93443/98A priority Critical patent/AU9344398A/en
Priority to EP98946380A priority patent/EP1005466A1/fr
Priority to CA002269502A priority patent/CA2269502C/fr
Publication of WO1999010337A1 publication Critical patent/WO1999010337A1/fr
Priority to US09/294,035 priority patent/US6316260B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D323/00—Heterocyclic compounds containing more than two oxygen atoms as the only ring hetero atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • 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.
  • 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.
  • the cosmetics industry produces liposome-containing skin creams that target active ingredients into the epidermis and deeper cell layers.
  • liposomes mainly natural Before soybean or egg yolk lecithins or defined natural or artificial phospholipids such as cardiolipin, sphingomyelin, lysolecithin and others are used.
  • 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.
  • 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.
  • 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.
  • liposome-forming lipid mixtures e.g. Lipofectamin®, Lipofectin® or DOTAP®
  • 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 Trans Stammio ⁇ seffizienzen can be achieved.
  • This makes transfections using commercial lipofection reagents very complex and cost-intensive.
  • large variations between the individual batches can be observed with the above-mentioned products, which makes them less reliable in practice.
  • TEL derivatives tetraether lipid derivatives
  • 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 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 ⁇ 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
  • ⁇ can be an integer between 0 and 10,
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • tetraether lipid derivatives according to the invention 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.
  • the substituents ⁇ 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • one of the radicals R 2 to R 6 can comprise an antibody against cell surface molecules or a ligand for cell surface receptors.
  • 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:
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • physiological saline pH 7.4
  • buffer systems for example Mcllvaine buffer
  • unbuffered solutions such as, for example, unbuffered potassium or sodium chloride solutions
  • 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.
  • large multilamellar vesicles with a size distribution in the ⁇ m 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:
  • Liposomes with a diameter of around 500 nm are formed.
  • 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.
  • a lipid film is first formed as described above. This is suspended in a detergent-containing buffer system (examples of dialysable detergents: octyl- ⁇ -D-glucopyranoside or octyl- ⁇ -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.
  • 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.
  • 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.
  • 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).
  • neutral lipids such as e.g.
  • DOPE dioleoylphosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • cholesterol and / or its derivatives are incorporated into the membrane.
  • 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.
  • 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.
  • TEL derivative liposome refers 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.
  • 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.
  • 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.
  • 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.
  • the active substance to be transported is nucleic acids, it is usually protected in the liposome or between the agglomerate layers.
  • 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.
  • 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.
  • preferred covalently bound active ingredients are, for example, antibodies, hormones, lectins and interleukins or active fragments of this group of substances.
  • the liposomes or lipid agglomerates according to the invention can interact particularly well with cell membranes.
  • 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.
  • 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.
  • TEL derivatives 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.
  • 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.
  • Figure 1 shows microscopic images of BHK cells that have been pre-incubated with rhodamine-coupled tetraether lipid derivative B (10 ⁇ g / 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.
  • Track 1 size marker ( ⁇ Hindlll: 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 ⁇ g / 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 ⁇ -galactosidase after incubation of BHK cells with tetraether lipid derivative complexes which contained the plasmid pSV-lacZ (Promega) coding for ⁇ -galactosidase.
  • 1.2 ⁇ g of DNA and 5 ⁇ g 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
  • 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).
  • a natural tetraether lipid e.g. isolated from Thermoplasma acidophilum
  • 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.
  • 1,3-diaminopropane is reacted to give the tetraetheriipid derivative (A).
  • reaction no. 4b the dicarboxylic acid chloride is reacted with 3-dimethylaminopropylamine to give the tetraetheriipid derivative B.
  • tetraetheriipid derivative B reacts with dimethyl sulfate to give the tetraetheriipid derivative C.
  • 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.
  • Thermoplasma acidophilum is preferably grown in 10 l or 50 l fermenters. All Ferme ⁇ termaschine 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).
  • All Ferme ⁇ termaschine 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).
  • a 10 l fermenter can be inoculated directly without prior bottle cultivation.
  • the normal conditions for fermenter cultivation are 59 ° C and pH 2.
  • 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.
  • 578 nm optical density
  • 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.
  • 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.
  • the pH is continuously measured during cultivation and regulated when there are changes in pH.
  • 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 Freu ⁇ dt'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.
  • 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 ⁇ m, some cells have pleio orphes appearance. According to laser light scatter measurements in the Malvern particle sizer, the maximum size distribution is 2.3 ⁇ m.
  • the cells usually appear round by freeze-fracture electron microscopy, pleio-morphic cells have longitudinal axes between 1, 1 and 2.7 ⁇ m and transverse axes around 0.6 ⁇ m.
  • 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).
  • Thermoplasma acidophilum is preserved at -80 ° C or in liquid N 2 .
  • an active culture (8-10 l) is adjusted to pH 5.0-5.5 by adding calcium carbonate.
  • 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.
  • cryocups are thawed with the cells in a water bath at 37 ° C.
  • Total lipid extraction is performed with freeze-dried material.
  • 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.
  • tetraether lipids 80 mg 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).
  • Derivative A is obtained by a similar process, in which 100 ⁇ l of 1,3-diaminopropane are used instead of 100 ⁇ l of 3-dimethylaminopropylamine.
  • Derivative C is obtained by dissolving derivative B in a solution of 10 ⁇ l 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%).
  • Fluorescence-labeled tetraetheriipid derivative is obtained by adding 2 mg rhodamine isothiocyanate and 5 ⁇ l 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.
  • the tetraether lipid derivatives A, B or C are used for the production of 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 ⁇ l 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.
  • DNA-lipofection agent complexes 3-5 ⁇ l of lipids (1 mg / ml) suspended in buffer A are dissolved in 100 ⁇ l of serum-free medium. 1-2 ⁇ g DNA (1 mg / ml) are diluted in 100 ⁇ l 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 ⁇ l of serum-free medium are added before each transfection, so that a final volume of 1 ml is reached.
  • DNA lipofection agent complexes were 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.
  • 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.
  • 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 ⁇ -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 ⁇ -galactosidase expression was observed 19 h after the start of the common incubation of the cells with the DNA-lipid complex.
  • the trafection efficiency under these conditions was 12%, based on the total number of cells used.
  • 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 ⁇ g / 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”.

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Abstract

Les liposomes classiques, utilisés pour véhiculer des principes actifs pharmaceutiques dans des cellules eucaryotes ou aux fins de lipofection, ont une durée de conservation limitée, une acidité instable et nécessitent la détermination de plusieurs paramètres pour pouvoir obtenir des résultats satisfaisants. Le développement de liposomes moins sensibles est donc souhaitable. Selon l'invention, on prépare des dérivés de tétraétherlipides qui sont très stables et parfaitement adaptés à la lipofection.
PCT/EP1998/005264 1997-08-22 1998-08-19 Derives de tetraetherlipides, liposomes contenant des derives de tetraetherlipides et agglomerats de lipidiques ainsi que leur utilisation WO1999010337A1 (fr)

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AU93443/98A AU9344398A (en) 1997-08-22 1998-08-19 Tetraether lipid derivatives and liposomes and lipid agglomerates containing tetraetherlipid derivatives, and use thereof
EP98946380A EP1005466A1 (fr) 1997-08-22 1998-08-19 Derives de tetraetherlipides, liposomes contenant des derives de tetraetherlipides et agglomerats de lipidiques ainsi que leur utilisation
CA002269502A CA2269502C (fr) 1997-08-22 1998-08-19 Derives de tetraetherlipides, liposomes contenant des derives de tetraetherlipides et agglomerats de lipidiques ainsi que leur utilisation
US09/294,035 US6316260B1 (en) 1997-08-22 1999-04-19 Tetraether lipid derivatives and liposomes and lipid agglomerates containing tetraether lipid derivatives, and use thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002053554A2 (fr) * 2000-12-28 2002-07-11 Bernina Biosystems Gmbh Derives de tetraetherlipides, liposomes contenant des derives de tetraetherlipides et agglomerats lipidiques ainsi que leur utilisation
EP2711369A1 (fr) 2012-09-20 2014-03-26 Bernina Plus GmbH Liposomes contenant des dérivés de tétraétherlipides
US10272041B2 (en) 2013-03-15 2019-04-30 The Penn State Research Foundation Acid stable liposomal compositions and methods for producing the same

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10204053A1 (de) * 2002-02-01 2003-08-14 Bernina Biosystems Gmbh Synthetische Tetraether und Herstellungsverfahren dafür
DE10228857B4 (de) 2002-06-26 2006-11-16 Surface & Interface Technologies Gmbh Rosenhof Tetraetherlipide mit kleinen Kopfgruppen, deren Herstellung und Verwendung
DE10249401A1 (de) * 2002-10-23 2004-05-13 Bernina Biosystems Gmbh Liposomen formende Zusammensetzung
FR3052361B1 (fr) 2016-06-09 2019-08-23 Centre National De La Recherche Scientifique Diethers d’archaea lipides synthetiques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993008202A1 (fr) * 1991-10-23 1993-04-29 National Research Council Of Canada Formation de liposomes stables a partir d'extraits de liposomes d'archeobacteries (archaea)
WO1997031927A1 (fr) * 1996-02-29 1997-09-04 Freisleben H J Tetraether lipides et liposomes les contenant, et utilisation de ceux-ci

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993008202A1 (fr) * 1991-10-23 1993-04-29 National Research Council Of Canada Formation de liposomes stables a partir d'extraits de liposomes d'archeobacteries (archaea)
WO1997031927A1 (fr) * 1996-02-29 1997-09-04 Freisleben H J Tetraether lipides et liposomes les contenant, et utilisation de ceux-ci

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
H. GERSHON ET AL., BIOCHEMISTRY, vol. 32, no. 28, 1993, pages 7143 - 51, XP000601477 *
H.-J- FREISLEBEN ET AL., APPL. MICROBIOL. BIOTECHNOL., vol. 40, 1994, pages 745 - 52, XP002087915 *
O. GRÄTHER ET AL., JOURNAL OF THE CHEMICAL SOCIETY, CHEMICAL COMMUNICATIONS, 1995, pages 405 - 6, XP002087914 *
P. L. FELGNER ET AL., PROC. NATL. ACAD. SCI., vol. 84, 1987, pages 7413 - 7, XP000602252 *
T. G. LANGWORTHY ET AL., SYSTEM. APPL. MICROBIOL., vol. 7, 1986, pages 253 - 7, XP002087913 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002053554A2 (fr) * 2000-12-28 2002-07-11 Bernina Biosystems Gmbh Derives de tetraetherlipides, liposomes contenant des derives de tetraetherlipides et agglomerats lipidiques ainsi que leur utilisation
WO2002053554A3 (fr) * 2000-12-28 2002-09-19 Bernina Biosystems Gmbh Derives de tetraetherlipides, liposomes contenant des derives de tetraetherlipides et agglomerats lipidiques ainsi que leur utilisation
EP2711369A1 (fr) 2012-09-20 2014-03-26 Bernina Plus GmbH Liposomes contenant des dérivés de tétraétherlipides
US10272041B2 (en) 2013-03-15 2019-04-30 The Penn State Research Foundation Acid stable liposomal compositions and methods for producing the same

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