WO2000015763A1 - Utilization of nanocells in final culture medium products - Google Patents

Utilization of nanocells in final culture medium products Download PDF

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Publication number
WO2000015763A1
WO2000015763A1 PCT/CH1999/000420 CH9900420W WO0015763A1 WO 2000015763 A1 WO2000015763 A1 WO 2000015763A1 CH 9900420 W CH9900420 W CH 9900420W WO 0015763 A1 WO0015763 A1 WO 0015763A1
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characterized
nano
vitamin
component
end products
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PCT/CH1999/000420
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German (de)
French (fr)
Inventor
Andreas Werner Supersaxo
Marc Antoine Weder
Hans Georg Weder
Hans Konrad Biesalski
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Vesifact Ag
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/107Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018Culture media for cell or tissue culture
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/30Organic components
    • C12N2500/36Lipids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/30Organic components
    • C12N2500/38Vitamins

Abstract

Disclosed is the utilization of a nanocells containing a preparation composition consisting of: (a) a membrane-forming molecule, (b) a co-emulsifier and (c) a lipophilic component in final culture medium products. The nanocells used according to the invention are non-toxic and suitable as solutizing agents for lipophilic substances in final culture medium products.

Description

Using Nano Cells in culture media-end products

The present invention relates to the use of nano Cells in culture media-finished products, end products culture media containing these nano-Cells as well as the various models uses culture media the end products.

Culture media the end products according to the present invention are, for example, media for culturing and analysis of cells, fungi, bacteria, viruses, bacteriophages, insect and plant, media for in vitro, respectively, ex vivo cultivation and analysis of organs and tissues, for freezing media of cells, fungi, bacteria, viruses, bacteriophages, insects, plants, organs and tissues, embryo transfer freezing media, the media for the therapy (adoptive immunotherapy treatment of cancer), media for perfusion of organs such as kidney or liver, media for storage of organs until their transplant and supplements for the above media.

The invention is based on the object lipophilic, ie to incorporate poorly water-soluble or water-insoluble substances such in culture media-end products that

Cells, fungi, bacteria, viruses, bacteriophages, insects, plants, tissues, organs, etc. be affected either in its growth nor in their biological functions.

The addition of lipophilic substances to culture media has not yet been satisfactorily resolved. this organic solvent such as ethanol, THF, or DMSO are used frequently. The disadvantage of this is that the dissolved substances may precipitate again when added to the medium as a result of dilute by the solvent. Precipitated hydrophobic substances frequently adherieren on the walls of the culture vessels and thus no longer available to the cells. In addition, the final concentrations commonly used in solvents of 0.1% already show negative effects on cells and other biological systems maintained in culture.

In addition to organic solvents and vehicles such as micelles, mixed micelles, liposomes or finely emulsified emulsions are used for the introduction of lipophilic substances today. Although physiologically effective than organic - solvent are also these vehicles, especially in high concentration, often toxic to cells, fungi, bacteria, viruses, bacteriophage, insects, plants, tissues and organs or affecting maintained in culture biological system in its function. The impossible functional influencing the culture held in biological systems or complicates the interpretation pharmacological gic, toxicological, metabolic and regulatory investigations. The low loading capacity with hydrophobic substances and the insufficient stability in certain culture media are further reasons that may limit the use of these vehicles.

Another often-used solubilizing agent is beta-cyclodextrin methyl. However, Methylbetacyclodextrin has the decisive disadvantage that it is "riddled" the cell membrane and so could be as stated repeatedly, the disregulated calcium flux; that is, it comes to the influx of calcium into the cell with all downstream processes activated by calcium.

also the task underlying the invention is to incorporate lipophilic compounds in culture media the end products, which are particularly suited for organ storage up to its transplantation. Various lipid-soluble compounds are suitable encryption to protect cells and tissues from transient ischemia, but especially also facing a resumption of reperfusion. Such a situation arises when organs removed, temporarily stored and later reimplanted. Here, there is initially an ischemic phase which is followed by bleeding from a so-called Wiederdurch-. Reperfusion with altered oxygen supply. These so-called. Ischemia / reperfusion results, due to the reduction of ATP during ischemia in an activation of enzyme systems, which are then metabolise the degradation products of ATP, depending on the availability of oxygen, significant amounts of reactive oxygen compounds are formed. In - Several studies have shown that different lipid-soluble antioxidants such as 3-carotene, vitamin E, but also the water-soluble vitamin C are suitable, including suppressing the formation of such occurring during ischemia / reperfusion syndrome reactive oxygen compounds derived products. The perfusion media used in this case were either fat solutions or the donor organs were by oral administration of vitamins ago

Removal enriched. Both solutions are unsuitable for organ preservation. to bring financing solutions another way to fat-soluble antioxidants in such retention or preservatives, it does not yet exist.

Surprisingly, it has been found that so-called nano-containing at Cells,

(A) a membrane-forming molecule

(B) a co-emulsifier and

(C) a lipophilic component, lipophilic, that is water-insoluble or poorly water-soluble substances in culture media-end products can be introduced, so that there the cells kept in culture, fungi, bacteria, viruses, bacteriophage, insect, plant or tissue either in your growth yet be adversely affected in their func- tions. The particular advantage of NanoCell lies in the fact that they represent an extremely physiological application form, which are bar unlike any other solubilizers fat-soluble compounds even under in vivo conditions easy insertion. The fact that endothelial cells, but also other cellular compartments such. record as fibroblasts or Darmmucosazellen vitamins well in this form also allows use for the application of vitamins in the buccal mucosa.

Surprisingly, it was possible with the help of NanoCell tech- nology to bring lipophilic compounds such as antioxidants in perfusion solutions which are suitable as

Organ preservation solutions are used. So far - present in vitro and in vivo studies were performed with such antioxidant formulations, show that there are actually ten in this way to an accumulation of antioxidant vitamins in the critical cellular compartments, particularly the endothelial cells occurs. This enrichment was not expected because the cells are normally supplied only by way of lipo-protein-bound antioxidants. With the present experimental approaches could also be shown so that the infusion of antioxidant vitamins by way of the nano-cell technology, a first pass effect in the body, that leads into the critical cells of the perfused organ. But that the organ using the so appli- ed antioxidants is protected in a physiological manner, so that the use of antioxidants in this form is suitable to improve the organ storage and preservation.

Other advantages of the Nano Cells is their high capacity Beladungs- with lipophilic substances and the good stability in culture media-end products.

The present invention provides the use of a nano Cell containing as preparative composition (a) is therefore a membrane-forming molecule

(B) a co-emulsifier and

(C) a lipophilic component in culture media-end products.

Preferably, the nano-Cell (a) as the membrane-forming molecules substances which are suitable, two-layer systems (so-called. "Bilayer") form,

(B) forming structures as coemulsifiers substances which preferably O / W and (c) a lipophilic component, a conventional culture media for lipophilic substance.

Preferably, the nano Cell contains as component (a) a phospholipid, a hydrogenated or partially hydrogenated phospholipid, a lysophospholipid or mixtures of these comparison compounds.

Very particular preference is given to a phospholipid of formula

CH ^ OR! (1)

R 2 -0-CH 0

CH 2 -0-P-0-R 3

OH wherein

R l C 10 -C 20 acyl;

R 2 is hydrogen or

Figure imgf000007_0001

R3 is hydrogen, 2-trimethylamino-l-ethyl, 2-amino-l- ethyl, unsubstituted or substituted by one or more

Carboxy, hydroxy or amino groups, substituted C j -Cς- alkyl; the inositol or glyceryl group, or a salt of these compounds.

Figure imgf000007_0002
is preferably a straight-chain
Figure imgf000007_0003
Alkanoyl having an even number of C atoms and straight-chain C 1Q -C2o _A l ken oyl with one or more double bonds and an even number of C-atoms.

Straight-chain

Figure imgf000007_0004
having an even number of C atoms are, for example, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl or n-octadecanoyl. Straight-chain
Figure imgf000008_0001
e i ner double bond and an even number of C atoms are, for example, 6-cis- or 6-trans-, 9-cis- or 9-trans-dodecenoyl, -Tetradece- noyl, -Hexadecenoyl, -Octadecenoyl or -Icosenoyl, in particular 9-cis-octa-decenoyl (oleoyl), also 9,12-cis-octadecadienoyl or 9, 12, 15-cis-octadecatrienoyl.

A phospholipid of the formula (1) wherein R is 2-Trimethylamino- means 1-ethyl is referred to by the trivial name lecithin, and a phospholipid of formula (1) wherein R 3 2-Amino-l-ethyl is the trivial name cephalin. Suitable are, for example, naturally occurring cephalin or lecithin, for example cephalin or lecithin from soybeans or chicken egg having different or identical acyl groups, or mixtures thereof.

But the phospholipid of formula (1) may also be of synthetic origin. The term synthetic phospholipid to define phospholipids, which with respect to R ^ and R2 have a uniform composition. Such synthetic phospholipids are preferably the above-defined lecithins and cephalins, wherein the acyl groups have RL and R2 a defined structure and are derived from a defined fatty acid with a degree of purity higher than approximately 95%. R ^ and R2 may be the same or different and unsaturated or saturated. Preferably, R ^ is saturated, for example, n-hexadecanoyl, and R unsaturated, for example 9-cis-octadecenoyl (oleoyl).

"Naturally-occurring" The term phospholipid defines phospholipids that do not have a uniform composition with respect to R] _ and R2. Such natural phospholipids are likewise lecithins and cephalins, wherein the acyl groups RL and R2 of naturally occurring fatty acid mixtures derived.

The requirement "substantially pure" phospholipid of formula (1) defines a degree of purity of more than 90 wt .-%, preferably more than 95 wt. the phospholipid of formula (1), which is detectable using suitable Bestimmungsmetho- to, for example, paper or thin-layer chromatography, HPLC or enzymatic Färbtest.

In a phospholipid of formula (1), R 3 with the meaning C ^ -C ^ j alkyl, for example methyl or ethyl. The ~~ is methyl is preferred.

R with the meanings of one or more carboxyl, hydroxyl or amino groups substituted C - ^ - C ^ alkyl, for example 2-hydroxyethyl, 2, 3-dihydroxy-n-propyl,

Carboxymethyl, 1- or 2-carboxyethyl, dicarboxymethyl, 2-carboxy-2-hydroxyethyl or 3-carboxy-2, 3-dihydroxy-n-propyl, 3-amino-3-carboxy-n-propyl or 2-amino-2 carboxy-n-propyl, preferably 2-amino-2-carboxyethyl.

Phospholipids of the formula (1) with these groups can, for example as the sodium or potassium salt, in salt form.

Phospholipids of the formula (1) wherein R3 represents the inositol or glyceryl group, are known under the names phosphatidylinositol and phosphatidylglycerol.

For the acyl groups in the phospholipids of the formula (1), the names given in brackets in use are: 9-cis-dodecenoyl (lauroleoyl), 9-cis-tetradecenoyl (Myristoleoyl), 9-cis-hexadecenoyl (palmitoleoyl), 6-cis - octadecenoyl (Petroseloyl), 6-trans-octadecenoyl (Petrose- laidoyl), 9-cis-octadecenoyl (oleoyl), 9-trans-octadecenoyl (elaidoyl), 9, 12-cis-octadecadienoyl (linoleoyl), 9.12, 15- cis-octadecatrienoyl (linolenoyl), 11-cis-octadecenoyl (Vaccenoyl), 9-cis-icosenoyl (Gadoleoyl), 5, 8, 11, 14-cis-eicosatetraenoyl (arachidonoyl), n-dodecanoyl (lauroyl), n-tetradecanoyl (myristoyl), n-hexadecanoyl (palmitoyl), n-octadecanoyl (stearoyl), n-icosanoyl (arachidoyl), n-docosahexaenoic sanoyl (behenoyl), n-tetracosanoyl (lignoceroyl). A salt of the phospholipid of formula (1) is acceptable. Salts are defined by the existence of salt-forming groups in the substituent R3 and by the free hydroxy group on the phosphorus. is also possible the formation of internal salts. Alkali metal salts, especially sodium salts are preferred.

In a particularly preferred embodiment, purified lecithin from soybeans quality LIPOID S 100 or S75 or a lecithin defined in the monograph of the USP23 / NF 18th

The component (a) is preferably used in a concentration of about 0.1 to 30 wt .-%, based on the total weight of components (a), (b) and (c), are used.

As component (b) an emulsifier or emulsifier is preferably used, the / the preferred O / W-formed structures (n).

Especially preferred emulsifiers are:

Alkali metal, ammonium and aminium salts of fatty acids. Examples of such salts are lithium, sodium, potassium, ammonium, triethylamine, ethanolamine, triethanolamine or Diet- hanolamin-. In particular, the sodium, potassium or ammonium are (NR2R2R3) - salts, where R ^, R and R3 are independently hydrogen, C ^ -C ^ alkyl or C ^ -C ^ hydroxy alkyl.

Saturated and unsaturated alkyl sulfates such as Natriumdocecylsulfat and alkanesulfonates such as sodium triumdocecansulfona; glycocholate bile salts such as sodium cholate, sodium glycocholate and sodium taurocholate;

Invert soaps (quats) like Zetylpyridiniumchlorid; Partial fatty esters of sorbitan such as sorbitan monolaurate; Zuckerester such as sucrose monolaurate; Alkylglucosides such as n-octyl glucoside or n-dodecyl cylglucosid;

Alkylmaltosides such as n-dodecyl maltoside;

Fatty acid such as Laurinsäuremonogly- cerid;

Cg-C ^ g-betaines, Cg-C24 alkyl amido C ^ -C4 alkylenbetaine and Cg-C ^ g-sulfobetaines;~~

Fettalkoholphosphorsäureester

Polyglycerol esters of fatty acids; - propylene glycol esters of fatty acids;

Milchsäureester of fatty acids such as Natriumstea- Royl-lactyl-2-lactate

Proteins such as casein. Emulsifiers of the polyoxyethylene type are particularly preferred. Examples of such emulsifiers are:

Polyethoxylated sorbitan fatty acid esters such as poly sorbate 80;

Polyethoxylated vitamin E derivatives such as vitamin

E polyethylene glycol 1000 succinate; - polyoxyethylene glycolated natural or hydrogenated

Vegetable oils such as polyoxyethylene glycolated natural or hydrogenated castor oils;

Polyethoxylated fatty acid partial such as

diethylene glycol; - polyethoxylated fatty alcohols such as oleth-20,

Polyethoxylated fatty acids such as stearic polyoxyl rat 20,

Polyethoxylated lanolin and its derivatives, such as

Laneth-20 - polyethoxylated carbohydrates;

Block polymers of ethylene oxide and propylene oxide such as Poloxamer 188

The component (b) is present in the used in the invention NanoCell in a concentration of about 1 to about 50% by weight, based on the total weight of components (a), (b) and (c). The component (c) is preferably a natural, a synthetic or a partially synthetic di- or tri- glyceride, a common culture media for lipophilic substance or mixtures of these substances.

For culture media-end products suitable substances are,. B. essential, the growth of cells, fungi, bacteria, ~~ viruses, bacteriophages, insects, plants, tissues, etc. supporting substances such as vitamins, amino acids, peptides, natural or recombinant proteins, carbohydrates, lipids, nucleic acids, ribonucleosides, Desoxyribonukleosi- de, inorganic salts and trace elements; furthermore natural, semisynthetic or synthetic active substances such as antioxidants, antibiotics, antifungal agents, hormones; Mixtures for screening and markers to investigate cell-biological functions.

Examples of vitamins are vitamin A, vitamin B lf Vitamin B 2, Vitamin B 6, nicotinamide, pantothenic acid, vitamin B 12, vitamin B 15, folic acid, biotin, vitamin C, vitamin D, vitamin E, vitamin F, vitamin K, and vitamin P.

Examples of amino acids and peptides are hionin alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, Met, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, Argi- nin, histidine, glycyl-L-glutamine, L-alanyl-L-glutamine and hydroxy-L-proline.

Examples of natural and recombinant proteins are growth factors such as Epidermal Growth Factor (EGF), ceramic tinocyte Growth Factor (KGF), Acidic and Basic Fibroblast Growth Factor (aFGF and bFGF), insulin-like growth factor-I and II (IGF-I and IGF-II), nerve growth factor (NGF),

Platelet-Derived Growth Factors (PDGF), Stem Cell Factors and transforming growth factors; Chemotactic factors such as macrophage / monocyte chemotactic and activating factor (MCAF); Colony-stimulating factors such as granulocyte-Macro- phage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF); Interferons and interleukins; Cell adhesion and extracellular matrix proteins such as collagens, fibronectins, integrins, laminins, Merosine, proteoglycans, adhesion peptides RGD, tenascins, platelet spondine and Vitronektine and enzymes such as collagenase, dispase and trypsin. Other examples of proteins are ~~ insulin, albumin and transferrin.

Examples of carbohydrates are glucose, galactose, fructose, sucrose, maltose, ribose, deoxyribose, Trehalo- se, tryptose and yeastolate.

Examples of lipids are saturated and unsaturated fatty acids and derivatives thereof. Examples of saturated fatty acids are palmitic acid, stearic acid. Examples of unsaturated fatty acids are omega-9 fatty acids such as oleic acid, omega-6 fatty acids such as linoleic acid, gamma-linolenic acid, dihommo-gamma linolenic acid and arachidonic acid, and omega-3 fatty acids such as alpha linolenic acid, stearidonic acid, eicosapentaenoic acid and docosahexaenoic sahexaensäure. Other examples of lipids which are cholesterol-rol and derivatives.

Examples of nucleic acids, ribonucleosides and ribonucleosides deoxy are adenine, guanine, thymidine, uracil, adenosine, guanosine, cytidine, uridine, 2 '-Desoxyadenosin, 2' -Desoxyguanosin, 2 'deoxycytidine, 2' -Desoxythymidin, 5- methyl deoxycytidine and 5-methylcytosine.

Examples of inorganic salts and trace elements are CaCl2, KC1, MgS0 4, NaCl, NaHC0 3, NaH 2 P0 4, Fe (N0 3), FeS0 4, KH 2 P0 4, MgCl 2, (NH 4) (Mo 7 0 24), CuS0 4, KN0 3, ZnS0 4, NiCl 2, Na 2 Si0 3, H 2 Se0 3, MnS0 4 and Ca (N0 3). 2

Examples of antioxidants are vitamin E and derivatives, tocotrienols, vitamin A and derivatives, vitamin C, beta carotene, carotenoids, ubiquinones such as coenzyme Q10, flavonoids de, isoflavones, polyphenols, phytoestrogens, Lycopene, Luteine, lipoic acids, glutathione.

Examples of antibiotics and antifungal drugs are Tylocin, gentamicin sulfate, kanamycin sulfate, neomycin sulfate, nystatin, polymixin B sulfate, streptomycin sulfate, Actino- mycin D, penicillin G, ampicillin, carbenicillin, Amphote- ~~ ricin B and mycophenolic acid.

Examples of hormones are hydrocortisone, progesterone, dexamethasone and Triamzinolon.

Examples of mixtures for screening are vegetable or animal extracts containing bioactive ingredients.

Examples of markers to study zellbiologiεcher functions are fluorescent dyes, immunohistochemical markers and vital markers.

Also examples of substances for culture media are as Coenzyme diphosphopyridine nucleotide (DPN), flavin adenine dinucleotide (FAD), Triphosphopyridin nucleotide (TPN) and uridine triphosphate (UTP), coenzyme A, cocarboxylase; Peptone as meat peptone, Kaseinpepton, soy peptone, Bacto-peptone and Laktalbuminhydrolysat and extracts such as yeast extract, bovine pituitary extract, chicken embryo extract and beef extract.

Other examples of materials for culture media are putrescine, phenol red, fumaric acid, malic acid, alpha-keto glutaric acid, succinic acid, lactic acid, acid-DL-68-Thiocitin-, para-aminobenzoic acid, HEPES, Ca-lactate, Na-Succi- nat, Na pyruvate, sodium glucuronate, sodium acetate, sodium mucate, D- Glukuronlacton, aminopterin, hypoxanthine, xanthine, choline chloride, choline bitartrate, taurine and Salmi sulfate.

The component (c) is used in accordance with the invention

NanoCell in a preferred concentration of from 0.1 to 70 wt .-%, based on the total weight of components (a), (b) and (c) present.

Optionally, the NanoCell used in the invention contains as an optional component (d) a solubilizer, preferably a C 2 -C 8 alcohol such as ethanol or propylene glycol.~~

In some special cases, the NanoCell used in the invention contains only the components (b) and (c).

A nano-Cell composition comprising components (a), (b) and (c) is characterized in dispersion in the aqueous medium by favorable phase properties. It can be seen where opalescence and transparency in the backlight only an extremely slight milky haze that the dispersion still has physical differences from the ideal state of a true molecular solution. Electron micrographs show that a population of more than 98% in a Gaussian distribution as a suspension of particles (nanoparticles) having a particle size less than about 100 nm (nano-dispersion), typically less than about 50 nm, is present. These

but differences from a real solution can be tolerated because of the particularly good homogeneity properties of the dispersion, for example, at a surprisingly high storage stability, eg. B. no separation after several months of storage at temperatures up to room temperature (to be expected by extrapolation stability for more than two years), can be detected.

For the production of the nano-Cells, the two components (b) and (c), optionally mixed with heating to a homogenous liquid phase. In this phase, the component (a), optionally dissolved with the aid of a solubilizer such as ethanol. The result is a clear liquid, the so-called Nano-Cell preliminary phase. This is added to the aqueous phase, optionally containing water-soluble substances with stirring. Stir at ordinary stirring apparatus, such as propeller, inclined blade or magnetic agitators. By special selection of the components (a), (b) and (c) are thereby directly corresponds ultrafine monodisperse nano Cells. It can be dispensed to a homogenization by means of nozzles, rotor-stator or ultrasonic homogenizers which produce high shear and cavitation forces ~~.

The addition of the nano-Cell preliminary phase to the aqueous phase is usually carried out <50 ° C at a temperatures.

The characterized by the production method described Nano Cells have an average particle diameter of less than 100 nm, typically less than 50 nm. The particle distribution is monodisperse and obeys a Gaussian distribution. Laser light scattering measurements and electron microscopy (cryo-TEM) confirm the very small size and excellent homogeneity of Nano Cells.

The Nano Cells are inventively used in Kulturmedien- end products.

These culture media-end products are preferably liquid or semi-solid, but can also be solid.

Examples of culture media end-products are animal sera such as fetal bovine serum, newborn calf serum, Kälberse- rum, sera of other animal species, serous fluids from cattle; synthetic culture media, such as serum-free / serum-reduced media, minus media for Radioactive incorporation studies, media for the molecular and cytogenetics, selection media, media for the plaque assay, media for the detection of mycoplasma media for freezing media for the tissue, organ and embryo transfer, media for therapy, media for the perfusion of organs, media organ storage until their transplant media for pharmacological, toxicological and metabolic studies, media for recording and transport studies, media to investigate functional and regulatory mechanisms , supplements such as amino acid and vitamin solutions enzyme; Lipidkonzen- would occur, growth-stimulating additives, serum replacement products containing growth factors, vitamins, binding proteins, hormones, adhesion factors and trace elements, bouillons, - peptones as meat peptone, Kaseinpepton, soy peptone, Bacto-peptone and Laktalbuminhydrolysat, extracts feextrakt as He, bovine pituitary, chicken embryo extract or beef extract, Pfufferlösungen and agarose gels. These culture media containing nano-end products Cells form a further subject of the invention.

For the preparation of liquid and semisolid Kulturmedien- end products nano Cells are incorporated into the aqueous portion of the final product. The incorporation of the nano-Cells is preferably carried out with stirring at room temperature, ordinary stirrers such as propeller, pitched-or magnetic agitators are used.

also the corresponding nano Cell preliminary phase in the aqueous portion of the Kulturmedien- end products can be incorporated in place of the nano Cells. The addition of the NanoCell- preliminary phase in the aqueous portion of the end products is carried out under stirring and preferably at a temperature <50 ° C.

Solid culture media end products such as powder media

Granules, tablets and lyophilizates be coated by spraying or soaking with nano Cells or loaded. For certain solid culture media the end products, it is advantageous to admix the NanoCell in dehydrated form the solid fabric mixture, wherein the dehydration of the nano Cell generally carried out by freeze drying or spray drying in the presence of adjuvants such as gebräuchhlicher Maltodextri- ne. The culture media the end products are preferably used for in vitro cultivation and analysis of cells, fungi, bacteria, viruses, insects and plants, respectively for the in vitro cultivation and ex vivo examination of tissues and organs, for freezing of organs, tissues, cells fungi, bacteria, viruses, insects and plants, for the transfer of organs, tissues and embryos, for the therapy ^ (adoptive immunotherapy treatment of cancer), cytogenetic for perfusion of organs such as kidney or liver, organ storage up to its transplantation, for used molecular genetic, pharmacological, toxicological and metabolic studies, for recording and transport studies or to study functional and regulatory mechanisms.

The invention is further illustrated by the following examples.

Nano Cell preparation examples:

Example 1: Soy lecithin 1.7% Nano Cell

Soy lecithin 1.70% Miglyol 812% 3:45

Polysorbate 80% 3:40

Ethanol 1.40%

10 mM phosphate buffer, pH 6.0 ad 100.00%

For the production of the nano Cells polysorbate 80 and Miglyol 812 are mixed to form a homogeneous liquid phase. At this stage, are added dissolved in ethanol soya lecithin. This results in a homogeneous solution, which is added with stirring to phosphate buffer. The NanoCell obtained is then rilfiltriert STE through a 0.2 micron filter. Example 2: egg lecithin 1.4% NanoCell

Egg lecithin 1.40%

Miglyol 812 2.85%

Polysorbate 80 4:00% ethanol 1.40%

10 mM phosphate buffer, pH 6.0 ad 100.00%

The production of the nano-Cells is carried out in, for example 1 analogously.

Example 3: Vitamin E 0.45% to NanoCell Vitamin E 0.45% to

Soy lecithin 1.20%

Miglyol 812% 3:00

Polysorbate 80 2.90%

1:00 ethanol% 10 mM phosphate buffer, pH 6.0 ad 100.00%

For the production of the nano Cells polysorbate 80, Miglyol 812 and vitamin E are mixed into a homogeneous liquid phase. At this stage, are added dissolved in ethanol soya lecithin. This results in a homogeneous solu- tion, which is added with stirring to phosphate buffer. The NanoCell obtained is then sterilized by filtration through a 0.2 micron filter.

Example 4: Vitamin E acetate 2% NanoCell

Vitamin E acetate 2:00% soy lecithin 00:49%

Miglyol 812 0.71%

Polysorbate 80 1.86%

Ethanol 0.40%

10 mM phosphate buffer, pH 6.0 ad 100.00%

The production of the nano-Cells is carried out in, for example, 3 analogous manner. Example 5: Vitamin E acetate 1.2% NanoCell

Vitamin E acetate 1.20%

Polysorbate 80 0.98%

10 mM phosphate buffer, pH 6.0 ad 100.00%

To produce the Nano Cells are vitamin E acetate and

Polysorbate 80 are mixed to form a homogeneous liquid phase.~~ The resulting clear solution is added with stirring to the water. The NanoCell obtained is then sterilized by filtration through a 0.2 micron filter.

Example 6: Vitamin A palmitate 1.8% NanoCell

Vitamin A palmitate (1.7 million IU / g) 1.80%

Vitamin E acetate 0.10% to

Soy lecithin 1.70%

Miglyol 812 1.80% Polysorbate 80 3.15%

Ethanol 1.40%

10 M phosphate buffer, pH 6.0 ad 100.00%

The production of the nano-Cells is carried out in, for example, 3 analogous manner.

Example 7: hydrocortisone 0.05% NanoCell

Hydrocortisone 0.05%

Soy lecithin 0.36% to

Miglyol 812 0.72%

Polysorbate 80 0.71% ethanol 0.30% to

10 mM phosphate buffer, pH 6.0 ad 100.00%

The production of the nano-Cells is carried out in, for example, 3 analogous manner. Example 8: Omega-3 fatty acids 3.5% NanoCell

Incromega E3322 (Croda) 5:50%

Vitamin E acetate 0.60%

Soya lecithin 0.30% to 3.60% polysorbate 80

10 mM phosphate buffer, pH 6 ad 100.00%

For the production of the nano Cells omega-3 fatty acids (Incromega E3322, Croda), vitamin E acetate, polysorbate 80 and soya are mixed to a homogeneous liquid phase overall lecithin. The resulting clear solution is added with stirring to phosphate buffer. The NanoCell obtained is then sterilized by filtration through a 0.2 micron filter.

Example 9: Coenzyme Q10 0.5% NanoCell

Coenzyme Q10 of 0.50% soy lecithin 1.10%

Miglyol 812% 4:50

Polysorbate 80% 3:40

Propylene glycol 0.48%

Ronoxan A (Roche) 0.02% to 10 mM phosphate buffer, pH 6 ad 100.00%

For the production of the nano Cells Miglyol 812, polysorbate 80, propylene glycol, Ronoxan A and soy lecithin are mixed to a homogeneous liquid phase. Coenzyme Q10 is added to this mixture with stirring and heating. The result is a clear liquid with stirring (magnetic stirrer for example) is the phosphate buffer which was previously heated to 50 ° C added. The NanoCell obtained is then sterilized by filtration through a 0.2 micron filter.

In Table 1, the particle sizes of Nano Cells are put together:

Figure imgf000022_0001

The particle diameter and the particle size distributions were (weighting Nicomp 370 Submicron Particle Sizer, Volume) by means of Laser Light Scattering determined as the following example shows, Nano Cells have also excellent storage stability (Table 2) g:

Vitamin E acetate 2% Nano Cell (Example 4)

Figure imgf000023_0001

The particle diameter and the particle size distributions were (weighting Nicomp 370 Submicron Particle Sizer, Volume) by means of Laser Light Scattering determined The content of Vitamin E acetate was determined by HPLC Examples of culture media containing nano-end products Cells

Example 10: Culture medium-finished product containing vitamin E acetate Nanocells

Cellular uptake of vitamin E, respectively. Vitamin E acetate microvascular endothelial cell line HMEC-1 were - used to confluency, then washed once and incubated in a medium of the following composition:

Vitamin E acetate NanoCell according to Example 4 0.0575%

EGF (Laboserv, Staufenberg, Germany) 0.0050% hydrocortisone (Sigma, Munich, Germany) 0.0001%

10.0000% fetal calf serum (Biochrom, Berlin, Germany)

MCDB 131 (Biochrom, Berlin, Germany) ad 100.0000%

After 96 hours, the cellular taken vitamin E amount was determined. For this, the cells were using

Trypsin / EDTA harvested, washed, extracted with hexane, and the concentration of vitamin E in hexane extract by HPLC. The remaining after the Hexanexktration pellets were used for DNA determination. The results presented in Figure 1 that vitamin E is taken from above medium efficiently into the cells. For comparison, the cellular concentrations of vitamin E are reported analogous experiments in which the same amount of vitamin E, respectively. Vitamin E acetate was not added to the medium complex in the form of nano-Cells, but as a solution in ethanol or DMSO as solubilizate in fetal calf serum, or as a cyclodextrin.

Antioxidant capacity of vitamin E, respectively. Vitamin E acetate microvascular endothelial cell line HMEC-1 were grown to confluence used, then washed once and incubated in a medium of the following composition for 96 hours Vitamin E acetate Nano Cell according to Example 4 0.0575%

EGF (Laboserv, Staufenberg, Germany) 0.0050%

Hydrocortisone (Sigma, Munich, Germany) 0.0001%

10.0000% fetal calf serum (Biochrom, Berlin, Germany)

MCDB 131 (Biochrom, Berlin, Germany) ad 100.0000%

To determine the antioxidant protective effect of vitamin E, respectively. Vitamin E acetate were determined in the generated natural metabolism of free radicals by means of a fluorome- trical method. For this purpose, the pre-incubated in the above medium for 96 hours, cells with a non-fluorescent dye (carboxy-H 2 DCFDA / AM, Molecular Probes) were loaded, the intracellularly with free radicals, in particular hydroperoxides, reacts and thereby becomes a fluorochrome. The resulting relative fluorescence is a measure of the amount of released radicals. In the present experiment, the relative fluorescence was measured over a period of 150 minutes. The results presented in Figure 2 that the vitamin E acetate Nanocells a very good antioxidant

has protective effect against intracellularly generated radicals. For comparison, the relative fluorescence values ​​analogous experiments are shown in which the same amount of vitamin E, respectively. Vitamin E acetate was not added to the medium complex in the form of nano-Cells, but as a solution in ethanol or DMSO as solubilizate in fetal calf serum, or as a cyclodextrin.

Cytotoxicity of Vitamin E acetate Nano Cells Microvascular endothelial cell line HMEC-1 were grown to confluency, then washed once and added to a medium of the following composition.

EGF (Laboserv, Staufenberg, Germany) 0.0050%

Hydrocortisone (Sigma, Munich, Germany) 0.0001%

10.0000% fetal calf serum (Biochrom, Berlin, Germany) MCDB 131 (Biochrom, Berlin, Germany) ad 100.0000%

The cell suspensions obtained were then supplemented with 5, 10, 25, 50 and 75 .mu.M vitamin E acetate, ACCORDING in the form of Vitamin E acetate Nano Cells Example 4 and incubated for 96 hours. The cytotoxic effect was

Using the LDA assays determined. The release is - of the enzyme lactate dehydrogenase measured from dying cells in the supernatant and presented as percent of control. The results presented in Figure 3 that the vitamin E acetate NanoCell, is non-toxic even at high concentrations.

Stability of vitamin E acetate Nanocells

Microvascular endothelial cell line HMEC-1 were grown to confluence used, then washed once and added to a medium of the following composition:

EGF (Laboserv, Staufenberg, Germany) 0.0050%

Hydrocortisone (Sigma, Munich, Germany) 0.0001%

10.0000% fetal calf serum (Biochrom, Berlin, Germany) MCDB 131 (Biochrom, Berlin, Germany) ad 100.0000%

The cell suspensions obtained were then supplemented with 5, 10, 25, 50 and 75 .mu.M vitamin E acetate, ACCORDING in the form of Vitamin E acetate Nano Cells Example 4. Fig. At time 0 and after 96 hours the content of vitamin E acetate was determined in the medium by means of HPLC. The decrease in the vitamin E acetate concentration fluctuated, depending on the concentration of added, 7-29%. This decrease is in contrast to other solubilizers ex- tremely low. Example 11: Culture medium-finished product containing vitamin A palmitate Nano Cells

Skin fibroblasts: Cellular uptake of vitamin A palmitate

Human dermal fibroblasts of the cell line HFP-1 were grown to confluence used, then washed once and incubated in egg ~~ nem medium of the following composition:

Vitamin A palmitate Nano Cell according to Example 6 0.0095% 1% penicillin / streptomycin, 0.0050%

(Biochrom, Berlin, Germany) Fetal calf serum 10.0000%

(Biochrom, Berlin, Germany)

DMEM ad 100.0000%

(Dulbecco's modified Eagle medium Biochrom, Berlin, Germany)

After 15 minutes, 2, 8 and 24 hours, the cellular recorded Vitamin A palmitate amount was determined. For this purpose, the cells were harvested at the indicated time points, washed, extracted with hexane, and the concentration of vitamin A palmitate in the hexane extract by HPLC. The remaining after the Hexanexktration pellets were used for DNA determination. The results presented in Figure 4 that vitamin A palmitate is added from above medium quickly and effectively into the cells. For comparison, the cellular vitamin A palmitate concen- trations of an analog experiment are shown in which the same amount of vitamin A palmitate was not added to the medium in the form of nano-Cells, but as a cyclodextrin complex.

Bronchial: Cellular uptake of vitamin A palmitate

Human bronchial epithelial cell line NHBE were grown to confluence used, then washed once and incubated in a medium of the following composition: Vitamin A palmitate Nano Cell according to Example 6 0.0095% BEGM BulletKit ad 100.0000%

(Bronchial epithelial growth media, Clonetics)

After 15 minutes, 2, 8 and 24 hours, the cellular recorded Vitamin A palmitate amount was determined. For this purpose, the cells were harvested at the indicated time points, - washed, extracted with hexane, and the concentration of vitamin A palmitate in the hexane extract by HPLC. The remaining after the Hexanexktration pellets were used for DNA determination. The results presented in Figure 5 that vitamin A palmitate is added from above medium quickly and extremely efficiently into the cells. For comparison, the cellular vitamin A palmitate are concentrations of an analog test reasonable results, in which the same amount of vitamin E was not added to the medium in the form of nano-Cells, but as Cyclodextrinkom- plex.

10 and 11 mentioned in the above examples figures of the drawing show in detail the following:

Figure 1: Cellular uptake of vitamin E, respectively. Vitamin E acetate from media containing different vitamin E_ respectively. Vitamin E acetate supplements. The amounts of vitamin E absorbed by the cells are expressed as pmol / ug DNA. The values ​​are mean values ​​of four independent experiments.

Figure 2: Antioxidant capacity of different vitamin E, respectively. Vitamin E acetate supplements against intracellular generated radicals. The values ​​are mean values ​​+/- SD of four independent experiments.

Figure 3: Cytotoxicity of Vitamin E acetate Nanocells against HMEC-1 cells. The values ​​are mean values ​​of four independent experiments.

Figure 4: Cellular uptake of vitamin A palmitate in media containing vitamin A palmitate or Vitamin A palmitate Cells Nano-cyclodextrin complexes. The fibroblasts of the Hautfi- (HFP-1) amounts of vitamin A absorbed

Palmitate are expressed as .mu.mol / ug DNA. The values ​​are mean values ​​+/- SD of three independent experiments.

Figure 5: Cellular uptake of vitamin A palmitate in media containing vitamin A palmitate or Vitamin A palmitate Cells Nano-cyclodextrin complexes. Of the bronchial epithelial (NHBE) received amounts of vitamin A palmitate are expressed as .mu.mol / ug DNA. The values ​​are mean values ​​+/- SD of three independent experiments.

Claims

Patentanspr├╝che
1. Use of a nano-Cell containing as präparative composition
(A) a membrane-forming Molek├╝l, (b) a co-emulsifier and
(C) a lipophilic component __ in culture media-end products.
2. Use according to claim 1, characterized in that the nano Cell enthält: (a) as a membrane-forming Moleküle substances which are adapted to form two-layer systems,
(B) co-emulsifiers are substances that preferably form O / W structures and
(C) as the lipophilic component a für culture media-end products gebräuchlichen lipophilic substance.
3. Use according to claim 1 or 2, characterized in that the nano Cell as component
(A) a natürliches or synthetic phospholipid, a hydrogenated or partially hydrogenated phospholipid or mixtures of these micro enthält.
4. Use according to any one of Ansprüche 1 to 3, characterized in that component (a) in erfindungsgemäss used NanoCell in a concentration of 0.1 to 30 wt .-%, relative (on the total weight of the components a), (b) and (c) is present.
5. Use according to any one of Anspr├╝che 1 to 4, characterized in that the nano Cell as component
(b) alkali metal, ammonium or Aminumsalze of C3-C20 Fettsäuren, gesättigte ungesättigte and Cg-C ^ g alkyl sulfates, Cg-C2o ~ Al alkanesulfonates, Fettalkoholphosphor- säureester, salts of Gallensäure, invert soaps (quats); Partialfettsäureester of sorbitan, Zuckerester of Fettsäuren, Fettsäurepartialglyceride, alkyl maltosides, alkyl, Cg-C ^ g-Betaine '^ 8 - (8 -l - sulfobetaines or Cg-C 2 4-alkylamido-C 1 -C 4 -alkylenbe- taine, polyglycerol esters of Fettsäuren, Propylengylko- lester before. Fettsäuren, Milchsäureester of Fettsäuren, proteins, emulsifiers' oxyethyler from poly. type, or mixtures of these substances enthält.
6. Use according to any one of Ansprüche 1 to 4, characterized in that the nano Cell as component (b) emulsifiers of the polyoxyethylene type enthält.
7. Use according to claim 6, characterized in that the nano Cell as component
(B) Polyethoxylated Sorbitanfettsäureester, polyethoxylated vitamin E derivatives, Polyoxye-hylenglykolierte natürliche Pflanzenöle or hydrogenated, polyethoxylated fatty alcohols, polyethoxylated Fettsäuren, polyethoxylated Fettsäurepartialglyceride, polyethoxylated lanolin and the derivatives thereof, polyethoxylated carbohydrates, block polymers oxide or of ethylene oxide and propylene enthält mixtures of these substances.
S. Use according to any one of Ansprüche 1 to 7, characterized in that component (b) irr. erfindur.gsger.äss used NanoCell in a concentration of 1 to 5C wt .-%, based on the total weight of components (ε)
(B) and (c) is present.
9. Use according to any one of Anspr├╝che 1 to 8, characterized in that component
(C) a natürliches, a synthetic or a partialεyn- thetic di- or triglyceride, a für culture media the end products gebräuchlichen lipophilic substance or Mischunger. these substances enthält.
10. Use according to claim 9, characterized in the cass Nanc ell as component (c) a fettlösliches vitamin, a carotenoid, a non ubiquitin, a Flavσnoid, an isoflavonoid, a polyphenol, a Phytoöstrogen , a lipophilic Aminosäure a gesättigte or ungesättigte Fettsäure a essentiel- le Fettsäure, a lipid, a lipophilic antibiotic or antifungal agent, a lipophilic hormone, a lipophilic markers to study biological functions ^ or mixture thereof enthält.
11. Use according to one of Ansprüche 1 to 10, characterized in that component (c) in erfindungsgemäss NanoCell used in a concentration of 0.1 to 70 wt .-%, based on the total weight of components ( a), (b) and (c), is present.
12. Use according to one of Anspr├╝che 1 to 11, characterized in that the nano Cell as component
(d) enthält a C 2 -C 8 alcohol.
13. Use according to any one of Anspr├╝che 1 to 12, characterized in that the culture medium final product fl├╝ssig, solid or semisolid.
14. Fl├╝ssige or semi-solid culture media the end products containing a NanoCell as defined in claim. 1
15. Solid culture media the end products containing a NanoCell as defined in claim. 1
16. Culture medium-finished product according to claim 14, characterized in that the nano Cell is present in the culture medium wässrigen phase of the end product.
17. The culture media the end products according to claim 15, characterized in that the nano Cell is present in dehydrated form.
18. A process for the preparation of the flüssigen or semi-solid culture media the end products according to claim 14, characterized in that the nano-Cells are incorporated in the wässrigen proportion of end products.
19. A method for preparing the solid Kulturmedien- end products according to claim 15, characterized in that the culture media the end products are ├╝berzogen with the NanoCell or loaded.
20. A process for preparing the solid Kulturmedien- end products according to claim 15, characterized in that the nano-Cells are mixed mixture in dehydrated form the solid fabric.
21. Use of the culture media the end products according to one of Anspr├╝che 13 to 17 for the in vitro cultivation and analysis of cells, fungi, bacteria, viruses, bacteriophages, insect and plant, respectively, for the in vitro ex vivo culturing and analysis of tissues and organs (for freezing of organs, tissues, cells, fungi, bacteria, viruses, bacteriophage, insect and plant, for Tranfer of organs, tissues and embryos, for the therapy adoptive
Immunotherapy, Krebsbehanclung) for perfusion of organs, organ storage until their transplant f├╝r cytogenetic, molecular genetic, pr.╬╡rmakolcgische, toxicological and metabolic studies, f├╝r up n├ñhme and Tr╬ ╡nsportstudien or to study functional tional and regulatory mechanisms.
22. Use of a nano Cell comprising
(A) a membrane-forming Molek├╝l,
(B) a co-emulsifier and (c) a lipophilic component in culture media end-products, wherein the nano-Cell is erh├ñltlich by (╬▒) mixing the components (a), (b) and (c) until a homogeneous, clear Fl├ ╝ssigkeit called Nano Cell preliminary phase arises and
(/ 3) adding in step (α) obtained in the Flüssigkeit wässrigen portion of the food-end products, characterized in that steps (α) and (3) take place without additional energy input. __
PCT/CH1999/000420 1998-09-14 1999-09-08 Utilization of nanocells in final culture medium products WO2000015763A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2417943A1 (en) 2002-04-24 2012-02-15 Biosensors International Group, Ltd. Drug-delivery endovascular stent and method for treating restenosis

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WO1993015736A1 (en) * 1992-02-18 1993-08-19 Pharmos Corp. Dry compositions for preparing submicron emulsions
EP0696452A1 (en) * 1994-08-08 1996-02-14 Laboratorios Cusi, S.A. Nanoemulsion of the oil in water type, useful as an ophthalmic vehicle and process for the preparation thereof
WO1997021428A1 (en) * 1995-12-12 1997-06-19 Vesifact Ag Cortisone spray for topical administration
EP0852941A1 (en) * 1996-12-13 1998-07-15 Vesifact Ag Nanodispersion cosmetic composition

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993015736A1 (en) * 1992-02-18 1993-08-19 Pharmos Corp. Dry compositions for preparing submicron emulsions
EP0696452A1 (en) * 1994-08-08 1996-02-14 Laboratorios Cusi, S.A. Nanoemulsion of the oil in water type, useful as an ophthalmic vehicle and process for the preparation thereof
WO1997021428A1 (en) * 1995-12-12 1997-06-19 Vesifact Ag Cortisone spray for topical administration
EP0852941A1 (en) * 1996-12-13 1998-07-15 Vesifact Ag Nanodispersion cosmetic composition

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2417943A1 (en) 2002-04-24 2012-02-15 Biosensors International Group, Ltd. Drug-delivery endovascular stent and method for treating restenosis

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