DD297170A5 - PROCESS FOR FIXING LIPID LAYERS ON POLYMER TRAUGER SURFACES - Google Patents

Abstract

The invention relates to a method for fixing lipid layers on polymeric Traegeroberflaechen. This novel composite material is used as a biocompatible matrix in medicine, in cell and tissue culture, as a carrier material for bioaffinity chromatography, or for immunodiagnostic procedures and after enzyme fixation in bioreactors for biotechnological conversion. The essence of the invention is to bind lipids covalently via their head group on the surface of monodisperse melamine / formaldehyde spheres by either the methylol groups on the Traegeroberflaeche or the lipid head groups are activated before the coupling and then the resulting lipid monolayer by adsorption of additional lipid to a Build up the bishops system {surface coating; Traegeroberflaeche, polymer; lipid; Lipid layer; Monolayers; bilayer; Proteins, membrane bound}

Description

Field of application of the invention

The invention relates to a method for fixing lipid layers on polymeric support surfaces.

Membrane-bound proteins can accumulate in a specific manner on such surface-modified polymer particles. Lipid-coated carrier materials are suitable for use in medical diagnostics, especially for immunological methods, they have been used as a biocompatible matrix in medicine and in cell and tissue engineering and are also suitable as carrier material for chromatographic methods (HPLC and bioaffinity chromatography).

After enzyme fixation on such lipid-coated polymer surfaces, they can be used in bioreactors for biotechnological material conversion.

Lipid-coated support materials can also be used as a membrane model in biochemical research.

Characteristic of the known technical solutions

Methods / 1,2,3 / for the superficial, covalent fixation of phospholipids and glycolipids via their head group to methyl methacrylate particles are known. Subsequent lipid adsorption achieved a bilayer coating.

This bilayer system resembles the structure of the normal cell membrane, it is regenerable, and it can stably incorporated into it membrane-bound proteins.

For use as a carrier, non-porous CALLOCRYL particles, microporous / CRYLEX material and macroporous perl cellulose have been described. As lipids phosphatidylethanolamine and some Glycolip Ie were used and as spacer only Pentaalanin was used so far. The introduction of a spacer is necessary if trans-embrane proteins are to be incorporated.

The disadvantages of the materials thus obtained are their size heterogeneity, low resistance to organic solvents, the high cost of manufacturing supports, lipid and spacer, and the extremely alkaline carrier derivatization conditions which result in non-uniform products.

Furthermore, the fixation of a lipid monolayer to aminopropyl-modified silica gel (Nucleosil-300) is known / 4 /.

However, the covalent coupling takes place via a terminal COOH group previously introduced into the HC chain of the lipid.

Thus, only a monolayer coating is possible.

Furthermore, adsorption-induced lipid deposits on polystyrene carrier beads were examined / 5 /. The long-term stability of such adsorbed lipid layers is unsatisfactory.

It is also known / 6,7,8 / to adsorb lipid layers on flat surfaces (glass, mica, silanized glass). Although ordered, regularly structured lipid mono- and bilayers were formed on these surfaces, these layers were sensitive to mechanical stress and could be destroyed by detergency,

Literature:

1. Rolhe, Ulrich Dissertation B (1989) MLU Halle

2. Rothe, U. and Aurich, H. Biotechnology and Applied Biochemistry 11 (1989) 18-30

3. Rothe, U. and Aurich, H. Wiss. Contributions of MLU WB 1989/32 (P38) pp. 40-61

4. Pidgeon, C. and Venkolaram, U.V. Analyte. Biochem. 176 (1989) p. 36-47

6. Reuinger, G.S. Meredith, CS .; Lau, S.H .; Kaiser, E.T .; Kezdy, F.J. Analyte. Biochem. 150 (1985) pp. 131-140

6. v.Tscharner.V .; McConnell.H.M.Biophys. J.36 (1981) S.421-427

7. Tamm, L.K .; McConnell, H.M. Biophys. J. 36 (1985) pp. 105-113

8. Sagiv, J., J.Amer.Chem.Soc.102 (1980) p.82-98

Object of the invention

It is the object of the invention to produce on the surface of suitable polymeric materials a tightly bound and densely packed lipid layer to which another lipid layer can be adsorbed so as to form a bilayer similar to the biological membrane.

-2- 297 170 Explanation of the nature of the invention

The object of the invention is a process for coating polymeric support surfaces with a densely packed and through

To develop covalent bond firmly fixed, monomolecular Lipldauflage, wherein the thus fixed lipid monolayer

Adsorption of additional lipid can be converted into a bilayer. According to the invention, this object is achieved in that methylol groups on the surface of monodisperse melamine / Formaldehyde polymer beads having particle sizes in the range of 1-10μηι be activated with fluoromethylpyridine and that

then lipids with Amlno head groups are coupled to the activated surface.

The functional groups of the particle surface also react without prior activation when lipids are used,

who were previously activated on their head group.

Since the carriers are solvent-resistant, the lipid coupling also succeeds in organic medium with high yields. The

The lipid layer obtained according to the invention can be modified in various ways. By subsequent attachment of various lipids or lipid mixtures, a bilayer is formed which may be constructed both symmetrically and asymmetrically. Likewise, hydrophilic linearly-built spacer molecules of variable length in the range of 10-50A can be inserted between the support surface and the inner lipid layer.

In the Lipiddoppelschlchten he produced in accordance with the membrane-bound proteins while preserving their function

be incorporated stably.

embodiment The invention will be described below with 6 examples.

1. Melamine resin and phosphate dithiol ethanol (FMP)

1 g of dry support material (melamine / formaldehyde latex) with a particle diameter of 2.1 μm and a surface area of about 2.8 m ² (Lerche, KH and Kretschmar, G. WP DD 224 602 A1) is dissolved in 10 ml of dry DIV .F stirred. The suspension is degassed and to the stirred batch is added 0.9 g (3.2 mmol) FMP. The activation superficial methylol istnach Sstündigem shaking at 2O ⁰ C ended. The activated polymer material is washed on a frit with DMF and transferred to the coupling batch after aspiration of excess liquid.

Coupling Approach A: 5 ml of a 50 mM carbonate buffer pH 8.3 which is 1% octylglucoside and 50 mg phosphatidylethanolamine

(Dihexadecyl etherlipld) and was 3 minutes with ultrasound dispersed.

Coupling B: 50mg PE dissolved in 5ml dry CHCI ₃ . In both cases, the suspension is left for 24 h at room temperature

shaken. The course of the reaction can be monitored photometrically on the basis of the reaction product methylpyridone detectable in the supernatant at 297 nm.

After washing the lipidated support, remaining FMP-activated OH groups are reacted with 0.1 M ethanolamine.

2. Melamlin resin and O-succinyl cardioliplin

15mg (10μΜοΙ) of prepared by reaction of cardiolipin with succinic anhydride in the presence of dimethylaminopyridine in pyridine O-succinyl-cardiolipins are suspended according to variant A in 5ml of 1% aqueous Oktylglucosidlösung and homogeneously dispersed by 3-minute ultrasound treatment. With 1N HCl the pH is adjusted to 3.3 and 10mg (50μΜοΙ) solid EDC is added. Under pH control, activate for 5 min. The activated lipid is then poured into the prepared particle suspension (0.25 g melamine / formaldehyde latex with 0 = 2 pm in 5 ml 0.1 M Borax pH 8.5) and shaken overnight. Variant B exploits the resistance of the carrier to organic solvents. 15 mg of O-Succ-CL are dissolved in 20 ml of chloroform and, while stirring, 0.25 g of melamine / formaldehyde latex and 0.5 g of dicyclohexylcarbodiimide are added. After 5 hours, the reaction is complete. Thorough washing frees the particles of adhering dicyclohexylurea.

3. Introduction of variable length PE (PEG) melamine spacers

The two terminal OH groups of 3 different PEG batches (PEG 200, 400 and 600) are converted to half ester with succinic anhydride and the free COOH groups are converted to the active imidazolide (Ferruti, P. Tanzi, MC and Vaccaroni, F. Macromol Chem.180 [1979] p.375-382). 0.2 g of the PEG-200 imidazolide (or equivalent amounts of the higher oligomers) are dissolved in 10 ml of dry chloroform, and with vigorous stirring to add 0.1 g of melamine / formaldehyde latex particles. The reaction of the imidazolide with superficial amino or rViethylolgruppen is complete after 4 h Lei 4O ⁰ C. The excess, unreacted PEG-imidazolide is washed out with chloroform. Subsequently, the carrier is resuspended in 10 ml of chloroform and 50 mg of phosphatidylethanolamine from chicken egg dissolved in 1 ml of CHCl _{3 are} added. It is shaken overnight, filtered and the carrier washed with chloroform, methanol and water.

4. Delphilization Rellpldlerverfahren for PE melamine resins

The lipid-coated carrier particles prepared according to Examples 1-3 can be freed from the only adsorptively bound lipid portion by successive washing with the 20-fold carrier volume of chloroform, methanol and water (delipidation).

By dialyzing a suspension of the delipidated particles together with any lipid or lipid mixture dispersed in a high CMC detergent (eg octylglucoside) against a suitable detergent-free buffer, a second lipid layer can be attached to the lipid monolayer covalently attached to the carrier (relipidation). , Surprisingly, the detachment and a renewed attachment of this outer bilayer half are easily repeated *, so that bilayer-coated particles can be regenerated. Since the adsorptive lipid accumulation of the upper layer does not depend on the nature of the firmly bound inner lipid portion, it is thus possible to produce asymmetrically constructed lipid layers in a simple manner.

5. Incorporation of Membrane-Bound Proteins Into the Supported Spacer Layer Membrane-bound proteins have hydrophobic anchor structures anchoring them in the biomembrane. They can be dissolved out of the membrane by detergent treatment. 1 mg of the isolated anchor form (or d-form) of Dipeptldytpeptidaee IV (DPP IV) was taken up in 1 ml of 1% octylglucoside solution (50 mM Trls / HCl pH 7.8) and co-prepared with 5 ml of carrier suspension (containing 100 mg of the delipidated carrier Example 4) and 20 mg of egg lecithin (dispersed in 1 ml of 1% aqueous solution of octylglucoside) dialyzed in a rotating dialysis tube for 24 h against a suitable detergent-free buffer. Unbound lipid and protoin are then removed by intensive washing of the particles. 72% of the enzyme activity used was firmly fixed to the carrier surface.

Claims (4)

A method for fixing lipid layers to polymeric support surfaces, characterized in that the methylol groups of monodisperse melamine / formaldehyde polymer beads having particle sizes in the range of 1-10 microns are activated with fluoromethylpyridine and then lipids are coupled via their amino head group to the activated surface or on the head group modified lipids are used, which react with functional groups on the support surface (-NHz, -CH2-OH) and that is attached to the resulting monolayer by adsorption a second lipid layer to form a bilayer. 2. The method according to claim 1, characterized in that different lipids or lipid mixtures are used to form the bilayer. 3. The method according to claim 1 and 2, characterized in that the bilayer is constructed symmetrically or asymmetrically. 4. The method according to claim 1, characterized in that the surface of the particles before the lipid coupling by Finfüh <ng hydrophilic linearly constructed spacer molecules with variable length in the range of 10-50Ä was modified.