Plasma Membrane Isolation

Abstract

The present invention relates to a population of monodisperse magnetic nanoparticles with a diameter between 1 and 100 nm which are coated with a layer with hydrophilic end groups. Herein the layer with hydrophilic end groups comprises an inner layer of monosaturated and/or monounsaturated fatty acids bound to said nanoparticles and bound to said fatty acids, an outer layer of a phospholipid conjugated to a monomethoxy polyethyleneglycol (PEG) comprising a hydrophilic end group, or comprises a covalently bound hydrophilic layer bound to said nanoparticles.

1 . A population of monodisperse positively charged magnetic nanoparticles which have a zeta potential in the range of 10-30 mV at pH 7 and a diameter between 1 and 100 nm which are coated with a layer with hydrophilic end groups, wherein said coating has an inner layer of monosaturated and/or monounsaturated fatty acids bound to said nanoparticles and bound to said fatty acids and an outer layer of a phospholipid conjugated to a monomethoxy polyethyleneglycol (PEG). 
     
     
         2 . The population according to  claim 1 , wherein said nanoparticles with an inner layer of fatty acids bound and an outer layer of a phospholipid do not comprise a peptide moiety. 
     
     
         3 . The population according to  claim 1 , wherein said hydrophilic end group is a phosphonate, an amine, a C 1 -C 20  alkane, a C 1 -C 20  alkene, a C 1 -C 20  alkyene, an azido, an epoxy, an NH 2 , a COOH, unsubstituted or substituted PEG, PDP, CHO or SH. 
     
     
         4 . The population according to  claim 1 , wherein the phospholipids in the outer layer are Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)-2000](DSPE-PEG-COOH), Distearoyl-sn-glycero-3-phosphoethanolamine-N-[Amine (polyethylene glycol)-2000] (DSPE-PEG-Amine), Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](DSPE-PEG), DSPE-Folate, DSPE-PEG(2000) Maleimide or DSPE-PEG(2000) Carboxyfluroscein. 
     
     
         5 . The population according to  claim 1 , wherein the covalently bound hydrophilic layer is silane, dimercaptosuccinic acid (DMSA) or ammonium chloride. 
     
     
         6 . A method of preparing a monodisperse population of magnetic nanoparticles with a diameter between 1 and 100 nm which are coated with a layer with hydrophilic end groups, comprising
 a) providing a magnetic material,   b) applying a layer of monounsaturated and/or monosaturated fatty acid to said material by thermal decomposition under conditions to provide magnetic nanoparticles with a diameter between 1 and 100 nm,   c) precipitating said fatty acid coated magnetic nanoparticles with an alcohol,   d) discarding from the nanoparticles of step c) the population of aggregated nanoparticles and collecting the population of monodisperse nanoparticles,   e) applying a layer of lipids comprising a hydrophilic end group to the monodisperse nanoparticles of step d).   f) selecting from the nanoparticles obtained in step e) the population of monodisperse nanoparticles in the presence of solvent,
 or instead of e) and f), 
   g) replacing the fatty acid coating with a hydrophilic layer in the presence of a nonaqueous solvent (chloroform), and   h) selecting from the nanoparticles obtained in step g) the population of monodisperse nanoparticles in the presence of said non aqueous solvent.   
     
     
         7 . The method of  claim 6 , wherein in g), said layer with hydrophilic end group is DMSA, Silane, Tetramethylammonium hydroxide (TMAOH) or ammonium chloride. 
     
     
         8 . A monodisperse population of magnetic nanoparticles obtained by the method according to  claim 6 . 
     
     
         9 . An isolated complex of a nanoparticle according to  claim 1  with the plasma membrane or with a plasma membrane derived organelle. 
     
     
         10 . A method for isolating a plasma membrane of a cell, a fraction thereof, or a plasma membrane derived organelle, comprising
 1. providing a population of intact and suspended cells at a temperature where endocytic uptake by a cell is inhibited,   2. contacting said intact cells with magnetic nanoparticles of  claim 1 , thereby allowing the binding of magnetic nanoparticles to and into the cell plasma membrane,   3. removing unbound magnetic nanoparticles,   4. disrupting the cells,   5. removing cellular organelles,   6. isolating from the disrupted cells by magnetic attraction the plasma membranes with magnetic nanoparticles.   
     
     
         11 . A preparation of a plasma membrane, wherein at least 60% of the proteins in said preparation are integral membrane proteins or proteins associated therewith. 
     
     
         12 . The population according to claim or  2 , wherein said hydrophilic end group is a phosphonate, an amine, a C 1 -C 20  alkane, a C 1 -C 20  alkene, a C 1 -C 20  alkyene, azido, epoxy, NH 2 , COOH, unsubstituted or substituted PEG, PDP, CHO or SH. 
     
     
         13 . The population according to  claim 2 , wherein the phospholipids in the outer layer is Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000](DSPE-PEG-COOH), Distearoyl-sn-glycero-3-phosphoethanolamine-N-[Amine (polyethylene glycol)-2000] (DSPE-PEG-Amine), Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](DSPE-PEG), DSPE-Folate, DSPE-PEG(2000) Maleimide or DSPE-PEG(2000) Carboxyfluroscein. 
     
     
         14 . An isolated complex of a nanoparticle according to  claim 8  with a plasma membrane or with a plasma membrane derived organelle. 
     
     
         15 . A method for isolating a plasma membrane of a cell, a fraction thereof, or a plasma membrane derived organelle, comprising:
 a) providing a population of intact and suspended cells at a temperature where endocytic uptake by a cell is inhibited,   b) contacting said intact cells with magnetic nanoparticles of  claim 8 , thereby allowing the binding of magnetic nanoparticles to and into the cell plasma membrane,   c) removing unbound magnetic nanoparticles,   d) disrupting the cells,   e) removing cellular organelles,   f) isolating from the disrupted cells by magnetic attraction the plasma membranes with magnetic nanoparticles.