Salipro particles

Abstract

The invention provides a nanoscale particle comprising a lipid binding polypeptide, lipids and a hydrophobic agent, wherein the hydrophobic agent is different from the lipids, and wherein the lipid binding polypeptide is a saposin-like protein or a derivative or truncated form thereof. The invention further provides a process for preparing a particle comprising a saposin-like protein or a derivative or truncated form thereof and lipids comprising the step of (a) contacting the saposin-like protein or a derivative or truncated form thereof with solubilized lipids in a liquid environment and (b) allowing for the self-assembly of the particle at a pH of from 5.0 to 10:0. In addition, pharmaceutical compositions and methods for using the particle of the invention are described.

1 . A particle comprising
 a lipid binding polypeptide,   lipids and   a hydrophobic agent,   wherein the hydrophobic agent is different from the lipids, and   wherein the lipid binding polypeptide is a saposin-like protein or a derivative or truncated form thereof.   
     
     
         2 . The particle according to  claim 1 , wherein the particle is substantially disc-shaped. 
     
     
         3 . The particle according to  claim 1 , wherein the particle has a maximum diameter of from 2 nm to 200 nm, in particular from 3 nm to 150 nm, preferably from 3 nm to 100 nm. 
     
     
         4 . The particle according to  claim 1 , wherein the hydrophobic agent is selected from the group consisting of a hydrophobic organic compound and a hydrophobic biomolecule. 
     
     
         5 . The particle according to  claim 4 , wherein the hydrophobic organic compound and/or the hydrophobic biomolecule is selected from the group consisting of a biologically active agent, a drug, an active ingredient of a drug, an active ingredient of a cosmetic product, an active ingredient of a plant protective product, a dietary and/or nutritional supplement, a diagnostic probe, a contrast agent, a label and an indicator. 
     
     
         6 . The particle according to  claim 4 , wherein the hydrophobic biomolecule is a protein comprising a hydrophobic moiety, in particular a protein selected from the group consisting of a membrane protein, an integral transmembrane protein, an integral monotopic membrane protein, a peripheral membrane protein, an amphitropic protein in a lipid-bound state, a lipid-anchored protein and a chimeric protein with a fused hydrophobic and/or transmembrane domain. 
     
     
         7 . The particle according to  claim 1 , wherein the lipids are lipid bilayer forming lipids and/or biocompatible lipids. 
     
     
         8 . The particle according to  claim 1 , wherein the lipids are selected from the group consisting of eukaryotic lipids, phospholipids and/or lipids present in the white and grey matter of the brain, in particular wherein the lipids are selected from the group consisting of phospholipids, glycosphingolipids, sterols, phosphatidylcholine, phosphatidylserine (PS), 2-oleoyl-1-pamlitoyl-sn-glycero-3-phosphocholine (POPC), 2-oleoyl-1-pamlitoyl-sn-glycero-3-glycerol (POPG), 2-oleoyl-1-pamlitoyl-sn-glycero-3-phosphoethanolamine (POPE), diacylglycerol, cholesterol, sphingomyelin, galactosylceramide, gangliosides, phosphatidylinositoles and sulphogalactoceramides or combinations thereof and particularly preferred wherein the lipids comprise phosphatidylserine (PS). 
     
     
         9 . The particle according to  claim 1 , wherein the lipid binding polypeptide is a chimeric polypeptide further comprising a functional moiety such as a targeting moiety or a bioactive moiety. 
     
     
         10 . The particle according to  claim 1 , wherein the lipid binding polypeptide is saposin A or a derivative or truncated form thereof, in particular a derivative of saposin A comprising an amino acid sequence with at least 60% sequence identity to SEQ ID NO. 1. 
     
     
         11 . A process for preparing a particle comprising a lipid binding polypeptide and lipids,
 wherein the lipid binding polypeptide is a saposin-like protein or a derivative or truncated form thereof,   the process comprising the step of   a) contacting the lipid binding polypeptide with solubilized lipids in a liquid environment;   b) allowing for the self-assembly of the particle at a pH of from 5.0 to 10.0.   
     
     
         12 . The process according to  claim 11 , wherein the lipids used in step a) are in a detergent-solubilized state. 
     
     
         13 . The process according to  claim 12 , wherein step b) comprises diluting the mixture obtained in step a) with a liquid containing less amounts of detergent than the mixture obtained in step a). 
     
     
         14 . The process according to  claim 11 , wherein the process comprises in step b) or as a subsequent step c) the purification of the particles by at least partial removal of free lipids and/or free lipid binding polypeptide, wherein optionally the purification is performed by chromatography, in particular size-exclusion chromatography; ultracentrifugation; dialysis; contacting with detergent-binding biobeads; use of concentrators; affinity chromatography, magnetic beads and/or membrane/filters to remove unbound/non-incorporated lipids and/or hydrophobic compounds. 
     
     
         15 . The process according to  claim 11 , wherein the particle is as defined in any one of  claim 2  or  3 , the lipids are as defined in  claim 7  or  8  and/or the lipid binding polypeptide is as defined in  claim 9  or  10 . 
     
     
         16 . The process according to  claim 11  for preparing a particle according to any one of  claims 1  to  10 , wherein in step a) the lipid binding polypeptide is contacted with lipids in a liquid environment comprising the hydrophobic agent that is to be incorporated into the particle, and optionally, wherein the hydrophobic agent is as defined in  claims 4  to  6 . 
     
     
         17 . A particle obtainable according to the process of  claim 11 . 
     
     
         18 . A pharmaceutical composition for delivering a hydrophobic agent to an individual in need thereof, comprising a particle according to any one of  claim 1  or  17  wherein the hydrophobic agent is an active ingredient and/or wherein in addition to the hydrophobic agent an active ingredient is present. 
     
     
         19 . A method for preventing, treating or lessening the severity of a disease in a subject in need or a method for diagnosing a disease in a subject in need or a method of performing a cosmetic treatment on a subject in need, the methods comprising administering an effective amount of a particle of  claim 1  or a particle of  claim 17  to each of the subjects in need of prevention, treatment or lessening the severity of said disease, or diagnosis of a disease or in need of cosmetic treatment. 
     
     
         20 . Use of a particle according to  claim 1  as hydrophobic agent delivery particle, as a tool for drug development, drug screening, membrane protein research or as vaccination formulation.