Method of isolating a selected population of exosomes

Abstract

The invention relates to isolating a selected population of exosomes with high specificity, thereby allowing accurate determination of exosomal protein content which is useful in the prediction and identification of a subject having Parkinson's Disease and in differentiating Parkinson's disease from atypical parkinsonian syndromes including MSA.

1 . A coated particle having a coating comprising a zwitterionic polymer coupled to a ligand having affinity for a selected population of exosomes. 
     
     
         2 . The coated particle of  claim 1 , wherein the zwitterionic polymer comprises carboxybetaine, sulfobetaine and/or phosphoryl choline moieties. 
     
     
         3 . The coated particle according to  claim 1 , wherein the zwitterionic polymer comprises poly(carboxybetaine methacrylate). 
     
     
         4 . The coated particle according to  claim 1 , wherein the ligand: (a) has affinity for neuron-derived exosomes, and/or (b) is an anti-L1CAM antibody. 
     
     
         5 . The coated particle according to  claim 1 , where the particle is: (a) from 30 nm to 5 μm in size, (b) from 100 nm to 5 μm in size, or (c) from 500 nm to 3 μm in size. 
     
     
         6 . The coated particle according to  claim 1 , wherein at least 80% of the particle surface is coated with polymer. 
     
     
         7 . The coated particle according to  claim 1 , wherein the polymer coating has a thickness of from 10 nm to 500 nm. 
     
     
         8 . The coated particle according  claim 1 , wherein the particle has a degree of nonspecific adsorption of less than 10%. 
     
     
         9 . The coated particle according to  claim 1 , wherein the polymer has a brush structure. 
     
     
         10 . The coated particle according to  claim 1 , wherein the polymer is obtainable by a RAFT polymerisation process or a RAFT process using bis(carboxymethyl)trithiocarbonate (BCMTTC) as a chain transfer agent. 
     
     
         11 . The coated particle according to  claim 1  which is obtained or obtainable by growing the zwitterionic polymer on the particle. 
     
     
         12 . A method of isolating exosomes from a sample, comprising steps of:
 contacting the sample with the coated particle of  claim 1 ;   removing unbound sample; and   separating the captured exosomes.   
     
     
         13 . A method for analysing a blood sample from a subject, comprising isolating neuron-derived exosomes from the sample according to the method of  claim 12 , and determining the levels of α-synuclein and/or clusterin in the neuron-derived exosomes in the blood sample. 
     
     
         14 . The method of  claim 13 , wherein the levels of α-synuclein and clusterin provide a diagnostic indicator of a subject susceptible to Parkinson's disease (PD) or of a subject having PD. 
     
     
         15 . The method of  claim 14 , wherein the levels of α-synuclein and clusterin provide a diagnostic indicator of a subject having prodromal PD. 
     
     
         16 . The method of  claim 14 , wherein an increase in the level of α-synuclein relative to a reference indicates that the subject is susceptible to PD or has PD, optionally wherein the reference is a threshold value of between 10-20 pg/ml. 
     
     
         17 . The method of  claim 14 , wherein a lack of increase in the level of clusterin relative to a reference indicates that the subject is susceptible to PD, optionally wherein the reference is a threshold value of between 7-17 ng/ml. 
     
     
         18 . A method of producing a coated particle according to  claim 1  which comprises the steps of:
 (a) growing a zwitterionic polymer on the surface of a particle using reversible addition fragmentation chain transfer (RAFT) to provide a particle having a coating comprising a zwitterionic polymer; 
 (b) optionally activating the zwitterionic polymer to provide active functional groups on the zwitterionic polymer; and 
 (c) conjugating a ligand having affinity for the selected population of exosomes to the zwitterionic polymer. 
 
     
     
         19 . The method of  claim 18 , wherein step (a) comprises using bis(carboxymethyl)trithiocarbonate (BCMTTC) as a chain transfer agent. 
     
     
         20 . The coated particle according to  claim 11  which is obtained or obtainable by a method which comprises steps of:
 (a) growing a zwitterionic polymer on the surface of a particle using reversible addition fragmentation chain transfer (RAFT) to provide a particle having a coating comprising a zwitterionic polymer; 
 (b) optionally activating the zwitterionic polymer to provide active functional groups on the zwitterionic polymer; and 
 (c) conjugating a ligand having affinity for the selected population of exosomes to the zwitterionic polymer; 
 
       wherein step (a) optionally comprises using bis(carboxymethyl)trithiocarbonate (BCMTTC) as a chain transfer agent.