Amine functionalized superparamagnetic nanoparticles for the synthesis of bioconjugates and uses therefor

Abstract

Amine functionalized magnetic nanoparticle compositions and processes for synthesizing the same are described. The process consists of obtaining a carboxylated polymer in substantially pure form, which is used to prepare a substantially size homogeneous, polymer coated carboxyl, functionalized magnetic nanoparticle. The carboxyl groups are converted to reactive primary amino groups by the use of a water-soluble carbodiimide followed by reaction of a large excess of a diamine. The amine-terminated nanoparticles are then reacted with bifunctional crosslinking agents and with various biomolecules to make nanoparticles for in vitro assays, cell sorting applications and target specific MR contrast agents.

We claim:  
     
         1 . A nanoparticle comprising: 
 (i) magnetic core having one or more magnetic metal oxide crystals; and    (ii) a noncrosslinked polymer coating associated with the core, wherein the polymer coating has a plurality of carboxyl groups and plurality of reactive primary amino groups, wherein a portion of the carboxyl groups are associated with the crystals.    
     
     
         2 . The nanoparticle of  claim 1  wherein reactive primary amino groups are associated with polymer through a peptidyl linkage of the formula: 
       —O—(CH 2 ) m —CONH—[X] wherein X is —(CH 2 ) n  NH 2 , —(CH 2 ) o CH NHCOO—, —(CH 2 ) 3 NH (CH 2 ) 4 NH 2  or —(CH 2 ) 3 NH(CH 2 ) 4 NH(CH 2 ) 3  NH 2 —   wherein m=1,2, or 3; n=2,3, 6, and o=3 or 4.    
     
     
         3 . The nanoparticle of  claim 1 , wherein the magnetic core comprises one or more superparamagnetic iron oxide crystals.  
     
     
         4 . The nanoparticle of  claim 3  wherein the superparamagnetic core has a diameter between about 1 nm and about 25 nm.  
     
     
         5 . The nanoparticle of  claim 4  where in the superparamagnetic core has a diameter between about 3 nm and about 10 nm.  
     
     
         6 . The nanoparticle of  claim 5  wherein the core has a diameter about 5 nm.  
     
     
         7 . The nanoparticle of  claim 1  wherein the nanoparticle has diameter between about 15 nm and 100 nm.  
     
     
         8 . The nanoparticle of  claim 7  wherein the nanoparticle has a diameter between about 20 nm and about 100 nm.  
     
     
         9 . The nanoparticle of  claim 1  wherein the coating is selected from the group of polymers consisting of natural polymers, synthetic polymers and derivatives thereof.  
     
     
         10 . The nanoparticle of  claim 1  wherein the polymer is carboxymethyldextran.  
     
     
         11 . The nanoparticle of  claim 1  wherein the nanoparticle is conjugated with a biomolecule.  
     
     
         12 . The nanoparticle of  claim 11  wherein the biomolecule has at least one sulfhydryl group wherein the sulfhydryl group reacts with an amino group on the coating.  
     
     
         13 . A nanoparticle-bioconjugate molecule comprising: 
 (i) a nanoparticle, the nanoparticle comprising 
 (a) a magnetic core having one or more magnetic metal oxide crystals;  
 (b) a noncrosslinked polymer coating associated with the core, wherein the polymer coating has a plurality of carboxyl groups and plurality of reactive primary amino groups, wherein a portion of the carboxyl groups are associated with the crystals;  
   (ii) a biomolecule linked to the nanoparticle, wherein the biomolecule has at least one sufhydryl group linked to the amino group.    
     
     
         14 . The a nanoparticle-bioconjugate of  claim 13  wherein the amino groups are associated with polymer through a peptidyl linkage of the formula: 
       —O—(CH 2 ) m —CONH—[X] wherein X is —(CH 2 ) n  NH 2 , —(CH 2 ) o CH NHCOO—, —(CH 2 ) 3 NH (CH 2 ) 4 NH 2  or —(CH 2 ) 3 NH(CH 2 ) 4 NH(CH 2 ) 3  NH 2      wherein m=1, 2, or 3; n=2, 3, 6, and o=3 or 4.    
     
     
         15 . A process for synthesizing an amine functionalized magnetic metal oxide nanoparticle comprising: 
 i. obtaining a polymer having a plurality of carboxyl groups attached thereto,    ii. contacting the polymer with magnetic metal oxide to produce a coated magnetic metal oxide wherein a portion of the carboxyl groups are associated with the metal oxide;    iii. reacting the coated magnetic metal oxide of step (ii) with a diamine.    
     
     
         16 . The process of  claim 15  wherein the diamine is ethylene diamine.  
     
     
         17 . The process of  claim 15  wherein the diamine is hexane diamine.  
     
     
         18 . The process of  claim 15  wherein step (ii) further comprises the steps of 
 (a) providing a solution of soluble iron salts;  
 (b) converting the iron salts into iron oxide crystals; and  
 (c) removing unassociated polymer.  
 
     
     
         19 . The process of  claim 18  wherein the step of converting the iron salts into iron oxides further includes the steps of: 
 (d) heating the iron salts to a temperature between about 4° C. and about 20° C.;  
 (e) adding an amount of base to raise the pH to about 8 or higher to form iron oxides.  
 
     
     
         20 . The process of  claim 19  further including the step of heating the iron oxides to a temperature about 60° C. or more for at least 30 minutes.  
     
     
         21 . A pharmaceutical composition comprising: 
 (A) A nanoparticle-bioconjugate molecule comprising: 
 (i) a nanoparticle, the nanoparticle comprising 
 (a) a magnetic core having one or more magnetic metal oxide crystals;  
 (b) a noncrosslinked polymer coating associated with the core, wherein the polymer coating has a plurality of carboxyl groups and plurality of reactive primary amino groups, wherein a portion of the carboxyl groups are associated with the crystals;  
 
 (ii) a biomolecule linked to the nanoparticle, wherein the biomolecule has at least one sufhydryl group linked to at least one amino group; and  
   (B) a pharmaceutically acceptable carrier.