Methods of forming targeted liposomes loaded with a therapeutic agent

Abstract

The present invention provides for liposomes loaded with a therapeutic agent, which liposomes are targeted to a cell of interest by incubation with a targeting protein protein conjugated to a linker molecule comprising a hydrophobic domain, a hydrophilic polymer chain terminally attached to the hydrophobic domain, and a chemical group reactive to one or more functional groups on a protein molecule and attached to the hydrophilic polymer chain at a terminus contralateral to the hydrophobic domain, for a time sufficient to permit the hydrophobic domain to become stably associated with the liposome.

1 . A method for providing liposomes loaded with a therapeutic agent, which liposome is targeted to a cell of interest, said method comprising attaching liposomes loaded with a therapeutic agent to a protein by means of a linker molecule, comprising the step of: 
 incubating said liposome loaded with a therapeutic agent with a protein conjugated to a linker molecule comprising a hydrophobic domain, a hydrophilic polymer chain terminally attached to the hydrophobic domain, and a chemical group reactive to one or more functional groups on a protein molecule and attached to the hydrophilic polymer chain at a terminus contralateral to the hydrophobic domain, for a time sufficient to permit the hydrophobic domain to become stably associated with the liposome loaded with a therapeutic agent, wherein said protein targets said liposome to said cell of interest.    
   
   
       2 . A method of  claim 1 , wherein said therapeutic agent is a chemotherapeutic agent.  
   
   
       3 . A method of  claim 1 , wherein less than 3.7% of said therapeutic agent leaks from said liposome loaded with said therapeutic agent during said incubation.  
   
   
       4 . A method of  claim 1 , wherein less than 2.0% of said therapeutic agent leaks from said liposome loaded with said therapeutic agent during said incubation.  
   
   
       5 . A method of  claim 1 , wherein no therapeutic agent leaks from said liposome loaded with said therapeutic agent during said incubation.  
   
   
       6 . A method of  claim 1 , wherein the protein is selected from the group consisting of an antibody, an Fab′ fragment of an antibody, a single chain Fv antibody, an enzyme, a hormone, a growth factor, and a nucleic acid binding protein.  
   
   
       7 . A method for preparing liposomes loaded with a therapeutic agent, which liposome is targeted to a cell of interest, said method comprising the step of: 
 incubating a protein comprising a terminally appended amino acid sequence comprising primarily amino acids with hydrophilic side chains, which sequence is followed by a lipid modification site with a synthetically appended lipid moiety, with a liposomes loaded with a therapeutic agent, which incubation is for a time sufficient to permit said lipid moiety to become stably associated with the liposome loaded with said therapeutic agent, wherein said protein targets said liposome to a cell of interest.    
   
   
       8 . The method of  claim 1 , wherein the protein is selected from the group consisting of an antibody, an Fab′ fragment of an antibody, a single chain Fv antibody, an enzyme, a hormone, a growth factor, and a nucleic acid binding protein.  
   
   
       9 . A liposome loaded with a therapeutic agent, which liposome is conjugated to a protein through a linker molecule comprising a hydrophobic domain, a hydrophilic polymer chain terminally attached to the hydrophobic domain, and a chemical group reactive to one or more functional groups on a protein molecule and attached to the hydrophilic polymer chain at a terminus contralateral to the hydrophobic domain, wherein the liposome does not bear unconjugated linkers.  
   
   
       10 . The liposome of  claim 1 , wherein the protein is selected from the group consisting of an antibody, an Fab′ fragment of an antibody, a single chain Fv antibody, an enzyme, a hormone, a growth factor, and a nucleic acid binding protein.