Biodegradable polyphosphoramidates for controlled release of bioactive substances

Abstract

The present invention is directed to a series of new polycationic biodegradable polyphosphoramidates. Process for making the polymers, compositions containing these polymers and bioactive ligands to enhance the cellular uptake ad intracellular trafficking, articles and methods for delivery of drugs and genes using these polymers are described. A gene delivery system based on these polymers is prepared by complex coacervation of nucleic acid (DNA or RNA) with polymers. Targeting ligands and molecules that could facilitate gene transfer can be conjugated to polymers to achieve selective and enhanced gene delivery. The current invention also provides a complex composition with buffering capacity.

1 . A positively charged biodegradable polyphosphoramidate composition formed by complexation in aqueous solution comprising:
 (a) at least one negatively charged bioactive molecule; and   (b) a water soluble and positively charged biodegradable polyphosphoramidate that is capable of forming a complex with negatively charged bioactive molecules in aqueous solution and comprises the recurring monomeric unit shown in Formula I:   
       
         
           
           
               
               
           
         
       
       wherein
 R 1  is a divalent aliphatic organic moiety; 
 R 2  and R 3  are each independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, heteroalicyclic, cycloalkyl, aralkyl, and cycloalkylalkyl groups; 
 each non-hydrogen occurrence of R 2  and R 3  is substituted with one or more positively charged groups; and 
 n is from 20 to 2,000; 
 wherein when R 1  is —(CH 2 ) a — and one of R 2  and R 3  is hydrogen, the other of R 3  and R 2  is selected from the group consisting of hydrogen, aryl, heteroaryl, heteroalicylcic, cycloalkyl, aralkyl, and cycloalkylaklyl; and 
 wherein α ranges from 2 to 6. 
 
     
     
         2 . The positively charged biodegradable polyphosphoramidate composition of  claim 1 , wherein each non-hydrogen occurrence of R 2  and R 3  is substituted with at least one of an amine group and an imidazoyl group. 
     
     
         3 . The positively charged biodegradable polyphosphoramidate composition of  claim 1 , wherein one or more of R 1 , R 2 , or R 3  is substituted with one or more groups capable of facilitating intracellular delivery of negatively charged bioactive molecules, selected from the group consisting of a lysosomalytic agent, an amphiphilic peptide, or a steroid derivative. 
     
     
         4 . The positively charged biodegradable polyphosphoramidate composition of  claim 3 , wherein the group capable of facilitating intracellular delivery of the negatively charged bioactive molecules is a cholesterol group. 
     
     
         5 . The positively charged biodegradable polyphosphoramidate of  claim 1 , wherein R 1  is defined in Formula II: 
       
         
           
           
               
               
           
         
         wherein each occurrence of R 3  and R 4  are independently selected from the group consisting of hydrogen or alkyl group; and 
         q is 2to 4. 
       
     
     
         6 . A positively charged biodegradable polyphosphoramidate composition formed by complexation in aqueous solutions comprising:
 (a) at least one negatively charged nucleic acid; and   (b) a water soluble and positively charged biodegradable polyphosphoramidate that is capable of forming a complex with negatively charged nucleic acid in aqueous solution and comprises the recurring monomeric unit shown in Formula I:   
       
         
           
           
               
               
           
         
       
       wherein
 R 1  is a divalent aliphatic organic moiety; 
 R 2  and R 3  are each independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, heteroalicyclic, cycloalkyl, aralkyl, and cycloalkylalkyl groups; 
 each non-hydrogen occurrence of R 2  and R 3  is substituted with one or more positively charged groups; and 
 n is from 20 to 2,000. 
 
     
     
         7 . The positively charged biodegradable polyphosphoramidate composition of  claim 6 , wherein each non-hydrogen occurrence of R 2  and R 3  is substituted with at least one of an amine group and an imidazoyl group. 
     
     
         8 . The positively charged biodegradable polyphosphoramidate composition of  claim 6 , wherein one or more of R 1 , R 2 , or R 3  is substituted with one or more groups capable of facilitating intracellular delivery of the negatively charged nucleic acid, selected from the group consisting of a lysosomalytic agent, an amphiphilic peptide, or a steroid derivative. 
     
     
         9 . The positively charged biodegradable polyphosphoramidate composition of  claim 8 , wherein the group capable of facilitating intracellular delivery of the negatively charged nucleic acid is a cholesterol group. 
     
     
         10 . The positively charged biodegradable polyphosphoramidate of  claim 6 , wherein R 1  is defined in Formula II: 
       
         
           
           
               
               
           
         
         wherein each occurrence of R 3  and R 4  are independently selected from the group consisting of hydrogen or alkyl group; and 
         q is 2 to 4. 
       
     
     
         11 . A method comprising:
 mixing a solution of a water soluble positively charged biodegradable polymer comprising the recurring monomeric unit shown in Formula I:   
       
         
           
           
               
               
           
         
       
       wherein
 R 1  is a divalent aliphatic organic moiety; 
 R 2  and R 3  are each independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, heteroalicyclic, cycloalkyl, aralkyl, and cycloalkylalkyl groups; 
 each non-hydrogen occurrence of R 2  and R 3  is substituted with one or more positively charged groups; and 
 n is from 20 to 2,000; 
 wherein when R 1  is —(CH 2 ) a — and one of R 2  and R 3  is hydrogen, the other of R 3  and R 2  is selected from the group consisting of hydrogen, aryl, heteroaryl, heteroalicylcic, cycloalkyl, aralkyl, and cycloalkylaklyl; and 
 wherein α ranges from 2 to 6; 
 
       with a solution of at least one negatively charged bioactive molecule that is able to complex with the polymer of Formula I to form complexes of the water soluble, positively charged biodegradable polymer of Formula I and at least one negatively charged molecule. 
     
     
         12 . The method of  claim 11 , wherein the at least one negatively charged bioactive molecule is selected from the group consisting of DNA, RNA, proteins, and polysaccharides. 
     
     
         13 . The method of  claim 11 , wherein the biodegradable polymer is capable of complexing about 20% to about 60% by weight of the at least one negatively charged bioactive molecule. 
     
     
         14 . The method of  claim 11 , wherein the water soluble, positively charged biodegradable polymer has between about 20 and about 200 phosphoramidate units. 
     
     
         15 . The method of  claim 11 , wherein the concentration of the water soluble, positively charged biodegradable polymer in the solution ranges from about 1 μg/ml to about 500 μg/ml. 
     
     
         16 . The method of  claim 11 , wherein the complexes are nanoparticles. 
     
     
         17 . The method of  claim 11 , wherein the complex is delivered to at least one of a biological fluid, tissue, or cell. 
     
     
         18 . The method of  claim 11 , wherein the complex is delivered to an animal. 
     
     
         19 . A method comprising:
 mixing a solution of a water soluble positively charged biodegradable polymer comprising the recurring monomeric unit shown in Formula I:   
       
         
           
           
               
               
           
         
       
       wherein
 R 1  is a divalent aliphatic organic moiety; 
 R 2  and R 3  are each independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, heteroalicyclic, cycloalkyl, aralkyl, and cycloalkylalkyl groups; 
 each non-hydrogen occurrence of R 2  and R 3  is substituted with one or more positively charged groups; and 
 n is from 20 to 2,000; 
 
       with a solution of at least one negatively charged nucleic acid molecule that is able to complex with the polymer of Formula I to form complexes of the water soluble, positively charged biodegradable polymer of Formula I and at least one negatively charged nucleic acid molecule. 
     
     
         20 . The method of  claim 19 , wherein the biodegradable polymer is capable of complexing about 20% to about 60% by weight of the at least one negatively charged nucleic acid. 
     
     
         21 . The method of  claim 19 , wherein the water soluble, positively charged biodegradable polymer has between about 20and about 200 phosphoramidate units. 
     
     
         22 . The method of  claim 19 , wherein the concentration of the water soluble, positively charged biodegradable polymer in the solution ranges from about 1 μg/ml to about 500 μg/ml. 
     
     
         23 . The method of  claim 19 , wherein the complexes are nanoparticles. 
     
     
         24 . The method of  claim 19 , wherein the complex is delivered to at least one of a biological fluid, tissue, or cell. 
     
     
         25 . The method of  claim 19 , wherein the complex is delivered to an animal.