Amphiphilic block copolymers for nucleic acid delivery

Abstract

The present invention provides a composition comprising vesicles and encapsulated within the vesicles, nucleic acid comprising less than 1000 nucleotides, wherein the vesicles comprise an amphiphilic block copolymer having a hydrophilic and a hydrophobic block. Methods of forming vesicles and methods of delivering nucleic acid, in particular, iRNA into cells, are also provided.

1 . A composition comprising nanovesicles having an aqueous core and encapsulated within the aqueous core of the nanovesicles, nucleic acid comprising less than 1000 nucleotides, wherein the nanovesicles comprise an amphiphilic block copolymer having a hydrophilic and a hydrophobic block, wherein the ratio of the degree of polymerization of the hydrophilic to hydrophobic block is in the range of 1:2.5 to 1:8,
 wherein the hydrophobic block has a degree of polymerization in the range 50 to 250 and the hydrophilic block has a degree of polymerization of at least 15, the hydrophilic block comprises ethylenically unsaturated radically polymerizable monomers comprising a zwitterionic monomer, and wherein the hydrophobic block comprises (diisopropylamino)ethyl methacrylate or (diethylamino)ethyl methacrylate monomers.   
     
     
         2 . A composition according to  claim 1 , wherein the nanovesicles have a diameter in the range 50-1000 nm. 
     
     
         3 . A composition according to  claim 1  wherein the nucleic acid is small iRNA and comprises 15 to 25 nucleotide pairs. 
     
     
         4 . A composition according to  claim 1  in which the zwitterionic monomer has the general formula
   Y B X  I
 
 in which Y is an ethylenically unsaturated group selected from H 2 C═CR—CO— 
 A-, H 2 C═CR—C 6 H 4 -A 1 -, H 2 C═CR—CH 2 A 2 , R 2 O—CO—CR═CR—CO—O, RCH═CH—CO—O—, RCH═C(COOR 2 )CH 2 —CO—O, 
 
       
         
           
           
               
               
           
         
         A is —O— or NR 1 ; 
         A 1  is selected from a bond, (CH 2 ) l A 2  and (CH 2 ) l SO 3 — in which 1 is 1 to 12; 
         A 2  is selected from a bond, —O—, O—CO—, CO—O, CO—NR 1 —, —NR 1 —CO, O—CO—NR 1 —, NR 1 —CO—O—; 
         R is hydrogen or C 1-4  alkyl; 
         R 1  is hydrogen, C 1-4- alkyl or BX; 
         R 2  is hydrogen or C 1-4  alkyl; 
         B is a bond, or a straight branched alkanediyl, alkylene oxaalkylene, or 
         alkylene (oligooxalkylene) group, optionally containing one or more fluorine substituents; 
         X is a zwitterionic group. 
       
     
     
         5 . A composition according to  claim 4  in which X is a group of the general formula II 
       
         
           
           
               
               
           
         
         in which moieties A 3  and A 4 , which are the same or different, are —O—, —S—, 
         —NH— or a valence bond, preferably —O—, and W +  is a group comprising an ammonium, phosphonium or sulphonium cationic group and a group linking the anionic and cationic moieties which is preferably a C 1-12 -alkanediyl group, 
         preferably in which W +  is a group of formula
   —W 1 —N + R 3   3 , —W 1 —P + R 4   3 , —W 1 —S + R 4   2  or —W 1 -Het +  in which:
 
 
         W 1  is alkanediyl of 1 or more, preferably 2-6 carbon atoms optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl (arylene), alkylene arylene, arylene alkylene, or alkylene aryl 
         alkylene, cycloalkanediyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene 
         cycloalkyl alkylene, which group W 1  optionally contains one or more fluorine substituents and/or one or more functional groups; and 
         either the groups R 3  are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such as phenyl, or two of the groups R 3  together with the nitrogen atom to which they are attached form an aliphatic heterocyclic ring containing from 5 to 7 atoms, or the three groups R 3  together with the nitrogen atom to which they are attached as heteroaromatic ring having 5 to 7 atoms, either of which rings may be fused with another saturated or unsaturated ring to form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups R 3  is substituted by a hydrophilic functional group, and 
         the groups R 4  are the same or different and each is R 3  or a group OR 3 , where R 3  is as defined above; or 
         Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring, for example pyridine. 
       
     
     
         6 . A composition according to  claim 4  in which the zwitterionic monomer is 2-methacryloyloxyethyl phosphorylcholine. 
     
     
         7 . A method for forming a composition according to  claim 1 , wherein one of the blocks is pH sensitive, comprising the steps:
 (i) dispersing the amphiphilic copolymer is an organic solvent;   (ii) acidifying the pH of the composition formed in step (i);   (iii) adding the nucleic acid to the composition; and   (iv) raising the pH to around neutral to encapsulate the nucleic acid.   
     
     
         8 . A method according to  claim 7  comprising a preliminary step before step (i), wherein the amphiphilic copolymer is dissolved in an organic solvent in a reaction vessel and the solvent is then evaporated to form a film on the inside of the reaction vessel. 
     
     
         9 . An in vitro method of delivering nucleic acid comprising less than 1000 nucleotides into a cell comprising contacting a composition according to  claim 1  with the cell. 
     
     
         10 . A method of treatment by therapy, comprising administering a composition according to  claim 1  to a human or animal body. 
     
     
         11 . A method of treatment by therapy according to  claim 10 , wherein nucleic acid comprising less than 1000 nucleotides is delivered into a cell. 
     
     
         12 . A composition comprising nanovesicles having an aqueous core and encapsulated within the aqueous core of the nanovesicles, nucleic acid comprising less than 1000 nucleotides, wherein the nanovesicles have a diameter in the range 50-5000 nm and comprise an amphiphilic block copolymer having a hydrophilic and a hydrophobic block, wherein the ratio of the degree of polymerization of the hydrophilic to hydrophobic block is in the range of 1:2.5 to 1:8,
 wherein the hydrophobic block has a degree of polymerization in the range 50 to 250 and the hydrophilic block has a degree of polymerization of at least 15, the hydrophilic block is formed from ethylenically unsaturated radically polymerizable monomers comprising a zwitterionic monomer, and   wherein the hydrophobic block comprises (diisopropylamino)ethyl methacrylate monomers.   
     
     
         13 . A composition according to  claim 12 , wherein the nanovesicles have a diameter in the range 50-1000 nm. 
     
     
         14 . A composition according to  claim 12  wherein the nucleic acid is small iRNA and comprises 15 to 25 nucleotide pairs. 
     
     
         15 . A composition according to  claim 12  in which the zwitterionic monomer has the general formula
   Y B X  I
 
 in which Y is an ethylenically unsaturated group selected from H 2 C═CR—CO— 
 A-, H 2 C═CR—C 6 H 4 -A 1 -, H 2 C═CR—CH 2 A 2 , R 2 O—CO—CR═CR—CO—O, RCH═CH—CO—O—, RCH═C(COOR 2 )CH 2 —CO—O, 
 
       
         
           
           
               
               
           
         
         A is —O— or NR 1 ; 
         A 1  is selected from a bond, (CH 2 ) l A 2  and (CH 2 ) l SO 3 — in which l is 1 to 12; 
         A 2  is selected from a bond, —O—, O—CO—, CO—O, CO—NR 1 —, —NR 1 —CO, O—CO—NR 1 —, NR 1 —CO—O—; 
         R is hydrogen or C 1-4  alkyl; 
         R 1  is hydrogen, C 1-4- alkyl or BX; 
         R 2  is hydrogen or C 1-4  alkyl; 
         B is a bond, or a straight branched alkanediyl, alkylene oxaalkylene, or 
         alkylene (oligooxalkylene) group, optionally containing one or more fluorine substituents; 
         X is a zwitterionic group. 
       
     
     
         16 . A composition according to  claim 15  in which X is a group of the general formula II 
       
         
           
           
               
               
           
         
         in which moieties A 3  and A 4 , which are the same or different, are —O—, —S—, 
         —NH— or a valence bond, preferably —O—, and W +  is a group comprising an ammonium, phosphonium or sulphonium cationic group and a group linking the anionic and cationic moieties which is preferably a C 1-12 -alkanediyl group, 
         preferably in which W +  is a group of formula
   —W 1 —N + R 3   3 , —W 1 —P + R 4   3 , —W 1 —S + R 4   2  or —W 1 -Het +  in which:
 
 
         W 1  is alkanediyl of 1 or more, preferably 2-6 carbon atoms optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl (arylene), alkylene arylene, arylene alkylene, or alkylene aryl 
         alkylene, cycloalkanediyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene 
         cycloalkyl alkylene, which group W 1  optionally contains one or more fluorine substituents and/or one or more functional groups; and 
         either the groups R 3  are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such as phenyl, or two of the groups R 3  together with the nitrogen atom to which they are attached form an aliphatic heterocyclic ring containing from 5 to 7 atoms, or the three groups R 3  together with the nitrogen atom to which they are attached as heteroaromatic ring having 5 to 7 atoms, either of which rings may be fused with another saturated or unsaturated ring to form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups R 3  is substituted by a hydrophilic functional group, and 
         the groups R 4  are the same or different and each is R 3  or a group OR 3 , where R 3  is as defined above; or 
         Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring, for example pyridine. 
       
     
     
         17 . A composition according to  claim 15  in which the zwitterionic monomer is 2-methacryloyloxyethyl phosphorylcholine. 
     
     
         18 . A method for forming a composition according to  claim 12 , wherein one of the blocks is pH sensitive, comprising the steps:
 (i) dispersing the amphiphilic copolymer is an organic solvent;   (ii) acidifying the pH of the composition formed in step (i);   (iii) adding the nucleic acid to the composition; and   (iv) raising the pH to around neutral to encapsulate the nucleic acid.   
     
     
         19 . A method according to  claim 18  comprising a preliminary step before step (i), wherein the amphiphilic copolymer is dissolved in an organic solvent in a reaction vessel and the solvent is then evaporated to form a film on the inside of the reaction vessel. 
     
     
         20 . An in vitro method of delivering nucleic acid comprising less than 1000 nucleotides into a cell comprising contacting a composition according to  claim 1  with the cell. 
     
     
         21 . A method of treatment by therapy, comprising administering a composition according to  claim 12  to a human or animal body. 
     
     
         22 . A method of treatment by therapy according to  claim 21 , wherein nucleic acid comprising less than 1000 nucleotides is delivered into a cell. 
     
     
         23 . A composition comprising nanovesicles and encapsulated within the aqueous core of the nanovesicles, nucleic acid comprising less than 1000 nucleotides, wherein the nanovesicles have a diameter in the range 50-1000 nm and comprise an amphiphilic block copolymer having a hydrophilic block comprising 2-methacryloyloxy ethyl phosphorylcholine and a hydrophobic block comprising 2-(diisopropyl)amino ethyl methacrylate, wherein the degree of polymerisation of the hydrophilic block is 20 to 25 and the degree of polymerisation of the hydrophobic block is 70 to 75, and wherein the nucleic acid is siRNA. 
     
     
         24 . A composition according to  claim 12  wherein the hydrophobic block consists of (diisopropylamino)ethyl methacrylate monomers. 
     
     
         25 . A composition according to  claim 12  wherein the hydrophobic block consists of 2-(diisopropyl)amino ethyl methacrylate.