Diblock copolymers and polynucleotide complexes thereof for delivery into cells

Abstract

Described herein are copolymers, and methods of making and utilizing such copolymers. Such copolymers have at least two blocks: a first block that has at least one unit that is hydrophilic at physiologic pH, and a second block that has hydrophobic groups. This second block further has at least one unit with a group that is anionic at about physiologic pH. The described copolymers are disruptive of a cellular membrane, including an extracellular membrane, an intracellular membrane, a vesicle, an organelle, an endosome, a liposome, or a red blood cell. Preferably, in certain instances, the copolymer disrupts the membrane and enters the intracellular environment. In specific examples, the copolymer is endosomolytic.

1 - 39 . (canceled) 
     
     
         40 . An endosomolytic copolymer comprising:
 (a) a first block, the first block being a hydrophilic block comprising a first chargeable species that is cationic at about neutral pH;   (b) a second block, the second block being a membrane destabilizing hydrophobic block comprising:
 (i) a second chargeable species that is anionic at about neutral pH; and 
 (ii) a third chargeable species that is cationic at about neutral pH; and 
   (c) an oligonucleotide associated with the first block;   wherein the first chargeable species is present on a first constitutional unit, the second chargeable species is present on a second constitutional unit, and the third chargeable species is present on a third constitutional unit.   
     
     
         41 . The copolymer of  claim 40 , wherein the copolymer is a diblock copolymer. 
     
     
         42 . The copolymer of  claim 40 , wherein the first chargeable species is selected from the group consisting of amino, alkylamino, guanidine, imidazole, and pyridyl. 
     
     
         43 . The copolymer of  claim 42 , wherein the first constitutional unit is a residue of dialkylaminoalkylmethacrylate (DMAEMA). 
     
     
         44 . The copolymer of  claim 40 , wherein the second chargeable species is selected from the group consisting of carboxylate, boronate, phosphonate, and phosphate. 
     
     
         45 . The copolymer of  claim 44 , wherein the second constitutional unit is a residue of ethyl acrylic acid or propyl acrylic acid. 
     
     
         46 . The copolymer of  claim 40 , wherein the third chargeable species is selected from the group consisting of amino, alkylamino, guanidine, imidazole, and pyridyl. 
     
     
         47 . The copolymer of  claim 46 , wherein the third constitutional unit is a residue of dialkylaminoalkylmethacrylate (DMAEMA). 
     
     
         48 . The copolymer of  claim 40 , wherein the second constitutional unit is a residue of propyl acrylic acid (PAA) and the third constitutional unit is a residue of dialkylaminoalkylmethacrylate (DMAEMA), and
 wherein the second block further comprises a residue of butyl methacrylate (BMA) as a fourth constitutional unit.   
     
     
         49 . The copolymer of  claim 40 , comprising the chemical Formula II: 
       
         
           
           
               
               
           
         
         wherein 
         A 0 , A 1 , A 2 , A 3  and A 4  are selected from the group consisting of —C—C—, —C(O)(C) a C(O)O—, and —O(C) a C(O)—, wherein a is 1-4; 
         Y 0  and Y 4  are independently selected from the group consisting of hydrogen, -(1C-10C)alkyl, -(3C-6C)cycloalkyl, —O-(1C-10C)alkyl, —C(O)O(1C-10C)alkyl, —C(O)NR 6 (1C-10C), -(4C-10C)hetroaryl and -(6C-10C)aryl, any of which is optionally substituted with one or more fluorine groups; 
         Y 1  and Y 2  are independently selected from the group consisting of a covalent bond, -(1C-10C)alkyl-, —C(O)O(2C-10C)alkyl-, —OC(O)(1C-10C)alkyl-, —O(2C-10C)alkyl-, —S(2C-10C)alkyl-, —C(O)NR 6 (2C-10C)alkyl-, -(4C-10C)hetroaryl- and -(6C-10C)aryl-; 
         Y 3  is selected from the group consisting of a covalent bond, -(1C-10C)alkyl-, -(4C-10C)hetroaryl- and -(6C-10C)aryl-; wherein tetravalent carbon atoms of A 0 -A 4  that are not fully substituted with R 1 -R 5  and Y 0 —Y 4  are completed with an appropriate number of hydrogen atoms; 
         R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  are independently selected from the group consisting of hydrogen, —CN, alkyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, any of which may be optionally substituted with one or more fluorine atoms; 
         Q 1  and Q 2  are residues which are positively charged at normal physiological pH; 
         Q 3  is a residue which is negatively charged at normal physiological pH, but undergoes protonation at lower pH; 
         m is a mole fraction of 0 to 0.49; 
         n is a mole fraction of 0.51 to 1.0; wherein m+n=1 
         p is a mole fraction of 0.2 to 0.5; 
         q is a mole fraction of 0.2 to 0.5; wherein p is substantially the same as q; 
         r is a mole fraction of 0 to 0.6; wherein p+q+r=1 
         v is from 1 to 25 kDa; and, 
         w is from 1 to 50 kDa. 
       
     
     
         50 . The copolymer of  claim 49 , wherein m is 0. 
     
     
         51 . The copolymer of  claim 49 , wherein R 2 -A 1 -Y 1 -Q 1  is a residue of a C1-6 dialkylamino(C1-6)alkylmethacrylate, C1-6 alkylamino(C1-6)alkylmethacrylate, amino(C1-6)alkylacrylate, C1-6 dialkylamino(C1-6)alkylethacrylate, C1-6 alkylamino(C1-6)alkylethacrylate, amino(C1-6)alkylethacrylate, C1-6 dialkylamino(C1-6)alkylacrylate, C1-6 alkyl amino(C1-6)alkylacrylate, or amino(C1-6)alkylacrylate. 
     
     
         52 . The copolymer of  claim 51 , wherein R 2 -A 1 -Y 1 -Q 1  is a residue of dialkylaminoalkylmethacrylate (DMAEMA). 
     
     
         53 . The copolymer of  claim 49 , wherein R 3 -A 2 -Y 2 -Q 2  is a residue of a C1-6 dialkylamino(C1-6)alkylmethacrylate, C1-6 alkylamino(C1-6)alkylmethacrylate, amino(C1-6)alkylacrylate, C1-6 dialkylamino(C1-6)alkylethacrylate, C1-6 alkylamino(C1-6)alkylethacrylate, amino(C1-6)alkylethacrylate, C1-6 dialkylamino(C1-6)alkylacrylate, C1-6 alkyl amino(C1-6)alkylacrylate, or amino(C1-6)alkylacrylate. 
     
     
         54 . The copolymer of  claim 53 , wherein R 3 -A 2 -Y 2 -Q 2  is a residue of dialkylaminoalkylmethacrylate (DMAEMA). 
     
     
         55 . The copolymer of  claim 49 , wherein R 4 -A 3 -Y 3 -Q 3  is a residue of ethyl acrylic acid or propyl acrylic acid. 
     
     
         56 . The copolymer of  claim 49 , wherein R 5 -A 4 -Y 4  is a residue of a C1-6 alkylacrylate, C1-C6 alkylmethacrylate, or C1-C6 alkylethacrylate. 
     
     
         57 . The copolymer of  claim 49 , comprising the chemical Formula IV3 or Formula IV5:
   [DMAEMA] v -[B p -/-P q -/-D r ] w   (IV3)
     [PEGMA m -/-DMAEMA n ] v -[B p -/-P q -/-Dr] w   (IV5)
   wherein B is butyl methacrylate residue; P is propyl acrylic acid residue; D and DMAEMA are dimethylaminoethyl methacrylate residue; and PEGMA is polyethyleneglycol methacrylate residue.   
     
     
         58 . The copolymer of  claim 40 , wherein the copolymer comprises a targeting moiety. 
     
     
         59 . The copolymer of  claim 40 , wherein the oligonucleotide is selected from the group consisting of an siRNA, a microRNA (miRNA), a short hairpin RNA (shRNA), an asymmetrical interfering RNA (aiRNA), and a dicer substrate.