Polymer nanoparticle and DNA nanostructure compositions and methods for non-viral delivery

1. A RAFT block copolymer comprising

a. a first terminus comprising a first capping unit derived from a first chain transfer agent in a RAFT copolymerization process;

b. a first block prepared from one or more monomer units covalently attached to the first reactive functional unit, and having a molecular weight (M n ) in the range of about 20 kDa to about 80 kDa and a degree of polymerization in the range of about 20 to about 400;

c. a second block prepared from one or more monomer units covalently attached to the first block, and having a molecular weight (M n ) in the range of about 5 kDa to about 80 kDa and a degree of polymerization in the range of about 10 to about 500; and

d. a second terminus comprising a second capping unit derived from a second chain transfer agent;

wherein the RAFT block copolymer has one or more of an overall molecular weight (M n ) in the range of about 25 kDa to about 160 kDa, and overall degree of polymerization in the range of about 30 to about 900, a size in the range of about of about 10 to about 60 nm, and a maximum corona-to-core ratio (CCR) of about 1 to about 4.

2. The RAFT block copolymer of claim 1 , wherein the first block is prepared from one or more monomer units selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

3. The RAFT block copolymer of claim 1 , wherein the first block is prepared from one of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, or methyl methacrylate.

4. The RAFT block copolymer of claim 1 , wherein the second block is prepared from one or more monomer units selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

5. The RAFT block copolymer of claim 1 , wherein the second block is a random copolymer prepared from two different monomer units independently selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

6. The RAFT block copolymer of claim 1 , wherein the second block is a random copolymer prepared from three different monomer units independently selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

7. The RAFT block copolymer of claim 1 , wherein each chain transfer agent is independently selected from the group consisting of bis(carboxymethyl)trithiocarbonate, bis(2-amino-2-oxoethyl) trithiocarbonate, bis[4-(2-hydroxyethoxycarbonyl)benzyl] trithiocarbonate, 4-cyano-4-(ethylsulfanylthiocarbonyl) sulfanylvpentanoic acid, 4-cyano-4-((phenylcarbonothioyl)thio)pentanoic acid, and 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid.

8. The RAFT block copolymer of claim 1 , wherein the first capping unit is of the formula

wherein * represents a point of covalent attachment to the first block.

9. The RAFT block copolymer of claim 1 , wherein the second capping unit is of the formula

wherein * represents a point of covalent attachment to the second block, and R is —SC 2 -C 12 alkyl or C 6 H 5 .

10. A RAFT block copolymer conjugate comprising a RAFT block copolymer according to claim 1 , and further comprising a biomolecule, drug, or label covalently attached to the RAFT block copolymer through a functional group on one of the first or second chain transfer agents that is incorporated into the RAFT block copolymer.

11. The RAFT block copolymer complex of claim 10 , wherein the payload comprises nucleic acids.

12. The RAFT block copolymer complex of claim 10 , wherein the nucleic acids comprise DNA or RNA.

13. The RAFT block copolymer complex of claim 10 , wherein the nucleic acids comprise a ribonucleoprotein.

14. The RAFT block copolymer complex of claim 10 , wherein the payload is a nucleic acid and the nucleic acid payload is of a size selected from the group consisting of 3 kB or more, 3.5 kB or more, 4 kB or more, 4.5 kB or more, 5 kB or more, 5.5 kB or more, 6 kB or more, 6.5 kB or more, 7 kB or more, 7.5 kB or more, 8 kB or more, and 8.5 kB or more.

15. A RAFT block copolymer complex comprising a RAFT block copolymer according to claim 1 , and further comprising a payload complexed to the RAFT block copolymer through electrostatic interaction.

16. The RAFT block copolymer complex of claim 15 , wherein the payload comprises nucleic acids.

17. The RAFT block copolymer complex of claim 15 , wherein the nucleic acids comprise DNA or RNA.

18. The RAFT block copolymer complex of claim 15 , wherein the payload is a nucleic acid and the nucleic acid payload is of a size selected from the group consisting of 3 kB or more, 3.5 kB or more, 4 kB or more, 4.5 kB or more, 5 kB or more, 5.5 kB or more, 6 kB or more, 6.5 kB or more, 7 kB or more, 7.5 kB or more, 8 kB or more, and 8.5 kB or more.

19. A method of preparing a RAFT block copolymer comprising:

i. contacting a first chain transfer agent, a first initiator and one or more monomer units to provide a first block;

ii. contacting a second chain transfer agent, a second initiator and one or more monomer units to provide a second block

iii. contacting the first block and the second block under conditions capable of coupling the blocks to provide the RAFT block copolymer.

20. The method of claim 19 , wherein the first chain transfer agent is a diamino or dihydroxy chain transfer agent, and the second chain transfer agent is a dicarboxylic acid chain transfer agent.

21. The method of claim 19 , wherein the first chain transfer agent is bis(2-amino-2-oxoethyl) trithiocarbonate or bis[4-(2-hydroxyethoxycarbonyl)benzyl] trithiocarbonate, and the second chain transfer agent is bis(carboxymethyl)trithiocarbonate.

22. The method of claim 19 , wherein the first chain transfer agent is a dicarboxylic acid chain transfer agent, and the second chain transfer agent is a diamino or dihydroxy chain transfer agent.

23. The method of claim 19 , wherein the first chain transfer agent is bis(carboxymethyl)trithiocarbonate, and the second chain transfer agent is bis(2-amino-2-oxoethyl) trithiocarbonate or bis[4-(2-hydroxyethoxycarbonyl)benzyl] trithiocarbonate.

24. The method of claim 19 , wherein the one or more monomer units in step (i) are independently selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

25. The method of claim 19 , wherein the one or more monomer units in step (i) is one of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, or methyl methacrylate.

26. The method of claim 19 , wherein the one or more monomer units in step (ii) are independently selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

27. The method of claim 19 , wherein the one or more monomer units in step (ii) are two different monomer units independently selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

28. The method of claim 19 , wherein the one or more monomer units in step (ii) are 2-dimethylaminoethyl acrylate, butyl methacrylate, and propyl acrylic acid; or 2-dimethylaminoethyl acrylate and butyl methacrylate; or 2-dimethylaminocthyl acrylate, butyl methacrylate, and ethyl acrylic acid.

29. The method of claim 19 , wherein the RAFT block copolymer has one or more of an overall molecular weight (M n ) in the range of about 25 kDa to about 160 kDa, and overall degree of polymerization in the range of about 30 to about 900, a size in the range of about of about 10 to about 60 nm, and a maximum corona-to-core ratio (CCR) of about 1 to about 4.

30. A method of preparing a library of RAFT block copolymers comprising:

i. providing an array of reaction mixtures in a multiwell plate, wherein each well comprises a mixture of a first chain transfer agent, a first initiator, one or more monomer units, and optionally a solvent or solvent mixture;

ii. reacting the mixture in each well under conditions that promote RAFT copolymerization to provide a series of first block copolymers in the wells of the multiwell plate;

iii. quenching the reactions in the wells of the multiwell plate;

iv. purifying the first block copolymer in each well of the multiwell plate;

v. optionally characterizing the first block copolymer in each well of the multiwell plate;

vi. optionally purifying the first block copolymer in each well of the multiwell plate;

vii. contacting to the first block copolymer in each well of the multiwell plate with a second array of reaction mixtures comprising a second chain transfer agent, a second initiator, and one or more monomer units, and optionally a solvent or solvent mixture;

viii. reacting the components of step (vii) under conditions that promote RAFT copolymerization to provide a series of RAFT block copolymers in the wells of the multiwell plate;

ix. quenching the reactions in the wells of the multiwell plate;

x. purifying the RAFT block copolymer in each well of the multiwell plate; and

xi. optionally characterizing the RAFT block copolymer in each well of the multiwell plate.