Crosslinked micellar spherical nucleic acids
Methods of preparing crosslinked spherical nucleic acids (SNAs) using thermoresponsive traceless templates, such as PEO-PPO-PEO block copolymers. The crosslinks can be added between oligonucleotides within the same SNA or oligonucleotides between multiple different SNAs.
1. A crosslinked micellar spherical nucleic acid (SNA) comprising a plurality of amphiphilic oligonucleotides, and polyethyleneoxide-polypropyleneoxide-polyethyleneoxide (PEO-PPO-PEO), wherein each amphiphilic oligonucleotide comprises (i) a lipid portion and (ii) a nucleobase portion, wherein the nucleobase portion includes at least one reactive group, wherein the lipid portion is conjugated to a termini of the nucleobase portion, wherein the lipid portion of the amphiphilic oligonucleotide intercalates with the PEO-PPO-PEO, and wherein the plurality of amphiphilic oligonucleotides are crosslinked with a PEGylated crosslinking agent that comprises at least two reactive sites, each of which is capable of forming a covalent bond with the reactive groups of the amphiphilic oligonucleotides and thereby crosslinking the amphiphilic oligonucleotides.
2. The crosslinked micellar SNA of claim 1 , having a diameter of 150 nm or less.
3. The crosslinked micellar SNA of claim 1 , having at least 200 strands of amphiphilic oligonucleotides.
4. The crosslinked micellar SNA of claim 1 , wherein each amphiphilic oligonucleotide comprises an oligonucleotide sequence of 10 to 100 nucleobases.
5. The crosslinked micellar SNA of claim 1 , wherein the nucleobase portion of each amphiphilic oligonucleotide comprises a section having a therapeutic sequence.
6. The crosslinked micellar SNA of claim 1 , wherein the lipid portion of each amphiphilic oligonucleotide comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), or 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE).
7. The crosslinked micellar SNA of claim 1 , wherein the lipid portion and the nucleobase portion of the amphiphilic oligonucleotide are connected via a triazolyl.
8. The crosslinked micellar SNA of claim 1 , wherein each amphiphilic oligonucleotide comprises RNA and the RNA is a non-coding RNA.
9. The crosslinked micellar SNA of claim 1 , wherein each amphiphilic oligonucleotide is DNA and the DNA is antisense-DNA.
10. The crosslinked micellar SNA of claim 8 , wherein the non-coding RNA is a microRNA.
11. The crosslinked micellar SNA of claim 1 , wherein the reactive group is at the 3′ end of the nucleobase portion.
12. The crosslinked micellar SNA of claim 1 , wherein the reactive group is at the 5′ end of the nucleobase portion.
13. The crosslinked micellar SNA of claim 1 , wherein the reactive group in an amine group.
14. The crosslinked micellar SNA of claim 1 , wherein the nucleobase portion comprises at least two nucleotides each having the amine reactive group.