Building a multifunctional aptamer-based DNA nanoassembly for targeted cancer therapy

Abstract

The ability to self-assemble one-dimensional DNA building blocks into two- and three-dimensional nanostructures via DNA/RNA nanotechnology has led to broad applications in bioimaging, basic biological mechanism studies, disease diagnosis, and drug delivery. However, the cellular uptake of most nucleic acid nanostructures is dependent on passive delivery or the enhanced permeability and retention effect, which may not be suitable for certain types of cancers, especially for treatment in vivo. To meet this need, we have constructed a multifunctional aptamer-based DNA nanoassembly (AptNA) for targeted cancer therapy. In particular, we first designed various Y-shaped functional DNA domains through predesigned base pair hybridization, including targeting aptamers, intercalated anticancer drugs, and therapeutic antisense oligonucleotides. Then these functional DNA domains were linked to an X-shaped DNA core connector, termed a building unit, through the complementary sequences in the arms of functional domains and connector. Finally, hundreds (~100-200) of these basic building units with 5'-modification of acrydite groups were further photo-cross-linked into a multifunctional and programmable aptamer-based nanoassembly structure able to take advantage of facile modular design and assembly, high programmability, excellent biostability and biocompatibility, as well as selective recognition and transportation. With these properties, AptNAs were demonstrated to have specific cytotoxic effect against leukemia cells. Moreover, the incorporation of therapeutic antisense oligonucleotides resulted in the inhibition of P-gp expression (a drug efflux pump to increase excretion of anticancer drugs) as well as a decrease in drug resistance. Therefore, these multifunctional and programmable aptamer-based DNA nanoassemblies show promise as candidates for targeted drug delivery and cancer therapy.

Figure 1

Schematic illustration of the multifunctional self-assembled nanoassembly building units and photocrosslinked nanoassembly structure. Multifunctional DNA sequences, including aptamers, acrydite-modified single-stranded DNA and antisense oligonucleotides are self-assembled to form Y-shaped functional domains, which further link via X-shaped connectors to form building units through the complementary arm sequences. Hundreds of these basic building units are then photocrosslinked into a multifunctional and programmable nanoassembly structure.

Figure 2

Characterization of aptamer-based DNA nanoassembly structures. (a) Transmission electron microscopy (TEM) image of spherical photocrosslinked nanostructures. Scale bar: 200 nm. (b) Size-dependent (in diameter) distribution of nanoassemblies based on controllable concentration of building unit.

Figure 3

Specific cancer cell recognition via sgc8-NAs. Analytical flow cytometry shows the selective binding of sgc8-modified nanoassemblies to target CCRF-CEM cells (a), but not nontarget Ramos cells (b). Black peak: Cells only; Green peak: Library DNA-NAs; Blue peak: sgc8 aptamer only; Red peak: sgc8-NAs.

Figure 4

Confocal laser scanning microscopy images of the colocalization of SYBR Green-stained sgc8-NAs and Tf-Alexa 633 (endosome marker), indicating the specific internalization of sgc8-NAs into CEM cells (a) rather than Ramos cells (b). Scale bar: 20 μm.

Figure 5

Selective cytotoxicity of Dox-loaded sgc8-NAs against targeted cancer cells. (a–d) Confocal fluorescence imaging shows Dox transport via Dox-loaded sgc8-NAs relative to the same concentration of free Dox to target CEM (a and c) and nontarget Ra-mos cells (b and d). CEM and Ramos cells were treated with free Dox (a and b) and sgc8-NAs Dox complex (c and d). Scale bar: 20 μm.

Figure 6

MTS assay was performed to assess the selective cytotoxicity of CEM and Ramos cells treated with sgc8-NA complexes.

Figure 7

Selective cytotoxicity of drug-resistant K562 cells treated with free Dox, KK1B10-AS NA-Dox, KK1B10-R NA-Dox and sgc8-AS NA-Dox.