Layer-by-layer assembled antisense DNA microsponge particles for efficient delivery of cancer therapeutics

Abstract

Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.

Keywords: DNA delivery; DNA nanotechnology; DNA oligonucleotide; antisense therapy; cancer; layer-by-layer; multifunctionality.

Figure 1

Design of the multifunctional DNA-based layer-by-layer assembled nanoparticle. Schematic illustration of the construction of multifunctional nanoparticle using three important strategies including the synthesis of antisense microsponge particles (ODN-MS), condensation process, and layer-by-layer assembly. A self-assembled microsponge-like structure of DNA containing a large amount of periodic antisense oligodeoxynucleotide (ODN) strand in the form of a long polymeric ssDNA was synthesized using rolling circle amplification (RCA) (Step 1). During condensation, ODN-MS were totally disrupted and then reconstructed into nanosized polyplexes by complexation with a selected polymer (Step 2). Next, keeping this complexation as the core, additional outer layer shells were formed through layer-by-layer (LbL) assembly technique (Step 3). Finally, these LbL-ODN nanoparticles (LbL-ODN-NPs) possessed multifunctionality due to the power of both functional DNA nanostructure and LbL assembly method.

Figure 2

Structural characterization of antisense microsponge particle. (A,B) SEM images of ODN-MS. Scale bars indicate 5 μm (A) and 1 μm (B), respectively. (C,D) TEM images of ODN-MS observed at low and higher magnification. Scale bars indicate 500 nm (C) and 100 nm (D), respectively. (E,F) Confocal microscopy images of ODN-MS, which was functionalized with Cy5-conjugated dUTP during the RCA process (red color). ODN-MS viewed at top point (E) and in the middle section (F). Scale bars indicate 1 μm.

Figure 3

Characterization of polymeric DNA after extraction from antisense microsponge particle. (A) SEM images of ODN-MS before and after EDTA treatment. Scale bars indicate 500 nm. (B) Gel electrophoresis analysis to verify the size of polymeric DNA disrupted by EDTA. Lane 1–2 indicate 1 and 5 kb DNA ladder, respectively. (C) Serum stability of polymeric DNA (top) and short ssDNA (bottom) after a preassigned incubation time in a 50% serum medium. (D) Enzyme stability of polymeric DNA (top) and short ssDNA (bottom) after a preassigned incubation time in DNase (3 units/μL) at 37 °C. (E) In vivo stability of polymeric DNA and short ssDNA. Biodistribution of polymeric DNA (top) and short ssDNA (bottom) prior to injection followed by 5 and 30 min postinjection in NCr nude. Images are representative of a photograph with fluorescent overlay (λ_ex = 640 nm, λ_em = 700 nm).

Figure 4

Characterization of polymeric DNA packaging by condensing process (A–C) SEM images of ODN-MS before condensing process (A) and after condensation with varying concentrations of PLL (B,C). Scale bars indicate 1 μm. SEM images show the reduced size of condensed nanoparticles with changed morphology. (D–G) Confocal microscopy images of ODN-MS after condensation with varying concentrations of PLL. Dual labeling was applied in this observation. PLL was functionalized with Cy5 (red color) and DNA was stained with SYBR II, ssDNA specific dyes (green color). Magnification was 60×, and scale bars indicate 5 μm. (H) Size of ODN-MS/PLL at varied concentrations of condensing polymer. (I) ζ Potential of ODN-MS/PLL at varying concentrations of condensing polymer.

Figure 5

Properties of layer-by-layer assembled antisense microsponge particles. (A) ζ Potential of ODN-MS, ODN-MS/PLL, ODN-MS/PLL/DNA, and ODN-MS/PLL/DNA/PEI. (B) Size of ODN-MS, ODN-MS/PLL, ODN-MS/PLL/DNA, and ODN-MS/PLL/DNA/PEI. (C,D) SEM images of ODN-MS (C) and ODN-MS/PLL/DNA/PEI (D). Scale bars indicate 5 and 0.5 μm, respectively. (E) TEM image of ODN-MS/PLL/DNA/PEI. Scale bar indicates 1 μm.

Figure 6

Intracellular and in vivo delivery of layer-by-layer assembled antisense DNA microsponge particles. (A) Intracellular delivery of layer-by-layer assembled antisense microsponge particles. Confocal microscopy image of cancer cells (e.g., SKOV3) treated with LbL-ODN-NPs. Nanoparticles were labeled red with Cy5. The actin cytoskeleton was stained green with phalloidin, and the nucleus was stained blue with DAPI. Scale bars are 10 μm. (B) Cellular uptake study of LbL-ODN-NPs in cancer cells (e.g., SKOV3) by using flow cytometry analysis. (C) The knockdown efficiency of target gene expression for firefly luciferase regulation using LbL-ODN-NPs was examined as a function of concentration in vitro. (D) Pharmacokinetics of nanoparticle clearance from IV-administered BALB/c mice with various LbL-ODN-NPs formulations fit with a 2-phase decay model (PRISM). ODN-MS/PLL/DNA (2L), ODN-MS/PLL/DNA/PEI/DNA (4L), ODN-MS/PLL/DNA/PEI/PEG (PEG).