Toll-Like Receptor-Mediated Recognition of Nucleic Acid Nanoparticles (NANPs) in Human Primary Blood Cells

Abstract

Infusion reactions (IRs) create a translational hurdle for many novel therapeutics, including those utilizing nanotechnology. Nucleic acid nanoparticles (NANPs) are a novel class of therapeutics prepared by rational design of relatively short oligonucleotides to self-assemble into various programmable geometric shapes. While cytokine storm, a common type of IR, has halted clinical development of several therapeutic oligonucleotides, NANP technologies hold tremendous potential to bring these reactions under control by tuning the particle's physicochemical properties to the desired type and magnitude of the immune response. Recently, we reported the very first comprehensive study of the structure⁻activity relationship between NANPs' shape, size, composition, and their immunorecognition in human cells, and identified the phagolysosomal pathway as the major route for the NANPs' uptake and subsequent immunostimulation. Here, we explore the molecular mechanism of NANPs' recognition by primary immune cells, and particularly the contributing role of the Toll-like receptors. Our current study expands the understanding of the immune recognition of engineered nucleic acid-based therapeutics and contributes to the improvement of the nanomedicine safety profile.

Keywords: NANPs; Toll-like receptors; immunotoxicity; infusion reaction; interferon; nanoparticles; nucleic acids.

Figure 1

Schematic design of the current study and experimental verification of nucleic acid nanoparticles’ (NANPs’) assemblies. (A) Blood from healthy donors was used as a source of peripheral blood mononuclear cells (PBMC), then treated with NANPs with and without prior exposure to the Toll-like receptor (TLR)-inhibiting siRNAs. NANPs were delivered into the cells either by lipofection or electroporation. Type I interferon (IFN) secretion was measured in the culture supernatants by enzyme-linked immunosorbent assay (ELISA). (B) RNA cubes, DNA cubes, RNA rings, and RNA fibers were used as model NANPs. All NANPs’ assemblies were confirmed by ethidium bromide total staining native-PAGE and AFM.

Figure 2

The response of dendritic cell (DC) subsets to delivered NANPs. NANPs were delivered to cells from major DC subsets purified by negative selection, and resulting supernatants were assayed for IFN production. The purified DC subsets tested were (A) plasmacytoid DCs, (B) monocytes, and (C) myeloid DCs. Additionally, isolated monocytes were differentiated into (D) monocyte-derived DCs, which were also tested for IFN induction. Some data from individual donors presented in this figure were adapted from our earlier study (1) with permission. ODN = ODN2216, an oligonucleotide, known to induce interferon response and used in our study as a positive control.

Figure 3

Effects of the inhibition of TLR7 and TLR9 expression on the IFN production by PBMCs treated with NANPs. Freshly isolated PBMCs were either untreated or treated with Accel control siRNA or siRNA specific to either TLR7 or TLR9. NANPs were delivered to cells 36 h after the exposure to siRNA, and the incubation continued for 24 h. At the end of the incubation time, supernatants were collected and analyzed for the presence of IFNα by ELISA, while cell lysates were analyzed for the expression of TLR7 and TLR9 by western blot. (A) Selection of donors whose cells responded to Accell SmartPool siRNA by downregulation of the TLR7 protein level. Beta-actin was used to control well loading. (B) Selection of donors whose cells responded to Accell siRNA by downregulation of the TLR9 protein level. Beta-actin was used to control well loading. (C) Densitometry analysis of western blots shown in A and B. Highlighted in red are the results of the individual donor cells demonstrating at least 25% reduction in TLR7 or TLR9 expression as compared to a respective control group exposed to the control siRNA. (D,E) Induction of IFNα by NANPs in PBMCs of donor Q7E8 and L9D7 treated with controls and TLR7 or TLR9 siRNA. (F) Induction of IFNα by NANPs in PBMCs of donor Y6O3 treated with controls or TLR7 siRNA. (G) Induction of IFNα by NANPs in PBMCs of donor F5R3 treated with controls or TLR9 siRNA. A statistically significant difference (p < 0.05) is highlighted above bar graphs showing the respective p-value. ODN2216, an oligonucleotide, known to induce interferon response via TLR9 and imiquimod, known to stimulate TLR7, were used in our study as positive controls. L2K is lipofectamine carrier, which was used as a baseline control to normalize for potential carrier-mediated effect.

Figure 4

Electroporation of PBMCs with NANPs with a 2350 V, 20 ms pulse. (A) Electroporation slightly reduced PBMC viability as measured by acridine orange (AO) and propidium iodide (PI) staining, and resulted in the uptake of AF488-labeled DNA and RNA cubes by both lymphocytes and monocytes, as measured in terms of (B) percentage of PBMCs that took up fluorescent nanoparticles, and (C) the fluorescence intensity of those cells. PBMC-associated NANPs fluorescence was maintained in PBMCs 20 h after electroporation. In figures (A–C), each symbol represents data from a single donor. PBMCs electroporated with unlabeled NANPs failed to induce (D) IFN-α, (E) IFN-β, (F) IFN-ω, and (G) IFN-λ. Additionally, mock-electroporated PBMCs lost their ability to respond to the positive control, ODN2216 (D–G).