Influence of the Charge Ratio of Guanine-Quadruplex Structure-Based CpG Oligodeoxynucleotides and Cationic DOTAP Liposomes on Cytokine Induction Profiles

Abstract

Cationic liposomes, specifically 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes, serve as successful carriers for guanine-quadruplex (G4) structure-based cytosine-guanine oligodeoxynucleotides (CpG ODNs). The combined benefits of CpG ODNs forming a G4 structure and a non-viral vector carrier endow the ensuing complex with promising adjuvant properties. Although G4-CpG ODN-DOTAP complexes show a higher immunostimulatory effect than naked G4-CpG ODNs, the effects of the complex composition, especially charge ratios, on the production of the pro-inflammatory cytokines interleukin (IL)-6 and interferon (IFN)-α remain unclear. Here, we examined whether charge ratios drive the bifurcation of cytokine inductions in human peripheral blood mononuclear cells. Linear CpG ODN-DOTAP liposome complexes formed micrometer-sized positively charged agglomerates; G4-CpG ODN-DOTAP liposome complexes with low charge ratios (0.5 and 1.5) formed ~250 nm-sized negatively charged complexes. Notably, low-charge-ratio (0.5 and 1.5) complexes induced significantly higher IL-6 and IFN-α levels simultaneously than high-charge-ratio (2 and 2.5) complexes. Moreover, confocal microscopy indicated a positive correlation between the cellular uptake of the complex and amount of cytokine induced. The observed effects of charge ratios on complex size, surface charge, and affinity for factors that modify cellular-uptake, intracellular-activity, and cytokine-production efficiency highlight the importance of a rational complex design for delivering and controlling G4-CpG ODN activity.

Keywords: CpG oligodeoxynucleotides; cationic liposomes; charge ratio; cytokine induction; guanine quadruplex.

Figure 1

Characterization of the G4-CpG ODN-DOTAP liposome complexes with various charge ratios. (a) GD2 loading efficiency onto DOTAP liposome. (b) Circular dichroism spectra of naked GD2 and GD2-DOTAP liposome complexes. (c) Hydrodynamic size (black bar), polydispersity index (red symbol), and (d) zeta potential of the GD2-DOTAP liposome complexes. Data are presented as mean ± SD (n = 3).

Figure 2

Nuclease resistance of naked GD2 and GD2-DOTAP complexes of various charge ratios in 50% fetal bovine serum after incubation for 1, 2, 4, and 24 h. (a–c) Images of polyacrylamide gel electrophoresis. (d) Quantitative results of the G4-CpG ODNs remaining after incubation in 50% serum for 24 h.

Figure 3

Effects of G4 structure and charge ratio of the CpG ODN-DOTAP liposome complexes on the production of IL-6 and IFN-α. (a,b) PBMCs, (c) B cells, and (d) pDCs were stimulated with 0.5 µM naked CpG ODNs or CpG ODN-DOTAP liposome complexes for 48 h. Data are represented as mean ± SD (n = 3–5). **** p < 0.0001 and *** p < 0.001 (one-way analysis of variance, followed by Tukey’s multiple comparisons test). # lower than the detection limit (3.9 pg/mL).

Figure 4

Internalization of naked GD2 and GD2-DOTAP complexes into Namalwa cells. The cells were incubated with 0.5 μM GD2^5′Cy5 or GD2^5′Cy5-DOTAP for 2 h. The complexes with low charge ratios of 0.5 and 1.5 underwent higher cellular uptake and were located inside the cells, as compared to naked GD2 or complexes with high charge ratios. Non-treated Namalwa cells were used as the negative control. GD2 was labeled with Cy5 (red). The cell membranes and nuclei were stained with MemBrite™ (green) and DAPI (blue), respectively. Scale bar: 10 μm.

Figure 5

Internalization of naked GD2 and GD2-DOTAP liposome complexes into PMDC05 cells after stimulation for 2 h. GD2-DOTAP liposome complexes of low charge ratios (0.5 and 1.5) were located inside the cells, while GD2-DOTAP of high charge ratios (2 and 2.5) caused aggregation of the cells. Non-treated cells were used as the control. Cy5 (red), MemBrite™ (green), and DAPI (blue) represent GD2, the cell membrane, and nuclei, respectively. Scale bar: 10 μm.

Figure 6

Cytokine induction by DOTAP liposome complexed with GD2, cytosine-methylated GD2 (methylated GD2), and GD2 with CG sequences replaced by GC sequences (GpC GD2) in hPBMCs after 48 h stimulation. (a) IL-6 production, (b) IFN-α production. Data are represented as mean ± SD (n = 5). * p < 0.05 (one-way analysis of variance, followed by Tukey’s multiple comparisons test); # = lower than the detection limit (3.9 pg/mL).