CpG incorporated DNA microparticles for elevated immune stimulation for antigen presenting cells

Abstract

As emerging evidence supports the immune stimulating capability of the CpG oligodeoxynucleotides (ODN), CpG-based adjuvants have been widely used. For efficient induction of immune responses, current issues affecting the use of nucleic acid-based adjuvants, e.g. stability in physiological conditions, delivery to immune cells, and successful release within the phagolysosome, should be addressed. Here, we present CpG-based DNA microparticles (DNA-MPs) fabricated by complementary rolling circle amplification (cRCA) as adjuvants for enhancing immune response and production of selective antibody production. Using cRCA method, the sizes of CpG-based DNA-MPs were finely controlled (0.5 and 1 μm) with superior and provided mismatched single stranded form of CpG ODN region for specific cleavage site by DNase II within the phagolysosome. Fabricated CpG-based 1 μm DNA-MPs (DNA-MP-1.0) were successfully internalized into primary macrophages and macrophage cell line (RAW264.7 cells), and elicited superior cytokine production e.g. TNF-α and IL-6, compared to conventional CpG ODNs. After in vivo administration of DNA-MP-1.0 with model antigen ovalbumin (OVA), significantly elevated OVA-specific antibody production was observed. With this in mind, DNA-MP-1.0 could serve as a novel type of adjuvant for the activation of macrophages and the following production of selective antibodies without any noticeable toxicity in vitro and in vivo.

Fig. 1. Schematic illustrations of the synthesis of the DNA-MPs and process for boosting immune response. (A) Complementary circular DNAs conjugated with primer DNAs replicating repeated CpG ODNs via complementary rolling circle amplification (cRCA) for producing self-assembled DNA-MPs with controlled sizes. (B) Phagocytosis of DNA-MPs by macrophages and subsequential release of CpG ODNs mediated by DNase II under acidic conditions, resulting in enhanced secretion of cytokines.

Fig. 2. Characterization of the DNA-MPs. SEM images (A and D), TEM images (B and E), TEM based EDX-mapping images (C and F) of DNA-MP-0.5, (A–C) and DNA-MP-1.0, (D–F) indicating their spherical structures (inset scale bars in (A and D) 250 nm). TEM and TEM based EDX-mapping images show the compactly packed insides, morphologies and chemical compositions of the DNA-MPs.

Fig. 3. DNA-MPs releasing CpG ODNs mediated by DNase II and their stability under physiological conditions. (A) Schematic illustration of the release of CpG ODNs from the DNA-MPs mediated by DNase II. (B) Gel electrophoresis result of DNA-MP-1.0 cleaved by increasing the amount of DNase II at pH 4.8 and pH 7.2 for 4 h, respectively. The arrow indicates the DNA-MPs stuck in the loading well due to their large sizes. (C) SEM images of DNA-MP-1.0 (inset scale bars: 300 nm). (D) SEM images of the DNA-MPs treated with serum containing medium with respect to the incubation time (scale bars: 1 μm). (E) Average diameters of DNA-MP-0.5 (orange) and the DNA-MP-1.0 (blue) based on the analysis of SEM images using ImageJ software (n = 30).

Fig. 4. (A) Intracellular uptake of POPO3-labeled DNA-MP-0.5 and DNA-MP-1.0 to live RAW264.7 cells. (B) Confocal microscopy images of internalized POPO3-labeled DNA-MPs in the BMDMs. (C) FACS analysis of internalized Cy5-DNA-MP-1.0 in the absence and presence of cytochalasin B (cytB).

Fig. 5. (A and B) The levels of released (A) TNF-α and (B) IL-6 in RAW264.7 cells treated with DNA-MP-0.5 and DNA-MP-1.0. (C) Released amounts of cytokines by the two types of DNA-MPs, CpG and GpC based microparticles in RAW264.7 cells. (D) Released amounts of TNF-α and IL-6 in the BMDMs.

Fig. 6. (A) Illustration of the immunization schedules for administration of DNA-MP-1.0 with OVA to C57BL/6 mice. (B) Quantification of the anti-OVA total IgG in mouse serum after intramuscular injection of different samples. *P < 0.05, ***P < 0.001; ns, not significant.