MIS416 as a siRNA Delivery System with the Ability to Target Antigen-Presenting Cells

Abstract

MIS416 is a microparticulate formulation derived from propionibacterium acnes cell wall skeletons with intrinsic adjuvant activity. Conjugates of MIS416-SS-peptide containing a disulfide linkage facilitate the cytoplasmic delivery and release of peptides in antigen-presenting cells (APCs). We hypothesized that MIS416-siRNA (small interfering RNA) conjugates, containing a disulfide linkage between MIS416 and the siRNA, would allow cytoplasmic release of siRNA in APCs. MIS416-SS-siStat3 conjugates added to cell culture medium of monolayers of DCs in culture flasks successfully targeted Stat3 mRNA in DCs in vitro without transfection, downregulating Stat3 mRNA and protein levels. These results suggest that MIS416-SS-siRNA conjugates can be used as a novel siRNA delivery system for the knockdown of mRNA levels in APCs.

Keywords: Stat3; delivery; dendritic cells; gene silencing; siRNA.

FIG. 1.

Internalization of MIS416-PE (MIS416-biotin-streptavidin-PE) by splenocytes, and BMDCs. (A–C) Graphs showing internalization of MIS416-PE after 1, 4, or 24 h, respectively, at 37°C. Splenocytes were pulsed with MIS416-PE (1, 5, or 10 μg) for 1, 4, or 24 h. After incubation, cells were washed in PBS, and then immunocytochemically stained with five different antibodies in PBS (LY6, B220, CD11c, F480, CD3) to identify five different cell populations (neutrophils, B cells, DCs, macrophages, and T cells), respectively. The gating strategies are explained in Supplementary Fig. S1. Error bars represent standard error of the mean (SEM). ns, not significant; *P < 0.05; **P < 0.005; ***P < 0.0005. The experiment was repeated three times. (D–F) Images showing BMDCs at 1, 4, and 24 h, respectively, after internalization of MIS416-PE. BMDCs (2 × 10⁵on coverslips in 250 μL of cell culture media) were treated with MIS416-PE (3 μg) for 1, 4, and 24 h. After incubation, cells were washed in PBS, and immunocytochemically stained with CD11c to identify DCs by incubating 1 μg/10⁶ cells in 100 μL of PBS for 30 min at 4°C. CD11c is shown in green, MIS416-PE in red, and DAPI (to stain nuclei) in blue. The images were taken using a Zeiss LSM 710 confocal microscope. BMDC, bone marrow-derived dendritic cell; PE, phycoerythrin.

FIG. 2.

Q-RT-PCR quantification of Stat3 mRNA levels. DCs were treated for 48 or 72 h with MIS416, MIS416-SS-Control_siRNA, and MIS416-SS-Stat3_siRNA using previously published sequences for Stat3-targeting siRNAs [27]. Analysis by Q-RT-PCR was performed on cDNA generated from total RNA extracted from treated cells. Downregulation of Stat3 mRNA levels occurs at 48 and 72 h in all samples treated with MIS416-SS-Stat3_siRNA compared with controls. The relative level of expression of Stat3 was set to 1 for MIS416-SS-Control_siRNA samples. The relative quantification was carried out using BioGazelle qBase+software. Error bars represent SEM. Results designated with ns were not significant. Results designated with * were significant (P < 0.05). This experiment was repeated three times.

FIG. 3.

Western blot quantification of Stat3 protein levels. (A) Graphs showing the relative Stat3 protein levels following treatment of DCs for 48 or 72 h with MIS416, MIS416-SS-Control_siRNA, or MIS416-SS-Stat3_siRNA. Relative Stat3 protein levels were quantified using ImageJ software. (B) Shows an example of western blot of data used to quantify Stat3 protein levels in (A). The numbers above the gel lanes represent the relative protein level, which was determined from the band intensity using ImageJ software, and normalized relative to the MIS416-SS-control_siRNA-treated samples. (C) Graph of IFN-γ cytokine levels following treatment of BMDCs for 24 h with MIS416, MIS416-SS-Stat3_siRNA, MIS416-SS-Control_siRNA, or no treatment. (D) Graph of ELISA assays of IL-6 cytokine levels in BMDCs at 6, 12, and 24 h following treatment with MIS416, MIS416-SS-Stat3_siRNA, MIS416-SS-control_siRNA, or no treatment. In all graphs, the error bars represent SEM, and the results designated with ns were not significant, whereas results designated with * were significant (P < 0.05), or ** (P < 0.005). These experiments were repeated three times.