SUPPLEMENTARY MATERIALS AND METHODS
Toxicity of LNP siRNA systems in vitro.
On day 8, bmMΦ and DCs were transferred in to 48-wells at 5x105 cells per well. The next day, free siRNA or siRNA encapsulated in DLinDAP, DLinDMA, DLinK-DMA and DLinKC2-DMA were added at 0.5, 1 and 5 μg/mL final concentrations and incubated for 72 h. Cell media was changed every other day while siRNA was maintained at required concentrations. Following treatment, cells were harvested, washed with FACS buffer and stained with Vybrant® Apoptosis Assay Kit #4 (Invitrogen) components A (Propidium Iodide) and B (to assess cell membrane permeability) for 30 min, as per manufacturers direction. Flow Cytometry was conducted to determine the apoptotic cells (FL1-green) exhibiting increased permeability, necrotic cells (FL3-red) assessed by incorporation of propidium iodide by fragmented DNA, cells expressing no fluorescence corresponding to live population and cells exhibiting increased permeability and necrosis. Data was acquired on a LSRII flow cytometer after gating 10000 events and analyzed using the FlowJo software.
Organ uptake and intracellular delivery in organ-resident APCs, B and T-cells.
To investigate organ uptake of fluorescently labeled siRNA, mice were injected i.v. with 3mg/kg siRNA-Cy3 formulated with DLinDAP, DLinDMA, DLinK-DMA and DLinKC2DMA, or PBS control. Thirty minutes later, liver, heart, lungs, inguinal lymph nodes (LNs) and bone marrow were harvested, fixed in 10% formalin, frozen and then cut at 10 μm thickness. Tissues were collected on to glass slides and examined using ICM to visualize and assess the organ uptake of Cy3-labeled siRNA. To examine the intracellular delivery of siRNA in organ-resident APCs, B cells, CD8 and CD4 T-cells, mice were injected i.v. with 1 mg/kg Cy5-labeled siRNA formulated with the most potent LNP DLinKC2-DMA and 24 hours later spleen, liver, inguinal LNs and bone marrow were harvested, digested using Collagenase D (Roche) 1 mg/mL and siRNA delivery was assessed in tissue-resident APCs by flow cytometry following staining of tissue-derived cell suspension with CD11b-FITC and CD11c-PE from AbLab (www.AbLab.ca) to detect MΦ and DCs respectively. B and CD8 T-cells were detected using the CD19-PE conjugated and CD8-FITC antibodies (BD Pharmingen) whereas CD4 T-cells were identified following staining with CD4-PE-Cy7 antibody (eBioscience). Data were analyzed after gating CD11bhigh or CD11chigh and other targeted cell populations and siRNA delivery was assessed based on Cy-5 expression in each population.
Dendritic cell induced T-cell activation and migration.
To investigate whether in vivo transfected DCs exhibit any functional impairment, mice were injected i.v. with 3 mg/kg siRNA formulated with DLinKC2-DMA or PBS and 72 hours later spleen DCs were isolated using magnetic beads and incubated overnight with 10 mg/mL ovalbumin (OVA) 1μM OVA257-264 peptide or PBS. Next, DCs were washed and co-cultured at 50:50 ratio in 48-well plates at 37°C with CFSE (Molecular Probes) labeled B3Z cells (kind gift from Nilabh Shastri at UC Berkeley), a T-cell hybridoma that becomes activated upon recognition of H-2Kb/OVA257–264 immuno-dominant complexes. After 72 hours incubation, the mixed cells were washed and stained with anti-CD8-PE labeled antibody (BD Pharmingen) to detect the T cells and analyzed by flow cytometry. The CFSE fluorescence (FL1) and CD8 (FL2) positive populations of B3Z T-cells were analyzed with FlowJo software to assess their proliferation. To study the migration potential of in vivo transfected DCs, GFP+ (C57Bl/6) mice were injected with 3 mg/kg siRNA-DLinKC2-DMA or PBS. Three days later spleen DCs were isolated and 2x107 cells were injected in the foot pad of C57Bl/6 mice. After further 72 hours observation, the presence of GFP+ DC population of PBS or siRNA-DLinKC2-DMA treated mice, was assessed in the inguinal LNs of C57Bl6 mice using flow cytometry.
Basha G., et al. 3