FIGURE 3.

RBCEVs deliver bi‐functional ASOs to simultaneously inhibit oncogenic miR‐125b and activate the RIG‐I pathway leading to cell death in cancer cells. (a) miR‐125b sequence and the design of ASO against miR‐125b with and without 5′ triphosphate. The seed sequence of miR‐125b is colored in red and underlined. A triphosphate group (PPP) was added to the 5′ end of 3p‐125b‐ASO whereas the first four nucleotides were replaced with GG. A short sequence (grey circles) was added to the 3′ end of 3p‐125b‐ASO during IVT. (b) qPCR analysis of miR‐125b relative to snoRNA234 in 4T1 cells treated with 0.1 μg/μl unloaded or NC‐RNA‐loaded, 125b‐ASO‐loaded and 3p‐125b‐ASO‐loaded RBCEVs for 24 h (n = 4, RNA loaded using REG1). (c) qPCR analysis of Ddx58 and its downstream effectors relative to Gapdh in 4T1 cells treated with RBCEVs as in (b). (d) qPCR analysis of miR‐125b relative to U6B in CA1a cells treated with RBCEVs as in (b). (e) qPCR analysis of DDX58 and its downstream effectors relative to GAPDH in CA1a cells treated with RBCEVs as in (b). (f) qPCR analysis of miR‐125b relative to U6B in H358 cells treated with RBCEVs as in (b). (g) qPCR analysis of DDX58 and its downstream effectors relative to GAPDH in H358 cells treated with RBCEVs as in (b). (h) Average luciferase activity in A549‐Dual™ and A549‐Dual™ RIG‐I^−/− cells treated with PBS, 0.05 μg/μl unloaded RBCEVs, NC‐RNA‐loaded RBCEVs and 3p‐125b‐ASO‐loaded RBCEVs for 24 and 48 h (n = 5–8). (i) Multiplex immunoassay analysis of cytokines in the conditioned media of 4T1 cells treated with 0.1 μg/μl unloaded, NC‐RNA‐loaded and 3p‐125b‐ASO‐loaded RBCEVs for 48 h (n = 3). (j–l) Flow cytometry analysis revealing the average percentage of ANXV⁺PI⁺ population in 4T1 cells (j), CA1a cells (k) and H358 cells (l) after a treatment with 0.1 μg/μl unloaded, NC‐RNA‐loaded, 125b‐ASO‐loaded and 3p‐125b‐ASO‐loaded RBCEVs for 72 h (n = 4). All bar graphs represent mean ± SEM. **p < 0.01, ***p < 0.001, and ****p < 0.0001 determined by Student's two‐tailed t‐test