Abstract| Volume 22, ISSUE 5, P585, May 2021

Development of an activated microglia cell line model for screening lipid nanoparticle formulations of BDNF siRNA for neuropathic pain therapy

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      Purpose Brain-derived neurotrophic factor (BDNF) released from activated spinal microglia via the ATP-P2 × 4R-BDNF signaling pathway plays a central role in initiating pain hypersensitivity in neuropathic pain. Therefore, knockdown of BDNF expression in microglia using molecules like siRNA can be a promising strategy for neuropathic pain therapy. Our lab is developing lipidoid nanoparticles (LNP) for the delivery of BDNF siRNA (siBDNF) for treating neuropathic pain. We characterized the Spontaneously Immortalized Microglia-A9 (SIM-A9) cell line and confirmed the expression of Iba1, P2 × 4R, and BDNF under resting (non-activated) and ATP-stimulated conditions. SIM-A9 cell line was cultured with and without external stimuli, adenosine triphosphate (ATP), and/or lipopolysaccharide (LPS) by varying parameters such as the concentration of stimuli, incubation time, and cell regeneration time post-activation. The cytocompatibility of SIM-A9 cells with ATP and LPS was determined using an ATP assay. Intracellular Iba1, P2 × 4R, and BDNF protein expression in the resting and stimulated cells were characterized using western blotting, in-cell western, and immunocytochemistry. In a pilot study, SIM-A9 cells were transfected with RNAiMAX (a commercial transfection agent)/siBDNF complexes and changes in BDNF expression were evaluated using western blotting. We present two novel findings: first, SIM-A9 cells expressed P2 × 4R and BDNF proteins. Second, ATP at a safe dose showed a time-dependent increase in Iba1 and BDNF expression without intracellular toxicity. At 40 nM siRNA concentration, RNAiMAX/siBDNF complexes showed about a 15% reduction in SIM-A9 BDNF expression. In preliminary studies, LNP/siBDNF at 40 nM were well-tolerated by SIM-A9 cells, however, they only showed negligible BDNF knockdown in activated SIM-A9 cells. Currently, we are optimizing LNP formulations and transfection conditions to increase the extent of BDNF gene silencing. We have developed an ATP-activated SIM-A9 cell line model system that can be utilized for screening of neuropathic pain therapeutics.
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