Analyze whether the delivery of an antisense RNA to miR-26a-5p, encapsulated in a lipid nanoparticle, will act to rescue PTEN levels and contribute to cytotoxic responses in vitro.

Glioblastoma (GBM) is the most common primary brain tumor in adults with a dismal prognosis of 18-24 months following standard-of-care treatment. The poor prognosis stems from (1) physical roadblocks to accessing the tumor microenvironment and (2) tumor heterogeneity. We are investigating the administration of lipid nanoparticles carrying an RNA-based therapeutic designed to disrupt molecules that contribute to GBM pathogenesis. We have identified a target miRNA, miR-26a, which putatively downregulates the tumor suppressor PTEN in GBM, thereby increasing its ability to proliferate. 

LN18 human GBM cells were cultured and transfected as an adherent monolayer with either anti-miR-26a or Dy547-tagged negative control miRNA (200 nM) encapsulated in a lipid nanoparticle, Lipofectamine 3000, then transferred to spheroid culture (FBS-free media in non-adherent plates). Spheroids were collected and processed for protein and RNA analysis. Concurrently, a microfluidic chamber was designed to produce size-controlled lipid polymer hybrid (LPH) nanoparticles.  

Spheroid culture of LN18 cells showed a reduction of PTEN protein and mRNA compared to LN18 adherent cells. Transfection of Lipofectamine:miRNA was confirmed via light microscopy which displayed an accumulation of Dy547-conjugated negative control miRNA inside of LN18 spheroids. Then, spheroids were transfected with an miR-26a inhibitor to rescue PTEN expression. Protein analysis displayed a 150% GAPDH-adjusted increase in PTEN protein expression in the miR-26a inhibitor group compared to the negative control group, corroborated with PCR. With our consult, the Steckl Nanolab designed a microfluidic chamber to create nanoparticles within a 40-60 nm size range for future combination with RNA therapeutics. 

Spheroid culture of LN18 cells decreased PTEN expression compared to adherent LN18 cells. Following growth as spheroids, an miR-26a inhibitor can rescue PTEN expression compared to control.