RALA-mediated dual delivery of pMIR-143 and gemcitabine triphosphate as a novel nanotherapy for the treatment of pancreatic cancer

Introduction: Pancreatic cancer has a low incidence compared to other types of solid tumours, however, it is an extremely lethal disease ranking among the top 5 most frequent causes of cancer related death in the UK1. Our group has previously shown that the RALA peptide can encapsulate negatively charged moieties and achieve efficient intracellular delivery 2-3. This project is designed to develop and characterise two novel RALA-based nanoparticles (NPs) that encapsulate a miR143-encoded plasmid and the active form of the first-line chemotherapeutic agent in pancreatic cancer, gemcitabine-triphosphate.

Experimental: The size and charge of the designed NPs were measured using DLS and the stability measured under different conditions. Consequently, pancreatic cancer cell lines (PANC-1 and MIA-PaCa2) were treated with RALA/pEGFP-N1 NPs to evaluate the cellular uptake efficiency and cell viability, via flow cytometry and MTS cytotoxicity assay 48 h post-transfection. qRT-PCR and western blot analysis were conducted on RALA/pCMV-MIR-143 treated cells, to evaluate the expression levels of miR-143 and K-RAS as a target gene respectively.

Results: NPs were formed with a diameter ≤ 150 nm and a charge of 20-40 mV. Cells treated with RALA/pEGFP-N1 and RALA/FITC-12-UTP expressed the highest fluorescence at N:P 10 and w:w 10 respectively in all cell lines with no discernible toxicity ˂10%. miR-143 showed increased levels in PANC-1 after 48 h of transfection while no change was recorded in MIA-PaCa2. As a result, the K-RAS target protein levels were decreased in PANC-1 after the transfection with RALA-pCMV-MIR-143 NPs.

Discussion and Conclusions: The designed NPs were formed in the optimal size and charge for cellular uptake with successful delivery of an intact therapeutic cargo which was translated by up-regulation of miR-143 after the transfection. Further studies will be conducted to assess the functionality and the therapeutic effect of the delivery system both in vitro and in vivo.