Co-formulation of IL-12 mRNA and doxorubicin in polymeric nanoparticles for simultaneous delivery in murine melanoma.

Abstract

Liposomal or polymeric nanoparticles have been instrumental in improving the delivery of poorly soluble chemotherapeutics and those with dose limiting toxicity such as doxorubicin (DOX). More recently, nanoformulations have been shown to enable simultaneous delivery of emerging biomolecules such as siRNA. However, for larger nucleic acids such as mRNA, this remains challenging. In this study, we developed a poly(β-amino ester) (PBAE) based platform, capable of co-formulating mRNA and doxorubicin into nanoparticles. To demonstrate proof of concept using therapeutically relevant cargo, immunomodulatory interleukin-12 (IL-12) was selected as a model mRNA. IL-12 is a pro-inflammatory cytokine that promotes anti-tumour immunity partly through amplifying effector cytokines such as interferon-γ (IFNγ). We found that PBAE complexed DOX and mRNA into positively charged nanoparticles of 120 nm and size-exclusion chromatography indicated a DOX loading efficiency of over 97%. Co-association of both DOX and mRNA was characterised at a single nanoparticle level by nano-flow cytometry. Following delivery to B16F10 murine melanoma cells, more than 95% of cells were double-positive for DOX and Cy5-labelled mRNA, and confocal microscopy confirmed co-localised regions of DOX with mRNA. Interestingly, nanoformulated DOX had increased nuclear accumulation by 1.7-fold relative to free DOX, which correlated with a significantly reduced cell viability of 12.9% with PBAE-DOX/mRNA, compared to 26.6% for free DOX at the same dose. Moreover, despite this strong cytotoxic effect, reporter mRNA translation remained robust, with luciferase expression approximately two orders of magnitude above non-transfected controls at the highest DOX doses. Co-formulation of IL-12 mRNA and DOX with PBAE demonstrated effective IL-12 protein secretion in transfected B16F10 cells with a simultaneous DOX dose dependent reduction in viability. Secreted IL-12 was bioactive, inducing dose-dependent STAT4 phosphorylation and IFNγ secretion in primary mouse splenocytes. Furthermore, in a syngeneic melanoma mouse model, intratumoural administration of PBAE-DOX/IL-12 mRNA achieved significantly elevated levels of IL-12 and IFNγ in the tumour compared to the saline control, confirming delivery of DOX, as well as IL-12 protein secretion, and immunostimulatory activity in vivo. These findings demonstrate that PBAE is a promising platform for co-delivery of cytokine encoded mRNA with DOX in a single formulation, establishing feasibility for advanced chemoimmunotherapy approaches.