P. Rychahou1,2, F. Haque4,5, Y. Shu4,5, Y. Zaytseva1, H. L. Weiss1, E. Y. Lee1,2,3, W. Mustain2, J. Valentino2, P. Guo1,4,5, B. M. Evers1,2 1University Of Kentucky,Markey Cancer Center,Lexington, KY, USA 2University Of Kentucky,Department Of Surgery,Lexington, KY, USA 3University Of Kentucky,Pathology And Laboratory Medicine,Lexington, KY, USA 4University Of Kentucky,Nanobiotechnology Center,Lexington, KY, USA 5University Of Kentucky,Department Of Pharmaceutical Sciences,Lexington, KY, USA
Introduction: The majority of deaths from all cancers, including colorectal cancer (CRC), is the result of systemic metastasis, usually to the liver and lung. If advances are to be made in the survival of patients with stage IV CRC, more selective, highly innovative, and better targeted therapies are required. The purpose of this study was to: (i) test primary and metastatic CRCs for expression of folate receptor α (FRα), which could be used to target nanoparticles, and (ii) construct multifunctional and chemically stable RNA nanoparticles, that take advantage of receptor targeting, to specifically target CRC liver and lung metastases.
Methods: (1) To determine FRα expression, we obtained primary CRCs (n=12), CRC liver metastases (n=22) and CRC lung metastases (n=10). All samples were tested for FRα expression by immunohistochemistry (IHC) and blindly analyzed by a pathologist. (2) Thermodynamically stable 3-way junction (3WJ) RNA nanoparticles were constructed and confirmed by atomic force microscopy (AFM) and gel electrophoresis; folate and the fluorescent dye Alexa647 were conjugated to the nanoparticles to serve as a ligand for the binding to CRC cells and to provide a fluorescent marker, respectively. Nanoparticle binding to KM20 and HT29 human CRC cells was evaluated in vitro with confocal microcopy. (3) The conjugated RNA nanoparticles were next evaluated in murine (athymic nude mice) models of human CRC liver and lung metastasis.
Results: (1) FRα expression was detected in 78% of primary CRCs, 91% of CRC liver metastases, and 80% of CRC lung metastases. (2) In vitro evaluation demonstrated that folate conjugated RNA nanoparticles are selectively taken up by CRC cells via receptor-mediated endocytosis. (3) In vivo evaluation showed, for the first time, that folate-conjugated RNA nanoparticles remained intact after systemic injection and strongly bound to CRC liver and lung metastases. Conversely, no accumulation of nanoparticles was detected in normal liver or lung parenchyma.
Conclusion: RNA nanoparticles specifically targeted metastatic liver and lung CRC cells, demonstrating the therapeutic potential of RNA-3WJ nanoparticles as a selective delivery system for the treatment of CRC metastasis. Our enthusiasm for current project is driven not only by its inherent scientific importance, but also by its translational potential, clinical impact, and the possibility to provide a more effective and less toxic delivery system targeting CRC metastases specifically in the liver and lung.