Pharmacodynamic Studies of Fluorescent Diamond Carriers of Doxorubicin in Liver Cancer Cells and Colorectal Cancer Organoids

Abstract

Ron Firestein,1,2,* Cezary Marcinkiewicz,3,4,* Linyan Nie,5 Hui Kheng Chua,1,2 Ines Velazquez Quesada,4 Marco Torelli,6 Mark Sternberg,3 Bojana Gligorijevic,4 Olga Shenderova,6 Romana Schirhagl,5 Giora Z Feuerstein3 1Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Victoria, 3168, Australia; 2Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria, 3800, Australia; 3Debina Diagnostics Inc., Newtown Square, PA, USA; 4College of Engineering, Department of Bioengineering, Temple University, Philadelphia, PA, USA; 5Groningen University, Groningen, 9727, the Netherlands; 6Adámas Nanotechnologies, Inc., Raleigh, NC, 27617, USA*These authors contributed equally to this workCorrespondence: Giora Z FeuersteinDebina Diagnostics Inc., 33 Bishop Hollow Road, Newtown Square, PA, 19073, USATel [phone]Email [email protected]: We recently reported on preferential deposition of bare fluorescent diamond particles FDP-NV-700/800nm (FDP-NV) in the liver following intravenous administration to rats. The pharmacokinetics of FDP-NV in that species indicated short residency in the circulation by rapid clearance by the liver. Retention of FDP-NV in the liver was not associated with any pathology. These observations suggested that cancer therapeutics, such as doxorubicin, linked to FDP-NV, could potentially serve for anti-cancer treatment while sparing toxicities of peripheral organs.Purpose: To generate proof-of-concept (POC) and detail mechanisms of action of doxorubicin-coated FDP-NV-700/800nm (FDP-DOX) as a prospective chemotherapeutic for metastatic liver cancer.Methods: FDP-DOX was generated by adsorption chemistry. Experimental design included concentration and time-dependent efficacy studies as compared with naïve (baren) FDP-NV in in vitro liver cancer cells models. Uptake of FDP-NV and FDP-DOX by HepG-2, Hep-3B and hCRC organoids were demonstrated by flow-cytometry and fluorescent microscopy. FDP-DOX pharmacodynamic effects included metabolic as well as cell death biomarkers Annexin V, TUNEL and LDH leakage. DOX desorpted from FDP-DOX was assessed by confocal microscopy and chemical assay of cells fractions.Results: FDP-DOX efficacy was dose- and time-dependent and manifested in both liver cancer cell lines and human CRC organoids. FDP-DOX was rapidly internalized into cancer cells/organoids leading to cancer growth inhibition and apoptosis. FDP-DOX disrupted cell membrane integrity as evident by LDH release and suppressing mitochondrial metabolic pathways (AlamarBlue assay). Access of free DOX to the nuclei was confirmed by direct UV-Visible fluorescent assay and confocal microscopy of DOX fluorescence.Conclusion: The rapid uptake and profound cancer inhibition observed using FDP-DOX in clinically relevant cancer models, highlight FDP-DOX promise for cancer chemotherapeutics. We also conclude that the in vitro data justify further investment in in vivo POC studies.Keywords: liver cancer cell-lines, human colorectal cancer organoids, fluorescent diamond particles-NV-700/800nm, doxorubicin, apoptosis

Keywords

• liver cancer cell-lines
• human colorectal cancer organoids
• fluorescent diamond particles-nv-700/800nm
• doxorubicin
• apoptosis