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. 2021 Sep 7:14:139-159.
doi: 10.2147/NSA.S321725. eCollection 2021.

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

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 Feuerstein  3
Affiliations

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

Ron Firestein et al. Nanotechnol Sci Appl. .

Abstract

Background: 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: apoptosis; doxorubicin; fluorescent diamond particles-NV-700/800nm; human colorectal cancer organoids; liver cancer cell-lines.

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Conflict of interest statement

Cezary Marcinkiewicz, Mark Sternberg, and Giora Z Feuerstein are employees of Debina Diagnostics Inc. Marco Torelli and Olga Shenderova are employees of Adámas Nanotechnologies, Inc. Dr Ron Firestein reports grants from Debina Diagnostics Inc, during the conduct of the study. The authors report no other conflict of interest in conducting this work.

Figures

Figure 1
Figure 1
Absorption and desorption of DOX from particles surface under different experimental conditions.
Figure 2
Figure 2
Zetasizer analysis of FDP coated or not with DOX.
Figure 3
Figure 3
Effect of FDP-DOX, on the HepG-2 cell metabolic activity measured by AlamarBlue method.
Figure 4
Figure 4
Effect of FDP-DOX on LDH release to the culture media by HepG-2 cells.
Figure 5
Figure 5
Effect of FDP-DOX and FDP-NV on the induction of apoptosis in HepG-2 cells detected by binding of FITC-annexin V and imaged with fluorescence microscope.
Figure 6
Figure 6
Effect of FDP-DOX and FDP-NV on the induction of apoptosis in HepG-2 cells detected by TUNEL assay in fluorescence microscopy imaging.
Figure 7
Figure 7
Effect of FDP-DOX and FDP-NV on induction of apoptosis in Hep-3B cells detected by TUNEL assay in fluorescence microscopy imaging.
Figure 8
Figure 8
Monitoring DOX in cytoplasm and nuclei fractions of HepG-2 and Hep-3B cells following treatment with FDP-DOX or free-DOX in equal concentrations (6 μM).
Figure 9
Figure 9
Effect of FDP-DOX and naïve FDP-NV on hCRC organoid (induced by 18SH112T cell line) metabolism and morphology.
Figure 10
Figure 10
Temporal flow cytometry analysis of FDP-DOX and FDP-NV uptake by hCRC organoids (induced by 18SH112T cell line).
See this image and copyright information in PMC

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