Effects of Fluorescent Diamond Particles FDP-NV-800nm on Essential Biochemical Functions of Primary Human Umbilical Vein Cells and Human Hepatic Cell Line, HepG-2 in vitro (Part VI): Acute Biocompatibility Studies

Abstract

Background

Recently, we reported the safety and biocompatibility of fluorescent diamond particles, FDP-NV-Z-800nm (FDP-NV) injected intravenously into rats, where no morbidity and mortality were noted over a period of 3 months. The acute effects of FDP-NV-800nm particles on cultured human endothelial and hepatic cells remain unexplored.

Purpose

In this study, we aimed to explore select cellular and biochemical functions in cultured human umbilical endothelial cells (HUVEC) and a human hepatic cancer cell line (HepG-2) exposed to FDP-NV-800 in vitro at exposure levels within the pharmacokinetics (Cmax and the nadir) previously reported in vivo.

Methods

Diverse cellular and biochemical functions were monitored, which cumulatively can provide insights into some vital cellular functions. Cell proliferation and migration were assessed by quantitative microscopy. Mitochondrial metabolic functions were tested by the MTT assay, and cytosolic esterase activity was studied by the calcein AM assay. Chaperons (CHOP), BiP and apoptosis (caspase-3 activation) were monitored by using Western blot (WB). MAPK Erk1/2 signaling was assessed by the detection of the phosphorylated form of the protein (P-Erk 1/2) and its translocation into the cell nucleus.

Results

At all concentrations tested (0.001–0.1mg/mL), FDP-NV did not affect any of the biomarkers of cell integrity of HepG2 cells. In contrast, the proliferation of HUVEC was affected at the highest concentration tested (0.1mg/mL, C_max). Exposure of HUVEC to (0.01 mg/mL) FDP-NV had a mild-moderate effect on cell proliferation as evident in the MTT assay and was absent when proliferation was assessed by direct cell counting or by using the calcein AM assays. In both cell types, exposure to the highest concentration (0.1 mg/mL) of FDP-NV did neither affect FBS-stimulated cell signaling (MAPK Erk1/2 phosphorylation) nor did it activate of Caspase 3.

Conclusion

Our data suggest that FDP-NV-800nm are largely biocompatible with HepG-2 cells proliferation within the pharmacokinetic data reported previously. In contrast, HUVEC proliferation at the highest exposure dose (0.1 mg/mL) responded adversely with respect to several biomarkers of cell integrity. However, since the C_max levels are very short-living, the risk for endothelial injury is likely minimal for slow rate cell proliferation such as endothelial cells.

Keywords:

• near infra-red
• HepG-2 cells
• HUVEC
• cell proliferation
• apoptosis
• MAPK kinase

Summary

This report expands our knowledge on the biocompatibility of FDP-NV-800nm with respect to endothelial (HUVEC) cells in vitro. Our study is, to our best awareness of publicly available information, distinct in that it probes biocompatibility within the realm of the pharmacokinetics of the particles in vivo (in a rat model). We conclude that HUVEC are more sensitive than HepG-2 cells to FDP-NV-800nm accumulation; this observation has not been described for any negative response at the top exposure level (C_max). Considering the mild to moderate interferences in certain biochemical functions in HUVEC and in the light of the pharmacokinetics profile of particles in vivo, it is plausible to predict limited aberrant consequences to the endothelium. The resilience of HepG-2 cells to all of the biochemical tests under the top dose of FDP-NV-800nm may support our in vivo data (with the caveat of these cells being abnormal, transformed cells) on normal liver function in spite of the prolonged hepatic retention of the particles. Overall, the results obtained in this paper indicate that further development of FDP-NV-800nm for in vivo imaging, and as a vehicle for the delivery of drugs and therapeutics is clearly warranted.

Disclosure

The authors report no conflict of interest in conducting this work.