Quantification of Nitric Oxide Produced By Breast Cancer Cells Stimulated By Persistent Luminescent Nanoparticles: A Study of Nanotoxicity Mechanism
Authors: Ramírez García Gonzalo , Gutierrez Granados Silvia , Martinez Alfaro Minerva , Varenne Anne , DxOrlye Fanny , Richard Cyrille , Mignet Nathalie 
Published: February 2020

Optical imaging is a rapidly developing field of research aimed at noninvasively interrogating animals for disease progression, evaluating the effects of a drug, assessing the pharmacokinetic behavior, or identifying molecular biomarkers of disease. The ZnGa1
.995O4:Cr0.005 nanoparticles (ZGO-NP) are innovative persistent luminescence materials interesting for optical imaging. This property allows overcoming the auto-fluorescence signals produced by biological tissues.

For nanoparticles, which can be seen as foreign materials by the cells, the generation of reactive oxygen and nitrogen species (ROS and NOS, respectively) as a reaction to these foreign species is quite standard. An increase in the presence of ROS and NOS in the cellular environment has the potential to damage or disrupt a host of key cellular processes. Generally, luminescent nanoparticles can be activated with light and generate reactive oxidative species, which cause cytotoxicity due to photo-oxidative processes. In addition, nitric oxide produced by inducible nitric oxide synthase is able to be released from the cytoplasm to the interstitial medium across the cellular membrane. For persistent luminescent nanoparticles, the induction of oxidative stress upon cell labeling has not before established. Systematic studies are essential and should be based on free radical quantification by stimulation of nanoparticles, driving to the understanding of their effects on cellular viability and function in relevant model systems.

On this work, the nitric oxide (NO) production by breast cancer cell line was analyzed and quantified by modified ultramicrolectrodes after interaction with ZGO persistent luminescent nanoparticles graphed with different functional groups.

Ultramicroelectrodes permits fast measures in highly resistive solutions, in steady-state conditions, and in vitro and in vivo performances. The catalytic activity of nickel tetrasulfonated phthalocyanine (NiTSPC) deposited over UME surface has been demonstrated for the NO oxidation, that joint to the selectivity provided by the polyphenol membranes, results in an electrochemical sensor with a wide field of applications.

The platinum working ultramicroelectrode (25 μm) was initially modified with a niquel tetrasulphonate phthalocyanine film, deposited in basic media by cyclic voltammetry (50 consecutive cycles, at ν=100 mV.s-1), to improve catalytic activity and specificity for the nitric oxide oxidation. Afterward, a permoselective polyphenol membrane was also electro deposited by cyclic voltammetry during 10 consecutive cycles at ν=10 mV.s-1 to discriminate possible interferences. A silver/silver chloride wire (Ag/AgCl) was used as counter electrode and pseudo-reference electrode.

The electrodes system was inserted into cell culture system to evaluate the NO production by cells in contact to modified-ZGO-NP in real time. Quantitative sensing of extracellular NO released from breast cancer cells treated with different concentrations of NP was per-formed by chronoamperometry at 800 mV. The measurements of NO molecules showed low detection and quantification limits of 12.8 and 38.4 nmol.L-1, respectively at 37 °C.

The influence of different functional groups as hydroxyl, aminosilane, and polyethylene glycol (negative, positive and neutral charge, respectively) presents in the nanoparticle surface was evaluated. The capillary electrophoresis analysis was also used to evaluate nanoparticle-cell interactions and complement the electrochemical measurements to have a best understanding of nanoparticle and cell interactions at interphase level. The level of induced NO is related to the total amount internalized NPs by the cells, which is greatly affected by the functional groups present in surface of the NPs.

The NO concentrations after treatment by nanoparticles continuously increased up until sub-micromolar levels. We confirmed that the coating of the NPs also plays an important role in interactions with cancerous cells in different way according to their specific surface properties. In conclusion, nitric oxide electrochemical biosensors are of great help for the toxicity assessment of nanomaterials, and for a major understanding of the reactions and mechanisms involved in nanotoxicity at cellular level.