Photo-stimulation of persistent luminescence nanoparticles enhances cancer cells death

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Abstract

Persistent luminescence nanoparticles made of ZnGa1.995Cr0.005O4 (ZGO-NPs) are innovative nanomaterials that emit photons during long periods of time after the end of the excitation, allowing their use as diagnosis probes for in vivo optical imaging. During the excitation process, a part of the energy is stored in traps to further emit photons over long time. However, we observed in this study that some of the energy reduces molecular oxygen to produce reactive oxygen species (ROS). Following this observation, theoxidative stress induction and cytotoxic effects of these NPs were investigated on human breast cancer cells. The results indicate that ROS production was stimulated by exposition of the hydroxylated ZGO-NPs to UV or visible light, and the oxidative stress induced in cells after internalization can be directly correlated to their dose-dependent inhibition of cell viability. On the contrary, PEGylated ZGOsingle bondNPs were not uptaken by cells and have no effect on the production of ROS. Thus, the cell viability was not altered by these nanoparticles. This study reveals the importance of considering light irradiation and surface coating of luminescent nanoparticles toxicity which open new perspectives for their use in photodynamic therapy.

Introduction

Nanoparticles have great potential for cancer therapy and diagnosis since they can allow the in vivo detection and monitoring of this pathology (Brigger et al., 2012, Fukumori and Ichikawa, 2006). Among the different imaging techniques available in preclinic, optic is an interesting one since it is not expensive, easy to use and allow doing real-time imaging. However, optical imaging is limited in sensitivity due to autofluorescence and light scattering in deep biological tissues. For these reasons more sensitive tools are necessary (Coll, 2011).

Persistent luminescence is an optical phenomenon in which light emission remains for extended periods of time (minutes to hours) after the irradiation source is switched off (le Masne de Chermont et al., 2009, Maldiney et al., 2011a). This property allows overcoming the main disadvantage of conventional optical markers such as quantum-dots and organic dyes responsible of non negligible autofluorescence signals, providing many conveniences for optical imaging (Palner et al., 2015, Richard et al., 2012).

To overcome this disadvantage, our group pioneered the use of persistent luminescent nanoparticles (PLNPs). A series of PLNPs have been developed and applied in the biomedical field as contrast agents for near infra-red in vivo imaging (Lecuyer et al., 2016, Maldiney et al., 2014a, Maldiney et al., 2012a, Maldiney et al., 2015, Maldiney et al., 2012b, Maldiney et al., 2013, Teston et al., 2015). Among them, the innovative chromium-doped zinc gallate ZnGa1.995Cr0.005O4 nanoparticles (ZGO-NPs) have denoted attractive optical properties due to the long persistent luminescence activated by UV and its capacity to be re-activated in vivo through living tissues using highly penetrating low-energy orange/red photons (Maldiney et al., 2014b). Due to its photonic emission at the region of the tissue transparency window, this material has demonstrated its application for optical imaging of vascularization, tumors and grafted cells with high sensitivity (Maldiney et al., 2014b).

The complexity and great diversity of new engineered nanomaterials and their properties imply the understanding of their complete mechanisms of nanoparticle toxicity, which are far from being comprehended (Djurišić et al., 2015). Actually, the side effects of most nanoparticles, including PLNPs (in living organisms) are not well known, and exhaustive toxicological studies are paramount (Hofmann-Amtenbrink et al., 2015).

One of the most important cause of nanoparticle toxicity is frequently attributed to reactive oxygen species (ROS) production, and the resulting oxidative stress (Soenen et al., 2011). In the literature, the most reported ways for ROS production by nanoparticles are the followings (Pisanic et al., 2009, Soenen et al., 2011): (i) direct generation of ROS as a result of nanoparticles exposition to an acidic environment (such in the lysosomes) (Jain et al., 2008, Stroh et al., 2004), (ii) alteration of subcellular functions by interaction of the nanoparticles with organelles such as mitochondria (Soto et al., 2007), (iii) interaction of nanoparticles with the redox active proteins such as NADPH oxidase, and (iv) interaction of the nanoparticles with cell surface receptors and activation of intracellular signaling pathways (Pisanic et al., 2009. In all these cases, the ROS production depends on direct interactions between cells and nanoparticles.

In recent studies it has been demonstrated that the irradiation of some metal oxide nanoparticles by light enhances ROS production. This property has been applied principally in photocatalytic antimicrobial treatments for water disinfection (Brunet et al., 2009, Li et al., 2012, Xiao et al., 2015). In these cases, the nanoparticle acts as photosensitizer (Levy, 1994, Zhang et al., 2015): upon irradiation, the activated nanoparticle transfers its excited-state energy to surrounding oxygen, resulting in ROS production, such as singlet oxygen (1O2), hydroxyl radical (radical dotOH) or superoxide radical (radical dotO2), which can to be cytotoxic in a well localized area (Kochevar and Redmond, 2000, Kolarova et al., 2008, Macdonald and Dougherty, 2001).

In this work, we evaluated the cytotoxic effect of ZGO-NPs on two cancer cell lines, MCF-7 and MDA-MB-231 derived from human breast cancer model. This study was focused on the evaluation of ROS production by photo-stimulated and non photo-stimulated nanoparticles with different coating, in vitro and in contact with cells, in order to reveal a possible mechanism of cancer cell death induced by exposure to ZGO-NPs.

Section snippets

Synthesis and functionalization of ZnGa1.995Cr0.005O4 nanoparticles

ZnGa1.995Cr0.005O4 nanoparticles were synthesized and functionalized according to a previously reported method (Maldiney et al., 2014b). After their synthesis, the ZGO-NPs were first hydroxylated (ZGO-OH) in aqueous 5 mM NaOH media. As an intermediary, aminosilane groups were covalently attached to hydroxylated nanoparticles by adding (3-aminopropyl)triethoxysilane (APTES) purchased from Sigma-Aldrich in DMF. Finally, pegylated nanoparticles (ZGO-PEG) were obtained by reacting aminosilaned

Results and discussion

In order to guarantee the quality of our synthesized nanoparticles, we used a conventional light scattering technique (Zetasizer) to measure the zeta potentials and hydrodynamic size of the ZGO-NPs. These data are summarized in Table 1. The same nanoparticle core, but with different molecules grafted on their surface (hydroxyl or polyethylene glycol groups) were tested. The initial ZGO-OH NPs with negative zeta potential and colloidal stability (due to electrostatic repulsions) were transformed

Conclusion

The determinant role of ZGO nanoparticles functionalization on in vitro effects has been shown: (i) UV and visible light stimulation of ZGO-OH yields superoxide production and (ii) the protective effect of nanoparticle functionalization by polyethylene glycol results in non-toxic ZGO-PEG nanoprobes towards cancer cells. This study opens new possible application of the ZGO-NPs in photodynamic therapy, in which this material can act as nanoprobes, and as photosensitizer, representing a bimodal

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

Authors thank the Mexican National Council for Science and Technology (CONACYT) for their support through doctoral fellowship. Complementary economical support from DAIP (University of Guanajuato) is also gratefully acknowledged. The authors extend their appreciation to Nydia Hernández (UNAM) for her technical assistance during confocal microscope observations. The authors are also thankful to the French National Research Agency (ANR) for a financial support (ANR PEPSI 14-CE-08-0016-01).

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