J. Nanopart. Res. 2015 November;17(12):461.
Biocompatible ZnS:Mn quantum dots for reactive oxygen generation and detection in aqueous media.
Daysi Diaz-Diestra, Juan Beltran-Huarac, Dina P. Bracho-Rincon, José A. González-Feliciano, Carlos I. Gonzalez, Brad R. Weiner, Gerardo Morell.
Molecular Sciences Research Center, University of Puerto Rico, San Juan, PR 00926, USA
We report here the versatility of Mn-doped ZnS quantum dots (ZnS:Mn QDs) synthesized in aqueous medium for generating reactive oxygen species and for detecting cells. Our experiments provide evidence leading to the elimination of Cd-based cores in CdSe/ZnS systems by substitution of Mn-doped ZnS. Advanced electron microscopy, X-ray diffraction and optical spectroscopy were applied to elucidate the formation, morphology and dispersion of the products. We study for the first time the ability of ZnS:Mn QDs to act as immobilizing agents for Tyrosinase (Tyr) enzyme. It was found that ZnS:Mn QDs show no deactivation of Tyr enzyme, which efficiently catalyzed the hydrogen peroxide (H2O2) oxidation and its eventual reduction (-0.063 V versus Ag/AgCl) on the biosensor surface. The biosensor showed a linear response in the range of 12 μmol/L-0.1 mmol/L at low operation potential. Our observations are explained in terms of a catalase-cycled kinetic mechanism based on the binding of H2O2 to the axial position of one of the active copper sites of the oxy-Tyr during the catalase cycle to produce deoxy-Tyr. A singlet oxygen quantum yield of 0.62 in buffer and 0.54 in water was found when ZnS:Mn QDs were employed as a photosensitizer in the presence of a chemical scavenger and a standard dye. These results are consistent with a chemical trapping energy-transfer mechanism. Our results also indicate that ZnS:Mn QDs are well tolerated by HeLa Cells reaching cell viabilities as high as 88% at 300 µg/mL of QDs for 24 h of incubation. The ability of ZnS:Mn QDs as luminescent nano-probes for bioimaging is also discussed.
Reactive oxygen species (ROSs) are radicals, molecules or ions that have a single unpaired electron in their outermost shell, and present in high levels can produce cellular oxidative stress, which damage cellular lipids, proteins and DNA, giving rise to degenerative or fatal lesions in cells and in turn causing cancer . Hydrogen peroxide (H2O2) and singlet oxygen (1O2) are the most well studied ROS in cancer ; however, their generation and detection in aqueous media using alternative photosensitizers (PSs), such as quantum dots (QDs), is still a big challenge, for instance in photodynamic therapy (PDT). Currently, core/shell QDs systems composed of a core Cd(Se,Te) and an enclosing ZnS shell are being used as PSs due to their highly refined chemistry and availability. Nevertheless, many investigations  have consistently reported the leeching of cytotoxic Cd2+ ions into biological settings when Cd-based/shell systems are exposed to body fluids, even though ZnS provides a physical barrier. It is generally accepted that the persistent Cd release is due to the erosion of the shell layer caused by the photo/chemical oxidation in intra- and extra-cellular fluids or acidic buffers. Our research group has been studying the possibility to avoid the use of toxic Cd-based cores and substitute their optical contribution for Mn‑doping (5%) in the ZnS system , for targeting ROS in PDT. Thus, Mn-doped ZnS (ZnS:Mn) QDs surface-treated with biomolecules represents an alternative biological nanoplatform for theranostics.
Through a photo-oxygenation pathway using a 1O2 sensor and a standard dye, our QDs dispersed in diluted solutions and exposed to light generate 1O2 with quantum yields of 0.62 ± 0.02 in buffer and 0.54 ± 0.03 in water. These values are significantly higher that those reported for Cd-based QDs, and are ascribed to a more efficient energy transfer from the multiplet state of QDs to the triplet ground state (To) of oxygen (3O2), which generates 1O2 and in turn irreversibly reacting with the scavenger (see Figure 1). Such reaction is favored by the large surface area of the QDs, which is further employed for the detection of another ROS, H2O2, upon immobilizing certain types of enzymes on the QDs surface. In this regard, the catalytic activity of QDs/enzyme can produce enhanced peak currents as high as 13.01 μA as H2O2 concentration gradually increases up to 75 μM (see Figure 1). This evidences that ZnS:Mn facilitates the reduction of H2O2 (increased binding sites of the QDs for enzyme loading) without inactivating the enzyme and following a catalase-cycled kinetic mechanism. The fact that the QD-macromolecule interaction did not cause enzymatic inactivation allowed us to assess their interaction with cancer cells. We observed that the QDs did not affect the metabolism of human cervical adenocarcinoma cells up to 1000 µg/mL after 24-h incubation. Moreover, when the cell-internalized QDs are irradiated with light their optical response is not significantly quenched by the cytoplasm/membrane and cellular culture medium (see Figure 1). Our findings evidence that ZnS:Mn QDs exhibit an efficient energy transfer mechanism, and bring forth new arenas toward the substitution of toxic Cd-based cores for Mn‑doping (5%) in the ZnS system. The generation and detection of ROS by ZnS:Mn QDs would be beneficial for further selectivity in tumor and non-tumor cells due to their redox signaling signature, which enables to choose ROS-elevating or -depleting therapy specific for certain type of cancer cells.
This work was supported in part by PR NASA EPSCoR (NASA Cooperative Agreement # NNX13AB22A) and the Institute for Functional Nanomaterials (NSF Grant 1002410).
Figure 1. Depiction illustrating the versatility of ZnS:Mn QDs for ROS generation and detection.
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Juan Beltran-Huarac, PhD
Molecular Sciences Research Center
University of Puerto Rico
San Juan, PR 00926, USA