Research Article Cite This: ACS Sustainable Chem. Eng. 2018, 6, 4661−4670
pubs.acs.org/journal/ascecg
Blood Dots: Hemoglobin-Derived Carbon Dots as Hydrogen Peroxide Sensors and Pro-Drug Activators Debayan Chakraborty, Saheli Sarkar, and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the Cultivation of Science, 2A & 2B Raja S. C. Mullick Road, Jadavpur, Kolkata − 700 032, India
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ABSTRACT: The present article highlights the preparation of hemoglobin-derived Fe2+-containing carbon dots, namely, blood dots (BD) and its simultaneous utilization in hydrogen peroxide (H2O2) sensing and pro-drug activation. The BD was characterized by different microscopic and spectroscopic techniques. The synthesized BD was highly water-soluble and exhibited strong blue emission under UV irradiation. This newly synthesized BD can efficiently split H2O2 to highly reactive hydroxyl/superoxide radicals which quench the intrinsic fluorescence of BD. Consequently, BD was utilized in H2O2 sensing with a limit of detection (LOD) of 1 μM through fluorimetric assay. Notably, the reactive oxygen species (hydroxyl and superoxide radicals) generated from H2O2, upon interaction with BD, can damage DNA by oxidation. In this context, high accumulation of H2O2 is known to occur in cancer cells, because of the high enzymatic metabolism, in comparison to noncancer cells. In a similar way, the Fe2+-enriched BD can catalyze reactive oxygen species (ROS) generation from H2O2 within the cancer cell, which causes selective killing of the cancer cell via oxidative DNA damage. In addition, BD also has been used in bioimaging by exploiting its intrinsic fluorescence to distinguish between cancer and noncancer cells. The inclusion of BD in noncancerous cells illuminates bright blue fluorescence, while no significant emission of BD was observed in cancer cells, because of radical induced quenching of BD fluorescence in the presence of a high content of H2O2. Hence, the synthesized blood dot can be used in multitasking applications, including biosensing, bioimaging, and pro-drug activation for the selective killing of cancer cells. KEYWORDS: Anticancer pro-drug therapy, Blood dot, Hydrogen peroxide, Oxidative DNA damage, Reactive oxygen species, Sensing
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INTRODUCTION Hydrogen peroxide (H2O2), which is a major reactive oxygen species, has immense importance in industry, food, medicine, biology, and clinical purpose.1 A high content of H2O2 is always known to be detrimental to the environment and living organisms, including mammalian cells.2,3 Overexpression of H2O2 is one of the major biochemical features of cancer cells.4−7 An accumulation of a high amount of H2O2 in cancer cells occurs, because of the presence of a high level of superoxide dismutase generated in mitochondria. This enzyme can transform superoxide ions into H2O2.8 Moreover, low concentrations of catalase and glutathione peroxidase also have been the cause of enhanced concentration of H2O2 in cancer cells.9 In the presence of a suitable activator, this high content of H2O2 within cancer cells may act as a pro-drug by producing hydroxyl/superoxide radical, which can kill those cancer cells through oxidative DNA damage.10−15 Considerable research efforts, including titrimetric determination, electrolytic analysis, high-performance liquid chromatography, and spectrophotometric methods, have been developed for the detection of H2O2.16−20 Among them, the fluorescence assay is found to be the most powerful sensing tool, because of its simplicity and rapid response.21 Nevertheless, a fluorescence assay using © 2018 American Chemical Society
organic dyes and metallic quantum dots often suffers from poor cytocompatibility and low water solubility.22 To this end, Lan et al. introduced a carbon-dot-based fluorescence sensor for H2O2 detection.23 Carbon dots are emerging nanomaterials with notable advantages, such as tunable intrinsic fluorescence, easy synthetic procedure, high water solubility, and others.24−30 Considering the high accumulation of H2O2 within cancer cells, it is highly desirable to make use of this overexpressed H2O2 as a pro-drug to kill the diseased cells. Hence, development of a bioprobe for simultaneous H2O2 detection and pro-drug activation for killing cancer cells is of high demand. Hydrogen peroxide potentially can serve as a pro-drug in the presence of transition-metal ions, such as Fe2+.10 In the presence of Fe2+, H2O2 produces hydroxyl anions and highly reactive hydroxyl radicals, which can kill the H2O2 overexpressed cells through oxidative DNA damage.10,31−34 To this end, various Fe2+-based complexes have been developed for the cancer treatment, on the basis of innate overproduction of H2O2 in cancer cells.35−37 Utilization of pro-drugs is a potential Received: October 12, 2017 Revised: February 5, 2018 Published: February 13, 2018 4661
DOI: 10.1021/acssuschemeng.7b03691 ACS Sustainable Chem. Eng. 2018, 6, 4661−4670
Research Article
ACS Sustainable Chemistry & Engineering
equimolar amount of NaOH solution (2 mL). To this, 2 mL of citric acid solution (3 g, 14 mmol) was added by maintaining a 1:1 molar ratio. This water-soluble mixture was dried at 100 °C and the obtained sticky mass was again dried in a hot oven. The dried mass that was obtained in both cases was crushed to a fine powder, followed by heating on a muffle furnace at 200 °C for 2 h. It then was allowed to cool to room temperature. The brownish-black material was extracted with water (25 mL). The aqueous suspension was centrifuged at 12 000 rpm for 30 min to discard the residual part. The supernatant was lyophilized to get the CCD and ACD with ∼73% yield. Synthesis of Porphyrin Dot. Protoporphyrin IX (30 mg) was dissolved in aqueous NaOH solution and was heated in a muffle furnace at 120 °C for 1 h. Thereafter, the sticky mass was dissolved in Milli-Q water and sonicated for 5 min. The resultant solution was then centrifuged at 12 000 rpm for 10 min to remove the insoluble part. The supernatant was collected, followed by lyophilization to obtain the porphyrin dot. The yield of porphyrin dot was ∼40%. Characterization. To prepare samples for transmission electron microscopy (TEM), a drop of the carbon dot solution was placed on a 300-mesh Cu-coated TEM grid, followed by drying under vacuum for 4 h before obtaining the image in JEOL JEM Model 2100F UHR TEM microscope. Samples for atomic force microscopy (AFM) was prepared by casting a drop of carbon dot solution on a freshly cleaved mica surface, followed by being air-dried overnight before imaging. A Veeco Model AP0100 microscope in noncontact mode was used for AFM imaging. To prepare samples for X-ray photoelectron spectroscopy (XPS), two drops of carbon dot solution were deposited on a rectangular Cu plate, followed by drying under vacuum for 8 h before the experiment and the experiment was performed in an Omicron (Series 0571) X-ray photoelectron spectrometer. A Bruker D8 Avance diffractometer was used to record X-ray diffraction (XRD) spectra of powdered carbon dots and the source was Cu Kα radiation (α = 0.15406 nm) with a voltage of 40 kV and a current of 30 mA. Xband electron paramagnetic resonance (EPR) measurement was performed in a JEOL Model JES-FA 200 instrument. Oxford make EXTREME INCA microscope was used to perform energy-dispersive X-ray (EDX) analysis. Thermogravimetric analysis (TGA) of the BD was carried out in a TA Instruments Model SDT Q600 system under a N2 atmosphere at a heating rate of 20 °C/min. Quantum Yield Measurement. The absorbance value of carbon dot solution was restricted to
