Blood Dots: Hemoglobin-Derived Carbon Dots as Hydrogen Peroxide

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Blood Dot: Hemoglobin Derived Carbon Dot as Hydrogen Peroxide Sensor and Pro-Drug Activator Debayan Chakraborty, Saheli Sarkar, and Prasanta Kumar Das ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03691 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018

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ACS Sustainable Chemistry & Engineering

Blood Dot: Hemoglobin Derived Carbon Dot as Hydrogen Peroxide Sensor and Pro-Drug Activator 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

*To whom correspondence should be addressed. E-mail: [email protected]

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ABSTRACT The present article highlights preparation of hemoglobin derived Fe2+ containing carbon dot, namely blood dot (BD) and its simultaneous utilization in hydrogen peroxide (H2O2) sensing and pro-drug activation. The blood dot 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 take place in cancer cells because of the high enzymatic metabolism in comparison to non-cancer cells. In a similar way, the Fe2+ enriched BD can catalyze reactive oxygen species (ROS) generation from H2O2 within cancer cell, which causes selective killing of cancer cell via oxidative DNA damage. Alongside, BD also has been used in bioimaging by exploiting its intrinsic fluorescence to distinguish between cancer and non-cancer cells. Inclusion of BD in non-cancerous cells illuminates bright blue fluorescence while no significant emission of BD was observed in cancer cells due to radical induced quenching of BD fluorescence in presence of high content of H2O2. Hence, the synthesized blood dot can be used in multi task applications including biosensing, bioimaging, pro-drug activation for selective killing of cancer cell.

KEYWORDS: Anticancer pro-drug therapy, Blood dot, Hydrogen peroxide, Oxidative DNA damage, Reactive oxygen species, Sensing

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INTRODUCTION. Hydrogen peroxide (H2O2), a major reactive oxygen species, has immense importance in industry, food, medicine, biology and clinical purpose.1 High content of H2O2 is always known to be detrimental to the environment and living organisms including mammalian cells.2,3 Over expression of H2O2 is one of the major biochemical features of cancer cells.4-7 Accumulation of high amount of H2O2 in cancer cells takes place due to the presence of high level of superoxide dismutase generated in mitochondria. This enzyme can transform superoxide ions into H2O2.8 Moreover, low concentration of catalase and glutathione peroxidase also have been the cause of enhanced concentration of H2O2 in cancer cells.9 In presence of 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, spectrophotometric methods have been developed for detection of H2O2.16-20 Among them fluorescence assay is found to be the most powerful sensing tool owing to its simplicity and rapid response.21 Nevertheless, fluorescence assay using 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 like 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 over expressed H2O2 as a prodrug 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.

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Hydrogen peroxide potentially can serve as a pro-drug in presence of transition metal ions, such as Fe2+.10 In presence of Fe2+, hydrogen peroxide produces hydroxyl anions and highly reactive hydroxyl radicals, which can kill the H2O2 over expressed 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 prodrugs is a potential approach for treating cancer that may reduce the adverse side effects related to conventional chemotherapy.10 Generally chemotherapeutic drugs have serious adverse effects non-diseased cells while treating the cancer cells.38 In contrast, pro-drugs are transformed into its active form of drugs by enzymatic or chemical activators which would minimize the detrimental effect towards healthy cells.39 In view of the above facts, we intrigued to develop a bioprobe which can simultaneously act as H2O2 sensor and pro-drug activator. Recently, various nitrogen containing biomass derived carbon dots are gaining extensive attention in different fields like biosensing, bioimaging and drug delivery.40-45 However, utilization of carbon dot simultaneously as sensor and pro-drug activator is still unexplored. Herein, we have synthesized carbon dot from hemoglobin powder and it is termed as blood derived carbon dot (blood dot, BD). A hemoglobin molecule consists of four globular protein subunits and a heme group. The heme group contains Fe2+ ion held within porphyrin ring. The metal ion (Fe2+) containing BD can be exploited as sensor and activator of hydrogen peroxide. The synthesized BD can sense H2O2 through the quenching of its intrinsic fluorescence by in situ generated hydroxyl/superoxide radicals with a limit of detection (LOD) of 1 µM. Notably, this newly synthesized BD can efficiently split H2O2 (pro-drug) to its active form (hydroxyl/superoxide radicals) that selectively kills cancer cells over non-cancerous cells. BD

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also has the ability to distinguish between cancer and non-cancer cells based on H2O2 content by exploiting the intrinsic fluorescence of BD in bioimaging. EXPERIMENTAL SECTION Materials. Hemoglobin, protoporphyrin IX, tris(hydroxymethyl)aminomethane, betaine hydrochloride, calf thymus DNA and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were bought from Sigma-Aldrich. Sodium hydroxide and ferrous sulfate were purchased from SRL India. All studies were performed using Milli-Q water. Perkin Elmer Lambda 25, Spectrum 100 and Varian Cary Eclipse luminescence spectrometer were used to record UV-vis absorption, FTIR and fluorescence spectra, respectively. Virtis 4KBTXL-75 freeze-drier was used for lyophilization. Thermo Scientific Espresso centrifuge and zetasizer Nano-ZS of Malvern Instruments Limited were used for centrifugation and measurement of zeta potential, respectively. Telsonic Ultrasonics bath sonicator was taken for Bath sonication. FBS (Heat inactivated fetal bovine serum), DMEM (Dulbecco’s Modified Eagles Medium), and trypsin (from porcine pancreas) were bought from HIMEDIA. HeLa and CHO cells were received from NCCS, Pune, India. Synthesis of carbon dot from hemoglobin powder (Blood Dot, BD). To synthesize blood dot (BD), 27 mg of hemoglobin powder was dissolved in Milli-Q water (2 mL) and the solution was heated on a muffle furnace at 120 °C for 1 h. The obtained sticky mass was washed three times with Milli-Q water to remove unreacted hemoglobin powder. The water insoluble mass was then dissolved in concentrated NaOH solution and sonicated for 5 min. The solution obtained was then centrifuged at 12000 rpm for 10 min to remove the insoluble part. The supernatant was collected and dried to get the blood dot with ~75% yield.

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Synthesis of cationic carbon dot (CCD) and anionic carbon dot (ACD). Both CCD and ACD were synthesized following the reported protocol.40 In case of cationic carbon dot (CCD), betaine hydrochloride (2.0 g, 14 mmol) was dissolved in water and tris(hydroxymethyl)aminomethane (1.2 g, 14 mmol) was added to this solution with shaking until complete dissolution. This mixed solution was dried by lyophilization. For anionic carbon dot (ACD), glycine (1.1 g, 14 mmol) was transformed to its carboxylate salt by adding an 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 hot oven. The obtained dried mass in both cases was crushed into a fine powder followed by heated on muffle furnace at 200 °C for 2 h. Then it was allowed to cool to room temperature. The brownish-black material was extracted with water (25 mL). The aqueous suspension was centrifuged at 12000 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 on 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 12000 rpm for 10 min to remove the insoluble part. The supernatant was collected followed by lyophilized 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 solutions was put on 300-mesh Cu-coated TEM grid followed by dried under vacuum for 4h

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before taking the image in JEOL JEM microscope (2100F UHR). Samples for atomic force microscopy (AFM) was prepared by casting a drop of carbon dot solution on a freshly cleaved mica surface followed by air-dried overnight before imaging. Veeco, model AP0100 microscope in non-contact mode was used for AFM imaging. To prepare samples for X-ray photoelectron spectroscopy (XPS), two drops of carbon dot solution was put on a rectangular Cu plate followed by dried under vacuum for 8 h before the experiment and the experiment was performed in Omicron (series 0571) X-ray photoelectron spectrometer. Bruker D8 Advance diffractometer was used to record X-ray diffraction (XRD) spectra of powdered carbon dots and the source was CuKα radiation (α = 0.15406 nm) with a voltage 40 kV and current 30 mA. X-band electron paramagnetic resonance (EPR) measurement was performed in JEOL JES-FA 200 instrument. Oxford make EXTREME INCA microscope was used to perform energy dispersive X-ray (EDX) analysis. Thermal gravimetric analysis (TGA) of blood dot was carried out in TA SDT Q600 under a N2 atmosphere at a heating rate of 20 °C/min. Quantum yield measurement. The absorbance value of carbon dot solution was restricted less than 0.01 and integrated emission intensities of those solutions were recorded in luminescence spectrometer.41 The quantum yield was obtained using the following equation, Q = Qst(Ism/Ist)(ODst/ODsm)(ηsm2/ηst2)

(1)

where Q is the quantum yield, I represents integrated emission intensity measured at excitation maxima 230 nm, OD represents optical density, η represents refractive index. The subscript 'sm' stands for sample and 'st' indicates standard fluorescence of known fluorophore. We have used quinine sulfate as a standard dissolved in 0.1 M H2SO4. Quantum yield of quinine sulfate is 0.54. H2O2 sensing using BD.

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The fluorescence response of the synthesized BD towards H2O2 was investigated by adding varying concentration of H2O2 (1-100 µM) within 25 µg/mL of BD. The fluorescence intensity was measured at 405 nm upon exciting the BD at 230 nm. EPR spectroscopy. The BD sample was prepared by dissolving the 5 mg of lyophilized BD mass in 1 mL water and consequently diluted up to 25 µg/mL. The same procedure was followed to prepare BD-H2O2 solution by adding 1 µM H2O2 into 25 µg/mL of BD solution. Next, 200 µL of these two solutions were taken in two EPR tube. EPR spectra of both solutions were measured at -80 °C. UV-vis spectroscopy calf thymus DNA. UV-vis spectra of native calf thymus DNA (CT DNA) and BD–CT DNA complexes were recorded at different H2O2 concentrations (1-25 mM). The absorbance was recorded using a quartz cell with a path length of 10 mm. Temperature of the sample was maintained at 25 °C using a Peltier thermostat. Concentrations of both CT DNA and BD were maintained at 25 µg/mL. Circular dichroism. JASCO J-815 spectropolarimeter was used to record CD spectra of native CT DNA and BD–CT DNA complexes at different H2O2. The spectra were recorded in the far- UV region (220–320 nm) in a quartz cell with a path length of 1mm. Three scans were collected at a scan speed of 50 nm/min. A peltier thermostat was used to maintain sample temperature at 25 °C. The concentration of CT DNA was 100 µg/mL and that of BD and H2O2 were 25µg/mL and 15µM, respectively. Media stability of blood dot.

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The media stability of the blood dot was investigated using [BD] = 100 µg/mL. BD solution was added to FBS containing DMEM media where FBS concentration was from 0–75% and the solutions were kept for 48 h. BD solution (100 µg/mL) was added to 10% FBS-DMEM media and kept undisturbed for 10 days to investigate the long time stability of BD. In each case, the supernatant of the solution was collected at different time intervals and the absorbance of the supernatant was recorded at 276 nm to measure the suspension stability index. Suspension Stability Index (SSI) = At/A0 × 100

(2)

where At = Absorbance of the solution after a specific time interval at 276 nm, A0 = Initial absorbance of the solution at 276 nm. Cell culture. HeLa and CHO cells were grown in 10% FBS containing DMEM medium in presence streptomycin (100 mg/L) and penicillin (100 IU/mL). The cells were cultured in a 25 mL cell culture flask and kept in 5% CO2 atmosphere at 37 °C. Sub-culture was performed in 2-3 days after 70-80% confluence of cultured cells. The media was changed after 48–72 h. Adherent cells were detached from the surface of the culture flask using trypsin. Further experiments were performed with these cells. Killing experiments. % killing of cancer cells and non-cancerous cells in presence of BD was investigated by the MTT assay.46 High content of H2O2 in cancer cells over non-cancer cells might play a key role in killing result of both cells in presence of BD. In this assay, mitochondrial dehydrogenase present in live cells convert tetrazolium salt to colored formazan which is insoluble in aqueous medium. Quantification of produced formazan was done by spectrophotometrically, which is proportional to the number of alive cells. The mammalian cells were cultured in a 96-well microtiter plate

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(15000 cells per well) for 18–24 h prior to the assay. The cells were incubated with varying concentration of BD (200 to 1000 µg/mL) for 24 h at 37 °C under 5% CO2. The cells were further incubated for 4h with MTT solution (5 mg/mL). The produced formazan was dissolved in DMSO and absorbance of the formazan was measured at 550 nm using BioTek Elisa Reader. The number of live cells were represented as percent viability (A550(treated cells) – background)/(A550(untreated cells) – background) × 100. Live dead viability assay. Cell viability of cancer cells and normal cells in presence of BD was further investigated by LIVE/DEAD assay kit (for eukaryotic cells).47 Considering high content of H2O2 in cancer cells over non-cancer cells, LIVE/DEAD assay may provide insight on the influence of BD. The kit contains mixture of two nucleic acid binding stains, Calcein AM (acetomethoxy) and ethidium homodimer-1. Calcein AM can penetrate the cell membrane while ethidium homodimer-1 is unable to penetrate cell membrane. The green fluorescence responsible for Calcein AM represents live cells while red fluorescence responsible for ethidium homodimer-1 represents dead cells. Before performing the assay, approximately 4 µL of the supplied 2 mM EthD-1 solution and 1 µL of the supplied 4 mM calcein AM stock solution were added to 2 mL of autoclaved, tissue culture-grade PBS buffer. This resulting solution was added to blood dot(500 µg/mL) containing pre-treated (12 h) HeLa and CHO cells. After 30 min of incubation, Olympus IX51 inverted microscope was used to investigate cells. BP460-495 nm excitation filter and a band absorbance filter covering wavelength below 505 nm for imaging of calcein AM intercalation whereas BP530-550 excitation filter and band absorbance filter covering wavelength below 570 nm were used for imaging of ethidium homodimer-1 intercalation. Bioimaging.

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High accumulation of H2O2 in cancer cells over non-cancer cells might play a crucial role in presence of BD in order to differentiate between cancer cells and non-cancer cells through bioimaging experiment. Before imaging experiment, HeLa and CHO cells were grown in a chamber slide for 18–24 h. The cells, pre-treated with 200 µg/mL of BD solutions for 6 h, were washed with phosphate-buffer saline followed by fixed with 4% paraformaldehyde for 30 min. Then the cells were mounted with 50% glycerol solution. Finally, the cells were covered with cover slip and kept for 24 h. Olympus IX51 inverted microscope was used to perform the imaging experiment. Excitation filter BP 330–385 nm and a band absorbance filter covering wavelength below 405 nm were used for blue images. During imaging,

Blood Dots: Hemoglobin-Derived Carbon Dots as Hydrogen Peroxide

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