Biomaterials 35 (2014) 4428e4435
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Photodynamic antibacterial effect of graphene quantum dots Biljana Z. Ristic a,1, Marina M. Milenkovic a,1, Ivana R. Dakic a,1, Biljana M. Todorovic-Markovic b, Momir S. Milosavljevic b, Milica D. Budimir b, Verica G. Paunovic a, Miroslav D. Dramicanin a, Zoran M. Markovic b, **, Vladimir S. Trajkovic a, * a b
Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade, Serbia Vinca Institute of Nuclear Sciences, University of Belgrade, 11000 Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2013 Accepted 9 February 2014 Available online 4 March 2014
Synthesis of new antibacterial agents is becoming increasingly important in light of the emerging antibiotic resistance. In the present study we report that electrochemically produced graphene quantum dots (GQD), a new class of carbon nanoparticles, generate reactive oxygen species when photoexcited (470 nm, 1 W), and kill two strains of pathogenic bacteria, methicillin-resistant Staphylococcus aureus and Escherichia coli. Bacterial killing was demonstrated by the reduction in number of bacterial colonies in a standard plate count method, the increase in propidium iodide uptake confirming the cell membrane damage, as well as by morphological defects visualized by atomic force microscopy. The induction of oxidative stress in bacteria exposed to photoexcited GQD was confirmed by staining with a redoxsensitive fluorochrome dihydrorhodamine 123. Neither GQD nor light exposure alone were able to cause oxidative stress and reduce the viability of bacteria. Importantly, mouse spleen cells were markedly less sensitive in the same experimental conditions, thus indicating a fairly selective antibacterial photodynamic action of GQD. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Graphene Quantum dot Photodynamic Antibacterial Oxidative stress
1. Introduction Different types of nanoparticles, ranging in size from 1 to 100 nm, have been investigated for their possible use in biomedicine [1]. Semiconductor quantum dots are nanoparticles with superior photo-physical properties suitable for biomedical imaging [2]. However, the potential toxicity resulting from the presence of heavy metal ions in conventional inorganic quantum dots (e.g. CdSe, CdTe) may impede their medical applications [3]. A new class of quantum dots, called graphene quantum dots (GQD), has recently been synthesized [4], displaying the special physicochemical properties of graphene, a single layer of carbon atoms in a honeycomb structure, endowed with large surface area and excellent thermal/chemical stability [5]. Compared to conventional inorganic quantum dots, GQD possess several advantages, including ease of production, high fluorescent activity, resistance to
* Corresponding author. Tel.: þ381 11 3643 233; fax: þ381 11 3643 235. ** Corresponding author. Tel.: þ381 11 2455 451; fax: þ381 11 3440 100. E-mail addresses: [email protected] (Z.M. Markovic), [email protected] (V.S. Trajkovic). 1 These authors equally contributed to the work. http://dx.doi.org/10.1016/j.biomaterials.2014.02.014 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
photo-bleaching, excellent solubility and biocompatibility [4]. Because of these favorable features, GQD are more suitable candidates for non-toxic bioimaging or biosensing agents than their inorganic counterparts. Despite similarities with semimetal graphene nanoparticles, semiconductor GQD, due to different electronic structure, display some unique physichochemical and biological properties. Unlike graphene and similarly to fullerenes (C60), another carbon allotrope [6], GQD in suspension are able to generate reactive oxygen species (ROS) upon photoexcitation [7]. Therefore, GQD are potential candidates for photodynamic therapy, in which the light-excited compound kills cells by ROS generated through energy or electron transfer to molecular oxygen [8]. Accordingly, we have recently reported that GQD exposed to blue light kill cancer cells in a ROS-dependent manner [9]. Photodynamic therapy can also target microbial pathogens, including bacteria, which is becoming increasingly relevant in light of the emerging antibiotic resistance and consequent reduction in effectiveness of conventional therapy [10,11]. While most carbon-based nanomaterials, including fullerenes, carbon nanotubes and graphene display antibacterial properties [12e14], the effects of GQD on bacteria have not been investigated so far.
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Fig. 1. Characterization of GQD. (A) Top view AFM image of GQD deposited on freshly cleaved mica substrate. (B) Height profile of GQD. (C) Size distribution of GQD nanoparticles calculated by AFM software (n ¼ 500). (D) Top view AFM image of typical GQD. (E) Low- and high resolution-TEM micrographs of GQD. (F) Photoluminescence spectra of GQD exposed to different excitation wavelengths (a.u. e arbitrary units).
In the present study, we assessed photodynamic antibacterial activity of electrochemically produced GQD. To that aim, we used as targets methicillin-resistant Staphylococcus aureus (MRSA), Grampositive cause of serious healthcare-associated and communityonset infections [15], and Escherichia coli, Gram-negative commensal of the human intestinal flora with pathogenic strains able to cause meningitis or urinary and gastrointestinal tract infections [16]. 2. Materials and methods 2.1. Preparation and characterization of GQD A stable suspension of GQD was prepared as previously described [9], using graphite rods as anode and cathode and NaOH/ethanol as electrolyte, followed by evaporation of the more volatile ethanol. The pH value of GQD suspension was adjusted to 7.0 by addition of hydrogen chloride and the total carbon particle and NaCl concentrations were adjusted to 1 mg/ml and 0.9%, respectively. We did not observe any visible aggregation of GQD in saline solution containing up to 5% of NaCl. A single GQD monolayer thin film was deposited on mica substrate (air-dried at 2000 C for 10 min) by spin coating and imaged after drying by atomic force microscopy (AFM). AFM measurements were performed using a Quesant AFM (Aguora Hills, CA) operating in tapping mode in air on room temperature, with
standard silicone tips (NanoAndMore Gmbh, Wetzlar, Germany) and with the constant force of 40 N/m. GQD were also characterized by transmission electron microscopy (TEM), using Philips CM200 microscope operated at 200 kV. Samples were prepared by drop casting of GQD dispersion on carbon coated copper grid with 300 mesh. The luminescence emission measurements were performed at room temperature on the Fluorolog-3 FL3-221 spectrofluorometer system (Horiba Jobin-Yvon S.A.S., Chilly Mazarin, France), utilizing a 450 W Xenon lamp as excitation source (328 nm) and R928P photomultiplier tube as a detector.
2.2. Bacterial suspension and treatment Stock cultures of a clinical isolate of MRSA [17] and the reference strain of E. coli (ATCC25922) were maintained on Columbia agar (BD, Franklin Lakes, NJ) supplemented with 5% sheep blood at 4 C. Prior to inoculation, the strains were transferred from the stock cultures to Columbia agar supplemented with 5% sheep blood and incubated aerobically at 37 C overnight, followed by subcultivation under the same conditions. The cultures were then used for preparation of bacterial suspensions (2 104 colony forming units/ml) in a phosphate-buffered saline (PBS). Subsequently, 200 ml of bacterial suspension were transferred to 15 ml glass centrifuge tube and 200 ml of GQD (final concentration 50e200 mg/ml) or PBS were added. After irradiation with blue light (465e475 nm, 1 W), bacterial suspensions were centrifuged at 4000 g for 10 min and resuspended in PBS for determination of cell death/ membrane damage, ROS measurement or AFM analysis.
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Fig. 2. Photoexcited GQD decrease bacterial cell viability. (A, B) Suspensions of E. coli (A) and S. aureus (B) were incubated with PBS (control) or GQD (200 mg/ml), and exposed to blue light (470 nm, 1W) for 15 min. After staining with PI, bacterial cells were visualized by optical (A, B, upper panel) and fluorescence microscopy (A, B, lower panel). (C) E. coli or S. aureus suspensions were treated with different concentrations of GQD and blue light for 15 min, or (D) S. aureus was incubated with 200 mg/ml GQD and exposed to blue light for different periods of time. The red fluorescent PI-permeable cells with membrane damage were enumerated and the data from a representative of three independent experiments are presented as mean SD values (*p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.3. Splenocyte isolation and treatment Spleens were aseptically removed from 6e8-week-old Balb/c mice sacrificed by cervical dislocation. Single cell suspensions were obtained by gently disrupting the spleen tissue through 70 mm pore mesh in cold RPMI 1640 containing 10% FCS. The cells were then centrifuged at 1500 rpm for 10 min and the erythrocytes were removed by incubation in the hypotonic ACK buffer (150 mM NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4). The cells were resuspended in PBS (3 106 cells/ml) and 200 ml of the cell suspension were mixed with 200 ml of GQD suspension or PBS. After exposure to blue light (465e475 nm, 1 W) suspensions were centrifuged and
resuspended in PBS for determination of cell death. The experiment was approved by the Local Animal Care Committee and conformed to the ethical guidelines stated in the “Principles of Laboratory Animal Care” (NIH publication #85-23, revised in 1985). 2.4. Cell death analysis Cell death was analyzed by propidium iodide (PI) staining for cell membrane damage [18]. Cells were incubated with 4 mM PI (BD, Franklin Lakes, NJ) for 15 min at 25 C in the dark. Red fluorescence of PI-stained cells with membrane damage was
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Fig. 3. Photoexcited GQD reduce the number of bacterial colonies. (A) E. coli and (B) S. aureus suspensions were incubated with PBS (control) or exposed simultaneously to GQD (200 mg/ml) and blue light (470 nm, 1 W) for 15 min. The number of CFU was determined by standard plate count method. The data in are mean SD values from a representative of three independent experiments (*p < 0.05).
visualized with a fluorescence microscope (Axiolab HB 50, Zeiss, Germany) under 100X/1.25 oil lens, using 536 nm excitation wavelength. The areas of observation were randomly photographed (5 view fields per sample) with a Nikon digital camera.
3. Results and discussion 3.1. Characterization of GQD
Intracellular ROS production was determined based on green fluorescence emitted by redox-sensitive dye dihydrorhodamine 123 (DHR) [19]. After treatment, bacterial suspensions were washed and stained with 5 mM DHR (Invitrogen/Life Technologies, Carslbad, CA) for 20 min in the dark at 25 C. Green fluorescent ROSproducing cells were visualized with a fluorescence microscope (Axiolab HB 50, Zeiss, Germany) under 100/1.25 oil lens, using 488 nm excitation wavelength. The areas of observation were randomly photographed (5 view fields per sample) with a Nikon digital camera.
AFM and TEM were used to visualize the size, morphology and structure of GQD. AFM images demonstrated the topographic morphology of nanosized GQD (Fig. 1A), whose average height was about 3 nm, corresponding to 3 graphene layers (Fig. 1B). AFM analysis indicated that GQD diameters were mainly distributed in the range of 20e67 nm, with the average diameter of 40.5 nm (Fig. 1C and D), which was consistent with the TEM images (Fig. 1E left). Both AFM and TEM demonstrated that typical GQD were round shape (Fig. 1D and E left). High resolution TEM analysis indicated high crystallinity of GQD, with a lattice parameter of 0.263 nm, also showing a zigzag edge structure of GQD (Fig. 1E right). The photoluminescence spectra have shown that GQD have highest luminescence in the visible part of the spectrum, with the maximum intensity reducing and shifting to higher wavelengths (494e548 nm) with the increase in the excitation wavelength (Fig. 1F).
2.7. AFM imaging of bacteria
3.2. Antibacterial effect of photoexcited GQD
2.5. Standard plate count method The plate count method was used to determine bacterial cell number. Bacterial suspensions at appropriate dilutions were inoculated onto Trypcase-soy agar (BioMerieux, Marcy l’Etoile, France) and the inoculated plates (3 per sample) were incubated aerobically at 37 C for 24 h. The number of colony forming units (CFU) was counted through visual inspection.
2.6. ROS measurement
Bacterial cells were fixed with 2.5% (vol/vol) glutaraldehyde solution (pH 6.8) for 2.5 h at 4 C and washed in PBS. AFM measurements were performed as described for GQD and the different AFM parameters were determined for at least 30 bacterial cells per sample.
2.8. Statistical analysis The statistical significance of the differences between treatments was assessed using one-way analysis of variance (ANOVA) followed by Student-Neuman-Keuls test for multiple comparisons. The value of p < 0.05 was considered significant.
We and others have previously shown that light absorbance by GQD was highest in the UV part of the spectrum, exponentially decreasing with the higher wavelengths [9,20]. Since UV irradiation itself induces DNA damage and cell death [21], for the photostimulation of GQD we used blue light (465e475 nm) as the wavelength-nearest non-cytotoxic alternative. The antibacterial effect of photoexcited GQD was assessed using a fluorescence assay for the cell membrane integrity, as well as standard plate count
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Fig. 4. AFM analysis of bacteria exposed to photoexcited GQD. (A, B) AFM images of E. coli (A) and S. aureus (B) incubated with PBS (control) or simultaneously exposed to GQD (200 mg/ml) and blue light (470 nm, 1 W) for 15 min.
method. PI staining demonstrated that neither GQD at the highest concentration (200 mg/ml) nor maximal duration (15 min) of blue light exposure alone were able to significantly affect the viability of E. coli or S. aureus (Fig. 2AeD). On the other hand, bacterial cells treated simultaneously with GQD and light exhibited a substantial loss in viability, as indicated by a significant increase in the number of PI-permeable red fluorescent bacteria (Fig. 2A, B). It is unlikely that the GQD themselves contributed to the observed fluorescence, as the photoluminescence spectra demonstrated that their capacity for red fluorescence emission is extremely low (Fig. 1F). The cytotoxic effect of GQD at maximal duration of photoexposure was concentration-dependent (Fig. 2C), with the comparable efficiency in both bacterial species (IC50 100 mg/ml). Also, the effect was
Table 1 AFM parameters of E. coli and S. aureus after exposure to photoexcited GQD. The data are presented as mean SD values (*p < 0.05 compared to control, untreated samples). E. coli
S. aureus
Control Length (mM) Width (mM) Height (nM) RMS roughness (nM)
5.75 0.53 99 33.2
1.13 0.07 16 5.6
Light þ GQD 7.2 1.02 300 84.0
3.1 0.15* 79* 19.2*
Control 0.74 0.56 148 27.4
0.08 0.09 21 4.9
Light þ GQD 0.70 0.40 450 63.0
0.07 0.08* 96* 14.1*
clearly dependent on the duration of photoexposure, as shown in S. aureus incubated with the fixed dose of GQD (Fig. 2D). In agreement with the fluorescence-based assessment of cell membrane damage, the plate count method demonstrated a significant reduction in the number of CFU in both E. coli and S. aureus suspensions exposed to photoexcited GQD (Fig. 3). It should be noted that inorganic QD have recently been shown to be toxic to various Gram-positive and Gram-negative bacteria, including S. aureus and E. coli [22e24], while amine-functionalized GQD exerted potent antimycoplasma activity [25] in the absence of overt photoexcitation. Therefore, light-induced antibacterial action described here could represent an additional modality for QD-mediated bacterial cell killing. 3.3. Morphological changes in bacteria exposed to photoexcited GQD AFM was used for visualization of the changes in bacterial cell morphology induced by photoexcited GQD. The control, untreated E. coli and S. aureus samples contained typically rodshaped (Fig. 4A) or near-spherical cells (Fig. 4B). After the treatment with blue light-irradiated GQD, bacterial cells displayed an increased tendency to aggregate (Fig. 4A and B). While the average length of bacteria did not change noticeably, the width of E. coli significantly increased and that of S. aureus
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decreased upon treatment (Table 1). The height, as well as the values of root mean square (RMS) roughness significantly increased in both bacterial species after exposure to lightirradiated GQD (Table 1). The observed increase in surface roughness and increase in height following exposure to photoexcited GQD indicate cell membrane damage and subsequent swelling of the bacterial cell, respectively, as recently demonstrated by AFM for other antibacterial agents [26e29]. Therefore, the extensive morphological changes associated with the
20 µM
increase in surface roughness further confirm the detrimental effects of photoexcited GQD on bacterial cells. 3.4. Production of ROS by bacteria exposed to photoexcited GQD Near-IR light-irradiated graphene and graphene oxide nanoparticles kill tumor cells by releasing heat [30e33]. However, as no significant temperature increase was observed in GQD-treated bacterial suspensions exposed to blue light (data not shown), we
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Fig. 5. Photoexcited GQD induce oxidative stress in bacterial cells. (A, B) Suspensions of E. coli (A) and S. aureus (B) were incubated with PBS (control) or GQD (200 mg/ml), and exposed to blue light (470 nm, 1 W) for 15 min. After staining with DHR, bacterial cells were visualized by optical (A, B, upper panel) and fluorescence microscopy (A, B, lower panel). (C) The green fluorescent ROS-producing E. coli and S. aureus were enumerated and the data from a representative of three independent experiments are presented as mean SD values (*p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.5. Photo-toxicity of GQD towards mouse splenocytes Having established that photoexcited GQD have bactericidal properties, it was important to examine the sensitivity of eukaryotic cells to the same treatment. We have previously shown that human cancer cells were sensitive to photodynamic action of GQD [9] at the same experimental conditions (GQD concentration and duration of light exposure) used in the present study. However, as fast-dividing cancer cells might be more sensitive to certain death stimuli than non-proliferating normal cells, we examined the effect of photoexcited GQD on primary mouse splenocytes as a model of normal eukaryotic cells. Fluorescence microscopy analysis of splenocytes exposed to conditions that killed more than 50% bacteria (100 mg/ml GQD þ 15 min photoirradiation; Fig. 2C) demonstrated only a moderate increase (from 5 to 17%) in the number of PIpermeable spleen cells (Fig. 6), while 200 mg/ml of photoexcited GQD killed almost all bacteria (Fig. 2), but less than 50% of splenocytes (Fig. 6). While the comparison with our previous study [9] indicates that cancer and normal cells (at least those examined in the two studies) are similarly sensitive to photodynamic toxicity of GQD, it appears that both cell types might be more resistant to GQD phototoxicity than bacteria. Although we did not compare the entry of GQD to bacterial and mammalian cells and their ROS production, the latter ingest GQD and subsequently produce ROS [9], so the apparent difference in sensitivity cannot be explained by the failure of mammalian cells to internalize GQD and/or produce ROS in response. On the other hand, bacteria and mammalian cells use different mechanisms to cope with oxidative stress [35], so the latter might somehow be more successful in performing this task. Nevertheless, this relatively selective photodynamic toxicity of GQD towards bacteria, as well as the underlying mechanisms, need to be confirmed in more detailed toxicological and mechanistic studies.
GQD (100 μg/ml) GQD (200 μg/ml)
irradiation
excluded the possibility that the antibacterial effect was due to photothermal cytotoxicity. Another plausible mechanism for the light-induced antibacterial effect of GQD was ROS-mediated photodynamic effect, as GQD are able to produce ROS, including singlet oxygen, when irradiated with blue light [7,9]. To examine this possibility, we measured intracellular ROS levels using DHR, a nonfluorescent cell-permeable indicator that can be oxidized to green fluorescent rhodamine 123. Fluorescent microscopy analysis revealed that no production of ROS was observed in untreated bacteria or those exposed to GQD or light alone (Fig. 5). On the other hand, a significant increase in the numbers of green fluorescent ROS-containing cells was demonstrated in both E. coli and S. aureus suspensions treated simultaneously with GQD and blue light (Fig. 5). When excited at 488 nm (DHR excitation wavelength), GQD themselves emit only low-intensity green fluorescence (Fig. 1F), but can produce ROS that oxidize DHR to a highly fluorescent product [7,9]. However, no DHR signal was detected in GQD-exposed bacteria in the absence of previous photoexcitation (Fig. 5), indicating that GQD might not be able to enter intact bacteria. In the likely chain of events, blue-light excited GQD photodynamically disrupt bacterial cell membrane integrity, enter the damaged bacteria and start producing additional ROS amounts due to excitation during DHR measurement. Moreover, damaged bacteria themselves can produce ROS in a form of “oxidative shielding” response [34]. To fully elucidate the time-kinetics of these complex events and their involvement in GQD-mediated photodynamic bacterial killing, a system for a real-time measurement of GQD internalization and ROS production is required. Nevertheless, by demonstrating the induction of oxidative stress in the absence of temperature increase, our results strongly indicate the photodynamic nature of the observed antibacterial effect.
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irradiation Fig. 6. The effect of photoexcited GQD on the viability of mouse splenocytes. Mouse splenocytes were left untreated (control) or exposed simultaneously to GQD (100 or 200 mg/ml) and blue light (470 nm, 1 W) for 15 min. After staining with PI, cells were visualized by optical and fluorescence microscopy. The red fluorescent PI-permeable cells with membrane damage were enumerated and the data from a representative of three independent experiments are presented as mean SD values (*p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Conclusion The data presented here demonstrate a relatively selective photodynamic antibacterial activity of GQD. Having in mind the low tissue penetrance of blue light and low absorbance of GQD at the higher wavelengths, such an approach would be presumably efficient for skin and mucosal infections, or water and surface disinfection. While photoexcited GQD might not be able to perform better than currently available broad spectrum antibiotics, they can be potentially valuable if antibiotic resistance is encountered. Therefore, our results support further development of GQD as antibacterial agents.
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Acknowledgments The study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant number 41025 and 172003). The authors thank Nikola Davidovac (Castanea, Belgrade, Serbia) for building the lamp used for photoexcitation and Ivana Cirkovic for providing the MRSA isolate.
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Photodynamic antibacterial effect of graphene quantum dots Biljana Z. Ristic a,1, Marina M. Milenkovic a,1, Ivana R. Dakic a,1, Biljana M. Todorovic-Markovic b, Momir S. Milosavljevic b, Milica D. Budimir b, Verica G. Paunovic a, Miroslav D. Dramicanin a, Zoran M. Markovic b, **, Vladimir S. Trajkovic a, * a b
Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade, Serbia Vinca Institute of Nuclear Sciences, University of Belgrade, 11000 Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2013 Accepted 9 February 2014 Available online 4 March 2014
Synthesis of new antibacterial agents is becoming increasingly important in light of the emerging antibiotic resistance. In the present study we report that electrochemically produced graphene quantum dots (GQD), a new class of carbon nanoparticles, generate reactive oxygen species when photoexcited (470 nm, 1 W), and kill two strains of pathogenic bacteria, methicillin-resistant Staphylococcus aureus and Escherichia coli. Bacterial killing was demonstrated by the reduction in number of bacterial colonies in a standard plate count method, the increase in propidium iodide uptake confirming the cell membrane damage, as well as by morphological defects visualized by atomic force microscopy. The induction of oxidative stress in bacteria exposed to photoexcited GQD was confirmed by staining with a redoxsensitive fluorochrome dihydrorhodamine 123. Neither GQD nor light exposure alone were able to cause oxidative stress and reduce the viability of bacteria. Importantly, mouse spleen cells were markedly less sensitive in the same experimental conditions, thus indicating a fairly selective antibacterial photodynamic action of GQD. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Graphene Quantum dot Photodynamic Antibacterial Oxidative stress
1. Introduction Different types of nanoparticles, ranging in size from 1 to 100 nm, have been investigated for their possible use in biomedicine [1]. Semiconductor quantum dots are nanoparticles with superior photo-physical properties suitable for biomedical imaging [2]. However, the potential toxicity resulting from the presence of heavy metal ions in conventional inorganic quantum dots (e.g. CdSe, CdTe) may impede their medical applications [3]. A new class of quantum dots, called graphene quantum dots (GQD), has recently been synthesized [4], displaying the special physicochemical properties of graphene, a single layer of carbon atoms in a honeycomb structure, endowed with large surface area and excellent thermal/chemical stability [5]. Compared to conventional inorganic quantum dots, GQD possess several advantages, including ease of production, high fluorescent activity, resistance to
* Corresponding author. Tel.: þ381 11 3643 233; fax: þ381 11 3643 235. ** Corresponding author. Tel.: þ381 11 2455 451; fax: þ381 11 3440 100. E-mail addresses: [email protected] (Z.M. Markovic), [email protected] (V.S. Trajkovic). 1 These authors equally contributed to the work. http://dx.doi.org/10.1016/j.biomaterials.2014.02.014 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
photo-bleaching, excellent solubility and biocompatibility [4]. Because of these favorable features, GQD are more suitable candidates for non-toxic bioimaging or biosensing agents than their inorganic counterparts. Despite similarities with semimetal graphene nanoparticles, semiconductor GQD, due to different electronic structure, display some unique physichochemical and biological properties. Unlike graphene and similarly to fullerenes (C60), another carbon allotrope [6], GQD in suspension are able to generate reactive oxygen species (ROS) upon photoexcitation [7]. Therefore, GQD are potential candidates for photodynamic therapy, in which the light-excited compound kills cells by ROS generated through energy or electron transfer to molecular oxygen [8]. Accordingly, we have recently reported that GQD exposed to blue light kill cancer cells in a ROS-dependent manner [9]. Photodynamic therapy can also target microbial pathogens, including bacteria, which is becoming increasingly relevant in light of the emerging antibiotic resistance and consequent reduction in effectiveness of conventional therapy [10,11]. While most carbon-based nanomaterials, including fullerenes, carbon nanotubes and graphene display antibacterial properties [12e14], the effects of GQD on bacteria have not been investigated so far.
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Fig. 1. Characterization of GQD. (A) Top view AFM image of GQD deposited on freshly cleaved mica substrate. (B) Height profile of GQD. (C) Size distribution of GQD nanoparticles calculated by AFM software (n ¼ 500). (D) Top view AFM image of typical GQD. (E) Low- and high resolution-TEM micrographs of GQD. (F) Photoluminescence spectra of GQD exposed to different excitation wavelengths (a.u. e arbitrary units).
In the present study, we assessed photodynamic antibacterial activity of electrochemically produced GQD. To that aim, we used as targets methicillin-resistant Staphylococcus aureus (MRSA), Grampositive cause of serious healthcare-associated and communityonset infections [15], and Escherichia coli, Gram-negative commensal of the human intestinal flora with pathogenic strains able to cause meningitis or urinary and gastrointestinal tract infections [16]. 2. Materials and methods 2.1. Preparation and characterization of GQD A stable suspension of GQD was prepared as previously described [9], using graphite rods as anode and cathode and NaOH/ethanol as electrolyte, followed by evaporation of the more volatile ethanol. The pH value of GQD suspension was adjusted to 7.0 by addition of hydrogen chloride and the total carbon particle and NaCl concentrations were adjusted to 1 mg/ml and 0.9%, respectively. We did not observe any visible aggregation of GQD in saline solution containing up to 5% of NaCl. A single GQD monolayer thin film was deposited on mica substrate (air-dried at 2000 C for 10 min) by spin coating and imaged after drying by atomic force microscopy (AFM). AFM measurements were performed using a Quesant AFM (Aguora Hills, CA) operating in tapping mode in air on room temperature, with
standard silicone tips (NanoAndMore Gmbh, Wetzlar, Germany) and with the constant force of 40 N/m. GQD were also characterized by transmission electron microscopy (TEM), using Philips CM200 microscope operated at 200 kV. Samples were prepared by drop casting of GQD dispersion on carbon coated copper grid with 300 mesh. The luminescence emission measurements were performed at room temperature on the Fluorolog-3 FL3-221 spectrofluorometer system (Horiba Jobin-Yvon S.A.S., Chilly Mazarin, France), utilizing a 450 W Xenon lamp as excitation source (328 nm) and R928P photomultiplier tube as a detector.
2.2. Bacterial suspension and treatment Stock cultures of a clinical isolate of MRSA [17] and the reference strain of E. coli (ATCC25922) were maintained on Columbia agar (BD, Franklin Lakes, NJ) supplemented with 5% sheep blood at 4 C. Prior to inoculation, the strains were transferred from the stock cultures to Columbia agar supplemented with 5% sheep blood and incubated aerobically at 37 C overnight, followed by subcultivation under the same conditions. The cultures were then used for preparation of bacterial suspensions (2 104 colony forming units/ml) in a phosphate-buffered saline (PBS). Subsequently, 200 ml of bacterial suspension were transferred to 15 ml glass centrifuge tube and 200 ml of GQD (final concentration 50e200 mg/ml) or PBS were added. After irradiation with blue light (465e475 nm, 1 W), bacterial suspensions were centrifuged at 4000 g for 10 min and resuspended in PBS for determination of cell death/ membrane damage, ROS measurement or AFM analysis.
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Fig. 2. Photoexcited GQD decrease bacterial cell viability. (A, B) Suspensions of E. coli (A) and S. aureus (B) were incubated with PBS (control) or GQD (200 mg/ml), and exposed to blue light (470 nm, 1W) for 15 min. After staining with PI, bacterial cells were visualized by optical (A, B, upper panel) and fluorescence microscopy (A, B, lower panel). (C) E. coli or S. aureus suspensions were treated with different concentrations of GQD and blue light for 15 min, or (D) S. aureus was incubated with 200 mg/ml GQD and exposed to blue light for different periods of time. The red fluorescent PI-permeable cells with membrane damage were enumerated and the data from a representative of three independent experiments are presented as mean SD values (*p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.3. Splenocyte isolation and treatment Spleens were aseptically removed from 6e8-week-old Balb/c mice sacrificed by cervical dislocation. Single cell suspensions were obtained by gently disrupting the spleen tissue through 70 mm pore mesh in cold RPMI 1640 containing 10% FCS. The cells were then centrifuged at 1500 rpm for 10 min and the erythrocytes were removed by incubation in the hypotonic ACK buffer (150 mM NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4). The cells were resuspended in PBS (3 106 cells/ml) and 200 ml of the cell suspension were mixed with 200 ml of GQD suspension or PBS. After exposure to blue light (465e475 nm, 1 W) suspensions were centrifuged and
resuspended in PBS for determination of cell death. The experiment was approved by the Local Animal Care Committee and conformed to the ethical guidelines stated in the “Principles of Laboratory Animal Care” (NIH publication #85-23, revised in 1985). 2.4. Cell death analysis Cell death was analyzed by propidium iodide (PI) staining for cell membrane damage [18]. Cells were incubated with 4 mM PI (BD, Franklin Lakes, NJ) for 15 min at 25 C in the dark. Red fluorescence of PI-stained cells with membrane damage was
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Fig. 3. Photoexcited GQD reduce the number of bacterial colonies. (A) E. coli and (B) S. aureus suspensions were incubated with PBS (control) or exposed simultaneously to GQD (200 mg/ml) and blue light (470 nm, 1 W) for 15 min. The number of CFU was determined by standard plate count method. The data in are mean SD values from a representative of three independent experiments (*p < 0.05).
visualized with a fluorescence microscope (Axiolab HB 50, Zeiss, Germany) under 100X/1.25 oil lens, using 536 nm excitation wavelength. The areas of observation were randomly photographed (5 view fields per sample) with a Nikon digital camera.
3. Results and discussion 3.1. Characterization of GQD
Intracellular ROS production was determined based on green fluorescence emitted by redox-sensitive dye dihydrorhodamine 123 (DHR) [19]. After treatment, bacterial suspensions were washed and stained with 5 mM DHR (Invitrogen/Life Technologies, Carslbad, CA) for 20 min in the dark at 25 C. Green fluorescent ROSproducing cells were visualized with a fluorescence microscope (Axiolab HB 50, Zeiss, Germany) under 100/1.25 oil lens, using 488 nm excitation wavelength. The areas of observation were randomly photographed (5 view fields per sample) with a Nikon digital camera.
AFM and TEM were used to visualize the size, morphology and structure of GQD. AFM images demonstrated the topographic morphology of nanosized GQD (Fig. 1A), whose average height was about 3 nm, corresponding to 3 graphene layers (Fig. 1B). AFM analysis indicated that GQD diameters were mainly distributed in the range of 20e67 nm, with the average diameter of 40.5 nm (Fig. 1C and D), which was consistent with the TEM images (Fig. 1E left). Both AFM and TEM demonstrated that typical GQD were round shape (Fig. 1D and E left). High resolution TEM analysis indicated high crystallinity of GQD, with a lattice parameter of 0.263 nm, also showing a zigzag edge structure of GQD (Fig. 1E right). The photoluminescence spectra have shown that GQD have highest luminescence in the visible part of the spectrum, with the maximum intensity reducing and shifting to higher wavelengths (494e548 nm) with the increase in the excitation wavelength (Fig. 1F).
2.7. AFM imaging of bacteria
3.2. Antibacterial effect of photoexcited GQD
2.5. Standard plate count method The plate count method was used to determine bacterial cell number. Bacterial suspensions at appropriate dilutions were inoculated onto Trypcase-soy agar (BioMerieux, Marcy l’Etoile, France) and the inoculated plates (3 per sample) were incubated aerobically at 37 C for 24 h. The number of colony forming units (CFU) was counted through visual inspection.
2.6. ROS measurement
Bacterial cells were fixed with 2.5% (vol/vol) glutaraldehyde solution (pH 6.8) for 2.5 h at 4 C and washed in PBS. AFM measurements were performed as described for GQD and the different AFM parameters were determined for at least 30 bacterial cells per sample.
2.8. Statistical analysis The statistical significance of the differences between treatments was assessed using one-way analysis of variance (ANOVA) followed by Student-Neuman-Keuls test for multiple comparisons. The value of p < 0.05 was considered significant.
We and others have previously shown that light absorbance by GQD was highest in the UV part of the spectrum, exponentially decreasing with the higher wavelengths [9,20]. Since UV irradiation itself induces DNA damage and cell death [21], for the photostimulation of GQD we used blue light (465e475 nm) as the wavelength-nearest non-cytotoxic alternative. The antibacterial effect of photoexcited GQD was assessed using a fluorescence assay for the cell membrane integrity, as well as standard plate count
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Fig. 4. AFM analysis of bacteria exposed to photoexcited GQD. (A, B) AFM images of E. coli (A) and S. aureus (B) incubated with PBS (control) or simultaneously exposed to GQD (200 mg/ml) and blue light (470 nm, 1 W) for 15 min.
method. PI staining demonstrated that neither GQD at the highest concentration (200 mg/ml) nor maximal duration (15 min) of blue light exposure alone were able to significantly affect the viability of E. coli or S. aureus (Fig. 2AeD). On the other hand, bacterial cells treated simultaneously with GQD and light exhibited a substantial loss in viability, as indicated by a significant increase in the number of PI-permeable red fluorescent bacteria (Fig. 2A, B). It is unlikely that the GQD themselves contributed to the observed fluorescence, as the photoluminescence spectra demonstrated that their capacity for red fluorescence emission is extremely low (Fig. 1F). The cytotoxic effect of GQD at maximal duration of photoexposure was concentration-dependent (Fig. 2C), with the comparable efficiency in both bacterial species (IC50 100 mg/ml). Also, the effect was
Table 1 AFM parameters of E. coli and S. aureus after exposure to photoexcited GQD. The data are presented as mean SD values (*p < 0.05 compared to control, untreated samples). E. coli
S. aureus
Control Length (mM) Width (mM) Height (nM) RMS roughness (nM)
5.75 0.53 99 33.2
1.13 0.07 16 5.6
Light þ GQD 7.2 1.02 300 84.0
3.1 0.15* 79* 19.2*
Control 0.74 0.56 148 27.4
0.08 0.09 21 4.9
Light þ GQD 0.70 0.40 450 63.0
0.07 0.08* 96* 14.1*
clearly dependent on the duration of photoexposure, as shown in S. aureus incubated with the fixed dose of GQD (Fig. 2D). In agreement with the fluorescence-based assessment of cell membrane damage, the plate count method demonstrated a significant reduction in the number of CFU in both E. coli and S. aureus suspensions exposed to photoexcited GQD (Fig. 3). It should be noted that inorganic QD have recently been shown to be toxic to various Gram-positive and Gram-negative bacteria, including S. aureus and E. coli [22e24], while amine-functionalized GQD exerted potent antimycoplasma activity [25] in the absence of overt photoexcitation. Therefore, light-induced antibacterial action described here could represent an additional modality for QD-mediated bacterial cell killing. 3.3. Morphological changes in bacteria exposed to photoexcited GQD AFM was used for visualization of the changes in bacterial cell morphology induced by photoexcited GQD. The control, untreated E. coli and S. aureus samples contained typically rodshaped (Fig. 4A) or near-spherical cells (Fig. 4B). After the treatment with blue light-irradiated GQD, bacterial cells displayed an increased tendency to aggregate (Fig. 4A and B). While the average length of bacteria did not change noticeably, the width of E. coli significantly increased and that of S. aureus
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decreased upon treatment (Table 1). The height, as well as the values of root mean square (RMS) roughness significantly increased in both bacterial species after exposure to lightirradiated GQD (Table 1). The observed increase in surface roughness and increase in height following exposure to photoexcited GQD indicate cell membrane damage and subsequent swelling of the bacterial cell, respectively, as recently demonstrated by AFM for other antibacterial agents [26e29]. Therefore, the extensive morphological changes associated with the
20 µM
increase in surface roughness further confirm the detrimental effects of photoexcited GQD on bacterial cells. 3.4. Production of ROS by bacteria exposed to photoexcited GQD Near-IR light-irradiated graphene and graphene oxide nanoparticles kill tumor cells by releasing heat [30e33]. However, as no significant temperature increase was observed in GQD-treated bacterial suspensions exposed to blue light (data not shown), we
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Fig. 5. Photoexcited GQD induce oxidative stress in bacterial cells. (A, B) Suspensions of E. coli (A) and S. aureus (B) were incubated with PBS (control) or GQD (200 mg/ml), and exposed to blue light (470 nm, 1 W) for 15 min. After staining with DHR, bacterial cells were visualized by optical (A, B, upper panel) and fluorescence microscopy (A, B, lower panel). (C) The green fluorescent ROS-producing E. coli and S. aureus were enumerated and the data from a representative of three independent experiments are presented as mean SD values (*p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.5. Photo-toxicity of GQD towards mouse splenocytes Having established that photoexcited GQD have bactericidal properties, it was important to examine the sensitivity of eukaryotic cells to the same treatment. We have previously shown that human cancer cells were sensitive to photodynamic action of GQD [9] at the same experimental conditions (GQD concentration and duration of light exposure) used in the present study. However, as fast-dividing cancer cells might be more sensitive to certain death stimuli than non-proliferating normal cells, we examined the effect of photoexcited GQD on primary mouse splenocytes as a model of normal eukaryotic cells. Fluorescence microscopy analysis of splenocytes exposed to conditions that killed more than 50% bacteria (100 mg/ml GQD þ 15 min photoirradiation; Fig. 2C) demonstrated only a moderate increase (from 5 to 17%) in the number of PIpermeable spleen cells (Fig. 6), while 200 mg/ml of photoexcited GQD killed almost all bacteria (Fig. 2), but less than 50% of splenocytes (Fig. 6). While the comparison with our previous study [9] indicates that cancer and normal cells (at least those examined in the two studies) are similarly sensitive to photodynamic toxicity of GQD, it appears that both cell types might be more resistant to GQD phototoxicity than bacteria. Although we did not compare the entry of GQD to bacterial and mammalian cells and their ROS production, the latter ingest GQD and subsequently produce ROS [9], so the apparent difference in sensitivity cannot be explained by the failure of mammalian cells to internalize GQD and/or produce ROS in response. On the other hand, bacteria and mammalian cells use different mechanisms to cope with oxidative stress [35], so the latter might somehow be more successful in performing this task. Nevertheless, this relatively selective photodynamic toxicity of GQD towards bacteria, as well as the underlying mechanisms, need to be confirmed in more detailed toxicological and mechanistic studies.
GQD (100 μg/ml) GQD (200 μg/ml)
irradiation
excluded the possibility that the antibacterial effect was due to photothermal cytotoxicity. Another plausible mechanism for the light-induced antibacterial effect of GQD was ROS-mediated photodynamic effect, as GQD are able to produce ROS, including singlet oxygen, when irradiated with blue light [7,9]. To examine this possibility, we measured intracellular ROS levels using DHR, a nonfluorescent cell-permeable indicator that can be oxidized to green fluorescent rhodamine 123. Fluorescent microscopy analysis revealed that no production of ROS was observed in untreated bacteria or those exposed to GQD or light alone (Fig. 5). On the other hand, a significant increase in the numbers of green fluorescent ROS-containing cells was demonstrated in both E. coli and S. aureus suspensions treated simultaneously with GQD and blue light (Fig. 5). When excited at 488 nm (DHR excitation wavelength), GQD themselves emit only low-intensity green fluorescence (Fig. 1F), but can produce ROS that oxidize DHR to a highly fluorescent product [7,9]. However, no DHR signal was detected in GQD-exposed bacteria in the absence of previous photoexcitation (Fig. 5), indicating that GQD might not be able to enter intact bacteria. In the likely chain of events, blue-light excited GQD photodynamically disrupt bacterial cell membrane integrity, enter the damaged bacteria and start producing additional ROS amounts due to excitation during DHR measurement. Moreover, damaged bacteria themselves can produce ROS in a form of “oxidative shielding” response [34]. To fully elucidate the time-kinetics of these complex events and their involvement in GQD-mediated photodynamic bacterial killing, a system for a real-time measurement of GQD internalization and ROS production is required. Nevertheless, by demonstrating the induction of oxidative stress in the absence of temperature increase, our results strongly indicate the photodynamic nature of the observed antibacterial effect.
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irradiation Fig. 6. The effect of photoexcited GQD on the viability of mouse splenocytes. Mouse splenocytes were left untreated (control) or exposed simultaneously to GQD (100 or 200 mg/ml) and blue light (470 nm, 1 W) for 15 min. After staining with PI, cells were visualized by optical and fluorescence microscopy. The red fluorescent PI-permeable cells with membrane damage were enumerated and the data from a representative of three independent experiments are presented as mean SD values (*p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Conclusion The data presented here demonstrate a relatively selective photodynamic antibacterial activity of GQD. Having in mind the low tissue penetrance of blue light and low absorbance of GQD at the higher wavelengths, such an approach would be presumably efficient for skin and mucosal infections, or water and surface disinfection. While photoexcited GQD might not be able to perform better than currently available broad spectrum antibiotics, they can be potentially valuable if antibiotic resistance is encountered. Therefore, our results support further development of GQD as antibacterial agents.
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Acknowledgments The study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant number 41025 and 172003). The authors thank Nikola Davidovac (Castanea, Belgrade, Serbia) for building the lamp used for photoexcitation and Ivana Cirkovic for providing the MRSA isolate.
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