Evaluation of the Photothermal Properties of a Reduced Graphene

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Evaluation of the Photothermal Properties of a Reduced Graphene Oxide/Arginine Nanostructure for Near-Infrared Absorption Mohadeseh Hashemi,†,‡,⊥ Meisam Omidi,∥ Bharadwaj Muralidharan,‡,§ Hugh Smyth,⊥ Mohammad A. Mohagheghi,∇ Javad Mohammadi,*,† and Thomas E. Milner*,‡ †

Biomedical Engineering Department, Faculty of New Sciences and Technologies, The University of Tehran, Tehran 14395-1561, Iran Biomedical Engineering Department, The University of Texas at Austin, Austin, Texas 78712, United States § Department of Electrical and Computer Engineering, The University of Texas at Austin, Austin, Texas 78712, United States ⊥ Division of Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, United States ∥ Protein Research Centre, Shahid Beheshti University, GC, Velenjak, Tehran 1985717443, Iran ∇ Cancer Research Center, Cancer Institute of Iran, Tehran University of Medical Sciences, Tehran 1419733141, Iran ‡

S Supporting Information *

ABSTRACT: Strong near-infrared (NIR) absorption of reduced graphene oxide (rGO) make this material a candidate for photothermal therapy. The use of rGO has been limited by low stability in aqueous media due to the lack of surface hydrophilic groups. We report synthesis of a novel form of reduced graphene-arginine (rGO-Arg) as a nanoprobe. Introduction of Arg to the surface of rGO not only increases the stability in aqueous solutions but also increases cancer cell uptake. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) images are recorded to characterize the morphology of rGO-Arg. Fourier transform infrared (FTIR), X-ray photoelectron spectra (XPS), Raman, and UV−vis spectroscopy are utilized to analyze the physiochemical properties of rGO-Arg. Interaction of rGO-Arg with 808 nm laser light has been evaluated by measuring the absorption cross section in response to periodically modulated intensity to minimize artifacts arising from lateral thermal diffusion with a material scattering matched to a low scattering optical standard. Cell toxicity and cellular uptake by MD-MB-231 cell lines provide supporting data for the potential application of rGO-Arg for photothermal therapy. Absorption cross-section results suggest rGO-Arg is an excellent NIR absorber that is 3.2 times stronger in comparison to GO. KEYWORDS: graphene oxide, laser, photothermal therapy, absorption cross section, breast cancer

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fluorophores is limited by low photothermal conversion efficiency and instability in aqueous solutions.5,11 Jingyi et al. investigated the application of immune gold nanocages for photothermal destruction of cancer cells. Their study showed that gold nanocages have excellent biocompatibility and tunable light absorption in the 500−880 nm spectral region.12 However, despite these advancements, tissue penetration depth of short wavelength light necessary for gold nanoparticles is limited by strong light scattering of tissues.12 As a solution, gold nanorods with an effective NIR absorbance around 808 nm have been utilized to obtain deeper tissue penetration. However, a primary drawback of this approach is the cytotoxic effect of gold nanorods due to the presence of cetyltrimethylammonium bromide (CTAB) which is used as the surfactant in gold rod-shaped structure formation.13−15 Despite the considerable effort to fabricate photoabsorbers for photothermal therapy, there remains a

INTRODUCTION Cancer is a family of diseases associated with defective control of cell proliferation. Despite many recent advances in cancer therapy, a need is recognized for an effective treatment of many cancer types.1 Among available efforts to improve therapeutic outcomes, hyperthermia offers a promising approach to treat deep seated tumors.2 Photothermal therapy is a medical procedure based on lightto-heat conversion for selective destruction of targeted cells.3 Considering the entire spectral range from ultraviolet (UV) to infrared (IR), the near IR (NIR) region provides some advantages for photothermal therapy since absorption by water is small and light has a relatively deep penetration depth in tissue.4 Driven by the need to carry out effective photothermal treatment, various nanoparticles have been employed as a NIR absorbers including organic fluorophores (e.g., indocyanine green, ICG),5 gold nanostructures (nanorods, nanoshells, and nanocages).6−10 Landsman et al. studied the photoabsorbing properties, spectral stabilization and stability of ICG in aqueous media. Their study demonstrated that the application of organic © 2017 American Chemical Society

Received: July 31, 2017 Accepted: August 25, 2017 Published: August 25, 2017 32607

DOI: 10.1021/acsami.7b11291 ACS Appl. Mater. Interfaces 2017, 9, 32607−32620

Research Article

ACS Applied Materials & Interfaces

In this work, a novel photoabsorber composed of rGO conjugated to Arg (rGO-Arg) has been synthesized as a photoabsorber. We have introduced an efficient method to prepare rGO-Arg, and also studied the effect of a functional group on the yield of reaction. The absorption cross section of both GO and rGO-Arg at 808 nm has been measured in the frequency domain by utilizing Fourier analysis. The in vitro effect of using rGO-Arg on cell toxicity was characterized using MTT and live/dead assays during NIR light irradiation. We also evaluated cellular uptake of rGO-Arg in cancer cell lines by multiphoton microscopy.

need to explore nanoparticles that provide a low cost, low toxicity, superior photothermal conversion ability, and optical absorption in the NIR region. In comparison to other potential photothermal agents, graphene nanostructures are promising candidates.16 Graphene oxide (GO) is a monolayer or few layer hexagonal or honeycomb lattice structure of sp2 bonded carbon atoms capable of absorbing NIR light and converting into thermal energy due to the delocalized electron orbitals.17−19 Hence GO, with a large number of functional groups, high aspect ratio, low production cost and effective thermal conductivity, can be considered as a versatile photoabsorber candidate for photothermal therapy application.18,20−26 To date, only a few studies report investigations of the potential application of GO for photothermal therapy. Yang et al., studied photothermal effect of polyethylene glycol (PEG) coated GO on cancer cells.20 In another study, Robinson et al., investigated the effect of PEG conjugated reduced graphene oxide (rGO) for photothermal therapy. Their research demonstrated by utilizing rGO instead of GO, NIR light absorption was increased by more than 6-fold leading to the restoration of π network of electrically insulated GO.21,27 Despite the promising properties of rGO for photothermal therapy applications, the use of rGO has been limited by low stability in aqueous media due to the absence of surface hydrophilic groups.28−32 To overcome these obstacles, numerous types of biological or synthetic polymers,33 such as hyaluronic acid derivatives,34 dextran,22 heparin,35 polyethylene glycol (PEG),36 phospholipid-polyethylene glycol,37 polystyrene-co-poly(acrylic acid),37 and polystyrene-co-poly 4-vinylpyridin,37 have been utilized for coating rGO nanosheets. Although, all these surface molecules increase the stability of rGO in aqueous media they are not optimized for targeted delivery to tumor cells. Hence, utilizing such molecules, which provide both better stability and improved targeting, is of interest. Small penetrating peptides, such as R9,38,39 TAT,40 pVEC,41 TP10,42 and polymers, such as poly-L-lysine and poly-L-arginine (Arg), have been used for targeting drug delivery systems.43,44 Arg is an important amino acid found in proteinaceous foods, which can effectively cross biological membranes and facilitate the uptake of small molecules to which they are attached.45 Yamin et al. modified the surface of graphene electrochemically by utilizing Nafion and arginine. As poly-L-arginine has electrocatalytic ability for terbutaline sulfate and graphene provides a large specific surface area, excellent electric conductivity and electrocatalytic activity was observed.46 In the other study, Sawosz et al. used Arg to prevent graphene agglomeration and increase the antitumor effect on glioblastoma multiform tumor in vivo.47 However, to the best of our knowledge, none of these studies applied Arg to improve the stability, targeting function, and nearinfrared cross section of GO. In spite of the excellent characteristic potential of GO as a photoabsorber, limited reports exist on the measurement of absorption cross section of GO. Optical absorption cross section is one of the fundamental properties of the materials,48 which characterizes an important aspect of light-matter interaction and provides a measure of photothermal potential. Beyond this approach, the absorption cross section of graphene sheets with a lateral size around 1 μm at 660 nm has been reported by Min Yi et al. and Khan et al. at low and high graphene concentrations, respectively.49,50 To the extent of our knowledge, no report attempts to calculate the absorption cross section of GO at 808 nm.

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EXPERIMENTAL SECTION

Chemicals and Materials. Graphene oxide (GO) was purchased from Graphene Supermarket (Reading, MA, USA). Sodium borohydride (NaBH4) and potassium hydroxide (KOH) were purchased from Sigma-Aldrich (USA). L-Arginine was obtained from Sigma-Aldrich (USA). Phosphate buffered saline (PBS), fetal bovine serum (FBS), Dulbecco’s modified eagle medium (DMEM), and cell proliferation reagent kits (MTT) were supplied by Sigma-Aldrich (USA). Live/dead assay kits were purchased from Molecular Probes (Eugene, OR, USA). PE (Lissamine Rhodamine B) was obtained from Sigma-Aldrich (USA). Cell tracker green and DAPI (4′,6-diamidino-2-phenylindole) were purchased from Molecular Probe Inc. (USA). Mounted media were ordered from Vector Laboratories (USA, California). All other chemicals were of analytical grade or better. Instrumentation. Polydispersity index (PDI), size, and zeta potential were determined using dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Atomic force microscopy (AFM) images were recorded by a SPM VEECO and transmission electron microscopy (TEM) images were recorded at a voltage of 80 kV using a FEI Tecnai. The UV absorption spectra were recorded by Tecan Infinite M200 PRO multimode microplate reader. Fourier transform infrared (FTIR) spectra were recorded using a Spectrum RX I (PerkinElmer). X-ray photoelectron spectroscopy (XPS) measurements were examined on an ultraspectrometer (Bestec, Germany) using an Mg Ka source and monochromatic Al Ka source. Raman spectra were obtained by RM 2000 microscopic confocal Raman spectrometer (Renishaw) by focusing 633 nm He−Ne laser light onto a sample with the scattered light input to a Raman spectrometer. Photothermal irradiation was carried out using a laser diode emitting at 808 nm. A laser diode controller (Thorlabs, Inc., LDC 240C) regulated the current into the laser diode and a temperature controller (Thorlabs, Inc., TED 200C) fixed the emission wavelength of the laser diode. The 808 nm laser light was coupled into a multimode optical fiber (Thorlabs, Inc., BFL48-1000). The tip of the fiber was imaged onto the solution surface using an aspherical lens (focal length of 20 mm) to produce a 6 mm diameter spot. Surface temperature of the suspension was measured using a temperature calibrated InSb IR camera (FLIR systems, Inc., SC4000 MWIR) sensitive in the 3−5 μm spectral range. Acquisition control signals for the IR camera and laser diode for triggering light emission and frame acquisition were generated by function generators (Agilent 33250A and Berkeley Nucleonics Corp., Model 645) and a digital delay/pulse generator (Stanford Research Systems, Inc., DG535). L-Arginine Functionalized GO. An aqueous GO suspension was probe sonicated for 2 h (60 W, 5 s on and 2 s off, Misonix, USA). Then, graphene-L-arginine (GO-Arg) was prepared by vigorous stirring of 8 mg of L-arginine, 2 mg of GO, and 10 mg of KOH in 10 mL of distilled water at 70 °C for 24 h. For reduction of GO-Arg (rGO-Arg), 1 mL of NaBH4 solution with 1 M concentration was added and kept for 2 h at 70 °C. Then, rGO-Arg suspension was collected by centrifugation (5000 rpm for 10 min, Hitachi Ltd., Tokyo, Japan) and washed with water several times to remove excess L-arginine and impurities (Scheme 1). Absorption Cross Section Determination. The experimental setup to measure absorption cross section consists of a laser diode emitting 808 nm light through a multimode optical fiber (Thorlabs, Inc., BFL48-1000) and aspheric lens (focal length of 20 mm) to image the 32608


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ACS Applied Materials & Interfaces Scheme 1. Representation of rGO-Arg Preparation Processa

Size of GO nanosheets are first decreased by 2 h of probe sonication and then by vigorous stirring of Arg in alkaline solution at 70 °C. After 24 h of stirring, rGO-Arg was synthesized.

a

1.7 W.cm−2 by applying an Arrhenius damage integral equation and using the recorded temperature versus time plots as input data. To investigate the in vitro toxicity of synthetic rGO-Arg and GO on breast cancer cell lines, MD-MB-231 cells were seeded at a density of 5 × 104 cells/well in a 96-well plate and left overnight. Then, the medium was replaced with a fresh medium (complete DMEM) containing different rGO and rGO-Arg concentrations (12.5, 25, 50, 100, 200, and 400 μg/mL). The plates were then incubated for 24 h. Then, a standard MTT assay was conducted to measure the viability of MD-MB-231 cells as described previously.52 The potential photothermal therapy properties of rGO-Arg and GO on MD-MB-231 cells were evaluated using an MTT assay. MD-MB-231 cells were seeded at a density of 5 × 104 cells/well in a 96-well plate and left overnight. Then the medium was replaced with fresh medium containing rGO-Arg (100 μg/mL) and GO (400 μg/mL) for 24 h. After incubation, the cell lines in the laser groups were exposed to pulsed NIR light at a fluence rate of 1.7 W.cm−2 for 120 and 240 s. Lastly, a standard MTT assay52 was performed to evaluate photothermal destruction of MD-MB-231 cells using GO and rGO-Arg agents. Each experiment was replicated four times. Photothermal cell toxicity of rGO-Arg was evaluated visually on MD-MB-231 cell lines by using a live/dead assay. MD-MB-231 cells were seeded at a cell density of 5000 cells/well in 96-well plates and incubated for 24 h prior to experimentation. The cells were then incubated with 100 μL of 100 μg/mL of rGO-Arg per well for 24 h. Then, cells were washed with PBS and subsequently exposed to NIR pulsed laser irradiation (4 min and 1.7 W.cm−2). After incubation for another 1 h, the cells were stained using calcein AM (2 M) and ethidium homodimer-1 (EthD−1) (4 M) for 1 h at room temperature. Calcein AM converts to fluorescent calcein (excitation ∼495 nm, emission ∼515 nm) becuase of the intracellular esterases present in live cells. While EthD-1 penetrates the damaged cell membrane and bind to the nucleic acids (excitation, ∼496 nm; emission, ∼635 nm). Fluorescence images were recorded using an inverted fluorescence microscope (Evos, Life Technologies). Multiphoton Laser Scanning Microscopy (MPLSM) Characterization of Cellular Uptake of rGO-Arg and GO. The MD-MB231 cells were cultured in a 6-well plate at a density of 2 × 105 cell/well. After 24 h, cells were incubated with lissamine rhodamine B sulfonyl chloride (Thermofisher) conjugated rGO and GO (20 μg/mL) for 24 h. Then, cells were stained with a cell tracker green fluorescent probe at a concentration of 4 μM in DMSO for 15 min, followed by washing three times with PBS (5 min each). Cells were then fixed with 4% paraformaldehyde (PFA) for 1 h at room temperature. Subsequently, nuclei were stained using 100 μL of 0.1 μg/mL of DAPI for 15 min followed by washings three times with PBS (5 min each). Cells were

fiber tip onto the GO solution. The thermal response of the material to laser excitation was measured using an IR camera. The laser diode controller controls the current and the operating temperature of the laser, whereas the duration of the laser pulse in pulsed mode operation and the IR camera acquisition rate is controlled by the timing controller. To minimize the effect of lateral heat diffusion through the sample holder (see Supporting Information 1), laser pulse duration was set to 250 ms with a pulse repetition rate of 2 Hz (see Supporting Information 1 and 2 (Figure S1)). To minimize the effect of optical scattering in the solution, time series of the spatial temperature variation were recorded by an InSb IR camera at three different concentrations of GO (1, 0.5, and 0.25 mg/mL) and rGO-Arg (0.05, 0.1, and 0.2 mg/mL) and compared to the corresponding profile of a black absorber (see Supporting Information 1, Figure S2, and Table S1 on black absorber characterization). The first images of each sample were recorded by the InSb camera within 25 ms after emission of the laser pulse and used to assess laser heating profile of suspensions without lateral thermal diffusion. Since the radial heat diffusion has azimuthal symmetry, the variation of the surface temperature along the diameter of the surface profile, were fitted to a Gaussian curve. The maximum concentration of rGO-Arg and GO that had a lateral thermal emission profile with a similar lateral spread to that of the black absorber was chosen, to minimize introduction of artifacts due to light scattering, in computation of the absorption cross section. Stability of GO and rGO-Arg. A quantitative method based on a DLS instrument38,51 has been carried out to evaluate the stability of GO and rGO-Arg in PBS, DMEM, and FBS. The average hydrodynamic diameters (HD) of the GO and rGO-Arg were measured primarily and after 1, 12, 24, 48, and 240 h. The stability is calculated as ⎛ HDt − HDt = 0 ⎞ stability (%) = ⎜1 − ⎟ × 100 HDt = 0 ⎝ ⎠ where HDt=0 and HDt are hydrodynamic diameter at initial time (t = 0) and the different intervals (t = 1, 12, 24, 48, and 240 h), respectively. Ex Vivo and in Vitro Photothermal Studies. GO, rGO, and rGO-Arg were transferred into a standard 96-well plate. 808 nm pulsed laser light with power densities of (0.3, 0.5, 1, and 1.7 W·cm−2), pulse repetition rate of 2 Hz and pulse duration of 250 ms was incident on solutions with various concentration of GO (50, 100, 200, and 400 μg/mL), rGO (25, 50, 100, 200 μg/mL), and rGO-Arg (25, 50, 100, 200 μg/mL). Increased temperature in response to laser irradiation was monitored with an InSb IR camera positioned above the sample and far from the laser spot (6 mm in diameter). Cell damage due to hyperthermia was predicted in response to an incident fluence rate of 32609


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ACS Applied Materials & Interfaces then mounted with VECTASHIELD mounting medium at room temperature overnight. MPLSM images were analyzed using Laser Scanning Microscope software (Prairie Technologie, Inc.).

intensity of C−O and CO peaks decreased leading to the elimination of oxygen functional groups. In rGO-Arg spectrum, the peak related to the C−O decreased and the peak related to CO disappeared completely. Moreover, a new peak at 285.7 and 288.5 eV, respectively, represent the presence of C-NH2 and C-NH groups in the spectrum and represent the conjugation of Arg to rGO.56 The Arg could be conjugated to the GO sheets through the epoxy57−59 or carboxylic acid60,61 functional groups on the GO surface. The effect of different GO functional groups on the rGO-Arg preparation was studied by chemically modified GO. Carboxylated graphene oxide (GO−COOH) and epoxylated graphene oxide (GO-O) were synthesized as described in the Supporting Information 8 (Figure S6A). The FTIR analyses confirmed the efficient carboxylation and epoxylation of GO (Figure S6B). As shown in Figure S6C, the mass ratio of Arg in rGO-Arg is 16 ± 2% and 3 ± 1% for the sample modified with GO-O and GO−COOH, respectively. This might be explained by a lower activation energy of epoxy as compared with carboxylic acid groups.61−63 Since the reaction of epoxy-amine possess many of the advantages to consider as a click reaction64 and the yield of rGO-Arg formation in our protocol is almost close (11.2 ± 1.5%) to the reaction of epoxy-amine (see Figure S6C),57−59 it is suggested that the presented reaction could be considered as a bifunctional reaction through clicking the epoxy group on the GO surface. To examine further defect formation during GO size reduction and rGO-Arg preparation, Raman spectroscopy was performed. As shown in Figure 2C, before GO sonication, two distinctive peaks were observed at ∼1370 and ∼1620 cm−1, which correspond to D and G bands, respectively.65 After 2 h sonication (Figure 2C), the G band has a small shift toward longer wavelengths, which may be due to increasing oxygen-contained functional groups.54 Moreover, the intensity of D-bonds are noticeably amplified suggesting more defects are present on graphene oxide sheets after 2 h probe sonication.54 After rGO-Arg formation, intensification of the D-bond (Figure 1C) may be due to the reduction of graphene oxide sheets.66 In addition, the ID/IG ratio is utilized as an appropriate index for demonstrating defects in graphene sheets.67 The increase in ID/IG ratio after 2 h GO sonication and also after rGO-Arg formation suggest an increase in the defects in graphene sheets in fragmented GO and in rGO-Arg (see inset of Figure 2C). To investigate the possibility of using rGO-Arg as a photothermal nanoprobe, the optical absorption spectra of GO, rGO, rGO-Arg, and Arg were examined (Figure 2D). Aqueous GO suspensions displayed a strong absorption peak at ∼233 nm (π−π of aromatic carbon bonds). After reduction, absorption peak shifted to ∼260 nm because of the removal of oxygen containing groups during reduction, also the optical absorption increased significantly over the entire IR spectral range with a significant increase around 800 nm leading to a partial restoration of π-network of electrically insulated GO.68 Further, increases in optical absorption of rGO-Arg as compared with rGO may be attributed to the further restoration of a π-network of electrically insulated GO resulting electronic conjugation among reduced graphene sheets.69 Increase in optical absorbance of rGO-Arg may enhance photothermal properties. Absorption Cross Section of rGO-Arg and GO. To evaluate the photothermal response of GO and rGO-Arg to 808 nm laser radiation, absorption cross section of rGO-Arg and GO have been obtained by calculating absorption coefficient

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RESULTS AND DISCUSSION Characterization of GO and rGO-Arg. DLS estimates lateral size and surface zeta potential of GO and rGO-Arg. The z average diameter and PDI for GO, sonicated GO, and rGO-Arg were 2930 ± 964.9, 217.7 ± 91.69, and 223.3 ± 108 nm and 0.54 ± 0.12, 0.3 ± 1.7, and 0.26 ± 0.9, respectively (Figure S3). DLS determined an equivalent hydrodynamic diameter, while GO and rGO-Arg are more accurately described as 2-D nanosheets.53 The formation of rGO-Arg also can be confirmed by zeta potential measurements. GO presents a zeta potential of −53.7 ± 6.57 mV and after conjugation of Arg the zeta potential increased by 107.5 to +53.8 ± 6.35 mV suggesting formation of rGO-Arg. The structure and size of GO and rGO-Arg was also characterized by AFM analysis (Figure 1A and B). AFM images indicate efficient size reduction of GO sheets after 2 h probe sonication. The average lateral size of GO sheets is reduced and consistent with increasing sonication time. The size of GO sheets before sonication ranged between 0.5 and 500 μm (57% of the nanoparticles are above 3000 nm, see Figure S4A) and after 2 h sonication the size decreased to 6−166 nm, with 70% of the nanoparticles under 66 nm (see Figure S4B). Size reduction of GO sheets may be due to the shear forces which are introduced as a microjet and shock waves through the samples.54 Furthermore, Figure 1A and B reveal that sheet thickness have decreased after 2 h probe sonication. While further decreased thickness of the sheets was observed after a reduction process (Figure 1C). Conjugation of Arg to rGO increased the sheet thickness from 1.1 to 1.57 nm and from 1.1 to 2.37 nm (Figure 1D). The greater thickness of rGO-Arg may be due to the attachment of Arg to the GO surface or the presence of intact functional groups on the surface after reduction. A similar result was found after conjugation of GO to PEG.55 In order to gain further morphological information, the GO, rGO, and rGO-Arg were examined by TEM. After reduction, morphology of the sheets changed to irregular and wrinkled flake. As shown in Figure 1G, functionalization of GO with Arg changed their appearance. After conjugation, thickness of the sheets increased and no visible free flake of rGO were observed. Similar results were reported by Ewa et al.47 To confirm covalent bonding of Arg to rGO, FTIR spectra of Arg, GO, and rGO-Arg are examined (Figure 2A). The GO spectrum showed a peak at 3440 cm−1, which corresponds to a O−H stretching vibration. Also, presence of peaks at 1733, 1614, and 1085 cm−1 maybe assigned to the CO, CC, and C−O bonds. The FTIR spectrum of rGO-Arg exhibits Arginine absorption features, such as N−H (at 3278 cm−1), C = O (at 1624 cm−1) and especially C−N (at 1313 cm−1) that confirm rGO was bound to Arginine. Also, the elimination of spectral peaks related to oxide groups indicate reduction of rGO-Arg. XPS was also conducted to further characterize rGO-Arg (Figure 2B). The XPS spectrum of GO has two peaks of C1s and O1s while in rGO the height of O1s decreased. RGO-Arg gave an additional N1s in the spectrum suggesting the presence of Arg on the GO sheets. C1s high resolution XPS spectrum of GO (Figure S5A) shows three distinctive peaks at 284.4, 287, and 288.5 eV which are attributed to C−C interaction, C−O, and CO groups, respectively. As presented in “figure S5 B” in rGO C1s spectrum, the intensity of C−C bonds has increased while the 32610


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Figure 1. AFM images of GO (A) before (scale bar is 2 μm) and (B) after 2 h of probe sonication (scale bar is 100 nm), (C) rGO (scale bar is 100 nm), and (D) rGO-Arg (scale bar is 100 nm). SEM images of (E) GO (scale bar is 200 nm), (F) rGO (scale bar is 500 nm), and (G) rGO-Arg (Scale bar is 500 nm.).

chosen, which had had the necessary well depth, assuming that the cross section absorption of GO and rGO-Arg samples will be close to graphene at this wavelength. Time necessary for heat loss through the lateral walls of the 96 well-plate was calculated according to the eq S1 (see Supporting Information 1). An appropriate laser pulse duration

of the sample volume and normalizing to the nanoparticle concentration. Laser radiation can interact with the material to produce different effects including reflection from the surface, lateral diffusion of heat along the side and bottom of the sample well (Figure 3A). On the basis of the absorption cross-section of graphene at 660 nm (36.00 mL/mg·cm),49 a 96-well plate was 32611


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Figure 2. (A) FTIR spectra of GO, Arg, and rGO-Arg, (B) XPS spectra of rGO-Arg, rGO and GO, (C) Raman spectra of GO sheet before (large GO) and after (small GO) 2 h of probe sonication and rGO-Arg, and (D) UV−vis absorption spectrum of rGO-Arg, GO, rGO, and Arg.

In this experiment, we calculate μa of GO and rGO-Arg by computing on a pulse-by-pulse basis. To measure the temperature per pulse, T0, 400 μL of GO with 0.25 mg/mL concentration and rGO-Arg with 0.1 mg/mL concentration (optimized concentration to reduce scattering effect) were placed in a 96 well plate and irradiated by 808 nm laser with pulse duration of 250 ms and pulse repetition rate of 2 Hz (to minimize heat loss due to the lateral diffusivity). The increasing temperature for 1 min was monitored with an IR camera. The profile of increasing temperature versus time of both GO and rGO-Arg (Figure S7A) was filtered with a band-pass filter centered at 2 Hz (Figure S7B). This calculation method ensures the slope of the temperature is more accurate and not affected by laser intensity noise. The steady state value of this filtered profile gives the contribution of increasing temperature per pulse, T0 with respect to the 2 Hz pulse repetition frequency. Thus, for rGO-Arg, T0 = 0.18 ± 0.024 °C, and for GO, T0 = 0.14 ± 0.015 °C. As expected, the Fourier transform of the temporal temperature profile data has a peak at a frequency of 2 Hz (Figure S7B). The laser fluence rate corresponding to 2 Hz pulse repetition frequency (φo) was measured by an optical power meter, φo = 1.77 W/cm2 by substituting density and specific heat capacity of water, ρ = 1001.77 kg/m3 and C = 4178.62 J/kg·K , and also f = 2 Hz, absorption coefficient of GO and rGO-Arg computed using eq 3

(250 ms) far shorter than the lateral thermal relaxation time was chosen to minimize the effect of lateral heat diffusion. The effect of scattering is reduced by optimizing GO and rGO-Arg concentrations. The temperature profile of various concentrations of GO (Figure 3B) and rGO-Arg (Figure 3C) were compared to a black absorber (Figure 3D). The maximum concentration of rGO-Arg and GO that had a profile spread similar to that of the black absorber was 0.1 and 0.25 mg/mL, respectively (see c parameter in Table 1). After minimizing the effect of scattering and lateral thermal diffusion the heat absorbed by the nanosheets is equal to heat generated by the laser. Corresponding to the equation dQ dT = mC dt dt

(1)

where Q is the heat generated during pulsed laser (J), m is the mass of the sample (kg), and C is the specific heat capacity (J/kg·K). By rearranging eq 1 and by dividing numerator and denominator by volume and substituting m/V = ρ and dQ/dt/V = μa × φ in eq 1

μ × φ (t ) d T (t ) = a dt ρc

(2)

where μa is the absorption coefficient (L·mol−1·cm−1), φ is laser beam fluence (W/m2), and ρ is density (kg/m3). By substituting analytic signals T = T0ei2πft and φ = φ0ei2πft into eq 2, the magnitude of absorption coefficient is

2πfρcT0 μa = φo

μa(GO) = 4.18 ± 0.45 1/cm μa(rGO‐Arg) = 5.35 ± 0.71 1/cm

The absorption cross section of the nanosheets were calculated using eq 4 μa = σ × c (4)

(3) 32612


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Figure 3. (A) Schematic of optical excitation of nanoparticles and its photothermal response for computing cross-section absorption of GO and rGOArg (left) prior and (right) posterior to minimization of scattering and lateral diffusion. (B) Temperature variation profile of different GO concentrations (1, 0.5, and 0.25 mg/mL), (C) rGO-Arg concentrations (0.05, 0.1, and 0.2 mg/mL), and (D) black paper (inset picture is the temperature profile of black absorber observed with an IR camera). The pixel resolution is 38.8 μm/pixel.

where σ is absorption cross section of the nanosheets (mL/mg·m) and C is the concentration of GO and rGO-Arg. In the experiment done here, they were 0.25 and 0.1 mg/mL for GO and rGO-Arg, respectively. Substituting the values of absorption coefficient of GO and rGO-Arg, the absorption cross section of the nano sheets are

σ(GO) = 16.65 ± 1.78

mL mg·cm

, σ(rGO‐Arg) = 53.52 ± 7.13 32613

mL mg·cm DOI: 10.1021/acsami.7b11291 ACS Appl. Mater. Interfaces 2017, 9, 32607−32620

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Table 1. Scattering Evaluation of GO and rGO-Arg at Various Concentrations by Fitting Gaussian Curve (95% Confidence for Coefficients) GO

rGO-Arg

parameters

0.25 mg/mL

0.5 mg/mL

1 mg/mL

0.05 mg/mL

0.1 mg/mL

0.2 mg/mL

black absorber

a b c d

0.50 ± 0.03 52.66 ± 1.92 13.84 ± 0.39 14.59 ± 0.38

1.16 ± 0.01 54.69 ± 2.97 17.52 ± 0.54 15.38 ± 0.48

2.02 ± 0.11 53.53 ± 2.78 27.81 ± 2.72 14.13 ± 0.07

2.38 ± 0.03 52.31 ± 1.58 18.33 ± 0.14 14.14 ± 0.02

1.27 ± 0.03 54.89 ± 2.36 15.24 ± 0.69 14.12 ± 0.11

4.17 ± 0.03 52.53 ± 1.51 22.82 ± 1.78 14.28 ± 0.32

45.32 56.16 14.92 18.22

Figure 4. Stability of GO, rGO, and rGO-Arg over time in (A) DMEM, (B) FBS, and (C) PBS. The time frames mentioned above arrows and in parentheses represent the time interval to capture individual photo.

their report, the absorption cross section of graphene at 660 nm wavelength is 36.00 mL/mg·cm.49 While in another study, Khan et al. showed that at higher concentration, absorption cross section of graphene varied from 24.60 to 36.20 mL/mg·cm.50,70 Herein, absorption cross section of GO and rGO-Arg are calculated at

Although numerous studies investigated the application of graphene oxide and its composites as a nanoprobe, the absorption cross section of graphene oxide was unclear. However, Min Yi et al. obtained absorption cross section of graphene at 660 nm at lower concentration based on the Lambert−Beer law. On the basis of 32614


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Figure 5. Temperature rise after 5 min irradiation with various incident laser (808 nm) fluences (0.3, 0.5, 1, 1.7 W.cm−2) at various concentration of (A) rGO-Arg (25, 50, 100, 200 μg/mL), (B) rGO (25, 50, 100, 200 μg/mL), and (C) GO (50, 100, 200, 400 μg/mL). Temporal profile of temperature response (plotted in black) and estimated cell damage (%) (plotted in red) of (D) rGO-Arg and (E) GO to NIR laser excitation (1.7 W.cm−2).

808 nm. The results suggest that the absorption cross section of GO is lower than that of graphene. The result may be due to the isolation of sp2 bonding in a GO structure in comparison to graphene.27 A consequence of reducing graphene oxide in rGO-Arg is that, new sp2 bonding is created through removal of oxygen, so the absorption cross section increases as a result of restoration of a π network.27,71 On the basis of the above results, for equivalent concentration of GO and rGO-Arg, the tendency of rGO-Arg to absorb light at 808 nm laser is 3.2 times stronger than GO. This result suggests rGO-Arg may be an alternative to GO for photothermal therapy. Stability of rGO and rGO-Arg. For biomedical applications, rGO-Arg should be stable in both a buffer solution and in the various physiological environments. The stability of rGO and rGO-Arg suspensions were estimated by assuming a spherical shape for GO and rGO-Arg (instead of 2D structure) and measuring

average hydrodynamic diameter and % stability of GO, rGO, and rGO-Arg at each time interval (Figure 4). The stability of GO in PBS decreased due to the aggregation of the larger sized sheets. As the smaller size of GO is more hydrophilic than the larger particles, this can result in higher charge density of the ionized oxygen functional groups on GO sheets.72,73 Moreover, the GO stability in FBS and DMEM is slightly less than PBS. This might be due to the electrostatic and salt effect to brake the equilibrium state of GO in suspension.74 In all media, the stability of rGO decreased by successful reduction of the functional group. However, rGO-Arg with the same concentration of rGO exhibited an excellent stability over 24 h with 92 ± 2%,78 ± 3%, and 75 ± 4% stability in PBS, DMEM and FBS, respectively. This phenomenon implied that the conjugation of hydrophilic Arg to rGO provides long-term stability by effectively preventing agglomeration of rGO-Arg in physiological fluids containing 32615


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Figure 6. Cell viability of MD-MB-231 cells with schematics depicting a petridish with the cell line. The GO/rGO-Arg nanoparticles are added to this petridish and incubated for 24 h, prior to laser irradiation. (A) Cell viabilities of MD-MB-231 cells after incubation with GO and rGO-Arg for 24 h, (B) cell viabilities of MD-MB-231 cell lines after NIR pulsed laser (1.7 W·cm−2) irradiation in the presence of 100 and 400 μg/mL of rGO-Arg and GO, respectively, (C) transmittance and fluorescent microscope images of live/dead assay of MD-MB-231 cell line in the presence of 100 μg/mL of rGO-Arg. Green and red channel of fluorescent microscope shows live cells and dead cells respectively (scale bar is 400 μm).

NIR laser irradiation. Comparing the rising temperature profile for rGO-Arg (Figure 5A), rGO (Figure 5B), and GO (Figure 5C), rGO-Arg exhibits substantially better photothermal energy conversion than GO and rGO. For example, after 5 min NIR pulsed laser irradiation with a power density of 1.7 W.cm−2, in a 400 μg/mL GO suspension temperature increased to 46.2 ± 2.2 °C, while in the suspension of 100 μg/mL of rGO-Arg the temperature increased to 53.83 ± 2.6 °C and for the suspension of 100 μg/mL of rGO the temperature increased to 42.7 ± 1 °C. However, as a control, water exhibited a small increase in the temperature (6.81 ± 1.3 °C) after 5 min pulsed laser irradiation with a power density of 1.7 W·cm−2. As the results suggest, modification of rGO with Arg leads to 30% increase in the photothermal temperature increase. While at the same condition Song et al. shows that NIR irradiation leads to a 20 and 30 °C increase in the temperature of 100 μg/mL of rGO and gold nanorods, respectively. In other words, utilizing gold nanorods

various protein, organic molecules and high salt concentration. Therefore, rGO-Arg possesses good stability for photothermal therapy. Characterization and Optimization of Ex Vivo Photothermal Study. Increased optical absorption cross section of rGO-Arg may lead to a strong photothermal effect. We explored the photothermal performance of GO, rGO, and rGO-Arg at various concentration of GO (50, 100, 200, 400 μg/mL), rGO (25, 50, 100, 200 μg/mL), and rGO-Arg (25, 50, 100, 200 μg/mL) suspensions, and temperature response was measured under NIR pulsed laser irradiation (with pulse duration of 250 ms and pulse repetition rate of 2 Hz) at various incident fluences (1.7, 1, 0.5, 0.3 W·cm−2) (Figure 5A−C). In all the samples temperature increased as incidence fluence increased. As shown in Figure 5A−C, all the samples demonstrated a concentration dependent (Figure 5A−C) and time-dependent (Figure 5D and E) temperature increase in response to pulsed 32616


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250 ms and pulse repetition rate of 2 Hz, 1.7 W·cm−2) was recorded using IR camera (Figure 5D and E). The Arrhenius rate damage equation (eq 5) was computed in each time interval to evaluate Ω (see Figure 5D and E (on the right y-access)). The estimated value for ΔE and A, according to Gaylor’s model parameters, were 2.4 × 105 J·mol−1 and 2.9 × 1037 s−1, respectively.78 Since temperature of suspensions were not increased sufficiently for protein denaturation at lower GO concentrations (50 and 100 μg·mL−1) and rGO-Arg (25 and 50 μg·mL−1), the Arrhenius equation was not evaluated for these cases. As shown in Figure 5D and E (red plot), the percentage of cell damage after 2 min laser irradiation for GO is 4.8% and 13.60% for a concentration of 200 and 400 μg·mL−1, respectively. While in the rGO-Arg suspensions, 2 min laser irradiation caused 46.23% and 98.49% cell damage for 100 and 200 μg/mL concentrations, respectively. By increasing the irradiation time to 4 min, the percentage of cell damage would reach 100% for both rGO-Arg concentrations. For further evaluation, the percentage of cell damage following NIR pulsed laser irradiation was measured using MTT as described in the next section. In Vitro Toxicity and Photothermal Study. To further investigate whether rGO-Arg can be used to target cells in biomedical applications, cell toxicity on MD-MB-231 cell line was evaluated by an MTT assay. As shown in Figure 6A, cells treated with rGO-Arg exhibited higher cellular viability (above 90.2 ± 3.6%) than that cells were treated with GO (above 72.1 ± 6%). Thus, the results suggest that rGO-Arg has better biocompatibility in comparison to GO. Higher cellular toxicity with GO may be due to a higher negative charge property, while conjugation of Arg peptide on the surface of rGO-Arg increased the viability even at higher concentration of rGO-Arg. Similar results were reported by Singh et al. Their studies demonstrated that the amine modified GO nanosheet does not have any longterm toxicity on a human monocyte cell line.30 Because of the concentration dependent toxicity (Figure 6A) and the percentage of cell damage results (Figure 5D and E), in vitro potential of photothermal therapy with rGO-Arg (100 μg·mL−1) and GO (400 μg·mL−1) was evaluated by MTT assays after 2 and 4 min NIR pulsed laser irradiation (Figure 6B). As the results suggest, the toxicity of GO and rGO-Arg were considerably increased with increasing NIR light exposure time. Also, the data suggests that after 4 min NIR laser irradiation, almost a 100 ± 3.1% and 75.4 ± 2.8% cells are dead in the groups treated with GO and rGO-Arg, respectively. The same concentration of rGO-Arg and GO without NIR light irradiation were used as a control. Also, MTT assays revealed negligible cell toxicity after 2 and 4 min NIR laser irradiation, with no GO and rGO-Arg in the cell culture medium. Even after a 4× increase in GO concentration, the amount of cell damage was still higher in the groups treated with rGO-Arg. Moreover, the cell lines treated with a 100 μg.ml−1 of rGO-Arg suspension showed 40 ± 4.7% cell death after 2 min NIR laser irradiation, which was close to that predicted by the Arrhenius equation using the parameters given by Gaylor (Figure 5 D). Similarly, in the groups treated with 400 μg·mL−1 GO suspensions, after 2 min NIR laser irradiation, the toxicity level was almost 12% less than that predicted by the Arrhenius equation (Figure 5 E). The results suggest that the Arrhenius equation can reasonably predict MD-MB-231 cell damage in response to 808 nm pulsed laser irradiation. As a supplementary examination of the photothermal therapy potential of rGO-Arg, we conducted a live/dead assay using calcein AM/EthD-1 (Figure 6C). Under inverted fluorescence

Figure 7. Two-photon microscopy images of MD-MB-231 after being exposed to 20 μg/mL of rGO-Arg and GO for 24 h. (A) Nucleus and cytoplasm, (B) nanoparticles, (C) cytoplasm and nanoparticles, and (D) merged image. Red, green, and blue colors represent nanoparticles, cytoplasm, and nucleus, respectively. Yellow regions contain both nanoparticles and cytoplasm. (Scale bar is 100 μm.).

instead of rGO gave a 34% increase in the photothermal effect.15 Gold nanorods are one of the more attractive nanoparticles for photothermal therapy but face some obstacles for clinical application such as high toxicity due to the presence of CTAB, and a low yield of reaction.13−15 Photothermal properties of rGO-Arg are comparative to gold nanorods and may be considered as an alternative candidate for photothermal therapy. To find the appropriate rGO-Arg and GO concentration and laser irradiation time duration for photothermal therapy, the percentage of cellular death resulting from thermal damage was calculated using the Arrhenius first order kinetics. Mathematically, the Arrhenius rate damage equation is based on the assumption that the rate of thermal damage is proportional to exp (−Ea/RT). The occurrence of coagulation injury at time τ is modeled as a dimensionless damage parameter Ω Ω(r , z , τ ) =

∫0

τ

Ae−[ΔE / RT(r , z , t )]dt

(5)

where τ is the time of interest (s), A is the pre-exponential frequency factor (s−1), ΔE is activation energy for irreversible damage reaction (J mol−1), R is the ideal gas constant (J/K mol), and T is the time-dependent absolute temperature (K).75−77 Ω is the damage parameter, where exp(−Ω) represents the fraction of undamaged tissue. A and ΔE are usually estimated experimentally and measurable for specific thermal injury. To predict the percentage of cell death from thermal injury, a temperature− time plot of various GO concentrations (50, 100, 200, 400 μg·mL−1) and rGO-Arg concentrations (25, 50, 100, 200 μg·mL−1) over 5 min of NIR pulsed laser irradiation (with pulse duration of 32617


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ACS Applied Materials & Interfaces microscope observation, dead cells exhibit red fluorescence (see red channel) while live cells exhibit green fluorescence (see green channels). Before photothermal therapy most cells were viable (Figure 6C) but after 4 min pulsed laser irradiation (808 nm, 1.7 W·cm−2) almost all cells were dead. Cell toxicity and live/dead assay results show the efficient potential of rGO-Arg to kill MD-MB-231 breast cancer cell lines in response to photothermal therapy. Multiphoton Laser Scanning Microscopy. Motivated by high absorption cross section and effective photothermal heating, the cellular internalization into MD-MB-231 cells of rGO-Arg and GO was examined (Figure 7). The red fluorescence intensity of rhodamine conjugated rGO-Arg was stronger than that of rhodamine conjugated GO, implying higher intracellular uptake capabilities of rGO-Arg. Kakoki et al. reported that Arg could be taken up by cells via y+ or y+L transporter systems.79,80 The results suggest that the ability of Arg to bind to its transporter was not affected by the covalent amine binding. On the basis of the data, rGO-Arg with high potential of cellular uptake could be considered as a good candidate as a photoabsorber.

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CONCLUSION In summary, rGO-Arg nanoprobes were successfully constructed via a facile and high efficient method. The yield of conjugation of Arg to rGO was 11.2 ± 1.5%. As successful fabrication of rGO-Arg requires administrating a protocol with high yield of reaction, we studied the effect of different functional groups on the yield of reaction. We found that the yield of reaction for fabricating rGO-Arg nanoparticles is close to the yield of reaction of epoxy-amine, which was reported as a click reaction. The rGO-Arg exhibited a very good colloidal stability with significant improvement in a wide range of solvents over GO and rGO owing to the small size range and effective conjugation of Arg. Moreover, the UV/vis results demonstrated that rGO-Arg has a stronger NIR absorption over GO and rGO with a significant photothermal efficiency, for an equal concentration of samples, indicating that rGO-Arg could be a better transducer for photothermal therapy. To the best of our knowledge, there is no reports on application of rGO-Arg as a viable transducer for photothermal therapy of cancer. The feasibility of rGO-Arg as a transducer was seconded by high in vitro photothermal cell toxicity and high cellular uptake over GO. To the best of our knowledge, we were the first to study one of the important aspect of light-matter interactions of GO and rGO-Arg, absorption cross-section at 808 nm. An experimental procedure, a modification to a standard therapeutical setup with a modulated laser light to minimize artifacts introduced by lateral diffusion, the material concentration chosen to match the scattering profile of an optical standard and a Fourier analysis to minimize laser intensity noise during computation, was utilized to compute the absorption cross-section. The results show that the absorption cross-section of rGO-Arg is 53.52 ± 7.13 mL/mg·cm, and it is 3.2 times higher than GO. Our results suggest that these integrated functionalities of rGO-Arg make it a promising candidate agent for photothermal destruction of cancer cells.

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schematic of the experimental setup with a diode laser modulated by a function generator to generate laser pulses and frame acquired by an IR camera with synchronized frame acquisition, characterization of black absorber, schematic of the setup to measure light scattering from black absorber, power of backscattered light of reflective standard and black absorber measured by power meter (steady state data acquired over 7 s of measurement), size and zeta potential evaluation of GO and rGO-Arg 8, size of GO before and after of 2 h probe sonication and rGOArg, and zeta potential of GO and rGO-Arg 9, particle size distribution of graphene before and after size reductionand before and after 2 h probe sonication, C1s core level spectra of GO, rGO, and rGO-Arg, calculation of the mass ratio of the component in rGO-Arg suspension, preparation of carboxylated graphene oxide (GO-COOH) and epoxylated graphene oxide (GO-O), schematic of GO-O and GO-COOH formation, FTIR spectra of GO, GO-O, and GO-COOH, comparison of Arg mass ratio in rGO-Arg suspension after conjugation of Arg to GO, GO-O, GO-COOH, calculation of cross section absorption, temporal profile of temperature versus time of GO and rGO-Arg, and filtering and FFT results (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +1-512-409-5874 (J.M.). *E-mail: [email protected]. Phone:+1-512-471-1332 (T.E.M.). ORCID

Hugh Smyth: 0000-0002-6582-5869 Javad Mohammadi: 0000-0001-9151-5242 Author Contributions

The manuscript was written through contributions of all authors. Notes

The authors declare no competing financial interest.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b11291. Calculation of heat loss through lateral diffusivity, instrumentation to measure optical absorption cross section, 32618


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Evaluation of the Photothermal Properties of a Reduced Graphene

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