Biomaterials 35 (2014) 4499e4507
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Magnetic and fluorescent graphene for dual modal imaging and single light induced photothermal and photodynamic therapy of cancer cells Ganesh Gollavelli, Yong-Chien Ling* Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 January 2014 Accepted 7 February 2014 Available online 3 March 2014
Developing a simple and cost-effective strategy to diagnose and treat cancer with single and minimal dosage through noninvasive strategies are highly challenging. To make the theranostic strategy effective, single light induced photothermal and photodynamic reagent with dual modal imaging capability is highly desired. Herein, a simple non-covalent approach was adopted to immobilize hydrophobic silicon napthalocyanine bis (trihexylsilyloxide) (SiNc4) photosensitizer onto water dispersible magnetic and fluorescent graphene (MFG) via pep stacking to yield MFGeSiNc4 functioned as a theranostic nanocarrier. Taking the advantage of broad near infra-red absorption (600e1200 nm) by graphene, photosensitizer of any wavelength within this range will facilitate the single light induced phototherapy. Phosphorescence spectra, singlet oxygen sensor green (SOSG) experiments, and 1,3-diphenyl isobenzofuran quenching studies confirm the generation of singlet 1O2 upon photoirradiation. Confocal microscopic images reveal successful internalization of MFGeSiNc4 in HeLa cells; whereas T2-weighted magnetic resonance images of MFG reveal a significant concentration dependent darkening effect. In vitro photodynamic/photothermal therapeutic studies on HeLa cells have demonstrated that the killing efficacy of MFGeSiNc4 using a single light source is w97.9%, presumably owing to the combined effects of generating reactive oxygen species, local heating, and induction of apoptosis. The developed MFGeSiNc4 may thus be utilized as a potential theranostic nanocarrier for dual modal imaging and phototherapy of cancer cells with single light source for time and cost effective treatments with a minimal therapy dose. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Graphene Fluorescence imaging Magnetic resonance imaging Photodynamic therapy Photothermal therapy
1. Introduction Photothermal (PTT) and photodynamic (PDT) therapies are considered to be emerging non-invasive modalities for the treatment of various cancers. Ideally, PTT reagents absorb the excitation light, leading to hyperthermia (>43 C) and causing photothermal ablation of cancer cells. PDT involves photosensitizers (PSs) to absorb light and transfers energy to surrounding tissue oxygen. The generation of highly reactive oxygen species (ROS) such as singlet oxygen and free radicals can oxidize cellular and sub-cellular compartments including plasma membrane, mitochondria, lysosomal, and nuclear membrane, finally leading to irreversible damage to tumor cells [1,2]. PDT has shown significant efficacy with PS such as Photofrin, a clinically approved first generation photodrug, which was normally activated at 630 nm to take maximum advantage of light penetration into biological tissues (1e3 nm), even though its absorbance is relatively low at this wavelength [3]. * Corresponding author. E-mail address: [email protected] (Y.-C. Ling). http://dx.doi.org/10.1016/j.biomaterials.2014.02.011 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
The drawbacks of poor penetration depths, excitation at short wavelengths (630 nm), and variation in chemical structure has opened a door in search of alternative PSs. The second generation PSs such as napthalocyanines are activatable at near infra-red (NIR) region (700e850 nm) with penetration depths of almost double than that of Photofrin [3]. Napthalocyanines are very good light absorbers in the first biological window (650e900 nm), where most of the molecules such as hemoglobin (Hb), oxyhemoglobin (HbO2), and several biological pigments do not absorb in this region [4]. In this perspective, napthalocyanines could be attractive as NIR excitable PSs (w775 nm), which is highly important for an effective phototherapeutic treatment [5]. The inherent hydrophobic nature of PSs limits their clinical usage. To overcome this barrier, several delivery approaches capable of stabilizing hydrophobic PDT drugs in aqueous solution including liposomes, polymeric micelles, gold, silica nanoparticles (NPs), and carbon dots have been demonstrated [6e11]. Among them Au nanorods (NRs) and NPs [6] as well as Au nanocages [12] have been extensively used to carry PSs, owing to their inherent photothermal capabilities, and thereby producing synergistic PTT and PDT (PTT/PDT) effects. In order to observe the
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synergistic therapeutic effects, two different light sources have to be adopted to excite PTT carrier and PS [13,14]. To this end graphene oxide (GO), folic acid-GO, and PEG functionalized GO loaded with hypocrellin A [15] and Chlorin e6 [16,17] were developed via pep stacking to perform PDT. The PEG functionalized GOeCe6 composite was excited using 660 and 808 nm lasers to observe the synergistic PTT/PDT therapeutic effects [16]. The improved outcome achieved by multi-laser treatments is very expensive and also prolongs the therapeutic time. Therefore, designing a phototherapeutic strategy excitable at a single wavelength is highly desired. To perform simultaneous PTT/PDT therapy using a single light source, following alternatives can be considered, (1) Use of two photon femtosecond laser systems to generate heat and singlet oxygen, (2) Design a PTT reagent in accordance with the excitation wavelength of PS, and (3) Selection of an appropriate PS to fit the absorption of PTT reagent. To this end, alternative 1 was successfully demonstrated by Gao et al., in which hypocrellin-loaded gold nanocages were used for PTT/PDT by two photon femtosecond laser excitation, in which simultaneous excitation of nanomaterial (NM) and PS is possible [12]. Alternative 2 was effectively achieved by Wang et al., in which the localized surface plasmon resonance band of gold nanostars were tuned to exactly match with the excitation wavelength of PS (Chlorin e6) [18]. However, the implementation of alternative 3 using a PTT reagent suitable for various PSs to achieve single light induced PTT/PDT is yet to come. Graphene is an emerging member in carbon family, owing to its extraordinary thermal and optoelectronic properties [19,20]. Its two-dimensional structure, high surface area, easy surface functionality [21], biocompatibility [22e24], high thermal stability, lower cost than Au NRs and carbon nanotubes (CNTs), and enhanced NIR absorption capability in first and second biological window (650e950 nm and 1000e1350 nm) make them as an ideal theranostic platforms for future nanomedicine [25,26]. Owing to its enhanced NIR absorption (w650e1300 nm), graphene could be a versatile platform to immobilize PSs with different excitation wavelengths, thereby achieving improved synergistic PTT/PDT effects. This type of strategy is advantageous by minimizing therapy time and avoiding the utilization of two laser systems. However, poor water dispersibility of graphene severely limits its biological applications. To overcome this problem, graphene was functionalized with various hydrophilic groups to enhance its water dispersibility [27e30]. However, it is necessary to have a balance between sp2 and sp3 structure of GO to impart water dispersibility as well as to hold hydrophobic drugs. Apart from simple noninvasive therapeutic strategy, it is also highly desired to possess multi-diagnostic capabilities, such as, fluorescence, magnetic resonance imaging (MRI), computed tomography, and positron emission tomography, which could offer cross validation of the results obtained from different methods, and also provide accurate and realiable disease detection ability to facilitate imaging-guided therapy [31]. This kind of theranostic nanoplatform will minimize the therapy time, cost, and toxicity associated by using different nanomaterials separately [32e35]. Developing cost effective theranostic NMs to treat cancer with minimal doses by means of non-invasive techniques is highly challenging. Here we report water dispersible magnetic and fluorescent graphene (MFG) functionalized with SiNc4 for dual modal imaging and single light induced PTT/PDT. We believe that MFGeSiNc4 would be advantageous in terms of simple preparation and costeffectiveness by using graphene. 2. Methods and materials 2.1. Synthesis of graphene oxide (GO) The GO was synthesized following modified Hummers process with slight modifications, by using pristine graphite flakes as starting material. The graphite
flakes (1 g) were added to 98% H2SO4 (23 mL) and stirred for w8 h. KMnO4 (3 g) was slowly added to the above mixture, while maintaining the temperature 0.2 mM. Indeed, Fig. 4A demonstrates that under photoirradiation, MFGeSiNc4 exhibited dramatic cytotoxic effects on cell viability. These results were in agreement with the CLSM image results (Fig. 3A), where most of the MFGeSiNc4 was uptaken by the cells, leading to induce toxicity upon photoirradiation. The decrease in cell viability by MFGeSiNc4 was mostly due to the toxicity induced by 1O2 and other ROS. The inherent photothermal capability of MFG, owing to its enhanced pelectrons and superior NIR absorption, also contributed to some extent of decrease in cell viability (Fig. 4B). However, the PTT effect might be less predominant at low power doses (300 mW/cm2) [16].
In general, PDT is more predominant at low power doses to generate 1O2 continuously. In contrast, at high power doses, rapid consumption of surrounding tissue oxygen can inhibit the efficiency of 1O2 formation, and also results in severe photobleaching of PS. Alternatively, PTT requires extremely high power doses in order to thermally ablate the tumors. The carbon-based NMs and gold nanostructures both bearing superior light absorbing capabilities have been widely explored for PTT. Often, GO has been used as a PTT reagent with high power doses in order to compensate its poor absorption in the NIR region. Later on, GO has been conjugated with PS, and two different light sources were adopted, a high power dose (4 W/cm2) to excite PTT reagent; and a low power dose (50 mW/cm2) to excite the PS [43]. On the other hand, gold nanostructures exhibit poor photostability as it endured morphological changes at an extent of local heating by pulsed lasers [44e46]. Moreover, PS functionalized with gold nanostructures requires two different light sources to generate heat and 1O2 [13]. On the other hand, graphene-based NMs can overcome these drawbacks and serve as excellent and cost-effective PTT reagents [25,47,48]. Most importantly, immobilizing SiNc4 onto graphene has facilitated the utilization of a single light source to simultaneously generate PTT/ PDT effects at the same time, owing to the absorption at w775 nm by both graphene and SiNc4 in MFGeSiNc4. The advantage of single light induced therapy is, while irradiation, the 1O2 (PDT) will destruct the cellular components such as membrane lipids,
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Fig. 4. (A) Comparative cell viabilities by MTT assay of HeLa cells treated with different concentrations of SiNc4/MFGeSiNc4 under dark/photoirradiation conditions. (B) Cell viabilities of MFG treated HeLa cells under dark/photo conditions.
proteins and DNA by oxidation pathway. However, there is an every chance of reoccurrence of tumor by partially oxidized DNA fragments and subsequent mutations. This kind of reoccurrence (mutations/DNA base excision repair) will be avoided by subsequent burning during PTT without any delay by using single light source, which might help to cause irrecoverable damage to the tumor cells [49]. Furthermore, utilization of a single light source to simultaneously generate PTT/PDT effects would reduce the cost, time, and excessive unwanted burning caused by different light sources. The highest cell deaths w97.9% (Fig. 4A) were obtained from HeLa cells treated with optimal SiNc4 solution (0.10 mM, corresponding to 100 mg/mL of MFGeSiNc4) imparted by PTT/PDT effects; among
which w64.7% from PDT and w33.2% from PTT effects (see Fig. 4B and also Table S1 for complete details). In addition to the HeLa cells, we have also performed the dark and phototoxicity experiments by using MTT assay in human fibroblast normal cells (MRC-5) and phagocytic cells (RAW-264.7 macrophages) with free SiNc4 and MFGeSiNc4 (Fig. S3). Upon comparison with HeLa cancer cells, the photo induced cellular deaths obtained from MFGeSiNc4 is lower for human fibroblast normal cells (70%) and macrophages (75%) which are similar to literature reports [50e52]. The reason for lower cellular deaths in human normal cells and phagocytic cells might be due to its better immune response to external heat as well as ROS, than HeLa cancer cells.
Fig. 5. (A) Intracellular ROS levels generated by SiNc4/MFGeSiNc4 solutions along with H2O2 as positive control. (B) NaN3 quenching of intracellular ROS levels by MFGeSiNc4 solutions. ROS experiments were carried out by using flow cytometry and the results were expressed as mean fluorescence intensity of DCF. (C) Percentage of apoptotic and necrotic HeLa cells induced by SiNc4/MFGeSiNc4 solutions under dark/photo conditions. (D) MMP in HeLa cells treated with SiNc4/MFGeSiNc4 solutions expressed as mean green to red fluorescence intensity ratio (FTTC/PE).
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Fig. 6. HSP 70 expression levels in HeLa cells treated with MFGeSiNc4 solutions under dark/photo conditions.
3.5. Intracellular effects and possible cell death pathway investigation To look at a deeper insight into the cellular deaths by MFGeSiNc4, several pathways were investigated in order to understand the mechanism. MFGeSiNc4 was internalized in HeLa cells first. Upon photoirradiation, a series of intracellular events such as generation of heat, 1O2, and increased intracellular ROS levels were triggered. The plausible causes include, inadequate buffering of tissue or cell antioxidant defense system and interactions with the proteins/DNA/ lipids, finally leading to cellular/tissue death [53]. To examine the oxidative stress induced by MFGeSiNc4, we monitored the intracellular ROS levels by flow cytometry using a reductive reagent, 20 ,70 -dichlorofluorescein diacetate (H2DCF-DA), which entrapped the intracellular ROS and subsequently being oxidized to fluorescent 20 ,70 -dichloro fluorescien (DCF). The intracellular ROS levels in HeLa cells treated with SiNc4/MFGeSiNc4 solutions under dark and photoirradiation conditions (Fig. 5A) reveal that the generated ROS levels apparently exhibited concentration dependent behavior under photoirradiation condition. The ROS levels obtained under photoirradiation conditions were w2 folds higher than that of dark conditions. To ensure that higher ROS levels were induced by MFGe SiNc4 internalized HeLa cells, we performed quenching experiments by pre-treating the HeLa cells with NaN3 solution (50 mM) before photoirradiation. The results in (Fig. 5B) reveal that under photoirradiation the intracellular ROS levels decreased after NaN3 pretreatment, demonstrating that 1O2 was indeed the key ROS responsible for the attributed cellular death by MFGeSiNc4. The apoptosis and necrosis events are considered as the most profound cause of cellular deaths in the therapeutic processes. The percentage of apoptotic and necrotic cells induced by SiNc4/MFGe SiNc4 (Fig. 5C) showed no obvious difference under dark conditions; whereas distinct increase was observed upon photoirradiation, revealing that induced ROS levels might lead to cellular death during photoirradiation and subsequently resulted in the destruction of cancer cells. Mitochondria is the power house of cells and plays an indispensable role in cellular death by regulating the apoptotic and necrotic pathways [1]. To evaluate the function of mitochondria in the presence of MFGeSiNc4 under dark/photoirradiation conditions, we have performed in vitro mitochondrial membrane potential (MMP) assay using JC-1 dye, which normally exists in either aggregate or monomer form. The permeabilization of JC-1 dye becomes much faster in healthy cells and readily enters into the mitochondria to display red fluorescence (PE channel) in aggregate form. For abnormal cells, only green color (FITC channel) in monomer form
can be visualized. The MMP expressed as the green to red fluorescence intensity ratio (FTTC/PE) (Fig. 5D) reveals increased FITC/PE (decreased MMP) with increased MFGeSiNc4 concentration under photoirradiation condition compared to that under dark. This implies that 1O2 generated by photoexcited MFGeSiNc4 can increase ROS levels, which leads to subsequent activation of apoptotic cascade, and finally directs to decrease in MMP. The decrease in MMP has been observed for pristine graphene treated RAW 264.7 cells and singled-wall CNT (SWCNT) treated EMT6 cells [54,55]. In contrast, increase in MMP was observed for GO-HA treated HeLa cells [15]. The photothermal property of MFGeSiNc4 partially contributes to the decreased cell viability under low light doses. During PTT/PDT, the photo-activated drug triggers local heating and thereby the cells may over-express the molecular chaperones of heat shock protein (HSP) family [56]. The HSP 70 expression levels in HeLa cells treated with different concentrations of MFGeSiNc4 under dark/photo conditions (Fig. 6) exhibited concentration dependent behavior and reached a threshold at w0.1 mM; whereas no any detectable expression level was observed under dark condition. This finding justifies that partial cell death observed in MTT assay from MFG (Fig. 4B) is due to the photothermal heating triggered by MFGeSiNc4 upon photoirradiation. The multi-walled CNTs (MWCNTs) treated tumors have shown increased HSP expression levels upon NIR laser irradiation owing to the elevation of sub-lethal temperature [57]. Western blot analysis of SWCNT-polyethylene glycol has also shown that HSP 70 expression levels were comparatively higher in the presence of light than in dark and SWCNTs treated cells alone [54].
4. Conclusions We have developed a theranostic platform based on noncovalent immobilization of SiNc4 onto MFG surface to form MFGeSiNc4 bearing good water dispersibility, fluorescence, and magnetic properties. The MFG serves as an excellent T2-weighted MRI contrast reagent owing to its superparamagnetic nature. The SiNc4 on MFG surface were readily activated by NIR light and furnished MFGeSiNc4 to offer 1O2 mediated PDT along with intrinsic photothermal effects for PTT. The broad NIR absorption (600e1200 nm) capability of graphene could facilitate the immobilization of diversified PSs in order to achieve PTT/PDT effects by single light source. Phosphorescence measurements, SOSG experiments, and DPBF quenching studies confirm the generation of 1O2 by MFGeSiNc4. Higher levels of intracellular ROS and NaN3 quenching experiments demonstrate the indispensable role of 1O2 in cellular deaths. Other biological cascades such as necrosis and over-expression of HSP 70 by photothermal effects might also contribute to cellular deaths. Overall, our in vitro results have demonstrated the simultaneous dual modal imaging and PTT/PDT capabilities of MFGeSiNc4 with appreciable cell killing efficacy of w97.9%. Among which, w64.7% was by PDT; whereas w33.2% was by PTT. The discovery of MFGeSiNc4 opens a new therapeutic window for the treatment of cancer and stands out as a cost effective theranostic reagent in the field of nanomedicine.
Acknowledgments We gratefully acknowledge the National Science Council of Taiwan (NSC 101-2627-M-007-005 and NSC101-2113-M-007-006MY3) and National Tsing Hua University (99N2454E1) for generous funding support. Constructive and fruitful discussions with Prof. Lih-Yuan Lin from the Department of Life Science, National Tsing Hua University was also gratefully acknowledged.
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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.02.011.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Magnetic and fluorescent graphene for dual modal imaging and single light induced photothermal and photodynamic therapy of cancer cells Ganesh Gollavelli, Yong-Chien Ling* Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 January 2014 Accepted 7 February 2014 Available online 3 March 2014
Developing a simple and cost-effective strategy to diagnose and treat cancer with single and minimal dosage through noninvasive strategies are highly challenging. To make the theranostic strategy effective, single light induced photothermal and photodynamic reagent with dual modal imaging capability is highly desired. Herein, a simple non-covalent approach was adopted to immobilize hydrophobic silicon napthalocyanine bis (trihexylsilyloxide) (SiNc4) photosensitizer onto water dispersible magnetic and fluorescent graphene (MFG) via pep stacking to yield MFGeSiNc4 functioned as a theranostic nanocarrier. Taking the advantage of broad near infra-red absorption (600e1200 nm) by graphene, photosensitizer of any wavelength within this range will facilitate the single light induced phototherapy. Phosphorescence spectra, singlet oxygen sensor green (SOSG) experiments, and 1,3-diphenyl isobenzofuran quenching studies confirm the generation of singlet 1O2 upon photoirradiation. Confocal microscopic images reveal successful internalization of MFGeSiNc4 in HeLa cells; whereas T2-weighted magnetic resonance images of MFG reveal a significant concentration dependent darkening effect. In vitro photodynamic/photothermal therapeutic studies on HeLa cells have demonstrated that the killing efficacy of MFGeSiNc4 using a single light source is w97.9%, presumably owing to the combined effects of generating reactive oxygen species, local heating, and induction of apoptosis. The developed MFGeSiNc4 may thus be utilized as a potential theranostic nanocarrier for dual modal imaging and phototherapy of cancer cells with single light source for time and cost effective treatments with a minimal therapy dose. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Graphene Fluorescence imaging Magnetic resonance imaging Photodynamic therapy Photothermal therapy
1. Introduction Photothermal (PTT) and photodynamic (PDT) therapies are considered to be emerging non-invasive modalities for the treatment of various cancers. Ideally, PTT reagents absorb the excitation light, leading to hyperthermia (>43 C) and causing photothermal ablation of cancer cells. PDT involves photosensitizers (PSs) to absorb light and transfers energy to surrounding tissue oxygen. The generation of highly reactive oxygen species (ROS) such as singlet oxygen and free radicals can oxidize cellular and sub-cellular compartments including plasma membrane, mitochondria, lysosomal, and nuclear membrane, finally leading to irreversible damage to tumor cells [1,2]. PDT has shown significant efficacy with PS such as Photofrin, a clinically approved first generation photodrug, which was normally activated at 630 nm to take maximum advantage of light penetration into biological tissues (1e3 nm), even though its absorbance is relatively low at this wavelength [3]. * Corresponding author. E-mail address: [email protected] (Y.-C. Ling). http://dx.doi.org/10.1016/j.biomaterials.2014.02.011 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
The drawbacks of poor penetration depths, excitation at short wavelengths (630 nm), and variation in chemical structure has opened a door in search of alternative PSs. The second generation PSs such as napthalocyanines are activatable at near infra-red (NIR) region (700e850 nm) with penetration depths of almost double than that of Photofrin [3]. Napthalocyanines are very good light absorbers in the first biological window (650e900 nm), where most of the molecules such as hemoglobin (Hb), oxyhemoglobin (HbO2), and several biological pigments do not absorb in this region [4]. In this perspective, napthalocyanines could be attractive as NIR excitable PSs (w775 nm), which is highly important for an effective phototherapeutic treatment [5]. The inherent hydrophobic nature of PSs limits their clinical usage. To overcome this barrier, several delivery approaches capable of stabilizing hydrophobic PDT drugs in aqueous solution including liposomes, polymeric micelles, gold, silica nanoparticles (NPs), and carbon dots have been demonstrated [6e11]. Among them Au nanorods (NRs) and NPs [6] as well as Au nanocages [12] have been extensively used to carry PSs, owing to their inherent photothermal capabilities, and thereby producing synergistic PTT and PDT (PTT/PDT) effects. In order to observe the
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synergistic therapeutic effects, two different light sources have to be adopted to excite PTT carrier and PS [13,14]. To this end graphene oxide (GO), folic acid-GO, and PEG functionalized GO loaded with hypocrellin A [15] and Chlorin e6 [16,17] were developed via pep stacking to perform PDT. The PEG functionalized GOeCe6 composite was excited using 660 and 808 nm lasers to observe the synergistic PTT/PDT therapeutic effects [16]. The improved outcome achieved by multi-laser treatments is very expensive and also prolongs the therapeutic time. Therefore, designing a phototherapeutic strategy excitable at a single wavelength is highly desired. To perform simultaneous PTT/PDT therapy using a single light source, following alternatives can be considered, (1) Use of two photon femtosecond laser systems to generate heat and singlet oxygen, (2) Design a PTT reagent in accordance with the excitation wavelength of PS, and (3) Selection of an appropriate PS to fit the absorption of PTT reagent. To this end, alternative 1 was successfully demonstrated by Gao et al., in which hypocrellin-loaded gold nanocages were used for PTT/PDT by two photon femtosecond laser excitation, in which simultaneous excitation of nanomaterial (NM) and PS is possible [12]. Alternative 2 was effectively achieved by Wang et al., in which the localized surface plasmon resonance band of gold nanostars were tuned to exactly match with the excitation wavelength of PS (Chlorin e6) [18]. However, the implementation of alternative 3 using a PTT reagent suitable for various PSs to achieve single light induced PTT/PDT is yet to come. Graphene is an emerging member in carbon family, owing to its extraordinary thermal and optoelectronic properties [19,20]. Its two-dimensional structure, high surface area, easy surface functionality [21], biocompatibility [22e24], high thermal stability, lower cost than Au NRs and carbon nanotubes (CNTs), and enhanced NIR absorption capability in first and second biological window (650e950 nm and 1000e1350 nm) make them as an ideal theranostic platforms for future nanomedicine [25,26]. Owing to its enhanced NIR absorption (w650e1300 nm), graphene could be a versatile platform to immobilize PSs with different excitation wavelengths, thereby achieving improved synergistic PTT/PDT effects. This type of strategy is advantageous by minimizing therapy time and avoiding the utilization of two laser systems. However, poor water dispersibility of graphene severely limits its biological applications. To overcome this problem, graphene was functionalized with various hydrophilic groups to enhance its water dispersibility [27e30]. However, it is necessary to have a balance between sp2 and sp3 structure of GO to impart water dispersibility as well as to hold hydrophobic drugs. Apart from simple noninvasive therapeutic strategy, it is also highly desired to possess multi-diagnostic capabilities, such as, fluorescence, magnetic resonance imaging (MRI), computed tomography, and positron emission tomography, which could offer cross validation of the results obtained from different methods, and also provide accurate and realiable disease detection ability to facilitate imaging-guided therapy [31]. This kind of theranostic nanoplatform will minimize the therapy time, cost, and toxicity associated by using different nanomaterials separately [32e35]. Developing cost effective theranostic NMs to treat cancer with minimal doses by means of non-invasive techniques is highly challenging. Here we report water dispersible magnetic and fluorescent graphene (MFG) functionalized with SiNc4 for dual modal imaging and single light induced PTT/PDT. We believe that MFGeSiNc4 would be advantageous in terms of simple preparation and costeffectiveness by using graphene. 2. Methods and materials 2.1. Synthesis of graphene oxide (GO) The GO was synthesized following modified Hummers process with slight modifications, by using pristine graphite flakes as starting material. The graphite
flakes (1 g) were added to 98% H2SO4 (23 mL) and stirred for w8 h. KMnO4 (3 g) was slowly added to the above mixture, while maintaining the temperature 0.2 mM. Indeed, Fig. 4A demonstrates that under photoirradiation, MFGeSiNc4 exhibited dramatic cytotoxic effects on cell viability. These results were in agreement with the CLSM image results (Fig. 3A), where most of the MFGeSiNc4 was uptaken by the cells, leading to induce toxicity upon photoirradiation. The decrease in cell viability by MFGeSiNc4 was mostly due to the toxicity induced by 1O2 and other ROS. The inherent photothermal capability of MFG, owing to its enhanced pelectrons and superior NIR absorption, also contributed to some extent of decrease in cell viability (Fig. 4B). However, the PTT effect might be less predominant at low power doses (300 mW/cm2) [16].
In general, PDT is more predominant at low power doses to generate 1O2 continuously. In contrast, at high power doses, rapid consumption of surrounding tissue oxygen can inhibit the efficiency of 1O2 formation, and also results in severe photobleaching of PS. Alternatively, PTT requires extremely high power doses in order to thermally ablate the tumors. The carbon-based NMs and gold nanostructures both bearing superior light absorbing capabilities have been widely explored for PTT. Often, GO has been used as a PTT reagent with high power doses in order to compensate its poor absorption in the NIR region. Later on, GO has been conjugated with PS, and two different light sources were adopted, a high power dose (4 W/cm2) to excite PTT reagent; and a low power dose (50 mW/cm2) to excite the PS [43]. On the other hand, gold nanostructures exhibit poor photostability as it endured morphological changes at an extent of local heating by pulsed lasers [44e46]. Moreover, PS functionalized with gold nanostructures requires two different light sources to generate heat and 1O2 [13]. On the other hand, graphene-based NMs can overcome these drawbacks and serve as excellent and cost-effective PTT reagents [25,47,48]. Most importantly, immobilizing SiNc4 onto graphene has facilitated the utilization of a single light source to simultaneously generate PTT/ PDT effects at the same time, owing to the absorption at w775 nm by both graphene and SiNc4 in MFGeSiNc4. The advantage of single light induced therapy is, while irradiation, the 1O2 (PDT) will destruct the cellular components such as membrane lipids,
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Fig. 4. (A) Comparative cell viabilities by MTT assay of HeLa cells treated with different concentrations of SiNc4/MFGeSiNc4 under dark/photoirradiation conditions. (B) Cell viabilities of MFG treated HeLa cells under dark/photo conditions.
proteins and DNA by oxidation pathway. However, there is an every chance of reoccurrence of tumor by partially oxidized DNA fragments and subsequent mutations. This kind of reoccurrence (mutations/DNA base excision repair) will be avoided by subsequent burning during PTT without any delay by using single light source, which might help to cause irrecoverable damage to the tumor cells [49]. Furthermore, utilization of a single light source to simultaneously generate PTT/PDT effects would reduce the cost, time, and excessive unwanted burning caused by different light sources. The highest cell deaths w97.9% (Fig. 4A) were obtained from HeLa cells treated with optimal SiNc4 solution (0.10 mM, corresponding to 100 mg/mL of MFGeSiNc4) imparted by PTT/PDT effects; among
which w64.7% from PDT and w33.2% from PTT effects (see Fig. 4B and also Table S1 for complete details). In addition to the HeLa cells, we have also performed the dark and phototoxicity experiments by using MTT assay in human fibroblast normal cells (MRC-5) and phagocytic cells (RAW-264.7 macrophages) with free SiNc4 and MFGeSiNc4 (Fig. S3). Upon comparison with HeLa cancer cells, the photo induced cellular deaths obtained from MFGeSiNc4 is lower for human fibroblast normal cells (70%) and macrophages (75%) which are similar to literature reports [50e52]. The reason for lower cellular deaths in human normal cells and phagocytic cells might be due to its better immune response to external heat as well as ROS, than HeLa cancer cells.
Fig. 5. (A) Intracellular ROS levels generated by SiNc4/MFGeSiNc4 solutions along with H2O2 as positive control. (B) NaN3 quenching of intracellular ROS levels by MFGeSiNc4 solutions. ROS experiments were carried out by using flow cytometry and the results were expressed as mean fluorescence intensity of DCF. (C) Percentage of apoptotic and necrotic HeLa cells induced by SiNc4/MFGeSiNc4 solutions under dark/photo conditions. (D) MMP in HeLa cells treated with SiNc4/MFGeSiNc4 solutions expressed as mean green to red fluorescence intensity ratio (FTTC/PE).
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Fig. 6. HSP 70 expression levels in HeLa cells treated with MFGeSiNc4 solutions under dark/photo conditions.
3.5. Intracellular effects and possible cell death pathway investigation To look at a deeper insight into the cellular deaths by MFGeSiNc4, several pathways were investigated in order to understand the mechanism. MFGeSiNc4 was internalized in HeLa cells first. Upon photoirradiation, a series of intracellular events such as generation of heat, 1O2, and increased intracellular ROS levels were triggered. The plausible causes include, inadequate buffering of tissue or cell antioxidant defense system and interactions with the proteins/DNA/ lipids, finally leading to cellular/tissue death [53]. To examine the oxidative stress induced by MFGeSiNc4, we monitored the intracellular ROS levels by flow cytometry using a reductive reagent, 20 ,70 -dichlorofluorescein diacetate (H2DCF-DA), which entrapped the intracellular ROS and subsequently being oxidized to fluorescent 20 ,70 -dichloro fluorescien (DCF). The intracellular ROS levels in HeLa cells treated with SiNc4/MFGeSiNc4 solutions under dark and photoirradiation conditions (Fig. 5A) reveal that the generated ROS levels apparently exhibited concentration dependent behavior under photoirradiation condition. The ROS levels obtained under photoirradiation conditions were w2 folds higher than that of dark conditions. To ensure that higher ROS levels were induced by MFGe SiNc4 internalized HeLa cells, we performed quenching experiments by pre-treating the HeLa cells with NaN3 solution (50 mM) before photoirradiation. The results in (Fig. 5B) reveal that under photoirradiation the intracellular ROS levels decreased after NaN3 pretreatment, demonstrating that 1O2 was indeed the key ROS responsible for the attributed cellular death by MFGeSiNc4. The apoptosis and necrosis events are considered as the most profound cause of cellular deaths in the therapeutic processes. The percentage of apoptotic and necrotic cells induced by SiNc4/MFGe SiNc4 (Fig. 5C) showed no obvious difference under dark conditions; whereas distinct increase was observed upon photoirradiation, revealing that induced ROS levels might lead to cellular death during photoirradiation and subsequently resulted in the destruction of cancer cells. Mitochondria is the power house of cells and plays an indispensable role in cellular death by regulating the apoptotic and necrotic pathways [1]. To evaluate the function of mitochondria in the presence of MFGeSiNc4 under dark/photoirradiation conditions, we have performed in vitro mitochondrial membrane potential (MMP) assay using JC-1 dye, which normally exists in either aggregate or monomer form. The permeabilization of JC-1 dye becomes much faster in healthy cells and readily enters into the mitochondria to display red fluorescence (PE channel) in aggregate form. For abnormal cells, only green color (FITC channel) in monomer form
can be visualized. The MMP expressed as the green to red fluorescence intensity ratio (FTTC/PE) (Fig. 5D) reveals increased FITC/PE (decreased MMP) with increased MFGeSiNc4 concentration under photoirradiation condition compared to that under dark. This implies that 1O2 generated by photoexcited MFGeSiNc4 can increase ROS levels, which leads to subsequent activation of apoptotic cascade, and finally directs to decrease in MMP. The decrease in MMP has been observed for pristine graphene treated RAW 264.7 cells and singled-wall CNT (SWCNT) treated EMT6 cells [54,55]. In contrast, increase in MMP was observed for GO-HA treated HeLa cells [15]. The photothermal property of MFGeSiNc4 partially contributes to the decreased cell viability under low light doses. During PTT/PDT, the photo-activated drug triggers local heating and thereby the cells may over-express the molecular chaperones of heat shock protein (HSP) family [56]. The HSP 70 expression levels in HeLa cells treated with different concentrations of MFGeSiNc4 under dark/photo conditions (Fig. 6) exhibited concentration dependent behavior and reached a threshold at w0.1 mM; whereas no any detectable expression level was observed under dark condition. This finding justifies that partial cell death observed in MTT assay from MFG (Fig. 4B) is due to the photothermal heating triggered by MFGeSiNc4 upon photoirradiation. The multi-walled CNTs (MWCNTs) treated tumors have shown increased HSP expression levels upon NIR laser irradiation owing to the elevation of sub-lethal temperature [57]. Western blot analysis of SWCNT-polyethylene glycol has also shown that HSP 70 expression levels were comparatively higher in the presence of light than in dark and SWCNTs treated cells alone [54].
4. Conclusions We have developed a theranostic platform based on noncovalent immobilization of SiNc4 onto MFG surface to form MFGeSiNc4 bearing good water dispersibility, fluorescence, and magnetic properties. The MFG serves as an excellent T2-weighted MRI contrast reagent owing to its superparamagnetic nature. The SiNc4 on MFG surface were readily activated by NIR light and furnished MFGeSiNc4 to offer 1O2 mediated PDT along with intrinsic photothermal effects for PTT. The broad NIR absorption (600e1200 nm) capability of graphene could facilitate the immobilization of diversified PSs in order to achieve PTT/PDT effects by single light source. Phosphorescence measurements, SOSG experiments, and DPBF quenching studies confirm the generation of 1O2 by MFGeSiNc4. Higher levels of intracellular ROS and NaN3 quenching experiments demonstrate the indispensable role of 1O2 in cellular deaths. Other biological cascades such as necrosis and over-expression of HSP 70 by photothermal effects might also contribute to cellular deaths. Overall, our in vitro results have demonstrated the simultaneous dual modal imaging and PTT/PDT capabilities of MFGeSiNc4 with appreciable cell killing efficacy of w97.9%. Among which, w64.7% was by PDT; whereas w33.2% was by PTT. The discovery of MFGeSiNc4 opens a new therapeutic window for the treatment of cancer and stands out as a cost effective theranostic reagent in the field of nanomedicine.
Acknowledgments We gratefully acknowledge the National Science Council of Taiwan (NSC 101-2627-M-007-005 and NSC101-2113-M-007-006MY3) and National Tsing Hua University (99N2454E1) for generous funding support. Constructive and fruitful discussions with Prof. Lih-Yuan Lin from the Department of Life Science, National Tsing Hua University was also gratefully acknowledged.
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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.02.011.
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