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Cite this: Chem. Commun., 2014, 50, 10815 Received 11th June 2014, Accepted 28th July 2014
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Facile fabrication of a C60–polydopamine– graphene nanohybrid for single light induced photothermal and photodynamic therapy† Zhen Hu,*ab Feng Zhao,a Yafei Wang,a Yudong Huang,*a Lei Chen,a Nan Li,a Jun Li,a Zhenhui Lia and Guoxing Yia
DOI: 10.1039/c4cc04416a www.rsc.org/chemcomm
A C60–polydopamine–graphene nanohybrid is prepared by a facile approach via polydopamine chemistry. The hybrid displays synergistic photodynamic and photothermal cancer cell killing effects under single light irradiation.
The growing demand for advancement in cancer therapy has triggered significant research efforts to construct smart nanomedicine.1 Photodynamic therapy (PDT) and photothermal therapy (PTT) are two different types of phototherapy methods commonly used for therapeutic purposes.2 Owing to the similar mechanism, combining the two phototherapy techniques may exceed the individual therapeutic effect and lead to a synergistic outcome. Because of high quantum yield of 1O2 generation, fullerenes (C60) and their derivatives have been investigated as candidates for PDT.3 Recently, a report suggested that C60 also has potential application in PTT.4 However, the clinical use of C60 for phototherapy has been limited because of the lack of hydrophilicity and specific targeting. Carbon nanomaterials have already shown extraordinary potential in the field of drug delivery.5 C60 will overcome its limitations by combining with other nanocarbons because of the similarity in chemical composition and difference in the geometry structure.6 Owing to its high surface area, easy surface functionality,7 biocompatibility,8 and photothermal activity,9 graphene will act as a versatile nanoplatform for C60 used in phototherapy. By conjugating with specific ligands that can recognize the cancer cell or magnetic nanoparticles,10 graphene will show great promise as a novel tumor targeting delivery system for C60. Furthermore, due to the intrinsic high near infrared (NIR) absorbance of graphene, the resulting C60–graphene hybrid will exhibit improved synergistic PTT/PDT effects. In the past several years, a
School of Chemical Engineering and Technology, State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150001, China. E-mail: [email protected], [email protected]; Fax: +86-451-86402403; Tel: +86-451-86413711 b Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, UK † Electronic supplementary information (ESI) available: Experimental details, compound characterization and supporting results. See DOI: 10.1039/c4cc04416a
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C60–graphene hybrids have been fabricated via nucleophilic reaction,11 coupling reaction,12 lithiation reaction,13 p–p interaction14 and chemical vapor deposition.15 However, it is still a great challenge to develop facile, mild, and efficient synthetic approaches for C60–graphene hybrids, which have specific targeting, biocompatibility and show improved combined PTT/PDT effects. Inspired by mussels, polydopamine (PDA) has moved into the spotlight as a novel coating material since 2007.16 The PDA can be easily capped on both sides of GO sheets with controllable thickness via self-polymerization.17 Furthermore, the redox capacity of dopamine (DA) produces stable reduced GO (rGO) in one step under mild conditions.18 The rGO is more suitable for PTT application since it has better NIR absorption than GO.19 More interestingly, PDA also exhibits strong absorption in the NIR region, making it very attractive as a new generation of PTT agents.20 Another valuable feature of PDA lies in its chemical structure that incorporates many functional groups. These functional groups not only display outstanding biocompatibility and hydrophilicity,20b,21 but also serve as starting points for covalent modification with desired molecules.18,22 Therefore, we think it worthwhile to develop a mild and facile route to fabricate C60–PDA–graphene nanohybrids. Moreover, to the best of our knowledge, while photodynamic activities of C60 and photothermal anticancer effects of graphene have been welldocumented, the possibility of photo-induced tumor cell killing by C60–PDA–graphene hybrids has remained unexplored. In this communication, GO was surface modified and reduced according to PDA chemistry. Then, we attempted to use PDA–rGO to react with nucleophilic amine groups of the folic acid–C60 derivative (FFA) by means of a Schiff base reaction or Michael addition. The FFA was synthesized by the method mentioned in our previous work.23 The C60–PDA–rGO nanohybrid displays targeting PDT/PTT synergistic activities against HeLa cancer cells. The synthetic route and the possible anti-cancer mechanism of a C60–PDA–rGO nanohybrid are shown in Scheme 1. The successful functionalization of the GO is confirmed by Fourier transform infrared spectroscopy (FT-IR). As shown in Fig. 1(a), noticeable absorptions are observed at B3402 cm1 (O–H), 1732 cm1 (CQO), and 1617 cm1 (CQC stretching) in
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Scheme 1 Approach for fabricating the C60–PDA–rGO nanohybrid, and the possible mechanism of the synergistic effect of combined PTT and PDT.
the IR spectrum of GO. For the PDA–rGO, the appearance of a typical absorption peak for the phenyl group at 1550 cm1 provides evidence for the formation of the PDA layer. In addition, the decrease of the CQO peak intensity at 1732 cm1 and the C–O peak at 1050 cm1 provides a solid indication of GO reduction. For C60–PDA–rGO, the characteristic absorption peaks of C60 (1425, 527 cm1) and FA (1690 cm1) simultaneously exist in its IR spectrum, confirming that C60–PDA–rGO has been
Fig. 1 (a) FT-IR spectra of GO, PDA–rGO, FFA and C60–PDA–rGO; (b) UV-Vis spectra of GO, PDA–rGO, FFA and C60–PDA–rGO; XPS C 1s spectra and fitted curves of (c) GO and (d) PDA–rGO; (e) TEM image of C60–PDA–rGO; (f) AFM image of C60–PDA–rGO.
10816 | Chem. Commun., 2014, 50, 10815--10818
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successfully prepared. As shown in the UV-vis spectra (Fig. 1(b)), the absorbance peak at 228 nm, characteristic of GO, has red shifted to 273 nm after PDA modification. Meanwhile, the PDA– rGO exhibits a boarder absorption range and higher absorbance. A peak at 228 nm disappeared while a new peak at B281 nm appeared due to the presence of FFA in the C60–PDA–rGO, which confirms the conjugation of FFA and PDA–rGO. The X-ray photoelectron spectroscopy (XPS) C 1s spectrum of GO (Fig. 1(c)) can be curved into four peak components with binding energies of 284.8, 286.6, 287.7, and 288.9 eV, attributable to C–C, C–O, CQO, and O–CQO, respectively. After reduction and surface modification by DA, the intensity of the CQO and O–CQO groups decreases dramatically, which indicates that most of the oxygen-containing functional groups are removed (Fig. 1(d)). Moreover, the appearance of the C–N peak at 285.5 eV is consistent with the presence of a surface capped PDA layer. Compared with the PDA–rGO, the peaks of C–N (285.6 eV), CQO (287.5 eV) and O–CQO (289.0 eV) for the C60–PDA–rGO are increased in intensity, which further proves that FFA is grafted onto the PDA–rGO successfully (Fig. S1, ESI†). As can be seen in Fig. 1(e), the representative TEM image of C60–PDA–rGO shows a rGO sheet attached with a PDA layer and some uniformly dispersed clusters on the whole sheet. As a comparison, the GO displays flake-like shapes with wrinkles and is free from any particulate contamination (Fig. S2, ESI†). Compared with pristine GO (Fig. S2, ESI†), the AFM image of C60–PDA–rGO (Fig. 1(f)) presents an apparently increased thickness of about 7.60 nm, likely owing to the covalent PDA that offers more condensed surface polymer coating on the rGO surface. In addition, the size of the FFA is measured to be B5.73 nm. The results confirm the attachment of the PDA and FFA on the graphene. Our previous study has indicated that FFA tended to form an aggregate with a size of B120 nm.23 In the present study, the rGO sheet is decorated with FFA with a size of B5 nm. It seems that the covalent modification firmly fixes the FFA on the graphene, which makes FFA unable to move freely and cannot form large aggregates. In this way, FFA will exert its biological activities more effectively. Furthermore, according to the results of thermogravimetric analysis (TGA), the content of FFA in C60–PDA–rGO can be calculated, which is 17.5 wt% (Fig. S3, ESI†). The representative images of the aqueous suspension of GO, PDA–rGO, and C60–PDA–rGO (50 mg mL1) show that the graphene hybrids are dispersed well in water (Fig. S4, ESI†), which indicates that they are suitable for biological applications. To perform simultaneous PTT/PDT therapy, a Xe lamp (50 W) equipped with a band pass filter (400–1100 nm) is used as the light source at an output power of 2 W cm2. The light source contains both the visible and NIR light, which can excite the C60–PDA–rGO to generate ROS and heat at the same time. To confirm the 1O2 generation capability of C60–PDA–rGO, p-nitroso-N,N 0 -dimethylaniline (RNO) is used as an indicator. As expected, C60–PDA–rGO shows a dramatic decrease in RNO absorption with time, which indicates that C60–PDA–rGO can efficiently generate 1O2 upon photo irradiation. The GO has been reported to quench the 1O2 formation, which significantly reduce the 1O2 production of the PSs.24 In the present work,
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Fig. 2 (a) The 1O2 production by PDA–rGO, FFA and C60–PDA–rGO after irradiation for different time periods. (b) Temperature elevation of water, PDA–rGO, FFA and C60–PDA–rGO aqueous solutions (50 mg mL1) as a function of irradiation time.
however, the 1O2 generation ability of C60–PDA–rGO is still appreciable and only slightly less than that of FFA. The reason may be related to the unique structure of the nanohybrid. As mentioned above, the hybridization process enhances the light absorption properties, and limits the aggregation of FFA, thereby showing an enhanced photodynamic effect of C60–PDA–rGO. After irradiating with a Xe lamp at 2 W cm2 for 15 min, the temperature of C60–PDA–rGO aqueous solution is increased by 17.2 1C at a concentration of 50 mg mL1 (Fig. 2(b)). In contrast, the temperature of pure water increased by only 2.1 1C. It is also worth noting that, the FFA converts the NIR light radiation to vibrational energy to elevate the temperature by 7.5 1C. To study the uptake by HeLa cells, C60–PDA–rGO is labeled with fluorescein isothiocyanate (FITC) via physical adsorption. As shown in Fig. 3(a–c), much stronger fluorescence can be seen in the HeLa cells after incubation with PDA–rGO–FITC
Fig. 3 Fluorescence images and corresponding fluorescence intensity curves of (a) GO–FITC, (b) PDA–rGO–FITC and (c) C60–PDA–rGO–FITC after incubation with HeLa cells for 12 h. (d) Cell viability of HeLa cells incubated with PDA–rGO, FFA and C60–PDA–rGO for different irradiation time. (e) Irradiation time-dependent intracellular ROS generation by PDA– rGO, FFA and C60–PDA–rGO. HeLa cells were incubated with nanoparticles for 12 h prior to irradiation (Xe lamp, 2 W cm2). Cell viability was measured by the conventional MTT reduction assay. The fluorescent probe DCF-DA was used to monitor the intracellular accumulation of ROS. Data are presented as mean S.D. (n = 3). *p o 0.05 compared to the blank group.
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than GO–FITC. Graphene sheets functionalized with PDA exhibit high solubility and biocompatibility in physiological solutions, which would enhance the endocytosis of PDA–rGO by cells. In the present study, C60–PDA–rGO–FITC shows stronger fluorescence inside cells than PDA–rGO–FITC, which suggests specific targeting in the presence of FA molecules. The corresponding intracellular fluorescence intensity is also given in Fig. 3(a–c) to make a quantitative comparison. The fluorescence intensity of the C60–PDA–rGO group (4.58) is much higher than GO (1.00) and PDA–rGO (2.97) after 12 h incubation. Phototoxicity of single light induced PDT–PTT combined treatment on HeLa cells is evaluated using MTT assay. Without light irradiation, PDA–rGO, FFA and C60–PDA–rGO do not show any significant dark toxicity towards HeLa cells (Fig. 3(d)), which proves that the C60–PDA–rGO is cytocompatible. However, upon irradiation, HeLa cell viability decreases significantly as the exposure time is increased, and only 8.6% of cells remain alive after treatment with 50 mg mL1 C60–PDA– rGO and 9 min light exposure. Meanwhile, the cell viability of FFA and PDA–rGO groups decreases to 26.3% and 90.3% after 9 min irradiation, respectively (Fig. 3(d)). It seems that the cytotoxic effects of C60–PDA–rGO are mostly due to the 1O2 and other ROS generated by the C60 component. On the other hand, owing to its drug delivery and superior NIR absorption, the PDA–rGO component also contributes to some extent of decrease in cell viability. The immobilization of FFA onto PDA–rGO has facilitated the utilization of a single light source to simultaneously generate PTT/PDT effects at the same time. The advantage of combined PTT/PDT is that, while irradiation, the 1O2 (PDT) will destroy the cellular components such as membrane lipids, proteins and DNA by the oxidation pathway. Meanwhile, the light-induced local heating (PTT) increases the incubation temperature, which may increase cell membrane permeability or cause cell membrane damage, and further increase cellular uptake and makes 1O2 attack the cells more easily.25 Furthermore, utilization of a single light source to simultaneously generate PTT/PDT effects will reduce the cost, time, and excessive unwanted burning caused by different light sources. In the present study, we also cultured HeLa cells with C60–PDA–rGO at different concentrations for MTT assay (Fig. S5, ESI†). Our results indicate that with the increasing concentrations of C60–PDA–rGO, the lethality increases, suggesting a dosedependent effect in vitro. The MTT assay results also show that 0–100 mg mL1 C60–PDA–rGO does not cause obvious cell death in HeLa and PC12 cells in the dark (Fig. S6, ESI†), which indicates the biocompatibility of the C60–PDA–rGO. The MTT results indicate that the photo induced cytotoxicity of C60–PDA–rGO is better than FFA, although the 1O2 generation ability of C60–PDA–rGO is slightly less than that of FFA. To understand why this happens, intracellular ROS accumulation is analyzed by using 2,7-dichlorofluorescein diacetate (DCF-DA). As shown in Fig. 3(e), while the PDA–rGO does not cause significant intracellular ROS production, the FFA and C60–PDA–rGO increase the intracellular ROS level from 1 to 3.67 and 7.13 after 9 min irradiation, respectively. The intracellular ROS production of C60–PDA–rGO presents a time and dose-dependent effect in vitro
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Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (no. QA201415), and the Fundamental Research Funds for the Central Universities (no. HIT. NSRIF. 2012034).
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Notes and references
Fig. 4 Hoechst 33342/propidium iodide (PI) double staining in HeLa cells. (a and a 0 ) Control cells; (b and b 0 ), (c and c 0 ), (d and d 0 ) HeLa cells were incubated with 50 mg mL1 PDA–rGO, FFA or C60–PDA–rGO for 12 h prior to irradiation (Xe lamp, 2 W cm2, 9 min), respectively. (a–d) PI staining; (a 0 –d 0 ) Hoechst 33342 staining.
(Fig. S5, ESI†). The results prove that anti-cancer activity of C60–PDA–rGO is mainly associated with the intracellular ROS production (PDT). Furthermore, the results also indicate that the enhanced uptake of C60–PDA–rGO based on the PDA–rGO platform and folate receptor transports more C60 molecules into the cells, which produces more ROS inside the cells and exerts enhanced phototherapy effects. The morphological changes of apoptotic HeLa cells induced by nanocarbons are observed by Hoechst 33342/PI staining. As shown in Fig. 4, a large number of cells showed nuclear condensation (Hoechst 33342 staining) or cell death (PI staining) after being exposed to nanocarbons under light irradiation, especially in FFA and C60–PDA–rGO groups. Treatment with C60–PDA–rGO significantly increases the percentage of apoptotic and dead cells, which is evidently more effective than individual FFA and PDA–rGO. For quantitative analysis of the apoptotic percentage, HeLa cells are double stained by annexin V and PI and determined by flow cytometry (Fig. S7, ESI†). The combined PTT/PDT effects induced by C60–PDA–rGO remarkably increase the percentage of apoptosis and death to 50.89% and 37.43% respectively, which is evidently better than the FFA (PDT) and PDA–rGO (PTT). Meanwhile, the results indicate that apoptotic cell death is predominant in all experiments. The work provides a facile way to synthesize a multifunctional C60–graphene nanohybrid with therapeutic efficiency for cancer. In this work, we have designed and developed a facile synthetic approach to prepare C60–PDA–rGO nanohybrids. The hybridization of graphene, PDA and FFA enhances the cellular uptake and light absorption, limits the aggregation of C60, and combines PTT and PDT effects in a single platform, thereby showing an enhanced phototherapy effect of C60–PDA– rGO. Under single light irradiation, C60–PDA–rGO generates PTT/PDT effects simultaneously, which causes a significant decrease in cell survival and elevation of oxidative stress, and induces apoptotic death. The study presents the potential of nanocarbons for synergistic phototherapy of cancer. We thank the National Natural Science Foundation of China (no. 51103031), the PhD Programs Foundation of Ministry of Education of China (no. 20112302120034), the Special Foundation of China Postdoctoral Science (no. 201003436), the Open
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Cite this: Chem. Commun., 2014, 50, 10815 Received 11th June 2014, Accepted 28th July 2014
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Facile fabrication of a C60–polydopamine– graphene nanohybrid for single light induced photothermal and photodynamic therapy† Zhen Hu,*ab Feng Zhao,a Yafei Wang,a Yudong Huang,*a Lei Chen,a Nan Li,a Jun Li,a Zhenhui Lia and Guoxing Yia
DOI: 10.1039/c4cc04416a www.rsc.org/chemcomm
A C60–polydopamine–graphene nanohybrid is prepared by a facile approach via polydopamine chemistry. The hybrid displays synergistic photodynamic and photothermal cancer cell killing effects under single light irradiation.
The growing demand for advancement in cancer therapy has triggered significant research efforts to construct smart nanomedicine.1 Photodynamic therapy (PDT) and photothermal therapy (PTT) are two different types of phototherapy methods commonly used for therapeutic purposes.2 Owing to the similar mechanism, combining the two phototherapy techniques may exceed the individual therapeutic effect and lead to a synergistic outcome. Because of high quantum yield of 1O2 generation, fullerenes (C60) and their derivatives have been investigated as candidates for PDT.3 Recently, a report suggested that C60 also has potential application in PTT.4 However, the clinical use of C60 for phototherapy has been limited because of the lack of hydrophilicity and specific targeting. Carbon nanomaterials have already shown extraordinary potential in the field of drug delivery.5 C60 will overcome its limitations by combining with other nanocarbons because of the similarity in chemical composition and difference in the geometry structure.6 Owing to its high surface area, easy surface functionality,7 biocompatibility,8 and photothermal activity,9 graphene will act as a versatile nanoplatform for C60 used in phototherapy. By conjugating with specific ligands that can recognize the cancer cell or magnetic nanoparticles,10 graphene will show great promise as a novel tumor targeting delivery system for C60. Furthermore, due to the intrinsic high near infrared (NIR) absorbance of graphene, the resulting C60–graphene hybrid will exhibit improved synergistic PTT/PDT effects. In the past several years, a
School of Chemical Engineering and Technology, State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150001, China. E-mail: [email protected], [email protected]; Fax: +86-451-86402403; Tel: +86-451-86413711 b Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, UK † Electronic supplementary information (ESI) available: Experimental details, compound characterization and supporting results. See DOI: 10.1039/c4cc04416a
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C60–graphene hybrids have been fabricated via nucleophilic reaction,11 coupling reaction,12 lithiation reaction,13 p–p interaction14 and chemical vapor deposition.15 However, it is still a great challenge to develop facile, mild, and efficient synthetic approaches for C60–graphene hybrids, which have specific targeting, biocompatibility and show improved combined PTT/PDT effects. Inspired by mussels, polydopamine (PDA) has moved into the spotlight as a novel coating material since 2007.16 The PDA can be easily capped on both sides of GO sheets with controllable thickness via self-polymerization.17 Furthermore, the redox capacity of dopamine (DA) produces stable reduced GO (rGO) in one step under mild conditions.18 The rGO is more suitable for PTT application since it has better NIR absorption than GO.19 More interestingly, PDA also exhibits strong absorption in the NIR region, making it very attractive as a new generation of PTT agents.20 Another valuable feature of PDA lies in its chemical structure that incorporates many functional groups. These functional groups not only display outstanding biocompatibility and hydrophilicity,20b,21 but also serve as starting points for covalent modification with desired molecules.18,22 Therefore, we think it worthwhile to develop a mild and facile route to fabricate C60–PDA–graphene nanohybrids. Moreover, to the best of our knowledge, while photodynamic activities of C60 and photothermal anticancer effects of graphene have been welldocumented, the possibility of photo-induced tumor cell killing by C60–PDA–graphene hybrids has remained unexplored. In this communication, GO was surface modified and reduced according to PDA chemistry. Then, we attempted to use PDA–rGO to react with nucleophilic amine groups of the folic acid–C60 derivative (FFA) by means of a Schiff base reaction or Michael addition. The FFA was synthesized by the method mentioned in our previous work.23 The C60–PDA–rGO nanohybrid displays targeting PDT/PTT synergistic activities against HeLa cancer cells. The synthetic route and the possible anti-cancer mechanism of a C60–PDA–rGO nanohybrid are shown in Scheme 1. The successful functionalization of the GO is confirmed by Fourier transform infrared spectroscopy (FT-IR). As shown in Fig. 1(a), noticeable absorptions are observed at B3402 cm1 (O–H), 1732 cm1 (CQO), and 1617 cm1 (CQC stretching) in
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Scheme 1 Approach for fabricating the C60–PDA–rGO nanohybrid, and the possible mechanism of the synergistic effect of combined PTT and PDT.
the IR spectrum of GO. For the PDA–rGO, the appearance of a typical absorption peak for the phenyl group at 1550 cm1 provides evidence for the formation of the PDA layer. In addition, the decrease of the CQO peak intensity at 1732 cm1 and the C–O peak at 1050 cm1 provides a solid indication of GO reduction. For C60–PDA–rGO, the characteristic absorption peaks of C60 (1425, 527 cm1) and FA (1690 cm1) simultaneously exist in its IR spectrum, confirming that C60–PDA–rGO has been
Fig. 1 (a) FT-IR spectra of GO, PDA–rGO, FFA and C60–PDA–rGO; (b) UV-Vis spectra of GO, PDA–rGO, FFA and C60–PDA–rGO; XPS C 1s spectra and fitted curves of (c) GO and (d) PDA–rGO; (e) TEM image of C60–PDA–rGO; (f) AFM image of C60–PDA–rGO.
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successfully prepared. As shown in the UV-vis spectra (Fig. 1(b)), the absorbance peak at 228 nm, characteristic of GO, has red shifted to 273 nm after PDA modification. Meanwhile, the PDA– rGO exhibits a boarder absorption range and higher absorbance. A peak at 228 nm disappeared while a new peak at B281 nm appeared due to the presence of FFA in the C60–PDA–rGO, which confirms the conjugation of FFA and PDA–rGO. The X-ray photoelectron spectroscopy (XPS) C 1s spectrum of GO (Fig. 1(c)) can be curved into four peak components with binding energies of 284.8, 286.6, 287.7, and 288.9 eV, attributable to C–C, C–O, CQO, and O–CQO, respectively. After reduction and surface modification by DA, the intensity of the CQO and O–CQO groups decreases dramatically, which indicates that most of the oxygen-containing functional groups are removed (Fig. 1(d)). Moreover, the appearance of the C–N peak at 285.5 eV is consistent with the presence of a surface capped PDA layer. Compared with the PDA–rGO, the peaks of C–N (285.6 eV), CQO (287.5 eV) and O–CQO (289.0 eV) for the C60–PDA–rGO are increased in intensity, which further proves that FFA is grafted onto the PDA–rGO successfully (Fig. S1, ESI†). As can be seen in Fig. 1(e), the representative TEM image of C60–PDA–rGO shows a rGO sheet attached with a PDA layer and some uniformly dispersed clusters on the whole sheet. As a comparison, the GO displays flake-like shapes with wrinkles and is free from any particulate contamination (Fig. S2, ESI†). Compared with pristine GO (Fig. S2, ESI†), the AFM image of C60–PDA–rGO (Fig. 1(f)) presents an apparently increased thickness of about 7.60 nm, likely owing to the covalent PDA that offers more condensed surface polymer coating on the rGO surface. In addition, the size of the FFA is measured to be B5.73 nm. The results confirm the attachment of the PDA and FFA on the graphene. Our previous study has indicated that FFA tended to form an aggregate with a size of B120 nm.23 In the present study, the rGO sheet is decorated with FFA with a size of B5 nm. It seems that the covalent modification firmly fixes the FFA on the graphene, which makes FFA unable to move freely and cannot form large aggregates. In this way, FFA will exert its biological activities more effectively. Furthermore, according to the results of thermogravimetric analysis (TGA), the content of FFA in C60–PDA–rGO can be calculated, which is 17.5 wt% (Fig. S3, ESI†). The representative images of the aqueous suspension of GO, PDA–rGO, and C60–PDA–rGO (50 mg mL1) show that the graphene hybrids are dispersed well in water (Fig. S4, ESI†), which indicates that they are suitable for biological applications. To perform simultaneous PTT/PDT therapy, a Xe lamp (50 W) equipped with a band pass filter (400–1100 nm) is used as the light source at an output power of 2 W cm2. The light source contains both the visible and NIR light, which can excite the C60–PDA–rGO to generate ROS and heat at the same time. To confirm the 1O2 generation capability of C60–PDA–rGO, p-nitroso-N,N 0 -dimethylaniline (RNO) is used as an indicator. As expected, C60–PDA–rGO shows a dramatic decrease in RNO absorption with time, which indicates that C60–PDA–rGO can efficiently generate 1O2 upon photo irradiation. The GO has been reported to quench the 1O2 formation, which significantly reduce the 1O2 production of the PSs.24 In the present work,
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Fig. 2 (a) The 1O2 production by PDA–rGO, FFA and C60–PDA–rGO after irradiation for different time periods. (b) Temperature elevation of water, PDA–rGO, FFA and C60–PDA–rGO aqueous solutions (50 mg mL1) as a function of irradiation time.
however, the 1O2 generation ability of C60–PDA–rGO is still appreciable and only slightly less than that of FFA. The reason may be related to the unique structure of the nanohybrid. As mentioned above, the hybridization process enhances the light absorption properties, and limits the aggregation of FFA, thereby showing an enhanced photodynamic effect of C60–PDA–rGO. After irradiating with a Xe lamp at 2 W cm2 for 15 min, the temperature of C60–PDA–rGO aqueous solution is increased by 17.2 1C at a concentration of 50 mg mL1 (Fig. 2(b)). In contrast, the temperature of pure water increased by only 2.1 1C. It is also worth noting that, the FFA converts the NIR light radiation to vibrational energy to elevate the temperature by 7.5 1C. To study the uptake by HeLa cells, C60–PDA–rGO is labeled with fluorescein isothiocyanate (FITC) via physical adsorption. As shown in Fig. 3(a–c), much stronger fluorescence can be seen in the HeLa cells after incubation with PDA–rGO–FITC
Fig. 3 Fluorescence images and corresponding fluorescence intensity curves of (a) GO–FITC, (b) PDA–rGO–FITC and (c) C60–PDA–rGO–FITC after incubation with HeLa cells for 12 h. (d) Cell viability of HeLa cells incubated with PDA–rGO, FFA and C60–PDA–rGO for different irradiation time. (e) Irradiation time-dependent intracellular ROS generation by PDA– rGO, FFA and C60–PDA–rGO. HeLa cells were incubated with nanoparticles for 12 h prior to irradiation (Xe lamp, 2 W cm2). Cell viability was measured by the conventional MTT reduction assay. The fluorescent probe DCF-DA was used to monitor the intracellular accumulation of ROS. Data are presented as mean S.D. (n = 3). *p o 0.05 compared to the blank group.
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than GO–FITC. Graphene sheets functionalized with PDA exhibit high solubility and biocompatibility in physiological solutions, which would enhance the endocytosis of PDA–rGO by cells. In the present study, C60–PDA–rGO–FITC shows stronger fluorescence inside cells than PDA–rGO–FITC, which suggests specific targeting in the presence of FA molecules. The corresponding intracellular fluorescence intensity is also given in Fig. 3(a–c) to make a quantitative comparison. The fluorescence intensity of the C60–PDA–rGO group (4.58) is much higher than GO (1.00) and PDA–rGO (2.97) after 12 h incubation. Phototoxicity of single light induced PDT–PTT combined treatment on HeLa cells is evaluated using MTT assay. Without light irradiation, PDA–rGO, FFA and C60–PDA–rGO do not show any significant dark toxicity towards HeLa cells (Fig. 3(d)), which proves that the C60–PDA–rGO is cytocompatible. However, upon irradiation, HeLa cell viability decreases significantly as the exposure time is increased, and only 8.6% of cells remain alive after treatment with 50 mg mL1 C60–PDA– rGO and 9 min light exposure. Meanwhile, the cell viability of FFA and PDA–rGO groups decreases to 26.3% and 90.3% after 9 min irradiation, respectively (Fig. 3(d)). It seems that the cytotoxic effects of C60–PDA–rGO are mostly due to the 1O2 and other ROS generated by the C60 component. On the other hand, owing to its drug delivery and superior NIR absorption, the PDA–rGO component also contributes to some extent of decrease in cell viability. The immobilization of FFA onto PDA–rGO has facilitated the utilization of a single light source to simultaneously generate PTT/PDT effects at the same time. The advantage of combined PTT/PDT is that, while irradiation, the 1O2 (PDT) will destroy the cellular components such as membrane lipids, proteins and DNA by the oxidation pathway. Meanwhile, the light-induced local heating (PTT) increases the incubation temperature, which may increase cell membrane permeability or cause cell membrane damage, and further increase cellular uptake and makes 1O2 attack the cells more easily.25 Furthermore, utilization of a single light source to simultaneously generate PTT/PDT effects will reduce the cost, time, and excessive unwanted burning caused by different light sources. In the present study, we also cultured HeLa cells with C60–PDA–rGO at different concentrations for MTT assay (Fig. S5, ESI†). Our results indicate that with the increasing concentrations of C60–PDA–rGO, the lethality increases, suggesting a dosedependent effect in vitro. The MTT assay results also show that 0–100 mg mL1 C60–PDA–rGO does not cause obvious cell death in HeLa and PC12 cells in the dark (Fig. S6, ESI†), which indicates the biocompatibility of the C60–PDA–rGO. The MTT results indicate that the photo induced cytotoxicity of C60–PDA–rGO is better than FFA, although the 1O2 generation ability of C60–PDA–rGO is slightly less than that of FFA. To understand why this happens, intracellular ROS accumulation is analyzed by using 2,7-dichlorofluorescein diacetate (DCF-DA). As shown in Fig. 3(e), while the PDA–rGO does not cause significant intracellular ROS production, the FFA and C60–PDA–rGO increase the intracellular ROS level from 1 to 3.67 and 7.13 after 9 min irradiation, respectively. The intracellular ROS production of C60–PDA–rGO presents a time and dose-dependent effect in vitro
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Notes and references
Fig. 4 Hoechst 33342/propidium iodide (PI) double staining in HeLa cells. (a and a 0 ) Control cells; (b and b 0 ), (c and c 0 ), (d and d 0 ) HeLa cells were incubated with 50 mg mL1 PDA–rGO, FFA or C60–PDA–rGO for 12 h prior to irradiation (Xe lamp, 2 W cm2, 9 min), respectively. (a–d) PI staining; (a 0 –d 0 ) Hoechst 33342 staining.
(Fig. S5, ESI†). The results prove that anti-cancer activity of C60–PDA–rGO is mainly associated with the intracellular ROS production (PDT). Furthermore, the results also indicate that the enhanced uptake of C60–PDA–rGO based on the PDA–rGO platform and folate receptor transports more C60 molecules into the cells, which produces more ROS inside the cells and exerts enhanced phototherapy effects. The morphological changes of apoptotic HeLa cells induced by nanocarbons are observed by Hoechst 33342/PI staining. As shown in Fig. 4, a large number of cells showed nuclear condensation (Hoechst 33342 staining) or cell death (PI staining) after being exposed to nanocarbons under light irradiation, especially in FFA and C60–PDA–rGO groups. Treatment with C60–PDA–rGO significantly increases the percentage of apoptotic and dead cells, which is evidently more effective than individual FFA and PDA–rGO. For quantitative analysis of the apoptotic percentage, HeLa cells are double stained by annexin V and PI and determined by flow cytometry (Fig. S7, ESI†). The combined PTT/PDT effects induced by C60–PDA–rGO remarkably increase the percentage of apoptosis and death to 50.89% and 37.43% respectively, which is evidently better than the FFA (PDT) and PDA–rGO (PTT). Meanwhile, the results indicate that apoptotic cell death is predominant in all experiments. The work provides a facile way to synthesize a multifunctional C60–graphene nanohybrid with therapeutic efficiency for cancer. In this work, we have designed and developed a facile synthetic approach to prepare C60–PDA–rGO nanohybrids. The hybridization of graphene, PDA and FFA enhances the cellular uptake and light absorption, limits the aggregation of C60, and combines PTT and PDT effects in a single platform, thereby showing an enhanced phototherapy effect of C60–PDA– rGO. Under single light irradiation, C60–PDA–rGO generates PTT/PDT effects simultaneously, which causes a significant decrease in cell survival and elevation of oxidative stress, and induces apoptotic death. The study presents the potential of nanocarbons for synergistic phototherapy of cancer. We thank the National Natural Science Foundation of China (no. 51103031), the PhD Programs Foundation of Ministry of Education of China (no. 20112302120034), the Special Foundation of China Postdoctoral Science (no. 201003436), the Open
10818 | Chem. Commun., 2014, 50, 10815--10818
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