Functionalized graphene/C₆₀ nanohybrid for targeting photothermally enhanced photodynamic therapy

Abstract

The unique structures and properties of nanocarbons will provide fascinating applications in biomedicine. Here we report a new strategy to combine C₆₀ with graphene for targeting phototherapy. Folic acid (FA) and polyethylene glycol (PEG) were conjugated onto graphene oxide (GO) via an imide linkage. Using nucleophilic addition, the FA–GO–PEG/C₆₀ nanohybrid was then prepared. The versatile platform of FA–GO–PEG for C₆₀ dramatically improved tumor targeting, which has been demonstrated by a cellular uptake assay. Owing to the doping effect and the inhibition of aggregation, the hybridization process also significantly enhanced the phototoxicity of FA–GO–PEG/C₆₀. More importantly, the photothermal therapy (PTT) of FA–GO–PEG/C₆₀ can cause obvious cell damage, which may further enhance the photodynamic therapy (PDT) efficacy against cancer cells. As expected, the combined PDT and PTT treatment with FA–GO–PEG/C₆₀ showed remarkably improved and synergistic effects compared to PTT or PDT alone. This study presents the potential of nanocarbons for synergistic phototherapy of cancer.

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1. Introduction

Phototherapy is an emerging technique for various diseases such as cancer. The use of phototherapy as a cancer therapy is particularly attractive because of its fundamental specificity and selectivity. The treatment is a feasible method to generate localized thermal/dynamic damage to malignant cells when surgical resection is not possible.^1,2 Because of their unique physicochemical properties, fullerenes (C₆₀) and their derivatives have been investigated as candidates for phototherapy. In response to light irradiation, fullerene generates cytotoxic singlet oxygen (¹O₂) or other reactive oxygen species (ROS), which is the main principle of photodynamic therapy (PDT). The ¹O₂ production yield of C₆₀ derivatives is even higher than those of traditional photosensitizers (PSs) such as rose bengal, methylene blue and eosin yellowish, makes it an ideal PSs used in PDT. A lot of pioneering work has focused on the use of fullerenes for PDT applications, including the cleavage of DNA strands,^3,4 photoinactivation of viruses,⁵ production of oxidative damage to lipids,⁶ PDT-induced killing of tumor cells,^7,8 and even a report of regressions after PDT in a mouse tumor model.⁹ Furthermore, a recent study suggests that multimeric C₆₀ has potential application in photothermal therapy (PTT).¹⁰ Despite their potential, most C₆₀ derivatives used in phototherapy have some limitations. Mainly, they are hydrophobic or easy to aggregate which leads to the decrease of singlet oxygen production and the efficiency of phototherapy. Moreover, the lack of specific target is also a principal challenge for C₆₀ derivatives used in phototherapy.

Owing to its unique physical and chemical properties,^11,12 graphene may overcome the limitations of C₆₀ used in phototherapy. A combination of these two kinds of nanocarbons will lead to remarkable synergies in certain properties because of the similarity in chemical composition and difference in geometry structure.^13,14 Some studies have paid attention to the fabrication of the graphene/C₆₀ hybrid nanomaterials. In these reports, C₆₀ derivatives are loaded onto the graphene via a coupling reaction,^15–17 nucleophilic reaction,^18,19 lithiation reaction,²⁰ π–π interaction²¹ and chemical vapor deposition.^13,22 The results indicate that the graphene/C₆₀ hybrid offers performance superior to that of the individual graphene and fullerene. In addition to the synergistic effect, graphene could also serve as an ideal carrier system for C₆₀ which is high capacity loading, efficient delivery and specific targeting. Ultra-high C₆₀ loading efficiency will be achieved owing to the extremely large surface area of graphene, which has every atom exposed on its surface.^23,24 Once the C₆₀ is coated on the graphene surface, its free movement is restricted. This effect will prevent the spontaneous aggregation of the C₆₀ and its derivatives, and make them stable in the solution. On the other hand, the presence of C₆₀ on a graphene sheet surface can effectively prevent the aggregation of the latter which in turn helps the dispersion of the former.^15,19 For targeting gene and drug delivery, much work has been carried out using graphene as the efficient nanocarrier.^25–27 A wise strategy to let graphene exhibits tumor targeting is to conjugate with specific ligands that can recognize the cancer cell or magnetic nanoparticles.^28,29 Therefore, graphene has shown great promise as a novel targeting delivery system for C₆₀ used in phototherapy.

Apart from the excellent drug carrier, graphene also can act as a photothermal agent and convert near infra red (NIR) light into heat efficiently and thus induce hyperthermia to cells and surrounding tissues.^30–36 In recent years, graphene is also used for combination cancer therapy. According to immobilize anticarcinogen or PDT PSs, several graphene based nanomaterials have shown excellent anti-cancer performance for combined chemotherapy/PTT^37–39 and PDT/PTT.^40–43 Previous studies have indicated that, combining C₆₀ and graphene in one system can exceed the individual phototherapeutic response of each nanocarbon and may lead to enhanced therapeutic outcome. However, it is still a great challenge to develop facile and efficient synthetic approaches for graphene/C₆₀ nanohybrid, which is specific targeting, biocompatibility and could effectively show hybridization effects between graphene and C₆₀. Moreover, to the best of our knowledge, while photodynamic activities of C₆₀ and photothermal anticancer effects of graphene have been well-documented, the possibility of direct tumor cell killing by graphene/C₆₀ hybrids has remained largely unexplored. Herein, we design and develop a multifunctional graphene/C₆₀ nanohybrid, which is synthesized by covalently grafting folic acid (FA) and polyethylene glycol (PEG) with graphene oxide (GO) via coupling reaction, and then pristine C₆₀ is loaded on the surface of graphene via the nucleophilic addition. Benefiting from the unique nanostructure, the hybrid integrates the multifunctions of PDT and PTT into a single nanoplatform, which is expected to improve the therapeutic efficiency.

2. Methods and materials

2.1. Materials

Chloroacetic acid, NaOH and FA were product of Sinoreagent Co. Ltd. C₆₀ was purchased from Wuhan University. Six-Armed PEG-amine (10 kDa) was provided by SunBio Inc. The N-hydroxysuccinimide (NHS), 2-(N-morpholino)ethanesulfonic acid (MES), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were provided by J&K Scientific Ltd. RMPI 1640 medium and the fetal calf serum were provided from Gibco BRL. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl (MTT) was provided from Sigma. 2,7-Dichlorofluorescein diacetate (DCF-DA) was obtained from Molecular Probe Inc. Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from Biosea Biotechnology Co. Ltd.

2.2. Synthesis of FA–GO–PEG

In our previous work, we have reported the synthesis of FA–GO.²⁸ In the present work, FA–GO was further modified by 6-armed PEG-amine for loading C₆₀, resulting in the synthesis of FA–GO–PEG. In brief, GO was carboxylated by ClCH₂COOH to obtain GO–COOH. Then GO–FA was covalently conjugated with the carboxylic acid group of GO–COOH using EDC/NHS chemistry. The resulting GO–FA was suspended in 20 mL water to give a concentration of 1 mg mL⁻¹. Then 100 mg EDC was added to the suspension and the mixture was sonicated for 30 min. 6-Armed PEG-amine (100 mg) was added in the mixture, allowed to react overnight at room temperature. The product (FA–GO–PEG) was purified by centrifugation (14 800 rpm, 10 min) and washed several times with deionized water to remove unreacted PEG and other reagents.

2.3. Preparation of FA–GO–PEG/C₆₀ nanohybrid

FA–GO–PEG (20 mg) was dispersed in 20 mL DMSO with the aid of sonication for 30 min. Then 50 mg C₆₀ in 20 mL toluene was added into above solution. The amino groups on FA–GO–PEG could undergo nucleophilic addition across the C=C bonds in C₆₀.^44,45 The mixture was stirred at 90 °C for 2 days and was filtered and washed with toluene and ethanol sequentially for several times to completely removing excess C₆₀. Followed by drying at 80 °C under vacuum for 12 h, 24 mg FA–GO–PEG/C₆₀ nanohybrid product was obtained.

2.4. Characterization

Fourier transform infrared spectra (FT-IR, Bruker, Tensor 27), Raman spectra (Horiba Jobin Yvon, France), and UV-visible light absorption spectra (UV-vis, Beijing Purkinje General Instrument Co., Ltd., TU-1901) were used to characterize the functionalized graphene. Thermogravimetric analysis (TG) was run under a nitrogen flow (40 mL min⁻¹) using a Q500 TG instrument (TA Instruments). The samples were heated from room temperature to 400 °C at a ramp rate of 5 °C min⁻¹. Transmission electron microscopy (TEM, JEOL, JEM-2100F) and atomic force microscope (AFM, Sounding Housing SPA 400) were used to characterize the morphology and structure of the samples. Two optical-fiber coupled power-tunable diode lasers (continuous wave) with wavelengths at 532 nm (maximal power = 2 W) and 808 nm (maximal power = 7 W) were both purchased from Beijing Stone Laser Ltd. The exact optical powers of lasers used in the experiments were calibrated and measured by a LPE-1B laser power/energy meter (Physcience Opto-Electronics Co. Ltd. Beijing).

2.5. Singlet oxygen generation in aqueous solutions

To measure the singlet oxygen (¹O₂) generation by FA–GO–PEG/C₆₀, p-nitroso-N,N′-dimethylaniline (RNO) was used as an indicator. Solutions of GO, folic acid C₆₀ derivative (FFA) or FA–GO–PEG/C₆₀ were mixed with RNO (25 μM), imidazole (25 μM), and phosphate-buffered saline (PBS, 10 mM, pH = 7.4) and then irradiated by 532 nm laser at the light power density of 0.1 W cm⁻² for different periods of time. The FFA served as comparison was synthesized by the method mentioned in our previous work.⁴⁶ The generation of ¹O₂ would result in the reduction of RNO absorption at 440 nm, which reflects the production of ¹O₂.

2.6. Photothermal activity of FA–GO–PEG/C₆₀ nanohybrid

Solutions of GO, FFA or FA–GO–PEG/C₆₀ (10 μg mL⁻¹) were taken in a 1 mL quartz sample cell. The solution was then illuminated with 808 nm NIR laser for different time periods with a power density of 2 W cm⁻². The increase in temperature was measured by a thermocouple immersed into suspension.

2.7. Cell treatment and cytotoxicity assay

HeLa cells were cultured in an atmosphere with 5% CO₂ and at 37 °C provided by a NAPCO CO₂ incubator in RMPI 1640 medium containing 10% heat-inactivated fetal calf serum. Then, the culture medium was replaced by the fresh medium containing 2% fetal calf serum and specific nano-materials. After 24 h of incubation, the cells were washed twice with PBS and irradiated using 808 nm NIR laser (2 W cm⁻², 3 min) for PTT treatment. The method of detecting the effect of PDT treatment was the same as that used in PTT treatment, except that 532 nm laser was used for irradiation (0.1 mW cm⁻², 5 min). For synergistically enhanced anti-cancer effect of the combination of PDT and PTT, the cells were irradiated using the 808 nm laser at a power density of 2 W cm⁻² for 3 min (360 J cm⁻²). After photothermally heating, cells were immediately irradiated by the 532 nm laser at a power density of 0.1 W cm⁻² for 5 min (30 J cm⁻²).

After another 24 h incubation, the cell viabilities of samples were determined by the MTT assay. In brief, HeLa cells (1 × 10⁵ per mL) were seeded in 96-well plates. 10 μL MTT solutions (final concentration, 0.5 mg mL⁻¹) were added and incubated for additional 4 h. The lysis buffer (20% sodium dodecylsulfate in 50% aqueous N,N-dimethylformamide) was added to solubilize the formazan crystal, and absorbance at 570/630 nm was measured with a microplate reader (Molecular Devices).

2.8. In vitro cellular uptake assay

HeLa cells were cultured in 24 well tissue plate at a density of 1 × 10⁵ cells per well. FITC was loaded on FA–GO–PEG/C₆₀ by sonicating FITC solution (0.05 mg mL⁻¹, 2 mL) with an aqueous suspension of FA–GO–PEG/C₆₀ (2.0 mg mL⁻¹, 1 mL) for 30 min to mix them together, followed by stirring in the dark for 12 h. Unbound FITC was removed by rinsing and centrifugation. As a comparison, GO and FA–GO were treated with FITC by exactly the same steps. Then, HeLa cells were exposed to GO–FITC, FA–GO–FITC and FA–GO–PEG/C₆₀–FITC with the final concentration of 10 μg mL⁻¹ at 37 °C for certain time, respectively. The cells were harvested and resuspended in PBS after washed for 3 times. To investigate the targeted uptake of FA–GO–PEG/C₆₀ by HeLa cells, cells were observed by confocal fluorescence microscopy equipped with 450–490 nm band-pass filter.

2.9. Measurement of intracellular ROS accumulation

The fluorescent probe DCF-DA was used to monitor the intracellular accumulation of ROS. After treatment, cells (1 × 10⁶ cells per 3 mL in 6-well plates) were rinsed with D-Hanks solution and 10 μM DCF-DA was loaded. After 20 min incubation at 37 °C, the cells were harvested after being washed with PBS 3 times. The intracellular ROS accumulation was measured by using a Becton–Dickinson fluorescence-activated cell analyzer while data analysis was performed with Modifit LT 2.0 (Becton–Dickinson). About 1 × 10⁴ cells were counted for each analysis. The fluorescence intensity was quantified with CELLQuest software.

2.10. Determination of apoptosis

Apoptotic cell death was analyzed by double staining with annexin V-FITC and propidium iodide (PI). Apoptosis of cells was evaluated using an annexin-V FITC apoptosis detection kit. Cells were harvested, washed and incubated at 4 °C for 30 min in the dark with annexin V-FITC and PI, then were analyzed on a FACS Vantage SE flow cytometer (Becton–Dickinson).

2.11. Statistical analysis

Values were reported as means ± S.D. Statistical comparisons were made by one-way ANOVA to detect significant difference using SPSS 13.0 for windows.

3. Results and discussion

3.1. Synthesis and characterization of FA–GO–PEG/C₆₀ nanohybrid

In this work, we design and employ a facile strategy to prepare FA–GO–PEG/C₆₀ nanohybrid via a three-step reaction using GO as the starting material. The notable difference from the previous work is that the nanocarbon hybrid would theoretically be expected to improve the targeting, biocompatibility and the phototherapy effects. The synthetic route to FA–GO–PEG/C₆₀ is explained in Scheme 1.

Scheme 1 The preparation of the FA–GO–PEG/C₆₀ nanohybrid.

As shown in Fig. 1a, noticeable absorptions are observed at ∼3406 cm⁻¹ (O–H), 2898 cm⁻¹ (C–H), 1715 cm⁻¹ (C=O), 1630 cm⁻¹ (C=C stretching) in the IR spectrum of GO. In the C₆₀ spectra, it shows several characteristic absorption peaks, respectively located at 1436, 1185, 577 and 527 cm⁻¹. The IR spectra of GO and FA–GO are almost same, however, the characteristic peak of FA at 1648 cm⁻¹ are clearly observed in the GO–FA spectra. This suggests that FA is successfully conjugated onto the GO surface. For FA–GO–PEG/C₆₀, the characteristic peaks of GO at ∼3406, 2989 and 1715 cm⁻¹ are observed. Moreover, the characteristic absorption peaks of C₆₀ (1436, 1185, 527 cm⁻¹) and FA (1648 cm⁻¹) are simultaneously exist in its IR spectrum, confirming that the FA–GO–PEG/C₆₀ have been successfully prepared.

Fig. 1 (a) The FTIR spectra of the GO, C₆₀, FA–GO and FA–GO–PEG/C₆₀; (b) the UV-vis spectra of the GO, FA–GO and FA–GO–PEG/C₆₀ in water; (c) the Raman spectra of the GO, C₆₀, FA–GO and FA–GO–PEG/C₆₀; (d) TG curves of C₆₀, GO, FA–GO–PEG and FA–GO–PEG/C₆₀ under N₂ atmosphere.

Fig. 1b gives UV-vis absorption spectra of GO, FA–GO and FA–GO–PEG/C₆₀ in H₂O. GO shows an absorption band at 228 nm, and the conjugation of FA to GO is confirmed by UV-vis spectra. A peak at 228 nm disappeared while a new peak at ∼281 nm appeared due to the presence of FA in the FA–GO. C₆₀ shows three typical absorption peaks located at 215, 265, 335 nm in n-hexane (Fig. S1†). As water is more polar than n-hexane, the absorption peaks of C₆₀ will show red shifts in water environment. The peak at ∼280 nm of FA–GO–PEG/C₆₀ may both due to the characteristic peak of C₆₀ (ref. 17) and FA. Meanwhile, FA–GO–PEG/C₆₀ shows strong absorbance at 200–400 nm, which may lead overlapping the characteristic peaks of C₆₀. The FA–GO–PEG/C₆₀ solution showed increased optical absorption values in the NIR and visible wavelengths (Fig. 2b). This increased optical absorption may attribute to the hydrolysis of esters and opening of epoxide groups on the GO sheets under basic conditions during the carboxylation process.⁴¹ Meanwhile, since all the samples have the same concentration and light path-length, the increase may also due to doping effect¹³ and the higher molar absorption coefficient of FA–GO–PEG/C₆₀. Owing to this enhancement of absorption, FA–GO–PEG/C₆₀ could be an excellent PS, thereby achieving improved synergistic PTT/PDT effects.

Fig. 2 (a) TEM image of GO, (b) TEM image of FA–GO–PEG/C₆₀, (c) AFM image of GO with height profile diagram, (d) AFM image of FA–GO–PEG/C₆₀ with height profile diagram.

Raman spectra of the GO, C₆₀, FA–GO and FA–GO–PEG/C₆₀ are also conducted, as shown in Fig. 1c. As expected, the GO shows an intense tangential mode (G band) at 1581 cm⁻¹, with a disordered-induced peak (D band) at 1368 cm⁻¹, while C₆₀ shows the A_g-breathing and the A_g-pinch modes at 478 cm⁻¹ and 1458 cm⁻¹, and additional H_g mode at 1523 cm⁻¹. Since the intensities for some H_g-modes are very weak in the room temperature spectra, some H_g lines of C₆₀ aren't detected in the present study. For the FA–GO and FA–GO–PEG/C₆₀, peaks are clearly observed at 1368 and 1581 cm⁻¹, which can be assigned to the D and G bands of the graphene. The peak at 2724 cm⁻¹ corresponding to the overtone of the D band and the peak at 2951 cm⁻¹ associating with the D + G band also indicates that increased disorder has appeared in graphene. The D-band to G-band intensity ratio (I_D/I_G) has been widely used as a measure for the disorder or the extent of covalent modification of the graphene surface. In the present work, a slight increase in the I_D/I_G ratio from 0.68 for the GO to 0.77 for FA–GO is observed. Moreover, the ID/IG of the FA–GO (0.77) is lower than FA–GO–PEG/C₆₀ (0.83). The results indicate that the grafting of FA, PEG and C₆₀ onto graphene produces more defects and introduces more functional groups. The small peak at 491 cm⁻¹ in the FA–GO–PEG/C₆₀ Raman spectrum should be assigned to the A_g-breathing mode of the C₆₀, which indicate the successful grafting of C₆₀. This A_g peak is shifted by 13 cm⁻¹ compared with the pure C₆₀, which suggests a strong interaction between the C₆₀ cage and the GO sheet.¹⁶ To further demonstrate the attachment of C₆₀, the fluorescence spectra of FA–GO–PEG/C₆₀ are provided (Fig. S2†). The results show week diagnostic fluorescence (excitation wavelength: 352 nm; emission wavelength: 438 nm) generated by C₆₀ component, which confirm the loading of C₆₀ to the graphene sheets.

To quantitatively determine the attaching of C₆₀, TG analysis is employed. The initial degradation temperature of GO (the temperature at 10 wt% mass loss, T_i) is 210 °C, and the mass residue is 59.0 wt% at 600 °C (Fig. 1d). After modified by FA and PEG, the T_i of FA–GO–PEG is delayed to 238 °C and its residue increases to 66.3 wt%. As the pristine C₆₀ is thermally stable, its mass residue is 98.1 wt% at 600 °C. Due to the higher thermal stability of C₆₀, the T_i of FA–GO–PEG/C₆₀ is 300 °C, while the mass residue at 600 °C is significantly increases to 74.5 wt%. By comparing the mass residue of C₆₀, FA–GO–PEG, and FA–GO–PEG/C₆₀, the content of C₆₀ in FA–GO–PEG/C₆₀ can be calculated, which is 25.5 wt%.

The morphology of GO and FA–GO–PEG/C₆₀ is characterized by TEM. As can be seen (Fig. 2a), the GO displays flake-like shapes with wrinkles and frees from any particulate contamination. By contrast, the corresponding TEM image for the FA–GO–PEG/C₆₀ given in Fig. 2b clearly shows the surface grafted by particles with an average size of 2–5 nm characteristic of C₆₀ aggregates. To observe and characterize the topography of the GO and FA–GO–PEG/C₆₀, AFM is applied as a suitable technique, as shown in Fig. 2c and d. Analysis of GO by AFM reveals the GO with ∼0.81 nm in topographic height (Fig. 2c), which is in good consistency with the typical thickness of the observed single-layer GO (∼0.8 nm). The sheet thickness of FA–GO–PEG/C₆₀ (∼3.29 nm) is much higher than that of GO (Fig. 2d), likely owing to the covalent PEGylation that offers more condensed surface polymer coating on GO surface. In addition, the size of the C₆₀ particles is measured ∼2 nm. The TEM and AFM results confirm the attachment of the C₆₀ on the surface of GO platelets.

As a matter of fact, C₆₀ is not able to solve in H₂O but solve in some nonpolar or low-polar solvents such as toluene. Modification of C₆₀ with hydrophilic addend produces an amphiphilic molecule, which tends to form aggregates, such as vesicles, rods, globules, membranes, and linear assemblies. Our previous study has demonstrated that the aggregate size influences the biological activities of water-soluble C₆₀ derivatives.^44–46 Generally speaking, the aggregation of C₆₀ would reduce surface-to-volume, enhance steric hindrance, and reduce the intracellular content, which further reduces the biological activities of C₆₀. In the present study, GO sheet is decorated with C₆₀ with a size of ∼2 nm. It seems that the nucleophilic addition firmly fixes the C₆₀ on the graphene, which makes C₆₀ unable to move freely and can't form large aggregates. In this way, C₆₀ will exert its biological activities more effectively.

3.2. Photodynamic and photothermal properties of FA–GO–PEG/C₆₀ nanohybrid

Singlet oxygen generation is the critical step in PDT. In this work, the ¹O₂ generation by GO, FFA and FA–GO–PEG/C₆₀ is measured under irradiation by a 532 nm laser (Fig. 3a). As expected, GO does not show any ¹O₂ production as there is no change in the absorption with increasing laser exposure time. FA–GO–PEG/C₆₀ shows a sharp decrease in RNO absorption with increasing time of laser exposure, indicating rapid generation of ¹O₂. Our previous work has been demonstrated that the FFA could generate ¹O when photoexcited.⁴⁶ In the present study, the FA–GO–PEG/C₆₀ shows similarly rapid absorption bleaching compared to the FFA, which indicates that the ¹O₂ production ability by FA–GO–PEG/C₆₀ is substantially high (80–90% of FFA). It has been shown previously that the GO would act as efficient quenchers, which significantly reduce the ¹O₂ production of PSs loaded on the GO.^41,42 In the present work, however, the ¹O₂ production ability of FA–GO–PEG/C₆₀ is similar with FFA. It seems that the quenching effect of ¹O₂ production from C₆₀ by FA–GO–PEG appears to be less drastic compared to previous study, and the reason may relate to the unique structure and properties of C₆₀ and graphene. On one hand, the aggregation of FFA (∼120 nm) reduces the ¹O₂ generation ability of C₆₀ core.⁴⁶ On the other hand, owing to the doping effect and aggregation prevention effect of GO, the C₆₀ on the FA–GO–PEG sheets will be more effective in light absorbing and produce more ¹O₂. The excellent ¹O₂ generation ability of C₆₀ attached on graphene allows us to use FA–GO–PEG/C₆₀ for PDT treatment.

Fig. 3 (a) Singlet oxygen (¹O₂) production by GO, FFA and FA–GO–PEG/C₆₀ after irradiation with 532 nm laser (0.1 W cm⁻²) for different time periods. (b) Photothermal effect of FFA and FA–GO–PEG/C₆₀ solution exposed to the 808 nm laser for different time periods at a power density of 2 W cm⁻².

The photothermal properties of FA–GO–PEG/C₆₀ are evaluated in the present study. Since PTT does not require oxygen to interact with the target cells or tissues, it is able to use longer wavelength light, which is less energetic and therefore less harmful. The biological tissues also exhibit a deep penetrability with very low absorption of NIR photons in the wavelength range of 700 to 1100 nm. Thus, 808 nm laser is selected as excitation light source in the present study. While the temperature of water only increases 2.3 °C after 808 nm laser irradiation, the solution temperature of FA–GO–PEG/C₆₀ rises rapidly from 26 °C to 44.8 °C in 3 min, showing a significant temperature increase of 18.8 °C (Fig. 3b). It is worth noting that, the water soluble C₆₀ derivative-FFA converts the NIR light radiation to vibrational energy to elevate the temperature for 6.4 °C. The photothermal activity of C₆₀ shows great promise to achieve excellent synergistic phototherapy effects of FA–GO–PEG/C₆₀. Following Roper's report,⁴⁷ the photothermal conversion efficiency of FA–GO–PEG/C₆₀, η, is calculated using the following equation.

where h is heat transfer coefficient, S is the surface area of the container, and the value of hS is obtained from the Fig. S3.† The T_max is the equilibrium temperature, T_surr is ambient temperature of the surroundings, and (T_max − T_surr) is 19.5 °C according to Fig. S3.† The Q_dis expresses heat dissipated from light absorbed by the quartz sample cell itself, and it was measured independently to be 10.6 mW. I is incident laser power (2 W cm⁻²), A₈₀₈ is the absorbance (0.3732) of FA–GO–PEG/C₆₀ at 808 nm (Fig. 1b). Thus, η of the FA–GO–PEG/C₆₀ can be calculated to be 44.4%. The previous studies have reported many nano materials used in PTT, especially gold nanoparticles. It is reported that the η of Au/SiO₂ nanoshells, Au/Au₂S nanoshells and Au nanorods is 30%, 59% and 55%, respectively.⁴⁸ Compared with the Au nanoparticles, FA–GO–PEG/C₆₀ exhibits considerable PTT effects.

3.3. FA–GO–PEG/C₆₀ induced phototoxicity and apoptosis in HeLa cells

Phototoxicity of PDT, PTT and PDT/PTT combined treatment on HeLa cells are evaluated in the present study. Without light irradiation, GO, FFA and FA–GO–PEG/C₆₀ does not show any significant dark toxicity towards HeLa cells (Fig. 4a). The cell viability is still 98.4% after treated with 20 μg mL⁻¹ FA–GO–PEG/C₆₀ in dark (Fig. 4b), which proves that FA–GO–PEG/C₆₀ is cytocompatible. As for the effect of PTT, the 808 nm laser irradiation with 10 μg mL⁻¹ GO and FA–GO–PEG/C₆₀ reduces the cell viability to 80.4% and 74.6% respectively, demonstrating the photothermal activities of graphene sheets (Fig. 4a). As for the effect of PDT, the cell viability of group FFA and FA–GO–PEG/C₆₀ decreases to 72.4% and 35.4% after irradiation of 532 nm laser, respectively (Fig. 4a). It seems that the cells are more sensitive to PDT in the present experimental conditions. The 808 nm laser irradiation at a low power only generates mild heating, which induces small amount of cell death. However, the light-induced local heating of graphene sheets may cause damage to the cell membrane, which further increases cellular uptake and makes ¹O₂ attacks the cells more easily. As expected, when the PTT and PDT are combined, the cell viability was remarkably reduced to 3.1% after treated with 10 μg mL⁻¹ FA–GO–PEG/C₆₀, which is evidently much lower than that by the individual ones (Fig. 4a). In the present study, we also cultures HeLa cells with FA–GO–PEG/C₆₀ at different concentrations for MTT assay (Fig. 4b). Our results indicate that with the increasing concentrations of FA–GO–PEG/C₆₀, the lethality increases, suggesting a dose-dependent effect in vitro. The cytotoxicity analysis shows that 0–20 μg mL⁻¹ GO, FA–GO and FA–GO–PEG/C₆₀ doesn't cause obvious cell death in dark, which indicates the biocompatibility of the nanocarbons (Fig. S4†). Besides, our observations demonstrate that the laser light irradiation doesn't cause significant cytotoxicity, which confirms the phototoxicity of FA–GO–PEG/C₆₀ are result from its ability to generate ¹O₂ and heating when photoexcited.

Fig. 4 FA–GO–PEG/C₆₀ induced phototoxicity in HeLa cells. (a) Cell viability data of HeLa cells incubated with GO, FFA and FA–GO–PEG/C₆₀; (b) cell viability of HeLa cells incubated with different concentrations of FA–GO–PEG/C₆₀ (0–20 μg mL⁻¹). Black, red, blue and purple colors represent cells without any light irradiation, with only 808 nm laser irradiation (light dose: 360 J cm⁻²), with only 532 nm laser irradiation (light dose: 30 J cm⁻²), and with both 808 nm and 532 nm laser irradiation, respectively. Cell viability was measured by the conventional MTT reduction assay. Data are presented as mean ± S.D. (n = 3).

It is interesting to notice that the PDT effect of FA–GO–PEG/C₆₀ is much better than FFA, although their light-induced ¹O₂ generation performances are similar (Fig. 4a). To understand why this happens, cellular uptake and intracellular ROS accumulation are analyzed.

Fig. 5 shows the fluorescence images of HeLa cells after being incubated with GO–FITC, FA–GO–FITC and FA–GO–PEG/C₆₀–FITC, respectively. Much stronger fluorescence can be seen in the HeLa cells after incubation with FA–GO–FITC than GO–FITC, which suggests specific targeting of FA–GO under the leading of FA molecules. In the present study, FA–GO–PEG/C₆₀–FITC shows stronger fluorescence inside cells than FA–GO–FITC. Graphene sheets functionalized with PEG exhibit high solubility and stability in physiological solutions, which would enhance the endocytosis of FA–GO–PEG/C₆₀ by cells. The corresponding intracellular fluorescence intensity is also given in Fig. 6a to make a quantitative comparison. The fluorescence intensity of FA–GO–PEG/C₆₀ group (1082) is much higher than GO (303) and FA–GO (746) after 24 h incubation. The result indicates that the FA–GO–PEG/C₆₀ can be quickly and effectively delivered into the targeted tumor cells. It is assumed that the enhanced uptake would make the FA–GO–PEG/C₆₀ kills tumor cells more effectively, and the intracellular ROS measurement proves the speculation. Accumulation of intracellular ROS is detected by using DCF-DA (Fig. 6b). The intracellular ROS generation presents the same trend as the cell viability decrease. The more intracellular ROS generated, the more decrease in cell viability. The results indicate that anti-cancer activity of FA–GO–PEG/C₆₀ is associated with the intracellular ROS production. The higher uptake of FA–GO–PEG/C₆₀ based on the folate receptor and PEG-induced endocytosis transports more C₆₀ molecule into the cells, which exerts the phototoxicity inside the cells and produces the enhanced phototherapy effects. The Fig. 6b also indicates that the intracellular ROS accumulation irradiated by 808 + 532 nm laser is higher than that of only 532 nm. The PTT effects cause overheating to cells, which induce cell death and apoptosis. ROS play an important role in the process of apoptosis in many cell types. Increased ROS generation is a key phenomena, which can be frequently observed in cells subjected to anticancer drugs treatment, that is, accumulation of ROS inside the cell often signalizes apoptosis.⁴⁹ The previous study has also indicated that PTT by NIR-excited graphene nanoparticles triggers apoptotic/necrotic cancer cell death associated with ROS.⁵⁰ While irradiated by 808 + 532 nm laser, the intracellular ROS level is both related with PDT and PTT effects. The cell damage caused by PTT elevates the ROS level in the cell, which further enhances the PDT effects of FA–GO–PEG/C₆₀.

Fig. 5 Cellular uptake of GO, FA–GO and FA–GO–PEG/C₆₀. Data are presented as confocal fluorescence images of cells incubated with GO, FA–GO and FA–GO–PEG/C₆₀ after different periods of time (1, 6, 12 and 24 h).

Fig. 6 (a) The corresponding fluorescence intensities of HeLa cells in Fig. 5; (b) intracellular ROS generation by GO, FFA and FA–GO–PEG/C₆₀. HeLa cells are exposed to 808 nm laser (light dose: 360 J cm⁻²), 532 nm laser (light dose: 30 J cm⁻²), or 808 nm and 532 nm laser for light irradiation, respectively. Cells incubated with GO, FFA and FA–GO–PEG/C₆₀ without irradiation served as dark group. The fluorescent probe DCF-DA was used to monitor the intracellular accumulation of ROS. The results from representative of at least three experiments are presented. Values are presented as mean ± S.D. (n = 3).

To explore the possible cell death mechanism, double staining of annexin V and PI is performed in the presence of laser irradiation. Cells are stained with annexin V which reacts against externalized phosphatidylserine, a characteristic of apoptotic cells, and PI dye which stains both late-stage apoptotic and necrotic cells displaying permeable membranes. Fig. 7 shows the dot plots of the flow cytometry analysis. In control group, cells show only very weak staining with annexin V and PI (Fig. 7a). As expected, the change trend of FA–GO–PEG/C₆₀ induced apoptosis is the same as cell viability. The PTT and PDT combining therapies remarkably increase the percentage of apoptosis to 54.27%, which is evidently better than the individual therapy (Fig. 4a). Meanwhile, the results indicate that the cell apoptosis or death is mainly dependent on the PDT effect, and apoptotic cell death is predominant in all experiments. The apoptosis, a preferred mode of killing the cancer cells in cancer therapy, is induced synergistically, resulting in distinguished improvement in phototherapy activities.

Fig. 7 FA–GO–PEG/C₆₀ induced apoptosis in HeLa cells. Data are presented as the flow cytometric histograms of (a) untreated cells, (b) cells exposed to 808 nm laser (light dose: 360 J cm⁻²), (c) cells exposed to 532 nm laser (light dose: 30 J cm⁻²), (d) cells exposed 808 nm and 532 nm laser.

Based on the above study, the hypothetical mechanism of PTT and PDT combined therapy of FA–GO–PEG/C₆₀ is shown in Scheme 2. The combination of these two inconceivable nanocarbons leads to remarkable synergies in phototherapy. First, the hybridization process prevents the restacking of graphene and the aggregation of C₆₀, which supply more surface active sites and increase the PDT efficiency of FA–GO–PEG/C₆₀. Second, owing to the doping effect, the FA–GO–PEG/C₆₀ exhibits boarder absorption range and higher absorbance. Third, the PTT effects of FA–GO–PEG/C₆₀ cause obvious cell damage and achieve synergistic enhancement of phototherapy effects. Fourth, the graphene carrier equipped with folic acid and PEG has specific targeting to tumor cells, which significantly enhances the cellar uptake of FA–GO–PEG/C₆₀. Once the FA–GO–PEG/C₆₀ is taken by tumor cells, it causes heating and significant intracellular ROS production under light irradiation. The photo generated ROS and heating cause a marked decrease in cell survival and elevation of oxidative stress. Finally, the phototherapy effects of FA–GO–PEG/C₆₀ apparently involves induction of the “programmed” cell death, known as apoptosis. The work provides a facile way to synthesize novel graphene/C₆₀ nanohybrid with excellent therapeutic efficiency for cancer. It should be valuable for developing highly effective nanocarbons-based cancer theranostics.

Scheme 2 The hypothetical mechanism of synergistic enhancement of FA–GO–PEG/C₆₀ in combined PTT and PDT.

4. Conclusions

In conclusion, we have developed a facile and efficient synthetic route to prepare a novel FA–GO–PEG/C₆₀ nanohybrid that combines the multiple functions and advantages of C₆₀ and graphene. In the present work, FA and PEG functionalized graphene is used as tumor targeting nanocarrier for C₆₀, which offers dramatically improved cell uptake. Furthermore, the hybridization process enhances the light absorption properties, prevents the restacking of graphene, and limits the aggregation of C₆₀, thereby shows an enhanced phototherapy effect of FA–GO–PEG/C₆₀. After light irradiation, FA–GO–PEG/C₆₀ causes a marked decrease in cell survival and elevation of oxidative stress, and induces apoptotic death. Compared with PDT or PTT alone, the combined treatment with FA–GO–PEG/C₆₀ shows higher cell apoptosis and death, indicating a synergistic effect of combination phototherapy. Our work reveals that the hybridization of the two nanocarbons represents a promising biomedicine for cancer therapy.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 51103031), the Research Fund for the Doctoral Program of Higher Education of China (no. 20112302120034), the Special Foundation of China Postdoctoral Science (no. 201003436), the Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (no. QA201415), the Fundamental Research Funds for the Central Universities (no. HIT. NSRIF. 2012034), and the China Scholarship Council (no. 201303070100).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra13427c

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