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Folic acid-conjugated graphene oxide for cancer targeted chemo-photothermal therapy
Journal of Photochemistry and Photobiology B: Biology 120 (2013) 156–162
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Folic acid-conjugated graphene oxide for cancer targeted chemo-photothermal therapy X.C. Qin, Z.Y. Guo ⇑, Z.M. Liu, W. Zhang, M.M. Wan, B.W. Yang MOE Key Laboratory of Laser Life Science & Laboratory of Photonic Chinese Medicine, College of Biophotonics, South China Normal University, Guangzhou 510631, China
a r t i c l e
i n f o
Article history: Received 19 July 2012 Received in revised form 3 November 2012 Accepted 10 December 2012 Available online 29 December 2012 Keywords: Graphene oxide Cancer Targeting Chemotherapy Photothermal therapy
a b s t r a c t Nanographene oxide (NGO), a new type of nanomaterial for anticancer drugs delivery and near-infrared (NIR)-mediated photothermal ablation of tumors, has been used in the combination of photothermal therapy and chemotherapy. Herein, targeted chemo-photothermal therapy based on polyvinylpyrrolidone (PVP) functionalized NGO was achieved. Folic acid (FA), a common target molecule to cancer cells, was conjugated to NGO via covalent amide bond. The obtained FA–NGO–PVP was proved to be an ideal pH-responsive nanocarrier for delivery of an anticancer drug doxorubicin (DOX) with the loading ratio more than 100%. In vitro experiments were then performed with the combination of chemotherapy and NIR photothermal therapy. The results demonstrated that the targeted chemo-photothermal therapy could specifically deliver drug and heat to tumor sites and showed excellent efficacy of anticancer therapy. Thus, FA–NGO–PVP could be used as a novel nanomaterial for selective chemo-photothermal therapy. Crown Copyright Ó 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Chemotherapy is an important clinical therapeutic modality for a wide variety of cancers such as acute promyelocytic leukemia [1,2] and lung cancer [3–5]. However, the low therapeutic efficacy because of the drug resistance, low cellular uptake efficiency, and high side-effects (such as liver and kidney damage [6,7], hair loss [8,9], nausea and cardiac toxicity [10–12]) limit the clinical applications of chemotherapy. Therefore, targetselective drug delivery with high efficiency, low toxicity and minimal side effects has been developed to improve the efficacy of chemotherapeutics [13,14]. On the other hand, near-infrared (NIR) photothermal therapy based on nanoparticles provides a promising treatment strategy for efficient tumor ablation with minor injury to the surrounding tissue. A wide variety of nanoparticles including graphene [15], carbon nanotubes [16–19] or gold nanospheres [20] have been employed to photothermal therapy due to their strong absorbance in the NIR region. Combined chemotherapy and photothermal therapy has been proven to be an effective strategy to improve the efficacy of cancer therapy and to reduce the drug resistance [21–24]. However, non-targeted delivery of drugs and heat to the tumor area can lead to undesired side effects to normal tissues.
⇑ Corresponding author. Tel./fax: +86 20 85211428. E-mail address: [email protected] (Z.Y. Guo).
Nanographene oxide (NGO), a two-dimensional material featured by a variety of reactive oxygen functional groups such as epoxy and hydroxyl groups on the basal plane and carboxylic acid groups at the sheet edges [25,26], has been widely used for cancer photothermal therapy, drug delivery, and biosensor [27–29]. For targeted chemotherapy, much work has been carried out using functionalized graphene oxide as the efficient nanocarrier [28,30,31]. In our latest work, we firstly demonstrated the ability of PEGylated NGO as a platform for combined chemotherapy and NIR photothermal tumor therapy, which exhibited obvious synergistic effects both in vivo and in vitro [32]. However, the construction of multifunctional graphene nanosheets for targeted chemo-photothermal therapy has not yet been carried out. Polyvinylpyrrolidone (PVP), a nonionic, nontoxic and biocompatible polymer surfactant, is often employed as a stabilizing agent and dispersant in the synthesis of metal nanostructures [33]. On the other hand, PVP can also serve as a biocompatible stabilizer of NGO in physiological environment [34]. In this work, PVPfunctionalized NGO was fabricated via non-covalent p–p stacking interaction. For targeted drug delivery, folic acid (FA) molecules were bond covalently with NGO–PVP for targeting the folate receptors, which were considered to be over-expressing on numerous cancer cell surfaces [17,35]. The obtained FA–NGO–PVP exhibited ultrahigh loading ratio of doxorubicin (DOX), which could specifically deliver both the heat and drug to the tumorigenic region. Targeted chemo-photothermal ablation of tumor cells were then performed using FA–NGO–PVP as the platform for drug delivery and heat transformation.
1011-1344/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.12.005
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unreacted materials were separated out by dialyzing against sodium bicarbonate solution (pH 8.0) for 48 h followed by dialysis against distilled water for another 24 h.
2. Methods and materials 2.1. Materials The major agents used in this research were as follows: Graphite flake, was purchased from Shanghai Yifan Graphite Co., Ltd.; Chloroacetic acid (ClCH2COOH), rhodamine B (Rho B), dimethyl sulfoxide (DMSO), N-(3-dimethylamino propyl-N0 -ethylcar-bodiimide) hydrochloride (EDCHCl), N-hydroxysuc-cinimide (NHS), doxorubicin (DOX) and folic acid (FA) were purchased from Sigma–Aldrich Co.; polyvinylpyrrolidone (PVP, K30) was purchased from MYM biological Technology Co., Ltd.; Trypsin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-ltetrazolium bromide (MTT), DMEM cell culture medium, penicillin, fetal bovine serum (FBS) and streptomycin were purchased from Gibco Invitrogen. Other reagents were purchased from China National Medicine Corporation and used as received. The dialysis bags (MWCO = 10,000) were purchased from Spectrum Laboratories Inc. 2.2. Synthesis of FA–NGO–PVP Graphene oxide (GO) was prepared from native graphite flake according to our previous work [32]. To obtain NGO, GO was cracked by ultrasonic probe at 570 W for 2 h. For carboxylation of NGO, NaOH (1.2 g) and ClCH2COOH (1.0 g) were added to NGO aqueous suspension (10 mL, 2 mg/mL), followed by bath sonication at 500 W for 3 h to convert OH groups to COOH. The NGO–COOH solution was neutralized with dilute hydrochloric acid and purified by repeated rinsing and filtrations, producing well dispersed NGO– COOH in deionized water. Preparation of NGO–PVP was carried out as following: typically, 20 mg of PVP was added into 10 mL NGO– COOH aqueous suspension (0.5 mg/mL) followed by a 30 min ultrasonication. The obtained NGO–COOH and PVP mixture solution was stirred at 50 °C for 18 h. The product was well dispersed NGO–PVP. FA was then conjugated with NGO–PVP by reaction between the COOH groups of the NGO–PVP and NH2 groups on the FA molecules. Briefly, EDC and NHS were added to the NGO–PVP suspension (10 mL, 1 mg/mL) and the mixture was sonicated for 2 h. Then, 0.5% FA (2 mL, adjusted to pH 8.0 using sodium bicarbonate solution) was added and the mixture was stirred overnight. The
2.3. Characterization The morphology and size of FA–NGO–PVP was performed by a JEM-2100HR transmission electron microscopy (TEM, JEOL, Japan) operated at 200 kV. Raman spectra of samples were measured using a confocal Raman microspectrometer (Renishaw InVia, Derbyshire, England) with 514 nm laser source. The optical properties of graphene oxide were characterized by ultraviolet–visible (UV– Vis) absorbance spectrometer (NanoDrop, ND-1000). FT-IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer. 2.4. Laser irradiation and temperature measurement Nanographene oxide suspensions were diluted to a desired final concentration (2.5, 5 or 10 lg/mL) in phosphate buffered saline containing 5% fetal calf serum (FCS; Sigma–Aldrich). The suspensions (500 lL) in 15 mL conical-bottom centrifuge glass tubes were illuminated with an 808 nm continuous-wave NIR laser (2 W/cm2, 1–5 min). The increase in temperature was measured by a thermocouple immersed into suspension. 2.5. DOX loading and release of FA–NGO–PVP Loading of doxorubicin (DOX) onto FA–NGO–PVP (or NGO–PVP) was carried out by simply mixing 0.4 mg/mL of DOX (dissolved in DMSO) with FA–NGO–PVP solution (0.2 mg/mL) at pH 8 overnight. The final DMSO concentration was kept at 50%. Unbound excess DOX was removed by repeated washing and ultrafiltration through a 100 kDa filter (Millipore). The resulting FA–NGO–PVP/DOX composites were resuspended and stored at 4 °C. The concentration of DOX loaded onto FA–NGO–PVP was measured by the absorbance peak at 490 nm after subtracting the absorbance of FA–NGO–PVP at that wavelength with a molar extinction coefficient of 1.05 104 mol/(L cm). The release of DOX from FA–NGO–PVP was investigated by placing FA–NGO–PVP/DOX samples into the dialysis bag, which
Fig. 1. Schematic illustration of the preparation of FA–NGO–PVP conjugates.
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were dialyzed in 50 mL phosphate buffered saline (PBS) at pH 7.4 and 5.5 respectively (37 °C). For measuring the DOX release mediated by the photothermal effect, FA–NGO–PVP/DOX (50 lg/mL, in terms of NGO concentration) was irradiated by NIR laser light (2 W/cm2 for 3 min) before being placed into the dialysis bag. The release reservoir was kept under constant stirring, and at various time points, the dialysis bags were taken out for characterization. The concentration of DOX released from FA–NGO–PVP into distilled water was quantified using UV–Vis spectroscopy. 2.6. Cell culture and cytotoxicity measurements Hela (FA receptor positive) and A549 (FA receptor negative) cells were cultured in Dulbecco0 s modified Eagle0 s medium (DMEM) at 37 °C in a humidified incubator atmosphere containing 5% CO2 and 95% air. The DMEM contained 10% fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (100 mg/mL). The cytotoxicity of FA–NGO–PVP/DOX under laser irradiation was performed by MTT assay. Briefly, Hela cells were plated into 96-well plates and allowed to adhere prior to addition of various concentrations of free DOX, NGO–PVP, FA–NGO–PVP, NGO–PVP/ DOX or FA–NGO–PVP/DOX. Then, the cells were or were not irradiated with an 808 nm continuous-wave NIR laser with the power density of 2 W/cm2 for 5 min. After that, the cells were incubated at 37 °C for a further 24 h. Cell viability was measured using the MTT assay according to the manufacturer suggested procedures. To study the cytotoxic effect of FA–NGO–PVP, Hela cells were
exposed to FA–NGO–PVP with different concentrations. Cell viability was measured after the incubation at 37 °C for 24 h. 2.7. Cellular uptake and internalization Confocal Raman scattering micro-spectroscopy was used to investigate the cellular uptake of FA–NGO–PVP. The composite of FA–NGO–PVP/Rho B was formed by simply mixing of FA–NGO– PVP (0.85 mg/mL, 5 mL) with Rho B (1 mg/mL, 0.3 mL) for 24 h at room temperature and then repeated washing to remove unbound Rho B. Hela cells (FA+) and A549 cells (FA) growing in 35-mm Petri dishes (1 104 per well) were incubated with FA–NGO–PVP/Rho B suspension (20 lg/mL) for 30 min, then rinsed three times with PBS. Laser-induced fluorescence spectra were recorded using Renishaw InVia Raman microspectrometer with a 514 nm laser as the excitation source. Fluorescence spectral maps were collected in a StreamLine mode at 1 s exposure time at wavelength center 580 nm using a 50 objective. The distribution of FA–NGO–PVP in cells was plotted by integrating the signals from 565 to 600 nm. 3. Results and discussions 3.1. Synthesis and characterization of FA–NGO–PVP Fig. 1 shows the synthetic route for the preparation of FA–NGO– PVP conjugate. The strategy involved sonication and carboxylation of GO resulted in NGO–COOH and then NGO–COOH was
Fig. 2. (a) TEM image of NGO. (b) Raman spectrum of NGO. (c) FTIR spectra of the NGO–PVP and FA–NGO–PVP measured in KBr pellets. (d) UV–Vis spectra of NGO–COOH, NGO–PVP and FA–NGO–PVP.
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solubilized in the aqueous phase by non-covalent interaction with PVP. On the base of this NGO functionalization, the conjugation of FA to NGO–COOH was achieved by the reaction between the NH2 groups of FA and COOH groups of NGO–COOH. As depicted in Fig. 2a, the sizes of resulting NGO nanosheets were mostly less than 100 nm according to TEM characterization. The covalent binding of NGO with FA was confirmed by FTIR measurements (Fig. 2c). The absorption peak at 1632 cm1 can be assigned to the vibration mode of the CO–NH in the FA–NGO–PVP [24]. Conjugation of FA to NGO was also confirmed by UV–Vis absorption spectra (Fig. 2d). A peak at 232 nm disappeared while a new peak at 276 nm appeared due to the presence of FA in the NGO [36]. Moreover, it was observed that the characteristic absorption peak at 250 nm in the UV–Vis spectrum of NGO–PVP which indicated the restored p–p conjugated structure of the graphene sheets due to the minor reduction of NGO (232 nm) [37,38]. In the Raman spectrum of NGO (Fig. 2b), the broaden graphite lattice (G band) at 1603 cm1 and disorder band (D band) at 1358 cm1 were observed. 3.2. Photothermal sensitivity of nanographene oxide The photothermal responsiveness of NGO–PVP and FA–NGO– PVP was compared using 2 W/cm2 NIR laser at 808 nm. As shown in Fig. 3, both of two suspensions demonstrated concentrationdependent and time-dependent temperature increases in response to NIR irradiation. Additionally, only a low concentration (2.5 lg/ mL) of NGO–PVP and FA–NGO–PVP were shown to demonstrate their extraordinary photothermal energy conversion efficiency. The temperatures were rapidly reached 50 °C within 5 min of
Fig. 3. NIR-induced heat generation. PBS suspensions containing various concentrations (2.5–10 lg/mL) of NGO–PVP (a), FA–NGO–PVP (b) were exposed to NIR laser (808 nm, 2 W/cm2) for 5 min and the temperature was measured by a thermocouple at the indicated time-points.
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irradiation. Moreover, the temperature of FA–NGO–PVP (10 lg/ mL) was above 80 °C at the end of irradiation. Importantly, FA– NGO–PVP in the same conditions generated heat more efficiently (DT 50 °C at 10 lg/mL, 5 min) than NGO–PVP (DT 40 °C at 10 lg/mL, 5 min). The more efficiently heat-generating capacity of FA–NGO–PVP may due to the minor reduction of NGO after the amidation process [37].
3.3. DOX loading and release of FA–NGO–PVP DOX was loaded onto FA–NGO–PVP (or NGO–PVP) by simply mixing 0.4 mg/mL of DOX solution with the FA–NGO–PVP solution (0.2 mg/mL) under controlled pH (pH 8.0) overnight. After removal of any unbound or undissolved DOX by centrifuge filtration and repeated rinsing, DOX loading on FA–NGO–PVP was evidenced by the deep red appearance of the FA–NGO–PVP/DOX suspension and was further confirmed by the UV–Vis spectra. The concentration of DOX loaded on FA–NGO–PVP was quantified by a strong absorption peak at around 490 nm over the FA–NGO–PVP background (Fig. 4a). The as-prepared FA–NGO–PVP/DOX was well solubilized and stable in water and physiological media for over 2 month without any significant aggregation. The loading of DOX onto FA– NGO–PVP could be attributed to simple p–p stacking and hydrophobic interactions according to previous studies [39–41].
Fig. 4. (a) UV–Vis absorption spectra of free DOX, FA–NGO–PVP, and FA–NGO–PVP/ DOX. The DOX concentration is 0.1 mg/mL for free DOX and FA–NGO–PVP/DOX. The FA–NGO–PVP concentration is 0.093 mg/mL for either FA–NGO–PVP or FA–NGO– PVP/DOX sample. Both free DOX and FA–NGO–PVP/DOX show a characteristic peak around 490 nm. The inset is the photographs of the aqueous dispersions (left: FA– NGO–PVP; middle: FA–NGO–PVP/DOX; right: free DOX). (b) The release profile of DOX from FA–NGO–PVP in buffer solutions with pH 5.5 and 7.4.
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The loading ratio of FA–NGO–PVP was calculated to be 107.5 wt% estimated from the weight ratio between drug and carrier. Such a value of loading was beyond the common drug carrier materials, which were always below 100%. The results indicated a great potential of FA–NGO–PVP as an excellent nanocarrier for drug delivery. The DOX release behavior from FA–NGO–PVP was investigated in PBS medium with different pH values (pH 7.4 and pH 5.5). As shown in Fig. 4b, a slow release of DOX was observed at pH 7.4. Only 13% of the DOX was released after 70 h dialysis. However, DOX became more water soluble at low pH value due to the protonated daunosamine group, which lead to the more release of DOX from FA–NGO–PVP. A rapid release of DOX from FA–NGO– PVP appeared at pH 5.5, and about 60% of the DOX was released after 70 h. This pH-dependent drug release mechanism from FA– NGO–PVP was important in the clinical application, because both the extracellular microenvironments of tumors tissues, intracellu-
Fig. 5. The release profile of DOX from FA–NGO–PVP in buffer solutions with pH 5.5 under laser irradiation (2 W/cm2, 3 min) at the concentration of 50 lg/mL (in terms of NGO concentration).
lar lysosomes and endosomes are acidic. Furthermore, the DOX release mediated by the photothermal effect under acidic condition (pH 5.5) was also studied. FA–NGO–PVP/DOX (50 lg/mL, in term of NGO concentration) was irradiated with NIR laser (2 W/cm2 for 3 min) before placing into the dialysis bag. As shown in Fig. 5, DOX released rapidly from FA–NGO–PVP, and more than 70% was released at 10 h after laser irradiation. The release rate gradually was almost unchanged in the following 60 h. The result indicated that laser irradiation could facilitate the drug release from the graphene nanocarrier. 3.4. In vitro cytotoxicity study The cytotoxic effect of FA–NGO–PVP was investigated by MTT assay. Human cervical carcinoma (Hela) cells were used as model. As shown in Fig. 6a, the viability of Hela cells remained above 98% even when the concentration of FA–NGO–PVP was increased to 100 lg/mL, which indicated the low cytotoxic of FA–NGO–PVP to Hela cells. To evaluate efficiency of FA–NGO–PVP for targeted chemophotothermal ablation of tumor cells, Hela cells were incubated with 2 lg/mL or 20 lg/mL of NGO–PVP, FA–NGO–PVP, free DOX, NGO–PVP/DOX and FA–NGO–PVP/DOX (in terms of DOX concentration) for 24 h prior to NIR irradiation. Fig. 6b showed a dosedependent cytotoxicity of these treatments. At the DOX concentration of 2 lg/mL, the inhibition rate of free DOX was 27%, which was somewhat higher than that of FA–NGO–PVP/DOX (18%). The lower cell-killing ability could be attributed to delayed DOX release from FA–NGO–PVP. The inhibition rate of FA–NGO–PVP/DOX + NIR group was significantly increased to 90% when the DOX concentration was 20 lg/mL, which was significantly higher than that of free DOX and FA–NGO–PVP/DOX groups (70.3% and 70.9%, respectively). It was demonstrated that FA–NGO–PVP/DOX under laser irradiation could selectively carry heat and drug to cancer cells and significantly enhance the therapeutic efficacy of chemophotothermal. The targeted uptake of FA–NGO–PVP/DOX was via receptor-mediated endocytosis. More importantly, the ability of FA to bind its receptor was not affected by the covalent amide bond, and the receptor-mediated endocytosis was unhindered [42].
Fig. 6. (a) The cell viability of Hela cells with different concentrations of NGO–PVP and FA–NGO–PVP. Cell viability remained at about 100% even at the concentration of FA– NGO–PVP up to 100 lg/mL, indicated that the FA–NGO–PVP was non-cytotoxic to Hela cells after 24 h incubation. (b) Relative cell viability of Hela cells for 24 h after treatment with various complexes with or without 2 W/cm2 of 808 nm NIR irradiation 5 min at different concentrations. The asterisks indicated P < 0.05 versus normal cells and N indicated P < 0.05 versus NGO–PVP/DOX plus laser group at the same concentration of DOX. When the P value was less than 0.05, differences were considered statistically significant.
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Fig. 7. Combined fluorescence and confocal Raman spectral imaging of internalized of FA–NGO–PVP in (a) A549 and (b) Hela cells. The fluorescence image was obtained by integrating the signals of Rho B from 565 to 600 nm.
3.5. Cellular targeting uptake and internalization of FA–NGO–PVP To study the uptake of FA–NGO–PVP by different cells with or without folate receptors, FA–NGO–PVP labeled with Rho B via physical adsorption was used. Then folate receptors positive cells (Hela) and folate receptors negative cells (A549) were incubated with FA–NGO–PVP/Rho B respectively. After 30 min of incubation, laser-induced fluorescence spectra of intracellular FA–NGO–PVP/ Rho B were collected using a confocal Raman microscope with a 514 nm semiconductor laser (10 mW, 100% laser intensity, 2 s exposure time) [43]. As shown in Fig. 7, the Hela cells treated with FA–NGO–PVP/Rho B showed strong fluorescence signals throughout the entire cell cytoplasm (Fig. 7a). However, no obvious fluorescence signals were detected in A549 cells (Fig. 7b), indicating the receptor-mediated specific uptake. The intracellular localization of FA–NGO–PVP was closely related to the threshold for photothermal damage effect. It has been reported that the internalization and mitochondrial accumulation of single-walled carbon nanotubes could enhance the sensitive of cells to NIR-mediated photothermal damage, and the property that temperature spike destroys the mitochondria membrane could lead to cell apoptosis factors, such as cytochrome c release and eventually trigger cell death [44]. However, evidences showed that folate conjugated gold nanorods were more effective in NIR-mediated thermal ablation when they were on the cell surface as compared to subcellular localization [45]. The mechanisms of graphene-mediated photothermal killing of cancer cells have been investigated to be induced cell apoptosis [46], but the subcellular localization of graphene nanoparticles was not clear. Briefly, the effective cellular uptake and internalization suggested the potential of FA–NGO–PVP as a transmembrane delivery carrier to promote the cellular specific uptake, decrease the drug side-effect, increase the drug intracellular accumulation and enhance the local photothermal killing effect.
4. Conclusion In summary, we have developed FA–NGO–PVP/DOX that could carry heat and drug specifically to cancer cells, and investigated its targeted chemo-photothermal therapeutic efficacy. The
as-prepared FA–NGO–PVP exhibited ultrahigh loading ratio of anticancer drug. Cellular uptake experiments demonstrated the internalization of FA–NGO–PVP into tumor cells via receptor-mediated endocytosis. Moreover, the folic acid conjugated NGO loaded with DOX under NIR irradiation exhibited the highest cytotoxicity to Hela cells compared with that of other treatments, indicating the efficient chemo-photothermal targeted therapy. Taken together, this work demonstrated functionalized NGO as the targeted nanocarrier for delivery of drug and heat to tumor cells, facilitating the cancer targeted chemo-photothermal therapy in one system, which might be an effective strategy to improve the efficacy of cancer therapy and reduce the drugs resistance.
Acknowledgments This work was supported by the National Natural Science Foundation of China (61275187), Specialized Research Fund for the Doctoral Program of Higher Education of China (20114407110001), the Natural Science Foundation of Guangdong Province (9251063101000009) and the cooperation project in industry, education and research of Guangdong province and Ministry of Education of P.R. China (2011A090200011).
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Folic acid-conjugated graphene oxide for cancer targeted chemo-photothermal therapy X.C. Qin, Z.Y. Guo ⇑, Z.M. Liu, W. Zhang, M.M. Wan, B.W. Yang MOE Key Laboratory of Laser Life Science & Laboratory of Photonic Chinese Medicine, College of Biophotonics, South China Normal University, Guangzhou 510631, China
a r t i c l e
i n f o
Article history: Received 19 July 2012 Received in revised form 3 November 2012 Accepted 10 December 2012 Available online 29 December 2012 Keywords: Graphene oxide Cancer Targeting Chemotherapy Photothermal therapy
a b s t r a c t Nanographene oxide (NGO), a new type of nanomaterial for anticancer drugs delivery and near-infrared (NIR)-mediated photothermal ablation of tumors, has been used in the combination of photothermal therapy and chemotherapy. Herein, targeted chemo-photothermal therapy based on polyvinylpyrrolidone (PVP) functionalized NGO was achieved. Folic acid (FA), a common target molecule to cancer cells, was conjugated to NGO via covalent amide bond. The obtained FA–NGO–PVP was proved to be an ideal pH-responsive nanocarrier for delivery of an anticancer drug doxorubicin (DOX) with the loading ratio more than 100%. In vitro experiments were then performed with the combination of chemotherapy and NIR photothermal therapy. The results demonstrated that the targeted chemo-photothermal therapy could specifically deliver drug and heat to tumor sites and showed excellent efficacy of anticancer therapy. Thus, FA–NGO–PVP could be used as a novel nanomaterial for selective chemo-photothermal therapy. Crown Copyright Ó 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Chemotherapy is an important clinical therapeutic modality for a wide variety of cancers such as acute promyelocytic leukemia [1,2] and lung cancer [3–5]. However, the low therapeutic efficacy because of the drug resistance, low cellular uptake efficiency, and high side-effects (such as liver and kidney damage [6,7], hair loss [8,9], nausea and cardiac toxicity [10–12]) limit the clinical applications of chemotherapy. Therefore, targetselective drug delivery with high efficiency, low toxicity and minimal side effects has been developed to improve the efficacy of chemotherapeutics [13,14]. On the other hand, near-infrared (NIR) photothermal therapy based on nanoparticles provides a promising treatment strategy for efficient tumor ablation with minor injury to the surrounding tissue. A wide variety of nanoparticles including graphene [15], carbon nanotubes [16–19] or gold nanospheres [20] have been employed to photothermal therapy due to their strong absorbance in the NIR region. Combined chemotherapy and photothermal therapy has been proven to be an effective strategy to improve the efficacy of cancer therapy and to reduce the drug resistance [21–24]. However, non-targeted delivery of drugs and heat to the tumor area can lead to undesired side effects to normal tissues.
⇑ Corresponding author. Tel./fax: +86 20 85211428. E-mail address: [email protected] (Z.Y. Guo).
Nanographene oxide (NGO), a two-dimensional material featured by a variety of reactive oxygen functional groups such as epoxy and hydroxyl groups on the basal plane and carboxylic acid groups at the sheet edges [25,26], has been widely used for cancer photothermal therapy, drug delivery, and biosensor [27–29]. For targeted chemotherapy, much work has been carried out using functionalized graphene oxide as the efficient nanocarrier [28,30,31]. In our latest work, we firstly demonstrated the ability of PEGylated NGO as a platform for combined chemotherapy and NIR photothermal tumor therapy, which exhibited obvious synergistic effects both in vivo and in vitro [32]. However, the construction of multifunctional graphene nanosheets for targeted chemo-photothermal therapy has not yet been carried out. Polyvinylpyrrolidone (PVP), a nonionic, nontoxic and biocompatible polymer surfactant, is often employed as a stabilizing agent and dispersant in the synthesis of metal nanostructures [33]. On the other hand, PVP can also serve as a biocompatible stabilizer of NGO in physiological environment [34]. In this work, PVPfunctionalized NGO was fabricated via non-covalent p–p stacking interaction. For targeted drug delivery, folic acid (FA) molecules were bond covalently with NGO–PVP for targeting the folate receptors, which were considered to be over-expressing on numerous cancer cell surfaces [17,35]. The obtained FA–NGO–PVP exhibited ultrahigh loading ratio of doxorubicin (DOX), which could specifically deliver both the heat and drug to the tumorigenic region. Targeted chemo-photothermal ablation of tumor cells were then performed using FA–NGO–PVP as the platform for drug delivery and heat transformation.
1011-1344/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.12.005
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unreacted materials were separated out by dialyzing against sodium bicarbonate solution (pH 8.0) for 48 h followed by dialysis against distilled water for another 24 h.
2. Methods and materials 2.1. Materials The major agents used in this research were as follows: Graphite flake, was purchased from Shanghai Yifan Graphite Co., Ltd.; Chloroacetic acid (ClCH2COOH), rhodamine B (Rho B), dimethyl sulfoxide (DMSO), N-(3-dimethylamino propyl-N0 -ethylcar-bodiimide) hydrochloride (EDCHCl), N-hydroxysuc-cinimide (NHS), doxorubicin (DOX) and folic acid (FA) were purchased from Sigma–Aldrich Co.; polyvinylpyrrolidone (PVP, K30) was purchased from MYM biological Technology Co., Ltd.; Trypsin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-ltetrazolium bromide (MTT), DMEM cell culture medium, penicillin, fetal bovine serum (FBS) and streptomycin were purchased from Gibco Invitrogen. Other reagents were purchased from China National Medicine Corporation and used as received. The dialysis bags (MWCO = 10,000) were purchased from Spectrum Laboratories Inc. 2.2. Synthesis of FA–NGO–PVP Graphene oxide (GO) was prepared from native graphite flake according to our previous work [32]. To obtain NGO, GO was cracked by ultrasonic probe at 570 W for 2 h. For carboxylation of NGO, NaOH (1.2 g) and ClCH2COOH (1.0 g) were added to NGO aqueous suspension (10 mL, 2 mg/mL), followed by bath sonication at 500 W for 3 h to convert OH groups to COOH. The NGO–COOH solution was neutralized with dilute hydrochloric acid and purified by repeated rinsing and filtrations, producing well dispersed NGO– COOH in deionized water. Preparation of NGO–PVP was carried out as following: typically, 20 mg of PVP was added into 10 mL NGO– COOH aqueous suspension (0.5 mg/mL) followed by a 30 min ultrasonication. The obtained NGO–COOH and PVP mixture solution was stirred at 50 °C for 18 h. The product was well dispersed NGO–PVP. FA was then conjugated with NGO–PVP by reaction between the COOH groups of the NGO–PVP and NH2 groups on the FA molecules. Briefly, EDC and NHS were added to the NGO–PVP suspension (10 mL, 1 mg/mL) and the mixture was sonicated for 2 h. Then, 0.5% FA (2 mL, adjusted to pH 8.0 using sodium bicarbonate solution) was added and the mixture was stirred overnight. The
2.3. Characterization The morphology and size of FA–NGO–PVP was performed by a JEM-2100HR transmission electron microscopy (TEM, JEOL, Japan) operated at 200 kV. Raman spectra of samples were measured using a confocal Raman microspectrometer (Renishaw InVia, Derbyshire, England) with 514 nm laser source. The optical properties of graphene oxide were characterized by ultraviolet–visible (UV– Vis) absorbance spectrometer (NanoDrop, ND-1000). FT-IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer. 2.4. Laser irradiation and temperature measurement Nanographene oxide suspensions were diluted to a desired final concentration (2.5, 5 or 10 lg/mL) in phosphate buffered saline containing 5% fetal calf serum (FCS; Sigma–Aldrich). The suspensions (500 lL) in 15 mL conical-bottom centrifuge glass tubes were illuminated with an 808 nm continuous-wave NIR laser (2 W/cm2, 1–5 min). The increase in temperature was measured by a thermocouple immersed into suspension. 2.5. DOX loading and release of FA–NGO–PVP Loading of doxorubicin (DOX) onto FA–NGO–PVP (or NGO–PVP) was carried out by simply mixing 0.4 mg/mL of DOX (dissolved in DMSO) with FA–NGO–PVP solution (0.2 mg/mL) at pH 8 overnight. The final DMSO concentration was kept at 50%. Unbound excess DOX was removed by repeated washing and ultrafiltration through a 100 kDa filter (Millipore). The resulting FA–NGO–PVP/DOX composites were resuspended and stored at 4 °C. The concentration of DOX loaded onto FA–NGO–PVP was measured by the absorbance peak at 490 nm after subtracting the absorbance of FA–NGO–PVP at that wavelength with a molar extinction coefficient of 1.05 104 mol/(L cm). The release of DOX from FA–NGO–PVP was investigated by placing FA–NGO–PVP/DOX samples into the dialysis bag, which
Fig. 1. Schematic illustration of the preparation of FA–NGO–PVP conjugates.
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were dialyzed in 50 mL phosphate buffered saline (PBS) at pH 7.4 and 5.5 respectively (37 °C). For measuring the DOX release mediated by the photothermal effect, FA–NGO–PVP/DOX (50 lg/mL, in terms of NGO concentration) was irradiated by NIR laser light (2 W/cm2 for 3 min) before being placed into the dialysis bag. The release reservoir was kept under constant stirring, and at various time points, the dialysis bags were taken out for characterization. The concentration of DOX released from FA–NGO–PVP into distilled water was quantified using UV–Vis spectroscopy. 2.6. Cell culture and cytotoxicity measurements Hela (FA receptor positive) and A549 (FA receptor negative) cells were cultured in Dulbecco0 s modified Eagle0 s medium (DMEM) at 37 °C in a humidified incubator atmosphere containing 5% CO2 and 95% air. The DMEM contained 10% fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (100 mg/mL). The cytotoxicity of FA–NGO–PVP/DOX under laser irradiation was performed by MTT assay. Briefly, Hela cells were plated into 96-well plates and allowed to adhere prior to addition of various concentrations of free DOX, NGO–PVP, FA–NGO–PVP, NGO–PVP/ DOX or FA–NGO–PVP/DOX. Then, the cells were or were not irradiated with an 808 nm continuous-wave NIR laser with the power density of 2 W/cm2 for 5 min. After that, the cells were incubated at 37 °C for a further 24 h. Cell viability was measured using the MTT assay according to the manufacturer suggested procedures. To study the cytotoxic effect of FA–NGO–PVP, Hela cells were
exposed to FA–NGO–PVP with different concentrations. Cell viability was measured after the incubation at 37 °C for 24 h. 2.7. Cellular uptake and internalization Confocal Raman scattering micro-spectroscopy was used to investigate the cellular uptake of FA–NGO–PVP. The composite of FA–NGO–PVP/Rho B was formed by simply mixing of FA–NGO– PVP (0.85 mg/mL, 5 mL) with Rho B (1 mg/mL, 0.3 mL) for 24 h at room temperature and then repeated washing to remove unbound Rho B. Hela cells (FA+) and A549 cells (FA) growing in 35-mm Petri dishes (1 104 per well) were incubated with FA–NGO–PVP/Rho B suspension (20 lg/mL) for 30 min, then rinsed three times with PBS. Laser-induced fluorescence spectra were recorded using Renishaw InVia Raman microspectrometer with a 514 nm laser as the excitation source. Fluorescence spectral maps were collected in a StreamLine mode at 1 s exposure time at wavelength center 580 nm using a 50 objective. The distribution of FA–NGO–PVP in cells was plotted by integrating the signals from 565 to 600 nm. 3. Results and discussions 3.1. Synthesis and characterization of FA–NGO–PVP Fig. 1 shows the synthetic route for the preparation of FA–NGO– PVP conjugate. The strategy involved sonication and carboxylation of GO resulted in NGO–COOH and then NGO–COOH was
Fig. 2. (a) TEM image of NGO. (b) Raman spectrum of NGO. (c) FTIR spectra of the NGO–PVP and FA–NGO–PVP measured in KBr pellets. (d) UV–Vis spectra of NGO–COOH, NGO–PVP and FA–NGO–PVP.
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solubilized in the aqueous phase by non-covalent interaction with PVP. On the base of this NGO functionalization, the conjugation of FA to NGO–COOH was achieved by the reaction between the NH2 groups of FA and COOH groups of NGO–COOH. As depicted in Fig. 2a, the sizes of resulting NGO nanosheets were mostly less than 100 nm according to TEM characterization. The covalent binding of NGO with FA was confirmed by FTIR measurements (Fig. 2c). The absorption peak at 1632 cm1 can be assigned to the vibration mode of the CO–NH in the FA–NGO–PVP [24]. Conjugation of FA to NGO was also confirmed by UV–Vis absorption spectra (Fig. 2d). A peak at 232 nm disappeared while a new peak at 276 nm appeared due to the presence of FA in the NGO [36]. Moreover, it was observed that the characteristic absorption peak at 250 nm in the UV–Vis spectrum of NGO–PVP which indicated the restored p–p conjugated structure of the graphene sheets due to the minor reduction of NGO (232 nm) [37,38]. In the Raman spectrum of NGO (Fig. 2b), the broaden graphite lattice (G band) at 1603 cm1 and disorder band (D band) at 1358 cm1 were observed. 3.2. Photothermal sensitivity of nanographene oxide The photothermal responsiveness of NGO–PVP and FA–NGO– PVP was compared using 2 W/cm2 NIR laser at 808 nm. As shown in Fig. 3, both of two suspensions demonstrated concentrationdependent and time-dependent temperature increases in response to NIR irradiation. Additionally, only a low concentration (2.5 lg/ mL) of NGO–PVP and FA–NGO–PVP were shown to demonstrate their extraordinary photothermal energy conversion efficiency. The temperatures were rapidly reached 50 °C within 5 min of
Fig. 3. NIR-induced heat generation. PBS suspensions containing various concentrations (2.5–10 lg/mL) of NGO–PVP (a), FA–NGO–PVP (b) were exposed to NIR laser (808 nm, 2 W/cm2) for 5 min and the temperature was measured by a thermocouple at the indicated time-points.
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irradiation. Moreover, the temperature of FA–NGO–PVP (10 lg/ mL) was above 80 °C at the end of irradiation. Importantly, FA– NGO–PVP in the same conditions generated heat more efficiently (DT 50 °C at 10 lg/mL, 5 min) than NGO–PVP (DT 40 °C at 10 lg/mL, 5 min). The more efficiently heat-generating capacity of FA–NGO–PVP may due to the minor reduction of NGO after the amidation process [37].
3.3. DOX loading and release of FA–NGO–PVP DOX was loaded onto FA–NGO–PVP (or NGO–PVP) by simply mixing 0.4 mg/mL of DOX solution with the FA–NGO–PVP solution (0.2 mg/mL) under controlled pH (pH 8.0) overnight. After removal of any unbound or undissolved DOX by centrifuge filtration and repeated rinsing, DOX loading on FA–NGO–PVP was evidenced by the deep red appearance of the FA–NGO–PVP/DOX suspension and was further confirmed by the UV–Vis spectra. The concentration of DOX loaded on FA–NGO–PVP was quantified by a strong absorption peak at around 490 nm over the FA–NGO–PVP background (Fig. 4a). The as-prepared FA–NGO–PVP/DOX was well solubilized and stable in water and physiological media for over 2 month without any significant aggregation. The loading of DOX onto FA– NGO–PVP could be attributed to simple p–p stacking and hydrophobic interactions according to previous studies [39–41].
Fig. 4. (a) UV–Vis absorption spectra of free DOX, FA–NGO–PVP, and FA–NGO–PVP/ DOX. The DOX concentration is 0.1 mg/mL for free DOX and FA–NGO–PVP/DOX. The FA–NGO–PVP concentration is 0.093 mg/mL for either FA–NGO–PVP or FA–NGO– PVP/DOX sample. Both free DOX and FA–NGO–PVP/DOX show a characteristic peak around 490 nm. The inset is the photographs of the aqueous dispersions (left: FA– NGO–PVP; middle: FA–NGO–PVP/DOX; right: free DOX). (b) The release profile of DOX from FA–NGO–PVP in buffer solutions with pH 5.5 and 7.4.
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The loading ratio of FA–NGO–PVP was calculated to be 107.5 wt% estimated from the weight ratio between drug and carrier. Such a value of loading was beyond the common drug carrier materials, which were always below 100%. The results indicated a great potential of FA–NGO–PVP as an excellent nanocarrier for drug delivery. The DOX release behavior from FA–NGO–PVP was investigated in PBS medium with different pH values (pH 7.4 and pH 5.5). As shown in Fig. 4b, a slow release of DOX was observed at pH 7.4. Only 13% of the DOX was released after 70 h dialysis. However, DOX became more water soluble at low pH value due to the protonated daunosamine group, which lead to the more release of DOX from FA–NGO–PVP. A rapid release of DOX from FA–NGO– PVP appeared at pH 5.5, and about 60% of the DOX was released after 70 h. This pH-dependent drug release mechanism from FA– NGO–PVP was important in the clinical application, because both the extracellular microenvironments of tumors tissues, intracellu-
Fig. 5. The release profile of DOX from FA–NGO–PVP in buffer solutions with pH 5.5 under laser irradiation (2 W/cm2, 3 min) at the concentration of 50 lg/mL (in terms of NGO concentration).
lar lysosomes and endosomes are acidic. Furthermore, the DOX release mediated by the photothermal effect under acidic condition (pH 5.5) was also studied. FA–NGO–PVP/DOX (50 lg/mL, in term of NGO concentration) was irradiated with NIR laser (2 W/cm2 for 3 min) before placing into the dialysis bag. As shown in Fig. 5, DOX released rapidly from FA–NGO–PVP, and more than 70% was released at 10 h after laser irradiation. The release rate gradually was almost unchanged in the following 60 h. The result indicated that laser irradiation could facilitate the drug release from the graphene nanocarrier. 3.4. In vitro cytotoxicity study The cytotoxic effect of FA–NGO–PVP was investigated by MTT assay. Human cervical carcinoma (Hela) cells were used as model. As shown in Fig. 6a, the viability of Hela cells remained above 98% even when the concentration of FA–NGO–PVP was increased to 100 lg/mL, which indicated the low cytotoxic of FA–NGO–PVP to Hela cells. To evaluate efficiency of FA–NGO–PVP for targeted chemophotothermal ablation of tumor cells, Hela cells were incubated with 2 lg/mL or 20 lg/mL of NGO–PVP, FA–NGO–PVP, free DOX, NGO–PVP/DOX and FA–NGO–PVP/DOX (in terms of DOX concentration) for 24 h prior to NIR irradiation. Fig. 6b showed a dosedependent cytotoxicity of these treatments. At the DOX concentration of 2 lg/mL, the inhibition rate of free DOX was 27%, which was somewhat higher than that of FA–NGO–PVP/DOX (18%). The lower cell-killing ability could be attributed to delayed DOX release from FA–NGO–PVP. The inhibition rate of FA–NGO–PVP/DOX + NIR group was significantly increased to 90% when the DOX concentration was 20 lg/mL, which was significantly higher than that of free DOX and FA–NGO–PVP/DOX groups (70.3% and 70.9%, respectively). It was demonstrated that FA–NGO–PVP/DOX under laser irradiation could selectively carry heat and drug to cancer cells and significantly enhance the therapeutic efficacy of chemophotothermal. The targeted uptake of FA–NGO–PVP/DOX was via receptor-mediated endocytosis. More importantly, the ability of FA to bind its receptor was not affected by the covalent amide bond, and the receptor-mediated endocytosis was unhindered [42].
Fig. 6. (a) The cell viability of Hela cells with different concentrations of NGO–PVP and FA–NGO–PVP. Cell viability remained at about 100% even at the concentration of FA– NGO–PVP up to 100 lg/mL, indicated that the FA–NGO–PVP was non-cytotoxic to Hela cells after 24 h incubation. (b) Relative cell viability of Hela cells for 24 h after treatment with various complexes with or without 2 W/cm2 of 808 nm NIR irradiation 5 min at different concentrations. The asterisks indicated P < 0.05 versus normal cells and N indicated P < 0.05 versus NGO–PVP/DOX plus laser group at the same concentration of DOX. When the P value was less than 0.05, differences were considered statistically significant.
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Fig. 7. Combined fluorescence and confocal Raman spectral imaging of internalized of FA–NGO–PVP in (a) A549 and (b) Hela cells. The fluorescence image was obtained by integrating the signals of Rho B from 565 to 600 nm.
3.5. Cellular targeting uptake and internalization of FA–NGO–PVP To study the uptake of FA–NGO–PVP by different cells with or without folate receptors, FA–NGO–PVP labeled with Rho B via physical adsorption was used. Then folate receptors positive cells (Hela) and folate receptors negative cells (A549) were incubated with FA–NGO–PVP/Rho B respectively. After 30 min of incubation, laser-induced fluorescence spectra of intracellular FA–NGO–PVP/ Rho B were collected using a confocal Raman microscope with a 514 nm semiconductor laser (10 mW, 100% laser intensity, 2 s exposure time) [43]. As shown in Fig. 7, the Hela cells treated with FA–NGO–PVP/Rho B showed strong fluorescence signals throughout the entire cell cytoplasm (Fig. 7a). However, no obvious fluorescence signals were detected in A549 cells (Fig. 7b), indicating the receptor-mediated specific uptake. The intracellular localization of FA–NGO–PVP was closely related to the threshold for photothermal damage effect. It has been reported that the internalization and mitochondrial accumulation of single-walled carbon nanotubes could enhance the sensitive of cells to NIR-mediated photothermal damage, and the property that temperature spike destroys the mitochondria membrane could lead to cell apoptosis factors, such as cytochrome c release and eventually trigger cell death [44]. However, evidences showed that folate conjugated gold nanorods were more effective in NIR-mediated thermal ablation when they were on the cell surface as compared to subcellular localization [45]. The mechanisms of graphene-mediated photothermal killing of cancer cells have been investigated to be induced cell apoptosis [46], but the subcellular localization of graphene nanoparticles was not clear. Briefly, the effective cellular uptake and internalization suggested the potential of FA–NGO–PVP as a transmembrane delivery carrier to promote the cellular specific uptake, decrease the drug side-effect, increase the drug intracellular accumulation and enhance the local photothermal killing effect.
4. Conclusion In summary, we have developed FA–NGO–PVP/DOX that could carry heat and drug specifically to cancer cells, and investigated its targeted chemo-photothermal therapeutic efficacy. The
as-prepared FA–NGO–PVP exhibited ultrahigh loading ratio of anticancer drug. Cellular uptake experiments demonstrated the internalization of FA–NGO–PVP into tumor cells via receptor-mediated endocytosis. Moreover, the folic acid conjugated NGO loaded with DOX under NIR irradiation exhibited the highest cytotoxicity to Hela cells compared with that of other treatments, indicating the efficient chemo-photothermal targeted therapy. Taken together, this work demonstrated functionalized NGO as the targeted nanocarrier for delivery of drug and heat to tumor cells, facilitating the cancer targeted chemo-photothermal therapy in one system, which might be an effective strategy to improve the efficacy of cancer therapy and reduce the drugs resistance.
Acknowledgments This work was supported by the National Natural Science Foundation of China (61275187), Specialized Research Fund for the Doctoral Program of Higher Education of China (20114407110001), the Natural Science Foundation of Guangdong Province (9251063101000009) and the cooperation project in industry, education and research of Guangdong province and Ministry of Education of P.R. China (2011A090200011).
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