Graphene in Mice: Ultrahigh In Vivo Tumor Uptake and Efficient

Center of Super-Diamond and Advanced Films (COSDAF) and Department of Physics and Materials Science, City University of Hong Kong, Hong Kong SAR, ... ...
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Graphene in Mice: Ultrahigh In Vivo Tumor Uptake and Efficient Photothermal Therapy Kai Yang,† Shuai Zhang,† Guoxin Zhang,‡ Xiaoming Sun,‡ Shuit-Tong Lee,§ and Zhuang Liu*,† †

Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials Laboratory (FUNSOM), Soochow University, Suzhou, Jiangsu, 215123, China, ‡ State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, China, and § Center of Super-Diamond and Advanced Films (COSDAF) and Department of Physics and Materials Science, City University of Hong Kong, Hong Kong SAR, China ABSTRACT Although biomedical applications of carbon nanotubes have been intensively studied in recent years, its sister, graphene, has been rarely explored in biomedicine. In this work, for the first time we study the in vivo behaviors of nanographene sheets (NGS) with polyethylene glycol (PEG) coating by a fluorescent labeling method. In vivo fluorescence imaging reveals surprisingly high tumor uptake of NGS in several xenograft tumor mouse models. Distinctive from PEGylated carbon nanotubes, PEGylated NGS shows several interesting in vivo behaviors including highly efficient tumor passive targeting and relatively low retention in reticuloendothelial systems. We then utilize the strong optical absorbance of NGS in the near-infrared (NIR) region for in vivo photothermal therapy, achieving ultraefficient tumor ablation after intravenous administration of NGS and low-power NIR laser irradiation on the tumor. Furthermore, no obvious side effect of PEGylated NGS is noted for the injected mice by histology, blood chemistry, and complete blood panel analysis in our pilot toxicity study. Although a lot more efforts are required to further understand the in vivo behaviors and the longterm toxicology of this new type of nanomaterials, our work is the first success of using carbon nanomaterials for efficient in vivo photothermal therapy by intravenous administration and suggests the great promise of graphene in biomedical applications, such as cancer treatment. KEYWORDS Graphene, in vivo tumor targeting, fluorescence imaging, photothermal therapy, toxicology

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ypical sp2 carbon nanomaterials, including fullerene, carbon nanotubes, and graphene, have many interesting physical and chemical properties that are potentially useful in biological and biomedical applications.1-3 Among those materials, carbon nanotubes have attracted the most attention in biomedicine.1,4 Well-functionalized carbon nanotubes stable in physiological environments have been shown to be nontoxic to cells in vitro and in vivo to mice.1,4-8 Carbon nanotubes have been widely used as drug delivery vehicles for in vitro drug and gene delivery as well as for vivo cancer treatment.9-14 In addition, the intrinsic optical properties of single-walled carbon nanotubes (SWNTs), including resonance Raman scattering, near-infrared (NIR) photoluminescence, and NIR optical absorption, can be utilized in biomedical imaging and phototherapy applications.15-18 As a rising star in material sciences, two-dimensional (2D) graphene has attracted tremendous attention in the past few years.19,20 Applications of graphene, including nanoelectronic devices,20,21 transparent conductors,22,23 and nanocomposites,24 are being actively pursued. Recently, we and others have explored biomedical applications of graphene at the cellular level.2,3,25 Nanographene sheet (NGS) functionalized with polyethylene glycol (PEG) exhibits high solu-

bility and stability in physiological solutions and has been used for in vitro drug delivery and imaging.2,3 However, the in vivo behaviors and applications of graphene have not yet been reported. In this work, for the first time we studied the behaviors of PEGylated NGS in mice by in vivo fluorescence imaging and observed surprisingly high tumor accumulation of NGS in a few different xenograft tumor models, likely owing to the enhanced permeability and retention (EPR) effect of cancerous tumors. We further utilized the strong NIR optical absorption ability of NGS for in vivo photothermal therapy of cancer and achieved highly efficient tumor destruction by intravenous NGS injection followed by lowpower NIR laser irradiation. Moreover, no obvious sign of toxicity was observed for NGS-PEG injected mice by the histology examination, blood chemistry, and whole blood panel analysis. NGS with a biocompatible coating may thus be a novel type of 2-D nanomaterials with great potential in cancer therapy. PEGylated NGS (NGS-PEG) was prepared starting from graphite oxide, following our previous protocol.2,3 Amine terminated six-arm branched PEG (10 kDa) was conjugated to graphene oxide sheets via amide formation (Figure 1a). Successful PEGylation was evidenced by the high stability of NGS-PEG in physiological solutions with high salt contents3 as well as infrared (IR) spectra (Supporting Information, Figure S1-S2). Amino groups at the PEG terminals

* Corresponding author. E-mail: [email protected]. Received for review: 03/21/2010 Published on Web: 08/04/2010 © 2010 American Chemical Society

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FIGURE 1. NGS functionalized with PEG. (a) A scheme of a NGS with PEG functionalization and labeled by Cy7. (b) An AFM image of NGS-PEG. (c) A UV-vis-NIR spectrum of a NGS-PEG solution at the concentration of 0.05 mg/mL. NGS has high optical absorption from UV to NIR regions. Inset: a photo of a NGS-PEG solution at the concentration of 0.5 mg/mL. (d) Temperature change curves of the NGS-PEG solution and the water exposed to the 808 nm laser at a power density of 2 W/cm2. Rapid raise of temperature was noted for the NGS-PEG solution, in marked contrast to the water temperature which showed little change during the laser irradiation.

were available for bioconjugation and fluorescent labeling (Figure 1a).2 Atomic force microscope (AFM) images showed that NGS-PEG were very small sheets with a size range of 10-50 nm (Figure 1b) and mostly single- or double-layered sheets (Supporting Information, Figure S3). Similar to carbon nanotubes, NGS exhibited high optical absorption (Figure 1c). To verify the potential of using NGS in photothermal therapy, a NGS-PEG solution was exposed to an 808 nm NIR laser at a power density of 2 W/cm2 with water as the control. In marked contrast to the water sample, the NGSPEG solution showed a rapid increase of temperature when exposed to the laser within a short time (Figure 1d). To study the in vivo behaviors of NGS, we labeled NGSPEG with Cy7, a commonly used NIR fluorescent dye (Figure 1a, Supporting Information, Figure S4). It was estimated that one NGS (assuming it has a sphere shape and an average diameter of 30 nm) was labeled by 14 Cy7 based on the Cy7 absorption peak (See Supporting Information for detailed estimation). Control experiments suggest that the majority of Cy7 dye was covalently conjugated to NGS-PEG via the formation of an amide bond instead of physical absorption by π-stacking, as evidenced by the fact that the deactivated Cy7 could not be attached to NGS-PEG (Supporting Information, Figure S4) by an appreciable amount. We first measured NGS levels in the blood over time (Figure 2a) after intravenous injection of NGS-PEG-Cy7 into Balb/c mice. Blood was drawn at different time points post injection, solubilized by a lysis buffer, and measured by a fluorometer to determine the NGS-PEG-Cy7 concentration © 2010 American Chemical Society

(blood autofluorescence was subtracted from the measured blood fluorescence intensity; see the Supporting Information for details). A blood circulation half-life of ∼1.5 h was observed for NGS-PEG-Cy7, similar to that of previously reported 5 kDa PEG coated SWNTs which showed limited passive tumor uptake in the absence of a targeting ligand.26 Next, Balb/c mice bearing 4T1 murine breast cancer tumors, nude mice bearing KB human epidermoid carcinoma tumors, and U87MG human glioblastoma tumors were intravenously injected with NGS-PEG-Cy7 (200 µL of 2 mg/mL solution for each mouse; a dose of 20 mg/kg) and then spectrally imaged by a Maestro EX in vivo fluorescence imaging system (CRi, Inc.) (Figure 2b, see Method section in the Supporting Information for detailed experimental parameters). The mouse autofluorescence was removed by spectral unmixing using the Maestro software, leaving pure Cy7 fluorescence shown in Figure 2b (Supporting Information, Figure S5). Widely dispersed among the whole mouse in the beginning (30 min post injection, p.i.), NGS-PEG-Cy7 tended to be enriched in the tumor over time. Prominent uptake of NGS was observed in the tumor (Figure 2, the first row) with relatively low signals in other parts of the mouse body at 24 h p.i. for all three types of tumor models. As the control, mice injected with PEG-Cy7 or free Cy7 solutions at the same dye concentrations were also imaged at various time points p.i., showing rapid decrease of fluorescent signals. The majority of PEG-Cy7 (∼10 kDa) and Cy7(∼1 kDa) was excreted within 6 h by fast renal clearance of those small molecules (Supporting Information, Figure S6), sug3319

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FIGURE 3. Semiquantitative biodistribution analysis of NGS-PEGCy7. 4T1 tumor-bearing mice were sacrificed at various time points post NGS-PEG-Cy7 injection with major organs collected for fluorescence imaging. (a) Spectrally resolved ex vivo fluorescence images of organs before injection and 1, 6, and 24 h after injection of NGSPEG-Cy7. SK: skin, M: muscle, I: intestine, H: heart, LU: lung, LI: liver, K: kidney, SP: spleen, ST: stomach, and T: tumor. (b) Semiquantitative biodistribution of NGS-PEG-Cy7 in mice determined by the averaged fluorescence intensity of each organ (after subtraction by the fluorescence intensity of each organ before injection). Error bars were based on three mice per group.

FIGURE 2. In vivo behaviors of NGS-PEG-Cy7. (a) The blood circulation curve of NGS-PEG-Cy7 determined by measuring Cy7 fluorescence in the blood at different time points post injection. The unit was a percentage of injected dose per gram tissue (% ID/g). Error bars were based on triplicated samples. (b) Spectrally unmixed in vivo fluorescence images of 4T1 tumor bearing Balb/c mice, KB, and U87MG tumor bearing nude mice at different time points post injection of NGS-PEG-Cy7. Mouse autofluorescence was removed by spectral unmixing in the above images. High tumor uptake of NGSPEG-Cy7 was observed for all of the three tumor models. Hairs on Balb/c mice were removed before fluorescence imaging.

Consistent with the in vivo imaging data, NGS-PEG-Cy7 levels in the majority of organs decreased over time as expected. Interestingly, the kidneys showed strong fluorescence even at 24 h p.i. This was unlikely, or at least not completely, due to the falling off of the Cy7 label from NGS considering the fact that free Cy7 was very rapidly cleared out from the mice, with the majority excreted within 6 h (Supporting Information, Figure S6). The high kidney uptake of NGS-PEG-Cy7 might indicate possible renal excretion of NGS with small sizes (Cy7 fluorescence was indeed detected in the mouse urine) but requires further validation. Nude mice bearing KB or U87MG tumors also showed high tumor and kidney uptake of NGS (Supporting Information, Figures S7 and S8). The ultrahigh tumor accumulation of NGS could be due to the EPR effect in cancerous tumors with tortuous and leaky vasculatures, which tend to trap materials in the nanosize range. The passive tumor targeting effect of the 2-D NGS appears to be much more efficient than that of the 1-D

gesting that the observed fluorescence on NGS-PEG-Cy7 injected mice was indeed from the labeled NGS instead of free or detached fluorescent dyes. To study the biodistribution of NGS-PEG-Cy7, we sacrificed 4T1 bearing Balb/c mice before injection and at 1, 6, and 24 h p.i. Various organs and tissues were spectrally imaged by the Maestro system (Figure 3a). The averaged Cy7 fluorescent intensity of each imaged organ (after removing the tissue autofluorescence and subtracting the background, if any, of each organ before NGS injection) was calculated for a semiquantitative biodistribution analysis (Figure 3b). The strongest fluorescent signal was observed in the tumor, suggesting high tumor accumulation of NGS. The NGS-PEGCy7 signals in reticuloendothelial systems (RES), including liver and spleen, were surprisingly low, which was different from the in vivo biodistribution of SWNTs reported earlier.6,26 © 2010 American Chemical Society

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FIGURE 4. In vivo photothermal therapy study using intravenously injected NGS-PEG. (a) Tumor growth curves of different groups after treatment. The tumor volumes were normalized to their initial sizes. There were 6 mice in the untreated, 10 mice in the ‘laser only’, 7 mice in the ‘NGS-PEG only’, and 10 mice in the ‘NGS-PEG + laser’ groups. While injection of NGS-PEG by itself or laser irradiation on uninjected mice did not affect tumor growth, tumors in the treated group were completely eliminated after NGS-PEG injection and the followed NIR laser irradiation. (b) Survival curves of mice bearing 4T1 tumor after various treatments indicated. NGS-PEG injected mice after photothermal therapy survived over 40 days without any single death. (c) Representative photos of tumors on mice after various treatments indicated. The laser irradiated tumor on NGS injected mouse was completely destructed. Error bars in (a) were based on standard deviations.

to ∼2 °C of surface temperature rise for irradiated tumors on uninjected mice. Another two control groups of mice with and without NGS injection were not irradiated. Tumor sizes were measured every 2 days after treatment (Figure 4a). All irradiated tumors on mice injected with NGS disappeared 1 day after laser irradiation, leaving the original tumor site black scars, which fell off about 1 week after treatment (Figure 4c). No tumor regrowth was noted in this treated group over a course of 40 days, after which the study was ended. In marked contrast, tumors in the control untreated group, the irradiation only group (no NGS injection), and the NGS only group (no laser irradiation) showed similarly rapid tumor growth, demonstrating that the NIR laser irradiation or NGS injection by itself did not affect the tumor development (Figure 4a and c). Importantly, mice in the three control groups showed average life spans of ∼16 days, while mice in the treated group were tumor-free after treatment (NGS injection, NIR laser irradiation) and survived over 40 days without a single death (Figure 4b), further demonstrating the excellent efficacy of NGS based in vivo photothermal therapy. A number of earlier studies have shown the promise of using carbon nanotubes for photothermal ablation of cancer cells in vitro.17,27 Recently, Ghosh et al. and Moon et al.

SWNTs.13,14,26 In a previous study, PEGylated SWNTs with a similar blood circulation half-life (∼1.5 h) showed dominant accumulation in RES organs and minimal passive tumor uptake.26 The unique shape and size of NGS could allow highly efficient tumor passive targeting of NGS by favoring the EPR effect. However, due to the limitations of fluorescence based in vivo/ex vivo imaging methods, including light absorption and scattering by tissues, potential photobleaching of fluorescent dyes as well as the nonquantitative nature of this method, other methodologies, such as radiolabeling methods, would be helpful and of great importance to further understand the in vivo behaviors of biocompatibly functionalized NGS in animals. Motivated by the ultraefficient passive tumor targeting of NGS and its strong NIR optical absorption ability, we then carried out an in vivo photothermal therapy study using the 4T1 tumor model on Balb/c mice. Ten mice bearing 4T1 tumor at the right shoulder were intravenously injected with 200 µL of NGS-PEG at 2 mg/mL (a dose of 20 mg/kg). Another 10 mice without NGS injection were used as the control. The tumor on each mouse was exposed to an 808 nm laser at the power density of 2 W/cm2 24 h after injection of NGS. The surface temperature of tumors on NGS injected mice reached to ∼50 °C after laser irradiation, in contrast © 2010 American Chemical Society

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separately reported that intratumoral injections of multiwalled carbon nanotubes (MWNTs) and SWNTs followed by NIR laser irradiations at powers of 2.5 and 3.8 W/cm2, respectively, were able to destroy tumors growing on mice.18,28 However, carbon nanotube solutions were directly injected into tumors prior to laser ablation in these two studies. In vivo photothermal therapy of cancer by intravenous systemic administration of carbon nanotubes or other carbon nanomaterials has not yet been reported to date. In this work, for the first time we have achieved highly efficient in vivo photothermal ablation of tumors in a mouse model by using intravenously administrated biocompatible nanographene. Achieving a therapy by systemic administration of the therapeutic agent has obvious advantages over that by local administration, by which not all lesions can be effectively reached. The power density we applied in this work (2 W/cm2) is among the lowest compared with that used by others when gold nanorods are utilized for in vivo photothermal therapy (2 ∼ 4 W/cm2),29,30 owing to the highly efficient tumor passive targeting of NGS likely via the EPR effect. Our results indicate that biocompatibly coated NGS is a powerful agent for photothermal therapy of cancer in vivo. The potential in vivo toxicity is always a great concern for nanomaterials used in biomedicine. In this work, we did not notice any obvious sign of toxic side effects for NGS-PEG injected mice at the dose of 20 mg/kg within 40 days. Neither death nor significant body weight drop was noted in the NGS-PEG + laser treated group (Figure 5a). Major organs of NGS-PEG treated mice whose tumors were eliminated by the photothermal therapy were collected 40 days after the treatment for histology analysis. No noticeable signal of organ damage was observed from haematoxylin and eosin (H&E) stained organ slices (Figure 5b), suggesting the promise of using PEGylated NGS for in vivo applications. We further carried out blood chemistry and complete blood panel analysis for healthy mice 3 months after injection of NGS-PEG at the treatment dose, observing no significant abnormality of the injected mice compared with the control group (Supporting Information, Tables S1 and S2). Although much work is needed to systematically study the potential short- and long-term toxicity of NGS at various doses with more animals used per group, our pilot small-scale toxicity study promises further explorations of using graphene for in vivo biomedical applications. In summary, we have studied the in vivo behaviors PEGylated nanographene sheets (NGS) in tumor bearing mice by in vivo fluorescence imaging. Highly efficient tumor passive targeting of NGS has been observed in several different tumor models without utilizing any targeting ligands, such as antibodies. PEGylated NGS appears to be an excellent in vivo tumor near-infrared (NIR) photothermal therapy agent without exhibiting noticeable toxicity to the treated mice. To our best knowledge, this work is the first to explore the in vivo behaviors, applications, and potential toxicology © 2010 American Chemical Society

FIGURE 5. No obvious toxic effect was observed for NGS-PEG treated mice. (a) Body weight curves after various treatments indicated. Neither NGS-PEG injection nor laser irradiation resulted in significant body weight drop to the treated mice. (b) H&E stained images of major organs. Mice survived after photothermal therapy with tumors eliminated were sacrificed 40 days after treatment. No noticeable abnormality was observed in major organs including kidney, liver, spleen, heart, intestine, and lung.

of graphene in animal models. This is also the first report to achieve in vivo photothermal therapy of cancer by systemic administration of carbon nanomaterials (including carbon nanotubes, C60, graphene, etc.) in general. Compared with carbon nanotubes, a typical kind of carbon nanomaterials widely explored in biomedicine, our PEGylated nanographene shows distinctive in vivo behaviors, such as reduced reticuloendothelial systems (RES) accumulation and notably improved tumor passive targeting effect.26 Although a lot more efforts are required to fully elucidate the mechanism, we hypothesize that the unique two-dimensional (2-D) shape, small size (10-50 nm in size), and biocompatible PEG coating may favor the enhanced permeability and retention (EPR) effect of NGS for high tumor passive uptake. Compared with gold nanomaterials, such as gold nanorods (AuNRs), extensively investigated as photothermal agents, the performance of PEGylated NGS appears to be comparable to that of PEGylated AuNRs in terms of administration routes (intravenous), injected doses (20 mg/kg), NIR laser densities (2 W/cm2), and irradiation durations (5 min).29 However graphene with all sp2 carbon 3322

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atoms exposed on its surface has an ultrahigh surface area available for efficient drug loading with an uniquely high molecular loading capacity.2,3,10 The graphene-based in vivo photothermal therapy, if combined with chemotherapeutic drugs also delivered by NGS as demonstrated in our earlier in vitro work,2,3 could bring novel opportunities to the next generation of combined cancer treatment. Although better understandings of graphene in vivo behaviors, including quantitative pharmacokinetics and biodistribution (e.g., by the radiolabeling method), as well as long-term toxicology are extremely important and demand further investigations, our work suggests that graphene as a novel class of nanomaterials has great potential in biomedical applications.

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Acknowledgment. This work was supported by a research start-up fund of Soochow University. We thank Dr. Haizhen Deng for her great help in the H&E staining and frozen sections, Profs. Quansheng Zhou and Yun Zhao for allowing us to use their fluorescence microscope, and the Shanghai Research Center for Biomodel Organism for the blood analysis.

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Supporting Information Available. Detailed experimental methods, materials characterization data, various control fluorescence imaging data, semiquantitative biodistribution data of KB and U87MG tumor-bearing mice, and blood analysis data. This material is available free of charge via the Internet at http://pubs.acs.org.

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