Article pubs.acs.org/Biomac
Graphene-Based Anticancer Nanosystem and Its Biosafety Evaluation Using a Zebrafish Model Chen-Wei Liu,† Feng Xiong,‡ Hui-Zhen Jia,† Xu-Li Wang,§ Han Cheng,† Yong-Hua Sun,*,‡ Xian-Zheng Zhang,† Ren-Xi Zhuo,† and Jun Feng*,† †
Key Laboratory of Biomedical Polymers (The Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072, China ‡ State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hyrobiology, Chinese Academy of Sciences, Wuhan, 430071, China § Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84108, United States S Supporting Information *
ABSTRACT: In this paper, a facile strategy to develop graphenebased delivery nanosystems for effective drug loading and sustained drug release was proposed and validated. Specifically, biocompatible naphthalene-terminated PEG (NP) and anticancer drugs (curcumin or doxorubicin (DOX)) were simultaneously integrated onto oxidized graphene (GO), leading to selfassembled, nanosized complexes. It was found that the oxidation degree of GO had a significant impact on the drug-loading efficiency and the structural stability of nanosystems. Interestingly, the nanoassemblies resulted in more effective cellular entry of DOX in comparison with free DOX or DOX-loaded PEGpolyester micelles at equivalent DOX dose, as demonstrated by confocal microscopy studies. Moreover, the nanoassemblies not only exhibited a sustained drug release pattern without an initial burst release, but also significantly improved the stability of formulations which were resistant to drug leaking even in the presence of strong surfactants such as aromatic sodium benzenesulfonate (SBen) and aliphatic sodium dodecylsulfonate (SDS). In addition, the nanoassemblies without DOX loading showed negligible in vitro cytotoxicity, whereas DOX-loaded counterparts led to considerable toxicity against HeLa cells. The DOX-mediated cytotoxicity of the graphene-based formulation was around 20 folds lower than that of free DOX, most likely due to the slow DOX release from complexes. A zebrafish model was established to assess the in vivo safety profile of curcumin-loaded nanosystems. The results showed they were able to excrete from the zebrafish body rapidly and had nearly no influence on the zebrafish upgrowth. Those encouraging results may prompt the advance of graphene-based nanotherapeutics for biomedical applications.
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INTRODUCTION
aging results obtained so far, yet in an infancy stage, adumbrate the promise that graphene materials may be developed as a suitable vehicle candidate for the construction of anticancer nanotherapeutics.6,14,15 Besides the high drug loading ability, the unique feature of graphene may offer possibilities to overcome the stability issue of nanotherapeutics. It is well-known that during the in vivo circulation, the aggregation/sedimentation of nanoparticles easily leads to premature clearance by the reticulo-endothelial system (RES) and potentially results in thrombosis upon intravenous administration. This physiological barrier deserves more concerns when it comes to graphene-based nanomaterials due to their exceptional adsorption capability to many biomolecules
Nanotherapeutics self-assembled from a variety of drug carriers, including proteins, amphiphilic block copolymers, lipids, and inorganic nanoassemblies, are attractive approaches to improve cancer therapies through the enhanced permeability and retention (EPR) effect.1,2 However, these drug carriers are often associated with drawbacks including undesirable burst drug release and premature drug leaking due to their limited stability. Very recently, graphene materials were shown to be capable of loading aromatic anticancer drugs such as doxorubicin (DOX) and camptothecin (CPT) with ultrahigh efficiency.3−8 The graphene’s large surface area as well as the combination of hydrophobic and π−π stacking interaction with the drug molecules was assumed to contribute much to the outstanding performance.9,10 In addition, graphene can be potentially used for photothermal ablation of tumors in the near-infrared tissue transparency window.11−13 Those encour© XXXX American Chemical Society
Received: September 29, 2012 Revised: December 4, 2012
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Scheme 1. Illustration of the Formulation of GO/NP/Drug Complexes and Their In Vivo Biotoxicity Assay in a Zebrafish Model
in the bloodstream.16−18 Another significant challenge against the in vivo applications of graphene materials arises from their safety concern.19−21 Unfortunately, the research in this area is very limited. Hence, it is highly desired to construct graphenebased drug delivery nanosystems which are compatible with the physiological environments. Given the nondegradable nature of graphene, the nanosystems in principle need to be ultimately secreted from the body so as to minimize the latent in vivo hazard of graphene nanomaterials.22,23 Chemical coating with hydrophilic polymers is one of mostly used strategies to improve the water-solubility and biocompatibility of materials.24−28 To acquire sufficient modification with hydrophilic polymers, vigorous oxidation toward graphene is usually required for the successive covalent conjugation. Nevertheless, that would dramatically destroy the surface structure of graphene, thus presumably disfavoring the drug loading and photothermal therapy. Additionally, the chemical modification process is quite complicated and thus hampering the control over the reproducibility and reliability regarding the property and structure of obtained graphene materials.23,25 With those in mind, stable incorporation of hydrophilic polymer onto GO by noncovalent interaction may be a better choice in terms of the fewer detriments to graphene surface.15,29 In the current study, we developed a graphene-based delivery nanosystem by simultaneously integrating biocompatible naphthalene-terminated PEG (NP) and anticancer drugs including DOX and curcumin, onto oxidized graphene (GO) through noncovalent self-assembly strategy (Scheme 1). We hypothesize that the terminal naphthalene could spontaneously attach onto GO surface, allowing for the building of GOcentered nanostructure surrounded by PEG chains, thus, preventing detection by immunogenic entities. The influence of the oxidation extent of GO was investigated with regard to the drug loading ability and structural stability of the nanoassemblies in both serum-free and serum-containing mediums. We also compared the cellular uptake efficiency of this DOX-loaded nanosystem with the controls including free DOX or DOX-loaded PEG-polyester micelles. Furthermore, the DOX release behavior from nanoassemblies was comparatively investigated in the presence of competitive model compounds including aromatic sodium benzenesulfonate
(SBen) and aliphatic sodium dodecylsulfonate (SDS). MTT assay was performed to compare the in vitro cytotoxicities of DOX drug, DOX-free, and DOX-loaded complexes. Biosafety of graphene-based biomaterials remains largely unknown and little information are currently available at in vivo level. Here we used zebrafish model to assess the in vivo biotoxicity of graphene-based nanosystem (Scheme 1), in hopes of providing preliminary biosafety profile of graphene-based nanosystems. Zebrafish has been established as a preclinical model system for the evaluation of novel drug and drug carrier candidates regarding the efficacy and biotoxicity because of the close homology with the human genome.30−33 In particular, zebrafish embryos are more prone to damage by chemical agents than are adult organisms.33,34 We have established the fabrication and biosafety evaluation of graphene-based nanosystems for anticancer nanotherapeutics. The experimental results demonstrated their improved stability, sustained drug release pattern without initial burst release, enhanced cellular entry in vitro and good biocompatibility in vivo. We expect that this facile and effective formulation strategy would be applicable for most hydrophobic aromatic drugs, which include but are not limited to anticancer drugs and, thus, prompting the advance of graphene-based therapeutic modalities toward practical application.
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EXPERIMENTAL SECTION
Materials. Graphite powder, 2-naphtol, KMnO4, PEG (Mn = 2 kDa), H2SO4 (98%), HCl, dimethyl sulfoxide (DMSO), curcumin, and H2O2 were purchased from Sinopharm Chemical Reagent Co. Ltd. in Shanghai China. 1,6-Diisocyantohexane was purchased from Acros Organics Co. U.S.A. Preparation of lGO. Lowly oxidized graphene (lGO) was synthesized by a modified Hummer’s method.35 A total of 1 g of graphite powder was ground with 50 g of NaCl for a period of 10 min. Then NaCl in the mixture was removed by dissolution in water and filtration. The ground graphite flakes were added to 20 mL of H2SO4 (98%) and stirred for 12 h. Then, 4 g of KMnO4 was added while keeping the temperature less than 20 °C with ice bath. The reaction system was then maintained at 40 °C for 30 min and then at 90 °C for 90 min under stirring. Next, 46 mL of distilled water was slowly added and the reaction temperature was raised to 105 °C and maintained for 25 min. Finally, 140 mL distilled water and 10 mL 30% H2O2 were added. After filtration, the isolated solid was washed with 5% HCl solution and then with deionized water for several times. B
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Figure 1. (A) Direct observations on the aqueous dispersion of lGO/NP complexes with different feed ratios during 8-day standing in comparison to controls including lGO alone and the mixture of lGO/mPEG. The initial lGO concentration was fixed at 0.2 mg/mL. The red rectangles mark the bottles with obvious deposition. (B) Direct observations on the aqueous dispersion of hGO/NP complex in 10% serum solution during 8-day standing in comparison to the controls. The initial hGO concentration was fixed at 0.1 mg/mL. (C) Direct observations on the aqueous dispersion of lGO/NP complex in 10% serum in comparison to the controls. The initial lGO concentration was fixed at 0.1 mg/mL. relationship: Relative Cell Viability (%) = (ODtreated/ODcontrol) × 100, ODtreated is obtained in the presence of samples and ODcontrol is obtained in the absence of samples. Fluorescence Assay. Fluorescence spectra were recorded on a LS55 luminescence spectrometer (Perkin-Elmer). A total of 16 mg (±0.05 mg) of NP was dissolved in 4 mL of DMSO. A total of 0.8 ± 0.05 mg of GO was added and then the mixture was subjected to ultrasonic oscillation for 1 h. After a 2 day standing at room temperature, the dispersion was diluted 800 times with water for measurement. The emission was carried out at 225 nm, and excitation spectra were recorded ranging from 300 to 650 nm. For comparison, the NP solution in the absence of GO was fabricated in the same manner and taken as the control with the identical NP concentration. DLS Analyses of GO and lGO/NP Complexes. The mean particle size and size distribution of the particles in aqueous medium was determined by dynamic light scattering (DLS) with a Nano-ZS ZEN3600 instrument. The GO/NP complexes with different w/w ratios prepared as the above-mentioned method and then subjected to ultrasonic oscillation for 10 min. The GO concentration in the dispersion was fixed at 4 μg/mL. TEM Observation. The samples were prepared by dripping a drop of the solution on the copper grid, then removing the liquid part after 5 min. TEM model was Tecnai G2 20S-TWIN. Drug Loading and In Vitro Drug Release. GO/NP complexes (2 mg) and 2 mg of doxorubicine (DOX) were dissolved in 2 mL of DMSO. The solution was put into a dialysis tube and subjected to dialysis against 1000 mL of distilled water for 24 h. The dialyzate after drug loading was measured by UV spectroscopy to determine the amount of unloaded drugs. After dialysis, the dialysis tube was directly immersed into 400 mL of 0.1 M PBS solution at 37 °C. Aliquots of 3 mL were withdrawn from the solution periodically. The volume of solution was held constantly by adding 3 mL of PBS after each sampling. The amount of DOX released from micelles was measured by UV absorbance at 490 nm, respectively. For comparison, the drug release behaviors in PBS solutions containing 0.5 mM sodium benzenesulfonate (SBen) and aliphatic sodium dodecylsulfonate (SDS) were also investigated, respectively. Biotoxicity Evaluation in Zebrafish Embryos and Larvae. Fertilized eggs were obtained from natural mating of adult zebrafish (Danio rerio AB line), which were bred at the Animal Center of Institute of Hyrobiology, Chinese Academy of Sciences. Newly fertilized eggs, approximately at the stage of 12 cells, were exposed to luminescent complexes (100 μg/mL) for 4 days. A total of 20 eggs
Preparation of hGO. Highly oxidized graphene (hGO) was synthesized in a similar way to that of lGO.36 In the preparation, a mixture of 4 g of KMnO4 and 10 g of NaNO3 were added after H2SO4 treatment. Synthesis of Naphthalene-Conjugated PEG (NP). A total of 3 mL of dimethyl sulfoxide (DMSO) solution containing 0.2 g of mPEG with the molecular weight of 2000 Da (0.1 mmol) was dropped into 1,6-diisocyantohexane (HDI; 34 mg, 0.2 mmol) solution in 10 mL of DMSO. The mixture was stirred for 24 h at 70 °C. Then 0.5 g of 2naphtol was added. After stirring at 70 °C for 20 h, the solution was dropped to 150 mL of ethyl ether to isolate the precipitated products. The precipitated products were immersed into THF (10 mL) with stirring for 1 h and then the mixture was subjected to centrifugation treatment at 10000 r/min for 5 min. The upper supernatant was collected. This dissolution-centrifugation treatment was repeated for three times to remove the residual insoluble 2-naphtol. Finally, the NP products were obtained by thoroughly removing the THF solvent under vacuum. The 1H NMR spectrum of the product was shown in Figure S1. Preparation of GO/NP Complexes in Water. Under ultrasonic oscillation, 0.5 mg of GO was dispersed in 50 μL of water. Then 0.5, 2.5, 5, and 25 mg (±0.05 mg) of NP in 1 mL of water was added with the w/w ratio of 1:1, 1:5, 1:10, and 1:50, respectively. After the treatment of ultrasonic oscillation for 3 h, 2.5 mL of water was added to the dispersion and the obtained solution was further treatment of ultrasonic oscillation for 1 h. Preparation of GO/NP Complexes in 10% Serum. The CO/ NP complexes in 10% serum were prepared in an identical way as described above. At last the solution volume in every bottle was maintained at 1.75 mL, and then 250 μL of pure serum was added in every bottle. Cell Culture and In Vitro Cytotoxicity Assay. HeLa cells were seeded in 96-well plates at a density of 5.0 × 104 cell per well and the cells were incubated in 100 μL DMEM containing 10% FBS for 1 day in an incubator (temperature, 37 °C; 5% CO2). After that, the culture medium was replaced with 100 μL of fresh medium containing the samples with predetermined weight, and the cells were incubated for another 48 h with the samples. Subsequently, 20 μL of MTT solution (5 mg/mL) was added to each well and further incubated for 4 h. After that, the medium was removed and 150 μL of DMSO was added, which was shaken briefly at room temperature. The optical density (OD) was measured at 570 nm with a microplate reader (BIO-RAD 550, U.S.A.). The relative cell viability was calculated as the following C
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Figure 2. DLS profiles of lGO (Z-average: 653.1 nm, PDI: 0.461; a) as well as lGO/NP complexes with the w(lGO)/w(NP) ratios of 1:5 (Zaverage: 595.7 nm, PDI: 0.267; b) and 1:10 (Z-average: 388.7 nm, PDI: 0.239; c), respectively. TEM images of lGO/mPEG mixture (w/w = 1:50; d) and lGO/NP complexes (w/w = 1:10; e); bar scale is 200 nm. were placed in each well of a six-well plate. During the exposure, 90% of each well was replaced by fresh complexes solution daily. After 3 days of static exposure, the larvae were rinsed with water and maintained in aerating water for further observation; meanwhile, 90% of the water was replaced daily. Embryos and larvae were maintained at 27 °C in a 14 h light and 10 h dark schedule. Each experiment was repeated three times. A group without the luminescent complexes was used as the control. All observations were done by an OLYMPUS CKX-31 inverted microscope. All of images were obtained using a Motic BA400 microscope with a Moticam 5000 device camera. Ethics Statement on the Use of Animals. The research animals are provided with the best possible care and treatment and are under the care of a specialized technician. All procedures were approved by the Institute of Hydrobiology, Chinese Academy of Sciences, and were conducted in accord with the Guiding Principles for the Care and Use of Laboratory Animals.
1:10, nearly no sediments were detectable during the entire process (Figure 1A). Such remarkably enhanced stability suggests there must have strong interaction between GO and naphthalene-containing NP. We speculated that GO surface might be tightly attached by freely extending PEG chains, which prevented the dispersed GO particles from aggregation owing to the high steric hindrance resulting from outer PEG shells.37,38 The NP-associated contribution to the IGO stability was also validated by the comparative investigations under the conditions of high salt concentration (150 mM NaCl) and high content of serum (50%; Figure S2). To validate our hypothesis, we employed transmission electron microscope (TEM) to explore the complexes morphology in an identical manner. As shown in the representative TEM pictures, lGO was well-dispersed as separate individuals with the diameters close to 200 nm caused by NP introduction (w/w, 1:10; Figure 2e) whereas interparticle aggregation appeared in lGO/mPEG control with a much higher mPEG feed ratio (w/w, 1:50; Figure 2d). Furthermore, dynamic light scattering (DLS) analyses were also conducted to obtain a rough measure about the complexes though their exact morphology in solution is not known.39 Interestingly, the DLS result correlated very well with the TEM observation. The lGO particles were determined to be around 653 nm in the mean size with the size polydispersity index (PDI) ∼ 0.461, which were immediately measured by DLS after ultrasonic oscillation. As shown in Figure 2, once added with NP, the mean diameter of lGO particles and the PDI exhibited a substantial decline. This tendency appears to be associated with the w/w ratio of NP:lGO. The mean DLS size of lGO/NP (1:5) and lGO/NP (1:10) complexes was around 595 and 386 nm, and the corresponding PDI was 0.267 and 0.239, respectively. Evidently, the introduction of NP rather than mPEG favors preventing GO particles from aggregation. When a designed drug nanocarrier is used in the blood circulating system, many biological barriers should be overcome thus to acquire high drug bioavailability in targeted tissue/cells as well as reduced systematic toxicity. One important hurdle is the surface adsorption of blood components and the following
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RESULTS AND DISCUSSION Stability of GO/NP Complexes in Serum-Free and Serum-Containing Mediums. Two established methods were used to prepare lowly oxidative GO (lGO) and highly oxidative GO (hGO), respectively.35,36 The weight percent of oxygen in hGO and lGO is 34.6% in 24.1%, respectively, as determined by elemental analysis. Under ultrasonic oscillation, the self-assembly of GOs and NP was completed by adding NP polymer into the GO dispersion in pure water. The stability of lGO/NP self-assemblies was visually inspected over an 8-day standing (Figure 1A). The special interaction between lGO and NP was demonstrated based on the comparison with two controls including lGO alone as well as the mixture of lGO and unmodified methyl-PEG-OH (mPEG; w/w, 1:50). For both of the controls, appreciable sediments appeared just after a 5 h standing (Figure 1A). Relatively, the tendency to sedimentation was presented more profoundly for the aqueous dispersion containing lGO alone. To some extent, the lGO dispersion in water was not thermodynamically stable owing to the low surface functionality of lGO. The addition of unmodified mPEG resulted in very slightly enhanced stability, which is believed to be mainly due to the increased solution viscosity upon mPEG introduction. In sharp contrast, even at the low w/w ratio of lGO versus NP of D
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interparticle aggregation accompanied by RES ingestion.40−42 Consequently, some nanotherapeutics, even though efficient in vitro, often fail to show the similarly high activity when applied in vivo or in the simulated physiological environment, serum.43 Therefore, the serum-conditioned experiment has been currently serving as a fundamental model for the predictive evaluation on the in vivo stability of most nanocarriers.44 In our study, the preliminary serum-tolerance assess was also conducted in 10% serum solution, which is a typical serum concentration in most in vitro cell culture studies.45 Similar to the serum-free case, a significant amount of sediments appeared just after 5 h standing in lGO and lGO/mPEG dispersions, whereas nearly no sediments were detected over the 8-day observation for the lGO/NP (1:10) dispersion (Figure 1C). For comparison purpose, such serum-conditioned evaluation was also conducted for highly oxidized hGO which has better water solubility and higher functional density. In consistence to the reported findings, hGO dispersion presented a dark yellow color (Figure 1B) whereas lGO was a black dispersion similar to that of reduced GO derivatives (Figure 1C).46 Despite the higher stability than lGO counterpart, considerable sediments still appeared at day 2 in the 10% serum solutions of both hGO and hGO/mPEG, owing to the intrinsically high absorption capability of GO to serum components (Figure 1B).9,17 Also, the noncovalent complexation with NP could effectively enhance the stability of the aqueous hGO dispersion. When the images between Figure 1B and C are compared, it was found that the PEGylation strategy proposed herein appeared to be less efficacious to stabilize hGO dispersion than that to stabilize lGO. This may be attributed to the higher oxidation extent of the former. This would lead to higher hydrophilicity and more destructed structure of hGO, presumably adversely affect the self-assembly between GO and NP via hydrophobic attraction and π−π stacking interaction. Drug Loading and Release of DOX/GO/NP Complexes. We further compared the fluorescence spectra of lGO/NP, hGO/NP complexes and NP alone to elucidate the possible underlying mechanism of the self-assembly of NP and GO. Under the condition with identical NP concentration, the intensity of the characteristic resonance of naphthalene moieties in NP was used as the indicator. Relative to that of NP alone, the intensity dropped by 50% with lGO/NP (w/w = 1:10) and 30% with hGO/NP (w/w = 1:10), respectively, indicating a marked fluorescence quenching (Figure 3). The result suggests that the state of naphthalene moieties, at least fractionally in GO/NP dispersion, was different from those in absolute NP solution. As well documented, graphene contains largely dislocated π-electrons that allow energy transfer from the nearby molecules, leading to fluorescence quenching.47,48 To a great extent, the observed fluorescence quenching reveals that the driving forces of self-assembly should involve π−π stacking interaction between lGO and the naphthalene moieties in NP molecules.49 The higher quenching efficiency of lGO is possibly correlated with the larger adsorption amount of NP owing to the less destroyed surface structure of lGO. We thus speculated that the oxidation extent may also affect the drug loading into GO/NP complexes. Doxorubicin (DOX) is one of the leading agents for chemotherapies which is now widely used in the treatment of a variety of solid tumors and hematologic malignancies.50 To minimize the dose-limiting toxicity, suitable delivery systems are needed to effectively load poorly water-soluble DOX and release it in a sustained manner, thus lowering doses required for efficacy as well as increasing
Figure 3. Fluorescence spectra of NP, lGO/NP (w(lGO)/w(NP) = 1:10) complexes and hGO/NP (w(hGO)/w(NP) = 1:10) complexes in pure water. The NP concentrations in all cases were identical (5 μg/ mL).
the therapeutic indices and safety profiles.51−54 As a representative example, this anticancer drug was herein used as the model drug to investigate how oxidation extent of GO affects drug loading capacity. As expected, the loading efficiency of lGO/NP complex was 27%, which is higher than that of hGO/NP complex (18%), further evidencing the important role of structural compatibility between GO and payload in determining the loading capability. Based on these results, it is concluded that the oxidation extent of GO would affect its complexation with hydrophobic aromatic compounds via hydrophobic and π−π stacking interaction. The in vitro DOX release from DOX/lGO/NP complexes was subsequently examined in PBS (pH 7.4, ionic strength 0.1 M) at 37.4 °C. The resulting data presented in Figure 4 showed a sustained DOX release. Initial burst release was not observed, which usually occurred in the case of drug-loaded polymeric micelles.55 Over a 3-day period, only about 35% drugs were released. A comparative test was further conducted in the presence of aromatic sodium benzenesulfonate (SBen) or aliphatic sodium dodecylsulfonate (SDS) in the solution.15,56
Figure 4. In vitro drug release of DOX loaded complexes in different environments. E
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regarding the strong potency to delivery DOX into cells by GO/NP nanocarrier are encouraging. In Vitro Cytotoxicity of lGO/NP Complexes. The in vitro cytotoxicity assay of free DOX, DOX-loaded, and DOXfree GO/NP complexes and NP alone was further performed. The high cell-biocompatibilities of DOX-free GO/NP complexes and NP were demonstrated by the high cell-viability above 90% of HeLa cells within the measured range of concentration up to 200 μg/mL (Figure 6A). Like other anthracyclines, the clinical application of DOX is associated with chronic cardiotoxicity, producing biventricular, progressive congestive heart failure.60 The toxicity is reported to be highly dose-dependent. To minimize these side effects, sustained release at the tissue of interest for the therapeutic effect may be a good solution to lower doses required for efficacy and increase the safety profiles.51−54 Free DOX showed expectedly high cytotoxicity with the rather low IC50 of 1.3 μg/ mL in HeLa cells. As for the DOX loaded in the nanoassemblies, the IC50 was promoted to 25 μg/mL, around 20fold higher than that of free DOX (Figure 6B). The reduced cytotoxicity of DOX after being loaded into the complexes is believed to have strong association with the slow release of DOX and delayed drug efficacy in the cultured cells. Biotoxicity Assay of NP/lGO Complexes Based on Zebrafish Model. The good correlation between in vivo data and those obtained from a zebrafish model has well demonstrated that zebrafish are effective in estimating possible biotoxicity.30−32 The genetic parallels to human impart physiological and pharmacological similarities.33,61 Furthermore, biology and optical clarity of zebrafish allow the visual screen of the whole organism at all stages of embryonic development.33,62 Noteworthy, a short-term zebrafish biotoxicity assessment could be more or less linked to the developmental biotoxicity during the long-term human growth.32,63 Herein, we investigated the biotoxicity of graphene-based nanomaterials using zebrafish as a vertebrate model from the embryo through the hatchling stage.62,64 Water-insoluble curcumin is a potentially anticancer drug, stemming from its ability to modulate growth of tumor cells through regulation of multiple cell signaling pathways.65,66 Because of its minimal toxic effect on healthy cells, curcumin was loaded into GO/NP complexes to serve as the fluorescent tracer. Two control groups including the group coincubated with free curcumin and the blank control were also examined for comparison purposes. Regardless of the oxidation extent, obvious precipitation immediately happened after mixing curcumin with lGOs. Nevertheless, no obvious change was observed during the complexation between lGO/NP and curcumin, further demonstrating the considerable contribution of NP to the stability of GO dispersion. Zebrafish was treated with the complexes for 3 days from the embryo stage. The zebrafish embryos hatched at the first day, and at the third day the larvae were transferred into the fresh water in the absence of lGO/NP-curcumin complexes. As a result, there appeared the curcumin-loaded complexes presenting green fluorescence inside the fish bodies upon the hatching on day 1 (Figure 7a4), and the signal intensity of green fluorescence (Figure 7a5) was relatively stronger (Figure 7a4) on day 3. It is noteworthy that the chorion surrounding the embryo during development is an acellular envelope made of three intercrossing layers that possess pores or channels with about 0.5−0.7 μm in diameter, allowing materials to pass
Those strong surfactants would competitively adsorb onto the GO surface by hydrophobic and π−π stacking interaction, thus, possibly facilitating the drug release rate. However, such strong competitors just led to a slightly accelerated drug release in both cases, indicating strong association/complexation of payload with GO. And after 8 days, around 9% drugs still remained in the complexes. Such strong binding appeared to be a favorable feature for nanotherapeutics, as the loaded drugs would be less likely leaching during the in vivo circulation before reaching the tumor by EPR effect, thus, minimizing the systematic toxicity against the normal tissue/cells.37,38 On the other hand, it means that appropriate mechanism may be pursued by further work to trigger the effective release in tumor tissues for the desirable chemotherapy performance.57 Cell Uptake of GO/NP/DOX Complex. Efficient cellular uptake of drug payloads is one of the crucial properties for nanotherapeutics for meaningful drug efficacy.58 To test the cellular uptake of GO/NP complex, we used the confocal fluorescence microscopy technique to observe cancerous HeLa cells after the coincubation with DOX-loaded GO/NP complex at the time of 0.5 and 2 h. We also used DOX-loaded PEGpolycaprolactone (PEG-PCL) micelle and soluble DOX·HCl as controls, and the initial DOX dose was identical in three cases. During the past decade, nanomicelles self-assembled from amphiphilic PEG-polyester block copolymers have been intensively demonstrated as effective drug carriers to improve the solubility, pharmacokinetics and efficacy of typical anticancer drugs.52 Compared with DOX/PEG-PCL control, apparently stronger fluorescence intensity was observed within the cells coincubated with GO/NP/DOX complexes (Figure 5). It is worthwhile noting that the cellular uptake of DOX
Figure 5. Laser-scanning confocal microscopy images of HeLa cells after exposure to lGO/NP/DOX complexes (b1−b2), free DOX·HCl (d1−d2), PEG-PCL/DOX (f1−f2), NP (h1−h3) for 0.5 or 2 h. Corresponding transmitted images were also shown (a1−a2, c1−c2, e1−e2, g1−g2) of b1−b2, d1−d2, f1−f2, h1−h2. Scale bars are 90 μm.
mediated by GO/NP complexes was enhanced as prolonging incubation time. This ascending trend was presented somewhat insignificantly in the case of two controls. Strategies with passively targeting nanoparticle formulations have been successfully established since mid-1980s as this approach can, to some degree, bypass and avoid frequently occurring P-gpmediated drug efflux.1,59 In this regard, the obtained results F
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Figure 6. In vitro cell viability assay in HeLa cells of NP and NP/lGO complexes (A); In vitro cell viability assay of free DOX·HCl and the DOX loaded in NP/lGO/DOX complexes in HeLa cells (B).
vessel, muscle and the internal organs (Figure S3).63 It is suggested that the zebrafish could grow healthily in the presence of lGO/NP/curcumin complexes. All the results showed that the complexes exerted negligible influence on the development of zebrafish from embryos to larvae, which is consistent with in vitro cytotoxicity assay. In comparison with the data available in the literature,62 a conclusion is drawn that NP attachment is able to dramatically improve the biosafety profiles of GO nanomaterials. In addition to acceptable biocompatibility, GO materials should in principle be potentially excreted from the body after the completion of biofunction, given the intrinsic nondegradability of GO. After the larvae were transferred into the fresh water at the third day, the fluorescence in larvae steadily faded and almost disappeared at the seventh day. Though the detachment of curcumin from the complexes cannot be ignored, the result still partly suggests that lGO/NP could be able to excrete from the body given the fact that just 36% drugs were detached from the complexes during a 3-day release experiment in vitro.68
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CONCLUSION In this paper, we have developed a novel graphene-based selfassembled nanosystem for effective loading and sustained release of hydrophobic anticancer drugs. Specifically, we simultaneously integrate biocompatible naphthalene-terminated PEG (NP) and DOX onto lowly oxidized graphene (lGO) by means of thoroughly noncovalent self-assembly. The obtained nanoassemblies were demonstrated with attractively long-term stability in the serum-free and serum-containing mediums. The nanoassemblies can effectively mediate cellular uptake of DOX at a high level. The complexes without DOX loading showed negligible in vitro cytotoxicity, whereas DOX loading led to considerable toxicity against cancerous HeLa cells. The nanoassemblies could release loaded DOX in a sustained manner. Due to this, the DOX-associated cytotoxicity of the formulation presented a substantial decline compared with that of free DOX. A zebrafish model was established to visually assess the in vivo safety of our proposed nanosystem, suggesting high biocompatibility as demonstrated with its fast excretion from the body. The proposed system may have considerable adaptability to accommodate different therapeutic compounds. Those promising results may prompt the advance of graphene-based therapeutic modalities toward practical application.
Figure 7. Full body shot of zebrafish group after treatment with lGO/ NP/curcumin complexes for 3 days (a1, a2, a3) and then with fresh water for 4 days (a8); the fluorescence image of the enterocoelia of this zebrafish group on the first day (a4), the third day (a5), the fifth day (a6), and the seventh day (a7). The images of b1−b7 were obtained under the same conditions after treatment with free curcumin. The images of c1−c7 were blank control.
through to the embryo via passive diffusion.30,31 The detected fluorescence means that the size of lGO/NP-curcumin complexes should not preclude passage through the chorion. In sharp contrast to it, no fluorescence could be detectable in the fish group treated with free curcumin, possibly due to its poor water-solubility. Assessment of biotoxicity can be semiquantified as sublethal toxicities (survival of the embryo and the severity of phenotypic and gross morphological differences).30−32,62,67 Compared with the blank control, neither sublethal toxicity nor drop of hatching rate was detectable in the presence of lGO/NPcurcumin complexes. Screening of phenotypic toxicity may be implemented because of opaque quality of tissues following cell death.62 In the enlarged view of zebrafish group after treatment with lGO/NP/curcumin complexes, we did not notice any obvious abnormality of the adult zebrafish regarding the blood G
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ASSOCIATED CONTENT
S Supporting Information *
1
H NMR spectrum of NP, additional pictures of stability observation, enlarged view of zebrafish group after treatment with lGO/NP/curcumin complexes. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected];
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Key Basic Research Program of China (2011CB606202 and 2009CB930301) and National Natural Science Foundation of China (Grant No. 21174110).
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