Biocompatible Graphene Oxide Nanoparticle-Based Drug Delivery

Aug 10, 2014 - ABSTRACT: A facile strategy was established to develop a drug delivery system (DDS) based on the graphene oxide nanoparticles (GON) wit...
21 downloads 9 Views 6MB Size
Download PDF
Article pubs.acs.org/Langmuir

Biocompatible Graphene Oxide Nanoparticle-Based Drug Delivery Platform for Tumor Microenvironment-Responsive Triggered Release of Doxorubicin Xubo Zhao, Lei Liu, Xiaorui Li, Jin Zeng, Xu Jia, and Peng Liu* State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China S Supporting Information *

ABSTRACT: A facile strategy was established to develop a drug delivery system (DDS) based on the graphene oxide nanoparticles (GON) with suitable size and shape to deliver drug effectively, by grafting the biocompatible PEGylated alginate (ALG-PEG) brushes onto the GON via the disulfide bridge bond. TEM analysis and drug-loading performance revealed that the 3-D nanoscaled, biocompatible, reduction-responsive nanocarriers (GON-Cy-ALG-PEG) were spherical in shape with diameters of 94.73 ± 9.56 nm. They possessed high doxorubicin (DOX)-loading capacity and excellent encapsulation efficiency, owing to their unique 3-D nanoscaled structure. They also had excellent stability in simulated physiological conditions and remarkable biocompatibility. Importantly, the in vitro release showed that the platform could not only prevent the leakage of the loaded DOX under physiological conditions but also detach the cytamine (Cy) modified PEGylated alginate (Cy-ALG-PEG) moieties, response to glutathione (GSH). Confocal microscopy and WST-1 assays provided clear evidence of the DOX-loaded GON-Cy-ALG-PEG endocytosis, whereas the drug-loaded nanocarriers exhibited high cytotoxicity to model cells. Furthermore, the cell apoptosis also was monitored via Flow cytometry. The results indicated that the DOX-loaded nanocarriers presented favorable efficiency of cell apoptosis. So these findings demonstrate that the accelerated release of the loaded DOX was realized in the presence of an elevated GSH that simulate the acidic endosomal compartments.

■

INTRODUCTION In the last decades, graphene has attracted considerable attention in the variety of different fields and created a revolution in chemistry and condensed matter physics, owing to their amazing unique physical and chemical properties since first appearance in 2004.1,2 Due to its unique structure of the sp2-hybrirdized carbon atom, graphene possesses excellent properties,3 which have opened remarkable pathways for its potential applications, ranging from transparent electrodes,4 semiconducting nanomaterials,5 electronics,6 to high-performance composites.7 Notably, the graphene oxide (GO) derivatives have emerged as effective treatments for hydrophobic anticancer drug delivery.8 In recent years, enormous efforts have been tried to optimize their biocompatibility, solubility, and stability in physiological media via noncovalent or covalent functionalization, especially as drug delivery system (DDS).9,10 Unfortunately, their application as DDS have been hindered by their bigger particle size, usually in micrometers. It has been revealed that the size of the nanocarriers plays an important role to control the in vivo fate11 and is a key parameter to overcome the barriers of reticuloendothelial system (RES) to deliver a chemotherapeutic drug deeply into tumors by means of intravenous injection.12−14 It is believed that the discontinuous © 2014 American Chemical Society

endothelium has many pores ranging in size from 200 nm to 2 μm with the average pore size approximately 400 nm on the tumor blood vessel walls; nanoparticles with size smaller than 100 nm are now regarded as being more favorable for extravasation from bloodstream into tumors through EPR effect and tumor tissue penetration.15−17 So the anticancer drug delivery vehicles with size smaller than 100 nm have drawn more and more attention in recent years, with the aim to enhance curative effect. Kataoka et al. have compared the accumulation of the longcirculating, drug-loaded polymeric micelles with different sizes (30, 50, 70, or 100 nm) in both highly and poorly permeable tumors. The results suggested that all samples penetrated highly permeable tumors in mice, but only the 30 nm samples could penetrate poorly permeable BXPC-3 pancreatic tumors to exhibit an antitumor effect.18 Thus, a major goal in developing anticancer GO-based DDS with small size may represent a considerable novel pattern for delivering anticancer drugs effectively. Received: April 9, 2014 Revised: July 31, 2014 Published: August 10, 2014 10419

dx.doi.org/10.1021/la502952f | Langmuir 2014, 30, 10419−10429

Langmuir

Article

Scheme 1. Schemtic Illustration of the Composition Preparation (A) and Drug Release of the Drug Delivery System by GSH and pH Trigger in the HepG2 Cell (B)

solution and rapidly released the encapsulated DOX at tumorrelevant glutathione (GSH) levels.21 Most recently, it has been reported that the PEGylated GO via SS bond may be gradually degraded by enzymes.22 Gan et al. prepared the chitosan grafted graphene oxide (GO−CS) with significantly improved aqueous solubility and biocompatibility, which could condense plasmid DNA into stable, nanosized complexes. The resulting GO−CS/pDNA nanoparticles exhibited reasonable transfection efficiency in HeLa cells at certain nitrogen/phosphate ratios.23 As compared with normal tissues, tumor tissues usually possess the unique microstructural features and physicochemical properties, such as weak acidity,24 abnormal temperature

Although these GO-based nanocarriers have excllent properties as DDS,19 the foremost problems has been retained: the biocompatible, stable dispersion in water, premature drug release during blood circulation, and low extent of nanocarrier dosage reaching the tumor tissues. To solve the adverse circumstance, many attempts have been successfully carried out. Dai et al. reported the first pioneered paper in 2008,20 in which the six-armed PEG-amine polymer had been grafted onto GO as a novel drug nanocarriers to load the water-insoluble cancer drugs via hydrophobic interactions and π−π stacking interactions. Shi’s groups prepared a PEGylated nanoscaled graphene oxide sheets (NGO-SS-mPEG) with redox-responsive detachable PEG shells. It had high stability in biological 10420

dx.doi.org/10.1021/la502952f | Langmuir 2014, 30, 10419−10429

Langmuir

Article

gradients,25 overexpressed proteins and enzymes,26 and so on. Most importantly, the different tumor intracellular microenvironments are expected to adjust the anticancer drug release, via the response to pH 4.5−6.5 inside endosomes and lysosomes,27 reductive microenvironments due to high-level cyteine or GSH in the cytoplasm and endolysosomes.28 Park explored the strategy to prepare stable graphene with significant advantages of applying redox-responsive Plu-SH to overcome the obstacles regarding the application of graphene in DDS.29 Most recently, Liu et al. designed the tumor microenvironmental responsive nano drug delivery system based on GO for combined chemo- and photothermal therapy overcoming drug resistance.30 Here, the biocompatible, 3-D nanoscaled, reductionresponsive graphene oxide nanoparticles (GON-Cy-ALGPEG) were prepared for the efficient loading and triggered release of DOX, by conjugating the biocompatible polymer brushes (ALG-PEG) onto the graphene oxide nanoparticles (GON) via disulfide bridge bond. To the best of our knowledge, this represents the first report on preparation of the 3-D GON for DDS (Scheme 1A). The nanocarriers have several important design aspects. They have highly DOXloading capacity and excellent encapsulation efficiency. The introduction of the PEGylated alginate onto the GON could prevent the leakage of the loaded DOX under physiological conditions. PEG moieties endow the nanocarriers with stealth features during blood circulation, owing to their proteinresistant ability and the bypassing of recognition and clearance by RES. Once being uptaken by the tumor tissues, the Cy-ALGPEG brushes could be detached to release the drug loaded.

■

3.2 mL of 30% H2O2 solution was subsequently added. The bright yellow mixture was stirred for 2 h at room temperature, centrifuged at 12 000 rpm for 30 min, and the GON was obtained in the supernatant. The precipitate was exfoliated repeatedly and the supernatant was collected in a 250 mL round-bottom flask. Afterward, most of the water in the suspension was evaporated by the rotary evaporator under the reduced pressure at 45 °C to approximately 30 mL and filtrated through a 0.45 μm nylon membrane. Then the surplus suspension was transferred into dialysis tubes (molecular weight cutoff of 12 000) extensively against 10% HCl solution for 2 days to remove residual metal ions, followed by dialysis against water for 2 days until neutral. The product was steadily dispersed in water as individual GON nanoparticles and would not precipitate for several months. Finally, the resulting GON nanoparticles were obtained by lyophilization. PEGylation of Alginate. The PEGylation of alginate with NH2− PEG2000 was conducted as reported previously.32 A 1 w/v % sodium alginate solution was prepared in a 0.10 mol/L MES buffered solution (pH 6) with 0.50 mol/L NaCl. 292.1 mg of NHS and 98.9 mg of EDCI were added into 100 mL of alginate solution to activate their carboxyl groups. The solution was agitated to homogeneous and 100 mL of 2 w/v% PEG solution (20 mol % of carboxyl groups in ALG) was added. After stirring for 24 h at room temperature, the mixture was dialyzed against water for 3 days to obtain the final PEGylated alginate (ALG-PEG) by lyophilization. Cytamine Modified PEGylated Alginate (Cy-ALG-PEG). The cytamine modified PEGylated alginate (Cy-ALG-PEG) was prepared by reacting ALG-PEG (0.8001g) with an excess amount of Cy (0.8294 g) in 80 mL of PBS (pH 7.4) solution at room temperature for 24 h (Scheme 1A), with EDCI (0.7126 g) and NHS (0.2139 g). The crude product was dialyzed against water for 2 days to obtain the Cy-ALGPEG followed by lyophilization. Conjugation of Cy-ALG-PEG with GON. Cy-ALG-PEG (40 mg) was sonicated in 200 mL of 1 mg/mL GON aqueous dispersion for 15 min. Then 0.1917 g (1 mmol) of EDCI was added, and the solution was sonicated for another 30 min, followed by addition of 0.7668 g (4 mmol) of EDCI and electromagnetic stirring for 12 h. The resultant product was centrifuged and washed with water, with several centrifugation and redispersion cycles, to remove any excess CyALG-PEG. The GON-Cy-ALG-PEG was freeze-dried and stored at 4 °C. Cell Toxicity Assays. WST-1 assay was performed to evaluate the biocompatibility of the GON-Cy-ALG-PEG hybrid complex with HepG2 cell. DOX was chosen as the model drug to evaluate the ability of inhibition growth to HepG2 cell about the DOX-loaded GON-CyALG-PEG in the absence or presence 10 mM GSH-OEt. For the WST-1assay, the cells were seeded into 96-well plates at densities of 1 × 105 cells per well for 24 h. Then, different concentrations of the GON-Cy-ALG-PEG, DOX-loaded GON-ALG-PEG in the absence or presence of 10 mM GSH-OEt, and free DOX were added and incubated for 24 h. Thereafter, the cells were washed three times with PBS and processed for the WST-1 assay to determine the cell viability. Cellular Uptake of DOX-Loaded GON-Cy-ALG-PEG. The cellular uptake was exhibited by confocal laser scanning microscopy (CLSM) (LYMPUS FV-1000) using HepG2 cells after 6 h incubation. The location of intracellular fluorescence was validated with excitation wavelengths of 480 nm for DOX and 405 nm for Hoechst. Flow Cytometric Analysis. Flow cytometry was used to monitor cell apoptosis of the DOX-loaded GON-Cy-ALG-PEG in the presence of 10 mM GSH-OEt by measuring the cell associated fluorescence by a flow cytometer (BD FACSCalibur), after 30 μg/mL DOX equiv/mL of free DOX or the DOX-loaded GON-Cy-ALG-PEG in the presence of 10 mM GSH-OEt and GON-Cy-ALG-PEG (30 μg/ml) was added to the HepG2 cell and incubated at 37 °C for 6 h. Drug Loading and Triggered Release. GON-Cy-ALG-PEG (10.0 mg) was added into 10.0 mL of 1.0 mg/mL DOX aqueous solution for drug-loading with the aid of ultrasound, and then the solution was adjusted to the desired pH, respectively. After being magnetic stirred and swung by a table concentrator for 24 h in the dark, the DOX-loaded GON-Cy-ALG-PEG was centrifuged to remove the excess DOX. The drug concentration in the supernatant solution

EXPERIMENTAL SECTION

Materials and Reagents. Graphite powder was purchased from Huatai Chemical Reagent Co. Ltd. (Shandong, China). Functional PEG (NH2−PEG2000) was provided by Beijing Kaizheng Biological Engineering Development Co., Ltd. (Beijing, China). Sodium alginate (viscosity of 30-80 cP for 1.0 mg/mL solution and viscosity-average molecular weight of 4.5 × 105) was obtained from Xvdong Chemical Plant (Beijing, China). Cytamine dihydrochloride and 2-N-morpholinoethanesulfonic acid (MES) were purchased from J & K Chemical Ltd. Glutathione was provided by Tianjin Heowns Biochem. Co., Ltd. (Tianjin, China). 1Ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDCI) was obtained from Fluorochem. N-Hydroxylsuccinimide (NHS) was provided by Aladdin Chemistry Co. Ltd. Doxorubicin hydrochloride (DOX) was purchased from Beijing Huafeng United Technology Co. Ltd. (Beijing, China). Glutathione reduced ethyl ester (GSH-OEt) was provided by J&K Chemical Ltd. WST-1 cell proliferation and cytotoxicity assay kits were purchased from Melonepharma. Other reagents were analytical reagent grade, purchased from Tianjin Chem. Co., Ltd. (Tianjin, China). Deionized water was used throughout the experiments. 3-D GON Nanoparticles. The 3-D GON nanoparticles were prepared with a modified Hummers method:31 2.01 g of graphite was pretreated in a mixture containing 20.00 mL 98.3% concentrated H2SO4, 3.00 g of K2S2O8, and 3.01 g of P2O5, with vigorous stirring at 80 °C for 24 h. The pretreated product was washed with water until neutral, filtrated, and dried at ambient temperature. Then it was subjected to oxidation by the Hummer method: 4.01 g of KMnO4 was added gradually into a mixture containing 1.01 g of pretreated graphite powder, 1.00 g of NaNO3, and 50.1 mL of concentrated H2SO4 at 0 °C with stirring about 1 h in an ice-water bath. After the mixture was stirred vigorously for 2 days at room temperature, and ultrasonicated for 2 h, 100 mL of 5 wt % H2SO4 aqueous solution was added in 1 h with stirring, and the temperature was kept at 98 °C. After additional stirring for 2 h at 98 °C, the temperature was reduced to 60 °C, and 10421

dx.doi.org/10.1021/la502952f | Langmuir 2014, 30, 10419−10429

Langmuir

Article

Figure 1. TEM images of GO with different oxidation times ((a) 24 h, (b) 36 h, and (c) 48 h), GON (d), and GON-Cy-ALG-PEG (e), AFM image (f), and SEM images (g) of GON. was monitored using an ultraviolet (UV) spectrophotometer at 485.00 nm to assess the drug-loading capacities and the encapsulation efficiency, by comparing the drug concentrations before and after loading. The DOX-loaded GON-Cy-ALG-PEG nanocarriers were dispersed into 10 mL of PBS (pH 7.4 without GSH, pH 7.4 with 10.0 μmol/L GSH, pH 7.4 with 10.0 mmol/L GSH, pH 5.0 without GSH, pH 5.0 with 5 mmol/L GSH, or pH 5.0 with 10.0 mmol/L GSH), transferred into dialysis tube (molecular weight cutoff of 10 000), and immersed into 120 mL of relevant PBS at 37 °C, respectively. Each aliquot of 5.0 mL was taken out at certain intervals, and the drug concentration was measured with a UV spectrophotometer to assess the cumulative release of the DOX-loaded GON-Cy-ALG-PEG. After each sampling, 5.0 mL of fresh relevant PBS was added to keep the total volume constant. The release mechanism of DOX from the drug-loaded GON-CyALG-PEG was discussed with the Higuchi and Korsmeyer−Peppas drug release equations as follows:

FT-IR spectra were recorded with a Bruker IFS 66 v/s infrared spectrometer, in the range of 400−4000 cm−1 with a resolution of 4 cm−1 with KBr pellet technique. Thermogravimetric analysis (TGA) were conducted with a TA Instrument 2050 thermogravimetric analyzer at 10 °C/min from 25 to 800 °C at nitrogen atmosphere. 1 H NMR spectra were recorded on 400 MHz with Bruker ARX 400 spectrometer (Bruker, Germany) using D2O as solvent with internal TMS as the reference (0 ppm). The ζ potentials of the GON, GON-Cy-ALG-PEG and DOXloaded GON-Cy-ALG-PEG were determined using a Zetasizer Nano ZS in media at pH 7.0. The elemental analyses of the samples were performed with an Elementar Vario EL instrument (Elementar Analysensysteme GmbH, Munich, Germany). The drug-loading and triggered release behavior of the GON-CyALG-PEG was assessed using a Lambda 35 UV−vis spectrometer at room temperature. The drug-loading capacity and the encapsulation efficiency were calculated by equations as follows:

M t = k · t 1/2

drug‐loading capacity (%)

M t /M∞ = k · t n

M t /M∞ < 0.6

= mass of DOX‐loaded/mass of the hybrid nanocarriers

where Mt is the amount of drug release at time t, k is the rate constant, Mt/M∞ is the fraction of drug release at time t, and n is the release exponent.33 When a plot of cumulative drug release of t1/2 yields a straight line with a slope that possesses a value ≥1, the particular system is considered to follow the Higuchi kinetics.34 As for the Korsmeyer−Peppas equation, the values of n, obtained from the slope of a plot of log Mt/M∞ vs log t,35 between 0.5 (diffusion-controlled drug release) and 1.0 (swelling-controlled drug release) can be regarded as an indicator for the superposition of both phenomena (anomalous transport). Analysis and Characterization. The morphology of the samples was characterized with a JEM-1200 EX/S transmission electron microscope (TEM) and SPA-300HV atomic force microscope (AFM). The samples were dispersed in water and then deposited on a copper grid covered with a perforated carbon film and deposited silicon wafer, dried at 45 °C in vacuum, respectively.

× 100% encapsulation efficiency (%) = mass of DOX‐loaded/mass of the feeding DOX × 100% The cumulative release (%) of drug at a particular time (t) can be calculated according to t−1

cumulative release (%) =

CtV0 + V ′ ∑i = 1 Ci M t otal

× 100%

where Mtotal represents the total content of DOX in dialysis tubes before release, V0 and V′ represent the initial volume of release media and the volume of the collected release media at specific time intervals (V0 of 120 mL and V′ of 5 mL in the present work), respectively; Ct 10422

dx.doi.org/10.1021/la502952f | Langmuir 2014, 30, 10419−10429

Langmuir

Article

Figure 2. FT-IR spectra of ALG, ALG-PEG, and Cy-ALG-PEG (A) and of GON and GON-Cy-ALG-PEG measured in KBr pellets (B). 1H NMR spectra of ALG, ALG-PEG, and Cy-ALG-PEG (in D2O, ppm) (C). TGA curves of the GON and GON-Cy-ALG-PEG at 10 °C/min in N2 (D). represents the obtained DOX concentration in release media at desired time point t.

■

A large number of nanoparticles were found in the AFM image of the GON nanoparticles. Moreover, the AFM image showed an interesting diameter around 50 nm for GON (Figure 1f). Furthermore, the SEM image (Figure 1g) of the GON exhibited the perfect 3-D morphology with an average diameter of 45.34 ± 9.84 nm. All these results are consistent with the TEM analysis. To improve the GON as DDS, functional polymer Cy-ALGPEG was synthesized via a versatile and straightforward route (Scheme 1 A). The strategy involved the PEGylation of ALG to obtain ALG-PEG, and then a conjugation of Cy to ALG-PEG by the amidation between the −NH2 group of Cy and the −COOH groups of ALG. A new characteristic peak at 1104 cm−1 appeared in the spectrum of ALG-PEG (Figure 2A), attributed to the C−O−C stretching vibration of PEG, indicating that PEG has been successfully grafted onto ALG. Furthermore, the FT-IR spectrum of Cy-ALG-PEG (Figure 2B) revealed the well-defined characteristic absorbance band of the primary amine at 1583 cm−1 (N−H, stretching vibration), indicating the incorporation of Cy. In addition, the PEG content (PEG%) in ALG-PEG and the cytamine content (Cy %) in Cy-ALG-PEG could be calculated to be 20.02% and 71.71%, respectively, from the N elemental analysis. And the Cy% was also determined as 73.79% based on the S elemental analysis. It demonstrated that almost all carboxyl groups of ALG had been substituted by PEG and Cy. Finally, the functional polymer Cy-ALG-PEG was grafted onto the 3-D nanoscaled GON via the amidation of the terminal amine groups of Cy-ALG-PEG and the carboxyl groups of the GON. Compared with the spectrum of the GON, the conjugation of Cy-ALG-PEG onto the GON through an ether bond could be confirmed by the new strong C−O−C

RESULTS AND DISCUSSION

Preparation of GON-Cy-ALG-PEG. The small size and biocompatibility is very important to ensure the application of the GON as DDS for cancer therapy. The key point in the present work is to realize the 3-D nanoscale of GO to enhance the drug-loaded capacity, encapsulation efficiency, and EPR effect. The biocompatible ALG-PEG brushes were conjugated onto the GON to prolong the circulation of blood and prevent the leakage of the loaded DOX from the DOX-loaded GONCy-ALG-PEG in the normal tissues, via the disulfide bridge between the −NH2 group of Cy and the −COOH of the GON, to provide the reduction-sensitive character. TEM was employed to trace the morphology and size during the different oxidation times (24, 36, or 48 h) (Figure 1a−c), respectively. The results supported sufficiently the forming process of the GON. Interestingly, the morphology of the GO changes from the sheet-like to the spherical shape, with increasing oxidation time. The product after oxidation for 24 h showed sheet in shape (Figure 1a). Comparatively, the product showed the undulating plate with similar protrusions with an oxidation time of 36 h (Figure 1b). And then the product changed into the aggregates of nanoparticles after oxidation for 48 h (Figure 1c). It reveals that the GO sheets have been transferred into GO nanoparticles (GON) under the strong oxidative condition with long oxidation time. After a further ultrasonication for 2 h, the nearly spherical shaped GON with near-monodisperse size of 43.36 ± 8.42 nm was obtained (Figure 1d). 10423

dx.doi.org/10.1021/la502952f | Langmuir 2014, 30, 10419−10429

Langmuir

Article

stretching vibration (1090 cm−1) in the spectrum of the GONCy-ALG-PEG (Figure 2B). The red shift of the C−O−C stretching vibration results from the hydrogen bond formed between the C−O−C units of PEG and the −OH and −COOH groups in the GON. The results suggested that the GON-Cy-ALG-PEG had been successfully designed via covalent attachment of the functional polymer brushes onto the surface of the GON. Compared with the TEM image of the GON, the GON-Cy-ALG-PEG has a large size, with the average diameter of approximately 94.73 ± 9.56 nm (Figure 1e) and tougher surface morphology, which also revealed the successful functionalization. In the 1H NMR spectrum of Cy-ALG-PEG (Figure 2C), the chemical shift at δ = 3.62 ppm is assigned to the inner methylene protons adjacent to the oxygen moieties (b, O− CH2), and the chemical shift at δ = 3.34 ppm is attributed to the terminal methyl protons (c, O−CH3). The chemical shifts at δ = 3.70−4.12 ppm (a) are assigned to the protons of ALG. Furthermore, the chemical shifts at δ = 2.85 ppm (e) and 2.72 ppm (d) are attributed to protons of Cy. Herein, the substitution values of PEG (PEG%) and Cy in ALG (Cy%) can be calculated to be 18.80% and 74.44%, based on the area ratios of signals at b and a, and e and a, respectively. The 1H NMR results are in accordance with the elemental analysis. The TGA technique was also used to verify the functionalization. Both the GON and GON-Cy-ALG-PEG exhibited small mass loss of about 9 wt % even below 150 °C (Figure 2D), which is attributed to the volatilization of stored water in the π−π stacking structure of graphene.36 The mass loss occurred around 200 °C, which is attributed to the pyrolysis of the labile oxygen-containing groups, such as hydroxyl, epoxy, and carboxyl groups in the carboxylated GON.37 The GON-Cy-ALG-PEG showed a 61 wt % weight loss at 450 °C, whereas the GON had a weight loss of 42 wt %. The mass loss of about 63.16 wt % of the GON-Cy-ALG-PEG in 210-450 °C may be due to the decomposition of the CyALG-PEG brushes. Therefore, it could be calculated that the GON-Cy-ALG-PEG hybrid contained about 23.45 wt % of the Cy-ALG-PEG brushes. Owing to the incorporation of the Cy-ALG-PEG moieties, the GON-Cy-ALG-PEG can be readily dispersed in PBS (pH 7.4) or water (pH 7.4) with the aid of slight ultrasound, as shown in Figure 3. Compared with the GON-Cy-ALG-PEG, it is interestingly found that the GON could not be well dispersed in pH 7.4 PBS and precipitated after stopping sonication for 20 min as seen in Figure 3A(a). Figure 3A(b) and Figure 3B(b) showed the dispersion of the GON-Cy-ALG-PEG in pH 7.4 PBS and water at the same concentrations after 48 h at room temperature, respectively. Figure 3B(a) showed the dispersion of the GON in pH 7.4 water at the same concentrations after 48 h at room temperature. The excellent solubility and dispersion stability of the GON-Cy-ALG-PEG in PBS is due to the benign solubility of the Cy-ALG-PEG polymer moieties, in comparison with the GON. These findings encourage us to explore the application of the GON-Cy-ALG-PEG nanocarriers as DDS. Reduction-Responsive Property of GON-Cy-ALG-PEG in PBS. The particle sizes of the GON and the GON-Cy-ALGPEG were tracked using the DLS technique. Both of them displayed narrow unimodal size distribution with average hydrodynamic diameter (Dh) of 87 and 207 nm (Figure 4A), respectively. The Dh of the GON was higher than that from TEM analysis, resulting from the unfolding of the GON in

Figure 3. Digital photographs of the aqueous dispersions of the GON (a) and the GON-Cy-ALG-PEG (b) at pH 7.4 PBS (A) or pH 7.4 water (B) with concentration of 0.01 mg/mL.

dispersion because of their surface carboxyl groups. There was no distinct change in the size of the GON-Cy-ALG-PEG in 0.1 mmol/L PBS (Figure 4B), suggesting its excellent dispersion stability and high solubility under physiological condition. The disulfide bonds were introduced between the grafted Cy-ALG-PEG moieties and the GON to design the GON-CyALG-PEG nanocarriers. The structural change is expected after cleaving the Cy-ALG-PEG moieties from the GON by reducing agents such as GSH. The GSH-induced size change of the GON-Cy-ALG-PEG was evident when dispersed in water with 10 mmol/L GSH (Figure 4C) and 0.1 mmol/L PBS with 10 mmol/L GSH (Figure 4 D), respectively. In water with the biological reducing agent (10 mmol/L GSH), its average diameter decreased from 223.47 to 79.64 nm during 32 h in a time interval of 4 h, as a result of detaching the Cy-ALG-PEG brushes (Figure 4C). Furthermore, the smaller fragment of the GON could be observed in 4 h. In contrast, upon exposure to 0.1 mmol/L PBS with 10 mmol/L GSH, the GON-Cy-ALGPEG significantly increased from 212.57 to 893.81 nm within 10 min (Figure 4D). Forming the lager aggregation (>890 nm) was more pronounced in 4 h, due to the poor stability of the GON in PBS.21 These phenomena demonstrated that the CyALG-PEG brushes could be detached from the GON-Cy-ALGPEG via cleaving the disulfide bonds with the reducing agents such as GSH in the tumor sites. Cell Toxicity and Reduction-Responsive Properties for HepG2 Cells. As DDS, it is important to retain low cytotoxicity in human body. The in vitro cytotoxicity of the GON-Cy-ALG-PEG hybrid was evaluated with the cultured HepG2 cells using WST-1 assays. As shown in Figure 5A, the viability of the HepG2 cells is close to 100% with treatment of GON-Cy-ALG-PEG during all the testing concentrations, meaning that GON-Cy-ALG-PEG have little toxicity on the HepG2 cells in the given concentration range after 24 h of incubation. The favorable biocompatibility was shown by increasing the concentration of the GON-Cy-ALG-PEG with a cell viability of 103.9% ± 4.1% to 93.4% ± 3.2% from 0 to 200 μg/mL for 24 h. To evaluate the inhibition growth of HepG2 for the reduction-responsive DOX-loaded GON-Cy-ALG-PEG, the DOX-loaded nanocarriers were used for the in vitro study with HepG2 cells by WST-1 assays. The results suggested that the DOX-loaded GON-Cy-ALG-PEG showed pronounced 10424

dx.doi.org/10.1021/la502952f | Langmuir 2014, 30, 10419−10429

Langmuir

Article

Figure 4. Typical Dh distributions of the GON and GON-Cy-ALG-PEG aqueous dispersion at pH 7.40 (A), the size of GON-Cy-ALG-PEG in 0.1 mM PBS (B), GON-Cy-ALG-PEG in water with 10 mM GSH (C), and the redox-induced size change of GON-Cy-ALG-PEG at 0.1 mM PBS with 10 mM GSH (D) in time measured by DLS.

Figure 5. Antitumor activity of GON-Cy-ALG-PEG, DOX-loaded GON-Cy-ALG-PEG without GSH-OEt, DOX-loaded GON-Cy-ALG-PEG with 10 mM GSH-OEt, and free DOX as a function of DOX dosages determined by standard WST-1 assay and cell toxicity. Data are presented as the mean ± standard deviation (SD; n = 5) (A). DOX release at pH 7.4 and 5.0 in the presence or absence of GSH by UV−vis analysis at 485.00 nm at room temperature. Each value represents the mean (SD < 1%, n = 3) (B).

GON-Cy-ALG-PEG in the absence GSH-OEt. Furthermore, compared with the free DOX, the DOX-loaded reductionsensitive GON-Cy-ALG-PEG possessed the same killing capacity for the HepG2 cell with an increase of its concentration. But even more crucial, the DOX-loaded reduction-sensitive GON-Cy-ALG-PEG in the presence of 10 mM GSH-OEt showed remarkable cell inhibition compared to that in the absence GSH-OEt. It is mainly attributed to the detachment of the Cy-ALG-PEG brushes from the GON

cytotoxic effects via reduction-sensitive properties to detach the Cy-ALG-PEG brushes from the GON in the presence of 10 mM GSH-OEt (Figure 5A). Introduction of DOX led to a reduction in cell viability, and significantly reduced cell viabilities are observed with increasing DOX concentration, compared to the case for the DOX-loaded nanocarriers in the absence of GSH-OEt. The order of efficacy as a killing agent was the DOX-loaded GON-Cy-ALG-PEG in the presence 10 mM GSH-OEt, then the free DOX, finally the DOX-loaded 10425

dx.doi.org/10.1021/la502952f | Langmuir 2014, 30, 10419−10429

Langmuir

Article

Figure 6. Cellular uptake of (A) HepG2 cells stained by Hoechst, DOX-loaded GON-Cy-ALG-PEG (30 μg/mL DOX equiv/mL) without GSHOEt (B), and DOX-loaded GON-Cy-ALG-PEG with 10 mM GSH-OEt (C) by CLSM using HepG2 cells after 6 h incubation. For each panel, images from left to right show cell nuclei stained by Hoechst (blue), DOX fluorescence in cells (green), and merged images. Bars represent 30 μm.

pH might be drastically acidic (pH 4.5−6.5) and the GSH level is about 10 mmol/L inside endosomes and lysosomes. Considering these differences in pH and the GSH level, the in vitro controlled release of DOX from the DOX-loaded GONCy-ALG-PEG was investigated at 37 °C under different conditions: (i) pH 7.4, (ii) pH 7.4 with 10 μmol/L GSH, (iii) pH 7.4 with 10 mmol/L GSH, (iv) pH 5.0 without GSH, (v) pH 5.0 with 5 mmol/L GSH, and (vi) pH 5.0 with 10 mmol/L GSH, respectively. The cumulative release ratio was 14.15% at physiological media (pH 7.4 with 10 μmol/L GSH, mimicking the intracellular trafficking pathway) within 32 h (Figure 5B). In contrast, it was only 8.23% at physiological pH about normal tissues within 32 h. Furthermore, it increased to 21.45% with increasing GSH concentration to 10 mmol/L at the same pH. This result showed that the Cy-ALG-PEG brushes maybe prevent the DOX to steal from the DOX-loaded GON-Cy-ALG-PEG nanocarriers. Within the higher reducing agent concentration, more Cy-ALG-PEG brushes were detached in a short time; therefore, the accelerated DOX release occurred. In addition, the cumulative release ratio of DOX was significantly accelerated at pH 5.0 with 30.68% of DOX released in 72 h under the same conditions in Figure 5B, maybe due to the increasing acidity favors the dissolution of DOX. To simulate the intracellular trafficking process, drug release studies were performed at pH 5.0 with 10 mmol/L GSH (mimicking the acidic endosomal compartments). Compared with the condition of pH 5.0 without GSH, the cumulative release ratios at pH 5.0 with 5 mmol/L GSH or pH 5.0 with 10 mmol/L GSH were 58.87% and 89.56%, respectively. These results clearly indicated that the biocompatible reductionresponsive GON-Cy-ALG-PEG hybrid presented a synergistic effect in DOX release. To understand the release mechanism of the loaded DOX from the DOX-loaded GON-Cy-ALG-PEG nanocarrier, the semiempirical equation of Higuchi and Korsmeyer−Peppas

nanoparticles to efficiently expose the loaded DOX in the nanocarriers via cleaving the disulfide bond between Cy-ALGPEG and GON by GSH-OEt of the HepG2 cells. This result demonstrated that the GON-Cy-ALG-PEG hybrid had reduction-sensitive properties to suit the tumor sites. Drug Loading and Controlled Release. The large π conjugated structure of the GON-Cy-ALG-PEG hybrid can form a π−π stacking interaction with the aromatic structure of DOX. Therefore, DOX is noncovalently loaded on the GONCy-ALG-PEG hybrid by a π−π stacking interaction in aqueous solution with the aid of slight sonication. The drug-loaded capacity and encapsulation efficiency of the GON-Cy-ALGPEG hybrid were measured in pH 7.4 DOX aqueous solution at room temperature, by UV−vis spectroscopy at 485.00 nm. The high DOX-loading capacities (%) and encapsulation efficiency (%) of the GON-Cy-ALG-PEG of 0.9764 mg/mg ±0.2358 mg/mg and 97.64% ± 3.36% (mean ± standard deviation, n = 5) resulted from combination of the π−π stacking interaction between the GON-Cy-ALG-PEG hybrid and DOX and the electrostatic interaction between ALG and DOX. As biocompatible and reduction-responsive nanocarriers, the GON-CyALG-PEG hybrid has not only a relatively stable structure but also a certain drug-loading capacity and encapsulation efficiency. The ζ potentials of the GON, GON-Cy-ALG-PEG, and DOX-loaded GON-Cy-ALG-PEG were also evaluated to track their surface charges. The data were −46.97 ± 1.26, −32.5 ± 1.73, and −14.87 ± 0.38 mV, respectively. The subsequent decrease in the absolute values of these ζ potentials demonstrated that the Cy-ALG-PEG brushes had been successfully immobilized and DOX had been effectively loaded onto the GON-Cy-ALG-PEG nanocarriers. So the investigation dealt with the in vitro controlled release performance of the drug-loaded nanocarriers. It is well-known that the extracellular pH and GSH level in normal tissues and blood is approximately 7.4 and 10 μmol/L, respectively, whereas in extracellular tumor tissues, the average 10426

dx.doi.org/10.1021/la502952f | Langmuir 2014, 30, 10419−10429

Langmuir

Article

Figure 7. Flow cytometric analyses of control experiment of HepG2 cells without any treatment (A), HepG2 cells incubated with GON-Cy-ALGPEG (30 μg/mL) (B), free DOX (C), DOX-loaded GON-Cy-ALG-PEG in the presence of 10 mM GSH-OEt (D) at the concentrations indicated for 6 h. The equivalent DOX dose is 30 μg/mL DOX, and the concentration of GON-Cy-ALG-PEG is 30 μg/mL.

were utilized to fit the accumulative release data (Figure S1, Supporting Information). Coefficients of correlation (R2) were used to evaluate the accuracy of the fitting. In addition, the unique conditions were chosen to research the release mechanism: pH 7.4 without GSH, pH 5.0 without GSH, and pH 5.0 with 10 mmol/L GSH at 37 °C. The plots for the Higuchi equation of the DOX-loaded GON-Cy-ALG-PEG resulted in linearity with the R2 and the k value of 0.9345 and 0.2779, 0.9610 and 0.7500, 0.9700, and 1.7970 under the above three conditions, respectively. However, the linearity (R2 = 0.9345, 0.9610, and 0.9700) of the Higuchi equation left much to be desired. However, the plots for the Korsmeyer−Peppas equation of the DOX-loaded GON-Cy-ALG-PEG also resulted in linearity with the R2 and the n value of 0.9731 and 0.3800, 0.9700 and 0.6207, and 0.9700 and 0.6870, respectively. The Korsmeyer− Peppas equation yielded comparatively good linearity (R2 = 0.9731, 0.9700, and 0.9700) and release exponent of around 0.5 (n = 0.3800, 0.6207, and 0.6870). This result suggested that the release mechanism was the Fickian diffusion at pH 7.4 without GSH.38 In contrast, the n values of otherwise samples were found to be 0.6207 and 0.6870, showing the transport process of DOX was anomalous, corresponding to a pseudo-Fickian or Case III mechanism.39 This phenomenon could be explained by considering the detachment of the Cy-ALG-PEG brushes and the increase of the pH value, which promoted the cumulative release of the loaded DOX. Cellular Uptake and Flow Cytometry Analysis. CLSM was employed to investigate the cellular uptake of the DOXloaded GON-Cy-ALG-PEG for HepG2 cells (Figure 6). The cell nuclei were stained with Hoechst (blue). After HepG2 cells were cultured in 24-well plate (1 × 105 cells/well), and incubated for 24 h, the cells were incubated in 100 μL of PBS of the DOX-loaded GON-Cy-ALG-PEG at 30.0 μg/mL of DOX equivalent dose, in the absence or presence of 10 mM GSHOEt. After 6 h incubation, the culture medium was removed and the cells were rinsed two times with PBS prior to the fluorescence assessment, stained with Hoechst 33258 and fixed with 4% paraformaldehyde. Therefore, the location of intracellular fluorescence was validated using a CLSM imaging system with excitation wavelengths of 480 nm for DOX and 405 nm for Hoechst. It is noteworthy that the strong DOX fluorescence was exhibited in the cells after 6 h incubation with DOX-loaded GON-Cy-ALG-PEG in the presence 10 mM GSH-OEt, indicating the internalization of the DOX-loaded GON-Cy-ALG-PEG and rapid release of DOX inside cells. It is clear from these fluorescence images that DOX has been

efficiently released from the DOX-loaded GON-Cy-ALG-PEG in the presence 10 mM GSH-OEt to cytosol. Remarkably, the CLSM studies showed that after 6 h incubation, most of the released DOX had also been transported into the cell nucleus (Figure 6C). Moreover, most of the HepG2 cells died in the field of vision. In comparison, the DOX-loaded GON-Cy-ALGPEG was mainly accumulated in cytoplasm in the absence of GSH-OEt (Figure 6B). Meanwhile, partial cells exhibited unbroken morphology. The results implied that the DOXloaded GON-Cy-ALG-PEG could efficiently carry DOX to the cell nucleus in the presence of 10 mM GSH-OEt (Scheme 1B). Notably, the stronger fluorescence of the latter emerged image indicating that the DOX escaped from the DOX-loaded GONCy-ALG-PEG in the presence of 10 mM GSH-OEt was mainly accumulated in cell nucleus by comparing with the DOXloaded GON-Cy-ALG-PEG in the absence of GSH-OEt (Figure 6B,C).21 It should be further noted that DNA of the cell nucleus was destroyed by intercalation of the released DOX from the DOX-loaded GON-Cy-ALG-PEG after 6 h incubation (Figure 6C). The CLSM analysis demonstrated that the DOXloaded GON-Cy-ALG-PEG were successfully internalized for HepG2 cells and the DOX escaped from DOX-loaded GONCy-ALG-PEG was mainly accumulated in cell nucleus and broken single- and double-strand of DNA. As shown in Figure 7, the percentages of apoptosis induced by the free DOX and the DOX-loaded GON-Cy-ALG-PEG in the presence of 10 mM GSH-OEt were 99.91%, and 99.96% after 6-h treatment, respectively. These results indicated that an enhanced cell apoptosis was correlated with reduction-sensitive properties to detach the Cy-ALG-PEG brushes of the GON in the presence of 10 mM GSH-OEt.40 Moreover, the percentages of apoptosis induced by the GON-Cy-ALG-PEG was only 7.31%, revealing the excellent biocompatibility arising from the polymeric moieties of ALG-PEG. The results of the cell apoptosis also validated the conclusion from the WST-1 assay and CLSM.

■

CONCLUSIONS In summary, we have demonstrated the three-dimensional (3D) nanoscaled, biocompatible, reduction-responsive GON-CyALG-PEG hybrid for efficiently loading and specific delivery of DOX for the first time. The GON-Cy-ALG-PEG hybrid offers several unique advantages: (i) 3-D nanoscaled graphene oxide nanoparticles (GON) with suitable scale and spherical shape can be successfully transferred from graphene oxide sheets for the first time; (ii) owing to the introduction of the ALG-PEG brushes, the phenomenon of the stealth of DOX is prevented 10427

dx.doi.org/10.1021/la502952f | Langmuir 2014, 30, 10419−10429

Langmuir

Article

(12) MacKay, J. A.; Chen, M.; McDaniel, J. R.; Liu, W.; Simnick, A. J.; Chilkoti, A. Self-assembling Chimeric Polypeptide−doxorubicin Conjugate Nanoparticles That Abolish Tumours After a Single Injection. Nat. Mater. 2009, 8, 993−999. (13) Gao, W. P.; Liu, W. G.; Christensen, T.; Zalutsky, M. R.; Chilkoti, A. In Situ Growth of a PEG-like Polymer from the C Terminus of an Intein Fusion Protein Improves Pharmacokinetics and Tumor Accumulation. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 16432− 16437. (14) Park, J. H.; Gu, L.; von Maltzahn, G.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Biodegradable Luminescent Porous Silicon Nanoparticles for In Vivo Applications. Nat. Mater. 2009, 8, 331−336. (15) Hobbs, S. K.; Monsky, W. L.; Yuan, F.; Roberts, W. G.; Griffith, L.; Torchilin, V. P.; Jain, R. K. Regulation of Transport Pathways in Tumor Vessels: Role of Tumor Type and Microenvironment. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 4607−4612. (16) MacEwan, S. R.; Callahan, D. J.; Chilkoti, A. Stimulusresponsive Macromolecules and Nanoparticles for Cancer Drug Delivery. Nanomedicine (London) 2010, 5, 793−806. (17) Ge, Z. S.; Liu, S. Y. Functional Block Copolymer Assemblies Responsive to Tumor and Intracellular Microenvironments for Sitespecific Drug Delivery and Enhanced Imaging Performance. Chem. Soc. Rev. 2013, 42, 7289−7325. (18) Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada, Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.; Nishiyama, N.; Kataoka, K. Accumulation of Sub-100 nm Polymeric Micelles in Poorly Permeable Tumours Depends on Size. Nat. Nanotechnol. 2011, 6, 815−823. (19) Yang, K.; Feng, L. Z.; Shi, X. Z.; Liu, Z. Nano-graphene in Biomedicine: Theranostic Applications. Chem. Soc. Rev. 2013, 42, 530−547. (20) Sun, X. M.; Liu, Z.; Welsher, K.; Robinson, J. T.; Goodwin, A.; Zaric, S.; Dai, H. J. Nano-Graphene Oxide for Cellular Imaging and Drug Delivery. Nano Res. 2008, 1, 203−212. (21) Wen, H. Y.; Dong, C. Y.; Dong, H. Q.; Shen, A. J.; Xia, W. J.; Cai, X. J.; Song, Y. Y.; Li, X. Q.; Li, Y. Y.; Shi, D. L. Engineered RedoxResponsive PEG Detachment Mechanism in PEGylated NanoGraphene Oxide for Intracellular Drug Delivery. Small 2012, 8, 760−769. (22) Li, Y. J.; Feng, L. Z.; Shi, X. Z.; Wang, X. J.; Yang, Y. L.; Yang, K.; Liu, T.; Yang, G. B.; Liu, Z. Surface Coating-Dependent Cytotoxicity and Degradation of Graphene Derivatives: Towards the Design of Non-Toxic, Degradable Nano-Graphene. Small 2014, 10, 1544−1554. (23) Bao, H. Q.; Pan, Y. Z.; Ping, Y.; Sahoo, N. G.; Wu, T. F.; Li, L.; Li, J.; Gan, L. H. Chitosan-functionalized Graphene Oxide as a Nanocarrier for Drug and Gene Delivery. Small 2011, 7, 1569−1578. (24) Gerweck, L. E.; Seetharaman, K. Cellular pH Gradient in Tumor versus Normal Tissue: Potential Exploitation for the Treatment of Cancer. Cancer Res. 1996, 56, 1194−1198. (25) Issels, R. D. Hyperthermia Adds to Chemotherapy. Eur. J. Cancer 2008, 44, 2546−2554. (26) de la Rica, R.; Aili, D.; Stevens, M. M. Enzyme-responsive Nanoparticles for Drug Release and Diagnostics. Adv. Drug Delivery Rev. 2012, 64, 967−978. (27) Murphy, R. F.; Powers, S.; Cantor, C. R. Endosome pH Measured in Single Cells by Dual Fluorescence Flow Cytometry: Rapid Acidification of Insulin to pH 6. J. Cell Biol. 1984, 98, 1757− 1762. (28) Go, Y. M.; Jones, D. P. Redox Compartmentalization in Eukaryotic Cells. Biochim. Biophys. Acta Gen. Subj. 2008, 1780, 1271− 1290. (29) Al-Nahaina, A.; Leeb, S. Y.; Inc, I.; Leed, K. D.; Park, S. Y. Triggered pH/redox Responsive Release of Doxorubicin from Prepared Highly Stable Graphene with Thiol Grafted Pluronic. Int. J. Pharm. 2013, 450, 208−217. (30) Feng, L. Z.; Li, K. Y.; Shi, X. Z.; Gao, M.; Liu, J.; Liu, Z. Smart pH-Responsive Nanocarrier Based on Nano-Graphene Oxide for

efficiently during the in vitro control release process under simulated physiological media in the normal tissues; (iii) importantly, the Cy-ALG-PEG polymer brushes are grafted onto the surface of the GON via reduction-sensitive disulfide bond, so the nanocarriers are response to cleave the disulfide bond to detach the Cy-ALG-PEG polymer moieties in reducing condition. Confocal microscopy, WST-1 assays, and flow cytometric experiments reveal that the DOX-loaded GONCy-ALG-PEG has efficient endocytosis, excellent biocompatibility, and high cytotoxicity to model cells. So it is believed that the biocompatible and reduction-responsive GON-Cy-ALGPEG hybrid can be used as advanced DDS for cancer therapy.

■

ASSOCIATED CONTENT

S Supporting Information *

Higuchi and Korsmeyer−Peppas equation drug release models. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*P. Liu. Tel./Fax: 86 0931 8912582. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This project was granted financial support from the National Nature Science Foundation of China (Grant no. 20904017), the Program for New Century Excellent Talents in University (Grant no. NCET-09-0441), and the Fundamental Research Funds for the Central Universities (No. lzujbky-2014-245).

■

REFERENCES

(1) Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Zhang, Y.; Dubonos, S. V.; Grigorieva, I. V.; Firsov, A. A. Electric Field Effect in Atomically Thin Carbon Films. Science 2004, 306, 666−669. (2) Novoselov, K. S.; Jiang, D.; Schedin, F.; Booth, T. J.; Khotkevich, V. V.; Morozov, S. V.; Geim, A. K. Two-dimensional Atomic Crystals. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 10451−10453. (3) Geim, A. K. Graphene: Status and Prospects. Science 2009, 324, 1530−1534. (4) Pang, S. P.; Hernandez, Y.; Feng, X. L.; Müllen, K. Graphene as Transparent Electrode Material for Organic Electronics. Adv. Mater. 2011, 23, 2779−2795. (5) Niyogi, S.; Bekyarova, E.; Itkis, M. E.; Zhang, H.; Shepperd, K.; Hicks, J.; Sprinkle, M.; Berger, C.; Lau, C. N.; de Heer, W. A.; Conrad, E. H.; Haddon, R. C. Spectroscopy of Covalently Functionalized Graphene. Nano Lett. 2010, 10, 4061−4066. (6) Stankovich, S.; Dikin, D. A.; Dommett, G. H. B.; Kohlhaas, K. M.; Zimney, E. J.; Stach, E. A.; Piner, R. D.; Nguyen, S. T.; Ruoff, R. S. Graphene-based Composite Materials. Nature 2006, 442, 282−286. (7) Kim, H.; Abdala, A. A.; Macosko, C. W. Graphene/Polymer Nanocomposites. Macromolecules 2010, 43, 6515−6530. (8) Liu, Z.; Robinson, J. T.; Sun, X. M.; Dai, H. J. PEGylated NanoGraphene Oxide for Delivery of Water Insoluble Cancer Drugs. J. Am. Chem. Soc. 2008, 130, 10876−10877. (9) Wang, Y.; Li, Z. H.; Wang, J.; Li, J. H.; Lin, Y. H. Graphene and Graphene Oxide: Biofunctionalization and Applications in Biotechnology. Trend Biotechnol. 2011, 29, 205−212. (10) Pan, Y. Z.; Bao, H. Q.; Sahoo, N. G.; Wu, T. F.; Li, L. WaterSoluble Poly(N-isopropylacrylamide)−Graphene Sheets Synthesized via Click Chemistry for Drug Delivery. Adv. Funct. Mater. 2011, 21, 2754−2763. (11) Mailander, V.; Landfester, K. Interaction of Nanoparticles with Cells. Biomacromolecules 2009, 10, 2379−2400. 10428

dx.doi.org/10.1021/la502952f | Langmuir 2014, 30, 10419−10429

Langmuir

Article

Combined Chemo- and Photothermal Therapy Overcoming Drug Resistance. Adv. Healthcare Mater. DOI: 10.1002/adhm.201300549. (31) Hummers, W. S.; Offeman, R. E. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 1958, 80, 1339−1339. (32) Meng, X. W.; Ha, W.; Cheng, C.; Dong, Z. Q.; Ding, L. S.; Li, B. J.; Zhang, S. Hollow Nanospheres Based on Self-assembly of Alginategraft-poly(ethylene glycol) and α-Cyclodextrin. Langmuir 2011, 27, 14401−14407. (33) Korsmeyer, R. W.; Gurny, R.; Doelker, E. M.; Buri, P.; Peppas, N. A. Mechanism of Solute Release from Porous Hydrophilic Polymers. Int. J. Pharm. 1983, 15, 25−35. (34) Higuchi, T. Rate of Release of Medicaments from Ointment Bases Containing Drugs in Suspension. J. Pharm. Sci. 1961, 50, 874− 875. (35) Siepmann, J.; Peppas, N. A. Modeling of Drug Release from Delivery Systems Based on Hydroxypropyl Methylcellulose (HPMC). Adv. Drug Delivery Rev. 2001, 48, 139−157. (36) Jung, I.; Dikin, D.; Park, S.; Cai, W.; Mielke, S. L.; Ruoff, R. S. Effect of Water Vapor on Electrical Properties of Individual Reduced Graphene Oxide Sheets. J. Phys. Chem. C 2008, 112, 20264−20268. (37) Shen, J.; Hu, Y.; Li, C.; Qin, C.; Ye, M. Synthesis of Amphiphilic Graphene Nanoplatelets. Small 2009, 5, 82−85. (38) Raval, A.; Parikh, J.; Engineer, C. Mechanism and in Vitro Release Kinetic Study of Sirolimus from a Biodegradable Polymeric Matrix Coated Cardiovascular Stent. Ind. Eng. Chem. Res. 2011, 50, 9539−9549. (39) Franson, N. W.; Peppas, N. A. Influence of Copolymer Composition on Non-fickian Water Transport through Glassy Copolymers. J. Appl. Polym. Sci. 1983, 28, 1299−1310. (40) Yu, B. J.; Tang, C.; Yin, C. H. Enhanced Antitumor Efficacy of Folate Modified Amphiphilic Nanoparticles through Co-delivery of Chemotherapeutic Drugs and Genes. Biomaterials 2014, 35, 6369− 6378.

10429

dx.doi.org/10.1021/la502952f | Langmuir 2014, 30, 10419−10429