Graphene oxide stabilized by PLA–PEG copolymers for the controlled delivery of paclitaxel

European Journal of Pharmaceutics and Biopharmaceutics 93 (2015) 18–26

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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Research Paper

Graphene oxide stabilized by PLA–PEG copolymers for the controlled delivery of paclitaxel A. Angelopoulou a, E. Voulgari a, E.K. Diamanti b, D. Gournis b, K. Avgoustakis a,⇑ a b

Department of Pharmacy, Medical School, University of Patras, 26 500 Patras, Greece Department of Materials Science and Engineering, University of Ioannina, 45110 Ioannina, Greece

a r t i c l e

i n f o

Article history: Received 12 November 2014 Revised 4 March 2015 Accepted in revised form 12 March 2015 Available online 24 March 2015 Keywords: Graphene oxide PLA–PEG Paclitaxel Colloidal stability Cytotoxicity

a b s t r a c t Purpose: To investigate the application of water-dispersible poly(lactide)–poly(ethylene glycol) (PLA– PEG) copolymers for the stabilization of graphene oxide (GO) aqueous dispersions and the feasibility of using the PLA–PEG stabilized GO as a delivery system for the potent anticancer agent paclitaxel. Methods: A modified Staudenmaier method was applied to synthesize graphene oxide (GO). Diblock PLA– PEG copolymers were synthesized by ring-opening polymerization of DL-lactide in the presence of monomethoxy-poly(ethylene glycol) (mPEG). Probe sonication in the presence of PLA–PEG copolymers was applied in order to reduce the hydrodynamic diameter of GO to the nano-size range according to dynamic light scattering (DLS) and obtain nano-graphene oxide (NGO) composites with PLA–PEG. The composites were characterized by atomic force microscopy (AFM), thermogravimetric analysis (TGA), and DLS. The colloidal stability of the composites was evaluated by recording the size of the composite particles with time and the resistance of composites to aggregation induced by increasing concentrations of NaCl. The composites were loaded with paclitaxel and the in vitro release profile was determined. The cytotoxicity of composites against A549 human lung cancer cells in culture was evaluated by flow cytometry. The uptake of FITC-labeled NGO/PLA–PEG by A549 cells was also estimated with flow cytometry and visualized with fluorescence microscopy. Results: The average hydrodynamic diameter of NGO/PLA–PEG according to DLS ranged between 455 and 534 nm, depending on the molecular weight and proportion of PLA–PEG in the composites. NGO/PLA– PEG exhibited high colloidal stability on storage and in the presence of high concentrations of NaCl (far exceeding physiological concentrations). Paclitaxel was effectively loaded in the composites and released by a highly sustained fashion. Drug release could be regulated by the molecular weight of the PLA–PEG copolymer and its proportion in the composite. The paclitaxel-loaded composites exhibited cytotoxicity against A549 cancer cells which increased with incubation time, in conjunction with the increasing with time uptake of composites by the cancer cells. Conclusion: Graphene oxide aqueous dispersions were effectively stabilized by water-dispersible, biocompatible and biodegradable PLA–PEG copolymers. The graphene oxide/PLA–PEG composites exhibited satisfactory paclitaxel loading capacity and sustained in vitro drug release. The paclitaxel-loaded composites could enter the A549 cancer cells and exert cytotoxicity. The results justify further investigation of the suitability of PLA–PEG stabilized graphene oxide for the controlled delivery of paclitaxel. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Graphene is a carbon material consisting of one atom thick two dimensional layers with a honeycomb structure. It is characterized by extraordinary mechanical, thermal, physical and electronic properties and is extensively investigated for applications in many ⇑ Corresponding author at: Department of Pharmacy, University of Patras, Rio 26504, Greece. Tel.: +30 2610 962317. E-mail address: [email protected] (K. Avgoustakis). http://dx.doi.org/10.1016/j.ejpb.2015.03.022 0939-6411/Ó 2015 Elsevier B.V. All rights reserved.

technological fields such as nanoelectronics, sensors, nanocomposites, batteries, supercapacitors and hydrogen storage [1]. However, graphene sheets, which have theoretically a high specific surface area, tend to form irreversible agglomerates or even restack to form graphite through van der Waals interactions [2]. The most common approach to graphite exfoliation is the use of strong oxidizing agents to yield graphene oxide (GO) [3], an oxygen rich derivative of graphite. The oxidation of flake graphite to produce GO can be affected using the Brodie [4], Staudenmaier [5] or the most commonly used today Hummers method [6]. In GO the

A. Angelopoulou et al. / European Journal of Pharmaceutics and Biopharmaceutics 93 (2015) 18–26

previously contiguous aromatic lattice of graphene is interrupted by epoxides, hydroxyls, ketone carbonyls, and carboxylic groups [7–9]. These functional groups are created by strong oxidation and distributed randomly on the basal planes and edges of the GO sheets, generating aliphatic regions (sp3-carbon atoms) within the sp2-hybridized matrix. The high surface area and the presence of functional groups, which would allow for the attachment of drugs, targeting moieties and anti-biofouling agents, render GO an attractive material for biomedical applications, such as drug delivery applications recently reviewed by Goenk et al. [10]. GO is dispersible in water, however quickly aggregates when dispersed in electrolyte solutions, such as buffered saline [11]. Such aggregation behavior significantly limits the applicability of graphene oxide in biological applications, since it would lead to inefficient coupling with biomolecules, limited cellular uptake, and diminished delivery efficiency [12]. To circumvent this aggregation problem, PEG molecules have been attached onto GO, either by covalently binding of suitable PEG derivatives on GO making use of the functional groups of GO [11,13–15] or by physically attaching amphiphilic PEG copolymers, such as pluronics, on GO [12,16,17]. The flexible, hydrophilic PEG chains sterically stabilize the GO dispersions in aqueous media. The latter approach is simpler; however, pluronics are not biodegradable raising concerns of toxicity upon repeated administration due to polymer accumulation in the body [18]. Besides pluronics, surfactants such as sodium dodecyl sulfate, sodium dodecyl benzene sulfonate and Triton X-100 have been applied to stabilize graphene aqueous dispersions [19], but the toxicity of surfactants limits the usefulness of such graphene systems for pharmaceutical applications. In this work, water dispersible poly(lactide)–poly(ethylene glycol) copolymers (PLA–PEG) were investigated for their ability to stabilize nanographene oxide (NGO) aqueous dispersions. PLA–PEG was selected because it is biocompatible and biodegradable, and FDA-approved copolymer, and PLA–PEG nanoparticles have entered clinical trials [20,21]. Also, the feasibility of using the NGO/PLA–PEG composites for the controlled delivery of the anticancer drug paclitaxel was studied. 2. Materials and methods 2.1. Materials Graphite flakes (purum, powder 60.2 mm) were obtained from Fluka and DL-lactide was purchased from Polysciences Inc. (USA). Monomethoxy poly(ethylene glycol) (mPEG) of molecule weight 10K and 20K, stannous 2-ethylhexanoate, N-ethyl-N0 (3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC, >98%), N-hydroxysuccinimide (NHS, 98%) and fluorescein isothiocyanate (FITC, >90% HPLC) were obtained from Sigma–Aldrich. Paclitaxel was purchased from LC Laboratories, USA. All other chemicals and solvents used were at least of analytical grade. 2.2. Synthesis and characterization of graphene oxide A modified Staudenmaier method was applied to synthesize graphene oxide (GO) [22]. In a typical synthesis, 10 g of powdered graphite was added to a mixture of concentrated sulfuric acid (400 ml, 95–97 wt%) and nitric acid (200 ml, 65 wt%) while cooling in an ice-water bath. Potassium chlorate powder (200 g, purum, >98.0%; Fluka) was added to the mixture in small portions while stirring and cooling. The reactions were quenched after 18 h by pouring the mixture into distilled water and the oxidation product washed until a pH 6. The sample was finally dried at room temperature.

19

The synthesized GO was characterized by powder X-ray diffraction (XRD) and infrared (FTIR) spectroscopy. The XRD patterns were collected on a D8 Advance Bruker diffractometer by using Cu Ka (40 kV, 40 mA) radiation and a secondary beam graphite monochromator. The patterns were recorded in the 2-theta (2H) range from 2° to 80°, in steps of 0.02° and a counting time of 2 s per step. Infrared spectra were measured with a Perkin–Elmer Spectrum GX infrared spectrometer, in the region of 400– 4000 cm1, equipped with a deuterated triglycine sulfate (DTGS) detector. Each spectrum was the average of 64 scans collected with 2 cm1 resolution. Samples were in the form of KBr pellets containing ca. 2 wt% sample. 2.3. Synthesis and characterization of PLA–PEG copolymers The diblock PLA–PEG copolymers were synthesized by ringopening polymerization of DL-lactide in the presence of monomethoxy-poly(ethylene glycol) (mPEG) of 10K and 20K molecular weights [23]. In brief, predetermined amounts of lactide and mPEG were dissolved in recently distilled dry toluene in a threenecked glass flask equipped with condenser and connected to a nitrogen source. The mixture was heated to 130 °C and agitated with a magnetic stirrer. Then, stannous 2-ethylhexanoate (0.25% w/w) was added as a catalyst and the polymerization was allowed to proceed for 4 h. The product was recovered by dissolution in dichloromethane and precipitation in excess diethyl ether. Finally, the product was washed and dried under vacuum to constant weight. The composition of the synthesized copolymers was confirmed by 1H NMR. The copolymers were dissolved in CDCl3 and the spectra were recorded in a Bruker spectrometer operating at 400 MHz. Fourier transform infrared spectra (FT-IR) of the synthesized copolymers were obtained using a DigiLab Excalibur series FTS 3000 spectrometer equipped with an attenuated total reflectance (ATR) and a class II laser. The spectra were recorded from 400 to 4000 cm1 using the KBr method. 2.4. Preparation and characterization of nano-graphene oxide (NGO) stabilized by PLA–PEG copolymers GO was dispersed in double-distilled water at a concentration of 0.2 mg/ml. The dispersion was bath sonicated for 1 h and then probe sonicated (50 kHz, 20 W) for an additional 1 h in an ice-bath. The GO suspension was centrifuged at 2500 rpm for 5 min at 15 °C and the supernatant was kept. This way, GO suspension of 85% yield was obtained. To the GO suspension PLA–PEG dissolved in water was added to generate GO/PLA–PEG 1/2 and 1/6 (w/w) mixtures. The mixtures were probe sonicated for 1 h (50 kHz, 20 W) and bath sonicated for 45 min. Then, they were centrifuged at 3000 rpm for 5 min at 15 °C and the supernatants were collected and stored in the fridge (8 °C). The same process was followed for GO without the addition of PLA–PEG (GO control). The yield of NGO/PLA–PEG composites preparation was determined by freeze-drying supernatant samples and weighing the solid residues and was found to be in the range 86–88%. The composition of the composites was investigated by thermal gravimetric analysis (TGA) on a TA Instrument Q500 series Thermogravimetric Analyzer at a heating rate of 10 °C/min from room temperature to 600 °C in air. The morphology and microstructure of NGO/PLA–PEG were investigated by atomic force microscopy (AFM). AFM images were obtained in tapping mode with a 3D Multimode Nanoscope, using Tap-300G silicon cantilevers with a tip radius <10 nm and a force constant of 20–75 N m1. Samples were deposited onto silicon wafers (P/Bor, single side polished) from aqueous dispersions by drop casting. The morphology of NGO/PLA–PEG was also investigated

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by transmission electron microscopy (TEM) using a JEOL JEM-2100 microscope at an acceleration potential of 200 kV, equipped with a GATAN camera Erlangshen ES500W, model 782. Specimens for TEM were prepared by spreading an aqueous solution of 0.1 mg/ ml concentration onto a carbon coated cooper MS200 grid, which was air dried before observation. The size (hydrodynamic diameter) distribution characteristics (average size and polydispersity index, Pdi) of NGO/PLA–PEG were evaluated using a ZetaSizer Nano series Nano-ZS (Malvern Instruments Ltd, Malvern, UK) equipped with a He–Ne Laser beam at a wavelength of 633 nm and a fixed backscattering angle of 173°. The colloidal stability of the composites was evaluated by recording the size of the composite particles with time and the resistance of composites to aggregation induced by increasing concentrations of NaCl.

2.5. FITC labeling In order to label NGO/PLA–PEG composites with the fluorescent dye fluorescein isothiocyanate (FITC), NGO/PLA–PEG (1.5 mg/ml) and EDC (1.5 mg/ml) were dispersed in phosphate buffer (1 mM, pH 7.4) at a final volume of 3.5 ml. The mixture was sonicated in ice-bath for 1 h and then the pH was adjusted to 8.0 with NaOH. FITC at a weight ratio with NGO/PLA–PEG of 1:1 was added and the reaction mixture was sonicated in ice-bath for 2 h in the dark. Following, the mixture was stirred at room temperature for 24 h in the dark. The product was purified by repeated centrifugation and extended dialysis against water. The purified NGO/PLA–PEG/FITC was stored in the fridge (4 °C). The amount of FITC label on GO was measured by spectrofluorometry (excitation 480 nm, emission 520 nm) using a Perkin Elmer, Precisely LS55 Fluorescence spectrofluorometer. For the measurement, a standard curve within a range of FITC concentrations 5–50 lg/ml (R2 = 0.9832) was constructed. 2.6. Loading of NGO/PLA–PEG with paclitaxel In order to load paclitaxel (PTX) on NGO/PLA–PEG 1 mg of NGO/ PLA–PEG was suspended in PB (pH 7.4) and 3 mg of PTX, dissolved in 0.5 ml of methanol, was added (dropwise). The mixture was bath sonicated for 3 h and then agitated gently for 3 days. After that, the mixture was centrifuged (5,000 rpm, 5 min) in order to remove PTX aggregates possibly formed and the supernatant was enclosed in a dialysis membrane with molecular weight cutoff of 12K and dialyzed for 24 h with excess water in order to remove free (not bound on GO) PTX. The purified product was stored in the fridge (4 °C). The quantity of drug loaded in NGO/PLA–PEG was determined by HPLC. Freeze-dried samples were dissolved in 1 ml of a 60:40 (v/v) mixture of acetonitrile:water, filtered through Millipore filters of 0.2 lm diameter and analyzed using a Waters 2695 Separations Module HPLC apparatus. A Kromasil 100-5-C18, 4.0  150 mm column was used. The mobile phase was a 60:40 (v/v) mixture of acetonitrile:water and a flow rate of 0.5 mg/ml was applied. Under these conditions, the retention time of the drug was 7.8 min. The PTX content of the samples was calculated based on a calibration curve (R2 = 0.9997). The limit of quantification was 0.005 lg/ml and the linear part of the standard curve used for PTX assay was from 0.005 to 200 lg/ml. The loading capacity and entrapment efficiency of PTX in NGO/ PLA–PEG were calculated from the following equations:

LC% ¼

W  100 WC þ W

ð1Þ

EE% ¼

W  100 W0

ð2Þ

where W, WC and W0 are the amount of entrapped drug according to the HPLC analysis, the amount of NGO/PLA–PEG carrier, and the amount of initially added drug, respectively. 2.7. PTX release from the NGO/PLA–PEG in vitro Samples of NGO/PLA–PEG/PTX were enclosed in a dialysis membrane with a molecular weight cutoff of 12K and transferred to 10 ml of PB (pH = 7.4). The release medium was retained in a water bath at 37 °C under gentle agitation. At predetermined time intervals the release medium was completely removed and replaced with fresh buffer. The released PTX was extracted from the release medium with 2 ml of dichloromethane. Then, the dichloromethane was allowed to evaporate at room temperature in a fume cupboard and the solid residue was dissolved in 1 ml of a 60:40 (v/v) mixture of acetonitrile:water. The samples were assayed for PTX by HPLC as described in the previous paragraph. 2.8. Cellular studies The human lung adenocarcinoma epithelial cells A549 (ATCC) were cultured in RPMI supplemented with 10% fetal bovine serum and 100 lg/ml penicillin–streptomycin at 37 °C in a humidified atmosphere with 5% CO2. The culture medium was changed three times a week. The in vitro anticancer activity of NGO/PLA–PEG/PTX against the A549 human lung cancer cells (ATCC) was determined using the propidium iodide (PI) fluorescence method [24]. The cells were plated into 24-well plates at a density 5  104 cells/well and allowed to attach and proliferate for 24 h under standard cell culture conditions. The supernatant in each well then replaced with medium containing various concentrations of NGO/PLA–PEG/PTX or control NGO/PLA–PEG. After the predetermined incubation period, the supernatant was removed and the cells were washed with phosphate buffered saline (PBS). The cells were detached with 0.25% trypsin, transferred to FACS tubes and then centrifuged (1600 rpm for 5 min) and the pellet washed with PBS. After washing, the cells in the pellet were incubated with 5 ll PI (propidium iodide) solution (1 mg/ml) for 1 min. The PI fluorescence (cell death) was determined with flow cytometry (excitation k = 488 nm, emission k = 620 nm), in a FACS Calibur, Coulter Epics XL-MCL apparatus. To calculate the background fluorescence of unlabeled cells, cells without any addition of studied particles were used as a negative control in every measurement. Data analysis was performed with the WinMDI cytometry analysis software. The uptake of FITC-labeled NGO/PLA–PEG by A549 cells was also quantitatively estimated with flow cytometry. A549 cells grown as a monolayer were harvested with 0.25% (w/v) trypsin and seeded in 24 well plates at a density of 5  104 cells per well and incubated for 24 h. FITC-labeled NGO/PLA–PEG (0.035 mg/ ml) was added in the wells and incubated for 6, 24, 48 and 72 h. The cells, after washing twice with PBS, were harvested and the fluorescence was measured using FACS (excitation k = 488 nm, emission k = 530 nm), as described above. In order to visualize cell uptake, the method proposed recently by Hezel et al. [25] was employed. A549 cells (4  105 cells/ml) were incubated with FITC-labeled NGO/PLA–PEG (100 lg/ml) for predetermined times on cover slips. The cells were washed thrice with PBS and fixed directly onto the glass coverslips using 4% paraformaldehyde (PFA) dissolved in PBS for 15 min at room temperature. After washing with PBS, the cells were incubated with 0.1% Triton X 100 for 10 min. The cells were then washed with PBS and incubated with PI for 15 min. The obtained specimens were imaged using a Leica

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Microsystems DMLB I/2001 microscope equipped with a Leica fluorescence source and a Leica DC 300 camera. 2.9. Statistical analysis Appropriate statistical procedures (Student’s t-test for comparison of means, Statgraphics plus 4.0 software) were applied in the statistical analysis of experimental data. 3. Results 3.1. Characterization of GO X-ray diffraction patterns of graphite and graphene oxide (GO) are shown in Fig. 1A. Pristine graphite shows a very sharp diffraction peak at 2h = 26.7°, which corresponds to the diffraction of the (0 0 2) plane, being the interplanar distance, d002 = 3.3 Å. After graphite oxidation to GO, the (0 0 2) reflection of graphite disappears and a diffraction peak at 2h = 11.9° is present, which corresponds to the diffraction of the 0 0 1 plane (d001 = 7.4 Å), indicative of the successful oxidation of graphite and the creation of oxygen-containing groups that are randomly distributed on the basal planes and edges of the graphene sheets. Contrary to graphite, which is an IR-inactive solid, the FTIR spectrum of GO presents many peaks attributed to the oxygen-containing groups of GO (Fig. 1B). The peak at 1630 cm1 is attributed to AC@O stretching, the peak at 1395 cm1 to OH vibration, the peaks at 1215 cm1 and 1060 cm1 to CAO stretching and the peak at 970 cm1 to epoxy groups [26]. Finally, the peak at 815 cm1 is attributed to the bending of C@O of carboxylic groups and the broad peak at 3420 cm1 to stretching vibrations of hydroxyls of the same groups. Both the XRD and FTIR data confirm the successful oxidation of the pristine graphite toward synthesis of GO. 3.2. Characterization of PLA–PEG copolymers

The morphology of NGO/PLA–PEG was examined thoroughly by AFM (Fig. 2A and B). The AFM images revealed that GO sheets have a thickness between 0.7 and 3.3 nm and, since the thickness of a single GO layer is about 0.61 nm [27], it indicates that the sample consists mostly of few layers graphene nanosheets and single graphene monolayers. In NGO/PLA–PEG sample various shape objects (polymer deposits) are shown to be attached on the surface of the GO sheets (indicated by arrows). In this case, the average height is increased and lies between 6.3 and 8.1 nm, due to the presence of the polymer chains on the surface and within the GO layers. TEM images (Fig. 2C) confirm that NGO/PLA–PEG has nanoscale sheetlike structure. TGA graphs for NGO/PLA–PEG composites and components are shown in Fig. 3. In the case of pure GO, little weight loss (about 5%) occurred before 200 °C, which can be ascribed to adsorbed water evaporation. The main weight loss (about 60%) occurred between 200 °C and 300 °C, attributed to the reduction of oxygen containing functional groups [28]. In the case of pure PLA–PEG copolymers, almost complete decomposition took place between 250 °C and 410 °C. The NGO/PLA–PEG composites exhibited an intermediate weight loss profile between that of pure GO and PLA–PEG. The composition of the NGO/PLA–PEG composites (Table 1) was estimated from the TGA graphs using the equation (1  M) n = W [29], where M is the polymer percent weight of the composite, n is the percent residual weight of GO and W is the percent residual weight of the composite. The hydrodynamic diameter of GO (control) and NGO/PLA–PEG according to DLS is presented in Table 1. The NGO/PLA–PEG samples exhibited lower size than GO control, probably because in the case of NGO/PLA–PEG the presence of PLA–PEG chains prevented aggregation of GO sheets. 3.4. Colloidal stability of NGO/PLA–PEG Contrary to GO, NGO/PLA–PEG exhibited high colloidal stability on storage at 8 °C (fridge) over the investigated period of 2 months and in the presence of increasing concentrations of NaCl (Fig. 4).

1625

(B)

d001=7.4 Å

1395 1215 1060 968 815

(A)

Absorbance

Intensity (a.u.)

d002=3.3 Å

3.3. Characterization of NGO/PLA–PEG

3420

The identity and purity of synthesized PLA(1.5K)–PEG(10K) and PLA(7.8K)–PEG(20K) copolymers was confirmed by 1H NMR and FT-IR spectroscopy. The proton 1H NMR spectra of PLA–PEG polymers (Fig. S1) are characterized by the presence of a signal at 3.65 ppm, which is ascribed to the methylene protons of the PEG units and the peaks at 1.55 ppm and 5.25 ppm, which are attributed to the methyl (ACH3) and methylene (ACH2A) protons of PLA, respectively. In the FTIR spectra (Fig. S2), the main peaks are as follows: the peak at 2887 cm1, which is attributed to CAH stretching

of PEG, the peak at 2500 cm1, which is attributed to the stretching vibrations of the hydrocarbon groups of ACH2A and ACH3, the peak at 1757 cm1, which is ascribed to the C@O stretching vibration of PLA, and the peaks at 1100 and 1338 cm1, which are assigned to the CAOAC stretching vibration of PEG.

GO

GO Graphite

Graphite 10

20

30

40

50 O

2θ ( )

60

70

80

4000

3500

3000

2500

2000

1500 -1

Wavenumbers (cm )

Fig. 1. XRD patterns (A) and FTIR spectra (B) of graphite and graphene oxide (GO).

1000

500

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Fig. 2. (A) AFM image of GO, (B) AFM image of NGO/PLA(7.8K)mPEG(20K), (C) TEM image of NGO/PLA(7.8K)mPEG(20K). Arrows in (B) indicate PLA–PEG structures deposited on GO sheets (red arrows indicate particulate structures, and blue arrows indicate continuous structures). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. TGA graphs for GO (control) and NGO/PLA–PEG composites, (A) NGO/PLA(1.5K)mPEG(10K) composites and (B) NGO/PLA(7.8K)mPEG(20K) composites.

There were no significant differences in the stability of NGO/PLA– PEG systems containing different proportions of PLA–PEG or prepared from PLA–PEG copolymers of different molecular weights. 3.5. Loading behavior and release profiles from PEGnano-GO-PTX complexes The loading capacity (LC, %w/w) and entrapment efficiency (EE, %w/w) of NGO/PLA–PEG for the anticancer agent paclitaxel are shown in Fig. 5. Loading capacities between 9% and 11% and entrapment efficiencies between 15% and 17% were obtained for the different copolymers and NGO/PLA–PEG proportions. The differences in loading and entrapment efficiency were not statistically

significant (Student t-test, p > 0.05) between the different copolymers and the different NGO/PLA–PEG proportions. Slightly lower drug loading was observed in the case of the higher molecular weight copolymer PLA(7.8K)–PEG(20K), probably because the higher molecular weight PLA chains covered higher proportion of graphene oxide surface posing a greater obstacle in paclitaxel attachment on graphene oxide surface. Sustained in vitro paclitaxel release over many hours was observed for the paclitaxel-loaded NGO/PLA–PEG composites (Fig. 6). An increase in the molecular weight of the PLA–PEG copolymer or in the proportion of PLA–PEG in the NGO/PLA–PEG composites caused a decrease of drug release rate, probably because both these changes increased the drug diffusion barrier

A. Angelopoulou et al. / European Journal of Pharmaceutics and Biopharmaceutics 93 (2015) 18–26 Table 1 The hydrodynamic diameter (average size and polydispersity index, Pdi, based on DLS) and the composition according to TGA of GO (control) and NGO/PLA–PEG. Three replicate experiments were conducted and the average ± standard deviation are presented. Sample

GO control NGO/PLA(1.5K)– PEG(10K) [1/6] NGO/PLA(1.5K)– PEG(10K) [1/2] NGO/PLA(7.8K)– PEG(20K) [1/6] NGO/PLA(7.8K)– PEG(20K) [1/2]

Average size (nm)

Pdi

Composition NGO (%)

PLA–PEG (%)

898.8 ± 19.92 501.6 ± 8.66

0.485 ± 0.034 0.335 ± 0.055

100 52

– 48

512.2 ± 12.15

0.418 ± 0.021

80

20

455.6 ± 5.71

0.358 ± 0.070

70

30

534.1 ± 9.24

0.430 ± 0.029

80

20

generated by the presence of copolymer chains on the surface of graphene oxide. 3.6. In vitro cytotoxicity and uptake by cancer cells NGO/PLA(7.8K)–PEG(20K) was selected for the cytotoxicity studies because it exhibited lower rate of drug release compared to NGO/PLA(1.5K)–PEG(10K) (Fig. 6). A low rate of drug release is

23

important in order to minimize drug loss from the particles in blood, i.e. before they reach the tumor site. The cytotoxicity of paclitaxel-loaded NGO/PLA–PEG against the A549 cancer cell line increased with concentration and time of incubation with the cells (Fig. 7A and B). At 48 h it became comparable with the cytotoxicity of free paclitaxel and at all concentrations there was not any statistical significant difference in cytotoxicity (Student t-test, p > 0.05) between free and entrapped paclitaxel. The cytotoxicity of blank NGO/PLA–PEG was low even at the maximum concentration of 400 ppm, which corresponded to the concentration of paclitaxel-loaded NGO/PLA–PEG composites generating the highest tested concentration of paclitaxel (40 ppm) (Fig. 7A and B). At paclitaxel concentrations equal or higher than 5 ppm, and at both incubation times, the cytotoxicity of paclitaxel-loaded NGO/PLA– PEG was significantly higher (Student t-test, p < 0.05) than that of blank NGO/PLA–PEG. The uptake of FITC-labeled NGO/PLA–PEG composites (3.2% w/w label load) was studied by FACS. It has been reported that FITC fluorescence quenches upon binding on GO [30]. However, we observed that although the fluorescence of FITC was reduced it was not completely quenched upon binding on the composites (Fig. S3), permitting the uptake of FITC-labeled NGO/PLA–PEG composites by cells to be studied by flow cytometry. The uptake of the composites by the A549 cells increased with time (Fig. 7C) and an almost linear relation was obtained when the % cytotoxicity was plotted against % cell uptake (Fig. 7D). The uptake of the FITClabeled NGO/PLA–PEG composites by the A549 cells at different incubation times was visualized with fluorescence microscopy using the post-fixation staining with PI method in order to stain nuclear and cytoplasmic nucleic acids of the cells [25]. The images obtained provide further evidence of the increasing with incubation time uptake of the composites by the A549 cells (Fig. 8).

4. Discussion Graphene is the most investigated material today due to its unique mechanical, thermal, optical and electronic properties which allow for its possible application in a wide range of fields. Intensive research is ongoing to investigate the quantum physics in this system and potential applications for nanoelectronic devices, transparent conductors, and composite materials [31–36]. Graphene applications in the biomedical field are still in nascent stages [37]. Graphene oxidation leads to the formation of graphene oxide (GO) which, in addition to the high surface area of parent

20

LC(%)

18

EE(%)

16

percent

14 12 10 8 6 4 2 0

Fig. 4. Variation of hydrodynamic diameter of NGO/PLA–PEG composites with storage time (8 °C, fridge) and at increasing concentrations of NaCl. Three replicate experiments were conducted and the averages and standard deviations are presented.

Fig. 5. Paclitaxel loading capacity and entrapment efficiency for the NGO/PLA–PEG composites. Three replicate experiments were conducted and the averages and standard deviations are presented.

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40 35

25 20 15 10

PLA(1.5K)mPEG(10K) [1/6]

5 0

PLA(1.5K)mPEG(10K) [1/2]

0

20

40

60

80

100

hours 40 35

NGO/PLA(7.8K)mPEG(20K) [1/6] NGO/PLA(7.8K)mPEG(20K) [1/2]

25

(A)

20 15 10 5 0

0

20

40

60

80

70

PCT

60

NGO/PLA(7.8K)-mPEG(20K)/PCT

50

% cytotoxicity

% release

30

NGO/PLA(7.8K)-mPEG(20K)

40

*

30 20

100

*

10

hours

0

Fig. 6. In vitro (PBS pH 7.4) release of paclitaxel from NGO/PLA–PEG composites. Three replicate experiments were conducted and the averages and standard deviations are presented.

0.5

1

5

25

40

ppm PCT

(B)

70

% cytotoxicity

60

PCT

48 hours

NGO/PLA(7.8K)-mPEG(20K)/PCT NGO/PLA(7.8K)-mPEG(20K)

50 40 30 20 10 0

0.5

1

5

25

40

ppm PCT

% cells uptake

material, bears many functional groups (epoxy, carboxyl, hydroxyl) that can be exploited for the attachment of functional and bioactive molecules. Thus, GO has great prospects in controlled drug delivery. However, GO dispersions in electrolytes and biological fluids are not stable and several approaches have been taken in order to overcome this problem. GO has been functionalized with chitosan [38] or sulfonic acid [39] in order to render it stable in physiological solution whereas Hong et al. [12] applied further oxidation of GO to a low C/O ratio, allowing it to be electrostatically stabilized in electrolyte solutions. However, the most commonly applied method is to sterically stabilize GO dispersions by attaching hydrophilic and flexible PEG chains on GO, either by covalently binding of suitable PEG derivatives on GO [11,13–15] or by physically attaching amphiphilic PEG copolymers, such as pluronics, on GO [12,17,40]. Stabilized with PEG GO has been investigated as a carrier for the delivery of anticancer drugs, such as doxorubicin [13,41,42] and a camptothecin analogue [11]. In this work, we investigated the application of water-dispersible PLA–PEG copolymers for the stabilization of GO aqueous dispersions and the feasibility of using the so stabilized GO as a delivery system for the potent anticancer agent paclitaxel (PTX). To the best of our knowledge, this is the first time that PLA–PEG copolymers are used to stabilize GO and that GO/PLA–PEG composites are investigated as possible controlled delivery carriers for paclitaxel. Probe sonication in the presence of PLA–PEG copolymers was applied in order to reduce the hydrodynamic diameter of GO to the nano-size range according to DLS (Table 1) and obtain NGO/ PLA–PEG composites. AFM indicated that the samples consist

24 hours

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mostly of few layers graphene nanosheets and single graphene monolayers (Fig. 2 A and B). Also, in NGO/PLA–PEG samples PLA– PEG structures are shown to be attached on the surface of the GO sheets (Fig. 2B). TEM (Fig. 2C) confirmed that NGO/PLA–PEG has sheet-like structure in the nanoscale size range. The composites exhibited high colloidal stability and no sign of aggregation was observed even in high electrolyte (NaCl) concentrations (Fig. 4). Paclitaxel (PTX) was effectively loaded on NGO/ PLA–PEG (Fig. 5) through the development of p–p stacking interactions between GO and PTX, due to the conjugated p electrons of the sp2 carbons and those in aromatic rings of PTX [43]. Sustained PTX release from the NGO/PLA–PEG composites was observed, with less than 15% being released the first 10 h (Fig. 6). The release rate appeared to be affected by PLA–PEG molecular weight and proportion in the composites (Fig. 6), providing the means to regulate the rate of drug release by judicious changes in these factors. The release data obtained would indicate the suitability of NGO/PLA– PEG composites as controlled delivery system for paclitaxel. The paclitaxel-loaded NGO/PLA–PEG composites exhibited in vitro anticancer activity against A549 lung cancer cells which

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y = 1,5149x - 16,486 R² = 0,955

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hours Fig. 7. Cytotoxicity of blank NGO/PLA(7.8K)mPEG(20K) composites, PCT-loaded NGO/PLA(7.8K)mPEG(20K)/PCT composites and paclitaxel (PCT) against A549 cancer cells (A) and (B), variation of uptake of NGO/PLA–PEG composites (10 ppm concentration) by the A549 cells with time (C), and relation of cytotoxicity and uptake for the incubation times between 6 and 72 h tested (D). Three replicate experiments were conducted (line sections on bars indicate standard deviation). The asterisks indicate significant (p < 0.05) differences in cytotoxicity between free PCT and NGO/PLA(7.8K)mPEG(20K)/PCT.

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with previous studies indicating the cytocompatibility of NGO [44– 46] and that NGO starts to exhibit cytotoxicity only at high concentrations (>100 mg/L) [13]. 5. Conclusions

0.5hrs

Graphene oxide aqueous dispersions were effectively stabilized by water-dispersible, biocompatible and biodegradable PLA–PEG copolymers. The graphene oxide/PLA–PEG composites were loaded with paclitaxel at satisfactory loadings. Sustained in vitro paclitaxel release was observed, which could be regulated by the molecular weight of the PLA–PEG copolymer and its proportion in the composite. The paclitaxel-loaded composites exhibited cytotoxicity against cancer cells in culture which increased with incubation time, in conjunction with the increasing with time uptake of composites by the cancer cells. The results justify further investigation of the suitability of PLA–PEG stabilized graphene oxide for the controlled delivery of paclitaxel. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejpb.2015.03.022. References

3hrs

24hrs Fig. 8. Fluorescence microscopy images of NGO/PLA(7.8K)–mPEG20K) composites uptake by A549 cells. Cells were incubated for different times (hours) with FITClabeled composites. PI was used to stain red the nuclear and cytoplasmic nucleic acids after incubation and overlay images are shown (magnification 20). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

increased with time, probably because the uptake of the composites by the cells also increased with time (Figs. 7 and 8). Although, free PTX appeared to be more effective (cytotoxic) than that loaded on the composites, their difference in cytotoxicity was diminished with incubation time (Fig. 7), presumably because cell uptake of the composites, and thus PTX loaded on the composites, is slower than that of free PTX and more time is needed for building effective intracellular PTX concentrations in the case of the composites. The blank (without drug) NGO/PLA–PEG composites exhibited low cytotoxicity (Fig. 7). This result is in accordance

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