Tailoring the interlayer interaction between doxorubicin-loaded graphene oxide nanosheets by controlling the drug content

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Tailoring the interlayer interaction between doxorubicin-loaded graphene oxide nanosheets by controlling the drug content Qi Zhang a, Weiwei Li a,b, Tao Kong a, Ruigong Su a,b, Ning Li Mingliang Tang a, Liwei Liu a, Guosheng Cheng a,b,*

a,b

, Qin Song a,

a

Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, 398 Ruoshui Road, Suzhou Industrial Park, Jiangsu 215123, China b Graduate University of the Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China

A R T I C L E I N F O

A B S T R A C T

Article history:

We experimentally and theoretically investigated the influence of a model drug, doxorubi-

Received 16 May 2012

cin (DOX) on GO interlayer interaction by evaluating the pyrolysis activation energy using

Accepted 13 August 2012

thermogravimetric analysis and a mathematical model. It was found that the pyrolysis

Available online 21 August 2012

activation energy of DOX-loaded GO decreased from 145.8 to 119.5 kJ/mol with DOX loading content increasing from 0 to 186.6 w/w%. Theoretical simulation showed that the reduced activation energy could be ascribed to the gradually decreased interlayer interaction with DOX molecule intercalation. This involved distorted p–p stacking originating from the enlarged interlayer distance, and partially blocked interlayer hydrogen bonding. Our study suggested the possibility of tailoring the interlayer interaction and macroscopic properties of GO composites by controlling the density of molecules on the individual sheet, and offered a better understanding of inserted molecules causing interlayer interaction changes.  2012 Elsevier Ltd. All rights reserved.

1.

Introduction

Owing to a unique structure, single or few-layered two-dimensional sp2-bonded carbon sheet, graphene has attracted considerable interests over the past few years, given their potential applications in electronic and optoelectronic devices [1], catalysis [2] and sensors [3]. Graphene oxide (GO), an oxygenated derivative of graphene, contains plenty of hydrophilic oxygen-containing groups such as carboxyl, carbonyl, hydroxyl and ether [4–6]. Thus remarkable advances have been made so far in the GO-based biosystems. Through physical adsorption or covalent bonding, GO have been modified with polymers, DNA, protein and other biomolecules with potential

use in DNA sensing [7], protein assay [8], and real-time monitoring [9]. In particular, due to an ultrahigh specific surface area, GO has attracted much attention for various biological deliveries, including gene and drug delivery [10–12]. The macroscopic properties, such as mechanical stiffness and strength of GO and GO composites, were strongly affected by the molecule/ion intercalation. For instance, Park et al. reported that the edge-bound divalent metal ions (e.g. Ca2+ and Mg2+) resulted in the improved stiffness and strength of GO papers due to cross-linking of neighboring sheets, whereas a metal ion intercalation between the GO sheets reduced the mechanical performance owing to the increased interlayer distance [13]. Gao et al. effectively tailored the

* Corresponding author at: Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, 398 Ruoshui Road, Suzhou Industrial Park, Jiangsu 215123, China. Tel.: +86 (512) 6287 2557. E-mail address: [email protected] (G. Cheng). 0008-6223/$ - see front matter  2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carbon.2012.08.025

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interlayer adhesions of the GO papers by introducing small molecules, i.e. glutaraldehyde and water molecules, into the gallery regions, thus both the tensile modulus and strength showed significant improvements for the glutaraldehydetreated GO papers, whereas decreased mechanical properties were observed for the H2O-treated GO papers [14]. Medhekar et al. found that water content controlled the interlayer hydrogen bonding networks, thereby affecting the interlayer spacing and elastic moduli of the composites [15]. These reports suggested that the changes of mechanical properties are probably due to the small-molecule-varied interlayer interaction between GO nanosheets. However, the way of small molecules affecting the interlayer interactions including p–p stacking [16–18] and hydrogen bonding [15] are still unclear. The computational methods, like molecular dynamics simulation, have been employed to theoretically investigate the potential energy and van der Waals energy for the interaction processes of biomolecule/graphene composite at different temperatures and pressures [19]. However, the experimental evaluation of interlayer interaction of GO or GO composite is still limited. Thermal analysis is an experimental method to quantify the activation energy for chemical reaction and evaluate the interaction strength. During thermal decomposition, the labile oxygen-containing functional groups decorated on the carbon plane are gradually destructed. As a result, the interlayer interaction between GO nanosheets is simultaneously tuned along with the decomposition. In thermogravimetric analysis (TGA), the activation energy (DE), calculated by the position of pyrolysis peak [20], could be utilized to estimate the interlayer interaction strength of GO composites, as the thermal decomposition energy is mainly contributed by the energy for destruction of the functional group-carbon plane bonding and interlayer interaction between GO sheets. Herein, we report combined experimental-theoretical studies to elucidate the interaction change in GO sheets with loading of a model drug-doxorubicin (DOX), by TGA, temperature-dependent X-ray diffraction (XRD) and modeling. The heat-pretreated GO was studied as a control to analyze the influence of interlayer distance, a key factor in controlling p–p stacking, and octadecyl isocyanate (ODI)-grafted GO was also studied as another control to fully block the interlayer hydrogen bonding and explore the effect of hydrogen bonding elimination during molecule insertion. Moreover, a theoretical simulation was performed to address the interaction change along with the DOX loading. These results provided direct evidence on how the molecule insertion affects the p–p stacking and interlayer hydrogen bonding between the GO nanosheets and how the interlayer interaction affects the pyrolysis process. These modeling results could facilitate modification of the macroscopic material properties of GO composites and pave a way for potential utilization in drug loading and release.

2.

Experimental

2.1.

Preparation of GO

The yellow brown GO was prepared from natural graphite powder (300 mesh, Alfa Aesar) by a modified Hummers method [21,22] in our previous work [23]. GO foams were obtained

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by desiccating the freeze-dried GO foams (2–3 mg/cm3) under vacuum for 48 h at RT, 50 C and 80 C, respectively.

2.2.

DOX-loaded GO

DOX-loaded GO was fabricated by mixing GO aqueous solution with DOX solution (Aladdin Reagents, China) and then vortexed for 24 h at room temperature in the dark [11,24]. The DOX-loaded GO was harvested by rinsing the precipitation (16000 · g, 30 min) three times in water. The foams were obtained by freeze-drying the aqueous solution and stored at 4 C when not in use.

2.3.

ODI-grafted GO

The ODI-grafted GO was synthesized by the reported method [25]. In brief, anhydrous graphene oxide foams (15 mg) were loaded into a 50-mL round-bottom flask equipped with a magnetic stir bar, followed by addition of 15 mL anhydrous N,Ndimethylformamide. The flask was then sonicated for 30 min under nitrogen to create a homogeneous suspension. The ODI (1 g, Sigma–Aldrich) was next added and the mixture was heated at 120 C with magnetic stirring under nitrogen for 24 h. The resulting product was further centrifugated, rinsed with anhydrous N,N-dimethylformamide and ethanol for six times, followed by drying under vacuum. To diminish the moisture, all the samples were stored in a P2O5 filled desiccator under vacuum for more than 48 h before use.

2.4.

Characterization

The morphology and microstructure were characterized using scanning electron microscopy (SEM, Quanta 400, FEG) and transmission electron microscopy (TEM, Tecnai G2 F20 S-Twin, FEI). Fourier transform infrared spectroscopy (FT-IR) spectra were measured by a Nicolet 6700 Fourier transform infrared spectrometer (Thermo) and samples were dispersed in pressed KBr disks. XRD (D8 Advance, Bruker equipped with a temperature control cell of HTK1200 N, Anton Paar) was employed to characterize the structural properties of GO foam ˚ . Temperausing the Cu Ka radiation wavelength of 1.5406 A ture-dependent XRD was conducted in a temperature range from RT to 300 C with an increase step of 20 C, a heating rate of 0.5 C/s and a duration of 5 min. TGA (TG/DTA 6200, Seiko) was performed under a nitrogen flow (50 cm3/min) on sample mass varying from 2 to 3 mg, and the weight loss was detected in a Pt crucible from RT to 800 C.

3.

Results and discussion

GO has an ultrahigh specific surface area (theoretical 2630 m2/g), which makes it an ideal candidate as aromatic drug carriers [10–12]. DOX, a potent antineoplastic agent against a wide range of human tumors [11,24], was used as a model drug in this work. DOX-loaded GO foams were prepared by mixing DOX with GO [24], followed by freeze-drying. The ultraviolet–visible (UV–Vis) spectra of GO, DOX and DOX-loaded GO are presented in Fig. S1 (see Supporting Information (SI)), in which a characteristic peak of DOX absorbance at 490 nm

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was appeared in that of DOX-loaded GO as well. The DOX content (D) was measured the absorbance at 490 nm and calculated using Eq. (1): D¼

m0  msup  100% mGO

ð1Þ

where m0 denotes the initial mass of DOX, msup, the mass of DOX in the supernatant after reaction, and mGO, the mass of GO. The calculated dose–response curve of DOX content according to the standard curve of absorbance versus DOX concentration (Fig. S1B) is plotted in Fig. 1. With an increase of the initial DOX solution concentration up to 4000 lg/mL, the DOX content increased and reached to 186.6 w/w% (pH 6.5). The increased DOX content at higher pH value was due to the decreased solubility of DOX and the changed distribution coefficient of DOX between GO and the medium (Fig. S2, SI). Though the DOX loading content was further improved to 382.7 w/w% at higher pH values (pH 8.0), the specimens prepared at pH 6.5 were selected for the subsequent investigation to avoid the inactivity of DOX at basic conditions. A macroscopic-structure change of DOX-loaded GO was remarkable since the composites became more fragile with the increase in DOX content. Fig. 2A shows the SEM image of sponge-like GO foam with pore sizes ranging from 1 to 100 lm. Fig. 2B illustrates the SEM image of GO foam loaded with 186.6 w/w% of DOX, in which a fluffy and small piecelike structure was observed. Comparing the TEM images in the bottom inset in Fig. 2, it clearly can be seen that the DOX-loaded GO film was much crumpled. These results suggested that the interlayer interaction of GO was disturbed by the insertion of DOX molecules. FT-IR spectroscopy characterization of DOX-loaded GO (Fig. 3) exhibits that the peak at 1730 cm1, corresponding to C@ @O carbonyl or carboxyl stretching of the DOX and GO, slightly shifted to a lower position at 1723 cm1 after the formation of DOX-GO complex, which might be due to the interaction between DOX and adjacent GO sheet [24]. TGA was performed to assess the thermal decomposition behavior of DOX-loaded GO foams. To clarify the influence of DOX content on the thermal properties of GO foams, we

Fig. 1 – The content of DOX loaded on GO at various DOX concentrations, measured by UV–Vis.

Fig. 2 – SEM micrographs of the freeze dried, sponge-like GO foams (A) and the fragile DOX-loaded GO (DOX content, 186.6 w/w%) debris (B). The upper inset SEM images and the lower inset TEM images show the magnified microstructures of (A) and (B), respectively.

Fig. 3 – FT-IR spectra of pure DOX (a), GO loaded with 186.6 w/w% of DOX (b) and pure GO (c). Characteristic peaks: (a1) C@O carbonyl stretching 1730 cm1; (b1) C–H methylene stretching 2921, 2849 cm1, (b2) C@O carbonyl or carboxyl stretching 1723 cm1; (c1) C@O carbonyl or carboxyl stretching 1730 cm1.

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first systematically studied the pyrolysis behavior of the foams with varied the DOX content from 0 to 186.6 w/w%. The TGA curves of the pure GO, DOX and DOX-loaded GO foams are shown in Fig. 4A. Pure GO decomposed at 200 C, with one mass loss feature (40%) of the original sample mass. The curve for pure DOX exhibited a three-step mass loss process: a small loss in 190–210 C, a major loss in 210–280 C and another loss in 300–400 C, leaving 52% of the original sample mass at 500 C. For DOX-loaded GO foams, the TGA curves gradually shifted with the increasing of DOX content. A similar thermal behavior was observed for GO loaded with 4–70 w/w% of DOX, and the main difference was that the weight loss rate (derivative weight, DW) at 125 C was augmented with the increasing of DOX content (Fig. 4 B). Whereas, when the drug content was more than 70 w/w%, the TGA curves of DOX-loaded GO were more close to that of pure DOX (Fig. 4 C). These results indicated that the thermal properties of DOX-loaded GO were dominated by the DOX content. Their thermal behavior was evolved from that of GO matrix to pure DOX with increasing of DOX content. With low DOX content (<70 w/w%), the GO nanosheets can still be viewed as continuous phase, and the intercalated DOX interrupted the interaction between GO nanosheets, leading to a lower decomposition temperature. Whereas, with high DOX content (>70 w/w%), the GO phase

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was tremendously disturbed by the intercalated drug molecules, providing a more DOX-like thermal behavior. Since the destruction of interaction networks occurs along with the thermal decomposition, activation energy, DE of pyrolysis can be employed to estimate the strength of interaction. Fig. 4D presents the activation energy of pyrolysis for GO and DOX-loaded GO, calculated by Ozawa method [26] (see Fig. S3, SI for Kissinger method [20], the calculated DE of pyrolysis consisted with that of Ozawa method). It was found that the activation energy gradually decreased with the increase in drug content D, by a second order polynomial regression equation, as shown in Eq. (2), suggesting that the interaction between GO nanosheets was reduced by the DOX molecule insertion. y ¼ 146:2  17:83  102 D þ 1:771  104  D2 ðR2 ¼ 0:9958Þ

ð2Þ

Experimentally, the interlayer interaction between GO nanosheets is mainly dominated by p–p stacking [16–18] and hydrogen bonding [15]. With intercalation of DOX molecules into GO phase, the GO-GO interaction was altered to the correlated GO-DOX and DOX-DOX interactions. Therefore, to further clarify how the DOX molecules affect interlayer interactions including p–p stacking and hydrogen bonding and deeply understand the physical interpretation of Eq. (2), two control samples, i.e. heat-pretreated GO and ODI-grafted

Fig. 4 – The weight (A) and the normalized derivative weight (DW) (B and C) of DOX-loaded GO at different drug loading contents as a function of annealing temperature with a heating rate of 10 C/min. Plot (D) presents the pyrolysis activation energy of the DOX-loaded GO versus DOX loading content calculated by the Ozawa method. The inset illustrates the three components of DE: (1) DEGO , of pure GO domain, (2) DEDOXGO , of single-side-loading-DOX-molecule domain, and (3) DE DOXGO , of dual-side-loading-DOX-molecule domain.

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GO were investigated by varying the p–p stacking and hydrogen bonding capacity, and exploring their effects on the pyrolysis behavior respectively. The heat-pretreated GO was employed to modulate the influence of the interlayer distance (d-spacing) since the d-spacing dramatically affects the p–p stacking interaction [18]. Fig. 5A presents XRD patterns of GO heated at room temperature (GO-preheated-RT), 50 C (GO-preheated-50 C) and 80 C (GO-preheated-80 C) for 48 h. The characteristic peak of anhydrous GO under air atmosphere was located at 2h = 10.4–10.5, corresponding to ˚ . Whereas, after the heat treatment, a d-spacing of 8.4–8.5 A its 2h peaks shifted to 11.0 for GO-preheated-50 C and 11.4 for GO-preheated-80 C, corresponding to d-spacings of ˚ and 7.8 A ˚ , respectively, even though no significant mor8.0 A phology change and mass loss (<1%) were observed after the heat pretreatment for 48 h. The effect of continuous temperature increase on d-spacing of GO was further investigated using the temperature-dependent XRD measurement. Fig. 5B illustrates the plots of d-spacing and 2h peak shift with respect to the temperature under air atmosphere. A systematic decrease in 2h peak value and a corresponding continuous reduction in the d-spacing were revealed with the temperature elevated from 25 C to 300 C. The reduced dspacing was attributed to the gradual removal of oxygen-containing functional groups and formation of partial reduced GO nanosheets [27]. To quantify the relationship between the interlayer distance and their pyrolysis activation energy, TGA was further performed. Fig. 5C,D present the TGA spectra of GO foams preheated at RT and 80 C, respectively. It was

found that the weight loss was largely determined by the heating rate. At a high heating rate (i.e. 20 C/min in Fig. 5C and 40 C/min in Fig. 5D), a sharp weight loss of 90% was observed due to the decomposition rate of the functional groups exceeding the diffusion rate of the evolved gases [28], which agreed well with the previous study [21]. These results demonstrated that a higher pyrolysis activation energy was needed for GO-preheated-80 C (3 kJ/mol, see Table S1 for Kissinger method), which has a reduced d-spacing featuring a stronger thermal stability. This effect validated that the p– p stacking, one of the interlayer interaction forces between GO nanosheets, can be reduced owing to extended interlayer distance, as reported [18], and vice versa. Furthermore, ODI-grafted GO was chosen to reveal the influence of interlayer hydrogen bonding elimination during organic molecule intercalation. As schematically illustrated in Fig. 6A, ODI-grafted GO was synthesized through the reaction between hydroxyl, phenol or carboxyl group of GO and isocyanate [25]. Theoretically, the interlayer hydrogen bonding should be totally blocked due to the substitution of hydrogen-containing functional groups by alkyl chains (60 times mass excess of ODI). In our experiment, it was found that the ODI-grafted GO became hydrophobic, suggesting that the hydrogen-containing functional groups of hydroxyl, phenol and carboxyl have been eliminated through the reaction. As shown in Fig. 6B, the ODI-grafted GO formed plate-like grains after the removal of solvent (e.g. N,N-dimethylformamide), and from TEM image, shown in the lower inset, folded graphene layers can be clearly visioned as compared to the

Fig. 5 – (A) XRD patterns of GO with variable heat pretreatments at RT, 50 C and 80 C. (B) d-spacing and 2h angles of GO as a function of annealing temperature using temperature-dependent XRD measurement. TGA analysis of the GO foams pretreated at RT and 80 C (C and D), the plots (C) and (D) show the weight as a function of annealing temperature.

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Fig. 7 – (A) TGA of GO, ODI and ODI-grafted GO at a heating rate of 10 C/min. (B) TGA analysis of ODI-grafted GO at the heating rates from 1 to 40 C/min.

Fig. 6 – Schematic chemical structure (A) and SEM micrographs (B) of ODI-grafted GO powders, the upper inset SEM image and the lower inset TEM image (in graph B) depict the wrinkled microstructure of ODI-grafted GO nanosheet. FT-IR spectra (C) of pure ODI (a), ODI-grafted GO (b) and pure GO (c). Characteristic peaks: (a1) O@C@N isocyanate stretching 2267 cm1; (b1) N–H amines stretching 3341 cm1, (b2) C–H methylene stretching 2923, 2848, 1470 cm1, (b3) carbamate or amide stretching 1616, 1570 cm1; (c1) C@O carbonyl or carboxyl stretching 1730 cm1.

relative flat GO sheet, shown in Fig. 2A. Additionally, the chemical grafting of ODI molecules onto GO sheets was verified by FT-IR spectra (Fig. 6C). Upon treatment with ODI, the 1 of GO became obC@ @O stretching vibration at 1730 cm scured by the appearance of two strong absorption peaks at 1616 and 1570 cm1 that can be attributed to the stretching

vibration of carbamate or amide. Significantly, the broad and intense peak of O–H (3200–3700 cm1) stretching vibration was substituted by the N–H stretching vibration at 3341 cm1, indicating that the hydrogen-containing functional groups of GO were totally removed after their reaction with ODI. Fig. 7 shows the TGA curve of ODI-grafted GO. The higher decomposition temperature and larger weight loss of ODIgrafted GO under the same heating rate presented a totally different pyrolysis process, as compared with that of pure GO (Fig. 7A). As shown in Fig. 7B, 97% of weight loss occurred at 220–370 C at either low (e.g. 1 C/min) or high heating rate (e.g. 40 C/min). Almost no residue was observed in the pyrolysis products. Moreover, for ODI-grafted GO, 14% of decrease (20 kJ/mol) in activation energy was observed with respect to the pure GO foams (Fig. 5C). These findings suggested that the interlayer interaction between adjacent layers was significantly reduced due to the insertion of ODI, resulting in a complete evaporation or decomposition of ODIgrafted GO nanosheets during the pyrolysis. Based on the preceding experimental observations, our views on how p–p stacking and hydrogen bonding change during the molecule insertion, and how the interlayer interaction affects the pyrolysis activation energy, are schematically illustrated in Fig. 8. The main interlayer interactions, p–p stacking and interlayer hydrogen bonding between GO nanosheets can be manipulated by adjusting the interlayer distance and/or

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Fig. 8 – A schematic for the interlayer interaction effect on the pyrolysis of GO and its organic complexes.

insertion of small organic molecules [6,29,30]. The interlayer hydrogen bonding strength of GO-preheated-RT and GO-preheated-80 C had negligible difference since the decomposition of functional groups can be ignored [31]. However, the interlayer distance was larger for GO-preheated-RT (Fig. 5A,B). Owing to the declined strength of p–p stacking with the larger layer distance [18], the overall interlayer interaction of GO-preheated-RT was lower than that of GO-preheated80 C, resulting in a weaker activation energy of pyrolysis (Fig. 5C,D). For an organic molecule-inserted GO, the interlayer distance was increased with the molecule loading [6]. Additionally, the interlayer hydrogen bonding between GO nanosheets can be destroyed and substituted by the van der Waals force between the modified organic molecules (e.g. octadecyl chains) [6]. Since the van der Waals force between organic molecules was usually weaker than the hydrogen bonding, the overall interaction between organic molecule-inserted GO sheets was reduced due to the replacement of hydrogen bonding with the van der Waals force, and the decrease in p–p stacking caused by enlarged distance. Their thermal behavior, therefore, was different from that of pure GO (Fig. 5C) and a decrease in the activation energy was observed (Fig. 4D and Fig. 7B). It is safe to conclude that the interlayer interaction between GO nanosheets can be modulated by the organic molecule loading. Together with the results from heat-preheated GO and ODI-grafted GO, we believe that the interlayer interaction between GO sheets can be gradually tailored by the amount of intercalated DOX molecules. The long-range p–p stacking

interaction between the GO nanosheets was gradually distorted by the GO-DOX and/or DOX–DOX p–p stacking. Meanwhile, the hydrogen bonding between GO nanosheets was blocked by increasing of the DOX molecules, and replaced by the hydrogen bonding of GO-DOX and/or DOX–DOX. Given that the DOX molecules are homogeneously loaded onto the GO sheets [19] and the coverage x (the proportion of surface area occupied by DOX on one side of GO sheet), is in the range of 0 6 x 6 1, the pyrolysis activation energy of DOX-loaded GO, DEDOXGO ðxÞ is composed of the following three contributions: (1) DEGO , the ideal activation energy of the domain containing no DOX molecule (viewed as pure GO), (2) DEDOXGO , the ideal activation energy of the domain in which only single side of GO sheet is covered by the DOX molecules (only one side contains DOX molecules while the facing side not, or vice versa), (3) DE DOXGO , the ideal activation energy of the domain in which dual sides are attached by the DOX molecules (see the inset in Fig. 4D). The equation is deduced as follows: 2 DEDOXGO ðxÞ ¼ DEGO  ð1  xÞ2 þ DEDOXGO  2xð1  xÞ þ DE DOXGO  x

¼ DEGO  2  ðDEGO  DEDOXGO Þ  x 2 þ ðDEGO  2  DEDOXGO þ DE DOXGO Þ  x

ð3Þ

where (1-x)2, 2x(1-x) and x2 are the relative probabilities of the three states. Moreover, DOX content D is proportional to the x when the molecules are homogeneously distributed on the GO surface [19]:

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D ¼ k  xð0 6 x 6 1Þ

5 1 (2 0 1 3) 1 6 4–17 2

ð4Þ

where k is a constant, which depends on the reaction parameters (e.g. temperature [19], pH values[24], GO-to-medium distribution coefficient, etc.), the density & volume of DOX molecule and GO domain. Comparison of Eqs. (2) and (3), a relationship can be derived by:  DEDOX < DE DOXGO < DEDOXGO < DEGO

ð5Þ

The values of DEDOXGO and DE DOXGO can be calculated according to equations (S2)-(S4). The detailed discussion of Eqs. (4) and (5) and calculation of DE with respect to different k values are shown in Table S2 and Section 2, SI. The Eq. (5) indicated that the ideal activation energy of DOX-loaded GO is reduced with the increasing of DOX coverage within GO sheets. From the Eqs. (2)–(5), it can be elucidated that with increasing of DOX content, the interlayer interaction between the GO sheets was gradually replaced by the GO sheet-DOX interaction, and therefore, the apparent activation energy was increasingly reduced. It could be the reason why a much more fragile morphology was observed for the DOX-loaded GO foams. Also, these findings re-validated the interlayer interaction mechanism of the heat-pretreated GO and the ODIgrafted GO. Furthermore, since the aromatic molecules can form one or several layers onto the graphene surface [32–35], it’s hard to clarify by conventional methods (e.g. atomic force microscopy [24]) whether the drug molecules prefer to occupy the empty spot or to stack into multi-layers with the increasing of drug content. Our work exhibited that the DOX molecule tended to occupy the empty spot for the relationship between activation energy and DOX content can be well fitted by Eq. (3). The simulated results offer the potential to reveal how biomolecules adsorb, interact and release on/from graphene, elucidating the bio-effects in terms of interactions at the atomic level. These findings are helpful for design of GO-based biomedical systems, since the thorough understanding of the biomolecule-GO interaction is essential for the interpretation and prediction of the macroscopic properties of biomolecule-graphene systems, such as the mechanical property and release behavior. Hence, our method opened a new door for structure design of GO complexes by tailoring the content of molecules on the individual sheet, providing insights on drug carriers and reinforcement materials.

4.

Conclusions

We have experimentally and theoretically demonstrated that the interlayer interaction between GO nanosheets can be adjusted by the amount of inserted organic molecules. The increase in interlayer distance was manifested to decrease the p–p stacking, and similarly, the insertion of ODI (or DOX) reduced the hydrogen bonding between GO nanosheets. The mechanism on how the interlayer interactions i.e. p–p stacking and hydrogen bonding change with the treatments of heat pretreatment, molecule grafting or loading, was schematically illustrated. We further developed a model demonstrating that with increasing of DOX content, the interlayer interaction between the GO sheets was gradually replaced

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by the GO-DOX and DOX-DOX interactions, and a decrease in activation energy was achieved. The results offered clear evidence that the DOX molecules prefer to occupy the empty spot rather than stack into multi-layers on GO surface during the drug loading. Our investigation into the interlayer interaction between GO sheets with biomolecules insertion paves a way to tailor the macroscopic properties of GO complexes by modulating the content of modified molecules for structure design, reaction optimization and quality assessment.

Acknowledgments This work was partially funded by National Basic Research Program of China under award numbers of 2011CB965004, 2009CB930802 and ‘‘100-Talents Program’’ of Chinese Academy of Sciences. This work was made possible by professional services by Nano-Fabrication & Nano-Characterization Platforms with Suzhou Institute of Nano-Tech and NanoBionics, Chinese Academy of Sciences.

Appendix A. Supplementary data The UV-Vis absorption spectra of pure GO, DOX and DOXloaded GO, TGA curves of the pure DOX, the calculated activation energy of Kissinger method, and the detailed discussion of DE varying with various k values. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbon.2012.08.025.

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CARBON

5 1 ( 2 0 1 3 ) 1 6 4 –1 7 2

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