Functionalized R9–reduced graphene oxide as an efficient nano-carrier for hydrophobic drug delivery

Abstract

Loading of hydrophobic drugs on smart carbon nano-carriers is a challenging issue for developing advanced drug delivery systems. We introduced a novel, stable, functionalized, and targeted graphene-based drug delivery system for smart transportation of hydrophobic agents. For this purpose, the planar size of graphene oxide (GO) sheets was initially engineered using ultra-sonic waves under controlled conditions. The sonication treatment not only tuned the GO sheet sizes, but also led to formation of desired reactive groups, appropriate for developing functionalized and targeted drug carriers. Afterwards, the hydrothermal reaction was simultaneously employed for both grafting R9 peptides and reduction of GO sheets. Therefore, the produced functional structure is an R9–rGO complex with proper stability in physiological solutions and also with a high-performance loading capability of Paclitaxel (PX). The in vitro experiments revealed that the R9–rGO–PX compound was efficiently uptook by Hela cancer cell lines, and reduced the viability of Hela and MCF-7 cells more than 90% after 72 hours. The proposed approach has the advantage of green production of an applicable graphene-based drug delivery system for improving the smart transportation of hydrophobic anti-cancer drugs.

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1 Introduction

Graphene has been employed in a number of devices and applications including batteries,^1,2 general organic and hybrid photovoltaics,^3–13 transistors,^14–16 biology,¹⁷ hydrogen storage¹⁸ and as field-emission cathodes.^19,20 In addition to its planar structure, the unique physical, chemical, and thermal properties have made graphene an ideal nano-carrier for novel drug delivery systems (DDSs).^21–23 Although the hydrophobic characteristics of graphene has led to appropriate drug loading,²⁴ this feature makes it unstable in physiological conditions,²⁵ which could hinder its therapeutical potential.²⁶ Therefore, various modifications have been accomplished to overcome this issue.^27–29 To stabilize reduced graphene oxide (rGO) for developing an anti-cancer DDS,^30,31 numerous types of bio- or synthetic polymers³² such as polyethylene glycol (PEG),³³ hyaluronic acid derivatives,³⁴ dextran,³⁵ and heparin³⁶ have been used for coating the surface of rGO nano-sheets. All these functionalizing molecules are effectively stabilizing the rGO sheets. However, they are not optimized for targeted and smart delivery of chemotherapeutic drugs into tumor cells. Hence, utilizing such molecules, which have the capability of both stabilization and improvement in targeted delivery of anti-cancer agents could be extremely valuable for developing of distinctive rGO-based DDSs. Small peptides such as R9 (ref. 37), TAT (48–60),³⁸ pVEC,³⁹ TP10 (ref. 40), and poly-L-lysine are recently introduced for targeting of drug delivery systems.⁴¹ R9 is a synthetic hydrophilic homopolyarginine (composed of nine arginine residues), which binds to cell membrane and penetrates into cytoplasm due to its positive charge.^42,43 Furthermore, modification of rGO sheet size has great impact on suitability of developed delivery system.²⁷ It has been shown that the planar size of rGO sheets could influence the transportation rate of nano-carrier via thin layers like blood brain barrier, cytotoxicity, and cellular uptake.^27,44–46 Our hypothesis in this study is that R9 could be an appropriate molecule for both stabilizing and targeting the rGO-based nano-carriers. We suggested a new method for manipulation of graphene oxide sheet size under organized conditions that led to improve the functionality of GO. To examine our idea, we first produced a broad range of GO sizes using sonication under controlled condition to generate new oxygen containing functional groups. Then, the R9 peptides were introduced to the functional groups of manufactured GO sheets under hydrothermal condition for reduction of GO sheets. Simultaneously, attachment of R9 led to production of R9 conjugated reduced graphene oxides (R9–rGO), as illustrated in Scheme 1. The stability of R9–rGO and the loading efficiency of Paclitaxel (PX) on produced R9–rGO complex were precisely evaluated. After examining the stabilization, cellular uptake of produced R9–rGO was studied. At the end, the toxicity of PX loaded R9–rGO was assessed on cancer cell lines.

Scheme 1 Schematic illustration for the preparation of tuned, functionalized, stabilized, and targeted rGO-based nano-carrier loaded with Paclitaxel (R9–rGO–PX complex). At first, the as prepared GO sheets were sonicated under controlled conditions (adjusting sonication time and power, temperature, pH, and excess acid). Then, the tuned GO sheets were hydrothermally incubated with R9 peptides resulted in R9–rGO compounds. Finally, the Paclitaxel agents were loaded on developed R9–rGO. The resultant R9–rGO–PX complex was administrated on cancer cell lines to evaluate the cell uptake and toxicity.

2 Experimental

2.1 Materials

All chemical materials in this study were used as received without further manipulation or purification. Graphite powder (mesh 325), K₂S₂O₈ (99%), P₂O₅ (99%), H₂SO₄ (98%), KMnO₄ (99%), H₂O₂ (35%), and HCl (37%) were purchased from Merck. Cell culture reagents including RPMI, fetal bovine serum (FBS), penicillin/streptomycin, and WST cell viability assay kit were purchased from Sigma-Aldrich. Hela and MCF-7 cancer cell lines were purchased from American Type Culture Collection (ATCC).

2.2 Synthesis of graphene oxide

Graphene oxide (GO) was synthesized by modified Hammer's method.⁴⁷ In brief, 3 g graphite powder was oxidized in (12 ml) concentrated H₂SO₄ in the presence of (2.5 g) of K₂S₂O₈ and (2.5 g) of P₂O₅ at 80 °C for 4.5 hours. Subsequently, the as prepared graphite oxide was neutralized and put into (120 ml) of pre-cooled concentrated sulfuric acid. After a short time, (15 g) of KMnO₄ was progressively added to the solution under vigorous stirring while the temperature was kept below 20 °C. Thereafter, the temperature was raised to 40 °C and kept stirring for 2 h, followed by sequential addition of (1) (250 ml) deionized water, (2) (700 ml) deionized water, and (3) (20 ml) H₂O₂ under strong stirring. In order to eliminate excess metal particles, the obtained mixture was washed and neutralized with (100 ml) of HCl (37%), (1000 ml) of HCl aqueous solution (1 : 10 v/v), and (1000 ml) of deionized water, respectively. Finally, the neutralized graphene oxide was dried at ambient condition, dispersed (0.5% wt) in deionized water, and purified using dialysis bag (7000 Da) for one week.

2.3 Sonication

The as prepared graphene oxide (1 mg ml⁻¹, 10 ml each batch) was modified through sonication process by varying pH, time, temperature, ultrasonic pulse and power, and also excess acids including sulfuric acid, HCl, and citric acid. Ultra-sonication process for all samples was applied with an Ultrasonic processor UP100H (100 W, 30 kHz) Hielscher Ultrasound Technology. Briefly, the pH was adjusted to 2, 4.5, 7, 9.5, or 12 by adding NaOH 1 M or HCl 1 M into the GO solution. The adjusted suspensions were sonicated at various time periods including 0, 15, 30, 45, 60, 75, 90, 105, and 120 minutes. Moreover, the effect of sonication pulse or power was further tested on the sonication process by varying the pulse modes to 0.25 s on/0.75 s off, 0.5 s on/0.5 s off, or 0.75 s on/0.25 s off, and the power modes to 25%, 50%, or 75%. The temperature was also adjusted to 20, 30, 40, or 50 °C. All the experiments were done at atmospheric pressure and under controlled conditions.

2.4 Hydrothermal reaction

The R9 peptides were grafted to GO immediately after sonication of the graphene oxide sheets through hydrothermal reaction. For this purpose, the R9 peptide solution (2 mg ml⁻¹) were mixed with GO samples (8 mg ml⁻¹) and incubated in humidified atmosphere at 95 °C for 3 hours.

2.5 Stability of R9–rGO

A quantitative technique based on Dynamic Light Scattering (DLS) analysis⁴⁴ has been used for evaluating the stability of formed R9–rGO in phosphate buffered saline (PBS), fetal bovine serum (FBS), and cell culture medium (RPMI). The average hydrodynamic diameters (HD) of the R9–rGO were measured primarily and after 1, 12, 24, 36, and 48 hours. The stability is calculated as:
where HD_t=0 and HD_t are hydrodynamic diameter at initial time (t = 0) and the different intervals (t = 1, 12, 24, 36, and 48 h), respectively.

2.6 Drug (PX) loading

For loading of PX on prepared tuned R9–rGO, the PX was first dissolved in DMSO (1.5 mg ml⁻¹) and mixed with R9–rGO solution (0.5 mg ml⁻¹) in different proportions of PX to rGO for 24 hours.⁴⁸ The unloaded PX was removed by serial centrifugation (4000 rpm). The amount of loaded PX on R9–rGO was measured by UV-vis spectroscopy at PX absorption peak (229 nm).

2.7 Cell uptake and toxicity

Hela (ATCC) and MCF-7 (ATCC) cancer cell lines were cultured in 24 well plates in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin overnight before each experiment. To evaluate the cell uptake of developed DDSs via Hela cancer cell line, the FITC-conjugated R9–rGO samples were added to each well (100 μg ml⁻¹) for 4 hours. The uptake of samples was evaluated by a Nikon TI-E fluorescence microscope. For quantitative calculation of FITC-positive cells to total cells ratio, the number of positive and negative cells in 10 visual fields per each well (10 wells per group) were counted. The mean proportion (%) of positive cells to total cells is reported for each group. The toxicity of R9–rGO samples loaded with PX on Hela and MCF-7 cancer cell lines was evaluated using WST assay kit, as described previously.²⁷ In brief, the R9–rGO–PX complexes (10 well per group) were added to cultured cells (106 cells per well) to the final concentration of (100 μg ml⁻¹). Then, the viability of cells was assessed after 0, 24, 48, and 72 hours by WST assay kit utilizing CCK-8. The rGO and free PX were also added as controls.

2.8 Characterization

The morphology of the samples was examined by transmission electron microscopy (TEM) on a Philips EM208 TEM and atomic force microscopy (AFM) SPM VEECO AFM. X-ray photoelectron spectroscopy (XPS) measurements were examined on an ultra-spectrometer (Bestec, Germany) using an Mg Ka source and monochromatic Al Ka source. FT-IR spectra were measured by a Spectrum RX I, PerkinElmer FT-IR. Ultraviolet-visible (UV-vis) spectra measurements were carried out on an Avaspec 2048 TEC spectrophotometer. Circular dichroic (CD) spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics). X-ray diffractions (XRD) were performed with a step size of 0.02 (2θ) per s with Cu Ka radiation by X'Pert PRO MPD (PANalytical) diffractometer. Raman spectra were recorded from 1000 to 2500 cm⁻¹ on a RM 2000 Microscopic using a 633 nm He–Ne laser beam. The zeta potential and Dynamic Light Scattering (DLS) analysis of the samples was measured using a Malvern ZEN 3600 zetasizer. Digital photos were taken by Nikon D5300 Kit18-55 VR II camera.

3 Results and discussion

3.1 Characterization of tuned graphene oxide sheets

The stability of GO in aqueous media could be modified by increasing edge-to-area ratio (RE) and functional groups at GO edges.^49–51 Sonication as a green, applicable, and facile technique was employed here to achieve a broad range of GO sheets, which simultaneously contained desirable functional groups and appropriate size (RE).⁵² AFM and TEM images proved successful size fragmentation of GO sheets after sonication (Fig. 1A–E). As shown in Fig. 1F, the average lateral size (ALS) and average thickness size (ATS) of graphene sheets are attenuated in consistent with increasing sonication time. The as-prepared graphene oxide sheets possess 2865 nm ALS and 2.1 nm ATS, whereas the ALS and ATS of GO sheets decreased to 232 nm and 1.1 nm after 60 minutes, respectively. Also, after 120 minutes of sonication, the quantity of ALS and ATS were found to be 24 and 0.92 nm, respectively. The WXRD analysis confirmed the alterations in lateral size and thickness of GO sheets after different times of sonication (Fig. S1†). As can be seen in Fig. S1,† the appearance of a broad peak at around 2θ ∼ 23° is observed after sonication of the graphene oxide sheets at different time periods. This peak can be attributed to partially reduction of graphene oxide sheets during the sonication process.⁵⁹ According to the XRD spectra, the intensity of these peaks has been increased by the passage of time. The peak broadening effect can be also described by distortion of graphene sheets which leads to stacking of graphene layers after sonication.⁵¹ It should be noted that remaining of the distinctive peaks at 2θ ∼ 10.3° for treated GO after 60 and 120 minutes, indicates the presence of graphene oxide.⁵¹ However, in the case of the rGO the broad peak at 2θ ∼ 23° exposes the complete reduction of graphene oxide and no peak at 2θ ∼ 10.3° is discernable.⁵³ Decreasing the size of GO sheets could be ascribed to physical effects of sonication, which was introduced as micro jets, shock waves, and shear forces.⁵³ Furthermore, it has been shown that sonication of GO sheets leads to formation of defects in GO structure.⁵² These defects could be verified by Raman spectroscopy. The Raman spectrums of the as-prepared and also sonicated GO for 120 minutes at different pH (2 and 10) are shown in Fig. 2A. In case of the as prepared graphene oxide, two distinctive peaks were observed at ∼1370 and ∼1620 cm⁻¹, which are assigned to D and G bands, respectively.⁵⁴ According to Fig. 2A-b (pH 2), the G band shows a small shift toward higher wavelength, which could be attributed to increasing oxygen-contained functional groups.⁵⁵ At pH 2 and 12, the intensity of D bands are noticeably amplified suggesting more defects on graphene sheets than those of the as-prepared graphene oxide.⁵⁵ Moreover, the more intensification of the D band at pH 12 versus pH 2 could be allocated to partially reduction of graphene oxide sheets.⁵³ In addition, the I_D/I_G ratio is known as an appropriate index for demonstrating defects in graphene sheets.⁵⁶ Increasing the I_D/I_G ratio after 120 minutes of sonication of GO in pH 2 or 12 suggested the formation of defects in fragmented GO sheets. However, owing to the fact that the Raman spectroscopy cannot provide prominent evidence for chemical alteration of graphene sheets;⁵⁶ hence, more extensive evaluations were carried out using XPS analysis. The XPS spectra of the as-prepared (Fig. 2B) and sonicated GO (120 minutes) at pH 2 (Fig. 2C) and pH 12 (Fig. 2D) revealed valuable information about underlying mechanisms mediating the modification of GO sheets by sonication at different pH. The distinctive peaks in XPS spectra at 284.4, 287, and 288.5 eV are attributed to C–C interaction, C–O, and C=O groups, respectively. The obtained results from XPS analysis led to calculation of O/C atomic ratio (O(1s) peak area to C(1s) peak area) for different samples. As presented in Fig. 2B and C, after 120 minutes of sonication at pH 12, the O/C ratio was reduced compared to the as prepared GO (0.56 vs. 0.71, respectively) due to partially reduction of graphene oxide sheets. Moreover, the intensity of C–O and C=O peak areas were declined leading to deterioration of oxygen functional groups. The reduction of GO sheets by sonication at pH 12 could be explained by the presence of NaOH, which used for adjusting the solution pH.^56–58 In our study, the chemical effect of sonication that is intermediated by high temperature cavitation bubbles,⁵⁹ resulted in reduction of graphene oxide at basic pH, and therefore less O/C atomic ratio.⁶⁰ On the other hand, the O/C atomic ratio increased after sonication of GO in pH 2 (O/C = 0.83) versus the as-prepared GO sheets (Fig. 2D). Additionally, the intensity of C=O peak area was evidently heightened in addition to a steady C–O peak area. The FT-IR and ζ-potential analyses confirmed the obtained data from XPS spectroscopy (Fig. S2A and B†). These results might be related to creation of new edge planes and defect structures,^61,62 which are emanated from ultra-sonication in acidic media (adjusted with HCl) and could be probably related to the formation of additional oxygen containing functional groups on the aforementioned edge planes.⁴⁹ Although our results revealed that the GO sheets are oxidized with sonication at pH 2, there are many reports that indicated the reduction of GO sheets in the presence of different kinds of acid solutions such as L-ascorbic acid,³¹ hydro iodic acid (HI),⁶³ hydro bromic acid (HBr),⁶⁴ and gallic acid.⁶⁵ To the best of our knowledge, there is no evident report about the effects of sonication at different pH on the graphene oxide functional groups. Thus, we examined the effects of different parameters including sonication time and power, pH, temperature (Fig. S3†), and extra acid solutions, e.g., sulfuric acid, HCl, and citric acid on the oxygenation/reduction of GO sheets (Fig. S4†). The obtained experimental results (O/C atomic ratio calculated by XPS analysis) relied on different sonication times and pH is presented in Fig. 2E. The outcomes exposed that the most oxygenated functional groups are generated after 90 minutes of sonication at pH 2, which is tend to reach a steady-state. Based on these results, it is concluded that the size and functional groups of graphene sheets, which are two prominent factors for developing graphene based drug delivery systems, could be engineered utilizing sonication under controlled conditions without using any hazardous chemical agents.

Fig. 1 AFM images of the as-prepared GO (A), after 60 minutes sonication (B), after 120 minutes sonication (C), and TEM image of the as-prepared GO (D), the effect of sonication time on the average lateral size (ALS) of graphene sheets (inset picture shows the impact of sonication time on the average thickness size (ATS) of graphene sheets) (F), TEM image of GO after 120 minutes sonication (E).

Fig. 2 Raman spectra (A) of the as-prepared GO (a), the GO sonicated at pH 2 (b), and the GO sonicated at pH 12 (c), both for 120 minutes (inset picture shows the D/G ratio for the samples). XPS spectra of the as-prepared GO (B), the sonicated GO at pH 2 (C), and the sonicated GO at pH 12 (D), both for 120 minutes. The simultaneous effect of sonication time and pH on the oxygen/carbon (O/R) ratio of prepared R9–rGO (E).

3.2 Characterization of R9–rGO

Graphene oxides with various sizes and functional groups were incubated with R9 peptide under hydrothermal condition. The UV-vis spectroscopy was applied for characterization of the formed R9–reduced graphene oxides (R9–rGO, Fig. 3A). The absorption peaks of R9 and graphene oxides sonicated under controlled conditions (90 minutes at pH 2) were at 201 nm and 230 nm, respectively. The production of hydrothermal incubation of GO with R9 reveals two peaks at 194 nm and 277 nm. The absorption peak of R9–rGO shifts from 230 nm to 277 nm. This shift suggests the restoration of electronic conjugation within the graphene sheets during the hydrothermal reaction.⁶⁶ In addition, the blue-shift of R9–rGO absorption peak from 201 nm to 194 nm proposed the interaction of rGO and R9 (ref. 67). The colour of solution changed from brown to dark after hydrothermal reaction, indicating the reduction of GO to rGO.⁶⁷ The FT-IR analysis confirmed covalent graft of graphene with R9 (Fig. 2B). The presence of O–H (at ∼3398 cm⁻¹), C=O (at ∼1721 cm⁻¹), C=C (at ∼1611 cm⁻¹), and C–O (at ∼1136 cm⁻¹) is shown in GO spectrum. The characteristic peaks of oxide groups are disappeared in FT-IR spectra of rGO and R9–rGO indicating the reduction of graphene oxide. R9 absorption peaks indicates that N–H (at ∼3279 cm⁻¹) and C=O (at ∼1630 cm⁻¹) bonds have been appeared in R9–rGO spectrum. Moreover, the representative peak of C–N binding with aromatic ring (at ∼1324 cm⁻¹, C–N stretch mode) is also exhibited in R9–rGO spectrum, which proves the successful covalent binding of R9 with graphene sheet. The XPS spectrum has been also applied to approve the formation of the R9–rGO complex. As Fig. 2C illustrates, the distinct peaks near 285.7 and 288.5 eV are assigned to C–NH₂ and C–NH, respectively. Additionally, the intensity of C–O bands tangibly decreased in comparison with the as prepared graphene oxide due to the creation of R9–rGO compounds. Moreover, the O/C atomic ratio after formation of R9–rGO complex was declined to 0.47 (O/C atomic ratio of sonicated GO under controlled condition = 0.83). The intensity of C–C bonds has also augmented. These results accommodate with UV-vis and FT-IR analyses implying the reduction of GO to rGO. The N/C atomic ratio (N(1s) peak area to C(1s) peak area) of formed R9–rGO is presented in Fig. 3D. According to this graph, the amounts of conjugated nitrogen containing groups (R9 peptides) are related to the primary sonication time and pH of GO sheets. The highest N/C ratio could be obtained by hydrothermal incubation of GO sheets, sonicated for 120 minutes at pH 2, with R9, which is related to more conjugation of R9 with rGO. These results can be assigned to smaller sizes of graphene sheets and also their more oxygen containing groups (Fig. 2E). The effect of hydrothermal process on secondary structures of R9 peptide was studied by circular dichroism (CD) spectroscopy in the far-UV region (Fig. S5†).⁷² As shown in Fig. S5a,† GO had positive peak around 230 nm and when GO was reduced the peak intensity was decrease (Fig. S5b†). The pure R9 in room temperature (25 °C) displayed a CD signature with characteristic minimum band near 200 nm and a positive one near 220 nm (Fig. S5c†). Upon heating the peptide, the amplitudes of molar ellipticities at 230, 210, and 200 nm were decreased (Fig. S5d†). Additionally, CD spectrum of R9–rGO (Fig. S5e†) exhibited minimum band near 200 nm and a positive one near 220 nm indicating the interaction of rGO and R9. It have been shown that increasing temperatures was induced a dynamic change in peptide secondary structure, and turn conformations without denaturing peptide structure. The decrease in the molar ellipticities in Fig. S5e† suggests that the non-covalent interactions of rGO and R9. The CD results indicate that the hydrogen bonding is one of main interaction mechanism in R9–rGO formation.⁷³ Generally, R9 arginine is chiefly composed of guanidine complex functional groups containing carboxylic, hydroxyl, and amide.³⁷ Due to the positive charge of (C=NH₂)⁺ complex in neutral environment, R9 arginine possess basic properties which can create multiple hydrogen bond by delocalization of positive charge emanating from the resonance of nitrogen ion pairs and double bond.⁴² Thus, it is anticipated that combination of R9 arginine with graphene oxide promotes the basicity of the mixture leading to formation of negatively charged oxygen functional groups which heighten the probability of electrostatic interaction.⁴⁶ Moreover, as can be seen in Fig. S6,† considering the presence of hydroxyl and amine groups in the structure of R9 peptide, we believe that either hydrogen bounding or C–N bond formation may occur during the hydrothermal reaction. In fact, arginine structure has a high capacity for the intramolecular and intermolecular hydrogen bonds formation.⁴² It should also be noted that, water molecules in hydrothermal condition can act like an acid catalyst by increasing H⁺ concentration. Hence, the existence of H⁺ facilitates the protonation of OH groups at the edge of graphene oxide sheets. The reduction of graphene oxide in water is a reversible reaction through which both of the intermolecular and intramolecular dehydration occurs simultaneously at the edge and basal planes as the desired sites for creation of oxygen containing functional groups. Owing to the reversibility of hydrothermal reduction of graphene oxide, the whole amount of oxygen functional groups cannot be removed. Therefore, at the end of the reaction, the remaining oxygen functional groups which are mainly negatively charged not only enhance the conjugation and grafting of R9 peptide chains with reduced graphene oxide through electrostatic interaction, but also play a pivotal role to increase the dispersion and stability of the partially reduced graphene sheets.⁵³ Additionally, it has been indicated that sonication leads to esterification of GO sheets, which might enhance the conjugation of R9 with graphene in hydrothermal conditions. Therefore, the amount of grafted R9 peptides with graphene sheets could be engineered for developing more suitable DDS.

Fig. 3 UV-vis spectroscopy (A) of R9 peptide (a), the as-prepared GO (b), and R9–rGO (c). XPS spectra of R9–rGO(120) (B). FT-IR spectra (C) of R9 (a), as-prepared GO (b), rGO (c), and R9–rGO(120) (d). The simultaneous effect of sonication time and pH on nitrogen/carbon (N/C) ratio of produced R9–rGO (D).

3.3 Stability of R9–rGO

A quantitative technique based on Dynamic Light Scattering (DLS) analysis⁶⁶ has been used for evaluating the stability of R9–rGO in phosphate buffered saline (PBS), fetal bovine serum (FBS), and cell culture medium (RPMI). The average hydrodynamic diameters of the R9–rGO were measured primarily and after 1, 12, 24, 36, and 48 hours. The quantitative stability of R9–rGO suspensions was estimated by assuming circular type GO sheets and measuring the average hydrodynamic diameter by DLS. The proportions of primary hydrodynamic diameter (t = 0) to diameters after each interval were summarized in Fig. 4. The as-prepared GO, which incubated hydrothermally with R9 (R9–rGO(0)), showed the lowest stability in all media. Sonication-tuned GO sheets for 90 (R9–rGO(90)) and 120 (R9–rGO(120)) minutes in pH 2 that hydrothermally conjugated with R9 were apparently stable in aqueous solutions for at least two weeks in physiological temperature (37 °C). Considering the size of GO sheets, the hydrophilicity of GO sheets increase by reducing the size of GO sheets owing to the higher charge density of the ionized oxygen functional groups on the edge planes of GO⁵² Hence, it can be concluded that the stability of graphene oxide in all type of colloids firmly depends on the size of GO sheets. Also the amount of grafted R9 peptides on the rGO is another parameter which results in the augmentation of stability parameter. Hydrophilic R9 peptides grafted to the rGO are the mediators of fine dispersion of the developed R9–rGO sheets, making this nano-carrier very suitable for in vivo applications.⁶⁸

Fig. 4 The effect of time on the stability of R9–rGO in phosphate buffered saline (A), fetal bovine serum (B), and RPMI (C). For all samples, (a), (b), (c) are denoted as 0, 90, 120 minutes' sonication times, respectively.

3.4 Loading of paclitaxel on R9–rGO

The Paclitaxel (PX) loading capacity of R9–rGO(0), R9–rGO(90), and R9–rGO(120) was measured using UV-vis spectroscopy at 229 nm (PX absorption peak). The amounts of loaded PX (wt%) were calculated at different PX : R9–rGO ratios (Fig. 5). The ultimate loading capacity of R9–rGO(0) was 5 wt%, whereas this capacity was increased to 11.1 wt% and 14.2 wt% by using R9–rGO(90) and R9–rGO(120) as drug carriers, respectively. PX is an aromatic and hydrophobic drug with very poor solubility.⁶⁹ Therefore, PX could be loaded properly on hydrophobic carriers such as rGO, which is mediated by π–π stacking and hydrophobic–hydrophobic interactions.⁴⁸ In our experiment, PX was more loaded on R9–rGO than rGO, which might be owing to stabilization and functionalization of R9–rGO by peptides. The R9–rGO(0) sheets are unstable in aqueous solutions and their aggregation leads to decrease the surfaces for absorption of PX.⁷⁰

Fig. 5 Investigation of Paclitaxel loading on developed R9–rGO nano-carriers by means of UV-vis absorbance at 299 nm in different Paclitaxel : rGO ratios. UV-vis spectra of Paclitaxel on R9–rGO(0) (A), R9–rGO(90) (B), and R9–rGO(120) (C). Inset diagrams illustrate the impact of different Paclitaxel : rGO ratios on Paclitaxel loading.

3.5 Cell uptake and toxicity

The representative images of Hela cells treated with FITC-conjugated R9–rGO(0), R9–rGO(90) and R9–rGO(120) for 4 hours are depicted in Fig. 6. The proportion of FITC-positive cells on total cells were quantitatively calculated. The results indicated that Hela cells uptook R9–rGO(120) (91%) more than R9–rGO(0) (11.4%) and R9–rGO(90) (64%). The more uptake of R9–rGO(120) could be attributed to the smaller sizes of these nano sheets and also more grafted R9 peptides. It has been shown that tumour cells efficiently uptake nano structures with >100 nm size.²⁷ In addition, the small size of the developed R9–rGO sheets and their convenient stability leads to better distribution and also entrancing in reticuloendothelial system.⁷¹ Moreover, various mechanisms were proposed for the act of R9 as a cell penetrating peptide. It has been suggested that carriers conjugated with arginine-rich peptides, e.g., R9 are entering into cells via direct penetration, pore formation, membrane perturbation, or endocytosis.⁴¹ After loading of PX on engineered nano-carriers, the effect of the developed DDS on viability of Hela and MCF-7 cancer cell lines were examined after 24, 48, and 72 hours (Fig. 7). The optimized carrier (R9–rGO(120)) loaded with PX exhibits more ability to reduce the viability of both tumour cell lines in comparison with rGO, R9–rGO(0), R9–rGO(90), and free PX. These results could be owing to instability of rGO sheets and hydrophobic characteristic of free PX in aqueous media. Also the enhanced cytotoxicity of R9–rGO(120) might be attributed to the enhanced cellular uptake of nanocarrier to the cancer cells followed by quick intracellular drug release. The high concentration of free drug in cytoplasm allowed more drugs distributed in acting site, so that a stronger cellular inhibition was obtained.⁷⁰ Furthermore, lower amounts of the loaded PX or grafted R9 on the R9–rGO(0) and R9–rGO(90) nano sheets versus R9–rGO(120) resulted in less toxicity.

Fig. 6 Representative images of uptaking developed FITC-conjugated R9–rGO nano-carrier systems by Hela cancer cells (fluorescent field). R9–rGO(0) (A), R9–rGO(90) (B), and R9–rGO(120) (C). Inset figures are bright field of each fluorescent field. The quantitative proportion of FITC-positive cells to total cells in each group (D). Scale bar, 50 μm.

Fig. 7 Relative cell viability (%) of Hela (A) and MCF-7 (B) cancer cell lines after 0, 24, 48, and 72 hours treatment with rGO, R9–rGO(0), R9–rGO(90), R9–rGO(120), and free paclitaxel (PX).

4 Conclusion

Loading of hydrophobic drugs on nano carriers is a challenging issue for developing novel drug delivery systems. Thus, it is beneficial to use hydrophobic carriers for loading hydrophobic drugs. On the other hand, hydrophobic carriers are not stable in physiological solutions. Moreover, targeting of nano-carriers exponentially improves the efficacy of cancer therapeutic strategies. In this study, we designed a novel graphene-based DDS by modulating graphene sheet sizes and functionality with following usage of R9 peptide as a convenient stabilizer and targeting agent. Sonication process was optimized in terms of modulating pH, time duration, power, temperature, and acid treatment for improving the size and functionality of GO sheets. We found that the sonication of GO sheets for 120 minutes at pH 2 under controlled conditions provides suitable ALS and ATS as well as oxygen-containing groups. Additionally, the hydrothermal introducing of R9 peptides to engineered GO sheets conduces to production of R9 grafted rGO. The produced R9–rGO was stable in physiological solutions for more than a week, and efficiently delivered Paclitaxel (as a hydrophobic chemotherapeutic drug) to cancer cells. In the present approach, simple methods were used for stabilization, functionalization, and targeting of rGO nano-carriers which could be economically scaled up. Due to the elimination of toxic chemical reagents, this protocol could be a great step toward commercialization of graphene based DDSs.

Acknowledgements

This work was supported by the grants from Shahid Beheshti University of Medical Sciences.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra13822e

‡ The first and second authors are contributed equally in this work.

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