cRGDyK-modified camretastain A4-loaded graphene oxide nanosheets for targeted anticancer drug delivery

Abstract

Functionalized graphene oxide (CGO–cRGDyK/POLO) nanosheets were designed and developed for targeted drug delivery to integrin expression. The anticancer drug cambretastain A4 (CA4) was conjugated onto the CGO–cRGDyK/POLO nanosheets. The tumor targeting ligands, cyclic arginine–glycine–aspartic acid–tyrosine–lysine pentapeptides (cRGDyK), were conjugated to carboxylated graphene oxide (CGO) nanosheets, and poloxamer 188 (POLO) was rendered to make the CGO–cRGDyK nanosheets stable. The CGO–cRGDyK/POLO nanosheets exhibited good cytocompatibility, high CA4-loading capacity (0.6872 ± 0.0121 mg mg⁻¹) via π–π stacking or hydrophobic interactions, and controlled release in vitro. The MTT assays showed that the CGO–cRGDyK nanosheets had greater antitumor effects on Hela cells than CGO/POLO nanosheets but lower cytotoxicity on the L02 cells than free CA4. In addition, the cellular uptake of CGO–cRGDyK/POLO nanosheets was increased significantly in human umbilical vein endothelial cells (the integrins-overexpressed cells) compared to the normal L02 cells. Thus, the CGO–cRGDyK/POLO nanosheets are an appealing platform for cancer chemotherapy.

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1. Introduction

Chemotherapy is commonly used to treat cancer patients in clinics.^1–5 However, the efficacy of many chemotherapeutic agents is often hindered by their low aqueous solubility, severe toxic side effects, drug resistance, and nonspecific action.⁶ In recent years, various types of drug delivery systems (DDS), such as nanoparticles,⁷ conjugated polymer⁸ and carbon nanotubes,⁹ have been investigated, a small number of which have already been commercialized or are in clinical studies. The properties of an ideal drug delivery system should include: targeted delivery, efficient loading and controlled release.^8–10

Graphene is a two dimensional monoatomic thick building block of a carbon allotrope with high specific surface area, π–π stacking and electrostatic or hydrophobic interactions that has been assessed as a new class of nano-material for drug delivery systems (DDSs). Dai et al. reported six-armed PEG-amine star polymer grafted graphene oxide (GO) as a novel drug nanocarrier for water-insoluble anti-cancer drugs via hydrophobic and π–π stacking interactions in 2008,¹¹ and GO has attracted considerable attention because of its excellent biocompatibility and well-dispersed stability in aqueous solutions. Drugs, such as 5-fluorouracil, ibuprofen, camptothecin, and doxorubicin, can be loaded onto GO via π–π stacking and van der Waals interaction. In addition, GO has abundant epoxide, hydroxyl and carboxyl functional groups that can form strong hydrogen bond with others.

By utilizing the antibodies or specific ligands, DDSs along with the therapeutic agent can bind selectively to the targeted cells, and then be delivered to the interior of a given type of cells.¹² Among the many active targeting strategies, receptor-mediated delivery has been studied extensively.^13–16 Cyclic arginine–glycine–aspartic acid–tyrosine–lysine pentapeptides cRGDyK have a high affinity to the integrin receptors over-expressed on angiogenic endothelial cells as well as tumor cells, such as malignant glioma cells, breast cancer cells, as well as tumor cells and prostate cancer cells, which render cRGDyK a unique molecular ligand for targeted cancer chemotherapy.

Tumor vascularisation is a critical process that determines tumor growth and metastasis.¹⁷ Cambretastain A4 (CA4) is a class of small molecular tubulin-binding agents isolated from the South African tree, Combretum caffrum, which exhibited favourable anti-cancer activities. CA4 is a class of novel drugs that exploit the unstable, immature characteristics of tumor blood vessels to selectively target and destroy the tumor vascular network, resulting in tumor ischemia and necrosis, and have been hailed as opening a new era in cancer therapy, as evidenced by some promising findings from clinical trials.^18–21 However, this prodrug has several undesirable side effects on many normal tissues.²² CA4 is a kind of hydrophobic drug. This drawback can be decreased by targeting the drug specifically to the tumor vasculature, while GO is an ideal nanomaterial for DDS and can provide a large specific surface area for insoluble drugs via π–π stacking or hydrophobic interactions. Therefore, an assessment of GO for drug delivery is recommended.

In this paper, we will describe how we explored and prepared a novel drug delivery system based on poloxamer 188 (POLO) and cRGDyK-modified graphene oxide nanosheets. In our strategy, cRGDyK was designed onto the carboxylated graphene oxide nanosheets (CGO) via classic amidation, POLO was deposited onto the CGO–cRGDyK nanosheets via physical absorption, and drug molecules, such as CA4, were loaded noncovalently onto GO via π–π stacking or hydrophobic interactions (Fig. 1). Through the transformation of the hydroxyl groups of the GO nanosheets into carboxylic acid groups, more POLO brushes as well as cRGDyK targeting moieties could be immobilized onto the functional graphene derivative. Therefore, excellent dispersibility, cyto-compatibility and targeting specificity are expected for the nanocarrier and the specifically targeted controlled release of the anti-cancer drug.

Fig. 1 Schematic of the synthesis of cRGDyK-modified CGO and preparation of nanocarrier CGO–cRGDyK/POLO/CA4. Top: scheme-A; scheme-B.

2. Experimental section

2.1 Materials and reagents

Graphite powder was purchased from Adamas Reagent Co., Ltd. in Shanghai, China.

Cyclic arginine–glycine–aspartic acid–tyrosine–lysine penta-peptides (cRGDyK) were purchased from China Peptides Co., Ltd. in Shanghai, China.

1-Ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydro-chloride (EDC) was purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). N-Hydroxylsuccinimide (NHS) was purchased from Aladdin Chemistry Co. Ltd. Cambretastain A4 (CA4) was provided by Shanghai Ecust Biomedicine Co., Ltd. Sulfuric acid (H₂SO₄, 98%), potassium permanganate (KMnO₄), sodium nitrate (NaNO₃) were analytical reagent grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Double distilled water was used throughout.

2.2 Preparation of graphene oxide (GO) and its characterization

GO nanosheets were prepared from graphite using the Hummers' method with a slight modification.²³ Briefly, graphite power (0.50 g) was added to cold H₂SO₄ (23.0 mL), and then stirred gradually with KMnO₄ (3.0 g) and NaNO₃ (0.50 g) on ice. The resulting mixture was stirred further at 35 °C for 1 h. Subsequently, 46 mL of distilled water was added and the temperature was increased to 90 °C and maintained for 1 h. The reaction was stopped with 140 mL of distilled water and 3.2 mL of 30% H₂O₂.²⁴ The reaction products were washed by repeated centrifugation, first with an aqueous 5% HCl solution, and then with distilled water until the solution became pH neutral. The products were dispersed in 40 mL of distilled water and sonicated at 400 W for 2 h to exfoliate the GO layers and form GO nanosheets, the unexfoliated GO nanosheets was collected and an extruder was used to filter it through a 0.45 μm polycarbonate membrane filter. The final solution was lyophilized and stored at −20 °C until use.

The shape and surface morphology of GO were investigated by transmission electron microscopy (TEM, JEOL and Tokyo, Japan) after negative staining with a uranyl methanol solution (1%, wt/vol). Raman spectra were recorded with a He–Ne laser (λ = 632.8 nm) as the excitation source. Powder X-ray diffraction was carried out on an X-ray diffractometer (XRD, Rigaku, Tokyo, Japan) using Ni-filtered CuKα radiation with 4 degrees per min scanning rate at room temperature. The Fourier transform infrared (FT-IR) spectra were recorded using a Nicolet NEXUS instrument with an attenuated total reflectance (ATR) accessory.

2.3 Preparation of carboxylated graphene oxide nanosheets (CGO) and its characterization

The aqueous suspension of GO (50 mL, 2.0 mg mL⁻¹) was ultrasonicated for 1 h to obtain a clear suspension. NaOH (5.00 g) and chloroacetic acid (ClCH₂COOH) (5.00 g) were then added to the GO suspension and ultrasonicated for another 3 h to convert the hydroxyl and epoxy groups on the GO nanosheets to carboxyl groups. The resulting carboxylated graphene oxide nanosheets (CGO) suspension was neutralized, and purified by repeating the cycle of rinsing and filtration. The CGO suspension was dialyzed against distilled water for more than 48 h to remove any ions, followed by drying at 65 °C.²⁵ The CGO was investigated by FT-IR spectroscopy.

2.4 Synthesis of CGO–cRGDyK and its characterization

The surfaces of the GO nanosheets were modified with cRGDyK peptide to increase their target ability. In brief, N-(3-dimethylaminopropyl-N′-ethylcarbodiimide)hydrochloride (EDC, 5 mg) and N-hydroxysulfosuccinimide sodium (NHS, 5 mg) were added to the GO nanosheets (1 mg mL⁻¹, 10 mL) in distilled water to activate the carboxyl groups in GO, adjusting the pH to 8.0–8.5 with N-methyl morpholine and stirring for 24 h at room temperature (20–25 °C). Subsequently, 5 mg of cRGDyK was added and the mixture was stirred for 24 h at room temperature. The resulting reaction mixture was washed eight times with a 3500 MWCO Millipore centrifuge filter at 3000g. The final solution was lyophilized and stored at −20 °C until use.²⁶

To test the number of cRGDyK-modified graphene oxide nanosheets, a standard curve and an equation were acquired by measuring the HPLC peak area of a series of cRGDyK in PBS solutions with known concentrations. A gradient high-performance liquid chromatography (HPLC, Shimazduo, Japan) method with a flow rate of 0.5 mL min⁻¹ was developed and used to quantify the conjugation efficiency. The mobile phase consisted of acetonitrile–H₂O–trifluoroacetic acid (2 : 98 : 0.05, eluent A) and acetonitrile–H₂O–trifluoroacetic acid (90 : 10 : 0.05, eluent B). The eluted gradient was set from 100% eluent A to 70% eluent A/30% eluent B over 30 min. The unreacted peptide was detected at 210 nm, and the column temperature was maintained at 30 °C.²⁷ A reversed phase column (Waters Symmetry C₁₈, 5 μm, 150 × 4.6 mm, Milford, MA, USA) was used and 20 μL samples were injected for all analyses. The CGO and CGO–cRGDyK nanosheets were examined by UV-vis spectroscopy on a Shimadzu UV-vis spectrophotometer. The CGO and CGO–cRGDyK nanosheets were investigated by XPS on a Thermo Escalab 250 XPS instrument with a monochromatic Al Kα X-ray source (hν = 1486.6 eV) (PHI Co., MN, USA).

2.5 Loading of poloxamer (POLO) onto CGO–cRGDyK and its characterization

To resuspend the solution, 2 mg mL⁻¹ of poloxamer 188 (POLO) was added to an aqueous suspension of CGO–cRGDyK nanosheets (0.5 mg mL⁻¹), and another 1 h sonication took place. Centrifugation was then done at 22 000g to remove any aggregates or multilayered nano GO sheets. The supernatant was collected after centrifugation and washed eight times with an 8000 MWCO Millipore centrifuge filter at 4000g.

The shape and surface morphology of the CGO–cRGDyK and CGO–cRGDyK/POLO nanosheets were investigated by transmission electron microscopy (TEM, JEOL and Tokyo, Japan) after negative staining with a uranyl methanol solution (1%, wt/vol). The zeta potential of CGO–cRGDyK and CGO–cRGDyK/POLO nanosheets were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, United Kingdom) at 25 °C.

2.6 Loading of cambretastain A4 (CA4) and its characterization

The loading of CA4 into the CGO–cRGDyK/POLO nanosheets was accomplished by dispersing CA4 (3 mL, 5 mg) in methanol and stirring at 37 °C in a water bath for 30 min. The CGO–cRGDyK/POLO nanosheets (20 mL) was placed into a funnel and added to the CA4 solution. With the drop-wise addition of the CGO–cRGDyK/POLO nanosheets, the temperature of the system was increased from room temperature to 60 °C and vortexed for 30 min. A part of the CA4 was immobilized onto the GO sheets, some was wrapped in the POLO, and the rest of the CA4 was dissolved in the mixture. The resulting solution was left to stand at room temperature for 12 h, and then put into a dialysis tube (MWCO = 8000) and dialyzed with 300 mL phosphate buffered saline (PBS, pH 7.4, 37 °C) solution with stirring for 24 h to remove the methanol, dissolved CA4, and CA4 on the surface of CGO–cRGDyK/POLO nanosheets. The loading efficiency of CA4 was quantified by HPLC analysis. In brief, the nanosheets solution was diluted 10 times with methanol/water (68 : 32, vol/vol) as the mobile phase with detection at 295 nm with an ultraviolet/visible detector. The released CA4 was measured using a validated HPLC method, as described above. The shape and surface morphology of CGO–cRGDyK/POLO/CA4 nanosheets were investigated by transmission electron microscopy (TEM, JEOL and Tokyo, Japan) after negative staining with a uranyl methanol solution (1%, wt/vol). The CGO–cRGDyK/POLO and CGO–cRGDyK/POLO/CA4 nanosheets were investigated by Raman spectroscopy using the method above. The particle size of the CGO–cRGDyK/POLO/CA4 nanosheets and the other nanosheets were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, United Kingdom) at 25 °C.

2.7 In vitro drug release assay

The loading efficiency was measured using a HPLC system because CA4 has a special absorbance at 295 nm. A reversed phase column (Waters Symmetry C₁₈, 5 μm, 150 × 4.6 mm, Milford, MA, USA) was used at 30 °C and 20 μL samples were injected for all analysis. The mobile phase consisted of methanol and water (68 : 32, v/v), and the flow rate was 0.8 mL min⁻¹. The retention time of CA4 was 12.9 min. To determine the relationship between the CA4 concentration (C) and peak area (A), a standard curve and an equation were acquired by measuring the HPLC peak area of a series of CA4 in PBS solutions with known concentrations. The mass of dissolved CA4 could then be calculated from the dissolved CA4 concentration (C) multiplied by the total volume. Therefore, the loading efficiency (f) can be measured using the following equation:
where W_d is the mass of the dissolved CA4 and W₀ is the total mass of CA4 added initially.

A dialysis tube containing the CGO–cRGDyK/POLO/CA4 nanosheets was washed with 37 °C DI water and then placed rapidly into a beaker with PBS containing 10% fetal bovine serum (FBS) at 37 °C (200 mL, pH 7.4). Keeping the release experiment at 37 °C, a 1.0 mL solution was taken out from the PBS dialysis solution at set intervals to measure the concentration of the released drug. At the same time, 1.0 mL PBS solution was added to the mixed solution to keep the volume constant. The amount of CA4 released from CGO–cRGDyK/POLO nanosheets was determined by HPLC. The cumulative release ratio (r) of CA4 at a particular time (t) was calculated using the following equation:

where C_i−1 and C_i are the concentration of released CA4 in (i − 1) times and (i) times, V₁ and V₀ are 1 and 200 mL, respectively. To compare with the drug release of CGO–cRGDyK/POLO nanosheets, two other controlled experiments including drug release from GO and POLO were carried out using the same process.

2.8 Cell culture

Hela cells and L02 cells were cultures in RPMI 1640 medium (Gibco, Invitrogen Co., USA) with 10% fetal bovine serum (FBS) (HyClone, USA) and antibiotics (100 U mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin) at 37 °C and 5% CO₂. The HUVECs were isolated from umbilical cords using the collagenase digestion method described by Jaffe et al. Briefly, freshly harvested cells were cultured in endothelial cell growth medium (Sigma-Aldrich, St. Louis, MO, USA) consisting of M199 (GIBGO, USA), 10 mM HEPES (GIBGO, USA), 10% FBS (HyClone, USA), 100 μg mL⁻¹ heparin (Sigma-Aldrich, St. Louis, MO, USA), 50 μg mL⁻¹ of endothelial cell growth supplements (ECGS) (Sigma-Aldrich, St. Louis, MO, USA), and penicillin/streptomycin at 37 °C and 5% CO₂.

2.9 In vitro cytocompatibility assays and confocal microscopy experiment

An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the cytocompatibility and targeting specificity of the CGO–cRGDyK/POLO nanosheets with Hela cells and L02 cells with CA4 as the model drug. Hela cells express some integrins, L02 cells are normal liver cells used as a control cell line. For the MTT assay,⁹ the cells were seeded into 96 well plates at densities of 1 × 10⁵ cells per well for 24 h. The CGO–cRGDyK/POLO nanosheets, drug-loaded CGO–cRGDyK and CGO–cRGDyK/POLO nanosheets with different concentrations, and CA4 were then added to the cells and incubated for 48 h. Subsequently, the cells were washed three times with a PBS solution (0.01 M, pH 7.4) and processed for the MTT assay to determine the cell viability. 100 μL of pH 7.4 PBS solutions containing 20 μL of 5 mg mL⁻¹ MTT was added to each well and incubated for an additional 4 h. The cell bound dye was dissolved with 100 μL DMSO in each well cell culture plate and swung on a table concentrator for 20 min. The absorbance of each well was read on a microplate reader using a test wavelength of 490 nm.

To examine the nanocarrier uptake by receptor-bearing cells, the HUVECs and L02 cells were incubated with CGO–cRGDyK/POLO nanosheets.³⁸ The CGO–cRGDyK/POLO nanosheets were labelled with FI-TC. A 1.0 mL aliquot of a human umbilical vein endothelial cells (HUVECs, 1 × 10⁵ cells per well) and L02 cells (1 × 10⁵ cells per well) suspension as seeded onto glass-bottom dishes. After 24 hours' incubation at 37 °C, 5% CO₂/95% air to allow cell growth, an aliquot of CGO–cRGDyK/POLO (20 ng mL⁻¹) treated with FI-TC was added to each dish and incubated for another 3 h at 37 °C. The medium was removed, and the cells were washed three times with cold PBS (0.01 M, pH 7.4) at room temperature. The confocal images of cells were then analyzed using a laser scanning confocal microscope (Leica Microsystems, Weltzer, Germany).

3. Results and discussion

3.1 Preparation of GO and CGO

Size is the key factor of the GO application for DDS. Transmission electron microscopy (TEM) was used to determine the morphology and size (Fig. 2(A)). The results revealed a large surface area for the assembly of POLO and CA4.

Fig. 2 Characterization of GO and CGO. (A)–(C) Typical (A) TEM image, (B) Raman, and (C) XRD spectra of synthesized GO nanosheets. (D) Typical FT-IR spectra of the GO and CG nanosheets measured in KBr pellets.

The Raman spectrum of GO nanosheets (Fig. 2(B)) indicated the disorder on the graphitic layer and the interlayer spacing of the GO, and the D peak at 1338 cm⁻¹ and G peak at 1587 cm⁻¹ were observed using 514 nm lasers as the excitation source. The strong D band is related to the high oxidation of graphite, which implies that many carboxyl and hydroxyl groups were generated on the surface of the graphitic layer.²⁸

The structure of GO nanosheets was characterized further by XRD, and the XRD pattern is shown in Fig. 2(C). The (002) peak at 2θ = 10.21° indicated that the interlayer distance in the GO was about 0.86 nm.²⁹ Compared to previous reports on the interlayer distance of pristine graphite (0.34 nm), the increase in the interlayer distance in GO was ascribed to the presence of epoxide, carboxyl groups, and water molecules between the GO layers, which proves that the oxidization is successful and the prepared GO nanosheets are pure.

The GO was synthesized using a modified Hummers method from natural graphite power and this was proven by the appearance of an absorbance peak at 1706 cm⁻¹ of the C=O stretch band of the carboxyl groups (Fig. 2(D)).²³ The absorbance peak at 1041 cm⁻¹, 1391 cm⁻¹, 1623 cm⁻¹, and 1706 cm⁻¹ can be attributed to C–O stretching (epoxy or alkoxy), O–H stretching (carboxyl), C=C skeletal vibrations of unoxidized graphite domains, and C=O in carboxylic acid and carbonyl moieties, respectively. There are many hydroxyl and epoxy groups in the GO nanosheets. It is necessary to convert these groups to COOH groups to improve the aqueous solubility and reaction sites of the graphene derivatives and to facilitate chemical binding of the functional cRGDyK to CGO via EDC chemistry. In the present study, the hydroxyl and epoxy groups of the GO were converted to –COOH groups by mixing the GO with ClCH₂COOH under strongly alkaline conditions according to the literature.²⁵ It is interesting that the color of the GO suspension changed from dark-brown to black during the conversion process, which may be due to the partial reduction of the GO under strongly alkaline conditions.³⁰ The presence of –CH₂COOH groups in the carboxylated graphene oxide (CGO) was confirmed by the FT-IR spectra (Fig. 2(D)). The peak at 1706 cm⁻¹ corresponding to the C=O bond of –COOH on GO shifted to 1700 cm⁻¹ in the CGO because the proportion of –CH₂COOH grew in the CGO. This indicated that the hydroxyl and epoxy groups of the GO were converted to –COOH groups.

3.2 Synthesis of CGO–cRGDyK and its characterization

As shown in Fig. 3(A), there was a small peak of free cRGDyK after 24 h at room temperature, indicating that a considerable amount of free peptide was conjugated at a weight ratio of 1 : 2 (cRGDyK to CGO). The free cRGDyK was eluted as a single peak with a retention time of 4.8 minutes. According to the standard curve and equation, the conjugation efficiency was determined to be approximately 98% (0.49 mg mg⁻¹ (W_cRGDyK/W_CGO)).

Fig. 3 Characterization of the CGO–cRGDyK nanocarrier (A)–(C). (A) HPLC image of cRGDyK, (B) UV-vis spectra of the CGO, CGO–cRGDyK nanosheets and cRGDyK in their aqueous dispersions, (C) N element analysis of GO and CGO–cRGDyK with XPS.

The new absorbance bands at 295 nm, which cannot be seen in the UV-vis absorption spectrum of the GO nanosheets, appeared in that of the CGO–cRGDyK due to the characteristic absorbance of the benzene in cRGDyK. However, GO only displayed a small absorbance peak at about 233 nm due to the π–π of C=C.³¹ This also verified the successful grafting of the CGO–cRGDyK onto the CGO nanosheets via facile amidation (Fig. 3(B)).

X-ray photoelectron spectroscopy (XPS) was performed to identify the successful modification of the cRGDyK peptide to the GO nanosheets. The simple N element analysis indicated that there was no N 1s peak appearing for GO but the N 1s peak at 399.85 eV was observed for the CGO–cRGDyK nanosheets (Fig. 3(C)).

3.3 Preparation of CGO–cRGDyK/POLO and its characterization

To make CGO–cRGDyK nanosheets stable in PBS solutions, poloxamer 188 (POLO) was absorbed physically to the CGO–cRGDyK, while the CGO–cRGDyK nanosheets solution was centrifuged at 12 000g to remove any aggregated, multilayer sheets. The surface modification on CGO–cRGDyK could be reflected by the change in zeta potential. The zeta potential is the mutual repulsion between particles or measuring the intensity of attraction, indicating the existence of a large number of hydroxyl groups. Fig. 4(A) and (B) showed a zeta potential of functionalized CGO–cRGDyK in phosphate buffered saline. Owing to the existence of hydroxyl and carboxyl groups on CGO–cRGDyK, the zeta potential of CGO–cRGDyK was −23.5 mV. Fig. 4(C) and (D) showed that the CGO–cRGDyK nanosheets were agglomerated and CGO–cRGDyK/POLO nanosheets were dispersed uniformly. Therefore, the zeta potential (positive or negative) is higher as the dispersed particles became smaller, the stabilizing system dissolved or dispersed resisted aggregation. In addition, because of the presence of hydroxyl groups on POLO,^32,33 the zeta potential of CGO–cRGDyK/POLO nanosheets was lower than that of the CGO–cRGDyK nanosheets. The result showed that, after grafting with POLO, the zeta potential of the CGO–cRGDyK/POLO nanosheets were increased to −36.4 mV, moreover, and the step of the CGO–cRGDyK/POLO nanocarrier averaging ∼100 nm, indicated that the system of CGO–cRGDyK/POLO was more stable than CGO–cRGDyK in PBS solutions.

Fig. 4 Characterization of CGO–cRGDyK and CGO–cRGDyK/POLO (A)–(D). Zeta potentials of CGO–cRGDyK and CGO–cRGDyK/POLO (A) and (B), TEM image of CGO–cRGDyK and CGO–cRGDyK/POLO nanosheets (C) and (D).

3.4 CA4 loading into CGO–cRGDyK/POLO and in vitro drug release assay

The large π conjugated structure of the CGO–cRGDyK/POLO nanosheets can form a π–π stacking interaction with the aromatic structure of CA4. Therefore, CA4 was loaded noncovalently on the CGO–cRGDyK/POLO hybrid by a π–π stacking interaction or hydrophobic in aqueous solution with the aid of slight sonication. The drug-loading capacity of the biocompatible and specifically targeted CGO–cRGDyK/POLO nanosheets were investigated at pH 7.4 values at 37 °C with a model hydrophobic anti-cancer drug (CA4), determined by HPLC at 295 nm. The CGO–cRGDyK/POLO nanocarrier exhibited a high CA4 loading capacity (0.6872 ± 0.0121 mg mg⁻¹) resulting from a combination of the π–π stacking interaction between the CGO–cRGDyK/POLO nanocarrier and CA4 and the electrostatic interaction between POLO and CA4. As a biocompatible and reduction-responsive nanocarrier, the CGO–cRGDyK/POLO nanocarrier has not only a relatively stable structure, but also a certain drug-loading capacity and encapsulation efficiency.

The shape was characterized by TEM. As shown in Fig. 5(A), the CGO–cRGDyK/POLO/CA4 nanosheets were roughly spherical in shape with a diameter of 100 nm. However, the unmodified GO sheets were adsorbed together due to π–π interactions and as a result, the size was far greater than 100 nm, which was the size of CGO–cRGDyK (Fig. 4(C)). The surfactant, poloxamer 188 (POLO), was adsorbed between each layer under an ultrasonic probe and finally reduced the sheet size and made CGO–cRGDyK nanosheets stable in solution.³⁴ TEM showed that there were no obvious changes in the shape of CGO–cRGDyK/POLO nanosheets (Fig. 4(D)) before and after loading the drugs.

Fig. 5 (A) TEM image of CGO–cRGDyK/POLO/CA4; (B) particle diameter distribution of CGO–cRGDyK/POLO/CA4; (C) Raman spectra (excitation at 514 nm) with the D and G bands of graphitic carbon.

The hydrodynamic diameter of the CGO–cRGDyK/POLO/CA4 nanosheets were measured by the dynamic light scattering (DLS) analysis with nanosheets dispersed in phosphate buffered saline (PBS, pH = 7.4). The average hydrodynamic diameter of the CGO–cRGDyK/POLO/CA4 nanosheets were about 120 nm (Fig. 5(B)), which is close to the particle size observed by TEM. The polydispersity index (PDI), reflecting the dispersity of the nanosheets was 0.248, indicated the monodisperse distribution of the CGO–cRGDyK/POLO/CA4 nanosheets. As shown in Fig. 5(C), a graphite-like band (G-band) at ∼1600 cm⁻¹ and a disorder-induced band (D-band) at ∼1380 cm⁻¹ were observed. The D-band is used to characterize the amorphous or disordered carbon. The G-band is related to the vibration of sp²-hybridized carbon atoms, which verifies the presence of graphitic domains.³⁵ The existence of a G-band in all samples suggests that well defined graphitic domains had developed. It has been reported that the G/D-band ratio is almost proportional to the degree of graphitization.³⁶ As observed, the ratios of the G/D-band were 1.12 and 0.97 for the CGO–cRGDyK/POLO and CGO–cRGDyK/POLO/CA4 nanosheets.

In previous studies, surfactants have been used as the recipient phase to evaluate the in vitro release kinetics of drugs from nanocarriers.^37,38 However, these release media cannot mimic the circulation systemic condition. Instead, fetal bovine serum (FBS) was used as a biomimetic recipient phase to maintain the sink condition for the release of CA4 from GO nanosheets. In addition, PBS (pH = 7.4) containing 10% FBS can also mimic the conditions employed for the cell culture. Fig. 6 includes the 72 h release profile for CA4 from targeted nanocarriers and nontargeted nanocarriers. The amount of cumulative drug release within 10 h was 42.4% and 40.8% for targeted nanocarriers and nontargeted nanocarriers, respectively.

Fig. 6 In vitro release of CA4 from the cRGDyK conjugated CGO nanosheets, CGO nanosheets and CA4 ethanol solution.

After 72 h, 79.3% and 79.1% of total drug were released from targeted nanosheets and nontargeted nanosheets, respectively. These results indicated that CGO–cRGDyK/POLO nanosheets could effectively load CA4 into the nanosheets and release drug in a sustained-release manner. CA4 in the nanosheets would be taken up into cells in the form of nanosheets rather than as the free drug during the first 10 h incubation.

3.5 In vitro cytotoxicity assays

The cytocompatibility of CGO–cRGDyK/POLO nanosheets with Hela cells and L02 cells was evaluated using MTT assays. A dose–response study was also performed by exposing the Hela cells and L02 cells to various concentrations of this nanocarrier.

As shown in Fig. 7(A), excellent cytocompatibility was demonstrated by increasing the concentration of the CGO–cRGDyK/POLO with Hela cell viability of 0.93 to 1.16 from 0 to 20 ng mL⁻¹ and the L02 cell viability was increased from 0.98 to 1.11 with the tested concentrations after 48 h of incubation. This indicates that the CGO–cRGDyK/POLO has low cyto-compatibility on tumor cells and normal cells, indicating good biocompatibility of this drug carrier under the current experimental conditions.

Fig. 7 Relative cell viability of Hela cells (target cells) and L02 cells (control cells) after treatment with CGO–cRGDyK/POLO (A), CGO–cRGDyK/POLO/CA4 (B), free CA4, CGO/POLO/CA4 and CGO–cRGDyK/POLO/CA4 (C) and (D) at different concentrations at 37 °C for 48 h.

To evaluate the cRGDyK peptide group-mediated targeting function of CGO–cRGDyK/POLO, the CGO/POLO/CA4 and CGO–cRGDyK/POLO/CA4 nanosheets were used for in vitro study with Hela cells and L02 cells. The two graphene-based nanosheets had a similar structure except that the POLO on the CGO–cRGDyK/POLO nanosheets was terminated with the cRGDyK peptide. The CA4-loading capacity of the CGO–cRGDyK/POLO nanosheets was 99.2% of the CGO/POLO under the same drug-loading conditions, and the marginal difference between the two nanocarriers was caused by the cRGDyK peptide in the CGO–cRGDyK/POLO nanosheets. Therefore, the two nanocarriers must have the same drug-loading mechanism, as well as the same releasing mechanism. As shown in Fig. 7(B), CGO–cRGDyK/POLO/CA4 nanosheets showed much greater cyto-compatibility to Hela cells than L02 cells. At a lower concentration of CA4 equivalent (10 ng mL⁻¹), the CGO–cRGDyK/POLO/CA4 nanosheets showed a much higher cytocompatibility to Hela cells than the CGO/POLO/CA4 nanosheets (Fig. 7(C)).

This might be due to the greater uptake of CA4 by Hela cells as a consequence of integrins receptor-mediated endocytosis. In contrast, the cytotoxicity of CGO–cRGDyK/POLO/CA4 to L02 cells was lower than that of either free CA4 or CGO/POLO/CA4 (Fig. 7(D)). At a low CA4 dosage (10 ng mL⁻¹), CGO–cRGDyK/POLO/CA4 nanosheets showed higher cytotoxicity to Hela tumor cells than either CGO/POLO/CA4 or free CA4, which were less cytotoxic to normal cells L02 because of their targeted and sustained drug release profiles. These results indicate that the CGO–cRGDyK/POLO/CA4 nanosheets drug system not only reduces the dosage of CA4 with the same cytocompatibility to tumor cells, but also reduced the side effects to normal cells, making the CGO–cRGDyK/POLO/CA4 nanosheets potentially promising for clinical targeted cancer therapy.

3.6 Confocal microscopy experiment

Cellular uptake of drug was evaluated by confocal microscopy after incubation of the cells and nanocarrier treated with FI-TC. To examine the cellular uptake efficiency, the primary cells, HUVECs, were used to mimic angiogenic tumor vascular cells. HUVECs overexpress integrins and have been used previously as an in vitro tumor vascular endothelial cells model.³⁹ L02 cells are normal liver cells without overexpressing integrins and have been used as a control cell line.

The results showed strong fluorescence in the HUVECs, which was significantly higher than that in the L02 cells, indicating that endocytosis mediated by the integrins receptor facilitates cellular uptake efficiency (Fig. 8). The selective drug uptake in the target cells provides the basis for targeted therapy mediated by CGO–cRGDyK/POLO nanosheets. These confocal microscopy observations confirm that the active targeting effect of CGO–cRGDyK/POLO nanosheets toward HUVECs is consistent with the in vitro antitumor activity indicated by the MTT assays.

Fig. 8 Confocal microscopy images showing the cellular uptake of CGO–cRGDyK/POLO nanosheets (concentration = 20 ng mL⁻¹) by HUVECs and L02 cells for 3 h.

4. Conclusions

In this investigation, we produced and characterized an innovative drug delivery system. The cRGDyK functionalized CGO/POLO nanosheets mediated targeted delivery, and controlled the release and efficient loading of CA4. The CGO–cRGDyK/POLO nanosheets have a small size and large specific surface in this system, resulting in a higher loading capacity for CA4 (68.7%) and sustained release. The cellular uptake of the CGO–cRGDyK/POLO nanosheets were increased significantly in human umbilical vein endothelial cells (the integrins-overexpressed cells) compared to the normal cells L02. Drug-loaded CGO–cRGDyK/POLO nanosheets exhibited greater cytotoxicity on the Hela cells than the CGO/POLO nanosheets, while lower cytotoxicity on L02 cells than free CA4. Based on these favorable results, we can conclude that this new system, which is biocompatible and easy to prepare, can be used in biomedical applications. Further work will be needed to investigate the details of the inhibition of tumor growth by the nanosheets to gain more information about how the delivery system works, and also to extend this application to several other small molecular weight chemotherapeutic agents of interest.

Acknowledgements

This project was granted financial support from Shanghai Excellent Young Teachers Program (no. 4521ZK11YQ02) and other programs (no. 21172148, 21472126).

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