A Robust Drug Delivery Carrier for Simultaneous Tumor MR Imaging

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Controlled Release and Delivery Systems

Biocompatible and stable GO-coated Fe3O4 nanocomposite: A robust drug delivery carrier for simultaneous tumor MR imaging and targeted therapy Dong Li, Mingwu Deng, Ziyou Yu, Wei Liu, Guangdong Zhou, Wei Li, Xiansong Wang, Da-Peng Yang, and Wenjie Zhang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00029 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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ACS Biomaterials Science & Engineering

Engineering Center of China, Shanghai, China, Department of Plastic and Reconstructive Surgery

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biocompatible and stable GO-coated Fe3O4 nanocomposite: A robust drug delivery carrier for simultaneous tumor MR imaging and targeted therapy Dong Li‡1; Mingwu Deng‡1; Ziyou Yu1, Wei Liu1, Guangdong Zhou1, Wei Li1, Xiansong Wang*1; Dapeng Yang*2; Wenjie Zhang*1 1

Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of

Tissue Engineering, National Tissue Engineering Center of China, Shanghai 200011, China 2

School of Chemical Engineering and Materials, Quanzhou Normal University, Quanzhou, Fujian, 362000, China

‡ Dong Li and Mingwu Deng contributed equally to this work. * Xiansong Wang, Dapeng Yang, and Wenjie Zhang are co-corresponding authors of this paper. E-mail addresses of corresponding authors are as follows: Xiansong Wang

[email protected]

Dapeng Yang

[email protected]

Wenjie Zhang

[email protected]

ABSTRACT: Combined targeted drug delivery and sustained drug release, 1


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through the application of nanomedicine, shows great potential in cancer therapy and diagnostics. Systems based on folic acid conjugated with graphene oxide-based magnetic nanoparticles (NPs) show distinct advantages for such chemotherapeutic applications. Herein, we prepared FA-Fe3O4@nGO-DOX magnetic nanoparticles (MNPs) with a uniform size distribution based on nanoscale graphene oxide (nGO) encapsulated Fe3O4, which was conjugated with folic acid (FA) and loaded with doxorubicin (DOX). The prepared MNPs were characterized by various biophysical methods and featured a uniform size distribution. The uniform size of the nGO resulted in a relative narrow size distribution of the Fe3O4@nGO MNPs, which contributed to the stability of the nanocarrier system. Cell viability and in vitro biocompatibility studies of the FA-Fe3O4@nGO-DOX NPs revealed their selective uptake by MGC-803 cells. The relative viability was maintained at ~90% after 48 h of incubation and the hemolysis ratio confirmed the low toxicity of our modified NPs. The pH-controlled drug release and selective uptake of FA-Fe3O4@nGO NPs by MGC-803 cells via the FA receptor ensured selective killing of tumor cells. Furthermore, the nanoparticles for magnetic resonance imaging were analyzed in vitro and their signal intensity decreased as the NP concentration was increased. The nanocomposite was highly effective for in vivo imaging. Additionally, our in vivo antitumor activity and histological analysis confirmed the selective anticancer activity of the FA-Fe3O4@nGO-DOX NPs. Notably, our NPs were highly active and mice treated with FA-Fe3O4@nGO-DOX showed lower weight loss compared with mice treated with Fe3O4@nGO-DOX. More necrotic tissue was observed in the tumors of 2



the FA-Fe3O4@nGO-DOX group compared with those observed in the control, Fe3O4@nGO-DOX, and DOX groups. Thus, FA-Fe3O4@nGO-DOX is an effective and stable candidate for targeted drug delivery. KEYWORDS: Nanoscale graphene oxide, iron oxides, magnetic particle imaging, targeted therapy

Introduction Chemotherapy is used widely in the treatment of cancer. However, many side effects of chemotherapy, such as myelosuppression, mucositis, and alopecia are associated with damage to normal cells. The delivery of chemotherapeutic agents specifically to tumor sites could potentially increase treatment efficacy and reduce side effects1-3. Graphene oxide (GO) exhibits good hydrophilicity4, has a large surface area, and is cost-effective to make5. Hence, GO has been widely investigated as an efficient drug-loading nanocarrier6-10. Song et al.6 and Zheng et al.6, 7 reported that chemotherapeutic agents such as doxorubicin hydrochloride (DOX) can be efficiently loaded onto GO via π–π stacking. The release of DOX is triggered under acidic conditions, where the release rate increases at lower pH8-10. In combination with magnetic nanoparticles (MNPs), GO-based nanocarrier systems are effective for magnetic resonance imaging (MRI). MRI typically offers excellent depth penetration and high spatial resolution. T2 contrast agents, such as the Fe3O4 nanoparticles used in this report, produce a negatively enhanced contrast, which contributes to effective imaging. After entering into cells, the magnetic nanoparticles remain stable for a relatively long time, which enables long-term tracking of the nanocarrier system. 3


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The folate receptor (FR) is overexpressed on the surface of many tumor cells, and this marker has been used to develop folic acid (FA)-mediated targeted chemotherapy 11-13

. Through conjugation with FA molecules, a GO-based nanocarrier system

provides a passive targeting mechanism for drug delivery to tumor cells. Many such GO-based nanocarrier systems combined with MNPs and/or folic acid have been prepared based on multifunctional nanocarrier systems9, 14-20. These materials have partially resolved certain issues such as low carrier permeability and weak ligand recognition. However, problems associated with the stability of the nanocomposites remain a major challenge. Few reports have focused on size control of GO particles to ensure a uniform and stable composite. It is particularly important to address the size and size distribution of nanoparticles because these features critically affect the stability and biocompatibility of nanocarrier systems

21, 22

. A previous report23 has

indicated that kaolinite-goethite plates preferentially aggregate with large GO species in dispersions containing both large and small GO species. This effect leads to heteroaggregation of GO, which limits its dispersibility in aqueous media and reduces circulation time in the blood owing to reticuloendothelial clearance

24

. Therefore,

there is an urgent need to develop a stable nanocarrier system of an appropriate and uniform size. Iron oxide-based nanocarriers enable magnetic targeting and MR imaging25-28. Uncoated iron oxide particles have a high surface activity and easily aggregate; however, after coating with GO, iron oxide nanocomposites are biocompatible and can be easily dispersed. Furthermore, functional groups can be introduced that enable conjugation with other bioactive molecules or drugs. There have been some previous reports on the combination of iron oxide with GO particles. For example, Lu et al.28 explored the application of multifunctional GO-carbon nanotube composites with a 4



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dual targeted ability. Song et al.27 studied a lactoferrin modified graphene oxide–iron oxide nanocomposite with particles in the size range of 200–1000 nm. Compared with previously

reported

iron

oxide

based

nanocarriers25-28,

our

nGO

coated

nanocomposites show the following advantages. 1) The superparamagnetic iron oxide particles in the nanocomposites enable MRI. 2) The functional groups on the nGO can be conjugated with targeting molecules, such as FA and antibodies. 3) The structure of GO can be loaded with DOX through π-π interactions, which enables controlled drug release. Therefore, our tailored nGO retains all the characteristics of GO, including the ability to be loaded with drugs, and our nanocarrier system has a narrow size distribution. Specifically, we obtained a highly stable and efficient therapeutic and diagnostic (theragnostic) system, through the use of an Ag+ tailoring method to obtain a nanoscale GO (nGO) with a narrow size distribution. This nGO was successfully conjugated with Fe3O4 nanoparticles (NPs) and FA to construct a well-defined targeted nanocarrier system. Here, Fe3O4 NPs were constructed in situ and encapsulated in a nGO sheet to form core/shell Fe3O4/nGO (Fe3O4@nGO) NPs (Fig. 1A). These Fe3O4@nGO NPs were evaluated in various aqueous media and found to exhibit uniform size with good stability. After modification with FA through covalent bonding and loading with the chemotherapeutic agent DOX through π-π interactions, the selectivity and antitumor effects of these NPs were investigated in vitro and in vivo. Cell assays showed the biocompatibility of this nanocarrier system together with its targeting ability. The systemic delivery of DOX loaded in FA-Fe3O4@nGO markedly increased the efficacy of drug delivery and inhibited tumor growth. Moreover, the tumor contrast was considerably increased, thereby improving the accuracy of cancer diagnosis. Our work provides a new approach to the synthesis of a stable and versatile theragnostic nanocarrier system based on 5


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Fe3O4@nGO NPs. These NPs are biocompatibility and show great potential for simultaneous imaging and targeted therapy in a clinical setting. Materials Natural graphite powder (800 mesh) was obtained from Beijing Chemical Reagents (Beijing, China). FA and DOX were purchased from Sigma-Aldrich (St Louis, MO, USA). Other chemicals were of analytical grade and used as received without further purification (purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). All aqueous solutions were prepared with ultrapure water (18 MΩ·cm), and other materials and solvents were chemical grade and used as received. Methods Preparation of nGO and synthesis of Fe3O4@nGO NPs The nGO was synthesized by slicing large-area graphene oxide sheets into nanoscale pieces with silver metal ions according to our previous report

29

. In a

typical synthesis, 20 mL of AgNO3 aqueous solution was gradually added to 20 mL of an aqueous GO dispersion (0.5 mg/mL) with vigorous stirring at room temperature (RT) for 30 min. Subsequently, the reaction mixture was allowed to stand for 48 h at RT. The sample was purified by centrifugation at 300 g for 10 min. The supernatant was then mixed with 10 mL of 2 mM HNO3 and concentrated in a rotary vacuum evaporator for 5 h to dissolve the silver NPs. The concentrated extract was centrifuged at 2,500 g for 10 min. The supernatant was then transferred to microcentrifuge tubes and centrifuged at 7,200 g for 30 min to collect the nGO. Finally, the precipitate containing nGO, with a particle size of approximately 100 nm, was resuspended in deionized water and examined with an atomic force microscope (AFM) and UV-Vis spectrometer. 6



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The Fe3O4@nGO NPs were prepared by a one-pot hydrothermal method 30. Briefly, 1.08 g of FeCl3⸱ 6H2O was dissolved in 30 mL of ethylene glycol to yield a clean yellow-brown solution under vigorous stirring for 30 min at RT. This was followed by the addition of 0.8 g of NaOH with stirring until the sample was completely dispersed. Subsequently, 5 mL of nGO (1.0 mg/mL) was added to the dispersion with stirring. Finally, the reaction mixture was stirred vigorously for 10 min and transferred to a 40 mL Teflon-lined stainless-steel autoclave, which was heated to 200 °C for 10 h. The autoclave was cooled to RT and the obtained black product was washed with ethanol and deionized water three times each with magnetic decanting of the un-bonded reactants in the washings. FA-conjugated Fe3O4@nGO NPs FA

molecules

were

conjugated

with

the

Fe3O4@nGO

NPs

through

carbodiimide-mediated covalent bond formation between the carboxyl groups in GO and the amine groups in FA, according to previous reports 8, 20, 31. Briefly, 1 mg of FA was

dispersed

in

1

mL

water,

and

3

mg

of

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 2 mg of N-hydroxysuccinimide (NHS) were added. The mixture was sonicated for 20 min to activate the amine groups of FA. The desired amount of the Fe3O4@nGO solution was added to the FA-containing solution and this mixture was allowed to stand at room temperature for 2 h for the carboxyl groups of nGO to react with the activated amine groups of FA. The product was washed three times with ddH2O to remove unreacted reagents with the assistance of a permanent magnet, then resuspended in ddH2O for further characterization. DOX loading and in vitro DOX release 7


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A

solution

containing

different

concentrations

of

DOX

and

0.9

mg

FA-Fe3O4@nGO aqueous solution was prepared and stirred at 4 °C for 24 h in the dark. The FA-Fe3O4@nGO-DOX NPs were precipitated with a permanent magnet. The product was collected after washing with water until the supernatant became color-free. The absorbance of the supernatant was measured at 490 nm to determine the remaining DOX concentration. The drug encapsulation efficiency (%) is defined as: encapsulation efficiency (%) = [weight of loaded DOX (mg)/weight of initial DOX (mg)] × 100%. For drug release, certain volumes of the DOX-loaded FA-Fe3O4@nGO NPs solution were washed with ddH2O and acetate buffer was added at pH 5.5 (endosomal pH), 6.5, and 7.4 (physiological pH) in separate glass bottles. The bottles were placed on a horizontal rotator shaker (40 rpm at 37 °C), followed by separation of FA-Fe3O4@nGO-DOX with a permanent magnet at predetermined time intervals

32,

33

. The supernatants were removed and replenished with an equal volume of each

sample for further drug release studies. The supernatants were measured at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader to quantify the concentration of DOX. Finally, the concentration of released DOX was calculated in a cumulative manner, according to the following equation 33: Cumulative DOX released (%) = (cumulative amount of DOX released/initial amount of DOX) × 100% Characterization of nanoparticles (NPs) X-Ray diffraction (XRD) patterns of samples were recorded with a Bruker-AXS micro-diffractometer (D8 ADVANCE) with Cu-Kα radiation (λ = 1.5406 Å) from 10 to 80° at a scanning speed of 0.33° min–1. The surface chemical groups of the 8



Fe3O4@nGO NPs were recorded by Fourier transform infrared spectroscopy (FT-IR, BrukerVector-22 FT-IR spectrometer, Bruker Corporation, Beijing, China). Magnetization versus magnetic field curves were measured at 300 K by a vibrating sample magnetometer (VSM; PPMS-9T (EC-II), Quantum Design, San Diego, CA, USA). The surface morphology and structure were observed by a field emission scanning electron microscope (FESEM; Zeiss, Shanghai, China) operating at an accelerating voltage of 5.0 kV and a transmission electron microscope (TEM; JEM-2010). AFM images were recorded on a Nanoscope Multimode V instrument (Veeco, Plainview, NY, USA). Commercially available AFM cantilever tips with a force constant of ~48 N/m and resonance vibration frequency of ~330 kHz were used. Absorption spectra were recorded on a UV-2550 UV-Vis spectrometer (Cary 50, Agilent Technologies Japan, Ltd., Tokyo, Japan). Aqueous suspensions of the Fe3O4@nGO NPs were used for the UV-Vis samples and pure water was used as the reference. Stability evaluation The stabilities of Fe3O4@nGO and FA-Fe3O4@nGO NPs in distilled water, saline, PBS and fetal blood serum (FBS) were analyzed by monitoring the homogeneity of the solution after standing at RT for 72 h. The sizes of nGO, Fe3O4@nGO, and FA-Fe3O4@nGO NPs were analyzed by dynamic light scattering (DLS, Zeta View, Meerbusch, Germany). Zeta potentials were measured for the Fe3O4@nGO and FA-Fe3O4@nGO nanocomposites. The particles stored in different media for different time periods were also examined by DLS. Biocompatibility of Fe3O4@nGO NPs

9


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The biocompatibility of the Fe3O4@nGO NPs was investigated with a cell counting kit (CCK) cytotoxicity assay and a hemolysis assay. Human gastric cancer cells (MGC-803) and human umbilical vein endothelial cells (ECs) were obtained from the Cell Bank, Chinese Academy of Sciences, and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and 1% gentamycin at 37 °C and in an atmosphere containing 5% CO2. The viability assay (CCK test) was performed according to the manufacturer’s instructions. Here, MGC-803 cells were seeded in 96-well microtiter plates (Falcon, BD, Franklin Lakes, NJ, USA) at a seeding density of 8000 cells/well and cultured for 12 h. Different concentrations of Fe3O4@nGO NPs were then added to the wells with six replicates and incubated for another 24 and 48 h in the cell culture incubator. CCK solution (10 µL) was added to each well at 24 and 48 h. After incubating at 37 °C for a further 2.5 h, the absorbance at 450 nm was measured for each well with an ELISA reader (StatFax-2100, Awareness Technology, Palm City, FL, USA). Hemolysis evaluations were performed according to a previous report

34

. Briefly,

red blood cells (RBCs) were separated from blood samples and washed at least four times and diluted with 10 mL of PBS. Then 200 µL of the diluted RBC suspension was mixed with 800 µL of deionized water (positive control), PBS (negative control), or Fe3O4@nGO NPs at different concentrations (10−200 µg/mL). After incubation for 2 h at 37 °C, the samples were centrifuged and the absorbance of the supernatants at 570 nm was measured. Finally, the percentage hemolysis of the RBCs was calculated as follows, Hemolysis (%) = OD value of different Fe3O4@nGO groups / OD value in ddH2O group × 100%. Targeted internalization of NPs 10



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We first assayed the FR expression level in MGC-803 cells and ECs by the quantitative real-time polymerase chain reaction. Prussian blue staining was used to evaluate the internalization of FA-Fe3O4@nGO and Fe3O4@nGO NPs within MGC-803 cells and ECs. Iron oxide NPs that were internalized by the cells were stained by a Prussian blue staining method with some modifications

35

. Typically,

~20,000 cells were seeded into each well of a 24-well plate. After incubating for 24 h at 37 °C and 5% CO2, the fresh medium was added containing different concentrations of FA-Fe3O4@nGO and Fe3O4@nGO. The cells were then incubated at 37 °C and 5% CO2 up to 12 h, with gentle shaking at 15 min intervals. At the indicated time points, the wells were washed five times with PBS to eliminate NPs that were not internalized by cells. Subsequently, 4% paraformaldehyde (PFA) was added to each well for 10 min before proceeding with the Perls Prussian blue reaction for 15 min. The cells were washed with ddH2O and imaged by light microscopy. In vitro cytotoxicity of FA-Fe3O4@nGO-DOX NPs MGC-803 cells and ECs were seeded in 96-well microtiter plates (Falcon, BD, USA) at a seeding density of 8000 cells/well and cultured for 12 h. The medium was exchanged with fresh medium containing different concentrations of DOX-loaded FA-Fe3O4@nGO NPs (DOX concentration: 5–80 µM). After incubation for 4 h, the medium was removed and washed with PBS before adding fresh medium supplemented with 10% FBS and a 1% gentamycin solution. After incubating for 24 h, the CCK assay was performed according to the manufacturer’s instructions, as described above. Cytotoxicity was expressed as the percentage cell viability compared with that of the untreated control cells. 11


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Animals and the gastric cancer xenograft model Male BALB/c nude mice weighing 20–24 g, obtained from the Shanghai SLAC Laboratory Animal (Shanghai, China), were housed under pathogen free conditions. All study protocols were approved by the Animal Care and Experiment Committee of Shanghai 9th People’s hospital. MGC-803 cells were harvested and resuspended in PBS and subcutaneously injected into athymic nude mice at a dose of 1 × 107 cells/mouse. MR imaging of FR-overexpressing gastric cancer in vivo In vitro MR phantom images were acquired with an MR scanner (0.55 T, MesoMR23-060H-I, Shanghai, China) to evaluate the effects of the Fe3O4@nGO NPs on the image contrast. Relaxation rates were calculated by fitting the 1/T2 relaxation times (s−1) versus Fe3O4@nGO concentration (µg/mL) curves. The targeting ability of FA-Fe3O4@nGO-DOX was evaluated in vivo by an MR scanner (7 T, Bruker, Biospec 70/20 USR, Germany). Briefly, a total of 10 gastric cancer xenograft mice (4 weeks after implantation) randomly received an intravenous injection (tail vein) with FA-Fe3O4@nGO-DOX NPs or Fe3O4@nGO-DOX (1 mg/mL) at a dose of 100 µL/mouse. After anesthetizing with 5% chloral hydrate, a mouse was placed in a scanning holder. The images were acquired before and after intravenous injection at given times (−5 min and 8 and 24 h). In vivo antitumor activity studies Twenty xenograft mice were randomly and equally divided into four groups. Three weeks later, when the tumor volumes reached approximately 100 mm3, the mice were 12



intravenously injected with 3.0 mg/kg of free DOX, 3.0 mg DOX/kg Fe3O4@nGO-DOX NPs, 3.0 mg DOX/kg FA-Fe3O4@nGO-DOX NPs, and PBS was used as a control. Injections were performed every 2 d until the 14th day. Tumor volumes and body weight were measured at 3 d intervals. The estimated tumor volume was calculated according to the formula: tumor volume (mm3) = ½ × length × width2. Finally, after three weeks all 20 mice were sacrificed by an anesthesia overdose and the tumor was excised for further analysis. Histotoxicity analysis On the 21st day of treatment, the 20 mice were sacrificed by an anesthesia overdose, their organs were removed, and tissues samples were taken. Samples of the heart, liver, spleen, lung, kidney, and cancer tissues were dissected and fixed in 4% PFA. All samples were dissected and embedded in paraffin and 5 µm slices were prepared before staining with hematoxylin and eosin (H&E). Statistical analysis Quantitative data were given as the mean ± standard deviation (SD) and analyzed with the aid of SPSS software (version 16.0). Means were compared by one-way ANOVA. Data were considered to be statistically significant at p < 0.05. Results and discussion Synthesis and characterization of FA-Fe3O4@nGO-DOX NPs We developed a new method to synthesize nanoscale graphene oxide-encapsulated Fe3O4 (Fe3O4@nGO) nanocomposites by assembly of positively charged Fe3+ and negatively charged nanoscale graphene oxide36-38 under solvothermal conditions (Fig. 13


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1A). First, GO was synthesized from graphite powder via the classic Hummer’s method 39. The as-synthesized GO featured a 2D sheet-like morphology that was too large for in vivo applications. To obtain small and uniform GO NPs, large pieces of 2D graphene oxide were cut into nanoscale pieces by the Ag+ tailoring method29, as previously reported40. Briefly, small GO fragments were obtained after a modified Ag+ process followed by centrifugation. AFM images showed that small graphene fragments were obtained through the Ag+ processing (Fig. 1B). After centrifugation, smaller graphene sheets with a relatively homogeneous size of ~100 nm and a thickness of ~1 nm were obtained (Fig. 1C). These dimensions indicated a single-layer structure, which may suitable for in vivo applications. The nanoscale Fe3O4@nGO nanocomposites were synthesized from these tailored graphene oxides through a one-pot hydrothermal method. The process was driven by the mutual electrostatic interactions between Fe3+ and nGO. The synthetic Fe3O4@nGO nanocomposites possessed ultrathin and flexible graphene shells, which encapsulated the Fe3O4 NPs. TEM images revealed that the as-obtained particles were spherical with an average size of ~50 nm and the particles were well dispersed (Fig. 1D). Moreover, high-resolution TEM images suggested that the Fe3O4@nGO NPs featured a core/shell structure (Fig. 1E). Lattice measurements showed that the typical lattice parameter for the Fe3O4 nanocrystals along the (311) lattice plane was 0.23 nm, and this value was unaffected by the nGO coating (white arrows Fig. 1E). The blurred region (yellow lines in Fig. 1E) around the iron oxide nanocrystals was attributed to the nGO shell. The FT-IR spectrum (Fig. S1, Electronic Supporting Information) 14



showed characteristic peaks of Fe3O4@nGO. The peaks at 586.90 and 631.17 cm–1 were related to an Fe-O stretching vibration derived from splitting of the υ1 band at 570 cm–1, which indicated the existence of Fe3O4. The phase purity and chemical composition of the Fe3O4@nGO NPs were examined by XRD. The diffraction peaks shown in Fig. 1F could be assigned to an Fe3O4 phase according to Joint Committee on Powder Diffraction Standards (Card No.65-3107) 41. The diffraction peaks could be used to estimate the particle size based on the Debye–Scherrer formula. The estimated crystalline size was ~50 nm, which was in accordance with the results from our TEM images. Raman spectroscopy is an important tool for studying ordered and disordered crystal structures of carbonaceous materials42. We further investigated the nanocomposites using Raman spectroscopy. As shown in Fig. 1G, the peaks at 1358 and 1577 cm–1 correspond to the D and G bands of GO particles, respectively. Super paramagnetic behavior was observed from the Fe3O4@nGO nanocomposites in the magnetic curves and the relative saturation magnetization reached 60.2 emu·g

–1

(Fig. 1H). The inserts in Fig. 1H show the

response of the Fe3O4@nGO nanocomposites under an external magnetic field. The magnetic nanoparticles (MNPs) were rapidly attracted to the magnet such that the remaining solution appeared colorless. This result confirmed the strong magnetic properties of our Fe3O4@nGO nanocomposites. As previously reported26, 43, 44, Fe3O4 MNPs possess good biocompatibility, good stability, and low toxicity, and have thus been widely used as MRI contrast agents to detect small tumors after modification with specific molecules. Strong magnetism is beneficial for tracking Fe3O4@nGO 15


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nanocomposites by in vivo MRI and also useful for manipulating the particles distribution45, 46. The ability of the Fe3O4@nGO MNPs to bind FA and DOX was also confirmed by standard UV-Vis spectroscopy. The pure nGO showed a characteristic absorption signal at ~230 nm. The purified FA and DOX absorption peaks occurred at 360 and 480 nm, respectively. The absorption curve of FA-Fe3O4@nGO-DOX exhibited absorption changes at 360 and 480 nm, further revealing that the FA and DOX molecules were conjugated with Fe3O4@nGO (Fig. 1I). Stability of FA-Fe3O4@nGO NPs The stabilities of Fe3O4@nGO and FA-Fe3O4@nGO NPs in different media, including ddH2O, saline, PBS, DMEM and FBS were assessed. No precipitation was observed for the nanocomposites in ddH2O and FBS; however, only the FA-conjugated NPs remained dispersed in the ion solutions (Fig. 2A), indicating that the FA-Fe3O4@nGO NPs showed the greatest stability in different media. The zeta potentials of these two different materials were also measured. These results showed that after combination of FA with the NPs, the zeta potential of FA-Fe3O4@nGO decreased to approximately −50 mV. Hence, conjugation with the FA molecules increased interactions between the particles and the liquid, thus promoting the stability of the dispersion (Fig. 2B). The hydrodynamic diameter of the particles was measured by DLS. As shown in Fig. 2C, the nGO particles featured a narrow distribution of ~100 nm which was consistent with the AFM results. After loading with Fe3O4, the diameter of the core/shell composite Fe3O4@nGO was approximately 50 nm, indicating that relatively small and uniform magnetic nanoparticles were 16



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formed. The FA-Fe3O4@nGO particles exhibited the same narrow size distribution of approximately 50 nm, indicating that FA molecules were successfully conjugated with Fe3O4@nGO NPs. Furthermore, Fig. 2D shows that the size of FA-Fe3O4@nGO did not increase over time and that no precipitation occurred in different media, confirming that FA-Fe3O4@nGO possessed good stability

under complex

physiological conditions. DOX loading efficiency and controllable DOX release of nanocomposites The drug loading efficiency saturated at a DOX concentration of 0.15 mg/mL (Fig.3A). The controlled release of DOX from FA-Fe3O4@nGO-DOX NPs was measured by quantifying the amount of DOX released in media of different pH. The in vitro drug release data are shown in Fig. 3B. At pH 7.4, the amount of DOX released reached 20%. However, this value was greater than 60% at pH 5.0. These results can be attributed to the π–π bonding interactions between DOX and GO, which control binding of the DOX under neutral conditions. The different release behaviors of DOX from the multi-functionalized GO at different pH values show that the release of

FA-Fe3O4@nGO-DOX

NPs

is

controllable.

This

feature

makes

FA-Fe3O4@nGO-DOX a good candidate material for intelligent drug delivery and release systems. Overall, the release profiles of DOX from the FA-Fe3O4@nGO-DOX NPs suggested that the drug will be stably bound during blood circulation and rapidly released in the acidic environment of the tumor 47, 48. Biocompatibility of FA-Fe3O4@nGO NPs Prior to in vivo applications, it was necessary to evaluate the biocompatibility and 17


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toxicity of our unloaded drug carrier, FA-Fe3O4@nGO NPs. As shown in Fig. 4A, when the cells were treated with the FA-Fe3O4@nGO NPs over a wide range of concentrations for 24 and 48 h, the results of the standard CCK assay showed no obvious cell toxicity, indicating good biocompatibility. Moreover, the relative viability of the cells was maintained at approximately 90% even after incubation for 48 h at a FA-Fe3O4@nGO NPs concentration of 200 µg/mL. Furthermore, the hemolysis ratio, as shown in Fig. 4B, was no more than 2.46±0.66% at the maximum concentration (400 µg/mL). These findings suggest that the FA-Fe3O4@nGO NPs are nontoxic and hem compatible. Thus, these NPs could be administered intravenously for in vivo cancer treatment. Targeting ability and antitumor activity of NPs The ability of the NPs to specifically target tumors is important for successful drug delivery. Prussian blue staining and the CCK-8 assay were used to assess the targeting ability and antitumor effects, respectively. Membrane-bound protein FR was selected as the target in this study, which is overexpressed in various malignant tumors

49

. First, the expression levels of FR in

MGC-803 cells and ECs were quantified by qRT-PCR analysis. The relative mRNA level of FR in MGC803 cells was statistically higher than the level in ECs (1905±40.47 vs. 1.000±0.1100, p < 0.0001; Fig. S2), suggesting that MGC803 cells will preferentially interacted with FA or FA-conjugated particles. Prussian blue staining was then performed to confirm selective uptake of the FA-Fe3O4@nGO NPs in MGC-803 cells. As shown in Fig. 5, a dose-dependent 18



Page 20 of 40

internalization of FA-Fe3O4@nGO was observed in MGC-803 cells after incubation for 4 h; however, no obvious internalization was observed in the Fe3O4@nGO treated cells. Additionally, no obvious internalization of FA-Fe3O4@nGO NPs or Fe3O4@nGO NPs was observed in ECs. The results demonstrate that uptake of FA-Fe3O4@nGO NPs by MGC-803 cells was FR selective and occurred over a very short period. We also observed that both the Fe3O4@nGO NPs and the EC groups showed a time-dependent internalization, which might be attributed to an enhanced permeability and retention (EPR) effect, as previously reported

50-53

. However,

MGC-803 cells exhibited a higher EPR effect compared with that of the ECs, which we attribute to the high metabolic rate of tumor cells. The results in Figure 5 also indicated that cellular uptake of these nanocomposites did not notably affect the viability or proliferation of cells. Additionally, no obvious changes in cellular morphology were observed after incubation with the nanocomposites for 12 h, which further suggested that the nanocomposites are non-toxic. The targeting ability was further confirmed by an antitumor activity assay. MGC-803 cells and ECs were treated with a series of concentrations of FA-Fe3O4@nGO-DOX NPs for 4 h. The medium was then replaced with regular cell culture medium without NPs. After an additional 24 h incubation period, the cell viability was measured by the CCK-8 assay. The activities of MGC-803 cells were inhibited by FA-Fe3O4@nGO-DOX treatment; however, the ECs were unaffected (Fig. 6), suggesting that FA-Fe3O4@nGO-DOX could selectively kill tumor cells but not normal cells. 19


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MR imaging of tumors In vitro studies confirmed the selectivity of FA-Fe3O4@nGO-DOX for MGC-803 cells. We also wanted to determine whether the NPs could target tumors formed by MGC-803 cells in nude mice and inhibit tumor growth (Fig. 7). Before injection, the magnetic sensitivity of the FA-Fe3O4@nGO-DOX solutions was investigated with a MR scanner. As shown in Fig. 8A, a gradual decrease in the T2 signal intensity was observed as the NPs concentration was increased, indicating that the NPs could be used as an MR contrast agent. FA-Fe3O4@nGO-DOX and Fe3O4@nGO-DOX composites were then injected intravenously into tumor-bearing mice, and MRI was performed 8 and 24 h after injection. The tumor MR signal in the FA-Fe3O4@nGO-DOX injected group decreased 8 and 24 h post injection (Fig. 8B, C, D, H). Although no significant changes were observed in the Fe3O4@nGO-DOX injected group at 8 and 24 h (Fig. 8E, F, G, I), the MR signal showed a decrease for the Fe3O4@nGO-DOX group, which might be attributed to the enhanced permeability and the retention effect, as reported previously50-53. The FA-Fe3O4@nGO-DOX NPs were more able to effectively target and become enriched in the solid tumor than the Fe3O4@nGO-DOX NPs. These results suggest that the FA-Fe3O4@nGO-DOX composite could be used as an MRI contrast agent, with a long lifetime, for diagnosis and detection of small tumors that express FR. According to a previous report54, negative contrast agents can be improved through modification with dextran to reduce the partial volume effect. In our study, we used a 20



high-resolution MRI machine to achieve the same improvement, as shown in Figure 8. Additionally, we verified that our nanocomposites could also be used as a targeted drug delivery system. However, this type of high-resolution MRI machine is expensive and not commonly used in hospitals. Thus, we intend to make further modifications to our nanocomposites to facilitate clinical applications54. Some of the latest literatures also described several different GO based drug delivery systems. Li and Cao55 developed a remote-controlled drug release system based on multi-functional Fe3O4/GO/Chitosan microspheres, with the help of NIR irradiation as well as ultrasound stimulation; Xu and Li56 synthesized a novel magnetic core-shell nanoparticles Fe3O4@La-BTC/GO which exhibited dual-modal imaging and pH-responsive drug release abilities. These latest studies were consistent with our results in terms of MRI as well as pH responsive drug release ability. In vivo antitumor activity The selective anticancer activity of FA-Fe3O4@nGO-DOX NPs was evaluated by determining the body weight and solid tumor volume. During the experiments, mice treated with PBS buffer gradually lost body mass (Fig. 9A). Similarly, body mass loss was also observed for both the free DOX and Fe3O4@nGO-DOX groups (Fig. 9A). Notably, the mass loss was less pronounced for the FA-Fe3O4@nGO-DOX group that that for the above three groups (Fig. 9A). This result suggests that selected binding of FA-Fe3O4@nGO-DOX to tumors reduced the side effects to some extent. Tumor volume changes represent a direct index to assess therapeutic effects. As shown in Fig. 9B, the tumors treated with FA-Fe3O4@nGO-DOX exhibited a 21


Page 22 of 40

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remarkable regression 3 d after treatment, whereas tumors treated with PBS (control), free DOX, or Fe3O4@nGO-DOX grew continuously over the 3-week period. Histological analysis confirmed that more necrotic tissues were observed in the FA-Fe3O4@nGO-DOX group than in the other groups (Fig. 9C). Many particles accumulated in the remaining solid tumors in the FA-Fe3O4@nGO-DOX group (Fig. 9C), which was consistent with the MRI results (Fig.8). Toxicity analysis of nanocomposites Histopathological analyses of important organs was performed to identify whether the FA-Fe3O4@nGO-DOX NPs had any serious histotoxic effects (Fig. 10). We observed that some nanocomposites accumulated in the liver and kidneys for both the FA-Fe3O4@nGO-DOX and Fe3O4@nGO-DOX nanocomposite groups; however, no nanoparticles were observed in other organs (Fig. 10). These results indicate that the GO based nanocomposites might be eliminated from the body through these organs, as previously demonstrated by Akhavan and Ghaderi57. No obvious structural abnormalities were observed in these organs for either the Fe3O4@nGO-DOX or FA-Fe3O4@nGO-DOX groups. Our FA conjugated nanocomposites showed a good targeting ability for solid tumors, based on the in vivo biodistribution of these nanocomposites in various organs, which suggests their relative safety. Comparing the uptake values between tumors (Fig. 9) and residual organs (Fig. 10), such as the liver and kidneys, we found that the FA-Fe3O4@nGO-DOX presented higher tumor uptake and lower residual organ uptake; hence, selective targeting of the tumors was achieved. These results are consistent with those of previously reported GO related 22



nanocomposites57. According to a previous report58, GO particles at a concentration of 100 µg/mL showed potential for cyto- and genotoxic effects on hMSCs in vitro. One mechanism for the cytotoxicity of graphene nanomaterials is based on damage to cell membranes through physical contact interactions with the extremely sharp edges of graphene59,

60

; another is through oxidative stress60-62. Our nGO based

nanocomposites were conjugated with FA molecules and nanosized with less pronounced sharp features and lower oxidative stress63, 64. Furthermore, the ratio of the blood volume for a 25 g weight mouse is in the range of 1.5–2.0 mL 57; hence, the overall distribution of our nanocomposites was ~66.6 µg/mL over the whole body of the mouse, which might reduce cell membrane damage to a certain extent. These results are in agreement with our in vitro studies, which showed no toxic effects in ECs at a high concentration of 200 µg/mL. Considering the potential for continuous drug delivery and the degradation products to have long-time side effects on mice hormones

65

, we are considering reducing the concentration of our

composites in future studies. The drug could be taken up and accumulated by the tumor.

Conclusions Well-dispersed Fe3O4@nGO NPs were successfully fabricated with good control over the GO size. The final NPs exhibited a core-shell structure with an average size distribution of approximately 50 nm. The high saturation magnetization values promoted effective MRI. The viability and proliferation of cells were not obviously affected after incubation with the nanocomposites, even at relatively high 23


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nanocomposite concentrations. Compared with traditional Fe3O4 nanoparticles, the GO coating provided active carboxyl groups that could be combined with different molecules, such as antitubercular agents and active enzymes, to treat various kinds of diseases through controlled release. These smart and stable NPs could be used as a novel drug delivery system and show promise as a candidate for diagnosis and treatment of different diseases. Supporting Information Figure S1 and S2 are listed as supporting information for the manuscript. Acknowledgements This work was supported by the National Natural Science Foundation of China (81671839 and 31400851), the Natural Science Foundation of Shanghai (15ZR1425300, 15JC1490600 and 16ZR1419800), the National Key Research and Development Program of China (2016YFC1101400) and sponsored by the Interdisciplinary Program of Shanghai Jiao Tong University (project number: YG2014MS01). We also thank Liwen Bianji, Edanz Editing China, for editing the English text of a draft of this manuscript. Disclosure The authors report no conflicts of interest in this work.

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For Table of Contents Use Only

Manuscript title

Biocompatible and stable GO-coated Fe3O4 nanocomposite: A robust drug delivery carrier for simultaneous tumor MR imaging and targeted therapy All authors Dong Li‡1; Mingwu Deng‡1; Ziyou Yu1, Wei Liu1, Guangdong Zhou1, Wei Li1, Xiansong Wang*1; Dapeng Yang*2; Wenjie Zhang*1

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Fig. 1 Design, fabrication and characterization of Fe3O4@nGO NPs. (A) Schematic illustration for the preparation of FA-Fe3O4@nGO-DOX; (B, C) AFM images of regular and nanoscale graphene oxides; (D, E) TEM and HR-TEM image; (F) typical XRD patterns; (G) Raman spectrum; (H) magnetization curves and (I) UV-Vis spectroscopy. 180x115mm (300 x 300 DPI)


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Fig. 2 Stability evaluation of the FA-Fe3O4@nGO NPs. (A) Stability of Fe3O4@nGO and FA-Fe3O4@nGO NPs in different media for 72 h;(B) Zeta potential of Fe3O4@nGO and FA-Fe3O4@nGO NPs in aqueous solution; (C) Particle size distribution of nGO, Fe3O4@nGO and FA-Fe3O4@nGO NPs; (D) Size change of FAFe3O4@nGO NPs in different media (ddH2O, PBS, Saline, DMEM and FBS). 163x127mm (150 x 150 DPI)



Fig. 3 DOX encapsulation efficiency and controllable release. (A) Plot of encapsulation efficiency of DOX to FA-Fe3O4@nGO NPs versus the concentration of DOX; (B) Plot of the release of DOX from FA-Fe3O4@nGODOX at pH 5.5, 6.5 and 7.4. 182x74mm (300 x 300 DPI)


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Fig. 4 In vitro biocompatibility. (A) Cell viabilities of MGC-803 cells incubated with different concentrations of FA-Fe3O4@nGO for 24 and 48 h;(B) Hemolysis of FA-Fe3O4@nGO NPs after incubation with red blood cells at various concentrations (0−200 µg/mL) for 2 h, PBS and deionized water were used as negative and positive controls, respectively. Inset: Hemolysis photo after centrifugation. 121x46mm (220 x 220 DPI)



Fig. 5 Comparison of the effects of Prussian blue staining of non-targeted Fe3O4@nGO NPs and targeted FAFe3O4@nGO NPs. 146x57mm (220 x 220 DPI)


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Fig. 6 In vitro cytotoxicity of FA-Fe3O4@nGO-DOX NPs toward (A) MGC-803 cells and (B) EC. 146x59mm (220 x 220 DPI)



Fig. 7 Schematic diagram showing the potential multi-functions of FA-Fe3O4@nGO-DOX NPs as MR imaging and targeted therapy in FR-overexpressing gastric cancer xenografted mice. 146x56mm (220 x 220 DPI)


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Fig. 8 In vitro MR images (A) of the Fe3O4@nGO composites at different concentrations. In vivo MR images of gastric cancer tissues in xenografted mice before (−5 min), and at 8 and 24 h post injection of the FAFe3O4@nGO-DOX composites (B, C, D) or Fe3O4@nGO-DOX composites (F, I, J) (n = 5 per group). (E) and (K) present the gray value of the tumor area at different time points before and after injection with FAFe3O4@nGO-DOX composites and Fe3O4@nGO-DOX composites, respectively. Notes: The dotted dash line indicates a tumor. *p < 0.05.

107x198mm (300 x 300 DPI)



Fig. 9 (A) Changes in body weight during treatment and (B) relative tumor growth curves; (C) Histological analysis of solid tumor tissues. 193x110mm (300 x 300 DPI)


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Fig. 10 Histopathological analysis of the main organs from xenografted mice treated with PBS, free DOX, Fe3O4@nGO-DOX and FA-Fe3O4@nGO-DOX NPs (n = 10 per group, scale bar = 100 µm). 154x147mm (300 x 300 DPI)


A Robust Drug Delivery Carrier for Simultaneous Tumor MR Imaging

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