Delivery of Oridonin and Methotrexate via PEGylated Graphene Oxide

Jun 5, 2019 - This nanosized GO-PEG10K-6arm was found to be of very low ... (16) Poly(ethylene glycol) (PEG) is available in a variety of pharmaceutic...
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Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 22915−22924

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Delivery of Oridonin and Methotrexate via PEGylated Graphene Oxide Dongdong Chai,†,∥ Bingjie Hao,‡,∥ Rong Hu,† Fang Zhang,*,§ Jia Yan,† Yu Sun,† Xiaoyu Huang,*,‡ Qingxiao Zhang,§ and Hong Jiang*,†

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Department of Anesthesiology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine, Center for Specialty Strategy Research of Shanghai Jiao Tong University China Hospital Development Institute, 639 Zhizaoju Road, Shanghai 200011, People’s Republic of China ‡ Key Laboratory of Synthetic and Self-Assembly Chemistry for Organic Functional Molecules, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, People’s Republic of China § The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, People’s Republic of China S Supporting Information *

ABSTRACT: Graphene oxide (GO) possessing plenty of hydroxyls and carboxyls is often used in the field of biomedicine. To improve its water solubility and biocompatibility, 6-armed poly(ethylene glycol) (PEG) was bonded on the surface of GO sheets via a facile amidation process to form the universal drug delivery platform (GO-PEG10K‑6arm) with a 200 nm size in favor of the enhanced permeability and retention effect. Herein, we prepared the stable and biocompatible platform of GO-PEG10K‑6arm under mild conditions and characterized the chemical structure and micromorphology via thermogravimetric analysis and atomic force microscopy. This nanosized GOPEG10K‑6arm was found to be of very low toxicity to human normal cells of 293T and tumor cells of CAL27, MG63, and HepG2. Moreover, oridonin and methotrexate (MTX), widely used hydrophobic cancer chemotherapy drugs, were compounded with GO-PEG10K‑6arm via π−π stacking and hydrophobic interactions so as to afford nanocomplexes of oridonin@ GO-PEG10K‑6arm and MTX@GO-PEG10K‑6arm, respectively. Both nanocomplexes could quickly enter into tumor cells, which was evidenced by inverted fluorescence microscopy using fluorescein isothiocyanate as a probe, and they both showed remarkably high cytotoxicity to the tumor cells of CAL27, MG63, and HepG2 within a broad range of concentration in comparison with free drugs. This kind of nanoscale drug delivery system based on GO-PEG10K‑6arm may have potential applications in biomedicine, and GO-PEG10K‑6arm would be a universal and available carrier for extensive hydrophobic anticarcinogens. KEYWORDS: graphene oxide, PEG, MTX, oridonin, drug delivery

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biocompatibility for biomedical applications.16 Poly(ethylene glycol) (PEG) is available in a variety of pharmaceutical formulations due to its low toxicity, biocompatibility, protein resistance, and good solubility in aqueous solution.17,18,33 The biofunctionalization of nanomaterials with water-soluble PEG was extensively used for drug delivery.19−23 PEGylated graphene oxide (GO-PEG) would be soluble and biocompatible in a physiological buffer so that GO-PEG would be a desirable platform for the delivery of hydrophobic antitumor drugs. Dai et al. originally employed PEG-functionalized nanoscale GO as a nanocarrier to load antitumor drugs like SN38 [a camptothecin

INTRODUCTION Graphene has emerged as a novel one-atom-thick two-dimensional graphitic material with interesting physical properties.1 Potential applications of graphene in diverse fields, such as nanoelectronic devices, sensors, conductors, solar cells, and nanocomposites, are widely explored.2−6 Furthermore, its oxidation derivative, graphene oxide (GO), is also deeply and systematically investigated in biological fields including drug loading and delivery.7−11 Indeed, hydrophilic functionalities of hydroxyl, carboxyl, and epoxy groups can be formed at the edge and basal plane of GO sheets so as to afford better dispersion in aqueous media and provide reaction sites for further functionalization through certain chemical reactions.7−15 Moreover, some researchers have indicated that fine-tuning of the lateral size of GO could provide a safer design approach to increase its © 2019 American Chemical Society

Received: March 5, 2019 Accepted: June 5, 2019 Published: June 5, 2019 22915

DOI: 10.1021/acsami.9b03983 ACS Appl. Mater. Interfaces 2019, 11, 22915−22924

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ACS Applied Materials & Interfaces Scheme 1. Chemical Structure of Nanoscale Drug Delivery System

improving its stability and biocompatibility.33 GO-PEG10K‑6arm nanocarrier could be effectively taken up by human renal epithelial 293T cells in a short time as shown by the intracellular imaging, and the results of the viability of 293 T renal epithelial cells indicated that GO-PEG10K‑6arm nanocarrier was of very low toxicity to 293T renal epithelial cells.39 Next, two different hydrophobic anticancer drugs of oridonin and MTX were physically loaded onto GO-PEG10K‑6arm nanovehicle through π−π stacking and hydrophobic interaction to provide two nanosized drug delivery systems via a suitable universal approach, oridonin@GO-PEG 10K‑6arm and MTX@GO-PEG 10K‑6arm (Scheme 1), respectively. It was proved that oridonin@GOPEG10K‑6arm is a very powerful killer of tumor cells in vitro with a higher cytotoxicity to human tongue squamous carcinoma CAL27, human osteosarcoma MG63, and human liver tumor HepG2 cells in comparison with free oridonin in the cell viability assay, which could obviously enhance the bioavailability of oridonin along with other hydrophobic drugs. It was also compliant for MTX@GO-PEG10K‑6arm in human tongue squamous carcinoma CAL27 cells. Thus, combined with our previously reported GO-PEG/PTX nanocomplex,33 GOPEG10K‑6arm is obviously an ideal universal platform for the delivery of hydrophobic antitumor drugs.

analogue] via noncovalent physisorption and investigated its cellular uptake.12,24 Oridonin, a highly oxygenated 7,20-epoxy-ent-kaurane-type diterpenoid isolated from the leaves of Isodon rubescens (a traditional Chinese herb), has attracted extensive attention in the last few years due to its wide activities including anticancer, antiviral, antibacterial, and anti-inflammatory effects, and it is widely employed in the treatment of various malignant tumors like lung cancer, which is the leading origin of cancer deaths worldwide.25,26 Furthermore, oridonin has also exhibited anti-inflammatory activity and protective role in colitis, sepsis, and neuroinflammation.25,26 Equally, methotrexate (MTX) is a kind of folic acid antimetabolic agent, commonly used as an antirheumatic drug, that it is widely employed in the treatment of various malignant tumors like osteosarcoma, which is the most common primary cancer of bone in children and the third most common cancer overall in adolescents, and it is also mostly used in autoimmune diseases such as rheumatoid arthritis, erythematosus, systemic lupus, and dermatomyositis.27 However, because of their poor solubility in aqueous media and low bioavailability, they have been restricted to a limited clinical application. Therefore, it is of great significance to find a suitable drug carrier to improve the bioavailability of these anticancer drugs. To enhance drug effects and reduce adverse drug reactions, some researchers combined the original drugs and drug nanocarriers to form the nanoparticle drug delivery system, including nanoparticles, nanospheres, nanomicelles, and nanoliposomes.28−31 More importantly, nanoparticles with a specific lateral size (50−200 nm) have an enhanced permeability and retention (EPR) effect on tumor tissues, which can infiltrate into and gather inside the tumor tissue for a better treatment effect.32 Since Dai et al. initially developed GO-PEG as a nanocarrier to load the anticancer drug of SN38,12 many other works using GO-PEG for drug delivery of diverse anticancer drugs have been reported.24,33−39 In striking contrast, none has reported the delivery of oridonin and MTX using GO-PEG as a nanocarrier. There are still lots of challenges in employing GO-PEG as a drug delivery platform. Herein, we proposed a drug delivery platform based on GO-PEG10K‑6arm (Scheme 1) to enhance the utilization rate of oridonin and MTX. GO was first prepared via modified Hummer’s method followed by ultrasonication to give GO with the certain size;16 next, 6-armed PEG (Mn = 10 000 g/mol) was attached onto the surface of GO sheets for

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EXPERIMENTAL SECTION

Preparation of GO-PEG10K‑6arm. GO was prepared according to the modified Hummer’s approach40 using graphite powder as the starting material. Graphite powder was oxidized by concentrated H2SO4, NaNO3, and KMnO4 at 0 and 35 °C each for 3 h followed by addition of H2O2 (30%) slowly while the color of the solution changed from black to yellow. The resulting suspension was extensively washed with distilled water by filtration and finally subjected to dialysis for removing residual salts and acids. The obtained GO aqueous suspension was then subjected to bath ultrasonication via a SONICS VCX750 instrument at 20 KHz for 1 h in an ice bath so as to provide the nanosized GO.41 The as-prepared nanosized GO was dispersed in double-distilled water followed by treatment with NaOH, and 6-armed PEG-NH2 and EDC·HCl were added to the suspension for sonication at ambient temperature for 0.5 h. The solution was stirred violently at ambient temperature for at least 12 h. The unreacted 6-armed PEG-NH2 was removed through dialysis (MWcutoff = 14 kDa) against ultrapure water for 7 days so as to afford the desired GO-PEG10K‑6arm nanocarrier. Preparation of GO-PEG10K‑6arm-FITC. GO-PEG10K‑6arm and FITC were added to double-distilled water, and the mixture was stirred violently in dark at ambient temperature for 1 day. The mixture was 22916

DOI: 10.1021/acsami.9b03983 ACS Appl. Mater. Interfaces 2019, 11, 22915−22924

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ACS Applied Materials & Interfaces Scheme 2. Preparation of GO-PEG10K‑6arm

Figure 2. TGA (in N2) profiles of GO, PEG10K‑6arm, and GOPEG10K‑6arm with a heating rate of 10 °C/min. mixture was stirred violently in dark at ambient temperature for 1 day. FITC was covalently bound to oridonin@GO-PEG10K‑6arm (or MTX@GO-PEG10K‑6arm) via the reaction between the end amino group of PEG and the SCN moiety of FITC. The mixture was purified by dialysis (MWcutoff = 3.5 kDa) against ultrapure water in dark for 7 days to remove free FITC.33 Cell Culture. Human renal epithelial 293 T, human tongue squamous carcinoma CAL27, human osteosarcoma MG63, and human liver tumor HepG2 cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. They were cultured in DMEM at 37 °C under a humid atmosphere (5% CO2) supplemented with 10% fetal bovine serum and 1% penicillin−streptomycin. Cellular Uptake of GO-PEG10K‑6arm-FITC. Human renal epithelial 293T cells were placed on a 20 mm glass round coverslip in six-well plates and allowed to adhere overnight. The cells were incubated with GO-PEG10K‑6arm-FITC for 3 h and washed with phosphate buffered saline (PBS) several times. The cells were then fixed in 4% paraformaldehyde (PFA) at 4 °C for 0.5 h and stained with 4′,6-diamidino2-phenylindole (DAPI) (2 μg/mL) under standard conditions (37 °C for 10 min). The stained cells were imaged under an inverted Nikon Ti−S fluorescence microscope.33

Figure 1. (A) AFM image and lateral dimension statistics of nanosized GO; (B) XRD patterns and (C) Raman spectra of graphite and GO. purified by dialysis (MWcutoff = 3.5 kDa) against ultrapure water in dark for 7 days to remove free FITC.33 Preparation of Oridonin@GO-PEG10K‑6arm and MTX@GOPEG10K‑6arm. Oridonin was dissolved in ethanol and mixed with GO-PEG10K‑6arm aqueous dispersion. The solution was stirred violently in dark at ambient temperature for 1 day. The mixture was purified by dialysis (MWcutoff = 3.5 kDa) against ultrapure water in dark for 7 days to remove the unabsorbed oridonin.33 MTX was dissolved in tetrahydrofuran (THF) and mixed with GO-PEG10K‑6arm aqueous dispersion. The solution was stirred violently in dark at ambient temperature for 1 day. The mixture was purified by dialysis (MWcutoff = 3.5 kDa) against ultrapure water in dark for 7 days to remove the unabsorbed MTX.33 Preparation of Oridonin@GO-PEG10K‑6arm-FITC and MTX@ GO-PEG10K‑6arm-FITC. Oridonin@GO-PEG10K‑6arm (or MTX@GOPEG10K‑6arm) and FITC were added to double-distilled water, and the 22917

DOI: 10.1021/acsami.9b03983 ACS Appl. Mater. Interfaces 2019, 11, 22915−22924

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Figure 3. (A) In vitro cytotoxicity of GO-PEG10K‑6arm at different concentrations of 293T cells. In vitro cytotoxicity of GO-PEG10K‑6arm and oridonin@GO-PEG10K‑6arm at different concentrations of (B) HepG2 cells, (C) CAL27 cells, and (D) MG63 cells. Cellular Uptake of Oridonin@GO-PEG10K‑6arm-FITC and MTX@GO-PEG10K‑6arm-FITC. Human liver tumor HepG2 cells were placed on a 20 mm glass round coverslip in six-well plates and allowed to adhere overnight. The cells were incubated with oridonin@GOPEG10K‑6arm-FITC for 3 h and washed with PBS several times. The cells were then fixed in 4% (PFA) at 4 °C for 0.5 h and stained with DAPI (2 μg/mL) under standard conditions (37 °C for 10 min). The stained cells were imaged under an inverted Nikon Ti−S fluorescence microscope.33 Human tongue squamous carcinoma CAL27 cells were placed on a 20 mm glass round coverslip in six-well plates and allowed to adhere overnight. The cells were incubated with MTX@GO-PEG10K‑6armFITC for 3 h and washed with PBS several times. The cells were then fixed in 4% PFA at 4 °C for 0.5 h and stained with DAPI (2 μg/mL) under standard conditions (37 °C for 10 min). The stained cells were imaged under an inverted Nikon Ti−S fluorescence microscope.33 In Vitro Cell Viability Assay. Human renal epithelial 293 T cells were placed in 96-well plates at a density of 5 × 103 cells per well in 100 μL of culture medium and injected with required concentrations of GO-PEG10K‑6arm. Human tongue squamous carcinoma CAL27, human osteosarcoma MG63, and human liver tumor cells HepG2 cells were plated in 96-well plates at a density of 5 × 103 cells per well in 100 μL of culture medium and added with desired concentrations of GO-PEG10K‑6arm, oridonin@GO-PEG10K‑6arm, and free oridonin (dissolved in dimethyl sulfoxide (DMSO) and diluted in PBS). Human tongue squamous carcinoma CAL27 cells were placed in 96-well plates at a density of 5 × 103 cells per well in 100 μL of culture medium and injected with required concentrations of MTX@ GO-PEG10K‑6arm and free MTX (dissolved in DMSO and diluted in PBS). The relative cell viability was determined by WST assay using CCK-8. After successive incubation for 12, 24, 36, 48, 60, and 72 h,

absorbance was measured at 450 nm using a BioTek Synergy H1 microplate reader.33 Statistical Analysis. The data were expressed as mean ± standard deviation (SD). Variables were compared by independent samples Student’s t test. Values of p < 0.05 were considered to be statistically significant.

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RESULTS AND DISCUSSION

Preparation and Characterization of GO-PEG10K‑6arm. On the basis of our previous work,33,44 we prepared a nanoscale drug carrier in the present work, GO-PEG10K‑6arm, for enhancing the utilization rate of water-insoluble drugs like oridonin and MTX in cancer therapy. Indeed, it is clear that GO-PEG10K‑6arm can be employed as a nanosized vehicle for the delivery of antitumor drugs because it has been verified to be not distinctly toxic in vitro and in vivo at the tested doses.8,40−45 The optimal size of nanosized carrier has been demonstrated to be in the range of 50−200 nm due to the EPR effect;46−49 therefore, GO-PEG10K‑6arm with a size between 50 and 200 nm could readily enter cells, which can be confirmed via intracellular imaging utilizing FITC as a probe.33,43,50 We prepared GO-PEG10K‑6arm by a two-step method at ambient temperature (Scheme 2). First, based on the modified Hummer’s method, we obtained GO possessing plenty of oxygen-containing functionalities such as carboxyls at the edge and hydroxyls and epoxy groups on the basal plane.44 The size of GO was regulated by sonication for meeting the demand of the EPR effect.47 The morphology of GO with a 22918

DOI: 10.1021/acsami.9b03983 ACS Appl. Mater. Interfaces 2019, 11, 22915−22924

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ACS Applied Materials & Interfaces specific size was observed via atomic force microscopy (AFM) (Figure 1A), and it can be found that the majority is loaded on the range of 120 ± 30 nm and the thickness is nearly 1.0 nm. The size of GO was also examined by dynamic light scattering (DLS), which revealed a mean hydrodynamic diameter of ∼140 nm (Figure S1), consistent with the result obtained from AFM. Furthermore, the chemical structure of nano-GO was characterized by X-ray diffraction (XRD) and Raman spectroscopy. XRD results show the increase of the interlayer spacing of GO (Figure 1B), and the D/G intensity ratio in the Raman spectrum also increased (Figure 1C), which were both consistent with the previously reported characteristics of GO.4,6,10,12 These data clearly illustrated that the chemical structure of nano-GO was not destroyed during the ultrasonication process. Second, 6-armed PEG-NH2 (Mn = 10 000 g/mol) was linked to GO via amidation reaction for better biocompatibility.44 The results of AFM and DLS demonstrated that the size of GO-PEG10K‑6arm is about 200 nm (Figure S2), which still met the demand of EPR. Thermogravimetric analysis (TGA) data could reveal the ratio of grafted PEG and thermal stability of GO-PEG10K‑6arm. It can be seen in Figure 2 that GO is not thermally stable and the mass loss of GO is obviously accelerated in the temperature range of 150−200 °C due to the pyrolysis of labile oxygen-containing groups.33 Meanwhile, the significant acceleration of the mass loss of GO-PEG10K‑6arm began from 300 °C, almost 150 °C higher than that of GO, accompanied with a slow mass loss around 180 °C similar to that of GO. This fact clearly showed that polymeric coatings on the surface of GO may be useful to enhance the thermostability of GO sheets.44 Besides, we can find that there is no obvious weight loss at 600 °C in TGA curves of GO, PEG10K‑6arm, and GO-PEG10K‑6arm, which meant that 600 °C is a suitable reference point for the estimation of percentage of grafted PEG. Differential thermal gravity (DTG) curves of GO, PEG10K‑6arm, and GO-PEG10K‑6arm are also presented in Figure S3. There are two apparent weight loss peaks in the DTG curve of GO-PEG10K‑6arm; one is located at ∼175 °C, which is similar to the DTG curve of GO, and the other one is located at ∼365 °C, which is similar to the DTG curve of PEG10K‑6arm. This phenomenon indicated that GO-PEG10K‑6arm contained both PEG10K‑6arm and GO. Additionally, there is no signal weight loss peak at ∼600 °C in DTG curves of GO, PEG10K‑6arm, and GO-PEG10K‑6arm. GO-PEG10K‑6arm (red line) was found to have a 75.8% weight loss at 600 °C in N2, whereas the corresponding weight losses for GO (blue line) and 6-armed PEG-NH2 (olive line) were 50.7 and 97.9%. Therefore, we can estimate the weight content of PEG via the following equation set (x and y are the weight contents of GO and PEG, respectively), and the result is that GO-PEG contains ∼53.2 wt % PEG and ∼46.8 wt % GO.33 Cellular Assay and Uptake of GO-PEG10K‑6arm. As for a drug nanocarrier, GO-PEG10K‑6arm should be hypotoxic to and compatible with cells. The cellular cytotoxicity of GO-PEG10K‑6arm was then investigated by incubating human renal epithelial 293T, human tongue squamous carcinoma CAL27, human osteosarcoma MG63, and human liver tumor HepG2 cells.39 We employed a WST-8-based colorimetric assay to check the cytotoxicity of the nanocarrier on 293T, CAL27, MG63, and HepG2 cells for confirming the toxicity of GO-PEG10K‑6arm to these cells. As shown in Figure 3, high cell viabilities (>85%) were obtained for 293T, CAL27, MG63, and HepG2 cells with different concentrations of GO-PEG10K‑6arm in the range of

Figure 4. (A) FITC, (B) DAPI, and (C) DIC images of 293T cells treated with GO-PEG10K‑6arm-FITC (scale bar = 200 μm).

0−100 mg/L, even after incubating for 3 days at a concentration as high as 100 mg/L. These data vividly evidenced that GO-PEG10K‑6arm is less toxic to these human cells, especially for normal 293T cells, i.e., GO-PEG10K‑6arm nanocarrier is not cytotoxic by itself. Moreover, as shown in Figure 3B−D, while the content of GO-PEG10K‑6arm in oridonin@GO-PEG10K‑6arm was about 50 mg/L, the relative cell viabilities of CAL27, MG63, and HepG2 cells were only as low as 1.5%, which meant that the oridonin@GO-PEG10K‑6arm drug delivery system has a strong lethal effect on tumor cells followed by GO-PEG10K‑6arm loaded with free oridonin. These results clearly indicated that GO-PEG10K‑6arm with low toxicity would be an efficient carrier for hydrophobic anticancer drugs like oridonin. The cellular uptake of GO-PEG10K‑6arm was examined by incubating 293T cells with FITC as a probe. We could 22919

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Figure 5. (A) UV/vis absorption spectra of GO-PEG10K‑6arm and oridonin@GO-PEG10K‑6arm, and (B) hydrodynamic diameter distribution and (C) AFM image of oridonin@GO-PEG10K‑6arm.

obviously notice the fluorescence of FITC in 293T cells after loading GO-PEG10K‑6arm-FITC (Figure 4A). Compared to the nuclei staining of DAPI (Figure 4B) and DIC (Figure 4C) images, the localization of FITC approximately matched with both of them, which definitely suggested that GO-PEG10K‑6arm could cross the membrane of cells to enter 293T cells.51,52 Finally, all of these data confirmed that GO-PEG10K‑6arm could penetrate the cells effectively and only cause a little injury to normal cells like 293T. Preparation and Characterization of Oridonin@GOPEG10K‑6arm and MTX@GO-PEG10K‑6arm. Based on previous reports, the π−π stacking interaction with drugs was produced from the large π-conjugated structure of GO to extend its application as a potential drug carrier,43,44,49,50,53 and PEGylated graphene sheets possessed appealing in vivo properties, such as excellent passive tumor targeting and comparatively low retention in reticuloendothelial systems.39 Therefore, two hydrophobic anticancer drugs of oridonin and MTX were loaded onto GO-PEG10K‑6arm to improve their bioavailability. Oridonin@GO-PEG10K‑6arm and MTX@GO-PEG10K‑6arm were prepared by mixing oridonin dissolved in ethanol (MTX dissolved in THF) with GO-PEG10K‑6arm aqueous suspension immediately. The free hydrophobic drug was discarded by dialysis in double-distilled water. The effective loading of oridonin on the nanocarrier was confirmed by the strong absorption peak located at 237 nm (derived from oridonin)15 in the UV/vis absorption spectrum of oridonin@GO-PEG10K‑6arm (blue line in Figure 5A), which was weak in GO-PEG10K‑6arm (red line in Figure 5A). Deducting the absorbance of the nanocarrier at 237 nm, the absorbance of the loaded oridonin in oridonin@GO-PEG10K‑6arm could be obtained. The load capacity (the weight content of loaded oridonin in oridonin@GOPEG10K‑6arm) was evaluated to be about 10% via the standard absorption of oridonin at 237 nm (Figure S4) in succession. The load capacity of MTX@GO-PEG10K‑6arm (about 5% from

the standard absorption of MTX at 302 nm in Figure S5) was also evaluated in the similar way (Figure S6).44 The micromorphologies of oridonin@GO-PEG10K‑6arm and MTX@GOPEG10K‑6arm were measured by AFM and DLS as shown in Figures 5 and S6, respectively. The size of oridonin@GOPEG10K‑6arm approached 250 nm according to the DLS data (Figure 5B), which was consistent with the lateral dimension of oridonin@GO-PEG10K‑6arm in the AFM image (Figure 5C). This size certainly could help the biomedical use of oridonin@ GO-PEG10K‑6arm for drug delivery because of the prominent EPR effect. The determined thickness of oridonin@GOPEG10K‑6arm via AFM kept at 1.0−1.5 nm accompanied by a smooth surface (Figure 5C), in accordance with a recent report.21 Similarly, the micromorphology of MTX@GOPEG10K‑6arm was roughly semblable with that of oridonin@ GO-PEG10K‑6arm (Figure S6); specifically, the lateral size of MTX@GO-PEG10K‑6arm was about 280 nm and the thickness was also 1.5−2.0 nm. Cell Uptake of Oridonin@GO-PEG10K‑6arm and MTX@ GO-PEG10K‑6arm. To affirm whether oridonin@GO-PEG10K‑6arm and MTX@GO-PEG10K‑6arm could readily enter tumor cells, FITC was employed as a fluorescent probe for intracellular imaging as shown in Figure 6A.44 Compared to the staining of the nuclei by DAPI (Figure 6B), the localization of FITC was approximately consistent with the DAPI image. This fact definitely illustrated the cell uptake of the drug delivery system and the arrival into those cells. Therefore, we chose HepG2 and CAL27 cells for the cellular uptake of oridonin@GOPEG10K‑6arm and MTX@GO-PEG10K‑6arm, respectively. We could distinctly see the fluorescence of FITC and DAPI in HepG2 cells only after incubating for 3 h, and the fluorescence spots in the images of DAPI and FITC were consistent with the sites in the DIC image (Figure 6C). All of these images confirmed that oridonin@GO-PEG10K‑6arm could enter HepG2 tumor cells smoothly. Correspondingly, we could observe the 22920

DOI: 10.1021/acsami.9b03983 ACS Appl. Mater. Interfaces 2019, 11, 22915−22924

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Figure 7. In vitro cytotoxicity of free oridonin and oridonin@GOPEG10K‑6arm at different concentrations after cultivation with (A) CAL27, (B) MG63, and (C) HepG2 cells for 72 h. Data are mean ± SD (n = 3).

For all of the three cells, when the concentration of oridonin was up to 20 μM, the cytotoxicity effect of oridonin@GO-PEG10K‑6arm was similar and the cell viabilities of all of the three cells were below 5%. Nevertheless, oridonin@GO-PEG10K‑6arm clearly presented a stronger cytotoxic effect on CAL27 and MG63 cells in comparison with HepG2 cells, which was resulted from the difference in the mechanisms of oridonin-induced CAL27, MG63, and HepG2 cell apoptosis.54−58 Moreover, oridonin@ GO-PEG10K‑6arm obviously presented a stronger cytotoxic effect on tumor cells in comparison with free oridonin with the increase in the concentration of oridonin. Especially for HepG2 cells, the cell viability was 74.6% for free oridonin and 7.3% for oridonin@GO-PEG10K‑6arm with the same oridonin concentration of 20 μM (Figure 7C). We further investigated the cytotoxicity effect of the MTX@GO-PEG10K‑6arm system and free MTX on CAL27 cells (Figure S7). The cell viability was 17.7% for free MTX and 9.2% for MTX@GO-PEG10K‑6arm with the same MTX concentration of 200 μM, which indicated that MTX@GO-PEG10K‑6arm displayed a stronger cytotoxic effect on CAL27 cells in comparison with free MTX with the

Figure 6. (A) FITC, (B) DAPI, and (C) DIC images of HepG2 cells treated with oridonin@GO-PEG10K‑6arm-FITC (scale bar = 200 μm).

same phenomenon in the DAPI and FITC images of CAL27 for treatment with MTX@GO-PEG10K‑6arm-FITC (Figure S7). Thus, we could verify the cellular uptake of oridonin@GOPEG10K‑6arm and MTX@GO-PEG10K‑6arm by HepG2 and CAL27 cells, respectively. In Vitro Cell Toxicity of Oridonin@GO-PEG10K‑6arm and MTX@GO-PEG10K‑6arm. Herein, a WST-8-based colorimetric assay was used to check the cytotoxicity effect of oridonin@ GO-PEG and free oridonin on CAL27, MG63, and HepG2 cells. CAL27, MG63, and HepG2 cells were initially incubated with free oridonin and oridonin@GO-PEG10K‑6arm for 3 days with various concentrations of oridonin (0−24 μM). CAL27, MG63, and HepG2 cells were all inhibited by both free oridonin and oridonin@GO-PEG10K‑6arm in a time-dependent manner, and the relative cell viability significantly decreased with the increase of the concentration of oridonin (Figure 7). 22921

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cell viability was 7.3% after 72 h. These results indicated that GO-PEG10K‑6arm could load oridonin to achieve a more effective treatment in comparison with free oridonin for enhancing drug efficiency and confirmed that oridonin@GO-PEG10K‑6arm was more harmful to CAL27 and MG63 cells than to HepG2 cells.

same MTX concentration. Thus, we can conclude from these data that the lethal effect of the original drug was enhanced after being loaded onto GO-PEG10K‑6arm. In addition, the cytotoxicities of free oridonin and oridonin@ GO-PEG10K‑6arm with the same oridonin concentration (20 μM) at different times (12−72 h) were also examined (Figure 8).

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CONCLUSIONS In summary, we prepared PEGylated GO, GO-PEG10K‑6arm, with excellent biocompatibility and physiological stability under mild conditions, which contained 53.2 wt % PEG10K‑6arm and 46.8 wt % GO. GO-PEG10K‑6arm can be applied as a nanocarrier to deliver water-insoluble anticancer drugs such as oridonin and MTX via π−π stacking and hydrophobic interactions. Fluorescence observations demonstrated that GO-PEG10K‑6arm-FITC can quickly enter 293 T cells. GO-PEG10K‑6arm nanocarrier was verified to be nearly nontoxic to human cells, especially to human renal epithelial 293 T cells. Oridonin@GO-PEG10K‑6arm and MTX@GO-PEG10K‑6arm were found to be potent against tumor cells, killing these in vitro as measured by the WST-8 assay. It is clear that oridonin@GO-PEG10K‑6arm inhibited CAL27, MG63, and HepG2 cells in a concentration- and timedependent mode, and it showed a stronger cytotoxicity effect in comparison with free oridonin for improving the bioavailability of oridonin, in addition MTX@GO-PEG10K‑6arm showed enhanced lethal effect in CAL27 cells compared to free MTX. Based on the above evidence, GO-PEG10K‑6arm could effectively improve the solubility in aqueous media and enhance the bioavailability of hydrophobic antitumor drugs so that it could be a favorable and universal nanomaterial in biological and nanomedical fields.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b03983. Materials; measurements; DLS of GO, GO-PEG10K‑6arm, and MTX@GO-PEG10K‑6arm; AFM images of GOPEG10K‑6arm and MTX@GO-PEG10K‑6arm; DTG curves of GO, 6-armed PEG-NH2, and GO-PEG10K‑6arm; UV/ vis absorption spectra of oridonin, MTX, and MTX@ GO-PEG10K‑6arm; DAPI and FITC images of CAL27 cells incubated with MTX@GO-PEG10K‑6arm-FITC; and relative cell viability of CAL27 cells after treatment with free MTX and MTX@GO-PEG10K‑6arm (PDF)

Figure 8. In vitro cytotoxicity of free oridonin and oridonin@GOPEG10K‑6arm with (A) CAL27, (B) MG63, and (C) HepG2 cells at a constant oridonin concentration of 20 μM for different times. Data are mean ± SD (n = 3).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86-21-64321673. Fax: +86-21-64322272 (F.Z.). *E-mail: [email protected]. Tel: +86-21-54925310. Fax: +86-21-64166128 (X.H.). *E-mail: [email protected]. Tel: +86-21-23271699. Fax: +86-21-63136856 (H.J.).

Both free oridonin and oridonin@GO-PEG10K‑6arm inhibited tumor cells in a time-dependent manner. For each interval, oridonin@GO-PEG10K‑6arm obviously presented a stronger effect on tumor cells in comparison with free oridonin. The enhanced cytotoxicity was originated from the increased cellular uptake of oridonin loaded on GO-PEG10K‑6arm. For instance, cell viabilities of CAL27 (Figure 8A) and MG63 (Figure 8B) cells after incubating with oridonin@GOPEG10K‑6arm for 60 h were both as low as 2%, whereas for those incubated with free oridonin for 60 h were both about 30%; the cell viability of HepG2 cells (Figure 8C) after incubating with free oridonin for 72 h was 74.6%, whereas oridonin@GO-PEG10K‑6arm only took 24 h to be 40.0% and the

ORCID

Fang Zhang: 0000-0002-9656-8893 Xiaoyu Huang: 0000-0002-9781-972X Author Contributions ∥

D.C. and B.H. contributed equally to this work.

Notes

The authors declare no competing financial interest. 22922

DOI: 10.1021/acsami.9b03983 ACS Appl. Mater. Interfaces 2019, 11, 22915−22924

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ACS Applied Materials & Interfaces

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Basic Research Program of China (2015CB931900), the National Key Research & Development Program of China (2016YFA0202900), the National Natural Science Foundation of China (81571028, 21677098, and 21632009), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB20000000), the Shanghai Scientific and Technological Innovation Project (16XD1401800, 17DZ1205402, 17DZ1205403, 18JC1410600, 18JC1415500, and 19ZR1476500), the Program of Shanghai Jiao Tong University School of Medicine (TM201715), and the Discipline Construction Fund of Shanghai Municipal Commission of Health and Family Planning (2016ZB0203-01).

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