Modification of graphene oxide by angiopep-2 enhances anti-glioma efficiency of the nanoscaled delivery system for doxorubicin

Characterization of ANG-Dox-GO

The color of commercial GO varies from yellow to brown (Figure 1A). Under a AFM, ANG-Dox-GO was 4-5 nm in thickness and 100-400 nm in diameter (Figure 1B), suggesting the formation of single or several layers of GO nanosheets. In addition, the optical absorption spectra of ANG-Dox-GO showed absorption peaks at approximately 230 nm (Figure 1C).

Figure 1. Characterization of angiopep-2 polypeptide-modified and doxorubicin-loaded graphene oxide (ANG-Dox-GO). (A) The appearance of commercial GO. (B) The representative images of ANG-Dox-GO using atomic force microscopy. (C) The ultraviolet visible absorption spectra of ANG-Dox-GO.

Cellular uptake of ANG-Dox-GO

Based on confocal imaging analysis, increased fluorescence intensity was detected in cells treated with ANG-Dox-GO, Dox-GO as compared with free Dox, and the strongest fluorescence intensity was found in cells treated with ANG-Dox-GO (Figure 2). These results indicated that the modification by angiopep-2 increased the cellular uptake of the delivery system. Moreover, the cellular uptake at 1 h of incubation time was higher than that at 0.5 h (Figure 2).

Figure 2. Cellular uptake of Dox, doxorubicin-loaded graphene oxide (Dox-GO) or angiopep-2 polypeptide-modified and doxorubicin-loaded graphene oxide (ANG-Dox-GO) by U87 MG cells. Confocal images of U87 MG cells treated with Dox, Dox-GO or ANG-Dox-GO at 10 μg/mL of Dox for 0.5 and 1 h.

Anti-proliferation effect of ANG-Dox-GO on U87 MG cells

Cell proliferation and apoptosis was utilized as the indicators to evaluate the antitumor effect of ANG-Dox-GO on U87 MG cells. Firstly, the cell viability was evaluated using MTT assay, and the results showed that compared with control cells, GO treatment exhibited few effects on cell viability in U87 MG cells; however, cell viability was significantly inhibited by Dox treatment (p < 0.05, Figure 3A) at 24 h and 48 h. In addition, compared with Dox treatment, U87 MG cells treated with Dox-GO exhibited lower cell viability, and ANG-Dox-GO treatment further inhibited cell viability (p < 0.05, Figure 3A). Similarly, colony formation assay also revealed that U87 MG cells treated with PBS had similar clone number compared with GO treatment, whereas the clone number was obviously reduced after free Dox treatment (p < 0.05, Figure 3B). Compared with cells treated with free Dox, the clone number was remarkably decreased in cells with Dox-GO, and cells treated with ANG-Dox-GO had reduced clone number compared with Dox-GO treatment (p < 0.05, Figure 3B).

Figure 3. Angiopep-2 polypeptide-modified and doxorubicin-loaded graphene oxide (ANG-Dox-GO) inhibits tumor growth in U87 MG cells. (A) Cell viability of U87 MG cells treated with PBS (control), GO, Dox (30 μg/mL), Dox-GO (containing 30 μg/mL of Dox), and ANG-Dox-GO (containing 30 μg/mL of Dox), respectively, for 24 h and 48 h by MTT assay. (B) Clone number of U87 MG cells treated with PBS (control), GO, Dox (30 μg/mL), Dox-GO (containing 30 μg/mL of Dox), and ANG-Dox-GO (containing 30 μg/mL of Dox), respectively, for 24 h by colony formation assay. *P < 0.05 vs control cells; #P < 0.05 vs Dox; &P < 0.05 vs. Dox-GO.

Pro-apoptosis effect of ANG-Dox-GO on U87 MG cells

Flow cytometry analysis revealed that the apoptotic rate (AR) of cells treated with free Dox was significantly increased as compared with control cells or cells treated with GO; meanwhile, compared with U87 MG cells treated with free Dox, cells treated with Dox-GO had increased AR, and the AR was further decreased in cells treated with ANG-Dox-GO (p < 0.05, Figure 4A). Meanwhile, the expression of apoptosis-related proteins, including Bax, Bid, Bim, Bcl-2, cleaved caspase-3 and cleaved caspase-9, was detected using western blotting analysis. The results indicated that compared with control cells or cells treated with GO, cells treated with free Dox had remarkably increased protein levels of Bax, Bid, Bim, cleaved caspase-3, and cleaved caspase-9, declined protein level of Bcl-2 (p < 0.05, Figure 4B). Moreover, when compared to cells treated with free Dox, cells treated with Dox-GO or ANG-Dox-GO had higher protein levels of Bax, Bid, Bim, cleaved caspase-3 and cleaved caspase-9 and declined protein level of Bcl-2, especially the cells treated with ANG-Dox-GO (p < 0.05, Figure 4B).

Figure 4. Angiopep-2 polypeptide-modified and doxorubicin-loaded graphene oxide (ANG-Dox-GO) induces cell apoptosis in U87 MG cells. (A) Cell apoptosis rate of U87 MG cells treated with PBS (control), GO, Dox (30 μg/mL), Dox-GO (containing 30 μg/mL of Dox), and ANG-Dox-GO (containing 30 μg/mL of Dox), respectively, for 24 h by flow cytometry analysis. (B) The expression of apoptosis-related proteins, including Bax, Bid, Bim, cleaved caspase-3, and cleaved caspase-9, in U87 MG cells treated with PBS (control), GO, Dox (30 μg/mL), Dox-GO (containing 30 μg/mL of Dox), and ANG-Dox-GO (containing 30 μg/mL of Dox), respectively, for 24 h by western blotting. *P < 0.05 vs control cells; #P < 0.05 vs Dox; &P < 0.05 vs. Dox-GO.

Anti-migration effect of ANG-Dox-GO on U87 MG cells

Wound healing assay showed that GO exhibited no effect on wound healing in U87 MG cells compared with control cells, while free Dox significantly inhibited the wound healing rate of U87 MG cells in time-dependent manner (p < 0.05, Figure 5A). In addition, compared with free Dox treatment, Dox-GO treatment was associated with decreased wound healing rate, and ANG-Dox-GO treatment further decreased the wound healing rate (p < 0.05, Figure 5A). Similarly, Transwell assay also revealed that cell migration and invasion were dramatically inhibited in U87 MG cells treated with free Dox compared with control cells or cells treated with GO (p < 0.05, Figure 3B). The rates of cell migration and invasion were remarkably decreased in cells treated with Dox-GO as compared with cells treated with free Dox, and cells treated with ANG-Dox-GO showed lower rates of cell migration and invasion than those treated with Dox-GO (p < 0.05, Figure 5B).

Figure 5. Angiopep-2 polypeptide-modified and doxorubicin-loaded graphene oxide (ANG-Dox-GO) inhibits metastasis in U87 MG cells. (A) The wound healing rate of U87 MG cells treated with PBS (control), GO, Dox (30 μg/mL), Dox-GO (containing 30 μg/mL of Dox), and ANG-Dox-GO (containing 30 μg/mL of Dox), respectively, at 0, 10, 12 h and 15 h by wound healing assay. (B) Cell migration and migration rates in U87 MG cells treated with PBS (control), GO, Dox (30 μg/mL), Dox-GO (containing 30 μg/mL of Dox), and ANG-Dox-GO (containing 30 μg/mL of Dox), respectively, by Transwell assay. *P < 0.05 vs control cells; #P < 0.05 vs Dox; &P < 0.05 vs. Dox-GO.

Effect of ANG-Dox-GO on glioma xenograft in nude mice

In vivo experiments showed that tumor size of mice xenografted with human glioma cells was decreased by Dox treatment compared with that of the control group in a time-dependent manner, and the tumor size was further reduced in Dox-GO and ANG-Dox-GO treated groups, especially in ANG-Dox-GO group (p < 0.05, Figure 6A). Moreover, compared to control groups, tumor weight of mice was the lowest in the ANG-Dox-GO group, followed by Dox-GO and Dox treated groups (p < 0.05, Figure 6B), indicating superior anti-tumor effect of ANG-Dox-GO on glioma xenograft in nude mice.

Figure 6. Angiopep-2 polypeptide-modified and doxorubicin-loaded graphene oxide (ANG-Dox-GO) exhibited better anti-tumor effect of glioma xenograft mice. (A) The tumor volumes of mice with different treatments, including PBS (control), Dox (2 mg/kg), Dox-GO (containing 2 mg/kg of Dox), and ANG-Dox-GO (containing 2 mg/kg of Dox), respectively, every two days for 20 days. (B) The tumor weights of mice with different treatments, including PBS (control), Dox (2 mg/kg), Dox-GO (containing 2 mg/kg of Dox), and ANG-Dox-GO (containing 2 mg/kg of Dox), respectively, on 20 days. *P < 0.05.

Preparation and characterization of ANG-Dox-GO

GO was purchased from Jiangsu XFNANO Materials Tech Co. Ltd (Nanjing, Jiangsu, China). In brief, 3 mg of GO was added into an Erlenmeyer flask containing 20 mL of Dox solution (40 μg/L), and then the flask were shaken at 130 rpm for 160 min in 30°C water bath. Afterwards, the mixture was adjusted to a corresponding pH value with 0.01-1.0 mol/L of hydrochloric acid and sodium hydroxide solution. Next, the supernatant was obtained using a 0.45 μm aqueous phase filter. Subsequently, Dox-GO was modified by angiopep-2 through covalent bonding of amino and carboxyl groups to obtain angiopep-2 polypeptide-modified GO drug delivery system (ANG-Dox-GO).

The ultraviolet (UV) visible spectra of ANG-Dox-GO was measured by UV visible absorption spectroscopy (Biochrom, Cambridge, UK). The morphology and thickness of ANG-Dox-GO were evaluated by atomic force microscopy (AFM, JPK Instruments AG, Berlin, Germany).

Cell culture and treatment

Human glioma cell line U87 MG was obtained from Shanghai Obio Technology Co., Ltd., and incubated in complete DMEM medium (Gibco, Carlsbad, CA, USA) at 37°C and in a 5% CO₂ incubator. To evaluate the anti-tumor effects of ANG-Dox-GO on U87 MG cells, the cells were exposed to PBS (control), GO, Dox (30 μg/mL), Dox-GO (containing 30 μg/mL of Dox) and ANG-Dox-GO (containing 30 μg/mL of Dox), respectively.

Observation of cell uptake

Cellular uptake of ANG-Dox-GO was evaluated using confocal imaging analysis. Briefly, U87 MG cells were seeded at the density of 2.5 × 10⁴ per well to 24-well plates for 24 h and incubated in the presence of Dox (10 μg/mL), Dox-GO (containing 10 μg/mL of Dox) and ANG-Dox-GO (containing 10 μg/mL of Dox) for 0.5h and 1 h, respectively. After being rinsed with PBS, the cells were observed using confocal fluorescence microscope with an argon-ion laser (488 nm).

MTT assay

U87 MG cells were grown on 96-well plates, and received the above treatments. After conventional incubation for 24 and 48 h, each well was added with 10 μl of MTT (5 mg/mL, Sigma) to incubate for another 4 h. And 100 μL of dimethyl sulfoxide was added. Finally, cell viability was evaluated using a microplate spectrophotometer to measure absorbance at 470 nm.

Colony-forming assay

U87 MG cells were seeded to 6-well plates at a density of 400 cells per well, and treated in the same way as described above for 14 days under standard culture conditions. Then the cells were fixed with absolute methanol, and incubated with crystal violet. Ultimately, the number of colonies was counted.

Cell apoptosis assay

FITC-Annexin V Apoptosis kit was used. U87 MG cells received the above treatments for 24 h. Trypsin was added to digest U87 MG cells, and then the cells were harvested. Next, cells were rinsed with PBS and re-suspended in binding buffer. After the cells were incubated in the presence of FITC-Annexin V and PI for 15 min, the number of apoptotic cells was calculated using a flow cytometer (BD, CA, USA).

Wound healing assay

U87 MG cells were inoculated in 6-well plates. After growing to 60% confluence, cells were wounded by scratching with sterile plastic pipette tips vertically against the well, followed by incubation only in DMEM without serum. Subsequently, cells were treated with PBS (control), GO, Dox (30 μg/mL), Dox-GO (containing 30 μg/mL of Dox) and ANG-Dox-GO (containing 30 μg/mL of Dox), respectively. The distance of the scratch was measured at 0, 10, 12 and 15 h using a light microscope (Olympus, Japan).

Transwell assay

Tumor cell invasion and migration were elevated by Transwell chambers (Corning). To be specific, the lower compartment was filled with DMEM supplemented with 10% FBS. The U87 MG cells with different treatments were cultured in the upper compartment coated with Matrigel Matrix in medium free of serum for 24 h. Subsequently, the cells in the lower compartment was fixed and stained with 4,6-diamidino-2-phenylindole. And cell migration and invasion were evaluated using an inverted microscope (Olympus, Japan).

Western blotting analysis

U87 MG cells were treated for 24 h with PBS (control), GO, Dox (30 μg/mL), Dox-GO (containing 30 μg/mL of Dox) and ANG-Dox-GO (containing 30 μg/mL of Dox), respectively. The harvested cells were lysed by RIPA lysis buffer (Gibco), and then proteins were extracted with a commercial kit (Pierce, Rockford, IL, USA). The extracted protein was resolved on PAGE gel, and the protein sample was transferred to PVDF membrane, which was blocked and reacted with Bax, Bid, Bim, Bcl-2, cleaved caspase-3, cleaved caspase-9 or β-actin primary antibody (1:300, Santa Cruz) overnight at 4°C, respectively.. After incubation in the presence of the second antibody (1:5000, Jackson, USA), the protein levels were determined using enhanced chemiluminescence (ECL, Millipore, USA).

Animal model and treatments

The experiments were approved by the local Animal Ethics Committee of Cangzhou Central Hospital prior to initiation. Healthy nude mice, weighting 18-22 g, purchased from Charles River, Beijing, China, were used for the following experiments after one week of acclimation. To construct the xenografted mouse model, 1×10⁶ of U87 MG cells were inoculated subcutaneously in each mouse. Then, xenografted mice were treated with PBS (control), Dox (2 mg/kg), Dox-GO (containing 2 mg/kg of Dox) and ANG-Dox-GO (containing 2 mg/kg of Dox) by rapid tail vein injection, respectively. The tumor size of each mouse was monitored every two days for 20 days. On day 20, mice were euthanized, and the tumor was resected for measurement of tumor weight.

Statistical analysis

Data was presented as mean ± SD. Data was compared using one-way ANOVA followed by multiple comparison tests based on SPSS software. P < 0.05 was considered statistically significant.