Photothermal/pH Dual-Responsive Drug Delivery System of Amino-Terminated HBP-Modified rGO and the Chemo-Photothermal Therapy on Tumor Cells
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In this paper, a simple method to prepare hydrophilic reduced graphene oxide (rGO) was proposed via reducing GO by amino-terminated hyperbranched polymer (NHBP), the as-prepared NrGO could present excellent dispersibility, near infrared (NIR) light absorbance, photothermal conversion ability and stability. Then, the doxorubicin hydrochloride (DOX) was conjugated with NrGO to prepare the drug-loading system, and a pH/photothermal dual-responsive drug delivery behavior was characterized. At acidic environment or under NIR laser irradiation, the drug release rate could be improved, which is beneficial to control release anti-tumor drug in tumor tissues. What is more, the in vitro cell experiments revealed that NrGO was well biocompatible, and in the tumor inhibition part, comparing to the control group without any treatment, DOX@NrGO gained efficient chemo-photothermal synergetic therapy, the inhibition rate of which was much higher than single chemotherapy of released DOX. Therefore, the as-prepared DOX@NrGO obtained great potential application in tumor therapy and an excellent candidate in other biomed applications.
KeywordsGraphene oxide Hyperbranched polymer Drug delivery Dual responsive Chemo-photothermal therapy
Confocal laser scanning microscopy
Methyl thiazolyl tetrazolium
Amino-terminated hyperbranched polymer reduced graphene oxide
Reduced graphene oxide
Scanning electron microscopy
Transmission electron microscope
Photothermal therapy (PTT) under near infrared (NIR) irradiation has attracted raising attention for tumor inhibition, due to the little side effect and minimal invasive properties . NIR light (700~1100 nm) penetrate deeper into body tissue without much absorption either any damage to healthy tissue or cells [2, 3]. Thus, under NIR laser irradiation, photothermal agent can raise the temperature in implanted location via its photothermal conversion ability. In addition, the applied photothermal agent requires good biocompatibility, photothermal conversion efficacy, and stability.
For recent years’ researches, variety of materials were designed and prepared to cure tumor tissues as PTT agents, such as precious metal (gold nanorods , gold nanoplates ), semiconductor nanomaterials (CuS , MoS2 , FeS ), organic materials (polydopamine , polypyrrole nanoparticles ), carbon nanomaterials (carbon nanotube , carbon nanoparticles , and graphene ). As a kind of promising carbon nanomaterial, graphene was widely used in tumor inhibition through PTT method due to its special two-dimensional nanosheets, which obtain ultra-high specific surface area and great potential for high drug loading efficiency [14, 15]. However, reduced graphene oxide (rGO) prepared via normal methods including urea and hydrazine hydrate, hydrothermal process always shows highly hydrophobicity, which is not beneficial to the application in water phenomenon of body tissue .
In this case, we proposed a novel idea to use water-soluble polymer with reductive ability to prepare hydrophilic rGO. In our previous work, we synthesized amino-terminated hyperbranched polymer (NHBP) and tried to use it to treat metallic oxide nanoparticles and prepare metal nanospheres, which is highly hydrophilic without obvious agglomeration, such as HBP-modified silver nanoparticles and its application in anti-bacteria field [17, 18].
Graphene oxide (GO, 0.8~1.2 nm in thickness and 0.5~5 μm in width) were supplied by XFNANO Co., Ltd. DOX were purchased from HuaFeng United Technology Co., Ltd. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin, penicillin (100 U/ml) and streptomycin (100 μg/ml) were all purchased from Thermo Fisher Scientific Inc. Methyl thiazolyl tetrazolium (MTT), 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) were obtained from Beyotime Biotechnology Co., Ltd. All other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. without further purification.
Preparation of Amino-Terminated Hyperbranched Polymer (NHBP)
The amino-terminated hyperbranched polymer was synthesized as our previous work . Tetraethylenepentamine (94 ml, 0.5 mol) was added to a 250-ml three-necked round-bottomed glass flask equipped with nitrogen gas protection and magnetic stirring. The reaction mixture was stirred with a heating magnetic agitator and cooled with an ice bath, while a solution of methyl acrylate (43 ml, 0.5 mol) in methanol (100 ml) was added dropwise into the flask. Then, the mixture was removed from the ice bath and left stirring for a further 4 h at room temperature. The mixture was transferred to an eggplant-shaped flask for automatic rotary vacuum evaporation, and the temperature was raised to 150 °C using an oil bath, and left for 4 h until yellowish viscid HBP scale was obtained with a weight average molecular weight about 7759.
Preparation of NHBP-Reduced GO (NrGO)
GO was first dispersed in deionized water and ultrasonicated mixed with appropriate HBP (weight ratio of GO and NHBP is 1:10, 1:20, and 1:30) for 10 min, kept stirring and reacted under 90 °C for 1 h. Then, the resultant (marked as NrGO-10, NrGO-20, and NrGO-30) was centrifuged and washed with deionized water for three times.
Preparation of DOX-Loaded NrGO (DOX@NrGO)
The as-prepared NrGO suspension was dispersed in DOX solution with weight ratio of 1:1, and kept stirring for 24 h under room temperature. Then, the composite solution was centrifuged and washed to collect DOX@NrGO.
The surface morphology was characterized via transmission electron microscopy (TEM, JEM-2100, JEOL, Japan). Fourier-transform infrared (FTIR, Nicolet iS10, Thermo Scientific, America) spectroscopy was performed to illustrate the chemical component change between GO and NrGO. All spectra were measured in a wavelength range of 400~4000 cm−1 with a resolution of 4 cm−1. The surface potential and particle size were investigated through Zeta potential-particle size analyzer (NanoBrook 90plus Zeta, Brookhaven, USA). The absorption of NrGO in NIR region was studied by UV-vis (Evolution 300, Thermo Fisher, USA) with wavelength range of 400~900 nm and resolution of 1 cm−1.
The photothermal properties were measured by using a NIR laser device (SFOLT Co., Ltd., Shanghai, China) and a thermocouple thermometer (DT-8891E, Shenzhen Everbest Machinery Industry Co., Ltd., China). The photothermal property of NrGO was measured under 808 nm laser irradiation. The spot area of the laser is about 0.25 cm2, and the temperature change of tested sample suspension was monitored in real time. Herein, pure water and GO suspension were applied as control groups: (1) 0.2 ml pure water, GO, and NrGO (NrGO-10, NrGO-20, and NrGO-30) suspension were put in 0.25 ml Eppendorf tube, then NIR laser was irradiated with power density of 1 W/cm2 for 5 min; (2) 0.2 ml NrGO-30 suspension with different concentration (100, 200, and 300 μg/ml) was irradiated (1 W/cm2) for 5 min; (3) 0.2 ml NrGO-30 suspension (200 μg/ml) was irradiated with different power density (1, 1.5, and 2 W/cm2) for 5 min; (4) 0.2 ml NrGO-30 (200 μg/ml) suspension was irradiated (1 W/cm2) for three on/off cycles.
The collected DOX@NrGO was divided into three groups for different treatment to investigate the drug delivery behavior: (1) dispersing in PBS solution with pH = 7.4, marked as control group; (2) dispersing in PBS solution with pH = 4.0, marked as acid group; (3) dispersing in PBS solution with pH = 7.4 and irradiated with NIR laser, marked as NIR group. The above three groups (each group was set three parallels) were put in dialysis bag (5 ml) with cut-off molecular weight of 8000, and then put into centrifuge tube with 20 ml corresponding PBS solution. After that, all tubes were put in 37 °C shaker with 100 rpm, 10 ml of PBS solution of each tube was withdrawn at predetermined time points for drug release analysis, and equal volume of corresponding fresh PBS was added back. In addition, the NIR group was treated as that NIR light was irradiated for 5 min after each predetermined time point. All withdrawn solutions were analyzed by UV-vis spectrophotometry, and the drug delivery profile was obtained.
The cytotoxicity of NrGO against tumor cells (HeLa) was investigated by MTT assay. Briefly, HeLa cells were seed in 96-well plates at a density of 5 × 103 cells per well and kept incubating till 80% of the well was covered. Then, the old medium was changed fresh medium with NrGO (3.125, 6.25, 12.5, 25, and 50 μg/ml), the medium without NrGO was set as control group. After incubating for 24 and 48 h, the MTT assay was used to measure the relative cell viability via the Eq. (1):
Then, chemo-photothermal synergetic therapy was investigated via treating HeLa cells with DOX@NrGO (3.125, 6.25, 12.5, 25, and 50 μg/ml) under NIR irradiation. After incubating with DOX@NrGO for 4 h, the HeLa cells were irradiated with NIR laser for 5 min and kept incubating for another 20 h. Afterwards, cell viability was tested via MTT assay again. For cells observation, HeLa cells were then stained with DAPI and PI, respectively, and observed under CLSM and fluorescence microscope.
Results and Discussion
Physical and Chemical Characterization
Photothermal Properties Measurement
Drug Delivery Behavior Test
Cytotoxicity of NrGO
Synergetic Inhibition of DOX@NrGO on Tumor Cells
Based on the biocompatibility of NrGO, the tumor inhibition efficacy of DOX@NrGO was studied in vitro. In order to exam the influence of photothermal behavior, NIR laser was irradiated on corresponding tumor cells for 5 min with power density of 0.5 W/cm2. As demonstrated in Fig. 10b, when tumor cells were treated with DOX@NrGO for 24 h, the viability decreased obviously with concentration increase, revealing the released DOX could inhibit tumor cell proliferation. Moreover, the viability decreased much more rapidly when NIR irradiation was applied as well, indicating the raised temperature and DOX release rate could play chemo-photothermal synergetic therapy.
In summary, novel hydrophilic NrGO was designed and successfully prepared via simple reaction of GO and amino-terminated HBP. Varied characterization showed that NrGO obtained stable and outstanding photothermal property. After DOX loading, the drug delivery presented pH and photothermal dual-responsive behavior, which could be accelerated at low pH value and NIR irradiation. In addition, in vitro cytotoxic experiment result showed that the as-prepared NrGO was well biocompatible. Due to the advantage, tumor cells could be effectively inhibited based on chemo-photothermal synergetic therapy, and anti-tumor drug-loaded NrGO obtained promising application in tumor therapy.
The present work was supported financially by the National Key Research and Development Program of China (no. 2016YFB0303101), National Natural Science Foundation of China (nos. 51303085, 51503105, and 51803094), Natural Science Research Project of Jiangsu Higher Education Institutions (17KJB540002).
Availability of Data and Materials
The datasets supporting the conclusions of this article are included within the article.
JD guided the experiments and test process, and revised the paper. WZ and DN designed and conducted the experiments, and wrote the manuscript. All authors discussed the results and commented on the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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