Graphene Quantum Dots for Radiotherapy - ACS Applied Materials

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Graphene Quantum Dots for Radiotherapy Jing Ruan, Ying Wang, Fang Li, Renbing Jia, Guangming Zhou, Chunlin Shao, Liqi Zhu, Malin Cui, Da-Peng Yang, and Shengfang Ge ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18975 • Publication Date (Web): 15 Mar 2018 Downloaded from http://pubs.acs.org on March 16, 2018

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ACS Applied Materials & Interfaces

Graphene Quantum Dots for Radiotherapy ⊥

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Jing Ruan† , Ying Wang#† , Fang Li† , Renbing Jia†, Guangming Zhou‡, Chunlin Shao†‡, Liqi Zhu* †, Malin Cui§, Da-Peng Yang*§, and Shengfang Ge* † †

Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, People's Republic of China. ‡ Department of Radiation Biology, School of Radiation Medication and Protection, Soochow University, Suzhou 215123, China. § College of Chemical Engineering and Materials Science, Quanzhou Normal University, Quanzhou 362000, China.

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These authors contributed equally to this work *Corresponding author E-mail: [email protected], [email protected]

[email protected],

Abstract Radiation therapy as a kind of tumor treatment that has been widely employed in clinics, but the therapeutic effect is largely hampered by various factors. Currently, great efforts have been made to look for effective and safe radiosensitizers. A nano-radiosensitizer is an ideal choice for improving the tumor radiotherapy effects due to its high degree of tumor tissue uptake and secondary electrons productivity. Herein, highly oxidized graphene quantum dots (GQDs) with a good oxidative stress response and significantly high phototoxicity were fabricated and purified via the photo-Fenton reaction of graphene oxide. The enhanced radio-sensitization effects were systematically evaluated by monitoring colorectal carcinoma cells cycle and apoptosis degree, and the possible mechanism of the GQD irradiating enhancement of cells apoptosis was preliminarily investigated. Our data show that the GQD synergy with ionizing radiation (IR) could noticeably enhance the G2/M stage arrest of cells, inhibit cells proliferation and improve apoptosis. This is mainly due to the overproduction of reactive oxygen species (ROS) by GQDs in combination with the IR that actives the apoptosis-related regulation proteins, and results in tumor cell apoptosis. This study suggests that the GQDs can act as a new nano radiosensitizer in tumor radiotherapy.

Keywords: graphene quantum dots; radiosensitizer; radiotherapy; apoptosis; reactive oxygen species.

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Introduction Colorectal carcinoma is a common gastrointestinal cancer, and its incident and mortality rates in China are gradually rising each year. This form of cancer, is mainly treated with radiation therapy1-2. Although the role of radiation therapy is increasingly prominent, its treatment effect is not ideal. This is because the radiation therapy efficacies are largely hampered by hypoxia which exists inside most solid tumors, because oxygen is essential to enhance radiation-induced DNA damages3-4. Additionally, the cure rate of radiotherapy remains low because it is highly dependent on the radiotherapy sensitivity of the patients5. Radiation resistance is not alleviated by large-dose radiotherapy treatments, and concurrent radiation enteritis can occur in some patients5-6. Therefore, improving the radiation sensitivity of the tumor and the lessening the side effects to surrounding normal tissues and organs has led to a quest to find a highly efficient radiosensitizer with a low toxicity, and this has gradually become a hotspot and focus in current radiation research. A radiosensitizer is a type of radiotherapy reagent that has none-to-few adverse effects with a single use, and they can enhance the results of tumor radiotherapy in combination with irradiation5. Traditional small molecule radiosensitizers have the characteristics of low toxicity and quick excretion; however, its uptake ratio in tumor tissues is low. With the development of nanomedicine, some research has reported the use of metal nanomaterials as radiosensitizers. Li et al designed a novel theranostic agent-Au@MnS@ZnS-PEG nanoparticle, and this radiosensitizer showed a significant enhancement in radiation treatment7. Song et al designed another MnSe@Bi2 Se3 core-shell nanostructures, and also found a promise concept to enhance radiotherapy8. Gold and gadolinium nanomaterials can effectively improve the radiotherapy effects owing to their high atomic numbers, which significantly enhance the photoelectric absorption and secondary electron productive ability9-12. However, the high preparation cost, difficult purification, and uncertain toxicity limit their clinical applications. Therefore, it is highly desirable to develop an economic, safe, and efficient radiosensitizer. Graphene quantum dots (GQDs) are graphene-based quasi-zero-dimensional nanomaterials, and it is a new member in carbon nanomaterials family, which has received much attention for their unique properties. Recently, Ge et al designed a novel C-dots using polythiophene phenylpropionic acid (PPA), which can be used as active agents in fluorescence (FL) imaging and photoacoustic (PA) imaging13. Besides, they also found the GQDs exhibited a broad absorption band spinning from the UV region to deep-red emission with superior performance in water dispersibility, photo- and pH-stability, and biocompatibility14-15. In addition, other researches also showed their biocompatibility, hydrophilicity, and photoluminescence (PL) properties hold great promise for many applications in drug delivery, bio-labeling, and imaging16-18. The abundant surface oxygen-containing groups endow GQDs with good chemical modification potential, and prolong the in vivo blood retention rates and improve tumor tissues uptake19. Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defenses of some enzymes. If the ROS amount exceeds the body scavenging capacity, it will lead to oxidative stress reactions and induce cell protein, DNA, and lipid damages. Although GQDs can induce some degree of oxidative stress reactions due to its special chemical structures, it still retains the pro-oxidant and antioxidant balance by acting as both an electron donor and acceptor. Therefore, its safety is dose dependent20-21. However, GQDs can exhibit a degree of photo toxicity due to their PL property. Since the GQDs with plenty of oxygen-containing groups could act as electron donor, moreover, the big π bond of GQDs is helpful in charge transfer and electron


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storage, when the GQDs are exposed to photoexcitation, they can generate a kind of ROS free radicals, including singlet oxygen via energy-transfer and electron-transfer pathways, hydroxyl radical and superoxide anions arising from electron−hole pairs and charge separation effects upon reacting with surrounding molecular oxygen or H2O, these amounts of ROS free radicals could enlarge or prolong the tumor radiotherapy effects22-23. Therefore, based on the good biocompatibility, blood retention, and photo toxicity of GQDs, we expect it to possess good sensitization effects in tumor radiotherapy. To the best of our knowledge, few studies have used GQDs as a radiosensitizer. Herein, to evaluate the synergy of GQDs with irradiation to improve colorectal carcinoma radiotherapy efficacy, GQDs with a high degree of oxidation were fabricated and purified through the photo-Fenton reaction of graphene oxide (GO). Then, the working concentration of the GQDs on colorectal carcinoma cells was optimized. Next, the effects of the enhanced GQDs on radio sensitization were systematically evaluated by monitoring cell cycles and apoptosis. Finally, the possible synergistic mechanism of GQDs with irradiation to enhance colorectal carcinoma cell apoptosis was presented by analyzing the cell ultra-microstructure, intracellular and mitochondrial ROS levels and DNA damage. Through a comprehensive evaluation of GQDs radio-sensitization effects, we hope to develop an economic, safe, and efficient nano-radiosensitizer to improve the results of tumor radiotherapy.

Results and discussion Characterization of the GQDs The fabrication of the GQDs included a facile top-down photo-Fenton reaction of GO to prepare GQDS, as the literature reported24-25. The as-generated GQDs have a good dispersity and are hydrophilic. The AFM image clearly depicts evenly dispersed GQDs with a thickness of ~ 2 nm, as presented in Figure 1A. The size distribution result for the GQDs also shows that they have an average lateral size of ~18 nm, as presented in Figure 1B. To better observe the morphology of the GQDs, transmission electron microscope (TEM) images revealed that the dispersity of the GQDs was uniform with a granular distribution, as shown in Figure 1C. The internal high-resolution TEM image showed that the GQDs have a crystallite structure as well as a lattice parameter of 0.23 nm along the graphitic lattice plane. GQDs are effective for photon harvesting within the short-wave length region26, so the UV-vis spectrum of the GQDs in Figure 1D shows a strong absorption peak (π-π* transition of C=C bonds) at 230 nm and a shoulder peak (n-π* transition of C=O bonds) at 290 nm.



Figure 1. Characterization of the GQDs. (A) Tapping mode AFM images of the GQDs. Inset: height profiles of the lined GQDs. (B) Size distribution of the as-synthesized GQDs in purified water. (C) TEM image of the as-generated GQDs. Scale bar: 20 nm. Inset: HR-TEM images of an individual GQD shown in the panel. Scale bar: 5 nm. (D) UV−vis spectra of the GQDs and GO. Many researchers groups have reported that the intensity ratio of the D band to the G band (ID/IG) can reflect the electron conjugate state and crystallinity27 of GQDs, so the carbon conjugate structure of the GQDs was further investigated with Raman spectroscopy, as shown in Figure 2A. Two prominent peaks of the GQDs appeared at approximately 1358 and 1595 cm-1, which are attributed to the D and G bands, respectively28-29. The GQDs exhibited a lower G peak frequency and improved ID/IG *(1.27) compared to those of GO (0.76). This phenomenon reflects that GQDs have a larger amount of sp3 conjugate regions in the graphene carbon structure due to the oxygen-containing groups and carbon holes that were introduced in the oxidation process.


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Figure 2. Physicochemical characterization of the GQDs. (A) Raman spectra of raw GO and the as-generated GQDs. (B) C 1s XPS spectrum of GO. (C) C 1s XPS spectrum of the GQDs. (D) Elemental analyses of the GQDs and GO by XPS. (E) Photoluminescence (PL) spectra of the GQDs aqueous suspensions acquired with emission wavelengths of 450 nm and 500 nm. (F) PL spectra of an aqueous suspension of GQDs acquired using different excitation wavelengths. To further investigate the oxidation degree of the GQDs, the XPS spectra of C1s of the GQDs was acquired, as shown in Figure 2B. The four peaks at 284.8, 286.4, 287.6, and 288.3 eV corresponds to the C=C/C-C in aromatic rings, C-O (epoxy and alkoxy), C=O (carbonyl), and COOH (carboxylic) groups, respectively. Compared with the spectrum of GO (Figure 2C), the intensities of the C1s XPS peaks of the carbonyl groups decreased, and the COOH peak increased, which implied that more COOH groups were generated after the C=O groups of GO were oxidized through the photo-Fenton reaction. Furthermore, there is a significant decrease in the C/O atomic ratios, from 2.77 of GO to 1.65 fot the GQDs (Figure 2D). This fully illustrated that the GQDs have more oxygen-containing groups and have a significantly higher degree of oxidation. The PL property of the GQDs assumes that the PL mechanism, which was similar to that of other nano sized carbon materials, mainly consisted of intrinsic state emissions (π-π* and σ-π* transition emission) and defect state emissions (n-π* transition emission), which correspond to the two emission peaks at 450 nm and 500 nm25. To verify the excitation region of the GQDs, the excitation spectrum of GQDs was observed under the emission wavelengths of 450 nm and 500 nm, as shown in Figure 2E. This fully illustrated that the total excitation region of the GQDs is between 300 nm to 400 nm. Next, the PL emission spectra of GQDs were also detected under different excitation wavelengths, as shown in Figure 2F. The results showed that the PL emission wavelength of the GQDs is strongly dependent on the excitation wavelength, and the emission peak of the GQDs is red-shifted from 450 nm to 500 nm, and the increased excitation wavelength shifted from 300 nm to 380 nm. The redshift phenomenon of the GQDs emission wavelength is mainly dependent on the basal surface and edge defects of the GQDs which are induced by the carboxylic groups content, higher GQDs oxidation degree, more surface defects, and higher intensity of the defect state emission. Finally, combined with the Raman and XPS analyze results above, we concluded that the GQDs have a high oxidation degree after the photon-Fenton reaction of GO.



To further test the stability of GQDs in different physiological environment including of PBS and serum solutions, the zeta potential of GQDs dispersed in different solutions were quantified as shown in Table S1, GQDs dispersion in water and PBS solutions showed strong negative potentials of -23.2 mV and -18.1 mV, which fully illustrated GQDs have good stability and hydrophilicity in physiological environment due to plenty of surface oxygen-containing groups existence in GQDs. But under the proteins-containing environment, GQDs could only keep relative stable status with negative potentials of -13.9 mV in low concentration of 0.1% FBS solution, and the electronegativity of GQDs increase to -8.3 mV by the FBS concentration reach to 1%, the increasing electronegativity of GQDs in serum environment mainly due to the π–π stacking interactions between the aromatic rings of protein molecule and the GQDs basal plane30-31. These results fully imply that the GQDs have good stability in physiological environment.

Cytotoxicity effects and subcellular localization of the GQDs Although the GQDs have good biocompatibility at a safe concentration, as reported in the literature24, considering this is the first use of GQDs in colorectal carcinoma irradiation therapy, we studied the GQDs cytotoxicity on colorectal carcinoma cells. To find a safe concentration of GQDs for use in colorectal carcinoma cells, we chose the SW620 and HCT116 cell lines to evaluate their growth after treating them with different concentrations of GQDs. The results showed that GQDs have a dose-dependent cytotoxicity on the SW620 and HCT116 cells, as shown in Figure 3A. Compared with the control group, there was no significant difference (p>0.05) in the cell viability after the SW620 cells were incubated with different concentrations of GQDs (50, 100 and 200 µg/mL) for 3 days. In contrast, the HCT116 cells exhibited a low sensitivity to the GQDs, and the growth curves were somewhat inhibited by the GQDs compared with that of the control group. The GQDs groups remained proliferative for 3 days, and there was no significant difference in the cell viability after the first 2 days of treatment with 50 µg/mL of GQDs compared with that of the control group. Based on the comprehensive analysis of the results for the viability of the two types of colorectal carcinoma cell, we concluded that 50 µg/mL of GQDs would be used for the following experiments. To evaluate excretion of GQDs, nude mice were injected with GQDs through tail vein and were monitored under a non-invasive manner by using IVIS fluorescence imaging system. The excretion of GQDs was determined by fluorescence imaging. As shown in Figure S1A, the control mouse showed weak autofluorescence in liver and the upper part of mice (panel a), while mice 4 h post-injection showed an increase of fluorescence in liver, which indicated that GQDs were preferentially accumulated in liver. At 24 h post-injection, the fluorescence of liver decreased and the kidney fluorescence increased, which indicated the GQDs would excrete from kidneys compared to other tissues. Consistent with the in vivo fluorescence imaging result, the ex vivo imaging also showed the same result, as shown in Figure S1B. As for the in vivo toxicity, whole blood was collected in tubes lined with EDTA to measure hemataology markers. Results in Figure S1C showed that white blood cell count (WBC) showed significant increase and platelet count（PLT）showed significant decrease day 1 after injection, compared with that of day 0 group without injection of GQDs. At day 3, WBC showed a little decrease compared with that of day 1, which was still higher than that of day 0 group, showing an


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acute inflammation. At day 7, WBC showed no significant difference between day 0 and day 7, indicating the resolution of acute inflammation32-33 We also measured alanine transferase (ALT), aspartate transferase (AST), alkaline phosphatase (ALP), albumin (ALB), blood urea nitrogen (BUN) and creatinine (Cr) to evaluate the liver and kidney function. Results in Figure S1D showed no significant difference between day 0 group and day 1, 3 and 7days after injection in ALT, AST, ALP, Cr, and ALB. This indicates the liver and kidney function still maintain normal after GQDs were injected. As for the decrease of BUN, it may be caused by the temporary slowing of metabolism of rats after injection through the tail vein. Thus, GQDs showed no obvious adverse effects on organ function.



Figure 3. Cytotoxicity evaluation and subcellular localization of the GQDs. (A) Cell viabilities of the SW620 and HCT116 cells treated with different concentrations (0, 50, 100 and 200 µg/mL) of GQDs for 3 days. (B) Representative confocal fluorescence (FL) images and merged images of SW620 and HCT116 cells incubated with 50 µg/mL the GQDs for 24 hours. The green fluorescence signal represents the cytoskeleton protein of actin that was labeled by the Alex flour


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594 Phalloidin, and the red fluorescence signals indicate internalized FITC-labeled GQDs, and the nuclear was stained blue with DAPI. (C) The cell viabilities of the SW620 and HCT116 cells treated with or without GQDs (50 µg/mL) under irradiations (3 and 6 Gy) for 3 days (n = 3, *P < 0.05, **P < 0.01 and ***P < 0.001). (D) Colony formation images of the cells treated with or without the GQDs (50 µg/mL) under irradiation (3 and 6 Gy) for 10 days. Aside from the toxicity of GQDs on colorectal carcinoma cells, the cytotoxicity of GQDs on normal tissue should also be considered. Through the analysis of the in vivo action pathway of GQDs, which are used as an irradiation sensitizer in colon cancer radiotherapy, the colon epithelial and blood vessel endothelium were used as the most accessible tissues after intravenous injection of the GQDs. The NCM460 and hUVEC cell lines were chosen to verify the safety of GQDs on normal tissues, as shown in Figure S2. The results showed that compared with that of the control groups, these two types of cells exhibited no significant differences in cell proliferation after treatment with 50 µg/mL of GQDs for 4 days. This confirms that 50 µg/mL of GQDs is a reasonable working concentration for colon cancer radiotherapy. The sub-cellular location of the GQDs plays an important role in explaining its action mechanism with cells. To further evaluate the GQD interactions with colorectal carcinoma cells, the subcellular localization of GQDs in the SW620 and HCT116 cell lines were also monitored, as shown in Figure 3B. The cytoskeleton of the cells was stained with red fluorescence, and the GQDs showed green fluorescence signals in the confocal laser scanning microscopy (CLSM) images. After the cells were incubated with 50 µg/ml of GQDs for 24 h, the results showed that the GQDs were mainly located in the cytoplasm and some accumulated inside the nucleus. These cellular internalization results illustrated that the GQDs could be endocytosed by colorectal carcinoma cells and exhibited similar phenomenon reported in the literatures24, 34. GQDs synergy with ionizing radiation (IR) and influence on colorectal carcinoma cell proliferation Because GQDs can generate an electron transition under irradiation, which exhibits a significant PL emission, the generated free radicals can enlarge and prolong radiotherapy effects. It is necessary to evaluate the synergetic effects of GQDs on tumor cells radiotherapy. To investigate the GQDs radio sensitization effects on colorectal carcinoma cells, the CCK-8 assay was first performed to evaluate the cell proliferation status. The cell viability of the SW620 and HCT116 cell lines were examined in the absence or presence of the GQDs followed by 3 Gy or 6 Gy γ-ray irradiation, as shown in Figure 3C. The results show that the SW620 and HCT116 cells growth rates when irradiated with 3 Gy in the presence of the GQDs (GQDs & 3 Gy ) decreased prominently in comparison to the group that was irradiated with 3 Gy alone (3 Gy), regardless of whether the irradiation treatment was 1 day (p

Graphene Quantum Dots for Radiotherapy - ACS Applied Materials

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