Electron Irradiation Effects on N and Fe3+ Doped Carbon Dots

Introduction Carbon dots (CDs) are novel carbon-based nanomaterials which exhibit excellent fluorescence properties such as strong light stability and wide excitation spectrum.1–4 Additionally, CDs have good biocompatibility, low toxicity, a broad range of raw materials, diverse synthesis methods, low production costs, and are conducive to functionalization and large-scale production.5–8 Consequently, CDs exhibit extensive application prospects and significant potential in a multitude of fields, including optoelectronic devices, environmental detection, photocatalysis, water treatment, biomedicine, and particularly in tumor treatment.8–10 At present, the conventional treatment methods for tumors mainly include surgery, radiotherapy, and chemotherapy. Electron beams are one of the commonly used radiation sources in radiotherapy. The killing of cells by electron beams is based on two mechanisms. One is the breaking of single or double strand DNA by ionization and thereby causing the cells to lose their ability to proliferate.11,12 The other is the generation of hydroxyl and other reactive oxygen species by ionization which would attacks the DNA to damage to the cells.13–15 However, the electron beams cannot distinguish the tumor cells from normal cells11,16 and will also damage the normal tissues and organs adjacent to the tumor during tumor treatment.17 One of the methods to control tumor growth is to irradiate the tumor site with a high dose of electron beams. For a large tumor, they require a higher radiation dose to control the growth of the tumor due to the severe hypoxia at the center of the tumor.18,19 However, there are dose limitations for the normal tissues and organs adjacent to the tumor. It would be difficult to increase the tumor dose during the therapy.20–23 And people expect to find new tumor treatment strategy with less damage to normal tissues and better therapeutic effects. Thus, the development of highly efficient tumor radiation sensitizers has become an important way to enhance the efficacy of radiotherapy. It is worth noting that the sensitization ratio (SER) can reach up to 1.23 by selectively sensitizing tumor hypoxic cells with electrophilic compounds or biological reducing agents.24 However, some drugs have potential toxicity to normal tissues.25,26 Targeting epidermal growth factor receptor (EGFR) to inhibit the DNA repair pathway can reverse radiotherapy resistance, and there are risks such as large individual treatment response differences and the generation of drug resistance.27 Importantly, CDs can penetrate the cell membrane and enter the cells to enhance the killing power against tumor cells by releasing reactive oxygen species (ROS) in coordination with radiation.28,29 Therefore, CDs can be used as sensitizers for radiotherapy of tumors and would have great research value and broad application prospects. In this paper, we prepared the nitrogen and N, Fe co-doped carbon dots (N-Fe-CDs) and evaluated the possibility for CDs used as tumor radiation sensitizers. The N-Fe-CDs aqueous solution was irradiated with radiation doses of 80 Gy and 160 Gy by using the 10 MeV electron beam generated by Elekta’s medical electron linear accelerator. The TEM, XRD, XPS, FT-IR, PL spectrum analysis, and cytotoxicity tests of N-Fe-CDs aqueous solutions before and after electron beam irradiation (EBI) were performed to investigate the electron irradiation effects and cytotoxicity of N-Fe-CDs. Finally, in vivo animal experiments were conducted based on tumor-bearing mice to verify the possibility of CDs as tumor radiation sensitizers. Materials and Methods Materials L-arginine (purity > 98%) was supplied by Shanghai Yuanye Biotechnology Co., LTD (Shanghai, China). Fe(OH)3 (AR grade, Analytical grade) was obtained from Tianjin Damao Chemical Reagent Factory (Tianjin, China). Dulbecco’s modified Eagle Medium (DMEM) was purchased from Thermo Fisher Scientific Biotechnology Co. (Massachusetts, USA). Fetal bovine serum (FBS) and penicillin-streptomycin mixed solution (100 units/mL penicillin and 0.1 mg/mL streptomycin) were provided by Nanjing Aofan Biotechnology Co., LTD (Nanjing, China). 10% FBS and 1% penicillin-streptomycin mixed solution were added to DMEM and mixed to form a complete medium for cell culture in this study. Cell Counting Kit 8 (CCK8) was provided by Sigma-Aldrich Inc. (St. Louis, USA). The deionized water was prepared using a Master-S15 deionized water production machine purchased from Shanghai Hetai Instrument Co., LTD. The experimental mice were provided by the Institute of Biology, Chinese Academy of Sciences. Preparation of N-Fe-CDs The preparation process of N-Fe-CDs is shown in Figure 1. 0.5 g of L-arginine and 0.125 g Fe(OH)3 were used as raw materials, and they were mixed and placed in the inner lining of the reaction vessel. Meanwhile, 55 mL of deionized water was injected and ultrasonically mixed for 20 minutes. Then, the reaction vessel was placed in a drying oven and continuously heated for 4 hours at 220°C. After the reaction vessel were natural cooling to room temperature, the reaction solution was filtered through a 0.22 μm filter and then centrifuged with 1200 r/min to obtain N-Fe-CDs’ aqueous solution. EBI Experiment and Characterization of CDs In this study, 10 MeV electron beam generated by the Elekta medical electron linear accelerator Synergy was used to irradiate the solution of N-Fe-CDs at doses of 80 Gy, 120 Gy and 160 Gy, respectively. The TEM, XPS, XRD, FT-IR and PL spectroscopy of the prepared solutions of N-Fe-CDs with and without radiation were investigated, respectively. The samples of prepared N-Fe-CDs aqueous solution samples were dropped onto a copper mesh, and then the copper mesh was dried under a high-power infrared lamp. The morphology and structure of the N-Fe-CDs particles were observed under a transmission electron microscope (TEM) (JEM-2100, JEOL, Japan). X-ray photoelectron spectroscopy (XPS) produced by Thermo Fisher scientific of USA (K-Alpha+) was performed to investigate the element composition of N-Fe-CDs before and after EBI. X-ray diffractometer (XRD) (Rigaku, Japan (TTRIII)) was used to detect the position (2θ angle) of the diffraction peaks of N-Fe-CDs before and after irradiation. Fourier Transform infrared spectroscopy (FTIR) (Nicoletis 10, Thermo Fisher scientific, USA) was used to evaluate the functional groups of N-Fe-CDs. Drop the aqueous solution of N-Fe-CDs (about 5 mL) into a mortar, and then place the mortar under a high-power infrared lamp to dry. Mixed potassium bromide powder with dried N-Fe-CDs and then pressed it into a transparent tablet on a tablet press. The spectra were recorded by the FTIR spectrometer. The infrared spectral resolution was 4 cm−1, the scanning range was 4000–400 cm−1, and the scanning times were 32. The fluorescence intensity of N-Fe-CDs was determined by a fluorescence spectrophotometer (F98, ShangHai Lingguang Technology Co., LTD., China). The fluorescence spectrum excitation range was 200 to 900 nm, with an interval of 10 nm, and the width of the excitation and emission slits was 10nm. Cell Culture Lewis lung carcinoma (LLC) cell line was purchased from Guangzhou Lide Biotechnology Co. (Guangdong, China). LLC cells were cultured at 37°C humidified incubator with 5% CO2 in the complete medium (DMEM medium supplemented with 10% FBS and 100 units/mL penicillin and 0.1 mg/mL streptomycin). The medium was changed every 2–3 days. Experimental Animal Models Single-cell suspensions were prepared using uncontaminated LLC cells in the logarithmic growth phase. Through steps of collecting cells, resuspend them, and adjusting the cell concentration to 1×106 to 5×106 cells/mL. Forty 6–8 weeks-old male C57 mice were purchased and raised adaptively in an SPF-level animal house for 7 days. Ear tags were made for forty mice. Isoflurane inhalation anesthesia was used. The skin on the back of the right hind limb of mice was selected as the inoculation site for skin and cell inoculation. Seven days after vaccination, obvious tumor nodules could be felt on the back of the right hind limb of 38 mice. The long and short diameters of the tumors were measured every two to three days, and the changes in tumor volume and body weight of each mouse were recorded in detail for about 20 days. When tumor long diameter was about 1.0 cm, selected 36 mice among 38 mice and randomly divided into 6 groups (6 mice per group) for experiments. Cells Cytotoxicity Assay Selected LLC cells that were in the logarithmic growth phase and in good condition. The LLC lung tumor cells were cultivated with normal and full cell morphology by an optical microscope. The cells were planted into two 96-well plates with a cell density of ≥104 cells per well for cells cytotoxicity assay of N-Fe-CDs before and after EBI, respectively. The N-Fe-CDs solutions with concentrations of 2.7×101, 2.7×100, 2.7×10−1, 2.7×10−2, 2.7×10−3, 2.7×10−4 mg/mL were prepared and added them to the 96-well plate with a volume of 200 μL per well. We set 6 parallel wells for each concentration as controls. The 96-well plate was placed in the cell culture box and culture for 24 hours. Removed the culture medium carefully after the cells adhere to the plate. Then, added 20 μL Cell Counting Kit-8 (CCK8) reagent and the culture medium to each well incubating for 4 hours and measure the cytotoxicity using an enzyme detector. Laser Confocal Imaging Experiment The biological imaging of N-Fe-CDs before and after EBI taken by LLC cells was respectively captured by confocal laser scanning microscopy. For laser confocal microscopy, the well-cultivated LLC cells were transplanted into the culture dish for further cultivation. After 12 hours of cultivation, when the cells adhered to the dish, the culture medium was gently removed, and then a culture medium with N-Fe-CDs at a concentration of 2.7×10−2 mg/mL was added. The cells were further cultivated for 4 hours, and the laser confocal microscope was used to observe the situation of N-Fe-CDs entering the LLC cells. Irradiation Sensitization Experiment in Tumor-Bearing Mice For radiation-sensitized animal experiment, 6 groups of C57 mice were named the control group, the group given the N-Fe-CDs sample, the 8 Gy radiotherapy group, the 16 Gy radiotherapy group, the 8 Gy with given sample radiotherapy group, and the 16 Gy with given sample radiotherapy group, respectively. For the convenience of the discussion in the following text, the names of the 6 groups would be abbreviated as “Control Group, CDs Group, 8 Gy Group, 16 Gy Group, 8 Gy+CDs Group and 16 Gy+CDs Group”. The physiological saline and N-Fe-CDs solution was injected by intraperitoneal injection to the mice with a dose of 200 μL and 2.7×10−2 mg/mL N-Fe-CDs solution. The tumor sites of the tumor-bearing experimental mice were irradiated using an electron beam generated by a medical linear accelerator, and the other parts of the mice’ bodies were shielded from the electron beam using lead blocks with notches. After the electron irradiation, the mice were fed for 120 hours, and the tumors were removed for measurements. Statistical Analysis The difference between groups was determined by the T test (SPSS 23.0 software). #p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 were considered to be statistically significant. Results and Discussion The microscopic morphology of N-Fe-CDs before and after electron irradiation are shown in Figure 2. As can be seen from Figure 2a, the N-Fe-CDs before electron irradiation have good dispersion and uniform size. Figure 2b shows that the N-Fe-CDs are quasi-circular plate-shaped, with the central region being crystalline and the lattice fringe spacing being 0.21 nm, corresponding to the (100) plane of graphite.30 The particle size distribution of N-Fe-CDs is given in the inset of Figure 2a, with an average diameter of 3.0 nm. After electron irradiation, N-Fe-CDs exhibit agglomeration (see Figure 2b), and the lattice fringe spacing in the inset is 0.24 nm, corresponding to the (112) plane of graphite.31 From Figure 3, we can see that the XRD diffraction peaks of N-Fe-CDs before and after electron irradiation are nearly the same, at 21.3° and 21.5°, respectively. The interplanar spacings are 0.43 nm and 0.41 nm, corresponding to the (002) plane of graphite. However, they are larger than the normal interplanar spacing of 0.34 nm of the (002) plane of graphite. This is because the N and Fe impurities, as well as the surface and edge groups in N-Fe-CDs causes the expansion of interplanar spacing. Therefore, the interplanar spacing of N-Fe-CDs would be larger than that of the ideal graphite crystal (002) plane with normal interplanar spacing. Additionally, the interplanar spacing after electron irradiation is slightly smaller than that before irradiation. This is because a large number of high-energy electrons deposit their energy on N-Fe-CDs, resulting in a thermal effect which reduces the doping of N and Fe as well as surface and edge groups during the electron irradiation process. Figure 4 shows the XPS spectra of N-Fe-CDs before and after electron irradiation. From Figure 4a, it can be seen that the atomic contents of C, O, N, and Fe in N-Fe-CDs before electron irradiation were 63.86%, 17.93%, 17.81%, and 0.4%, respectively. In Figure 4b, after electron irradiation, the atomic contents of C, O, N, and Fe in N-Fe-CDs were 73.0%, 14.8%, 12.0%, and 0.2%, respectively. By comparing the changes in the atomic contents of C, O, N, and Fe in N-Fe-CDs, we obtained that the atomic content of C in N-Fe-CDs increased after irradiation, while the atomic contents of O, N, and Fe decreased. During electron irradiation, a large number of high-energy electrons deposit their energy on N-Fe-CDs and anneal the N-Fe-CDs to increase temperature. Thus, as shown in Figures 4c and d, the electron irradiation process can reduce the impurity defects and surface groups C-N, COOH in N-Fe-CDs. Therefore, the relative content of C atoms in N-Fe-CDs increased while the relative contents of O, N, and Fe decreased after electron irradiation. In Figures 4e and f, the content of Pyridinic N of N-Fe-CDs decreased and the contents of Fe-N4,32 Pyrrolic N and Graphitic N increased which are attributed by the thermal effect caused by electron irradiation. In addition, in Figures 4g and h we also see that electron irradiation also led to a reduction in C-O groups and an increasing of C=O groups on the surface of N-Fe-CDs. This was due to the conversion of C-O to C=O via absorbing electrons,33 and the ionization of some -COOH groups on the surface of N-Fe-CDs by electron irradiation, which transformed into C=O and ·OH,11 as illustrated in Figure 1, Figures 4c and d. Figure 5 gives the Fourier Transform Infrared Absorption Spectra (FT-IR) of N-Fe-CDs before and after electron irradiation. It can be seen that the infrared absorption peak positions of N-Fe-CDs before and after electron irradiation are basically the same, suggesting that the types of surface functional groups of N-Fe-CDs have not undergone significant changes before and after irradiation. However, the intensity of the C=O/C=N33 stretching vibration absorption peak at 1643 cm−1 has been significantly weakened. In addition, the intensities of the stretching vibration absorption peaks of C-H, N-H and O-H groups33 at 2943 cm−1, 3275 cm−1 and 3350 cm−1 decreased significantly, suggesting that the hydroxyl, carboxyl and amino groups34 on the surface or edge of N-Fe-CDs have significantly decreased due to the generation of free radicals such as ·OH and H·11 caused by electron irradiation. The main reason for these changes is the energy deposition caused by electron irradiation, which leads to the breakage of chemical bonds of hydroxyl, carboxyl and other groups on the surface and edge of N-Fe-CDs, generating ·OH and H· and other free radicals. Figure 6 shows the photoluminescence (PL) spectra of N-Fe-CDs before and after electron irradiation. We can see that the fluorescence peak positions of N-Fe-CDs before and after electron irradiation are both at 360 nm. In Figure 6a, as the electron beam irradiation dose increases, the fluorescence intensity of the CDs continuously decreases. When the electron irradiation dose reaches 160 Gy, the fluorescence intensity of N-Fe-CDs is about half of the fluorescence intensity of N-Fe-CDs before irradiation. From Figures 4g and h, we can see that nearly half of the C-O bonds on the surface and edges of N-Fe-CDs are converted into C=O bonds during electron irradiation. According to our previous research results,35 CDs have more C-O bonds and fewer C=O bonds, which would result in a stronger fluorescence. While for N-Fe-CDs after electron irradiation, there are fewer C-O bonds and more C=O bonds and the fluorescence becomes weaker. Besides, the hydroxyl, carboxyl, O-H, N-H, C-H bonds on the surface of N-Fe-CDs could be knocked off by the electron beam and transform to free radicals such as ·OH and H·, etc.11 The surface passivation layer of N-Fe-CDs would be destroyed to reduce the fluorescence efficiency.36,37 The deionized water used to disperse N-Fe-CDs can be ionized to generate active substances such as ·OH, H·, and H2O2 which can increase in the size of N-Fe-CDs.38,39 At the same time, electron radiation changes the surface charge distribution of N-Fe-CDs to induce the occurrence of aggregation (seen in Figure 2b) and weak the fluorescence intensity. The electron irradiation can aggregate N-Fe-CDs and increase the size of N-Fe-CDs. As the larger of the size, quantum confinement effect would decrease, leading to a decrease in PL intensity.40,41 The excitation and emission contour maps of N-Fe-CDs in Figures 6b–d also shown the decreasing of fluorescence intensity as increasing the electron irradiation dose. In Figure 7a, the absorption spectrum (black curve) of Fe-N-CDs has two main absorption peaks at 284 nm and 360 nm, corresponding to the π-π* electron transition of the C=C bond in the aromatic conjugated structure of the carbon core and the n-π* transition of the surface functional group C=O, respectively. The excitation spectrum (blue curve) of Fe-CDs also has two peaks, with the main excitation peak at 300 nm and the secondary excitation peak at 610 nm. The PL spectrum (red curve) of Fe-N-CDs also has two emission peaks, with the stronger down-conversion emission peak at 360 nm and the weaker up-conversion emission peak at 710 nm. Figure 7b shows the variation curve of the up-conversion emission peak of N-Fe-CDs at an excitation wavelength of 610 nm with different electron irradiation doses. Figures 7c and d show the variation curves of the down-conversion fluorescence of N-Fe-CDs under an excitation wavelength at 300 nm. The PL peaks are at wavelengths of 360 nm and 710 nm with different electron irradiation doses. We can obtain that the PL intensities for both of up-conversion and down-conversion become weaker with increasing the electron irradiation dose and the surface functional groups such as C=O and C-O in N-Fe-CDs have a significant impact on their luminescence performance.42–44 Using the Olympus/FV3000 laser confocal microscope, we give the imaging of LLC cells with adding N-Fe-CDs in Figure 8. In Figure 8a–c are the cell bioimaging of N-Fe-CDs before EBI taken by LLC cells, and Figure 8d–f are the cell bioimaging of N-Fe-CDs after EBI taken by LLC cells. In Figure 8a there is a bright-field image where the LLC cells present an irregular shape with clear boundaries. The general morphology of the cells can be observed. Figure 8b shows that LLC cells with N-Fe-CDs are emitting blue fluorescence under an excitation wavelength of 405 nm. When the excitation wavelength is 488 nm, the LLC lung cancer cells with N-Fe-CDs emit green fluorescence as shown in Figure 8c. It can be seen that N-Fe-CDs can be well taken up by LLC and can form a clear bioimaging under the excitation of laser. It is difficult to detect the difference in light intensity by comparing Figure 8a–f, which is inconsistent with the significant decrease in PL intensity after EBI in Figure 6. The reason is that the light intensity of bioimaging is influenced by multiple factors, such as the state of the cells, the amounts of N-Fe-CDs taken up by the cells, the imaging conditions, etc. Figure 9 shows the results of the LLC cytotoxicity test with CCK8. Among them, Figure 9a is the result of LLC cytotoxicity of N-Fe-CDs before EBI, and Figure 9b is the result of LLC cytotoxicity of N-Fe-CDs before EBI. And comparing Figure 9a with Figure 9b, it shows that there is no significant difference in the cytotoxicity of N-Fe-CDs before and after EBI. It can be seen that when the concentration of the N-Fe-CDs solution was 2.7×101 mg/mL, the cell survival rate was approximately 80%. Thus, it can be expected that the toxicity of N-Fe-CDs would have an inhibitory effect on LLC growth.45,46 As the concentration of the N-Fe-CDs solution gradually decreased from 2.7×100 mg/mL to 2.7×10−4 mg/mL, the cell survival rate generally showed an upward trend. When the concentration was 2.7×10−2 mg/mL, the cell survival rate was close to 100% which indicates the N-Fe-CDs solution have almost no toxicity to LLC cells with the concentration ≤2.7×10−2 mg/mL. It can be found from Figure 9 that when the concentration of CDs was <2.7×10−2 mg/mL, the survival rate of cells was >100%. This was mainly due to the proliferation of cells. When the added CDs solution was nontoxic for cells, the cells proliferation was in their normal cycle. The toxicity assessment was carried out 24 hours after the addition of CDs solution and co-culture with the cells. During these 24 hours, cell proliferation increased the number of cells with a cell survival rate >100%. The statistical analysis in Figure 9 shows that when the concentration of CDs is 2.7×10−2 mg/mL, there are statistically significant differences in the cell survival fraction compared with that of other most groups (p<0.05). Based on the previous analysis, it can be known that the statistical differences among the groups with concentrations >2.7×10−2 mg/mL are mainly caused by the cytotoxicity of CDs. The statistical differences with each group with a concentration of <2.7×10−2 mg/mL are mainly caused by the normal proliferation of cells. To verify the experimental results of CCK8, flow cytometry was used to detect the cytotoxicity of two samples with a concentration of 2.7×10−2 mg/mL before and after EBI in this study. The results are shown in Figure 9c and d, indicating that the results are consistent with those of CCK8. Figure 10 shows the sensitizing effect of N-Fe-CDs on tumor-bearing mice during electron beam radiotherapy. The tumor-bearing experimental mice model established using the LLC cell is given in Figure 10a. In Figure 10b the Control group represents the experimental results of the tumor-bearing mice with injection normal saline. The tumor volumes of the six control samples were relatively large and plump. The tumor growth was vigorous, and there were no obvious signs of inhibition. The N-Fe-CD group shows the experimental results of injecting an aqueous solution of N-Fe-CDs at a concentration of 2.7×10−2 mg/mL into the tumor-bearing mice. We can see that the tumor volumes do not have too much change and the inhibitory effect of injecting N-Fe-CDs into the tumor was not obvious. The paired t-test of the two groups (Control group vs CDs group), p>0.05, also confirmed this result. When the concentration of CDs is 2.7×10−2 mg/mL, CDs are almost non-toxic to tumors in vivo. The 8 Gy group and 16 Gy group are the results of radiotherapy with 8 Gy and 16 Gy doses irradiated by 10 MeV electrons. We can see that the tumor volumes were significantly smaller than those of the Control group and the CDs group. The results indicate that radiotherapy has a significant inhibitory effect on tumor growth and the inhibitory effect of electron beam radiotherapy on the tumor increases with increasing the radiotherapy dose, 16 Gy group vs 8 Gy group, p<0.01. The 8 Gy+CDs group and 16 Gy+CDs group show the results of tumor radiotherapy after injecting N-Fe-CDs into the tumor-bearing mice by using 8 Gy and 16 Gy doses of 10 MeV electrons radiation. We can see that the tumor volumes of these two groups were smaller than the previous groups, 8 Gy group vs 8 Gy+CDs group, p<0.05, and 16 Gy group vs 16 Gy+CDs group, p<0.05. The 16 Gy+CDs group has the smallest volume, and some tumor shapes were severely irregular and show a state of atrophy. Compared the results of 16 Gy+CDs group and 8 Gy+CDs group in Figure 9d, p<0.01. It indicates that the sensitization effect of N-Fe-CDs on electron beams may increase with the increase of dose. According to the theory of radiation biology, the inhibition of tumor growth is due to the attack and breakage of the DNA double helix structure by the irradiation. This would result in a decline in the reproductive ability of tumor cells and cause their death.11,47 In this study, there are two types of DNA breakage. As can be seen in Figure 10b, the 8 Gy and 16 Gy groups show that the DNA breakage can be directly caused by electron beam irradiation and the effect increases with the increasing of the irradiation dose.48,49 The second type is caused by the ionization of certain substances (such as H2O2) within the cells by electron radiation, generating free radicals such as ·OH and H·. These charged free radicals will attack DNA, causing the breakage of the double helix structure and resulting in a decline in the reproductive ability of tumor cells and even death.50 Comparing the experimental results 8 Gy group and 8 Gy+N-Fe-CDs group, or 16 Gy group and 16 Gy+N-Fe-CDs group in Figure 10b, we can obtain that N-Fe-CDs can strengthen the DNA breakage in tumor cells. The N-Fe-CDs prepared in this study are small and can penetrate the cell membrane and enter the tumor cells. Electron beam irradiation can ionize the functional groups such as hydroxyl and carboxyl on N-Fe-CDs to form free radicals in the cells which can cause more DNA double helix structure breakage to significantly inhibit the tumor growth. Thus, N-Fe-CDs can effectively enhance the sensitivity of electron beam therapy for lung tumors. Conclusion