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In vitro studies on radiosensitization effect of glucose capped gold nanoparticles in photon and ion irradiation of HeLa cells
Nuclear Instruments and Methods in Physics Research B 301 (2013) 7–11
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Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
In vitro studies on radiosensitization effect of glucose capped gold nanoparticles in photon and ion irradiation of HeLa cells Harminder Kaur a, Geetanjali Pujari a, Manoj K. Semwal b, Asitikantha Sarma a, Devesh Kumar Avasthi a,⇑ a b
Radiation Biology Group, Inter University Accelerator Centre, Post Box 10502, New Delhi 110067, India Army Hospital (R & R), Delhi Cantonment, New Delhi 110010, India
a r t i c l e
i n f o
Article history: Received 2 December 2012 Received in revised form 24 February 2013 Available online 7 March 2013 Keywords: Glucose capped gold nanoparticles HeLa cells c-Radiation 12 6+ C irradiation Radiosensitization
a b s t r a c t Noble metal nanoparticles are of great interest due to their potential applications in diagnostics and therapeutics. In the present work, we synthesized glucose capped gold nanoparticle (Glu-AuNP) for internalization in the HeLa cell line (human cervix cancer cells). The capping of glucose on Au nanoparticle was confirmed by Raman spectroscopy. The Glu-AuNP did not show any toxicity to the HeLa cell. The c-radiation and carbon ion irradiation of HeLa cell with and without Glu-AuNP were performed to evaluate radiosensitization effects. The study revealed a significant reduction in radiation dose for killing the HeLa cells with internalized Glu-AuNPs as compared to the HeLa cells without Glu-AuNP. The Glu-AuNP treatment resulted in enhancement of radiation effect as evident from increase in relative biological effectiveness (RBE) values for carbon ion irradiated HeLa cells. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Nanoparticles have attractive properties due to their large surface to volume ratio which renders them suitable for various applications. Noble metal nanoparticles have been of great interest in biotechnology for their role in (i) bio-sensing due to surface plasmon resonance [1,2], (ii) medical diagnostics due to better imaging contrast [3–5] and (iii) therapy [6–8]. The role of gold nanoparticles (AuNPs) in enhancement of radiation effect for cancer therapy is of vital importance [9]. Radiation (photon or ion beam) is one of the most effective and widely used modes for the treatment of cancer. Photon beam from 60Co source or linear accelerators, proton and carbon ion beam from ion accelerator are currently being used in clinical applications. In radiotherapy, it is desirable to deliver the radiation dose in such a way that the cancer cells are killed whereas there is minimal effect on the surrounding healthy cells. This is possible if one can enhance the effect of irradiation on the malignant cells for a given dose which is under tolerable limit of the surrounding normal cells. There have been efforts to develop the strategies to optimize the therapeutic index of radiotherapy using agents that can enhance the effect of radiation with the aim of controlling tumor growth. The incorporation of high Z elements has been proposed as a technique for radiosensitization (enhancement in effect of irradiation) of tumors [10–13]. There are several reports on the use of ⇑ Corresponding author. Tel.: +91 011 2689 3955; fax: +91 011 2689 3666. E-mail address: [email protected] (D.K. Avasthi). 0168-583X/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nimb.2013.02.015
AuNPs to enhance the radiation effect (X-rays and c-rays) on cancer cells [14–20]. Chithrani et al. quantified DNA double-strand breaks using radiation induced foci of gamma-H2AX and 53BP1, and observed a modest increase in number of foci per nucleus in irradiated cell populations with internalized gold nanoparticles [21]. Glu-AuNPs showed significant increase in radiosensitivity in prostate cancer cells [22]. Although non-functionalized AuNPs have shown attachment with cancer cell [23] and reasonable level of uptake by cancer cells, it is desirable to synthesize AuNPs with suitable functional group for efficient targeting and internalization. Glu-AuNPs have been reported to show significant increase in cellular uptake as compared to bare AuNP [24]. It has been reported that Glu-AuNPs uptake was seven times more in DU145 cells compared to AuNPs without glucose binding, indicating that glucose helps in delivery of more number of AuNPs into cells. It has also been reported that Glu-AuNPs do not enhance radiosensitivity in non-cancerous cells [25]. Since Glu-AuNPs has higher efficiency of internalization in cancer cell and they are non toxic, therefore, the Glu-AuNPs were chosen for internalization in the cells. In this work, we report the effect of c and 12C6+ ion beam irradiation on human cervical cancer cell line, HeLa, which were pretreated with Glu-AuNPs. It is seen that irradiation of Glu-AuNPs treated HeLa cells leads to a steeper survival curve as compared to that of irradiation of untreated cells. The result suggests an enhancement in the effect of radiation on HeLa cell in presence of Glu-AuNPs.
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H. Kaur et al. / Nuclear Instruments and Methods in Physics Research B 301 (2013) 7–11
2. Methodology 2.1. Synthesis of Glu-AuNPs The Glu-AuNPs were synthesized by the method proposed by Liu et al [26], using HAuCl4.3H2O (from Sigma, US) as the AuNP source and b-D-Glucose (from Sigma, US) as reducing agent. The synthesized Glu-AuNPs were characterized by UV-Vis spectroscopy and transmission electron microscopy (TEM) using electron beam accelerated at 200 kV (15,000 magnification), as shown in Fig. 1. The UV-Vis spectroscopy was performed using Hitachi U3300 spectrophotometer at Inter University Accelerator Centre (IUAC), New Delhi (India). Glucose capping of AuNPs was evaluated by Raman spectroscopic analysis. This required removal of unbound glucose from the solution of Glu-AuNPs. For this, Glu-AuNPs were centrifuged at 6000 rpm (Eppendorf centrifuge, USA) till GluAuNPs settled at the bottom separated by liquid at top. The liquid was pippetted out without disturbing settled Glu-AuNPs. This was further suspended in deionized water and this process of removing traces of unbound glucose was repeated thrice. The Raman spectroscopy was performed on the sample containing a drop of triply washed and as prepared Glu-AuNPs solution on Si substrate using InVia Raman Microscope (Renishaw, UK). The Raman spectrum obtained was compared with that of a drop of glucose on the Si substrate as shown in Fig. 2.
Fig. 2. Raman spectra of (i) a drop (10 ll) of glucose solution on Si substrate; (ii) a drop (10 ll) of triply washed glucose capped AuNPs solution on Si substrate.
card the unabsorbed Glu-AuNPs. The flasks were filled with fresh medium supplemented with FBS and penicillin streptomycin.
2.2. HeLa cell treatment with Glu-AuNPs
2.3. TEM imaging
HeLa cell line was procured from National Centre for Cell Science (NCCS), Pune, India. The cells were routinely cultured in DMEM (Dulbecco’s Modified Eagle Medium, Hi-Media, India) supplemented with 10% heat inactivated FBS (fetal bovine serum, Hyclone, US) and 1% penicillin streptomycin (Hyclone, US). The cells were maintained at 37 °C, 5% CO2 and 95% humidity. To evaluate the localization of Glu-AuNPs, the HeLa cells were incubated with 5.5 lM/ml of Glu-AuNPs for six hours. For this, asynchronous HeLa cells were cultured in 75 cm2 culture flask. At 80% confluency, cells were treated with Glu-AuNPs at concentration of 5.5 lM/ml for six hours. After incubation, the medium was decanted in order to dis-
After Glu-AuNP treatment, the cell fixation was done in 2.5% glutaraldehyde in 0.1 M Phosphate buffer (pH 7.2). The ultra-thin sections of fixed HeLa cells were prepared by ultramicrotomy. Thereafter, ultra thin sections of cells were stained with PTA (Phospho Tungstic Acid) prior to TEM viewing. The TEM images of ultrathin section of HeLa cells treated with Glu-AuNPs is shown in Fig. 3. The TEM imaging of Glu-AuNP treated HeLa cells was performed with electron beam accelerated at 120 kV (2500 magnification). The TEM measurements (using JEOL 2100F TEM apparatus) were performed at Advance Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi, India.
Fig. 1. (a) UV-Vis absorbance spectrum of Glu-AuNPs, showing characteristic surface plasmon resonance peak of AuNPs at 540 nm, (b) TEM image of Glu-AuNPs and (c) Histogram showing the size distribution of Glu-AuNPs.
H. Kaur et al. / Nuclear Instruments and Methods in Physics Research B 301 (2013) 7–11
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Cells were counted using Countess cell counter (Invitrogen) and pre-counted number of cells was seeded in 60 mm petriplates for colony forming assay. Since it is expected that the survival fraction would decrease exponentially as the dose of radiation is increased [28,29], thus 1000, 5000, 10,000, 15,000 and 30,000 cells for (1.8, 4.6, 7.4, 9.2 and 13.8 Gy of c-irradiated as well as 0.9, 1.9, 2.8, 3.7 and 4.6 Gy of 12C6+ irradiated HeLa cells respectively) were plated in the petridish to score a clonogenic survival data with reasonable statistics. The cells were incubated at 37 °C, 5% CO2 and 95% humidity for 10–12 days to form colonies. After staining with 0.25% methylene blue, visible colonies were counted with a criterion that a colony must have at least 50 cells. The survival fraction, for each dose level, was determined by dividing the number of colonies by number of cells seeded in the treated samples and was normalized with plating efficiency calculated from the control sample. For control HeLa cells (asynchronous population), the plating efficiency was calculated to be 73%. 3. Result and discussion Fig. 3. TEM image of ultra-thin section of HeLa cells treated with 5.5 lM GluAuNPs/ml for 6 h.
2.4. Cell irradiation The c-irradiations were carried out at the Army Hospital (Research and Referral), New Delhi, India using Theratron 1000E Telecobalt machine. For c irradiation, cells were maintained in 75 cm2 cell culture flasks and these properly sealed flasks were taken to Army Referral Hospital for c irradiation. For each data point, three flasks were irradiated and then irradiated cells were reseeded in 60 mm petriplate for colony forming assay (at IUAC). One set of HeLa cells was subjected to c-radiation at dose rate of 0.5 Gy/min with doses of 1.8, 4.6, 7.4, 9.2 and 13.8 Gy. 12C6+ ion beam irradiation was performed at Radiation Biology beam line of Pelletron accelerator at IUAC, New Delhi, India. It is equipped with the irradiation system called ASPIRE [Automatic Sample Positioning for Irradiation in Radiation Biology Experiments]. This is a computer controlled system that enables one to irradiate cells grown on 35 mm petri dishes. In a single run, ten of 35 mm petriplates are kept in sample tray filled with cell culture medium (without serum) in a sterile condition. The petriplates are picked up pneumatically and are irradiated at horizontal position, one after another at quick succession with predetermined particle dose. The uniformity of carbon ion beam was found to be 98% (with 2% SD) across field of 40 mm diameter as measured by counting the etched tracks in a polymer foil CN85. Dose was determined using the value of fluence. Another set of HeLa cells with and without Glu-AuNPs were seeded onto 35 mm petriplates to carry out irradiation in sterile irradiation chamber with 62 MeV 12C6+ having LET of 290 keV/lm (with particle flux rate of 2 105 particles/cm2/ sec). The irradiation was performed at the 12C6+ ion doses of 0.9, 1.9, 2.8, 3.7 and 4.6 Gy. While irradiation cells remaining unirradiated under the eclipse of medium at lower end of petriplate, were scrapped off. 2.5. Colony forming assay Colony forming assay was employed to assess survival fraction of HeLa cells with and without Glu-AuNPs treatment following c as well as 12C6+ beam irradiation as per standard method [27]. For each dose level, three petriplates were irradiated and cells from each petriplates were seeded into three culture petriplates (i.e. 9 petriplates in total for each dose point). After irradiation, the cells were detached from 35 mm petriplates using trypsin treatment.
The absorbance at 540 nm in UV-Vis spectrum of Glu-AuNPs suspension in water, shown in Fig. 1(a), is characteristic surface plasmon resonance of Au nanoparticles, providing an evidence for Au nanoparticles. It is clear from the Fig. 1(b) and (c) that most of the Glu-AuNPs are within 5–9 nm in diameter. Data from seven image frames was extracted for size distribution. To ensure the capping of AuNPs by glucose, Raman spectroscopy has been employed. The Raman spectrum of triply washed Glu-AuNP shown in Fig. 2 confirms the presence of glucose on AuNP. The Raman spectrum of a drop of glucose solution is also shown in Fig. 2 as a reference. The presence of vibrational modes at 1120 cm1 due to bending of C–O–H is the signature of glucose [30]. To confirm internalization of Glu-AuNPs in HeLa cells, the TEM imaging of ultrathin section of Glu-AuNPs treated HeLa cells was carried out. From TEM image of ultrathin section as shown in Fig. 3, it is observed that the size of Glu-AuNPs is 50–60 nm inside the cell. This might be due to agglomeration of nanoparticles in the cytoplasm within the cell [31]. Such agglomeration of AuNPs in the cell has also been observed by Nativo et al. [32]. It has been found that the AuNPs with size smaller than 50 nm find the endosome mediated path to get internalized into HeLa cells as reported by Wang et al. [33]. It is well established from study carried out by Rudlowski et al. that the up-regulation of Glut-1 receptors in HeLa cells plays an important role in higher transport of glucose inside HeLa cells [34]. Therefore Glut-1 receptors on HeLa cells are considered to be responsible for internalization of glucose functionalized AuNPs in HeLa cells. The toxicity of Glu-AuNPs was studied for HeLa cells and they were found to be non-cytotoxic as evident by the colony forming assay (Fig. 4). The results of colony forming assay of 12C6+ ion irradiated as well as c-irradiated HeLa cells with and without GluAuNPs pre-treatment in form of a survival curves are shown in Fig. 5. The experimental survival fraction [S/So] data points for cirradiated HeLa cells were fitted with linear quadratic dose [D] dependent relation given by
S=So ¼ exp ½aD þ bD2
ð1Þ
where a and b are constants. The fitted values of a and b for c-irradiated HeLa cells are given in Table 1. These data points are average values obtained by nine data points per dose level with standard deviation as error bars. It is well known that for heavy ions (with LET higher than 100 keV/lm), the b term is excluded due to higher value of error than b value itself [35]. In present study as well, using the linear quadratic fit, the value of error is found to be higher than b value for 12C6+ ion irradiated HeLa cells as evident from data given
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H. Kaur et al. / Nuclear Instruments and Methods in Physics Research B 301 (2013) 7–11 Table 3 REF and dose reduction values. Radiation type
REF value
Dose reduction (%)
c-Radiation
1.52 1.39
34 29
C6+ radiation
Table 4 RBE values for 90% cell killing. Radiation type
C6+ radiation *
Fig. 4. Survival fraction of HeLa cells treated with different concentration of GluAuNPs for 6 h.
RBE
Increment in RBE (%)
HeLa only
HeLa with GNP*
3.03
4.27
41
Here GNP refers to Glu-AuNPs.
S=So ¼ exp ½aD
ð2Þ
The values of a for the linear fit for HeLa cells (with and without Glu-AuNPs) irradiated with 12C6+ ions, are 0.710 ± 0.002 and 0.941 ± 0.009 respectively. The interpolated values from the fitted curves (Fig. 5) shows that there is a significant decrease in survival fraction on combined treatment of Glu-AuNPs and radiation (c or 12C6+ ions) in comparison to those cells exposed to radiation only for 90% cell killing as shown in Table 2. The data from Table 2 shows the dose reduction of 29% and 34% for carbon ion and c-irradiation to achieve 90% cell killing in the present in vitro experiment. Radiosensitivity Enhancement Factor, REF, [36] corresponding to 90% cell killing is calculated to quantify the radiosensitization due to the presence of Glu-AuNPs using Eq. (3):
REF ¼ Radiation dose for 90% Killing =Radiation dose in presence of AuNP for 90% killing ð3Þ
Fig. 5. Survival fraction of HeLa cells and the c/12C6+ irradiated HeLa cells pretreated with Glu-AuNPs for 6 h. In the figure, dotted arrows are given to show radiation dose corresponding to 10% cell survival. Block arrows are drawn to indicate radiation dose reduction in the presence of Glu-AuNPs inside the HeLa cells.
Table 1 Fitting parameters a and b for survival assay data. Radiation type
a (Gy1)
b (Gy1)
60
0.181 ± 0.025 0.321 ± 0.017 0.691 ± 0.021 1.011 ± 0.017
0.007 ± 0.003 0.008 ± 0.003 0.005 ± 0.006 0.024 ± 0.025
Co-c 60 Co-c + GNP* 12 6+ C 12 6+ C + GNP* *
Here GNP refers to Glu-AuNP.
Table 2 Radiation dose (c-radiation & 12C6+ ion beam) required for 90% cell killing with and without GNP (Glu-AuNP), extracted from Fig. 5. Radiation type
Dose (Gy) for 90% cell killing without GNP*
Dose (Gy) for 90% cell killing with GNP*
c-Radiation
9.4 3.1
6.2 2.2
12 6+
C
*
radiation
Here GNP refers to Glu-AuNP.
in Table 1. Since in the present case LET is 290 keV/lm, therefore data for 12C6+ ion irradiation (shown in Fig. 5) has been fitted by
The REF values for c and 12C6+ irradiation of Glu- AuNPs treated HeLa cells are shown in Table 3. Polf et al. observed enhanced relative radiobiological effectiveness (RBE) of proton beam in DU145 prostrate carcinoma cells with internalized AuNPs and their results demonstrated 15–20% enhancement in proton therapy at different doses of proton beam. RBE is defined as ratio of dose of 60Co c-radiation to that of test radiation for a given survival fraction (SF) value and is expressed as shown in Eq. (4) [37].
RBEðSFÞ ¼ D60 CoðSFÞ=D12 C6þ ðSFÞ
ð4Þ
We observed an increase in RBE value from 3.03 to 4.27 for 90% cell killing in 12C6+ irradiated HeLa cells when treated with GluAuNPs as shown in Table 4. From these RBE values, it is evident that Glu-AuNPs treatment of HeLa cells results in increase in RBE (by 41%) due to radiosensitization effect induced by Glu-AuNPs. This increment of RBE value in HeLa cells after combined treatment with Glu-AuNPs and 12 6+ C radiation, indicates the increase in the ionization density induced by 12C6+ radiation in the presence of Glu-AuNPs. The observed enhanced effect of radiation on HeLa cells in the presence of Glu-AuNPs is attributed to the consequence of interaction of photons (from c-radiation) and 12C6+ ion with AuNPs. The photons in 60Co source have energies of 1173 keV and 1332 keV. Such high energies of photon interact with matter dominantly via the process of Compton scattering. The presence of Glu-AuNPs in the cell causes enhancement in the number of Compton recoil electrons which subsequently interacts with cellular matter and results in higher radiolysis and DNA damage as compared to the cells without Glu-AuNPs. This mechanism of cell damage by Comp-
H. Kaur et al. / Nuclear Instruments and Methods in Physics Research B 301 (2013) 7–11
ton recoils by c-irradiation is well established [9]. In case of C ion irradiation, interaction of energetic 12C6+ ion with Glu-AuNPs treated HeLa cells produces increased number of secondary electrons during traversal inside the cells due to presence of Glu-AuNPs. These secondary electrons generate radiolysis products and bring about clustered DNA damage which is difficult to repair [38,39]. 4. Conclusion In the present study, we synthesized Glu-AuNPs and confirmed the glucose capping on AuNPs by Raman spectroscopy. The cell survival studies were performed on c and C ion irradiatied Hela cells with and without Glu-AuNP, which revealed radiosensitization of HeLa cells due to the presence of Glu-AuNP. A dose reduction of 34% and 29% for c-irradiation and 12C6+ irradiation respectively is demonstrated for Glu-AuNP treated Hela cells as compored to untreated cells. We also observed enhancement by 41% in RBE value for 12C6+ irradiation of HeLa treated with GluAuNPs. Acknowledgements The authors acknowledge Advance Instrumentation Research Facility (AIRF), JNU, New Delhi for carrying out cell sectioning by ultramicrotome and TEM imaging of the Glu-AuNPs samples. We are thankful to Pelletron operation group at IUAC for beam tuning and support during 12C6+ irradiation. Authors also extend thanks to Dr. Fouran Singh for Raman spectroscopy of Glu-AuNPs at IUAC. References [1] J.C.Y. Kah, K.W. Kho, C.G.L. Lee, C.J.R. Sheppard, Z.X. Shen, K.C. Soo, M.C. Olivo, Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles, Int. J. Nanomed. 2 (2007) 785–798. [2] T.A. Taton, G. Lu, C.A. Mirkin, Two-color labeling of oligonucleotide arrays via size-selective scattering of nanoparticle probes, J. Am. Chem. Soc. 123 (2001) 5164–5165. [3] I.H. El-Sayed, X. Huang, M.A. EI-Sayed, Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer, Nano. Lett. 5 (2005) 829–834. [4] O. Veiseh, C. Sun, J. Gunn, N. Kohler, P. Gabikian, D. Lee, N. Bhattarai, R. Ellenbogen, R. Sze, A. Hallahan, J. Olson, M. Zhang, Optical and MRI multifunctional nanoprobe for targeting gliomas, Nano. Lett. 5 (2005) 1003– 1008. [5] W. Jiang, E. Papa, H. Fischer, S. Mardyani, W.C.W. Chan, Semiconductor quantum dots as contrast agents for whole animal imaging, Trends Biotechnol. 22 (2004) 607–608. [6] J. Panyam, V. Labhasetwar, Biodegradable nanoparticles for drug delivery gene delivery to cells and tissue, Adv. Drug Delivery Rev. 5 (2003) 329–347. [7] P. Yang, X. Sun, J. Chiu, H. Sun, Q. He, Transferrin mediated gold nanoparticle cellular uptake, Bioconjugate Chem. 16 (2005) 494–496. [8] N. Kohler, C. Sun, J. Wang, M. Zhang, Methotrexate-immobilized poly(ethylene glycol) magnetic nanoparticles for MR imaging and drug delivery, Langmuir 21 (2005) 8855–8864. [9] K.T. Butterworth, J.A. Coulter, S. Jain, J. Forker, S.J. McMahon, G. Schettino, K.M. Prise, F.J. Currell, D.G. Hirst, Evaluation of cytotoxicity and radiation enhancement using 1.9 nm gold particles: potential application for cancer therapy, Nanotechnol 21 (2010) 295101. [10] R.S. Mello, H. Callisen, J. Winter, R. Kagan, A. Norman, Radiation dose enhancement in tumors with iodine, Am. Assoc. Phys. Med. (1983) 75–78. [11] T.D. Solberg, K.S. Iwamoto, A. Norman, Calculation of radiation dose enhancement factors for dose enhancement therapy of brain tumors, Phys. Med. Biol. 37 (1992) 439–443. [12] A.V. Mesa, A. Norman, T.D. Solberg, J.J. Demarco, J.B. Smathers, Dose distributions using kilovoltage x-rays and dose enhancement from iodine contrast agents, Phys. Med. Biol. 44 (1999) 1955–1968. [13] J.L. Robar, S.A. Riccio, M.A. Martin, Tumour dose enhancement using modified megavoltage photon beams and contrast media, Phys. Med. Biol. 47 (2002) 2433–2449.
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In vitro studies on radiosensitization effect of glucose capped gold nanoparticles in photon and ion irradiation of HeLa cells Harminder Kaur a, Geetanjali Pujari a, Manoj K. Semwal b, Asitikantha Sarma a, Devesh Kumar Avasthi a,⇑ a b
Radiation Biology Group, Inter University Accelerator Centre, Post Box 10502, New Delhi 110067, India Army Hospital (R & R), Delhi Cantonment, New Delhi 110010, India
a r t i c l e
i n f o
Article history: Received 2 December 2012 Received in revised form 24 February 2013 Available online 7 March 2013 Keywords: Glucose capped gold nanoparticles HeLa cells c-Radiation 12 6+ C irradiation Radiosensitization
a b s t r a c t Noble metal nanoparticles are of great interest due to their potential applications in diagnostics and therapeutics. In the present work, we synthesized glucose capped gold nanoparticle (Glu-AuNP) for internalization in the HeLa cell line (human cervix cancer cells). The capping of glucose on Au nanoparticle was confirmed by Raman spectroscopy. The Glu-AuNP did not show any toxicity to the HeLa cell. The c-radiation and carbon ion irradiation of HeLa cell with and without Glu-AuNP were performed to evaluate radiosensitization effects. The study revealed a significant reduction in radiation dose for killing the HeLa cells with internalized Glu-AuNPs as compared to the HeLa cells without Glu-AuNP. The Glu-AuNP treatment resulted in enhancement of radiation effect as evident from increase in relative biological effectiveness (RBE) values for carbon ion irradiated HeLa cells. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Nanoparticles have attractive properties due to their large surface to volume ratio which renders them suitable for various applications. Noble metal nanoparticles have been of great interest in biotechnology for their role in (i) bio-sensing due to surface plasmon resonance [1,2], (ii) medical diagnostics due to better imaging contrast [3–5] and (iii) therapy [6–8]. The role of gold nanoparticles (AuNPs) in enhancement of radiation effect for cancer therapy is of vital importance [9]. Radiation (photon or ion beam) is one of the most effective and widely used modes for the treatment of cancer. Photon beam from 60Co source or linear accelerators, proton and carbon ion beam from ion accelerator are currently being used in clinical applications. In radiotherapy, it is desirable to deliver the radiation dose in such a way that the cancer cells are killed whereas there is minimal effect on the surrounding healthy cells. This is possible if one can enhance the effect of irradiation on the malignant cells for a given dose which is under tolerable limit of the surrounding normal cells. There have been efforts to develop the strategies to optimize the therapeutic index of radiotherapy using agents that can enhance the effect of radiation with the aim of controlling tumor growth. The incorporation of high Z elements has been proposed as a technique for radiosensitization (enhancement in effect of irradiation) of tumors [10–13]. There are several reports on the use of ⇑ Corresponding author. Tel.: +91 011 2689 3955; fax: +91 011 2689 3666. E-mail address: [email protected] (D.K. Avasthi). 0168-583X/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nimb.2013.02.015
AuNPs to enhance the radiation effect (X-rays and c-rays) on cancer cells [14–20]. Chithrani et al. quantified DNA double-strand breaks using radiation induced foci of gamma-H2AX and 53BP1, and observed a modest increase in number of foci per nucleus in irradiated cell populations with internalized gold nanoparticles [21]. Glu-AuNPs showed significant increase in radiosensitivity in prostate cancer cells [22]. Although non-functionalized AuNPs have shown attachment with cancer cell [23] and reasonable level of uptake by cancer cells, it is desirable to synthesize AuNPs with suitable functional group for efficient targeting and internalization. Glu-AuNPs have been reported to show significant increase in cellular uptake as compared to bare AuNP [24]. It has been reported that Glu-AuNPs uptake was seven times more in DU145 cells compared to AuNPs without glucose binding, indicating that glucose helps in delivery of more number of AuNPs into cells. It has also been reported that Glu-AuNPs do not enhance radiosensitivity in non-cancerous cells [25]. Since Glu-AuNPs has higher efficiency of internalization in cancer cell and they are non toxic, therefore, the Glu-AuNPs were chosen for internalization in the cells. In this work, we report the effect of c and 12C6+ ion beam irradiation on human cervical cancer cell line, HeLa, which were pretreated with Glu-AuNPs. It is seen that irradiation of Glu-AuNPs treated HeLa cells leads to a steeper survival curve as compared to that of irradiation of untreated cells. The result suggests an enhancement in the effect of radiation on HeLa cell in presence of Glu-AuNPs.
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2. Methodology 2.1. Synthesis of Glu-AuNPs The Glu-AuNPs were synthesized by the method proposed by Liu et al [26], using HAuCl4.3H2O (from Sigma, US) as the AuNP source and b-D-Glucose (from Sigma, US) as reducing agent. The synthesized Glu-AuNPs were characterized by UV-Vis spectroscopy and transmission electron microscopy (TEM) using electron beam accelerated at 200 kV (15,000 magnification), as shown in Fig. 1. The UV-Vis spectroscopy was performed using Hitachi U3300 spectrophotometer at Inter University Accelerator Centre (IUAC), New Delhi (India). Glucose capping of AuNPs was evaluated by Raman spectroscopic analysis. This required removal of unbound glucose from the solution of Glu-AuNPs. For this, Glu-AuNPs were centrifuged at 6000 rpm (Eppendorf centrifuge, USA) till GluAuNPs settled at the bottom separated by liquid at top. The liquid was pippetted out without disturbing settled Glu-AuNPs. This was further suspended in deionized water and this process of removing traces of unbound glucose was repeated thrice. The Raman spectroscopy was performed on the sample containing a drop of triply washed and as prepared Glu-AuNPs solution on Si substrate using InVia Raman Microscope (Renishaw, UK). The Raman spectrum obtained was compared with that of a drop of glucose on the Si substrate as shown in Fig. 2.
Fig. 2. Raman spectra of (i) a drop (10 ll) of glucose solution on Si substrate; (ii) a drop (10 ll) of triply washed glucose capped AuNPs solution on Si substrate.
card the unabsorbed Glu-AuNPs. The flasks were filled with fresh medium supplemented with FBS and penicillin streptomycin.
2.2. HeLa cell treatment with Glu-AuNPs
2.3. TEM imaging
HeLa cell line was procured from National Centre for Cell Science (NCCS), Pune, India. The cells were routinely cultured in DMEM (Dulbecco’s Modified Eagle Medium, Hi-Media, India) supplemented with 10% heat inactivated FBS (fetal bovine serum, Hyclone, US) and 1% penicillin streptomycin (Hyclone, US). The cells were maintained at 37 °C, 5% CO2 and 95% humidity. To evaluate the localization of Glu-AuNPs, the HeLa cells were incubated with 5.5 lM/ml of Glu-AuNPs for six hours. For this, asynchronous HeLa cells were cultured in 75 cm2 culture flask. At 80% confluency, cells were treated with Glu-AuNPs at concentration of 5.5 lM/ml for six hours. After incubation, the medium was decanted in order to dis-
After Glu-AuNP treatment, the cell fixation was done in 2.5% glutaraldehyde in 0.1 M Phosphate buffer (pH 7.2). The ultra-thin sections of fixed HeLa cells were prepared by ultramicrotomy. Thereafter, ultra thin sections of cells were stained with PTA (Phospho Tungstic Acid) prior to TEM viewing. The TEM images of ultrathin section of HeLa cells treated with Glu-AuNPs is shown in Fig. 3. The TEM imaging of Glu-AuNP treated HeLa cells was performed with electron beam accelerated at 120 kV (2500 magnification). The TEM measurements (using JEOL 2100F TEM apparatus) were performed at Advance Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi, India.
Fig. 1. (a) UV-Vis absorbance spectrum of Glu-AuNPs, showing characteristic surface plasmon resonance peak of AuNPs at 540 nm, (b) TEM image of Glu-AuNPs and (c) Histogram showing the size distribution of Glu-AuNPs.
H. Kaur et al. / Nuclear Instruments and Methods in Physics Research B 301 (2013) 7–11
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Cells were counted using Countess cell counter (Invitrogen) and pre-counted number of cells was seeded in 60 mm petriplates for colony forming assay. Since it is expected that the survival fraction would decrease exponentially as the dose of radiation is increased [28,29], thus 1000, 5000, 10,000, 15,000 and 30,000 cells for (1.8, 4.6, 7.4, 9.2 and 13.8 Gy of c-irradiated as well as 0.9, 1.9, 2.8, 3.7 and 4.6 Gy of 12C6+ irradiated HeLa cells respectively) were plated in the petridish to score a clonogenic survival data with reasonable statistics. The cells were incubated at 37 °C, 5% CO2 and 95% humidity for 10–12 days to form colonies. After staining with 0.25% methylene blue, visible colonies were counted with a criterion that a colony must have at least 50 cells. The survival fraction, for each dose level, was determined by dividing the number of colonies by number of cells seeded in the treated samples and was normalized with plating efficiency calculated from the control sample. For control HeLa cells (asynchronous population), the plating efficiency was calculated to be 73%. 3. Result and discussion Fig. 3. TEM image of ultra-thin section of HeLa cells treated with 5.5 lM GluAuNPs/ml for 6 h.
2.4. Cell irradiation The c-irradiations were carried out at the Army Hospital (Research and Referral), New Delhi, India using Theratron 1000E Telecobalt machine. For c irradiation, cells were maintained in 75 cm2 cell culture flasks and these properly sealed flasks were taken to Army Referral Hospital for c irradiation. For each data point, three flasks were irradiated and then irradiated cells were reseeded in 60 mm petriplate for colony forming assay (at IUAC). One set of HeLa cells was subjected to c-radiation at dose rate of 0.5 Gy/min with doses of 1.8, 4.6, 7.4, 9.2 and 13.8 Gy. 12C6+ ion beam irradiation was performed at Radiation Biology beam line of Pelletron accelerator at IUAC, New Delhi, India. It is equipped with the irradiation system called ASPIRE [Automatic Sample Positioning for Irradiation in Radiation Biology Experiments]. This is a computer controlled system that enables one to irradiate cells grown on 35 mm petri dishes. In a single run, ten of 35 mm petriplates are kept in sample tray filled with cell culture medium (without serum) in a sterile condition. The petriplates are picked up pneumatically and are irradiated at horizontal position, one after another at quick succession with predetermined particle dose. The uniformity of carbon ion beam was found to be 98% (with 2% SD) across field of 40 mm diameter as measured by counting the etched tracks in a polymer foil CN85. Dose was determined using the value of fluence. Another set of HeLa cells with and without Glu-AuNPs were seeded onto 35 mm petriplates to carry out irradiation in sterile irradiation chamber with 62 MeV 12C6+ having LET of 290 keV/lm (with particle flux rate of 2 105 particles/cm2/ sec). The irradiation was performed at the 12C6+ ion doses of 0.9, 1.9, 2.8, 3.7 and 4.6 Gy. While irradiation cells remaining unirradiated under the eclipse of medium at lower end of petriplate, were scrapped off. 2.5. Colony forming assay Colony forming assay was employed to assess survival fraction of HeLa cells with and without Glu-AuNPs treatment following c as well as 12C6+ beam irradiation as per standard method [27]. For each dose level, three petriplates were irradiated and cells from each petriplates were seeded into three culture petriplates (i.e. 9 petriplates in total for each dose point). After irradiation, the cells were detached from 35 mm petriplates using trypsin treatment.
The absorbance at 540 nm in UV-Vis spectrum of Glu-AuNPs suspension in water, shown in Fig. 1(a), is characteristic surface plasmon resonance of Au nanoparticles, providing an evidence for Au nanoparticles. It is clear from the Fig. 1(b) and (c) that most of the Glu-AuNPs are within 5–9 nm in diameter. Data from seven image frames was extracted for size distribution. To ensure the capping of AuNPs by glucose, Raman spectroscopy has been employed. The Raman spectrum of triply washed Glu-AuNP shown in Fig. 2 confirms the presence of glucose on AuNP. The Raman spectrum of a drop of glucose solution is also shown in Fig. 2 as a reference. The presence of vibrational modes at 1120 cm1 due to bending of C–O–H is the signature of glucose [30]. To confirm internalization of Glu-AuNPs in HeLa cells, the TEM imaging of ultrathin section of Glu-AuNPs treated HeLa cells was carried out. From TEM image of ultrathin section as shown in Fig. 3, it is observed that the size of Glu-AuNPs is 50–60 nm inside the cell. This might be due to agglomeration of nanoparticles in the cytoplasm within the cell [31]. Such agglomeration of AuNPs in the cell has also been observed by Nativo et al. [32]. It has been found that the AuNPs with size smaller than 50 nm find the endosome mediated path to get internalized into HeLa cells as reported by Wang et al. [33]. It is well established from study carried out by Rudlowski et al. that the up-regulation of Glut-1 receptors in HeLa cells plays an important role in higher transport of glucose inside HeLa cells [34]. Therefore Glut-1 receptors on HeLa cells are considered to be responsible for internalization of glucose functionalized AuNPs in HeLa cells. The toxicity of Glu-AuNPs was studied for HeLa cells and they were found to be non-cytotoxic as evident by the colony forming assay (Fig. 4). The results of colony forming assay of 12C6+ ion irradiated as well as c-irradiated HeLa cells with and without GluAuNPs pre-treatment in form of a survival curves are shown in Fig. 5. The experimental survival fraction [S/So] data points for cirradiated HeLa cells were fitted with linear quadratic dose [D] dependent relation given by
S=So ¼ exp ½aD þ bD2
ð1Þ
where a and b are constants. The fitted values of a and b for c-irradiated HeLa cells are given in Table 1. These data points are average values obtained by nine data points per dose level with standard deviation as error bars. It is well known that for heavy ions (with LET higher than 100 keV/lm), the b term is excluded due to higher value of error than b value itself [35]. In present study as well, using the linear quadratic fit, the value of error is found to be higher than b value for 12C6+ ion irradiated HeLa cells as evident from data given
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H. Kaur et al. / Nuclear Instruments and Methods in Physics Research B 301 (2013) 7–11 Table 3 REF and dose reduction values. Radiation type
REF value
Dose reduction (%)
c-Radiation
1.52 1.39
34 29
C6+ radiation
Table 4 RBE values for 90% cell killing. Radiation type
C6+ radiation *
Fig. 4. Survival fraction of HeLa cells treated with different concentration of GluAuNPs for 6 h.
RBE
Increment in RBE (%)
HeLa only
HeLa with GNP*
3.03
4.27
41
Here GNP refers to Glu-AuNPs.
S=So ¼ exp ½aD
ð2Þ
The values of a for the linear fit for HeLa cells (with and without Glu-AuNPs) irradiated with 12C6+ ions, are 0.710 ± 0.002 and 0.941 ± 0.009 respectively. The interpolated values from the fitted curves (Fig. 5) shows that there is a significant decrease in survival fraction on combined treatment of Glu-AuNPs and radiation (c or 12C6+ ions) in comparison to those cells exposed to radiation only for 90% cell killing as shown in Table 2. The data from Table 2 shows the dose reduction of 29% and 34% for carbon ion and c-irradiation to achieve 90% cell killing in the present in vitro experiment. Radiosensitivity Enhancement Factor, REF, [36] corresponding to 90% cell killing is calculated to quantify the radiosensitization due to the presence of Glu-AuNPs using Eq. (3):
REF ¼ Radiation dose for 90% Killing =Radiation dose in presence of AuNP for 90% killing ð3Þ
Fig. 5. Survival fraction of HeLa cells and the c/12C6+ irradiated HeLa cells pretreated with Glu-AuNPs for 6 h. In the figure, dotted arrows are given to show radiation dose corresponding to 10% cell survival. Block arrows are drawn to indicate radiation dose reduction in the presence of Glu-AuNPs inside the HeLa cells.
Table 1 Fitting parameters a and b for survival assay data. Radiation type
a (Gy1)
b (Gy1)
60
0.181 ± 0.025 0.321 ± 0.017 0.691 ± 0.021 1.011 ± 0.017
0.007 ± 0.003 0.008 ± 0.003 0.005 ± 0.006 0.024 ± 0.025
Co-c 60 Co-c + GNP* 12 6+ C 12 6+ C + GNP* *
Here GNP refers to Glu-AuNP.
Table 2 Radiation dose (c-radiation & 12C6+ ion beam) required for 90% cell killing with and without GNP (Glu-AuNP), extracted from Fig. 5. Radiation type
Dose (Gy) for 90% cell killing without GNP*
Dose (Gy) for 90% cell killing with GNP*
c-Radiation
9.4 3.1
6.2 2.2
12 6+
C
*
radiation
Here GNP refers to Glu-AuNP.
in Table 1. Since in the present case LET is 290 keV/lm, therefore data for 12C6+ ion irradiation (shown in Fig. 5) has been fitted by
The REF values for c and 12C6+ irradiation of Glu- AuNPs treated HeLa cells are shown in Table 3. Polf et al. observed enhanced relative radiobiological effectiveness (RBE) of proton beam in DU145 prostrate carcinoma cells with internalized AuNPs and their results demonstrated 15–20% enhancement in proton therapy at different doses of proton beam. RBE is defined as ratio of dose of 60Co c-radiation to that of test radiation for a given survival fraction (SF) value and is expressed as shown in Eq. (4) [37].
RBEðSFÞ ¼ D60 CoðSFÞ=D12 C6þ ðSFÞ
ð4Þ
We observed an increase in RBE value from 3.03 to 4.27 for 90% cell killing in 12C6+ irradiated HeLa cells when treated with GluAuNPs as shown in Table 4. From these RBE values, it is evident that Glu-AuNPs treatment of HeLa cells results in increase in RBE (by 41%) due to radiosensitization effect induced by Glu-AuNPs. This increment of RBE value in HeLa cells after combined treatment with Glu-AuNPs and 12 6+ C radiation, indicates the increase in the ionization density induced by 12C6+ radiation in the presence of Glu-AuNPs. The observed enhanced effect of radiation on HeLa cells in the presence of Glu-AuNPs is attributed to the consequence of interaction of photons (from c-radiation) and 12C6+ ion with AuNPs. The photons in 60Co source have energies of 1173 keV and 1332 keV. Such high energies of photon interact with matter dominantly via the process of Compton scattering. The presence of Glu-AuNPs in the cell causes enhancement in the number of Compton recoil electrons which subsequently interacts with cellular matter and results in higher radiolysis and DNA damage as compared to the cells without Glu-AuNPs. This mechanism of cell damage by Comp-
H. Kaur et al. / Nuclear Instruments and Methods in Physics Research B 301 (2013) 7–11
ton recoils by c-irradiation is well established [9]. In case of C ion irradiation, interaction of energetic 12C6+ ion with Glu-AuNPs treated HeLa cells produces increased number of secondary electrons during traversal inside the cells due to presence of Glu-AuNPs. These secondary electrons generate radiolysis products and bring about clustered DNA damage which is difficult to repair [38,39]. 4. Conclusion In the present study, we synthesized Glu-AuNPs and confirmed the glucose capping on AuNPs by Raman spectroscopy. The cell survival studies were performed on c and C ion irradiatied Hela cells with and without Glu-AuNP, which revealed radiosensitization of HeLa cells due to the presence of Glu-AuNP. A dose reduction of 34% and 29% for c-irradiation and 12C6+ irradiation respectively is demonstrated for Glu-AuNP treated Hela cells as compored to untreated cells. We also observed enhancement by 41% in RBE value for 12C6+ irradiation of HeLa treated with GluAuNPs. Acknowledgements The authors acknowledge Advance Instrumentation Research Facility (AIRF), JNU, New Delhi for carrying out cell sectioning by ultramicrotome and TEM imaging of the Glu-AuNPs samples. We are thankful to Pelletron operation group at IUAC for beam tuning and support during 12C6+ irradiation. Authors also extend thanks to Dr. Fouran Singh for Raman spectroscopy of Glu-AuNPs at IUAC. References [1] J.C.Y. Kah, K.W. Kho, C.G.L. Lee, C.J.R. Sheppard, Z.X. Shen, K.C. Soo, M.C. Olivo, Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles, Int. J. Nanomed. 2 (2007) 785–798. [2] T.A. Taton, G. Lu, C.A. Mirkin, Two-color labeling of oligonucleotide arrays via size-selective scattering of nanoparticle probes, J. Am. Chem. Soc. 123 (2001) 5164–5165. [3] I.H. El-Sayed, X. Huang, M.A. EI-Sayed, Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer, Nano. Lett. 5 (2005) 829–834. [4] O. Veiseh, C. Sun, J. Gunn, N. Kohler, P. Gabikian, D. Lee, N. Bhattarai, R. Ellenbogen, R. Sze, A. Hallahan, J. Olson, M. Zhang, Optical and MRI multifunctional nanoprobe for targeting gliomas, Nano. Lett. 5 (2005) 1003– 1008. [5] W. Jiang, E. Papa, H. Fischer, S. Mardyani, W.C.W. Chan, Semiconductor quantum dots as contrast agents for whole animal imaging, Trends Biotechnol. 22 (2004) 607–608. [6] J. Panyam, V. Labhasetwar, Biodegradable nanoparticles for drug delivery gene delivery to cells and tissue, Adv. Drug Delivery Rev. 5 (2003) 329–347. [7] P. Yang, X. Sun, J. Chiu, H. Sun, Q. He, Transferrin mediated gold nanoparticle cellular uptake, Bioconjugate Chem. 16 (2005) 494–496. [8] N. Kohler, C. Sun, J. Wang, M. Zhang, Methotrexate-immobilized poly(ethylene glycol) magnetic nanoparticles for MR imaging and drug delivery, Langmuir 21 (2005) 8855–8864. [9] K.T. Butterworth, J.A. Coulter, S. Jain, J. Forker, S.J. McMahon, G. Schettino, K.M. Prise, F.J. Currell, D.G. Hirst, Evaluation of cytotoxicity and radiation enhancement using 1.9 nm gold particles: potential application for cancer therapy, Nanotechnol 21 (2010) 295101. [10] R.S. Mello, H. Callisen, J. Winter, R. Kagan, A. Norman, Radiation dose enhancement in tumors with iodine, Am. Assoc. Phys. Med. (1983) 75–78. [11] T.D. Solberg, K.S. Iwamoto, A. Norman, Calculation of radiation dose enhancement factors for dose enhancement therapy of brain tumors, Phys. Med. Biol. 37 (1992) 439–443. [12] A.V. Mesa, A. Norman, T.D. Solberg, J.J. Demarco, J.B. Smathers, Dose distributions using kilovoltage x-rays and dose enhancement from iodine contrast agents, Phys. Med. Biol. 44 (1999) 1955–1968. [13] J.L. Robar, S.A. Riccio, M.A. Martin, Tumour dose enhancement using modified megavoltage photon beams and contrast media, Phys. Med. Biol. 47 (2002) 2433–2449.
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