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Energy Dependence of Gold Nanoparticle Radiosensitization in Plasmid DNA Stephen J. McMahon,*,†,‡ Wendy B. Hyland,‡,§ Emilie Brun,|| Karl T. Butterworth,† Jonathan A. Coulter,† Thierry Douki,^ David G. Hirst,§ Suneil Jain,†,# Anthony P. Kavanagh,r Zeljka Krpetic,O Marcus H. Mendenhall,[ Mark F. Muir,‡ Kevin M. Prise,† Herwig Requardt,z Leon Sanche,||,†† Giuseppe Schettino,† Fred J. Currell,‡ and Cecile Sicard-Roselli|| †
Centre for Cancer Research and Cell Biology, School of Medicine, ‡Centre for Plasma Physics, School of Mathematics and Physics, Experimental Therapeutics Research Group, School of Pharmacy, and #Northern Ireland Cancer Centre, Queen’s University, Belfast, Northern Ireland Laboratoire de Chimie Physique, CNRS UMR 8000, Universite Paris-Sud, B^atiment 350, 91405 Orsay Cedex, France ^ Lesions des Acides Nucleiques, INAC/SCIB UMR-E3 CEA-UJF CNRS FRE 3200/CEA-Grenoble, 17 avenue des Martyrs, 38054 Grenoble Cedex 9, France r Advanced Technology Development Group, ORCRB, University of Oxford, Old Road Campus, Oxford, United Kingdom O Centre for Nanoscale Science, Department of Chemistry, University of Liverpool, Crown Street, Liverpool L69 7ZD, United Kingdom [ Department of Electrical Engineering and Computer Science, Vanderbilt University, P.O. Box 351824B, Nashville, Tennessee 37235-1824, United States z European Synchrotron Radiation Facility (ESRF), 6 Rue Horovitz, F-34043 Grenoble, France †† Groupe en Sciences des Radiations, Departement de medecine nucleaire et radiobiologie, Faculte de medecine, Universite de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada
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ABSTRACT: Gold nanoparticles (GNPs) are of considerable interest for use as a radiosensitizer, because of their biocompatibility and their ability to increase dose deposited because of their high mass energy absorption coefficient. Their sensitizing properties have been verified experimentally, but a discrepancy between the experimental results and theoretical predictions suggests that the sensitizing effect does not depend solely on gold’s superior absorption of energetic photons. This work presents the results of three sets of experiments that independently mapped out the energy dependence of the radiosensitizing effects of GNPs on plasmid DNA suspended in water. Incident photon energy was varied from 11.8 to 80 keV through the use of monochromatic synchrotron and broadband X-rays. These results depart significantly from the theoretical predictions in two ways: First, the sensitization is significantly larger than would be predicted; second, it does not vary with energy as would be predicted from energy absorption coefficients. These results clearly demonstrate that the effects of GNP-enhanced therapies cannot be predicted by considering additional dose alone and that a greater understanding of the processes involved is necessary for the development of future therapeutics.
’ INTRODUCTION The aim of radiotherapy is to deliver a lethal dose to tumor volumes while at the same time avoiding exposure to healthy tissue. However, this task is significantly complicated by the similar mass energy absorption characteristics of healthy and cancerous tissues. To overcome this difficulty, most modern radiotherapy is delivered using many spatially modulated fields that combine to create dose profiles that conform tightly to tumor volumes. Unfortunately, these approaches are still often limited by the toxicity that results from the dose delivered to healthy tissue. As a result, there is considerable interest in techniques that could increase the sensitivity of cancerous cells to radiation. r 2011 American Chemical Society
One such approach that has gained attention in recent years is the use of heavy-element contrast agents. These agents significantly increase the dose deposited in their vicinity because of their high energy absorption coefficients. Some of the earliest observations of this effect came from negative effects seen near metallic implants following radiotherapy and after contrast-enhanced imaging.1,2 However, selective delivery of such agents to tumors is increasingly being investigated as a possible therapeutic approach.35 Received: July 18, 2011 Revised: September 13, 2011 Published: September 13, 2011 20160
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for the reconciliation of many results in the literature and for the design of novel GNPs as future therapeutic agents. To address this need, experiments were carried out to measure the effect of GNPs on the sensitivity of DNA to a range of X-ray energies using synchrotron X-rays. Two independent sets of experiments were carried out, one at the Diamond Light Source and one at the European Synchrotron Radiation Facility (ESRF), together with supporting experiments using a tunable broadband Therapax X-ray source. Although these experiments were originally designed and carried out independently, they are presented together in this work to highlight the broad agreement in energy dependence found between the different studies.
’ MATERIALS AND METHODS Figure 1. Comparison of photon mass energy absorption coefficients of gold and soft tissue, along with their ratio, as a function of photon energy. Data from Hubbell and Seltzer.36 A similar relationship exists with water, whose energy absorption coefficient differs by less than 4% from that of soft tissue.
Although there are many potential heavy-atom contrast agents, gold nanoparticles (GNPs) are of particular interest because of the combination of gold’s high atomic number and the apparent biocompatibility of GNPs.69 The additional observation that such particles tend to accumulate in tumor cells in vivo and in vitro1012 makes them an ideal therapeutic agent. A comparison of the mass energy absorption coefficients of gold and soft tissue is shown in Figure 1. This plot clearly shows the rationale for the use of contrast agents, as gold absorbs between 10 and 150 times more energy per unit mass than soft tissue for photons in the kilovolt range. This corresponds to a significant increase in dose: For example, the addition of 1% by mass of gold to a tissue would almost double the amount of energy deposited by a kilovolt X-ray source. Similar values hold for water volumes, whose mass energy absorption coefficient differs by less than 4% from that of soft tissue across this energy range. These simple predictions of increased dose deposition have been validated by a series of theoretical studies.1317 However, when measured experimentally both in vitro1822 and in vivo,10,11,23 the sensitizing effects of GNPs significantly differ from the predictions and do not appear to be well described by physical predictions of the additional dose deposited as a result of the presence of GNPs. Instead, many studies report substantial radiosensitization at gold concentrations that are orders of magnitude smaller than those that are needed to significantly increase the dose deposited in the sample. This might be due to the significant dose inhomogeneities that are introduced by the addition of GNPs, which one recent study suggested might significantly increase the biological effectiveness of a given concentration of GNPs.24 Additionally, whereas some studies have reported a degree of dependence on X-ray energy, such findings are also in poor agreement with theoretical predictions and have typically been made using broadband X-ray sources.19,25 Of particular interest are experiments that have observed enhancement with photons in the megavolt range.20,21 At these energies, there is negligible difference between the mass energy absorption of gold and soft tissue, which suggests that the radiosensitization is the result of a different phenomenon. The development of an accurate description of the underlying mechanisms of GNP radiosensitization is very important, both
DNA Preparation. Three different plasmids were studied in the three different sets of experiments presented. The plasmid used at the Diamond synchrotron was the 5400base-pair pcDNA3.1 (Invitrogen, Paisley, U.K.), which was propagated in E. coli TOP10 cells and purified using a Qiagen plasmid maxi kit (Qiagen, Hilden, Germany). The purified DNA was resuspended in Tris-EDTA buffer [10 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl), 1 mM disodium ethylenediaminetetraacetic acid (EDTA), pH 8.0] at a concentration of 700 μg/mL and stored at 20 C until used. pUC18 (2686 base pairs) and pGEM-3Zf() (3197 base pairs) (Promega, Southampton, U.K.) plasmids were used at the ESRF and with the Therapax source, respectively. Both plasmids were extracted from E. coli DH5α and purified with the QIAfilter Plasmid Giga Kit (Qiagen). The DNA pellet was resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 8) and stored at 50 μg/mL at 80 C. Before use, these plasmids were desalted using Sephadex G-50 purification as previously described.25 For all DNA preparations, agarose gel electrophoresis showed that g95% of the extracted plasmid was in the supercoiled form. DNA purity was checked by comparing the absorbance at 260 and 280 nm; a ratio of 1.98 indicated the absence of any protein contamination. Concentrations were determined by measuring DNA absorption at 260 nm. GNP Preparation. In this work, the radiosensitizing effects of two kinds of GNP were investigated, namely, citrate-reduced particles with diameters of 11.9 and 37.5 nm, both prepared through the TurkevichFrens citrate reduction procedure.26,27 For use in the Diamond experiments, 11.9-nm-diameter GNPs were prepared as follows: First, 0.2 mmol (78.8 mg) of HAuCl4 3 3H2O was dissolved in 200 mL of milli-Q water, and the solution was heated to boiling. Subsequently, 20 mL of 40 mM aqueous trisodium citrate solution, previously warmed to 70 C, was added, and the mixture was refluxed for 1 h under vigorous stirring. The solution was then allowed to cool to room temperature under vigorous stirring overnight. The colloid was then filtered through a 0.45-μm syringe filter and stored at 4 C. The GNPs produced by this process were found to have a mean diameter of 11.9 nm and a concentration of 61 μg/mL. The 37.5-nm-diameter GNPs, used for ESRF and Therapax exposures, were synthesized by mixing 4.2 mL of a 1% (w/v) solution of trisodium citrate with 0.1 mmol of KAuCl4 in 100 mL of aqueous solution. The final solution was then heated under moderate stirring for 10 min after the solution had turned purple. To remove most of the free citrate, the GNPs were subjected to centrifugation (2300g, 20 min) three times, and the pellet of concentrated GNPs was resuspended in water at a concentration 20161
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The Journal of Physical Chemistry C of 8000 μg/mL (corresponding to a nanoparticle molar concentration of 25 nM) and kept at 4 C. GNP Characterization. GNPs were characterized both by transmission electron microscopy (TEM) and by measurement of their absorption characteristics in the visible spectrum. For TEM analysis, 10 μL of the GNP solution was deposited on a copper grid (400 mesh) and imaged with a JEOL 100CXII TEM instrument (JEOL, Tokyo, Japan) or with a Philips CM120 electron microscope, both operating at 80 keV. GNP size was measured from these images using ImageJ 1.41 software.28 The absorbance of the GNP solution was measured from 400 to 800 nm (using a Varian Cary 50 or a Thermo Electron corporation Evolution 500 spectrometer), spanning the well-characterized range where GNPs absorb most strongly because of their plasmon resonance.29 The height of this peak provided a measurement of the GNP concentration, and the location provided an additional check on the GNP size. In both cases, previously determined absorption characteristics were used to verify GNP properties.30 Sample Preparation. Samples were prepared immediately before irradiation, by combining DNA and GNP stocks and diluting to reach the target concentration. For experiments carried out at the Diamond synchrotron, a DNA concentration of 17.5 μg/mL (5 nM) was used, together with a 13 μg/mL concentration of GNPs, corresponding to a molar nanoparticle concentration of 1.25 nM. This gave a ratio of 1 GNP per 4 plasmids, or 1 GNP per 21600 base pairs. These solutions were diluted in distilled water to reach the target concentrations. This gave a final Tris-EDTA concentration of 0.25 mM in the irradiated samples, which provided only a weak scavenging capacity. This was necessary to provide sufficient sensitivity to damage to allow for radiation exposures carried out at the synchrotron to be completed on practical time scales. The ESRF and Therapax experiments were performed in water with DNA concentrations of 2 μg/mL (1 nM), together with a GNP concentration of 1600 μg/mL (5 nM nanoparticles) at ESRF and 320 μg/mL (1 nM) with the Therapax source. These concentrations correspond to 1 GNP per 640 base pairs and 1 GNP per 3197 base pairs, respectively. These GNP concentrations were selected based on previous work.25 It should be noted that these concentrations are, in all cases, substantially less than the 1% by mass (10000 μg/mL) that is necessary to significantly increase the dose deposited in the sample based on theoretical studies. Additional experiments investigating the effects of varying scavenging capacities on the GNP dose enhancement were also carried out at Queen’s University Belfast (QUB), with samples diluted in varying combinations of Tris-EDTA and water to provide a range of scavenging capacities, ranging from the low 0.25 mM concentration used at Diamond to 5 mM, both with and without GNPs. Diamond and QUB Irradiations. Beamlines B16 and I15 were used at the Diamond Light Source for this work, providing X-rays with energies below and above 20 keV, respectively. Both beamlines provide extremely intense radiation on a small spot, with a maximum dose rate of 195 Gy s1 being obtained for a 0.5mm-diameter spot at 11.8 keV. A sample translation system was developed that enabled samples to be rastered through the beam at high speeds, allowing for the delivery of low, uniform doses over large areas through relatively short exposure times.31 Uniformity of the dose profile was verified by attaching Gafchromic film (Gafchromic EBT film,
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International Specialty Products) to the translation stage during irradiations. Unirradiated controls were placed on the translator along with corresponding irradiated samples. At Diamond, samples were exposed to X-rays with energies of 11.8, 14.5, 20, 25.6, 38.5, 52.3, and 60 keV. These energies were chosen to encompass gold’s L-edge and to span the peak around 38.5 keV where the greatest contrast in mass energy absorption ratios was expected. Experiments were carried out at 20 keV using both B16 and I15, to ensure reproducibility between the two beamlines. A dose range of 050 Gy was selected to provide a balance between damaging the plasmid DNA sufficiently to provide quantifiable observations and completing irradiations on practical time scales. Because of the reduced dose rates available at higher energies, this range was reduced to 25 Gy for samples exposed to 52.3 and 60 keV X-rays. At high energies, samples were stored in polymerase chain reaction (PCR) tubes, mounted in a horizontal holder to accommodate the beam produced by the synchrotron. However, at low energies, the attenuation of the plastic tubes became a significant factor. As a result, for energies of less than 20 keV, samples were loaded on a 384-well plate (Thermo Scientific, Rochester, NY) that was covered with a thin film of cellophane to prevent excess evaporation of sample while still permitting sufficient transmission of X-rays. ESRF and Therapax Irradiations. Irradiations were performed in Lindemann glass capillary tubes (Glas Technik & Konstruktion, Berlin, Germany) to avoid sample-holder beam attenuation. Broadband X-rays of different effective energies were generated with a superficial therapy X-ray unit (Pantak Therapax 3 series) at the Universite de Sherbrooke Hospital Centre. The effective energies provided were 14.8, 24.4, 29.8, 42.4, 49, and 70.1 keV. Doses ranging from 0 to 5 Gy were measured by ionization chamber. Samples were exposed in a treatment cylinder (2.5-cm diameter, 15-cm source-to-surface distance). ESRF monochromatic irradiation was performed with X-rays from 30 to 80 keV from the ID17 2-crystal Si(111) bent Laue monochromator. The beam was line-shaped, 1 mm in height and 52 mm in width. The sample was horizontally adjustable to the beam and fixed on a vertical translation stage and scanned through the beam over 31 mm for X-ray exposure. The X-ray dosimetry was done using a calibrated PTW semiflex chamber coupled to a PTW Unidos electrometer that was scanned vertically through the beam as if it were one of the cell samples. This dosimetry was done to cross-calibrate ID17’s standard flux monitors used to ensure exposure of the cell samples to the correct dose. The dose rates for experiments were about 0.0057 Gy/s for 30 keV, 0.027 Gy/s for 50 and 60 keV, 0.05 Gy/s for 70 keV, and 0.037 Gy/s for 80 keV. Irradiations with Variable Scavenging Conditions. Additional work was carried out at QUB to investigate the effects of scavenger concentration on GNP sensitization. In these experiments, samples were prepared as they were for experiments carried out at Diamond, but additional Tris-EDTA was added, with concentrations varying from 0.25 to 5 mM Tris (0.025 to 0.5 mM EDTA). Samples were prepared and irradiated in PCR tubes, as with the higher-energy experiments carried out at Diamond. Dose ranges were chosen to accommodate the reduced DNA sensitivity in higher scavenging concentrations, ranging from 50 to 500 Gy for the most radio-resistant samples. These samples 20162
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double-strand breakage were fitted to these forms using a rate equation analysis, as has been described elsewhere.32 Because of the limited dose ranges necessitated by the available dose rate, in many cases, the yield of the linear form was only a few percent of the total DNA, leading to a very poor characterization of the double-strand break (DSB) rate. As a result, single-strand breaks (SSBs) were used to quantify the dose enhancement upon the addition of GNPs. The yield of single-strand breaks (reflected in the slope of the loss of supercoiled DNA) was quantified for each radiation energy with and without GNPs, and the ratio of this value was taken as the corresponding enhancement value. For radical scavenging experiments, enhancement was calculated separately for each Tris-EDTA concentration by comparing the SSB yield with gold to that in the absence of gold at matching scavenging capacity, to that ensure the change in overall radiation sensitivity with varying scavenging capacity was taken into account. All results of radiation damage exposures presented herein are the means of at least three independent repeats, with an uncertainty given by the standard error.
’ RESULTS
Figure 2. Characterization of GNPs. (a) TEM image of 37.5-nmdiameter GNPs, recorded at 89000 magnification. (b) Absorbance measurement of 11.9-nm-diameter GNPs at visible wavelengths, including the characteristic absorbance peak around 520 nm.
were irradiated using a Faxitron CP-160 X-ray source. This is a broadband source, operating with a peak voltage of 160 keV, filtered with 0.4-mm Cu, and producing a spectrum with a mean energy of 78 keV. Irradiations were carried out both with and without gold at each Tris-EDTA concentration, to account for the overall change in radiation sensitivity with changing scavenging capacity of the solution. Damage Quantification. DNA damage was quantified through agarose gel electrophoresis. Samples were separated in a 1% w/v agarose gel by running at 90 V for 1.5 h; stained with either ethidium bromide, SYBR Green I (Molecular Probes), or SYBR Gold; and imaged. The resulting images were analyzed using ImageJ software, with a correction factor of 1.4 used to correct for the weaker binding of ethidium bromide or SYBR to the supercoiled form of DNA. Yields of three plasmid forms, namely, supercoiled, open circular, and linear, were quantified, corresponding to undamaged DNA, DNA bearing only single-strand breaks, and DNA bearing double-strand breaks, respectively. Rates of single- and
GNP Characterization. GNP size, monodispersivity, and concentration were verified through the use of TEM and visible-light spectrometry, the results of which are illustrated in Figure 2. Figure 2a presents a section of a TEM image of 37.5-nmdiameter GNPs used at the ESRF, showing them to be generally circular and with sizes on the order of 3040 nm. The diameters of a large population of GNPs were measured in ImageJ and were used to determine that the mean diameter of this population of GNPs was 37.5 nm ( 5.6 nm. Similar analysis for the smaller nanoparticles measured a mean diameter of 11.9 ( 1.2 nm. The absorbance of 11.9-nm GNPs in the visible range is plotted in Figure 2b. A characteristic absorption peak can be seen near 520 nm, which is known to depend on the GNP size and concentration. Based on previously measured data from other well-characterized GNP samples, this was used to verify the size measured by TEM and the concentration of the GNPs. GNP Energy Dependence. To investigate the energy dependence of GNP radiosensitization, samples were exposed to a series of radiation energies, at several different sources. At the Diamond synchrotron, plasmids were irradiated with monochromatic X-rays with energies from 11.8 to 60 keV, with and without 11.9-nm-diameter GNPs. At the ESRF, the effects of 37.5-nm-diameter GNPs were studied, with monoenergetic exposures from 30 to 80 keV. The 37.5-nm GNPs were also investigated using a clinical Therapax broadband kiloelectronvolt X-ray source at the Sherbrooke Hospital Centre, tuned to generate X-rays with mean energies from 14.8 to 70.1 keV. Dose ranges were determined by timing considerations associated with each source and energy range. In all cases, the maximum dose was sufficient to induce single-strand breaks in at least 50% of plasmids, allowing for SSB rates to be thoroughly quantified. Figure 3 shows an example doseresponse curve, for plasmid DNA exposed to 25.6 keV X-rays at the Diamond Light Source, with and without 11.9-nm GNPs at a concentration of 13 μg/mL. It can be seen that the introduction of the GNPs significantly increases the sensitivity of DNA to the radiation, leading to an increase in the rate of single-strand breaks by 25%. 20163
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Figure 3. Example plasmid doseresponse curves, showing change in undamaged supercoiled (SC), open circular (OC), and linear (Lin) plasmid forms as a function of dose for DNA irradiated with 25.6 keV photons with and without 11.9-nm-diameter GNPs at a concentration of 13 μg/mL.
Figure 4. Energy dependence of GNP enhancement of single-strand breaks. (a) Enhancements seen on a broadband Therapax source tuned to a range of mean energies (data taken from Brun et al.25). (b) Measurements of GNP enhancement carried out using monochromatic X-rays at the ESRF and Diamond synchrotrons, showing similar energy dependence. An approximate cubic spline33 was plotted through each data set.
Although this experiment involved a relatively high dose range, the total number of double-strand breaks remained small (less than 3% linear DNA in the control plasmids), preventing accurate quantification of the change in the rate of double-strand breaks.
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The variation of enhancement as a function of energy is plotted for the broadband Therapax source in Figure 4a and for the two monochromatic sources in Figure 4b. In all cases, significant dose enhancements are observed. This is in stark contrast with theoretical predictions based on the energy absorption of the gold nanoparticles. Even at the conditions of maximum additional absorption (ESRF experiments carried out at 40 keV), the additional energy deposited by the gold was less than one-quarter of the energy deposited in the surrounding water according to mass energy absorption coefficients, and only negligible changes in energy deposition would be expected at the concentrations used at Diamond. This suggests that the observed mechanism is not driven purely by additional dose deposited by the GNPs. Indeed, the dose enhancement observed does not appear to even be proportional to the change in dose deposited in the samples, as the variation with energy differs significantly from that shown in Figure 1. The most striking manifestation of this is the local minimum in enhancement seen in all experimental conditions near 40 keV, where the maximum difference in dose deposited would be predicted. To illustrate this dependence, approximate cubic splines33 were fitted. A single spline was fitted to all points measured at the two monochromatic sources, excluding the 30 keV ESRF and 60 keV Diamond observations. Although both of these points show high sensitization, each has a large associated error, each is at an extreme point in their energy range, and their apparent enhancement is not reflected in the Therapax results. This suggests that their disagreement with the other data points could be due to experimental error, rather than a real effect. However, it should be noted that, even if they are included, the minimum near 40 keV remains clear. There is good agreement between the energy dependency of GNP radiosensitization across all of the experiments presented in this work, with a clear local minimum in enhancement around 3540 keV. This indicates that the energy dependence is a property of GNP dose enhancement and not an artifact related to the type of DNA, GNP, radiation source, or experimental setup being used. Despite the good agreement in the energy dependence of the different sources, GNP radiosensitization does not appear to scale linearly with GNP mass concentration, as the Diamond and ESRF experiments see similar levels of enhancement, despite the ESRF work using a substantially greater mass concentration of GNPs. Similarly, the Therapax work uses a lower concentration of GNPs than the ESRF work, but sees substantially larger enhancements. Overall, these results make it clear that the relationship between beam energy and enhancement is not a simple one and that a complex set of processes contribute to the large-scale and unexpected energy dependence that is observed. Radical Scavenging Effects. The experiments that investigated the energy dependence of GNP radiosensitization were all carried out in water or solutions that contained very low scavenger concentrations. Under such conditions, the majority of radiation damage is expected to be mediated by free radicals (created either through direct interactions between radiation and water or following ionizing events in GNPs), rather than direct radiation interaction with DNA. Experiments were carried out under higher scavenging conditions to determine the contribution of free radicals to GNP radiosensitization. 20164
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Figure 5. Variation in the enhancement of DNA strand breaking at a series of Tris-EDTA concentrations. Sensitization was measured by comparing damage caused by 160 kVp X-rays to plasmid DNA with and without a 13 μg/mL concentration of 11.9-nm-diameter GNPs at each Tris-EDTA concentration.
As these experiments required much higher doses to achieve the same levels of strand breakage, these experiments could not be carried out using synchrotron radiation because of time constraints. Instead, these effects were investigated at QUB using a broadband X-ray source (Faxitron CP-160). In these experiments, the Tris-EDTA content of the solution was varied from 0.25 to 5 mM. Increasing the Tris-EDTA content of the solution leads to an increase in the scavenging capacity, with a corresponding reduction in the levels of DNA damage observed for a given dose. To correct for this difference, samples both with and without GNPs were irradiated at each concentration. The enhancement in SSB yield upon the addition of gold was then calculated for each Tris-EDTA concentration, as shown in Figure 5. It can be seen that the ratio of SSBs seen in with- and withoutgold samples is significantly reduced at higher Tris-EDTA concentrations. At low scavenging capacities, there is roughly a factor of 1.5 difference between the damage caused with and without gold, but at a 5 mM concentration of Tris-EDTA, the addition of gold produced no significant increase in the observed levels of damage. This shows that the radiosensitizing effects of GNPs in the plasmid system can be effectively quenched by moving to a more highly scavenging buffer, highlighting the sensitivity of this effect to the chemical environment.
’ DISCUSSION As has been reported elsewhere, there is a striking difference between these observations and predictions of increased dose based on mass energy absorption coefficients. This can be seen in two ways. First, the scale of the effect is dramatically greater than the dose increase caused by the addition of GNPs. As noted above, even at the conditions where the maximum dose is deposited in gold, this is less than one-quarter of the dose deposited in the surrounding water volume. This is even more pronounced in the experiments carried out at Diamond, where a gold concentration of 13 μg/mL corresponds to an increase in dose on the order of 0.1%. By contrast, enhancements of tens of percent are measured in the plasmid system, suggesting a mechanism more complex than simple changes in energy deposition.
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Moreover, if GNP are considered as the sum of individual gold atoms, the increased energy absorbed by the sample, and thus the dose enhancement, should be much higher in the case of larger nanoparticles if molar concentrations are similar. However, these experiments demonstrate that enhancement is not related to GNP mass by a simple scaling factor, as the relationship between enhancement and mass of gold present varies significantly between the different sets of experiments. For example, although comparable molar concentrations were used in the different experiments, the 37.5-nm GNPs are roughly 30 times more massive than the 11.9-nm GNPs, leading to a significant difference in the amount of dose deposited by the gold. However, when comparing the results obtained at the Diamond Light Source and the ESRF, very little variation in the magnitude of enhancement is observed. These results are supported by previous work on this system,25 which showed that enhancement is not proportional to the mass of GNP but rather exhibited a linear dependence of enhancement with GNP diameter. Based on these previous data, an increase of 38% in enhancement factor would be predicted when going from 11.9- to 37.5-nm GNP, at the same molar concentration. However, in this work, no significant difference is seen between the enhancements observed in the Diamond and ESRF synchrotron experiments. There are several differences in conditions between the two experiments that could be responsible for this, such as the differences between the two plasmids used in the experiments. In addition, the extremely low scavenging environments used in the experiments could point to a possible explanation for this variation. The smaller nanoparticles were irradiated in the presence of 0.25 mM TE buffer, which is known to be a radical scavenger, whereas the larger GNPs were irradiated in water. The low overall scavenging environment means that even small shifts in scavenger concentration can significantly affect the overall sensitivity of the DNA by modifying its structure or by affecting free-radical lifetimes. These results show that, although dose enhancement is seen across all experiments with a similar energy dependence, the absolute magnitude of this enhancement is not simply proportional to the mass of added GNPs. This indicates that care must be taken when attempting to make absolute comparisons between different conditions. The second way these results depart from the predictions of physical dose is in the behavior of the enhancement with energy. Energy absorption coefficients (Figure 1) show that the greatest effect is expected at about 40 keV and that no effect is expected for much higher energies. Nevertheless, significant radiosensitization by GNPs has been reported in the literature for a large range of photon energies. For example, Jain et al.20 reported significant radiosensitization of MDA cells for 160 kVp, 6 MV, and 15 MV X-rays. Similarly, Liu et al.22 reported that enhancement was obtained with megaelectronvolt X-rays and that 6.5 and 8 keV photons were more efficient than 73 keV photons. Comparing these energy dependencies is not easy, as several systems, particles, and energies were studied. The novelty of these present experiments is that, although particles (size and coating), plasmids, and experimental conditions (buffer) were different, enhancement was quantified for the same energy range, using monochromatic X-rays. The observed energy dependence strongly disagrees with the theoretical predictions, with a local minimum in the vicinity of the maximum of the mass energy absorption ratio curve. 20165
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The Journal of Physical Chemistry C Both monochromatic beams agree very well, whereas the Therapax minimum energy is slightly shifted to higher values. This can be explained by the fact that this medical beam is not monoenergetic, so that its average value is expected to be overestimated, as Bremsstrahlung, which is not taken into account, represents a non-negligible contribution. Although it is a significant departure from predictions, this minimum is present in all of the observed results, suggesting that it is a real effect and independent of the variations in GNPs, DNA, and experimental conditions that affect the absolute magnitude of the enhancement. Taken together, these results show that considering GNPs as simple contrast agents that act by increasing dose is misleading. The radiosensitizing effects of GNPs can potentially be explained by considering the variety of interactions that can occur with a GNP during radiation exposure. Over most of the energy range considered in this work, the Compton effect is the dominant mechanism by which photons lose energy in water. This typically involves the ejection of a single outer-shell electron from water with moderate energy, which leads to further ionizations, sparsely distributed along the electron’s track. By contrast, the dominant interaction with gold is the photoelectric effect. This leads to the ejection of an inner-shell electron, which is then followed by the production of a large number of low-energy Auger electrons, with over a dozen electrons potentially being liberated by the absorption of a single photon. As a result, a photon interaction with a gold atom will create a much higher density of secondary electrons (and subsequent ionizations) than a similar photon interacting with a water molecule. These secondary electrons have relatively short ranges in water (with most traveling 200 nm or less24). This leads to high ionization densities in the vicinity of the GNP, but means that, if they are to cause single- or double-strand breaks directly, the GNPs must be in close association with plasmid molecules. If plasmids and GNP are well separated, then the vast majority of these electrons will instead ionize water molecules in the surrounding solution and lead to the formation of hydroxyl radicals. These radicals cause the majority of DNA damage observed in this system, and as a result, a significant overproduction of these species would lead to a corresponding increase in damage. The suggestion that these sets of ionizations lead to an overproduction of free radicals is consistent with other measurements of free-radical production in the presence of GNPs, which suggests that they have the capacity to increase the quantity of free radicals generated by radiation, even at relatively low concentrations.17,34 Misawa and Takahashi34 reported that the addition of 20-nmdiameter GNPs at a concentration of 10 μg/mL increased OH• production in solution following irradiation by a 100 kVp X-ray source by a factor of 1.46, in agreement with the magnitude of the enhancement observed in these experiments, suggesting that this greater-than-expected increase in free-radical yield makes a significant contributor to the sensitizing properties of GNPs. With this in mind, the results in Figure 5 can be interpreted as the additional scavengers reducing the rate at which DNA reacts with GNP-generated radicals. This could occur either through inhibition of the production of free radicals or through a reduction in the likelihood of their interaction with DNA. The former could occur if, for example, the scavengers associate with the additional GNPs and modify their surface
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properties, which is likely to play a significant role in free-radical production because of the very short range of low-energy secondary electrons. Even if there is no association of this type, increasing the scavenger concentration would lead to a reduction in the lifetime and diffusion range of free radicals, which would potentially reduce the probability of GNP-induced radicals reaching plasmids to cause damage. If the observed enhancement does result from the production of additional free radicals, then the fact that the mass energy absorption ratio is a poor predictor of the sensitizing effects of GNPs is less surprising, as it is well-known that the free-radical yield in water varies with energy.35 The free-radical production from GNPs following X-ray exposure would also be expected to vary as a function of energy, with a behavior that would be significantly different from that seen in water because of the different processes by which ionization typically occurs in the two materials. Whereas the abundance of secondary electrons produced following ionization is the most obvious method by which additional damage could be generated, it is possible that GNPs might induce further ionizations by other processes: for example, through localized heating, or by capturing electrons from nearby water molecules to neutralize the positive charge that remains after an ionizing event. All of these effects can vary with energy in different fashions and might contribute further to the observed energy dependency in these results. Furthermore, these observations do not rule out the possibility that increasing gold concentrations could see further enhancement from higher energy deposition within the sample, as the gold concentrations used in this work mean that the change in energy deposition is small compared to the observed enhancement. The dependence on free-radical production or other processes following initial ionizations means that it is not possible to accurately predict the enhancing effects of GNPs based on macroscopic absorption coefficients alone. Rather, a much more indepth consideration of how dose is deposited, rather than simply the amount, is required to explain experimentally observed enhancements and allow for an understanding of the parameters that must be optimized to provide the greatest therapeutic benefit. These results also suggest that the absolute magnitude of the dose enhancement that is observed is very sensitive to the chemical environment present during the irradiation. As a result, care must be taken to ensure that predictions of the scale of GNP radiosensitization translate meaningfully into more complex systems and between different GNP preparations. Further investigations are required to accurately characterize the changes in radical chemistry brought about by the combination of GNPs and radiation, and investigate the effects of these in more complex biological systems before truly robust, general predictions can be made about the effects of GNP-enhanced therapies.
’ CONCLUSIONS This work presents measurements of the energy dependence of the radiosensitizing effects of GNPs using monochromatic X-rays and provides a clear demonstration that these effects cannot be simply explained by a consideration of the increased energy deposition that results from the presence of gold nanoparticles. 20166
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The Journal of Physical Chemistry C Additional experiments suggest that this effect is dominated by the contribution of free radicals generated by the gold in this system and that the energy dependence of GNP radiosensitization is dominated by the change in efficiency of free-radical production and other processes, rather than dose. Further investigations are required to confirm this observation and to quantify this process to enable more general predictions to be made about the radiosensitizing potential of GNPs in a therapeutic context.
’ AUTHOR INFORMATION Corresponding Author
*Tel.: +44 (0)2890972779. E-mail:
[email protected].
’ ACKNOWLEDGMENT S.M.M. and W.B.H. contributed equally to this work and should be considered as joint first authors. L.S. thanks the European Commission for the reception of a Marie Curie international incoming fellowship. C.S.-R. and L.S. thank the Triangle de la Physique for financial support. Some of this work was carried out with the support of the Diamond Light Source. From among the many Diamond Light Source staff who have helped us with those measurements, we particularly acknowledge the help and support of Andrew Malandain, Kawal Sawheney, ManoJ. Tiwari, Igor Dolbnya, Andrew Jephcoat, Annette Kleppe, and Alan Ross. We thank Mathias Brust for advice on gold nanoparticle synthesis in connection with the experiments performed at the Diamond Light Source. Part of this work was supported by a Cancer Research UK grant (Grant C1278/A9901). ’ REFERENCES (1) Castillo, M. H.; Button, T. M.; Doerr, R.; Homs, M. I.; Pruett, C. W.; Pearce, J. I. Am. J. Surg. 1988, 156, 261–263. (2) Norman, A.; Adams, F. H.; Riley, R. F. Radiology 1978, 129, 199–203. (3) Robar, J. L.; Riccio, S. A.; Martin, M. A. Phys. Med. Biol. 2002, 47, 2433–49. (4) Rousseau, J.; Boudou, C.; Barth, R. F.; Balosso, J.; Esteve, F.; Elleaume, H. Clin. Cancer Res. 2007, 13, 5195–201. (5) Corde, S.; Joubert, A.; Adam, J. F.; Charvet, A. M.; Le Bas, J. F.; Esteve, F.; Elleaume, H.; Balosso, J. Br. J. Cancer 2004, 91, 544–51. (6) Hainfeld, J. F.; Slatkin, D. N.; Focella, T. M.; Smilowitz, H. M. Br. J. Radiol. 2006, 79, 248–53. (7) Hainfeld, J. F.; Dilmanian, F. A.; Slatkin, D. N.; Smilowitz, H. M. J. Pharm. Pharmacol. 2008, 60, 977–85. (8) Lewinski, N.; Colvin, V.; Drezek, R. Small 2008, 4, 26–49. (9) Khlebtsov, N.; Dykman, L. Chem. Soc. Rev. 2010, 40, 1647–1671. (10) Hainfeld, J.; Dilmanian, F.; Zhong, Z.; Slatkin, D.; Kalef-Ezra, J.; Smilowitz, H. Phys. Med. Biol. 2010, 55, 3045. (11) Chang, M.-Y.; Shiau, A.-L.; Chen, Y.-H.; Chang, C.-J.; Chen, H. H.-W.; Wu, C.-L. Cancer Sci. 2008, 99, 1479–84. (12) Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. W. Nano Lett. 2006, 6, 662–8. (13) McMahon, S. J.; Mendenhall, M. H.; Jain, S.; Currell, F. Phys. Med. Biol. 2008, 53, 5635–51. (14) Robar, J. L. Phys. Med. Biol. 2006, 51, 5487–504. (15) Cho, S. H. Phys. Med. Biol. 2005, 50, N163–73. (16) Garnica-Garza, H. M. Phys. Med. Biol. 2009, 54, 5411–25. (17) Carter, J. D.; Cheng, N. N.; Qu, Y.; Suarez, G. D.; Guo, T. J. Phys. Chem. B 2007, 111, 11622–5. (18) Butterworth, K. T.; Coulter, J. A.; Jain, S.; Forker, J.; McMahon, S. J.; Schettino, G.; Prise, K. M.; Currell, F. J.; Hirst, D. G. Nanotechnology 2010, 21, 295101.
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