Size-Tuning Ionization To Optimize Gold Nanoparticles for

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Size-Tuning Ionization to Optimize Gold Nanoparticles for Simultaneous Enhanced CT Imaging and Radiotherapy Yan Dou, Yanyan Guo, Xiaodong Li, Xue Li, Sheng Wang, Lin Wang, Guoxian Lv, Xuening Zhang, Hanjie Wang, Xiaoqun Gong, and Jin Chang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b07473 • Publication Date (Web): 27 Jan 2016 Downloaded from http://pubs.acs.org on January 31, 2016

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Illustration of the approach for optimizing particle sizes of spherical AuNPs as X-ray theranostic adjuvants. 237x165mm (300 x 300 DPI)

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Size-Tuning Ionization to Optimize Gold Nanoparticles for Simultaneous Enhanced CT Imaging and Radiotherapy Yan Dou†, Yanyan Guo‡, Xiaodong Li‡, Xue Li‡, Sheng Wang†, Lin Wang‡, Guoxian Lv†, Xuening Zhang‡, Hanjie Wang†, Xiaoqun Gong†, Jin Chang† †

School of Material Science and Engineering, School of Life Sciences, Tianjin

University,

Tianjin

Engineering

Center

of

Micro-Nano

Biomaterials

and

Detection-Treatment Technology, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, 300072, P.R. China. ‡Department of Radiation Oncology and Department of Radiology, The Second Hospital of Tianjin Medical University, Tianjin, 300211, P.R. China. Address correspondence to E-mail: [email protected]

ABSTRACT Computed tomography (CT) contrast and radiosensitization usually increase with particle sizes of gold nanoparticles (AuNPs), but there is a huge challenge to improve both by adjusting sizes under requirements of in vivo application. Here, we report that AuNPs have great size-dependent enhancements on CT imaging as well as radiotherapy (RT) in the size range of 3-50 nm. It’s demonstrated that AuNPs with size 1


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of ~ 13 nm could simultaneously possess superior CT contrast ability and significant radioactive disruption. Monte Carlo (MC) method is further used to evaluate this phenomenon and indicate that the inhomogeneity of gold atom distributions caused by sizes may influence secondary ionization in whole X-ray interactions. In vivo studies further indicate that this optimal sized AuNPs improve real-time CT imaging and radiotherapeutic inhibition of tumors in living mice by effective accumulation at tumors with prolonged in vivo circulation times compared to clinical used small-molecule agents. These results suggested ~ 13 nm AuNPs may serve as multifunctional adjuvants for clinical X-ray theranostic application.

KEYWORDS: AuNPs· ·Size-tuning ionization· ·CT imaging· ·Radiosensitization· ·MC simulations· ·X-ray theranostics

2


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Nanometer-sized materials as exogenous agents demonstrate their potential on biomedical applications from medical diagnostics to radiation oncology,1-5 owing to efficient tumor localization by passive or active targeting effects compared with conventional small-molecule reagents.6-8 Over the past two decades, gold nanoparticles (AuNPs) with high X-ray photon capture cross-section of gold, have been

intensively

nanoparticles

as

designed computed

and

fabricated

tomography

among (CT)

heavy

contrast

atom-based agents

or

radiosensitizers.9-12 Despite recent advances about different shapes of AuNPs in improving CT attenuation differences or radiotherapy damage of tumors, few studies have been reported to systematically investigate the size effect of spherical gold nanoparticles on CT imaging and radiosensitization.13-18 In most cases, concentration gradients have been tested to judge these enhancement performances.15-18 There are great differences between results even under the same concentrations due to different sizes studied.13,19-21 Several studies have demonstrated that the enhancement performances increase with particle sizes increasing.22,23 However, there still needs to explore how to simultaneous achieve CT enhanced imaging and radiosensitization by adjusting sizes. In addition, sizes of nanoparticles applied in vivo should be limited in the nanometer range for taking advantage of the enhanced permeability and retention (EPR) effects to achieve enrichment in tumors and metabolism.24-26 3


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Solving this problem is crucial, not only to select the proper AuNPs for future clinical applications, but also to evaluate preclinical carcinogenic risks associated with imaging dose and radiation exposure. Recently, size effects of a single AuNP on radiosensitisation irritated by photo beams with different energy were studied by Monte Carlo (MC) methods,27, 28 which can simulate photoelectron transport processes with the most accurate calculations and effectively handle the change of density and atomic number in non-uniform tissue with the least approximation.29 However, the interaction between X-rays and a single particle could not represent the reality. For accurate assessments, the inhomogeneity of gold atom distributions which is caused by particles with various sizes should be taken into account. By precisely evaluating the entire interactions between primary X-rays, secondary X-rays and particles, how sizes of AuNPs influence CT attenuation and radiosensitization could be speculated theoretically according to X-ray radiation physics. Here we present an approach to explore and effectively confirm the distinctive size-dependent effects based on a series of spherical AuNPs for enhanced CT imaging and radiotherapy (Scheme 1). We succeeded in finding that there was a common enhancement tendency in both X-ray attenuation and radiosensitization effect induced by AuNPs (< 50 nm) at small internals under same concentration. To achieve accurate explanations and evaluations, MC simulations were carried out under clinical X-ray radiation with KeV energy for CT 4


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and MeV energy for radiotherapy respectively, based on the inhomogeneity of AuNPs solutions which could be closer to the actual conditions caused by different sizes. We have achieved excellent imaging sensitivity and significant tumor inhibition by ~ 13 nm AuNPs among all sizes studied, compared with clinically used small-molecule agents in cells and animal models. These results provide a highly versatile research method that may facilitate to evaluate X-ray caused

diagnostic

or

therapeutic

properties

for

selecting

appropriate

nanoparticles from thousands of sizes or structures before clinical applications, thereby reducing the experimental cost and thus supporting AuNPs as multifunctional theranostic platform in future clinical applications. RESULTS AND DISCUSSION Although some studies have shown that the enhancement performances including CT imaging and radiosensitization are directly proportional to sizes, size distribution and size intervals were usually randomly selected.22, 23, 30 To discover a more accurate tendency in size-dependent effect, spherical AuNPs among 50 nm with slight disparities of particle sizes were first studied to explore the optimal properties, considering in vivo application as X-ray theranostic adjuvants (Scheme 1). We synthesized spherical AuNPs with seven kinds of size in range of 3-50 nm at interval of 4-10 nm. AuNPs were uniform and monodispersable as characterized by transmission electron microscopy (TEM) (Figure 1a-n and 5


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Supporting Figure S1) (hereafter, size expressions refer to here). There was a red shift in the UV-visible light spectrum of AuNPs when the particle size increases (1 o, p). PEGylated surface modification of as-prepared AuNPs was then performed for in vivo applications. The colloidal stability of PEGylated AuNPs (Supporting Figure S2, 3, 4, 5 and Supporting Table S1) against different pH (5, 6, 7, and 8) and temperature (4, 25, 37, and 50 °C) were characterized by UV-visible light spectrum. No significant changes in the absorption value or absorption peak position confirmed that the PEGylated AuNPs were quite stable and dispersible in the above mentioned condition (Supporting Figure S6, 7). A slight red shift about 2-5 nm of absorption peak location was observed in all sizes of the PEGylated AuNPs after aging for 240 days in all the buffer solutions indicating the PEGylated AuNPs were quite stable (Supporting Figure S8). Furthermore, cytotoxicity effect of each AuNPs size was evaluated. The results showed that at all concentrations used, the AuNPs of sizes below 20 nm showed more than 90% cell viability while AuNPs of sizes above 20 nm showed less than 80% cell viability indicating the bigger AuNPs were more toxic (Supporting Figure S9c-i). We next investigated CT enhanced contrast and radiosensitization of different-sized PEGylated AuNPs in vitro to explore if there is size-dependent effect. First, CT contrast properties of AuNPs were analyzed compared with clinical used CT contrast agent (Idohexol) at different molar concentrations of the 6


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active element (Au or iodine) (Supporting Figure S10). We observed that AuNPs elicited strong fluctuation of attenuation intensity compared with Idohexol in a size–dependent

manner.

We

then

evaluated

these

size-dependent

enhancements at concentration of 0.02 M. CT contrast phantom images (Figure 2a up) revealed that 13.2 nm, 34.8 nm and 41.1 nm AuNPs showed greater X-ray attenuation than others, around 13.2 nm AuNPs held almost 1.5-fold CT values more than sizes before and after which were similar with sizes larger than 34.8 nm, confirmed by further quantitative analysis (Figure 2b). Good distribution of measured CT values in the calibration curve indicated the accuracy of the measured data (Supporting Figure S11a). It was observed that only the AuNPs with size of 13.2 nm and size of larger than 34.8 nm showed the brighter CT contrast phantom images than Idohexol (Figure 2c), by the extent of ~ 20% in the attenuation intensity (indicated by HUAu - HUI/ HUI) (Figure 2d). Considering cytotoxicity and in vivo application, 13.2 nm PEGylated AuNPs could be more suitable to be used as CT contrast agents among all the sizes we have studied (Supporting Figure S9a, e, h-i). In addition to CT contrast enhancements, we assessed radiosensitization properties in vitro by examining accelerator dose distributions (Figure 2a bottom) of the aqueous solutions with the same reference point fixed for all particle sizes. There is an upward trend in the monitor unit with size of AuNPs increasing except for a turning point at the 17.5 nm (Figure 2b). We then compared the 7


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radiosensitization capability of AuNPs with the clinical used radiosensitizer (glycididazolum natrium (CMNa)) (Supporting Figure S 9b, j). The viability of cells treated with seven kinds of particle sizes decreased rapidly and differently in response to radiation doses compared with radiation alone (Supporting Figure S12a-d). The survival fraction curves for different-sized AuNPs presented obvious radiation enhancements effect (P < 0.05) at AuNPs concentration of 50µM while 13.2 nm AuNPs showed the most significant enhancement of radiation effect (P < 0.001) compared with radiation alone and CMNa (Figure 2e). To evaluate the enhancement efficiency of various sized-AuNPs, the sensitization enhancement ratios (SER), defined as the ratio of eradicating tumor cells with and without presence of AuNPs, were calculated under various AuNPs concentration at different radiation dose (Supporting Figure S13a-d). There was no obvious size-dependent effect in the sensitization enhancement ratios under both lower (below 4 Gy) and higher irradiation dose (above 8 Gy) no matter what concentrations used (Figure 2f and Supporting Figure S13a, b, d). Only under 6 Gy irradiation, there was a clear size-dependent effect in sensitization enhancement ratio when the AuNPs concentration reaches 50 µM (Figure 2g). Furthermore, the sensitization enhancement disparity (indicated by SERAu SERCMNa/ SERCMNa) indicated that among all the sizes of AuNPs, only 13.2 nm AuNPs showed a radiosensitization ratio that is twice of the value of CMNa (Figure 2h), which was quantitatively identified by cell apoptosis analysis 8


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(Supporting Figure S14). Taken together, the size-dependent enhancements of AuNPs for CT contrast and radiosensitization showed similar trend that also coincided with the curves of AuNPs relative electron density at different sizes (Supporting Figure S11b and Supporting Table S2). This result suggested that particle sizes have influence in relative electron density of the AuNPs, and therefore could further influence X-ray based enhancement performances. In order to explain and evaluate the size-dependent results, we further implemented Monte Carlo (MC) simulations to study the physical mechanisms, which might help in the further size-selection before in vivo theranostic application. X-rays interact with a simulated phantom through primary electromagnetic radiation processes (primary effects) including photoelectric effect, Rayleigh scattering, Compton scattering, and electron pair effect, resulting in the reduction in beam intensity and subsequent secondary electromagnetic radiation processes (secondary effects).19, 30-33 The phantom was designed and filled with “modeling vesicle” containing two gold nanoparticles, representing the inhomogeneity of aqueous solutions due to the presence of simulated particle sizes (Figure 3a). Based on high atomic number (Z) of gold, different types of primary radiation occurred at kilovoltage or megavoltage X-ray energies.30, 34, 35 X-ray attenuation originates from photoelectric effect which is the predominant process at Kilovoltage radiation energy of clinical CT imaging from 9


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10 to 500 KeV (Figure 3b, c).36, 37 We simulated attenuation properties for all particle sizes by measuring the photon fluence before and after X-rays pass through the modeling vesicle. The higher X-ray attenuation properties were representative of better CT imaging properties to display the lower photon fluence in MC simulations. Total emitted photon fluence for all sized AuNPs dropped much more (P < 0.05) than the control (pure water without AuNPs) below incident fluence level, with the greatest decrease of ~ 15% for ~ 14 nm AuNPs (Figure 3d). The attenuation enhancement ratio, defined as the ratio of emitted photon fluence compared with incident fluence, exhibited a strong sensitivity to AuNPs sizes. We found that the ~ 14 nm AuNPs exhibited the greatest photon fluence attenuation among all the AuNPs sizes we studied (Figure 3d, e) in accordance with the experimental results of size-dependent CT contrast enhancements (Figure 2a-d). We observed similar effect for radiosensitization properties (Figure 2e-h) by analyzing dose absorption distributions when electron pair effect dominated at megavoltage clinical radiotherapy energies (Figure 3f, g).36, 38 Average absorbed doses were calculated layer-by-layer along X-ray incident direction. There were two kinds of dose absorption peaks. The higher absorption peaks represent primary effects determined dose absorption level (Figure 3h and Supporting Figure S15) while the lower absorption peaks represent secondary effects. Dose enhancement factors (DEF) were calculated, defined as the ratio of the average dose in water phantom with and without AuNPs (Figure 3h). We found that DEF 10


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of simulated 2 nm AuNPs was high but useless because of lowest dose absorption energy level. DEF existed meaningful only under the same order of magnitudes of two dose absorption peaks in the size range of 10-18 nm (Figure 3h and Supporting Figure S15c-e). To account for actual effectiveness of radiosensitization properties, the absorbed doses per specific surface area were evaluated (Figure 3i) to reflect the actual dose absorption changes. Absorbed doses per specific surface area varied with sizes further indicating that around ~ 14 nm AuNPs possess unique dose absorption effectiveness among particle sizes less than 18 nm (Figure 3h, i). As shown from experimental discovery and MC evaluations (Figure 2, 3), AuNPs with sizes around ~ 13.2 nm should be preferentially selected among other particle sizes studied for obtaining the better of CT enhanced imaging combined with radiosensitization properties in vitro. To assess the cellular enhancement effects, PEGylated AuNPs with this optimized size were incubated with HeLa cells, as a comparison to clinical used agents including Idohexol and CMNa respectively. ~ 13 nm PEGylated AuNPs showed sufficiently brighter signals for CT imaging. When the concentration of contrast agents increase to 2.5 mM, ~ 13 nm AuNPs showed a 1.2-fold enhancement (Figure 4a) in attenuation intensity comparing to Idohexol (Figure 4b). After incubating with AuNPs for 24 hours, the cell morphologies remained healthy (Supporting Figure S18a) while the cells 11


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incubated with Idohexol showed some abnormal irregularity (Supporting Figure S18b), suggesting cell death induced by cytotoxicity of Idohexol (Supporting Figure S9a, e). Next, the ability of ~13 nm PEGylated AuNPs as efficient radiosensitizers on the cellular level was evaluated. More than 90% of the cells treated with the optimized AuNPs or CMNa alone at a high concentration of 50 µM were still alive (Figure 4d, e and Supporting Figure S19). However, when subjected to X-ray radiation of 6 Gy, cell viability greatly declined to 23.1% for the cells treated with AuNPs and 47.3% for those treated with CMNa at concentration of 50µM while the control cells only received radiotherapy (RT) still showed 82% viability (Figure 4d). SER of AuNPs were measured at various concentrations. At concentration of 50 uM, SER of AuNPs were twice of that of CMNa (Figure 4c), supported by cell apoptosis studies. Apoptosis and necrotic ratios were 82.76%, 52.89% and 21.5%, corresponding to ~ 13 nm AuNPs, CMNa and RT alone, respectively (Figure 4f). Finally, we explored the feasibility of PEGylated AuNPs with the optimized sizes around ~ 13 nm as in vivo theranostic agent candidate for simultaneous CT enhanced imaging and radiosensitization. The intratumoral injection studies showed higher tissue attenuation signals for CT imaging than Idohexol (Supporting Figure S20). PEGylated AuNPs with sizes around ~ 13 nm were administrated intravenously into living mice bearing HeLa xenograft tumors for 12


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immediately acquiring sequential CT contrast phantom images in different organs over time (Supporting Figure S21). It was clear that the strongest enhancements in the heart, brain, kidney and liver regions were presented within 15 to 30 min post injection for Idohexol compared with 30 to 45 min for AuNPs (Figure 5a and Supporting Figure S22). Idohexol displayed much brighter CT images (P < 0.01) of bladder than that of AuNPs during 90 min post-injection observation (Figure 5a and Supporting Figure S21b), indicating PEGylated AuNPs possessing longer in vivo circulating time than Idohexol. By quantitatively detecting the gold and iodine amounts in different tissues, in vivo biodistribution demonstrated that Idohexol possessed faster metabolism than PEGylated AuNPs (Supporting Figure S23). At the tumor sites, CT signals of AuNPs were found gradually increased and significantly elevated (P < 0.001) to 1.8-fold higher than that of Idohexol at 90 min post injection, revealed by quantitative analysis (Figure 5a, b and Supporting Figure S21e). To further estimate the antitumor efficacy, HeLa xenograft tumors loaded with intravenously injected AuNPs after CT imaging were then subjected to high-energy X-ray radiation at a dose of 6 Gy. Different radiotherapy groups had significant effects (P < 0.001) on inhibiting tumor growth compared with PBS as a negative control (Figure 5c, d and Supporting Figure S24). For clinical radiosensitizer CMNa, radiation within 30 min after intravenous injection presented remarkably higher (P < 0.05) inhibition efficacy towards tumor growth 13


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than radiation performed at 4 h post-injection, which primarily resulted from the rapid excretion of small-molecule agents (Figure 5c, e and Supporting Figure S24c, e). More importantly, the tumor-growth inhibition by AuNPs was enhanced dramatically when apply radiation at 4 h post-injection, and exhibited much more sensitivity (P < 0.05) to radiation than CMNa suggesting the efficient localization of AuNPs in tumors by passive targeting with longer in vivo circulation (Figure 5c, e and Supporting Figure S24c-f). The body weights of the mice had no significant change during different treatments (Figure 5f). Moreover, the histological changes were examined using hematoxylin and eosin staining showing that after applying corresponding radiosensitization treatments, different levels of cancer cell death occurred in the tumor tissue (Figure 5g), while producing no obvious pathological abnormalities in other organs, such as cardiomyopathy (Supporting Figure S25). The similar damages were found in the tumor tissues treated with radiotherapy after injection of CMNa and AuNPs for 30 min, while the complete eradication of cancer cells was achieved when the radiation was applied after AuNPs were injected for over 4 h, which was consistent with the results of tumor growth inhibition, further demonstrating the persistent optimal radiosensitization effects (Figure 5c, e, g). From above, the size-dependent enhancements are largely determined by the particle size and size intervals implemented. Micro-sized particles are obviously not suitable for in vivo application because effective EPR effects and 14


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renal clearance of nanoparticles are normally achieved for sizes less than 100 nm.39-41 In this study, we used AuNPs with sizes below 50 nm. We showed that AuNPs could reflect distinctive CT attenuation enhancements similar to radiosensitization within our size range, revealing the hidden changes due to slight disparities of particle sizes (Figure 2). Sizes around 13.2 nm could exhibit the

greatest

enhancement

effects

for

CT

contrast

combined

with

radiosensitization among all the sizes we have tried. AuNPs of larger than 30 nm also possess similar CT contrast enhancement but showed more serious cytotoxicity issues (Supporting Figure S9). The ability to accurately simulate the actual conditions is one of the advantages of Monte Carlo methods and will enable us to evaluate our size-dependent experimental results.29 The simulations of size-dependence based on a single particle have been widely studied; however, the actual case is that the solutions are never composed of only one nanoparticle. To reorganize the inhomogeneity of AuNPs solutions appears to be crucial because different gold atom distributions in the actually used solutions were caused by different sizes of AuNPs under the same concentration. Therefore, we designed the AuNPs solutions composed of “modeling vesicle” containing two nanoparticles to represent the inhomogeneity to investigate the entire ionization interactions with X-rays (Figure 3a). We observed that simulations based on two nanoparticles could certainly exhibit the distinctive size-dependent effects (Figure 3) rather 15


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than simulations based on only one nanoparticle (Supporting Figure S16 and Supporting Figure S17). We noticed that for CT imaging simulations, the attenuation enhancement ratio calculated by photon fluence before and after X-rays traversed the modeling vesicle could directly reflect X-ray attenuation enhancements (Figure 3d, e). However, for radiosensitization simulations, the absorbed doses per specific surface area could reflect the actual enhancements other than dose enhancement factors (DEF) calculated by the ratio of the average dose in water phantom with and without AuNPs because the absorbed energy level should be combined for evaluation (Figure 3h, i). Although DEF is high, low dose absorption energy level remains little enhancements of the actual effectiveness, such as 2 nm. When dose absorption energy level makes primary effects and secondary effects with the same order of magnitudes of energy, DEF existed meaningful to show the actual enhancements with sizes (Figure 3h, i and Supporting Figure S15c-e). The optimized size actually works around sizes of 13 nm, corresponding to the deviation range of nanoparticle size prepared in the experiments. From the perspective of radiation physics, we made the following speculation to explain theoretically and evaluate the size-dependent enhancements we found. These enhancement performances depend on the ionization interations between X-rays and gold atoms. The primary interactions occur firstly due to original X-ray 16


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irradiation. The characteristic rays produced by photoelectric effect at kilovoltage energy and electron-positron pairs produced by electron pair effect at megavoltage energy may trigger the secondary effects with nearby other atoms if energy was sufficient. Under the same concentrations, primary effects were same determined by the same numbers of gold atoms per volume in aqueous solutions regardless of sizes. It is worth noting that different sizes cause the inhomogeneity of gold atom distributions resulting in different frequency of secondary interactions between atoms. The inhomogeneity of gold atom distributions may be indirectly indicated by relative electron density (Supporting Figure S11). Sparse distribution caused by small sizes makes low secondary interactive frequency because secondary photoelectrons lose energy when passing the long path. More effective secondary interactions could be realized when dense distributions caused by large sizes makes high frequency by shortening the interactive path. We first found that secondary interactions may not be neglected within relatively small nano-sized range with slight size interval, which could compete with primary effects to make a contribution to the size-dependent enhancement performances. Generally, secondary interactions were ignored by previous works due to large size range studied from more than nanometers to dozens of micrometers sizes with large size interval set.22, 23 Because these sizes beyond the scope of our size-dependent enhancements so that atoms are packed more closely, more 17


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frequent secondary interactions leading to secondary rays or electron-pairs with little energy largely evanishing in water result in secondary effects negligible. These ideas may also explain why increasing the concentration can improve the X-ray-based properties (Supporting Figure S10), because dense packed atoms make primary effects enlarged, and approximately assuming the enhancement performances increase with atom numbers only. X-rays with high frequency in electromagnetic spectrum have been used, which can be expected to trigger the interaction with AuNPs concentrated in the deeply localized tumors (Figure 1). In vivo studies clearly demonstrate that PEG modification of AuNPs could prolong their in vivo circulation time (Figure 5a, e).12,15,23,42,43 These advantages towards high contrast CT imaging and efficient radiotherapy may be adoptable for clinical applications, where avoid of repeated injection to patients is favored. Moreover, the biosafety of the injected PEGylated AuNPs should be considered for potential medical applications. Synthetic AuNPs are generally recognized as safe and PEG is widely used in cosmetics and also used as a food additive approved by the US Food and Drug Administration.44-47 The current administration route (intravenous injection) and dosage (236 μg Au/g) of AuNPs used in our studies did not cause local inflammation, side effects or animal mortality after 30 days post-injection for various treatments, indicative of the biocompatibility of this technology (Supporting Figure S25). One potential barrier to AuNPs used in daily clinical applications is the high market cost of 18


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gold.33, 45 CONCLUSIONS In summary, PEGylated AuNPs with the optimized sizes at ~ 13 nm could be applied as X-ray theranostic agents for simultaneous enhanced CT imaging and radiosensitization. The optimized AuNPs could present significant in vivo efficacy with better CT contrast than Idohexol and suppressing the growth of tumors more drastically than CMNa. This study demonstrates that the optimal sizes of spherical AuNPs for simultaneous excellent CT imaging and radiosensitization could be selected and evaluated based on the distinctive size-dependent enhancement effects we found. In addition, we propose Monte Carlo simulations to theoretically evaluate our experimental discovery that is different from previous works. This approach eliminates subjective errors with providing sufficient and reliable basis for future clinical applications. Looking forward, this approach could be further applied to other materials (such as lanthanide, etc.) and other morphologies other than spheres that can interact with X-rays. The actual properties of the optimized AuNPs could be further tuned, through the coupling of surface ligands to enhance their active-targeting capability and designing biocompatible multifunctional tools combined with drug therapy or gene therapy to achieve better theranostic effects. Consequently, the actual effects of nanometer range combined with Monte Carlo simulations could be used in selection and evaluation of AuNPs to develop 19


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clinical X-ray theranostic fields.

20


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EXPERIMENTAL SECTION Synthesis and characterization of gold nanoparticles (AuNPs). AuNPs modified with PEG were prepared following the previous reports.48,

49

To

synthesize smaller-sized AuNPs (< 10 nm), typically, 10 mL of 0.5 mM trisodium citrate solution (Aldrich) was gently mixed with 10ml of 0.5 mM tetrachloroauric acid trihydrate (HAuCl4, Aldrich) solution and were stirred vigorously by adding 600 µl of ice-cold, freshly prepared 0.1 M sodium borohydride (NaBH4, Sigma) at 25 °C for 2 min, followed by aging at 25 °C for 2 h. Then 2.5 mL the resulting brownish yellow solution (3.9 nm ± 0.6 nm) were used as seed solution and was added to 200 ml growth solution composed of 0.08 M cetyltrimethylamonium bromide (CTAB, Sigma) solution with 0.25 mM HAuCl4 solution after mixing 0.05 mL of freshly prepared 0.1 M L-ascorbic acid (Aldrich) solution for stirring at 25 °C for 10 min to obtain deep red nanoparticles solution (9.1 nm ± 0.7 nm). To prepare larger-sized AuNPs (10 nm - 50 nm), 150 mL of 2.2mM trisodium citrate solution were mixed with 1 mL of 24 mM HAuCl4 solution at 100°C for 10 min under vigorous stirring. The resulting red-wine solution (13.2 nm ± 0.7 nm) was then maintained at 90 °C after mixed with 53 ml of deionized water. 55 mL of above solution were mixed with 2 mL of 60 mM trisodium citrate solution and then was added by 1 mL of 24 mM HAuCl4 solution at 90 °C for 30 min to increase AuNPs particle size (17.5 nm ± 1.1 nm). The above steps were repeated for fabricating AuNPs with other larger sizes (28.3 nm ± 2.2 nm, 34.8 nm ± 2.2 nm 21


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and 41.1 nm ± 1.9 nm). All the as-prepared particles were then washed by centrifugation in order to extract the surfactant and re-dispersed in deionized water. For PEGylation, following a previous report, we added 1mL of 2mM SH-PEG-COOH (number average molecular weight (Mn) = 2000g mol−1, Sigma) aqueous solution to 1mL of as-prepared AuNPs. After incubation at room temperature for 12 h, the modified gold nanoparticles were separated by centrifugation and re-dispersed in deionized water. The morphology of all above AuNPs was measured by Transmission electron microscopy (TEM) carried out using a JEOL 2010F analytical electron microscope at an operating voltage of 200 kV. Particle size and size distribution were measured through statistics histograms from TEM images.100 particles for statistics were measured for all sizes. The particle size of AuNPs is described as mean ± standard deviation. Maximum absorption wavelength and surface zeta-potential charges were analyzed using UV-visible spectrophotometer (Perkin-Elmer, United States) and a Malvern Zetasizer Nano ZS model ZEN 3600 (Worcestershire, UK) equipped with a standard 633 nm laser. Gold nanoparticles stability study. The stability of the PEGylated AuNPs was estimated by UV-visible spectroscopy. For pH stability, four sets of the PEGylated AuNPs solution (2.5 mM) possessed different pH conditions (5, 6, 7, and 8, respectively) via adjusting by HCl (0.1 M) or NaOH (0.1 M) and were kept at room temperature for 30min before measurements. For temperature stability, 22


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PEGylated AuNPs dispersed in deionized water were divided into four sets to maintain at different temperatures conditions (4, 25, 37, and 50 °C, respectively) for 30 min before measurements. The colloid stability of the PEGylated AuNPs was further evaluated after dispersed in water, phosphate-buffered saline (PBS), the Dulbecco's Modified eagle's Medium (DMEM, Welgene) and DMEM with 10% (v/v) fetal bovine serum (FBS, Gibco) for 240 days incubation at room temperature. In vitro attenuation and dose distributions measurement. PEGylated AuNPs of different particle size or idohexol (Omnipaque 350 mg I/mL, GE Healthcare) were diluted by PBS for different concentrations of Au or iodine (0 - 60 mM) were placed in 1.5-mL Eppendorf tubes and put in a self-designed scanning holder. In addition, PBS was used as blank control. CT images were obtained using a clinical Light Speed VCT CT imaging system (GE Medical Systems, Milwaukee, WI, USA). CT scanning parameters were as follows: slice thickness, 2.5 mm; pitch, 1:1; the tube voltage of 120 kV, the tube current of 200 mA; field of view, 512 × 512, gantry rotation time, 1 s. The X-ray attenuation measurements of PEGylated AuNPs were evaluated by loading the digital CT images in a standard display program. And then, Hounsfield units (HU) which were used to quantify the CT contrast enhancement were obtained from a uniform circular region of interest on the resultant CT image for each sample. Dose distributions measurements of PEGylated AuNPs were performed using accelerator planning system by 23


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calculating the accelerator monitor units (MU) with localizing the same reference point which supplies the same dose standard level for all samples to fix matching position. The standard lines and calculations of relative electron density were obtained also using calibrating procedure of accelerator planning system. Cell culture and cytotoxicity assessment. Human cervical carcinoma HeLa cells were cultured in DMEM which supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin in an incubator (Thermo Scientific) under an atmosphere of 5% CO2 and 90% relative humidity at 37 °C. The cells were sub-cultivated approximately every 3 days at 80% confluence using 0.25% (w:v) trypsin at a split ratio of 1:5. The cytotoxicity assessment was measured by the typical 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay, which is based on the mitochondrial conversion of the tetrazolium salt into a dye with absorption in the visible region. HeLa cells were seeded in 96 - well plates at 1 × 104 per well. After culture for 24 h, the cells were exposed to the solutions of different-sized

PEGylated

AuNPs,

Iohexol

and

CMNa

with

different

concentrations (0-100 µM) of gold or iodine for 24 h, followed by adding 20 µL of 5 mg/mL MTT solution. After further 4 h incubation, the medium was carefully removed, and the cells were mixed with 200 µL of dimethyl sulphoxide (DMSO). The absorbance was measured at a test wavelength of 560 nm using a microplate reader/Thermo Scientific Multiskan MK3 ELISA reader (Thermo Scientific, USA). Cell viability (%) was calculated by (A of tested cells /A of control 24


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sets) × 100%, where A represent the amounts of formazan determined for treated-cells and non-treated cells, respectively. Mean and standard deviation calculated from the quintuplicate wells were reported for all concentrations. In vitro CT imaging. Cell imaging experiments were performed in culture plates seeded with density of 1 × 106 HeLa cells per plate. After culture for 24 h, PEGylated AuNPs with the optimized sizes of around 13.2 nm and Idohexol were dispersed into DMEM solutions with a concentration of 200 µg/mL and then added into the culture dish. After co-incubation for 24 h, the cells were rinsed twice with PBS to remove free particles and were collected. The cells were treated with 3 mL 1% nitric acid (HNO3) aqueous solution with further sonication for 30 min in a hot water bath to completely disrupt the cell membranes. For converting to ions, gold nanoparticles and Idohexol were dissolved by successively adding 1.0 mL 37% (vol.) hydrochloric acid (HCl) and 0.3mL 70% (vol.) HNO3, followed by sonication for 20 min in a hot water bath for completely digesting the cells. The resultant solution was diluted with 1 mL of deionized water, followed by ICP-MS (DRCII, Perkin Elmer) analysis to determine of gold/iodine element contents in the cells. Cell suspensions with different concentrations (gold/iodine) of 0.5 - 10 mM were transferred to 0.2 mL Eppendorf tubes and placed in a self-designed scanning holder. CT images and corresponding attenuation measurements were acquired using a clinical CT imaging system with 120 kV, 200 mA. Moreover, cell morphology after incubation 25


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was examined under bright field using an inverted Olympus fluorescence microscope (IX-70). In vitro radiosensitization evaluation. HeLa cells were seeded into several 96-well plates at 105/well and then incubated for 24 h at 37 °C under 5% CO2. First, DMEM solutions of different-sized PEGylated AuNPs and CMNa with different concentrations of 5, 10, 25, 50, and 100 µM were added to the wells and co-incubated for another 4 h. PBS incubation was used as blank control. Excess particles were removed by cold PBS washing, followed by 0 (control), 2, 4, 6, or 8 Gy of X-ray radiations and incubated 20 h again. All the treatments were carried out only once. Cell viability was determined by MTT assay. Second, for colony formation assay, the cells were exposed to different-sized PEGylated AuNPs and CMNa solutions at 50 µM concentrations for another 24 h. After remove of excess particles, the cells were irradiated at doses of 0 (control), 2, 4, 6, and 8 Gy, respectively. All the treatments were given only once. After irradiation, HeLa cells were trypsinized, counted, and seeded in 6 cm dishes at appropriately same concentrations. There were five dishes for each dose. The cells were incubated for 12 days and then then stained with 0.4% crystal violet (Sigma-Aldrich, MO, USA). The colonies formations were fixed and colonies with more than 50 cells were counted for calculation of the surviving fraction (SF) by the proportion of seeded cells following irradiation to form colonies relative to untreated cells as described. The cell survival curve was estimated by a multitarget single-hit model 26


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(S = 1-(1-eD/D0) N) (L-Q) and then D0 was calculated, where S is the surviving fraction and D is the radiation dose. The sensitization enhancement ratio (SER) was determined by ratio of 50% survival of the cells with and without the presence of radiosensitizer (AuNPs or CMNa). Cell apoptosis assay. HeLa cells were incubated at a density of 1×105 cells per well in six-well plates for 24 h at 37 °C with 5% CO2, and the cells were divided into four groups: control (no AuNPs and no radiation), different-sized AuNPs or CMNa (only radiation), RT (only 6 Gy of X-ray radiation), different-sized AuNPs or CMNa +RT (6 Gy of X-ray radiation simultaneously with AuNPs or CMNa). 2 mL DMEM solutions of AuNPs and CMNa (50 µM) were added into the plates and co-incubated for 24 h. All the radiation treatments were given only once. Then after continuously culture for another 24 h, Annexin V binding was performed using Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, US). The subsequent procedures were performed in accordance with the manufacturer’s protocol. Briefly, the cells were washed twice with cold PBS and then suspended in 195 µL binding buffer at the concentration of 5×105 cells/mL. After incubation with 5 µL Annexin V-FITC for 10 min, the cells were re-suspended in 190µL binding buffer with adding 10 µL propidium iodide. After incubation for 15 min at room temperature in the dark, cell apoptosis were analyzed by flow cytometry (BD FACSCalibur). The Monte Carlo simulations. The EGSnrc Monte Carlo code toolkit was used 27


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in this study for radiation transport simulations using the Geant 427 Monte Carlo toolkit (version 3.3.0, National Research Council Canada).50 The medium algorithm was carried out for setting medium composition with all elements including gold, hydrogen and oxygen by weight fractions and coupled to actual solutions with same mass density for all particle sizes. The simulation geometry designed by “modeling vesicle” consisted of one or two gold nanoparticle (whose size could be varied at runtime), placed at the central axis of a cube of water with sides of 200 nm. Simulations were carried out with nanoparticles ranging in diameter from 2 to 40 nm. To give accurate statistics, the number of random histories was required 1 × 108 events using a condensed random walk method of particle transport with kerma approximation for all cases. 51, 52 The energy physics processes are capable of simulating secondary events to energies from low energy (KeV) to high energy (MeV) because the production threshold of secondary events are less than the 1 keV cutoff of the standard electromagnetic physics list. For CT attenuation simulations, photon fluence was calculated by FLURZnrc extension code were simulated followed by incident X-ray beams with monoenergetic energy of 120 keV mainly from point source off axis. The interaction

types

radiosensitization

were

settled

simulations

by

as

mainly

DOSRZnrc

photoelectric extension

effect.

code,

For

incident

monoenergetic energy was redefined as 1MeV depending mainly on electron pair effect. Energy absorption in the water volume surrounding AuNPs was recorded 28


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according to position and sizes. The output data is written to text files which are recorded includes: average total photon energy, total photon fluence/incident fluence(incl. secondaries), Total dose (Gray/(incident fluence)) and Total dose minus stoppers. The simulation output data were analyzed in order to determine the attenuation enhancement ratio and the dose enhancement ratio (DER) of one or two AuNPs geometries. In this study, the attenuation enhancement ratio is defined as the ratio of emitted photon fluence and incident fluence, and the dose enhancement factors (DEF) is defined as the ratio of the absorbed dose deposited in water phantom with and without AuNPs. The statistical errors are less than 1%. Animals and tumour xenograft models. All animals were performed in accordance with relevant laws and institutional guidelines, approved by the institutional committee for animal care and the National Ministry of Health. All procedures were carried out under sterile conditions. To set up the tumor xenograft model, Balb/c female nude mice (6 weeks, average body weight: 22 g; Animal Medical Laboratory Institute of Chinese Academy of Medical Sciences, Beijing) were subcutaneously injected in the bottom back with 5 × 107 HeLa cells/mouse. After approximately 3 weeks post injection, the tumor nodules reached a volume of 0.9 cm3. The tumor size was monitored by a vernier caliper and the tumor volume (V) was calculated as V = L × W 2/2, where L and W were the length and width of the tumor, respectively. 29


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In vivo CT imaging study. When the tumors reached to 200 – 400 mm3, PEGylated AuNPs with the optimized sizes of around 13.2 nm and Idohexol dispersed in PBS at gold or iodine dose of 60 nmol kg-1 were delivered to mice via subcutaneous injection or intravenous injection through the tail vein, respectively. Each nude mouse was anesthetized by intraperitoneal injection of 1% pentobarbital sodium (4 mL/kg). CT images were taken from GE Light Speed VCT clinical imaging system (USA) at 5 min, 15 min, 30 min, 45 min, 60 min, and 90 min post injection under same CT scanning parameters with in vitro attenuation measurements. In vivo radiosensitization evaluation. The tumor-bearing mice were weighed and randomly divided into different groups when the tumor volume reached to around 100 mm3. The mice were then divided into six groups: PBS (negative control), Radiotherapy (RT) alone, CMNa + RT within 30 min (effective time of CMNa), the optimized AuNPs + RT within 30 min, CMNa + RT over 4 h (ineffective time of CMNa) and the optimized AuNPs + RT over 4 h. Each group included five mice. From Day 0, the mice were intravenously injected with a gold dose of 60 nmol kg-1as same as CMNa. Then the mice received the 6 Gy of X-ray radiation according to group requirements on a Siemens Primus clinical linear accelerator (6 MeV) using a 1.5 cm × 1.5 cm radiation field to cover the entire tumor without the need of critical anatomies at a source-to-skin distance (SSD) of 100 cm. All the treatments were given only once. During the next 30 days, the 30


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tumor volume and the whole body weight of each mouse were measured by vernier caliper every other day. Histology analysis. At Day 30, the mice were euthanized, and the tumor as well as major organs was collected, weighed, washed with saline thrice and fixed in the 10% neutral-buffered formalin. For the hematoxylin and eosin staining, the formalin-fixed tumors were embedded in paraffin blocks and visualized by optical microscope (DM5500B, Leica). Biodistribution. The tissue biodistribution were studied after 24 h post-injection of AuNPs and Idohexol. The mice were euthanized and organs including brain, heart, lung, liver, spleen, kidneys, bladder, and tumor were collected, weighed, and digested in aqua regia solution for 4 h. The mouse with injection of PBS was used as blank control. Gold (Au) and iodine (I) contents in tissue samples was quantified by using inductively coupled plasma-atomic emission spectroscopy (ICP-AES; Perkin Elmer OPTIMA 4300 DV, MA). Statistical analysis. All values in the present study were expressed as mean ± s.d. Sample sizes were calculated, using InStat software, to allow the statistical significance of differences of 50% or greater to be determined. Statistical analysis was performed using OriginPro 9.0 and Microsoft Excel. Sample variance was tested using the F-test. For samples with equal variance, the significance between the groups were analyzed by a, two-tailed, Student’s t-test. For samples with unequal variance, a two-tailed Welch’s t-test was performed. In all cases, the 31


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data were indicated with (*) for p < 0.05, (**) for p < 0.001, and (***) for p < 0.0001, respectively. 0.05 of p value was considered significant. Author contributions. Yan Dou conceived the idea and designed the experiments. Yan Dou and Yanyan Guo performed the MC simulations. Yan Dou, Xue Li and Guoxian Lv performed the experiments. Xiaodong Li and Lin Wang conducted

radiotherapy

measurements.

Xuening

Zhang

conducted

CT

measurements. Yan Dou and Sheng Wang analyzed the data. Yan Dou wrote the manuscript. Jin Chang, Hanjie Wang and Xiaoqun Gong directed the research. All authors discussed the results and improved the manuscript. The principal investigator is Jin Chang. Conflict of Interest: The authors declare no competing financial interests. Acknowledgment. This work was supported by National Natural Science Foundation of China (51373117, 51573128, 51303126), Key Project of Tianjin Natural Science Foundation (13JCZDJC33200), National High Technology Program of China (2012AA022603), and Doctoral Base Foundation of Educational Ministry of China (20120032110027). Supporting Information Available: This included Supplementary TEM images, statistics histograms and UV-visible absorption spectra for AuNPs after PEGylation, the stability of PEGylated AuNPs under different PH, temperature conditions and storage times, cell viability tests, X-ray attenuation intensity and relative electron density test, cellular radiosensitization results, Monte Carlo 32


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simulations results, real-time CT imaging by intratumoral and intravenous injection, in vivo radiosensitization therapy, in vivo biodistribution and histology assessments. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org.

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REFERENCES AND NOTES 1.

Canellos, G. P. Clinical Oncology: Onward and Upward. J. Clin. Oncol. 2008, 26, 2-2.

2.

Villanueva, M. T. Imaging in Oncology: Lighting the Way. Nat. Rev. Clin. Oncol. 2012, 9, 667.

3.

Butof, R.; Baumann, M. Time in Radiation Oncology-Keep it Short! Radiother. Oncol. 2013, 106, 271-275.

4.

Cheng, L.; Yuan, C.; Shen, S.; Yi, X.; Gong, H.; Yang, K.; Liu, Z. Bottom-Up Synthesis of Metal-Ion-Doped WS2 Nanoflakes for Cancer Theranostics. Acs Nano 2015, 9, 11090-11101.

5.

Song, G.; Liang, C.; Gong, H.; Li, M.; Zheng, X.; Cheng, L.; Yang, K.; Jiang, X.; Liu, Z. Core–Shell Mnse@Bi2Se3 Fabricated via a Cation Exchange Method as Novel Nanotheranostics for Multimodal Imaging and Synergistic Thermoradiotherapy. Adv. Mater. 2015, 27, 6110-6117.

6.

Choi, H. S.; Liu, W.; Liu, F.; Nasr, K.; Misra, P.; Bawendi. M. G.; Frangioni, J. V.

Design

Considerations

for

Tumour-Targeted

Nanoparticles.

Nat.Nanotechnol. 2009, 5, 42-47. 7.

Lee, D.-E.; Koo, H.; Sun, I.-C.; Ryu, J. H.; Kim, K.; Kwon, I. C. Multifunctional Nanoparticles for Multimodal Imaging and Theragnosis. Chem Soc Rev. 2012, 41, 2656-2672.

8.

Kukowska-Latallo.J. F.; Candido, K. A.; Cao, Z.; Nigavekar, S. S.; Majoros, 34


ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 55

I. J.; Thomas,T. P.; Balogh, l. P.; Khan, M. K.; Baker-Jr, J. R. Nanoparticle Targeting of Anticancer Drug Improves Therapeutic Response in Animal Model of Human Epithelial Cancer. Cancer Res. 2005, 65, 5317-5324. 9.

Xing, H.; Zheng, X.; Ren, Q.; Bu, W.; Ge, W.; Xiao, Q.; Zhang, S.; Wei, C.; Qu, H.; Wang, Z.; Hua, Y.; Zhou, L.; Peng, W.; Zhao, K.; Shi, J. Computed Tomography Imaging-Guided Radiotherapy by Targeting Upconversion Nanocubes

with

Significant

Imaging

and

Radiosensitization

Enhancements. SCI. REP-UK. 2013, 3, 1751. 10.

Xiao, Q.; Zheng, X.; Bu, W.; Ge, W.; Zhang, S.; Chen, F.; Xing, H.; Ren, Q.; Fan, W.; Zhao, K.; Hua, Y.; Shi, J. A Core/Satellite Multifunctional Nanotheranostic for in Vivo Imaging and Tumor Eradication by Radiation/Photothermal Synergistic Therapy. J. Am. Chem. Soc. 2013, 135, 13041-13048.

11.

Jakhmola, A.; Anton, N.; Vandamme, T. F. Inorganic Nanoparticles Based Contrast Agents for X-Ray Computed Tomography. Adv. Healthc. Mater. 2012, 1, 413-431.

12.

Peng, C.; Zheng, L.; Chen, Q.; Shen, M.; Guo, R.; Wang, H.; Cao, X.; Zhang, G.; Shi, X. Pegylated Dendrimer-Entrapped Gold Nanoparticles for in Vivo Blood Pool and Tumor Imaging by Computed Tomography. Biomaterials 2012, 33, 1107-1119.

13.

Deng, H.; Zhong, Y.; Du, M.; Liu, Q.; Fan, Z.; Dai, F.; Zhang, X. Theranostic 35


Page 37 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Self-Assembly Structure of Gold Nanoparticles for NIR Photothermal Therapy and X-Ray Computed Tomography Imaging. Theranostics 2014, 4, 904-918. 14.

Hainfeld, J. F.; Smilowitz, H. M.; O’Connor, M. J.; Dilmanian, F. A.; Slatkin, D. N. Gold Nanoparticle Imaging and Radiotherapy of Brain Tumors in Mice. Nanomedicine 2013, 8, 1601-1609.

15.

Liu, H.; Wang, H.; Xu, Y.; Shen, M.; Zhao, J.; Zhang, G.; Shi, X. Synthesis of Pegylated Low Generation Dendrimer-Entrapped Gold Nanoparticles for CT Imaging Applications. Nanoscale 2014, 6, 4521-4526.

16.

Wen, S.; Li, K.; Cai, H.; Chen, Q.; Shen, M.; Huang, Y.; Peng, C.; Hou, W.; Zhu, M.; Zhang, G.; Shi, X. Multifunctional Dendrimer-Entrapped Gold Nanoparticles for Dual Mode CT/MR Imaging Applications. Biomaterials 2013, 34, 1570-1580.

17.

Zhang, X.-D.; Chen, J.; Luo, Z.; Wu, D.; Shen, X.; Song, S.-S.; Sun, Y.-M.; Liu, P.-X.; Zhao, J.; Huo, S.; Fan, S; Fan, F.; Liang, X.-J.; Xie, J. Enhanced Tumor Accumulation of Sub- 2nm Gold Nanoclusters for Cancer Radiation Therapy. Adv. Healthc. Mater. 2014, 3, 133-141.

18.

Huang, P.; Bao, L.; Zhang, C.; Lin, J.; Luo, T.; Yang, D.; He, M.; Li, Z.; Gao, G.; Gao, B.; Fu, S.; Cui. D. Folic Acid-Conjugated Silica-Modified Gold Nanorods for X-Ray/CT Imaging-Guided Dual-Mode Radiation and Photo-Thermal Therapy. Biomaterials 2011, 32, 9796-809. 36


ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

19.

Page 38 of 55

Jeong, S. Y.; Park, S. J.; Yoon, S. M.; Jung, J.; Woo, H. N.; Yi, S. L.; Song, S. Y.; Park, H. J.; Kim, C.; Lee, J. S.; Choi, E. K. Systemic Delivery and Preclinical Evaluation of Au Nanoparticle Containing Beta-Lapachone for Radiosensitization. J. Control. Release. 2009, 139, 239-245.

20.

Lee, N.; Choi, S. H.; Hyeon, T. Nano-Sized CT Contrast Agents. Adv. Mater. 2013, 25, 2641-2660.

21.

Maltzahn, G. V.; Park, J. H.; Agrawal, A.; Bandaru, N. K.; Das, S. K.; Sailor, M. J.; Bhatia, S. N. Computationally Guided Photothermal Tumor Therapy Using Long-Circulating Gold Nanorod Antennas. Cancer Res. 2009, 69, 3892-3900.

22.

Xu, C.; Tung, G. A.; Sun, S. Size and Concentration Effect of Gold Nanoparticles

on

X-ray Attenuation as Measured on

Computed

Tomography. Chem. Mater. 2008, 20, 4167-4169. 23.

Zhang, X. D.; Wu, D.; Shen, X.; Chen, J.; Sun, Y. M.; Liu, P. X.; Liang, X. J. Size-Dependent Radiosensitization of PEG-Coated Gold Nanoparticles for Cancer Radiation Therapy. Biomaterials 2012, 33, 6408-6419.

24.

Jones, C. F.; Grainger, D. W. In Vitro Assessments of Nanomaterial Toxicity. Adv. Drug. Deliver. 2009, 61, 438-456.

25.

Brannon-Peppas, L.; Blanchette, J. O. Nanoparticle and Targeted Systems for Cancer Therapy. Adv. Drug. Deliver. 2012, 64, 206-212.

26.

Suh, W. H.; Suslick, K. S.; Stucky, G. D.; Suh, Y. H. Nanotechnology, 37


Page 39 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Nanotoxicology, and Neuroscience. Prog. Neurobiol. 2009, 87, 133-170. 27.

McMahon, S. J.; Hyland, W. B.; Muir, MF.; Coulter, J. A.; Jain, S.; Butterworth, K. T.; Schettino, G.; Dickson, G. R.; Hounsell, A. R.; O'Sullivan, J. M.; Prise, K. M.; Hirst, D. G.; Currrell, F. J. Biological Consequences of Nanoscale Energy Deposition Near Irradiated Heavy Atom Nanoparticles. SCI. REP-UK. 2011, 1, 18.

28.

Jeynes J. C.; Merchant, M. J.; Spindler, A.; Wera, A. C.; Kirkby, K. J. Investigation of Gold Nanoparticle Radiosensitisation Mechanisms Using a Free Radical Scavenger and Protons of Different Energies. Phys. Med. Biol. 2014, 59, 6431-6443.

29.

Metropolis, N.; Ulam, S. The Monte Carlo Method. J. Am. Stat. Assoc. 1949, 44, 335-341.

30.

Douglass, M.; Bezak, E.; Penfold, S. Monte Carlo Investigation of the Increased Radiation Deposition Due to Gold Nanoparticles Using Kilovoltage and Megavoltage Photons in a 3D Randomized Cell Model. Med. Phys. 2013, 40, 071710.

31.

Leung, M. K. K.; Chow, J. C. L.; Chithrani, B. D.; Lee, M. J. G.; Oms, B.; Jaffray, D. A. Irradiation of Gold Nanoparticles by X-Rays: Monte Carlo Simulation of Dose Enhancements and the Spatial Properties of the Secondary Electrons Production. Med. Phys. 2011, 38, 624-631.

32.

Yu, S.B.; Watson, A. D. Metal-Based X-ray Contrast Media. Chem. Rev. 38


ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 55

1999, 99, 2353-2378. 33.

Lusic, H.; Grinstaff, M. W. X-Ray-Computed Tomography Contrast Agents. Chem. Rev. 2013, 113, 1641-1666.

34.

Nilsson, B. Electron Contamination from Different Materials in High Energy Photon Beams. Phy.s Med. Biol. 1985, 30, 139-151.

35.

Biggs, P. J.; Russell, M. D. An Investigation into the Presence of Secondary Electrons in Megavoltage Photon Beams. Phy.s Med. Biol. 1983, 28, 1033-1043.

36.

Lin, Y.; McMahon, S. J.; Scarpelli, M.; Paganetti, H.; Schuemann, J. Comparing Gold Nano-Particle Enhanced Radiotherapy with Protons, Megavoltage Photons and Kilovoltage Photons: A Monte Carlo Simulation. Phy.s Med. Biol. 2014, 59, 7675-7689.

37.

Keall, P.; Monti, d. S. F.; Beckham, W.; Delaney, G. Comparison of Kilovoltage X-Ray and Electron Beam Dose Distributions for Radiotherapy of The Sternum. Med. Dosim. 1999, 24, 141–144.

38.

Robar, J. L. Generation and Modelling of Megavoltage Photon Beams for Contrast-Enhanced Radiation Therapy. Phy.s Med. Biol. 2006, 51, 5487-5504.

39.

Yu, M.; Zheng, J. Clearance Pathways and Tumor Targeting of Imaging Nanoparticles. ACS nano 2015, 9, 6655-6674.

40.

Zhang, X. D.; Wu, D.; Shen, X.; Liu, P. X.; Fan, F. Y.; Fan, S. J. In Vivo 39


Page 41 of 55

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Renal

Clearance,

Biodistribution,

Toxicity

of

Gold

Nanoclusters.

Biomaterials 2012, 33, 4628-4638. 41.

Cho, W.-S.; Cho, M.; Jeong, J.; Choi, M.; Han, B. S.; Shin, H.-S.; Hong, J.; Chung, B. H.; Jeong, J.; Cho, M.-H. Size-Dependent Tissue Kinetics of PEG-Coated Gold Nanoparticles. Toxicol. Appl. Pharm. 2010, 245, 116–123.

42.

Cho, E. C.; Zhang, Q.; Xia, Y. The Effect of Sedimentation and Diffusion on Cellular Uptake of Gold Nanoparticles. Nat. Nanotechnol. 2011, 6, 385-391.

43.

Mout, R.; Moyano, D. F.; Rana, S.; Rotello, V. M. Surface Functionalization of Nanoparticles for Nanomedicine. Chem. Soc. Rev. 2012, 41, 2539-2544.

44.

Khlebtsov, N.; Dykman, L. Biodistribution and Toxicity of Engineered Gold Nanoparticles: A Review of in Vitro and in Vivo Studies. Chem. Soc. Rev. 2011, 40, 1647-1671.

45.

Weintraub, K. Biomedicine: The New Gold Standard. Nat. Nanotechnol. 2013, 495, 128-129.

46.

Pelegri-O'Day,

E.M.;

Lin,

EW.;

Maynard,

H.

D.

Therapeutic

Protein-Polymer Conjugates: Advancing beyond Pegylation. J. Am. Chem. Soc. 2014, 136, 14323-14332. 47.

Delgado, C.; Francis, G. E.; Fisher, D. The Uses and Properties of 40


ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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PEG-Linked Proteins. Crit. Rev. Ther. Drug. 1992, 9, 249-304. 48.

Bastús, N. G.; Comenge, J.; Puntes, V. Kinetically Controlled Seeded Growth Synthesis of Citrate-Stabilized Gold Nanoparticles of Up to 200 nm: Size Focusing Versus Ostwald Ripening. Langmuir 2011, 27, 11098-11105.

49.

Jana, N. R.; Gearheart, L.; Murphy C. J. Seeding Growth for Size Control of 5-40 nm Diameter Gold Nanoparticles. Langmuir 2001, 17, 6782-6786.

50.

Kawrakow, I. Accurate Condensed History Monte Carlo Simulation of Electron Transport. I. Egsnrc, the New EGS4 Version. Med. Phys. 2000, 27, 485-498.

51.

Botta, F.; Mairani, A.; Hobbs, RF.; Gil, A. V.; Pacilio M.; Parodi, K. Use of the FLUKA Monte Carlo Code for 3D Patient-Specific Dosimetry on PET-CT and SPECT-CT Images. Phy.s Med. Biol. 2013, 58, 8099-8120.

52.

Xuejun, W.; Yulong, Y.; Huazhong, S.; Jiawang, W.; Jiang, S. B.; Limin L. A Vectorized Monte Carlo Code for Radiotherapy Treatment Planning Dose Calculation. Phy.s Med. Biol. 2003, 48, N111-N120(110).

41


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Scheme 1 The approach for optimizing particle sizes of spherical AuNPs as X-ray theranostic adjuvants. Size-dependent enhancements were discovered by examining CT attenuation and radiosensitization from a size range of 3-50 nm by in vitro experiments. After the evaluations by MC simulations, AuNPs with the optimal sizes at around 13 nm were modified with biocompatible molecules, and then intravenously injected into mice for in vivo assessments. X-rays with high frequency have the deeper tissue penetration compared to the lights commonly used in electromagnetic spectrum, which are expected to trigger the interactions with AuNPs aggregated in the tumors through EPR effects for simultaneously enhanced CT imaging and radiotherapy. 43


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Figure 1 Basic characterization of AuNPs with different particle size. TEM images showing seven types of AuNPs: 3.9nm ± 0.6 nm (a), 9.1 nm ± 0.7 nm (c), 13.2 nm ± 0.7 nm (e), 17.5 nm ± 1.1 nm (g), 28.3 nm ± 2.2 nm (i), 34.8 nm ± 2.2 nm (k), 41.1 nm ± 1.9 nm (m). The 50 nm scale bar applies to all images. Statistics histograms (b, d, f, h, j, l, n corresponded with a, c, e, h, i, k, m, respectively) of particle size and particle-size distribution by measuring the diameter of the particles through TEM images to obtain the results as a rough approximation of (the mean diameter ± the standard deviation). 100 particles for statistics were measured for all particle size. 44


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UV-visible absorption spectra (o) of as-prepared AuNPs for seven types together with the color change from brown yellow to mauve as the particle size increases. Variations of UV-visible maximum absorption wavelength (p) show an increase to confirm the growth of particle size.

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Figure 2 Size-dependent enhancements and characterization. (a) Representative CT contrast (up) and dose distribution (down) phantom images of various size of AuNPs under same concentration ([Au] = 0.02 M) with PBS as control. (b) Accurate measurements of CT attenuation Hounsfield units and Accelerator monitor units changes against particle size indicating size-dependent effect (n = 3). (c) Representative CT contrast phantom images of different sizes of AuNPs (up) and Idohexol (down) at concentration of [Au or I] = 0.02 M. (d) The attenuation enhancement disparity between AuNPs and Idohexol (indicated by HUAu - HUI/ HUI) under 0.02 M element concentrations for different particle sizes (n = 3). (e) Clonogenic survival curves of all sizes of AuNPs compared with CMNa and PBS as control ([Au or CMNa] = 50 µM), under various irradiation doses (n = 4). (f,g)The sensitization 46


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enhancement ratio (SER) of all sizes of AuNPs with various concentrations under 2 Gy radiation (f) and under 6 Gy radiation (n = 5) (g). (h) The sensitization enhancement disparity between AuNPs and CMNa (indicated by SERAu - SERCMNa/ SERCMNa) for various particle sizes at 50 µM concentrations under 6 Gy radiation (n = 3). Biological replicates were used, and studies were repeated at least two times in the laboratory. Error bars, mean ± s.d. *P < 0.05, * *P < 0.001.

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Figure 3 Monte Carlo simulations to evaluate size-dependent enhancements. (a) A schematic showing a phantom filled with AuNPs aqueous suspensions which may trigger completely different secondary radiation depending on primary radiation energies irradiated from X-ray point source for CT detection or radiotherapy. A simulated “modeling vesicle” containing two particles randomly distributed representative of the system inhomogeneity based on particle sizes. (b) X-ray attenuation mainly comes from Photoelectric effect under the kilovoltage energy radiation for CT imaging expect little influence by other interactions including Rayleigh scattering, Compton scattering, and Electron pair effect. (c) Photoelectric effect generation. The inner-shell electrons receive energy from the incident X-rays, which are subsequently ejected from the atom as photoelectron, and the vacancy left in the electron shell (typically K or L shell) is quickly filled by outer-shell electrons, producing characteristic X-rays. (d) Total emitted photon fluence variation with simulated particle sizes compared to water absence of particles under incident fluence level. (e) Total emitted photon fluence and attenuation

enhancement

ratio

of

simulated

particle

sizes,

indicating

size-dependent effects respectively. (f) Dose absorption mainly comes from Electron pair effect under the megavoltage energy radiation for radiotherapy expect little influence by other interactions including Photoelectric effect, Rayleigh scattering, and Compton scattering. (g) Electron pair effect generation. The incident X-ray photons with high enough energy pass from the nucleus, and then 49


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are transformed into a positron and a negative electron resulting from nuclear coulomb field. (h) Dose absorption levels (left) and dose enhancement ratios (DER) (right) of simulated particle sizes. Inset, DER disparity is highlighted for sizes over 5nm. (i) Absorbed dose per specific surface area of simulated particle sizes with water as control. Simulations were repeated at least three times using the Monte Carlo code EGSnrc. Error bars, mean ± s.d. *P < 0.05.

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Figure 4 In vitro characterizations of the optimized AuNPs with 13.2 nm ± 0.7 nm diameters as X-ray theranostic agents. (a, b) CT contrast phantom images (a) and X-ray attenuation (b) of 13.2 nm ± 0.7 nm PEGylated AuNPs compared with Idohexol measured through endocytosis into HeLa cells at varying concentrations of 0.5-10 mM (n = 3). (c) The sensitization enhancement ratio (SER) of ~ 13 nm AuNPs and CMNa with different concentrations (n = 3). (d) The cell viability and the sensitization enhancement ratio (SER) for cells treated with ~ 13 nm PEGylated AuNPs, CMNa at concentration of 50 µM, and blank control (PBS) before and after 6 Gy radiation (n = 5). (e,f) Representative flow cytometry plots to quantitatively analyze cellar apoptosis and necrosis induced by ~ 13 nm 51


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AuNPs, CMNa and blank control (PBS) with (e) and without (f) 6 Gy X-ray irradiation (n = 3). Biological replicates were used, and studies were repeated at least two times in the laboratory. Error bars, mean ± s.d. *P < 0.05, * *P < 0.001.

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Figure 5 In vivo characterizations of the optimized AuNPs with 13.2 nm ± 0.7 nm diameters as X-ray theranostic agents. (a) Representative real-time in vivo CT contrast phantom images of heart, bladder and tumor regions of the HeLa xenograft tumor-bearing balb/c nude mice at 0, 5, 15, 30 and 60 min after intravenous injection of 13.2 nm AuNPs (left) and Idohexol (right) respectively with the same dose of 0.6 nmol/g ([Au or I]) at different time points post-injection (n = 3). Arrows indicate the interesting regions. (b) Region-of-interest quantitative analysis of X-ray attenuation signals from the tumors and other tissues as a function of time post-injection of 13.2 nm AuNPs and Idohexol (n = 3). (c) 53


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Representative images of in vivo volume and ex vivo weight of the tumors harvested from the euthanized the HeLa xenograft tumor-bearing nude mice after treatment with Radiotherapy for different groups as follows: Control with PBS injection, Radiotherapy alone, Radiotherapy within 30 min after intravenous injection of CMNa (0.6 nmol/g), Radiotherapy within 30 min after intravenous injection of AuNPs (0.6 nmol/g), Radiotherapy over 30 min after intravenous injection of AuNPs (0.6 nmol/g) and Radiotherapy over 30 min after intravenous injection of AuNPs (0.6 nmol/g). (d,e) In vivo tumor growth inhibition curves from different groups for 30 days after various treatments (n = 5). (f) The body weight variation of HeLa xenograft tumor-bearing mice during treatment (n = 5). (g) Representative histology (hematoxylin and eosin staining (H&E)) of the tumors of mice after treatment (top), with corresponding image enlargements (bottom) (n = 5). Error bars, mean ± s.d. *P < 0.05, ** P < 0.01 and *** P < 0.001 (two-tailed Student’s t-test).

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Size-Tuning Ionization To Optimize Gold Nanoparticles for

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