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. Author manuscript; available in PMC: 2024 Jan 1.
Published in final edited form as: Int J Radiat Biol. 2022 Jun 21;99(2):308–317. doi: 10.1080/09553002.2022.2087931

Microdosimetric and Radiobiological Effects of Gold Nanoparticles at Therapeutic Radiation Energies

Tara Gray 1, Shaquan David 1, Nema Bassiri 2, Devanshi Yogeshkumar Patel 1, Neil Kirby 2, Kathryn M Mayer 1
PMCID: PMC10089366  NIHMSID: NIHMS1871972  PMID: 35709481

Abstract

Purpose:

The purpose of this study was to quantify the microscopic dose distribution surrounding gold nanoparticles (GNPs) irradiated at therapeutic energies and to measure the changes in cell survival in vitro caused by this dose enhancement.

Methods:

The dose distributions from secondary electrons surrounding a single gold nanosphere and single gold nanocube of equal volume were both simulated using MCNP6. Dose enhancement factors (DEFs) in the 1 μm3 volume surrounding a GNP were calculated and compared between a nanosphere and nanocube and between 6 MV and 18 MV energies. This microscopic effect was explored further by experimentally measuring cell survival of C-33a cervical cancer cells irradiated at 18 MV with varying doses of energy and concentrations of GNPs. Survival of cells receiving no irradiation, a 3 Gy dose, and a 6 Gy dose of 18 MV energy were determined for each concentration of GNPs.

Results:

It was observed that dose from electrons surrounding the gold nanocube surpasses that of a gold nanosphere up to a distance of 1.1 μm by 18.5% for the 18 MV energy spectrum and by 23.1% for the 6 MV spectrum. DEFs ranging from approximately 2–8 were found, with the maximum DEF resulting from the case of the gold nanocube irradiated at 6 MV energy. Experimentally, for irradiation at 18 MV, incubating cells with 6 nM (0.10% gold by mass) GNPs produces an average 6.7% decrease in cell survival, and incubating cells with 9 nM (0.15% gold by mass) GNPs produces an average 14.6% decrease in cell survival, as compared to cells incubated and irradiated without GNPs.

Conclusion:

We have successfully demonstrated the potential radiation dose enhancing effects in vitro and microdosimetrically from gold nanoparticles.

Keywords: Gold nanoparticles, Monte Carlo simulations, Dose enhancement factor, Radiation therapy, Microdosimetry

1. Introduction

Over the past decade, gold nanoparticles (GNPs) have emerged as a possible radiosensitization agent in clinical practice.(Schuemann et al., 2016) This radiosensitization effect originates from low-energy electron production from the surface of GNPs and the deposition of their energy at the subcellular or DNA level. Although the general phenomenon of dose enhancement by gold has been well known for decades, the nanoparticle-mediated microscopic and radiobiological enhancements are not yet well understood. Understanding these questions remains important to study before gold nanoparticle-enhanced radiation therapy can fully become clinically viable. Interdisciplinary research between medical physics, nanoscience, and biology is needed to understand the true radiobiological effects of nanoparticle-enhanced radiation.

As of late, there has been great demand for microdosimetry studies indicating the impact of gold nanoparticle radiosensitization at the cellular level. Due to many experimental difficulties measuring low-energy electron doses on a nano- or micrometer scale, Monte Carlo studies have primarily been the source for quantitative information about the dose distribution on this level.

In our previous study exploring the effects of megavoltage energies on GNP dose enhancement for different concentrations of GNPs, it was shown via both experiments and macroscopic Monte Carlo simulations that the effect of physical, macroscopic dose enhancement using GNPs can be up to about 2.15%, using a GNP mass percent of about 1.3% with an energy of 18 MV.(Gray et al., 2020) Dose enhancement due to GNPs has also been characterized via electron spin resonance (ESR) in the presence of alanine, a method which detects radiation-induced free radicals. In a 2021 paper, this technique measured DEFs of up to ~3; however, this was for lower energies of kVp = 42 keV.(Lima et al., 2021) An earlier report using the ESR method found macroscopic DEFs of up to 1.27 at 6 MV energies for dry powder samples.(Wolfe et al., 2015b) The simulations in the present study were carried out to gain insight on the microscopic dose distribution, which is not possible to measure directly by any existing standard method. We show here via Monte Carlo simulations that much larger physical dose enhancements are seen microscopically, with ranges of secondary electrons enough to penetrate through a DNA molecule. This comparison is important in that it shows the large contribution that gold nanoparticles can potentially make to cancer cell DNA damage and decreased cell viability in vivo. In addition, we computationally investigate the effects of GNP surface area to volume ratio on the radial dose distribution of secondary electrons and the dose enhancement immediately surrounding a single GNP for therapeutic energies of 6 MV and 18 MV. We also experimentally explore the dose enhancement effects at a cellular level in vitro for 18 MV photons and different concentrations of GNPs taken up by C-33a cervical cancer cells, as measured in terms of cell viability. Irradiation at 18 MV was chosen for the study because it is a clinically relevant irradiation method where we also expect to see gold nanoparticle enhancement. Radiation enhancement due to gold nanoparticles is seen in the keV range due to the photoelectric effect; however, enhancement is also seen in the range of 18 MV irradiation due to pair production. It is the latter effect that we exploit in the present study.

To the authors’ knowledge, this is the first study to demonstrate the effect of PEGylated GNPs on C-33a cells irradiated at 18 MV energy. It is also the first Monte Carlo simulation study to demonstrate the effects on dose enhancement surrounding a single GNP provided by increasing the surface-area to volume ratio of a single GNP by keeping volume constant and changing the surface area by changing the shape of the GNP from a sphere to a cube. This study is also the first of its kind to utilize the Trypan Blue assay to evaluate cell survival after irradiation with high energy gamma rays. Another unique data analysis technique explored in our present study is the evaluation of synergistic effects of cell survival with cells containing GNPs and given radiation as compared with cells given radiation only. Most importantly, this study provides evidence for microscopic dose enhancement and radiobiological (radiosensitization) enhancement due to gold nanoparticles which are significantly higher than the previously measured macroscopic dose enhancement.

2. Materials and Methods

2.1. Microdosimetric Gold Nanoparticle Dose Distribution using MCNP6.2

Radial dose distributions for 6 MV and 18 MV external beam energies surrounding a single GNP were simulated using MCNP6.2. A photon source definition for these microscopic simulations was first generated by estimating an energy spectrum using the F2 tally (photons/cm2) from macroscopic simulations of GNP dose enhancement (Tara Gray, 2020) as shown in Figure 1a at a depth in the center of a GNP-containing volume for both 6 MV and 18 MV energy beams. In a subsequent microscopic simulation, this energy spectrum was used for a set of six point source evenly distributed around a single GNP as seen in Figure 1b. Each point source was placed 1 nm from the surface of the GNP. Approximately 4 × 109 photons were simulated for each run. A phase-space file was scored on the surface of the GNP, using the SSW/SSR cards in MCNP. The surface-source write (SSW) card in MCNP records particle energy, position, and direction angle to use as a source (using the surface-source read (SSR) card in a subsequent simulation. This phase space file was used as a source of electrons only in a subsequent microscopic simulation, where dose was scored in concentric spherical shells of radii ranging from 20 nm to 2200 nm, in 1 nm increments, surrounding a 15 nm radius GNP sphere, using the F6 tally (MeV/g). The new MCNP6.2 physics library, eprdata12, based on the Evaluated Nuclear Data File/B-VI.8 (ENDF/B-VI.8), was used to simulate electrons down to an energy threshold of 100 eV. The eprdata12 physics library includes cross-section data for coherent and incoherent scattering and photoelectric absorption down to 1 eV. A new single-event method to use these data for low energy electron transport was developed to replace the normal condensed history method used by MCNP6.2 and was implemented in each simulation run.(Werner, 2017) The dose distribution surrounding a gold nanosphere and a gold nanocube of the same volume (each side of the gold nanocube was approximately 24 nm) were both simulated in the above fashion and compared to determine the difference in radial dose distribution corresponding to a change in surface-area to volume ratio.

Figure 1.

Figure 1.

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(a) MCNP6.2 simulation set-up to estimate energy spectrum in center of GNP solution. (b) Simulation set-up with surrounding isotropic sources emitting photons to calculate dose from electrons emitted from surface of GNP.

The DEF for electron dose in a 1 μm3 volume surrounding each GNP (sphere or cube) at each energy spectrum (6 MV or 18 MV) was computed by first estimating the dose per unit volume to an equal volume (1 μm3) composed of water only from a macroscopic simulation. The F6 tally was utilized to tally the dose from secondary electrons from the surface of the GNP contributing to dose inside the 1μm3 volume in a separate simulation, using the phase-space file generated in the previous simulations as the source. Dose tallied to the volume containing a GNP was divided by dose tallied to water without a GNP to get a DEF for the 1 μm3 volume. This DEF was compared between a sphere and a cube for each energy.

2.2. Gold Nanoparticle Synthesis and PEG Functionalization

A standard citrate reduction method was utilized to synthesize GNPs in this study.(Kimling et al., 2006) After the GNPs were formed in solution, they were functionalized with polyethylene glycol (PEG). Carboxy-PEG-thiol (10 mg, molecular weight 5000) was dissolved in 1 mL of ultrapure water. In order to achieve a final PEG concentration of 0.0036 mg/mL(Manson et al., 2011), 2.348 mL of the PEG solution was added to 650 mL of the as-synthesized GNP solution. The solution was stirred rapidly at room temperature for 2 hours continuously, using a magnetic stir rod, to ensure full reaction. This was followed by a wash step, during which the GNP + PEG solution was placed in 50 mL centrifuge tubes and centrifuged for 20 minutes, supernatant was disposed of (with GNPs remaining) and water was added in place of supernatant on top of GNPs in each centrifuge tube. The GNPs + PEG + water solution was then centrifuged again for another 20 minutes, again removing the supernatant. This wash step was performed twice.

The GNPs were characterized through scanning electron microscopy (SEM) on a Hitachi S5500 SEM microscope (Figure 2), and the approximate size of the GNPs observed was found to be 29.8 ± 2.8 nm in diameter. GNP solution was concentrated by aliquoting the GNP solution in 15 mL centrifuge tubes and centrifuging at 4,000 rpm for 25 minutes. After centrifuging initially, 14 mL supernatant was removed. The 1 mL remaining in the tubes was re-suspended in ultrapure water and re-centrifuged using the same rpm and time. After centrifugation, the supernatant was removed a second time from each centrifuge tube. The remaining GNP solution in each tube was combined into one tube and centrifuged a final time in order to obtain the desired GNP solution concentration for use in the experiment. A fixed volume of GNP solution was weighed using an analytical balance to determine mass percent of gold in each solution.

Figure 2.

Figure 2.

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SEM image of GNPs.

2.3. Cell Culture

C-33a cervical cancer cells were sub-cultured and maintained in complete Eagle’s Minimal Essential Medium (EMEM), with 10% Fetal Bovine Serum (FBS) and 0.1 mg/mL penicillin at a temperature of 37°C and 5% CO2. Both cells and culture media were purchased from the American Type Culture Collection (ATCC).

For the GNP experiments, GNPs in PBS buffer were added to each of the four plates labeled for GNP solution in each of the three 6-well plates. In summary, each plate contained two wells with no GNPs, two wells containing a final concentration of 0.10% GNPs by mass (6 nM) and two wells containing a final concentration of 0.15% GNPs by mass (9 nM). The C-33a cells and C-33a cells + GNPs were incubated for 24 h after addition of GNPs to ensure GNP uptake by cells.

2.4. Irradiation of Cells

Cultures of the C-33a cells were irradiated using a Varian 23EX linear accelerator at the UT Health MD Anderson Mays Cancer Center (San Antonio, TX). Cells were plated in three 6-well plates, each containing two control wells with cells only, two wells containing a concentration of 0.10% GNPs by mass + cells and two wells containing 0.15% GNPs by mass + cells as shown in Figure 3. An energy of 18 MV was utilized to irradiate the cells at doses of 3 Gy and 6 Gy with a dose rate of 600 cGy/min. Two cell culture plates were set up in an irradiation geometry as shown in Figure 4. A 20 × 20 cm2 field size and 100 cm SSD were used to irradiate each cell plate from underneath the set-up at a 180° gantry angle. The gantry angle of 180° (i.e., irradiating from beneath) was chosen in order to avoid an air gap between the solid water buildup slab and the cell media within the well plate. A solid water build up slab of 3 cm thickness and a backscatter slab of 5 cm thickness were placed below and above the cell well plate, respectively. Attenuation of the table was accounted for in the total delivered dose.

Figure 3.

Figure 3.

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Diagram of experimental set-up to irradiate C-33a cells and C-33a cells + GNPs.

Figure 4.

Figure 4.

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Experimental set-up for irradiation of two 6-well plates containing C-33a cells and C-33a cells + GNPs.

2.5. Cell Viability Assay and Cell Survival

Cell viability before and after irradiation was evaluated using Trypan Blue. Cells were incubated at a temperature of 37°C with 5% CO2 for 6 hours following irradiation for the radiation dose to take effect on cell viability and to ensure accurate cell counting. Approximately 20 μL of cells or cells + GNPs were added to a small Eppendorf tube and mixed with 20 μL of 0.4% Trypan Blue assay solution (VWR). Then, 10 μL of this mixture was placed inside a cell counting chamber slide. The chamber slide containing the cells or cells + GNPs were placed in an Invitrogen Countess II Automated Cell Counter (ThermoFisher Scientific). The concentration and percent of live and dead cells from each sample were recorded. A total of 10 measurements were taken for each sample.

The dose of radiation vs. the fraction of cells surviving was plotted for three cases: radiation only, radiation + 0.10% GNPs by mass, and radiation + 0.15% GNPs by mass. These cell survival curves were fit using a linear quadratic (LQ) cell survival model. The log of cell survival versus dose was fit with a quadratic equation. The resulting fits were used to predict the dose resulting in a given cell survival percentage for each condition (radiation only, 0.10% GNPs by mass + radiation, 0.15% GNPs by mass + radiation). Radiosensitization enhancement factor (REF) is the ratio of the dose producing a given cell survival percentage in the presence of GNPs to the dose producing the same cell survival percentage in the absence of GNPs. The REF was calculated for both 30% and 60% cell survival for each GNP concentration tested (0.10 and 0.15% by mass) to quantitatively determine the enhanced effect of the GNPs on the C-33a cells.

The synergistic effect of the GNPs + radiation vs. radiation only vs. GNPs only was also quantified. This was performed by comparing the percent decrease in cell survival obtained for each combination of radiation dose (3 Gy and 6 Gy) and GNP concentration (0.10 mass % and 0.15 mass %) against the sum of the effects obtained for the corresponding radiation dose alone and GNP concentration alone.

3. Results

3.1. Microdosimetric Monte Carlo simulations

MCNP6.2 simulations show that the radial dose distribution of electrons generated from the surface of a gold nanocube exhibits a slightly higher dose closer to the surface of the GNP than that from the nanosphere at each dose interval up to about 1.1 μm from the center of the GNP for both 18 MV and 6 MV energies. Dose from electrons surrounding the gold nanocube surpasses that of a gold nanosphere up to a distance of 1.1 μm by about 23.1% for the 6 MV spectrum (Figure 5a) and by 18.5% for the 18 MV energy spectrum (Figure 5b).

Figure 5.

Figure 5.

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(a) Dose deposition of secondary electrons up to 1.1 μm from center of GNP for 6 MV spectrum. (b) Dose deposition of secondary electrons up to 1.1 μm from center of GNP for 18 MV spectrum.

Comparing the dose of secondary electrons to a 1 μm3 region surrounding a GNP with the dose to a 1 μm3 region containing water, we find large DEFs at each energy for both the nanocube and the nanosphere as shown in Figure 6. The DEF in a 1 μm3 volume is generally higher for 6 MV than for 18 MV between both the nanosphere and nanocube. As the surface area to volume ratio increases, the DEF increases. As surface area to volume ratio increases (sphere to a cube), the DEF in 1 cm3 volume increases by 20% at 6 MV and 18% for 18 MV.

Figure 6.

Figure 6.

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Surface area to volume ratio vs. DEF for a 1 μm3 region containing a GNP.

3.2. Experimental Cell Survival Study

The results of the trypan blue assay with the varying doses of GNPs shows a significant decrease in cell survival with radiation therapy as the concentration of GNPs is increased. Figure 7 depicts the cell survival curve for each dose of radiation: 0 Gy, 3 Gy and 6 Gy. Cells treated with 0.10% GNPs by mass + radiation showed an average 6.7% reduction in cell survival compared to cells treated with radiation only across the two radiation doses tested. Cells treated with 0.15% GNPs by mass + radiation showed an average 14.6% reduction in cell survival when compared to cells treated with radiation only across the two radiation doses tested. Figure 7a shows the curve fitted to the standard LQ model for cell survival. Cell viability decreased by an average of 2–3% for cells treated with GNPs only (no radiation). Figures 7b and 7c show cell survival as a function of the GNP dose for each dose of radiation (3 Gy and 6 Gy, respectively).

Figure 7.

Figure 7.

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(a) Surviving Fraction of C-33a cells treated with radiation alone (blue), radiation + 0.10% GNPs by mass (orange), and radiation + 0.15% GNPs by mass (gray) with LQ model fits, (b) Cell survival as a function of GNP concentration for 3 Gy radiation dose at 18 MV energy, and (c) Cell survival as a function of GNP concentration for 6 Gy radiation dose at 18 MV energy.

Cell survival decreased by approximately 8.3% and then by 15% total between 0 and 0.10% GNPs by mass and then 0 and 0.15% GNPs by mass, respectively for a 3 Gy dose. Cell survival decreased by approximately 5% and then by 14% total between 0 and 0.10% GNPs by mass and then 0 and 0.15% GNPs by mass, respectively for a 6 Gy dose. The radiosensitization enhancement for each concentration of GNPs was evaluated at 30% and 60% cell survival to further quantify the effect of the GNPs. These ratios are shown in Table 1 . These particular surviving fractions were used because they fall within the center, with 30% being more towards the low end and 60% being more towards the high end of the cell survival curve. This allows for better robustness of the data.

Table 1.

REF for 0.10% GNPs by mass and 0.15% GNPs by mass vs. surviving fraction (SF).

Surviving Fraction (SF) 0.10% GNPs by mass REF 0.15% GNPs by mass REF
0.3 1.056 1.107
0.6 1.066 1.115
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These results show that the dose increase required to produce the same effect on cell viability using no GNPs compared with that using GNPs, is on average 6.1% for the 0.10% by mass GNP concentration and 11.1% for the 0.15% by mass GNP concentration. Table 2 quantifies the synergistic effect of dosing the C-33a cells with GNPs and irradiating them with a dose of 3 Gy of radiation and 6 Gy of radiation. There is clearly a synergistic effect seen for the addition of GNPs to the cells, as the percent change between the GNPs + radiation and GNPs only + radiation only cases is positive. In general, the higher concentration of GNPs (0.10 mass%) produced an overall greater synergistic effect than the lower concentration (0.15 mass%), which is expected.

Table 2.

Synergistic effect of GNPs and radiation. Values shown indicate percent decrease and standard deviation in cell survival from a baseline measurement taken before GNP + C-33a cell irradiation and after incubation of GNPs with C-33a cells for both a dose of 6 Gy and 3 Gy.

Change in survival (% decrease)
GNP concentration (% by mass) GNPs only Radiation Only – 6 Gy GNPs + Radiation Sum of GNPs only and Radiation only
0.10 (6 nM) 1.9 ± 0.4 71.4 ± 2.2 76.6 ± 4.5 73.3 ± 2.5
0.15 (9 nM) 2.6 ± 0.6 73.7 ± 2.1 82.5 ± 3.5 76.3 ± 2.3
GNP concentration (% by mass) GNPs only Radiation Only – 3 Gy GNPs + Radiation Sum of GNPs only and Radiation only
0.10 (6 nM) 2.0 ± 0.5 58.2 ± 4.5 65.5 ± 7.2 60.2 ± 4.5
0.15 (9 nM) 2.7 ± 0.3 70.9 ± 5.6 80.5 ± 5.4 73.6 ± 5.2
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4. Discussion

Understanding the effects of gold nanoparticle enhanced radiation at the microscopic, cellular level is essential to the development of nanoparticle-enhanced radiation therapy into a viable clinical technique. In this study, we perform in vitro studies to determine the effects of nanoparticle-enhanced radiation on cell viability, measured via the radiosensitization enhancement factor (REF). We also perform microdosimetric Monte Carlo simulations to gain insight into our in vitro results. It is important to distinguish REF, measured in terms of effects on cell viability, from dose enhancement factor (DEF), measured in terms of physical dose enhancement. The enhancing effect in terms of cell viability will be highly based on biological factors, such as the quantity of tumor DNA damage due to secondary electrons generated from the GNPs, how many GNPs are taken up by the cell, etc. When GNPs enter a cell, GNP concentration can increase on a microscopic level due to decreased container volume, thus resulting in a greater effect measured in terms of cell viability. REF is also enhanced due to the fact that the GNPs may be close to the nucleus, with cell DNA within the range of secondary electrons generated from the surface of a single GNP when irradiated. This increases the probability for secondary electrons generated from the GNPs to create more single or double strand breaks in the tumor cell DNA. In general, the greater the concentration of GNPs that can be added to a tumor volume with least toxic effects and the more efficient any surface coating or targeting moieties are at helping the GNPs enter tumor cells, the larger the effect on cell viability. Computationally investigating the microdosimetric effects including how much the dose is enhanced immediately surrounding a GNP, and how the surface area to volume ratio of a single GNP affects local dose, can help us to understand the effects of GNPs on cell viability. Evaluating the range of secondary electrons from a single GNP and DEF in a volume immediately surrounding the GNP can also be helpful in determining how far from the nucleus a GNP can be to still produce a significant amount of tumor cell DNA damage. Through this information, we gain knowledge of the optimal parameters (GNP concentration, size, shape, and surface coating) that we can utilize in future in vivo and clinical studies to maximize effects on tumor cells.

To provide a basis for understanding the results of the in vitro experiments, microdosimetric Monte Carlo studies concerning dose distribution of electrons produced by a GNP during radiation were performed. With this data, we can estimate the range of secondary electrons for purposes of predicting the amount of DNA damage that may be incurred on a cell depending on where the GNP is located in the cell and help guide biological studies on GNP functionalization to target the GNP to a specific location in the tumor cell.

There have previously been many different types of microdosimetric studies using Monte Carlo to explore dose enhancement due to GNPs.(Champion et al., 2014, Bordes et al., 2017, Chow et al., 2012, Leung et al., 2011, Lin et al., 2014, Sung et al., 2017, Sung et al., 2016) Among the most prominent studies in microdosimetric dose enhancement using GNPs is that by Leung et al.(Leung et al., 2011), who investigated the range and radial dose distribution of secondary electrons from varying photon beam energies, including 50 kVp, 250 kVp, Co-60 and 6 MV. Results of secondary electron production for the GNP were compared with those of water only and showed a 10- to 2000-fold increase in secondary electron production from the GNPs. This study also showed that range of secondary electrons from a single GNP increases with energy up to 6 MV.(Leung et al., 2011) Another study by Jones et al.(Jones et al., 2010) showed the effect of radial dose distribution from a similar set of energies, including 125I, 103Pd, 169Yb, 192Ir, 50 kVp and 6 MV. Authors of this study observed that dose is enhanced 2 to 20 times within 5 μm from the surface of the GNP. Lastly, Jung et al.(Jung et al., 2018) compared radial dose distributions for 2 nm and 15 nm GNPs at energies of 100 eV, 1 keV and 10 keV energies for PENELOPE2014, MCNP6.1 and Geant4 Monte Carlo Codes. All these studies suggest that with increasing energy, the range of secondary electrons generated from the surface of a GNP increases. In the present study, we observe that electrons generated from the nanocube at both energies traveled further without losing their energy as fast as those generated from the sphere as evidenced from the increased dose from electrons to more distant surrounding concentric spheres around the gold nanocube. So, we see that increasing the surface area while keeping volume constant (changing from a sphere to a cube shape) directly increases the range of secondary electrons and increases the dose from secondary electrons at each range interval surrounding the GNP. We also observed that the difference in effect between the cube and the sphere of the same volume is smaller for the 18 MV energy than for the 6 MV energy. This is likely because the average energy for the 6 MV spectrum generates a higher probability for the photoelectric effect to occur with gold than for the 18 MV spectrum. The DEF in a 1 μm3 volume surrounding a single GNP compared with an equal volume of water is greater at 6 MV than at 18 MV for both the sphere and the cube geometries due to the photoelectric effect being more pronounced at 6 MV. The DEF is greater for the cube than the sphere at both energies, due to the surface area to volume ratio increasing, resulting in greater secondary electron yield. It is concluded that the lower the energy, and the higher the surface area to volume ratio of a GNP, the greater the production of secondary electrons and the higher the DEF in a volume close to the surface of a single GNP.

Many past studies have also experimentally investigated the in vitro effects of GNP-enhanced radiation therapy using different cell lines, as well as some in vivo effects.(Bodgi et al., 2019, Sazgarnia et al., 2013, Oesten et al., 2019b, Oesten et al., 2019a, Kim, 2017, Lacoste-Collin et al., 2015, Nagle et al., 2016, Moiseenko et al., 2007, Ebrahimi Fard et al., 2017, Zellweger et al., 2002) To assess the potential in vivo toxicity of the GNPs used in this study for purposes of future studies, we can compare it with GNP concentrations used in an in vivo study conducted by Hainfeld et al., who reported an LD50 (lethal dose, 50%; the dose killing 50% of mice) of > 5g Au/kg body weight of mice for 11.2 ± 8.6 nm diameter GNPs.(Hainfeld et al., 2013) In the present study, the equivalent concentration of GNPs in g/kg amounts to 0.98–1.5 g Au/kg for 6 nM GNPs and 3.5 g Au/kg for 9 nM GNPs. Zhang et al. performed an in vitro and in vivo radiosensitization study of 4.8, 12.1, 27.3 and 46.6 nm PEG-coated gold nanoparticles. The toxicity to cells of PEG-coated GNPs of different sizes were analyzed after 24 and 48 h treatments and evaluated using cloning formation and cell apoptosis. Authors of this study reported that pathology, immune response and blood biochemistry indicate that PEG-coated GNPs have not caused spleen and kidney damage and that 12.1 and 27.3 nm PEG-coated GNPs show high radiosensitivity. Smaller PEG-coated GNPs show a greater toxicity effect than the 12.1 or 27.3 nm PEG GNPs.(Zhang et al., 2011) Chithrani et al. used 14–74 nm GNPs to estimate the radiosensitization enhancement factor (REF) for lower (105 kVp) and higher (6 MVp) energy photons and demonstrated that as the energy cells are irradiated with is increased, the REF decreases. This study also showed GNPs that are 50 nm in diameter showed the most radiosensitization.(Chithrani et al., 2010) The concentration of GNPs used in Chithrani et al. was 7 × 109 nanoparticles/mL.(Chithrani et al., 2010) In our study, the 6 nM and 9 nM concentrations convert to 3.6 × 1012 and 5.4 × 1012 nanoparticles/mL, respectively. The trend of decreasing REF with increasing energy seen in Chithrani et al. agrees with the present study. Chithrani et al. report a REF of 1.17 at the highest energy used in that particular study, 6 MV, and at 10% survival with REF decreasing with increasing energy. A relatively recent study by Kim et al. demonstrates that the REF can be different across different cell lines and demonstrated how the REF is affected when neutrons are used to irradiate cells and cells + GNPs. Kim et al. found that the REFs for Huh7 and HepG2 hepatocellular cancers were 1.41 and 1.16, respectively using a 137Cs gamma ray source at a dose of 3.81 Gy/min. GNP size and concentration used in this study was 5 nm and 1 mM, respectively. REFs changed to 1.80 and 1.35 for a neutron beam (9.8 MeV average energy, 30–40 keV/μm LET).(Kim et al., 2017) Further studies in in vitro GNP dose enhancement could potentially focus on evaluating the effect on cell viability when changing radiation energy, radiation particle type or cell line.

Past studies have also demonstrated the effects on cell viability in vitro when cells are exposed to radiation. Wang et al. exposed SW1990 and PANC-1 pancreatic cancer cell lines to 0, 2, 4, 6 and 8 Gy doses of radiation using 125I radioactive seeds and a 12.13 Gy/h initial dose rate. A clonogenic survival experiment, cell-cycle and apoptosis analyses were conducted, and it was observed that survival fractions of PANC-1 and SW1990 cells irradiated with 125I seeds decreased exponentially as radiation dose was increased. The 125I seeds also induced a higher percentage of apoptosis than observed in the control for both cell lines, and no significant differences were observed in the dosimetric results of the two cell lines when exposed to the same dose of radiation.(Wang et al., 2015) In a recent study conducted by Hau et al., the synergy between GNPs and radiation on the kilo- or mega-voltage scale to cause increased cytotoxicity was investigated. 10 nm diameter GNPs at up to 10–25 μg/mL were shown to be nontoxic in both 2D and 3D in vitro cultures of colon cancer cells. Significant increases in reduction to cell survival fractions following exposure to 1 Gy of kilovoltage and 2 Gy of megavoltage radiation were observed when the cells were incubated with 50 μg/ml of GNPs.(Hau et al., 2016) The potential to improve radiation therapy when combining GNP mediated radiation sensitization with chemoradiation as compared with chemoradiation alone was studied by Yang et al.. 2 Gy of 6 MV radiation with GNPs resulted in a 19 ± 6% decrease in MDA-MB-231 cell survival. Monte Carlo simulations also demonstrated dosimetric differences in the presence of GNPs in radiation. Survival of cells exposed to GNPs, cisplatin, and radiation decreased by 84% ± 0.7%, while survival of cells treated with cisplatin and radiation only decreased by 77% ± 1.1%. The presence of GNPs resulted in a 30 ± 6% decrease in the survival.(Yang et al., 2018) A recent study presented by Wolfe et al. reported the in vitro and in vivo radiosensitization of prostate cancer using gold nanorods conjugated with gosrelin acetate. The cells and nanorods were irradiated using 6 MV energy. Tumor sizes in mice were allowed to reach about 7–8 mm on the longest axis before receiving i.) no treatment (control), ii.) radiation therapy alone, iii.) gold nanorods plus radiation therapy, iv.) PEGylated gold nanorods plus radiation therapy. Biodistribution data showed only slight increases in absolute quantity of gold within tumors after conjugation. Gold nanorods, in combination with radiation, were shown to drastically decrease cell survival by up to 35%.(Wolfe et al., 2015a) Radiosensitization enhancement factor (REF) in this work was calculated by dividing D20% (control) by D20% (AuNR). D20%(control) was the dose necessary to reduce the fraction of viable cells after radiation alone to 20% of that when treated without radiation, and D20%(AuNR) represented the dose necessary to reduce the fraction of viable cells after radiation and nanoparticles to 20% of that when treated with nanoparticles without radiation.

Our in vitro study was performed to evaluate the efficacy and synergistic effects of GNPs with radiation therapy at a high energy. The effect of different radiation doses combined with different concentrations of GNPs on cancer cell viability was evaluated. Results indicate that there is a potentially significant benefit, up to an average 14.6% decrease in tumor cell survival, by the addition of biocompatible and non-toxic doses of GNPs to a tumor. GNPs used to treat tumors using radiation therapy would be especially useful in place of alternative therapies, such as proton therapy, when the alternative therapies are not available to a patient due to increased cost, availability of equipment, etc. One of the most interesting results of this study is the fact that when compared against a similar macroscopic study, showing up to a 2.15% dose enhancement (Behrouzkia et al., 2019), microdosimetric effects of GNPs show a significant increase in localized dose enhancement of up to 11.5%. This effect is a result of the increased amount of secondary electrons emanating from the immediate surface of a GNP, which can lead to significant DNA damage due to the range of these secondary electrons and ultimately decreases in cell viability. This also shows that localized dose enhancement plays a large role in tumor cell DNA damage and decreased cell viability and should be investigated further in future studies.

It is also important to evaluate the synergistic effect of GNPs and radiation on the cell viability. Obtaining the greatest therapeutic benefit while minimizing toxicity constitutes a great benefit of multi-component cancer treatments. The final effect of a multi-component treatment combination may be synergistic, additive or antagonistic. If these effects are not scientifically evaluated, they can potentially cause patients therapeutic and toxic risks due to interactions.(Yuan and Chen, 2019) In this study, GNPs + radiation used together produced a synergistic effect of 3–7% compared with radiation alone + GNPs alone. Thus, we predict an overall beneficial effect for GNP use in radiation therapy clinically in the future. Future in vitro and in vivo studies should evaluate synergistic effects of GNPs of different surface coatings, targeting moieties, and concentrations as well as different GNP sizes and shapes, to evaluate the best combination of GNP type and radiation to be used in a clinical setting.

5. Conclusion

We have successfully predicted, using MCNP6.2, the radial dose distribution from a single GNP as the energy changes and as the surface-area to volume ratio changes. Microscopic DEFs ranging from approximately 2–8 were found, with the maximum DEF resulting from the case of a gold nanocube irradiated at 6 MV energy. We have also successfully determined the effect of GNP-enhanced radiation on cell survival across multiple doses of radiation and GNPs. We observed a 6.7% decrease in cancer cell survival for cells treated with 0.10% GNPs by mass + radiation, and a 14.6% decrease in cell survival for cells treated with of 0.15% GNPs by mass + radiation, as compared with cells receiving radiation alone. Future studies would involve observing cell viability at multiple time intervals for different doses of GNPs and radiation, as well as testing different types of GNPs, including GNPs with different surface-area to volume ratios, surface coatings, and antibody attachments for cell-specific targeting.

Acknowledgements

The authors would like to thank Dr. Kelly Nash at the University of Texas at San Antonio for all of her advice on cell culture and cell survival assays. The authors would also like to thank Alejandro Morales Betancourt for his assistance involving lab equipment for cell culture and cell culture supplies as well as all of his advice on cell culture care. The authors acknowledge the University of Texas at San Antonio Kleberg Advanced Microscopy Center for support during this work.

Funding

This work was supported by the San Antonio Medical Foundation, San Antonio Life Sciences Institute, and the National Institutes of Health under Grant R25-GM060655–09.

Footnotes

Disclosure Statement

No potential competing interest was reported by the authors.

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