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Intratumoral Injection of Low-Energy Photon-Emitting Gold Nanoparticles: a Microdosimetric Monte Carlo-Based Model Myriam Laprise-Pelletier, Yunzhi Ma, Jean Lagueux, Marie-France Côté, Luc Beaulieu, and Marc-André Fortin ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b08242 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018
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Intratumoral Injection of Low-Energy PhotonEmitting Gold Nanoparticles: a Microdosimetric Monte Carlo-Based Model Myriam Laprise-Pelletier,†,‡ Yunzhi Ma, ┴Jean Lagueux,† Marie-France Côté,† Luc Beaulieu, §,┴ Marc-André Fortin †,‡,* †
Centre de recherche du Centre hospitalier universitaire de Québec – Université Laval
(CR-CHU de Québec), axe Médecine Régénératrice, Québec, G1V 4G2, Qc, Canada ‡
Department of Mining, Metallurgy and Materials Engineering and Centre de recherche
sur les matériaux avancés (CERMA), Université Laval, Québec, G1V 0A6, Qc, Canada §
Département de physique, de génie physique et d’optique et Centre de recherche sur le cancer (CRC), Université Laval, Québec, G1V 0A6, Qc, Canada ┴
Département de radio-oncologie et axe Oncologie du Centre de recherche du Centre
hospitalier universitaire de Québec – Université Laval (CR-CHU de Québec), Québec, G1R 2J6, Qc, Canada *
Corresponding author :Marc-André Fortin/
[email protected] Phone: 1-418-525-4444 ext: 52366
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ABSTRACT
Gold nanoparticles (Au NPs) distributed in the vicinity of low-dose rate (LDR) brachytherapy seeds, could multiply their efficacy thanks to the secondary emissions induced by the photoelectric effect. Injections of radioactive LDR gold nanoparticles (LDR Au NPs), instead of conventional millimetre-size radioactive seeds surrounded by Au NPs, could further enhance the dose by distributing the radioactivity more precisely and homogeneously in tumors. However, the potential of LDR Au NPs as an emerging strategy to treat cancer is strongly dependent on the macroscopic diffusion of the NPs in tumors, as well as on their microscopic internalization within the cells. Understanding the relationship between interstitial and intracellular distribution of NPs, and the outcomes of dose deposition in the cancer tissue is essential for considering future applications of radioactive Au NPs in oncology. Here, LDR Au NPs (103Pd:Pd@Au-PEG NPs) were injected in prostate cancer tumors. The particles were visualized at time-points by computed tomography imaging (in vivo), transmission electron microscopy (ex vivo) and optical microscopy (ex vivo). These data were used in a Monte Carlo-based dosimetric model to reveal the dose deposition produced by LDRAu NPs both at tumoral and cellular scales.
103
Pd:Pd@Au-PEG NPs injected in tumors produce a strong dose
enhancement at the intracellular level. However, energy deposition is mainly confined around vesicles filled with NPs, and not necessarily close to the nuclei. This suggests that indirect damage caused by the production of reactive oxygen species might be the leading therapeutic mechanism of tumor growth control, over direct damage to the DNA.
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KEYWORDS: radioactive nanoparticles, prostate cancer, brachytherapy, low-dose rate brachytherapy, microdosimetry, Monte Carlo simulations, intratumoral injections of nanoparticles
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Low-dose rate brachytherapy (LDR-BT) is a radiotherapeutic treatment based on the implantation of radioactive seeds into localized cancers (e.g. prostate, breast).1-3 In general, the seeds contain low-energy photon emitters (e.g.
103
Pd and
125
I; ~20 keV,
Figure 1A). This approach, compared with external beam radiotherapy (EBRT), maximizes the dose delivery inside tumoral tissues and minimizes irradiation to the surrounding healthy tissues and organs at risk.4 In particular, prostate brachytherapy is associated to fewer urinary and gastrointestinal long-term side effects.5 It is also less expensive and less time-consuming in terms of number of hospital visits per patient. However, certain aspects of the LDR-BT treatment can impede its overall efficacy. In LDR-BT, about 50 - 100 seeds are implanted in the prostate via catheters. The number of seeds implanted depends on the volume and shape of the prostate gland in each patient. The procedure itself is invasive, while causing bleeding and patient fatigue. The patient is likely to experience urinary obstruction and/or a sensation of burning when he urinates. Dosimetric accuracy in LDR-BT is directly related to the capacity of the oncologist to precisely position each seed implant. Given the large number of seeds implanted in a small volume (~35 cc on average), trauma and edema are likely to occur, which leads to positioning errors. Over time, the real dose often deviates from the planned dose due to post-implant displacement, the rotation of seeds, and to the loss of seeds through urination. Seed positioning errors developing over time in the prostate, are likely to cause severe hot and cold spots. Overall this causes a degradation in treatment precision.6 This is a paradox of LDR-BT where the sharp dose fall-off around the seeds is considered as a benefit which, in principle, should make sparing the organs at risk an easier task. In fact,
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deviations to the original seed placement plan deteriorate the dose distribution in the prostate gland. Recent studies have demonstrated the dose enhancement effect of gold nanoparticles (Au NPs) as radiosensitizers in brachytherapy.7-15 The presence of Au NPs in tumors enhance energy deposition: x-rays penetrating the tissue either by EBRT or from BT seeds, interact with the Au atoms which convert them into electrons through photoabsorption mechanisms. Under irradiation with photons emitted by LDR-BT seeds, in the case of BT, Au NPs generate a whole cascade of emission products (photoelectrons, Auger electrons, or characteristic X-rays) through photoabsorptions and de-excitations occurring in molecules and atoms in their immediagte vicinity. The probability of photoabsorption to occur increases with atomic number (Z), and decreases with the energy (E). Therefore, Au NPs are much more efficient at generating photoabsorption events for the low-energy photons (e.g.
103
Pd: 20.1, 23.0 keV) emitted
by LDR-BT, compared with the high-energy photons used in EBRT.8-10, 12-15 In fact, the mass attenuation coefficient for 20 keV photons with gold is in the order of 100 cm2/g (dominated by the photoelectric effect), whereas for 1 MeV, it is in the order of 0.1 cm2/g (dominated by the Compton effect).16 Then, charged particles emitted through the photoelectric effect and the Auger process, deposit energy in the vicinity of Au NPs and the therapeutic dose is thereby enhanced.9, 13-15, 17-19
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Figure 1. Schematic representation of prostate cancer brachytherapy using conventional millimetre-sized seeds (A) and using LDRAu NPs (B). In (A), the green arrows correspond to photons emitted by 103Pd. The black tracks represent the path of secondary electrons. In (B), representation of the microscopic brachytherapy dosimetry using LDRAu NPs (103Pd:Pd@Au-PEG NPs). Interactions between 103Pd photons and Au atoms generate a large amount of secondary photoelectric products (low-energy photons, photoelectrons, Auger electrons), and this in turn increases the radiotherapeutic treatment. Image adapted from Laprise-Pelletier et al.20 Radioactive Au NPs, gold nanoparticles that enclose radioactive isotopes such as 198Au or 103Pd, represent a promising alternative to radiosensitizers-enhanced LDR-BT.20-26 The treatment would consist of simple injections of Au NP-containing radioactive fluids: it would require thin needles, thinner in fact than the current millimetre-sized radioactive seeds). This would minimize blooding and discomfort. It would also eliminate the risks associated to seed displacement, rotation and loss: overall, this would improve dose uniformity. Unlike the LDR-BT seeds, Au NPs carried in an aqueous solution could slightly diffuse into the tissue, and this could attenuate to a certain extent, the occurrence of hot and cold spots. The radiosensitization effect of Au could also decrease the total radioisotope activity needed per injection, for an equivalent level of energy deposition.
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Our research team has recently transferred this theoretical concept in vivo, by demonstrating the efficiency of
103
Pd-doped Au NPs (103Pd:Pd@Au-PEG NPs) for
controlling the growth of prostate cancer tumors in vivo.20, 27 More specifically, tumors treated with single injections of
103
Pd:Pd@Au-PEG NPs (1.7 mCi) performed in 200 –
400 mm3 tumors, were ∼56% smaller than untreated controls 4 weeks after the injection. More investigation is needed to better understand the respective impacts of primary and secondary photon emissions, as well as low-dose rate gold nanoparticles (LDRAu NPs) distribution, on the resulting efficacy of tumor volume control. At this step of the research, a relevant dosimetry model based on both primary and secondary emissions must be developed to quantify how energy deposition occurs at the scale of the tumor tissue down to the cancer cell level. An accurate prediction of cellular and subcellular dose distribution in LDRAu NPs-treated tumoral tissue should take into account the microscopic distribution of Au NPs in vivo, the complex geometry of cells and the complexity of NP-to-nuclei distribution. The computational model should include all the diversity of radiation energy deposition processes susceptible to occur at the microscopic level. Finally, more and more studies point to the impact of toxic reactive oxygen species (ROS) on tumor growth as one of the leading factors of the radiosensitization effect induced by Au NPs.28-29 Theoretically, the microscopic distribution of LDRAu NPs in the tumor tissue should have an impact on the intracellular energy deposition profile, and thereby on the total dose delivered to nuclei. Unfortunately, most of the current dosimetric models pertaining to Au NPs applications in radiotherapy, do not take into account the complexity of intracellular NP distribution profiles. In fact, cells have diverse and complex 7 ACS Paragon Plus Environment
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morphologies, in particular when they are part of macroscopic constructs such as tumors. In addition to this, the spatial distribution expected for Au NPs diffusing between and ingested by cells, is very heterogeneous and this reality is dramatically different from the simplistic geometrical assumptions taken in most of the theoretical models of microdosimetry published until now in the litterature. In a recent study, Zygmanski et al. have clearly stated the following factors as determinant in the correlation between relative biological effectiveness values to theoretically calculated dose enhancement values: the number of Au NPs per cell and their clustering status, the proximity of Au NPs to cellular or subcellular targets (entire cell, cell nucleus, DNA, clonogenic cancer or endothelial cells), and the heterogeneity of dose distribution within the target volume.30 The authors proposed a general mathematical model for a macroscopic population of cells with nonuniform distribution of Au NPs. Several parameters were taken into account. However, the geometry of cells was rather simplistic and is yet to be confronted with more realistic cell microscopy data. In fact, tumor cells are most of them highly irregular: they present a wide diversity of morphological shapes, which are also dynamic. Therefore, biological cells and subcellular structures in general cannot be modeled using simple geometries (spheres or ovoids), which calls for in vivo observation of the cell shapes. There is a scarcity of studies in the literature reporting on the energy deposition at the cellular level based on microscopic data acquired in vivo. An ideal approach is to acquire the three dimensional image of (sub-)cellular structures and these accurate geometries in vivo should serve as an essential input for dosimetry models.
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In the present study,
103
Pd-doped Au NPs were injected in human prostate cancer
tumors grown in a murine model. The tumors were harvested at different time-points (2 h, 24 h and 8 days), sliced and visualized by optical microscopy and imaged by transmission electron microscopy (TEM). These images confirmed that
103
Pd:Pd@Au-
PEG NPs are ingested by the tumor cells very rapidly where they strongly agglomerate in vesicles. The highly inhomogeneous intracellular distribution of Au NPs must be taken into account in the dosimetric approach. However, the majority of theoretical assumptions taken until now in microdosimetric studies, used very simple spherical or ovoid shapes which are not biologically relevant.12,
31-35
In the present study, TEM
images were numerically converted into maps showing the distribution of Au NPs into tumor tissues at the scale of tumor cells grown in vivo. These were used to generate a Monte Carlo-based macrodosimetric and microdosimetric model of dose deposition. More specifically, we aimed at mapping dose enhancement to the nuclei, which has been considered until now as the main mechanism of cell proliferation control in radiosensitization-enhanced brachytherapy.28 RESULTS Synthesis of radioactive gold nanoparticles (103Pd:Pd@Au-PEG NPs) The synthesis of radioactive core-shell
103
Pd:Pd@Au-PEG NPs was adapted from a
methodology previously reported by our group, based on a one-pot technique using ascorbic acid as a reducing agent.27
103
Pd:Pd NPs were first obtained from the reduction
of palladium chloride by ascorbic acid in presence of 2,3-meso-dimercaptosuccinic acid (DMSA) as a capping agent. Then, a shell of Au was formed around the
103
Pd:Pd core,
thus leading to spheroidal 103Pd:Pd@Au-PEG NPs (mean diameter: 50 ± 8 nm; Figure 2, 9 ACS Paragon Plus Environment
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A, B, C). A relatively narrow particle size distribution was obtained (Figure 2C), which is one of the challenges when using nanoparticle synthesis techniques based on direct chemical reduction techniques. Finally, the biocompatible molecule polyethylene glycol (PEG) was grafted at the surface of the NPs to provide steric hindrance (Figure 2A).
Figure 2. Schematic representation of (A) the synthesis of 103Pd:Pd@Au-PEG NPs; (B) representative TEM image, (C) size distribution profile (diameter), and (D) hydrodynamic diameter measured by DLS before and after capping with Au + PEG (number weighted). The effect of gold and PEG addition was revealed in the dynamic light scattering (DLS) profiles (Figure 2D) obtained from colloidal suspensions of Pd NPs before (10.4 ± 3.3 nm) and after addition of Au and PEG (59.3 ± 15.1 nm). All results showed polydispersity indexes inferior to 0.3, without evidence of large clusters or aggregates. Injection of radioactive gold nanoparticles and fate in vivo Human prostate cancer tumors were grown in the mouse model (flank), until they reached a volume of at least 200 mm3. Each tumor was injected with ∼ 7 µCi of 103
Pd:Pd@Au-PEG NPs (2 µL). The total quantity of gold per injection was kept well
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below 6 mg Au/kg body weight (b.w.). As an indication, the LD50 lethal dose for intravascular injections of Au NPs is in the order of ~3.2 g Au/kg b.w. for BALB/c mice.36 The diffusion of NPs into the tumors was followed-up by micro computed tomography (CT) scanning (Figure 3). For CT reconstruction, a numerical density threshold was applied to reveal the volume of highest NP concentration at t = 2 h , 24 h, 4 days and 8 days. At t = 2 h and t = 24 h, the mean apparent volume of the injection was 4.1 ± 1.6 mm3 (n = 4) and was clearly visualized in the reconstructed images (Figure 3D). After several hours, the diffusion of the NPs in the tissue translates into an apparent increase in the volume of highest NPs concentration, along with a decrease in X-ray attenuation potential (Figure 3B, C). Four days after the injection, the mean apparent volume was 3.1 ± 1.2 mm3 (n = 4) and remained constant for up to 8 days. Between the first scan and the last scan at t = 8 days, a decrease of 24% was observed in the mean volume of the injection site (Figure 3C, F).
Figure 3. X-ray attenuation images (A-C) and corresponding CT (3D) reconstruction images (D-F) of a tumor injected with 103Pd:Pd@Au-PEG NPs at t = 2 h (A, D), t = 4 days (B, E), and t = 8 days (C, F). (Micro CT voxel resolution : 89 µm) 11 ACS Paragon Plus Environment
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Organs and tumors were harvested at 2 h, 24 h and 8 days post-injection, and radioactivity-counted (Atomlab 950 gamma counter). More than 80 % of the total activity measured in the animals at the time of harvest, was found in the tumors (see biodistribution data in the supporting info). This confirmed that the apparent X-ray attenuation decrease seen in the CT images, was not due to a drainage of NPs out of the tumor site, but rather to the slight diffusion of NPs in the tumor tissue, from the injection site. A significant fraction of NPs were also found in the liver (˂ 15 %), and in the spleen (˂ 3 %). After 8 days, the total activity retained in the tumors was 92% of the initial dose. In brief, the NPs were strongly retained in the tumor tissues several days after their administration, which is an essential condition to deliver an effective therapeutic dose with a any LDR-BT approach. Histological slices of the injected tumors were observed in optical microscopy in order to reveal the diffusion of
103
Pd:Pd@Au-PEG NPs in the cancer tissue over time (Figure
4). The area of the tumor histology slice corresponding to the end of the needle track, and therefore to the site of NPs administration, appeared colored in pink. After 2 h, the NPs appeared densely distributed around the injection site. The torn made by the needle was clearly visible in the center of the tumors and was highlighted by the presence of blood cells (small spherical blue dots, present at the injection site, Figure 4A, D). The latter were identified as erythrocytes due to their abundance, shape (roundness) and the absence of nucleus.37-38 After 24 h, evidences were found that NPs were found as clusters in the cytoplasm, probably in vesicles. The images taken on the samples harvested after 24 h, also confirm the evidence of NPs diffusion slightly away from the injection site, as suggested by the CT data (Figure 3E, F). These observations suggest that, following their 12 ACS Paragon Plus Environment
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intratumoral (i.t.) injection, NPs transit in the extracellular matrix before being taken up by cells. The optical microscopy images (Figure 4A, D) were processed by Image J in order to identify the pixels corresponding ot the NPs (black dots). Then, a numerical analysis was performed to mesure, for each one of these black pixels, the distance separating them from the center of injection. The number of pixels was then converted in a number distribution of particles (see experimental section). After 2 h, most of the NPs are still very close to the injection site
