Modulation of nanoparticle uptake, intracellular distribution, and retention with docetaxel to enhance radiotherapy

Abstract

Objective:

One of the major issues in current radiotherapy (RT) is the normal tissue toxicity. A smart combination of agents within the tumor would allow lowering the RT dose required while minimizing the damage to healthy tissue surrounding the tumor. We chose gold nanoparticles (GNPs) and docetaxel (DTX) as our choice of two radiosensitizing agents. They have a different mechanism of action which could lead to a synergistic effect. Our first goal was to assess the variation in GNP uptake, distribution, and retention in the presence of DTX. Our second goal was to assess the therapeutic results of the triple combination, RT/GNPs/DTX.

Methods:

We used HeLa and MDA-MB-231 cells for our study. Cells were incubated with GNPs (0.2 nM) in the absence and presence of DTX (50 nM) for 24 h to determine uptake, distribution, and retention of NPs. For RT experiments, treated cells were given a 2 Gy dose of 6 MV photons using a linear accelerator.

Results:

Concurrent treatment of DTX and GNPs resulted in over 85% retention of GNPs in tumor cells. DTX treatment also forced GNPs to be closer to the most important target, the nucleus, resulting in a decrease in cell survival and increase in DNA damage with the triple combination of RT/ GNPs/DTX vs RT/DTX. Our experimental therapeutic results were supported by Monte Carlo simulations.

Conclusion:

The ability to not only trap GNPs at clinically feasible doses but also to retain them within the cells could lead to meaningful fractionated treatments in future combined cancer therapy. Furthermore, the suggested triple combination of RT/GNPs/DTX may allow lowering the RT dose to spare surrounding healthy tissue.

Advances in knowledge:

This is the first study to show intracellular GNP transport disruption by DTX, and its advantage in radiosensitization.

Introduction

Integrating the systemic effects of chemotherapy with the local and targetable treatments available with radiotherapy (RT) has been shown to greatly increase the cure rates of solid tumours.¹ However, the combined and simultaneous normal-tissue toxicity in such treatments is a major barrier to their effectiveness. Improving the therapeutic ratio by reducing the impact of side-effects while enhancing the treatment effectiveness, is a major goal of current research. Among other approaches, the use of high Z materials such as gold nanoparticles (GNPs) concurrently with RT has shown improved results but has yet to see significant clinical adoption.² Nanoparticles can passively accumulate in tumors by diffusing through gaps in their hastily grown vasculature.³ GNPs enhance radiation damage by producing a shower of secondary electrons when exposed to X-ray beams. The distribution of added dose is extremely peaked, falling off 100-fold within a few hundred nanometers of the surface according to Monte Carlo simulations.⁴ Radiation dose enhancement properties due to GNPs is dependent on their localization within cells, due to the short range of these electrons. For example, having GNPs closer to the nucleus, if not within the nucleus, is expected to produce optimum radiation damage to cells during RT.⁵ We have found that a smart combination of GNPs and the anticancer drug, docetaxel (DTX) could modify the distribution of GNPs in favor of RT as described in this manuscript. The presence of DTX is expected to affect the phase of cell cycle and vesicle transport of GNPs within cells which could have a direct impact in RT.

DTX is a commonly used chemotherapy drug, used to treat a number of cancers including breast, stomach, prostate, and non-small-cell lung cancer.^6,7 It disrupts division of cells by causing unregulated formation of microtubules (MTs). DTX acts as a radiosensitizer by blocking the phase of the cell cycle in one of the most radiosensitive phases. The eukaryotic cell cycle can be divided into four major phases; G1, S, G2, and M. The genetic information is duplicated during the synthesis (S) phase and the cell divides into two daughter cells during mitosis (M). S phase and mitosis are separated by Gap phases, G1 and G2.

In a normal cell, MTs are arranged radially, nucleating from a microtubule organizing center (MTOC) near the nucleus and extending towards the cell membrane (Figure 1a(i)). This allows for directed traffic by motor proteins within the cell. At mitosis, a normal cell forms a mitotic spindle out of microtubules, stretching between two "asters" originating at centrosomes at either pole (Figure 1a(ii)). The DNA is arranged at a metaphase plate between the asters before the chromosomes are evenly separated into daughter cells (Figure 1a(iii)). However, the presence of DTX within cells affects cell division significantly as discussed next.

Figure 1.

Triple combination of RT/GNPs/DTX in cancer therapy. (a–b) Schematic diagrams and confocal images of MT structures in control cells and ones treated with DTX, respectively. a (i–iii) cell division in control cells: (i) quiescent control cell with MTs (green) originating from the microtubule organizing centre, (ii) normal mitotic spindle, (iii) normal pair of daughter cells. b (i–iii) cell division in the presence of DTX: (i) stabilizing of MTs into bundles, (ii) Multiple aster formation in non-dividing cell with DTX, and (iii) mitotic arrest with multinucleation under 50 nM DTX. (c) Summary of combined treatment with GNPs, DTX, and RT. (d) Schematic diagram of the functionalized GNP surface. DTX, docetaxel; GNP, gold nanoparticle; MT, microtubule; RT, radiotherapy.

DTX sequesters MTs in bundles hindering the formation of a mitotic spindle which is necessary for cell division (Figure 1b(i)).^8,9 With DTX treatment, asters and bundles are formed independently of centrosomes, creating multiple cleavage planes (Figure 1b(ii)). For example, a DTX concentration of 50 nM is sufficient to cause a "mitotic catastrophe": the cell cannot enter anaphase and remains locked in mitosis or becomes multinucleate as the nuclear envelope reforms around the multiple asters (Figure 1b(iii)). This results in blocking the cell cycle at the G2/M phase which is most sensitive to radiation. DTX alone has shown remarkable radiosensitization in vitro, in vivo, and in clinical trials.^6,7,10–14

One of the major issues in current radiotherapy is the normal tissue toxicity. Our goal was to combine both GNPs and DTX with RT in an effort to reduce the radiation dose by combining two radiosensitizers with different mechanisms of action to yield a higher damage to tumor cells as outlined in schematic Figure 1c. DTX rearranges the MT network and we expect changes in the GNP uptake and transport since GNPs are transported in vesicles along MTs within the cell.¹⁵ Finally, the goal of our study was to address two important challenges to the effective use of GNPs in future RT with DTX, a commonly used chemotherapy agent:

(a) How does the presence of DTX modulate uptake, distribution, and retention of GNPs?

(b) How does the modulation of GNP uptake and distribution in the presence of DTX affect outcome in RT? Does this agree with theoretical model predictions of GNP dose enhancement?

We believe that this study lays the foundation to understand the effect of DTX on GNP uptake, transport, and exocytosis processes in order to optimize a GNP-mediated chemoradiation (RT plus chemotherapy) regimen in future cancer therapy.

Methods and materials

Gold nanoparticle preparation and characterization

All listed reagents for GNPs were obtained from Sigma–Aldrich unless otherwise specified. Spherical GNPs were prepared via a modified citrate reduction method.¹⁶ 11.5 mL of 100 nM NaOH (Fisher Scientific) was added dropwise to 1.29 mL of 10% (w/v) chloroauric acid (HauCl₄ ·3H₂O). This was added to a beaker containing 117.5 mL of double distilled water. It was brought to 90°C while stirring and 19.7 mL of 1% sodium citrate tribasic dihydrate (HOC(COONa)(CH₂COONa)₂ · 2H₂O) was added. The temperature was reduced to 85°C and maintained for 20 min while stirring. The solution first became a dark black/purple color, and gradually changed to a wine or cherry red color. The solution was then brought to room temperature while stirring. The NP solution was diluted 1:10 with double-distilled water, then characterized by measurement of hydrodynamic diameter, ζ potential (LiteSizer 500 (Anton-Paar)), ultraviolet-visible spectrum (PerkinElmer λ 365 spectrophotometer).

Nanoparticle surface modification

PEGylation was performed using polyethylene glycol (PEG)-thiol (2 kDa, Nanocs, Boston, MA). A ratio of 600 PEG molecules/GNP was used to PEGylate 15 nm NPs (GNP_PEG). For confocal imaging, a mix of the above PEG and a PEG-thiol-Cy5 complex were used in equal proportion, with the same ratio of PEG/GNP. A peptide with the integrin binding domain RGD (NH₂-Cys-Lys-Lys-Lys-Lys-Lys-Lys-Gly-Gly-Arg-Gly-Asp-Met-Phe-Gly-COOH; AnaSpec, CA,USA) was added to the solution at a ratio of 300 molecules/GNP_PEG. A schematic diagram of the PEGylated and RGD-modified GNP construct used in this study is illustrated in Figure 1d.

Docetaxel and GNP inoculation

MDA-MB-231 and HeLa cells were maintained in high-glucose DMEM (HyClone) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). 6-well dishes were plated with 150 k cells/well (MDA-MB-231) or 100 k cells/well (HeLa). The next day, wells were inoculated with DTX [diluted from dimethyl sulfoxide (DMSO) in phosphate buffered saline (PBS) and media, DMSO concentration 0.04% v/v] to a final concentration of 50 nM. Control wells were inoculated with DMSO carrier diluted via the same method. Appropriate wells were inoculated with 15 nm GNPs diluted in media to a final concentration of 0.2 nM. Inoculation with DTX and functionalized GNPs occurred concurrently, and exposure was carried out for at 37°C. Both MDA-MB-231 and HeLa cells were bought from ATCC in 2019.

GNP uptake and retention dynamics

To measure uptake of GNPs, batches of cells were prepared and incubated with GNPs alone, DTX alone, and GNPs plus DTX. Final concentration of GNPs and DTX in the media was 0.2 and 50 nM, respectively. After the incubation time period, each dish was gently washed three times with PBS to remove any GNPs which were not incorporated within cells.^17–19 To detach cells from the dish for processing, 1 ml of 0.25% Trypsin-EDTA (Gibco) was added and left in the incubator for 5 min. For retention studies, cells were gently washed three times with PBS followed by adding fresh media and were incubated for a further 24 h. For both uptake and retention studies, cell concentrations were measured using an automatic cell counter (Z2 Coulter from Beckman Coulter) with 100 µL of the cell suspension after the incubation time period. A known number of cells were processed with aqua regia (3:1 mixture of HCl and HNO₃ (VWR)) in a heated mineral oil bath until solutions were clear with no visible debris or turbidity and diluted to 4% v/v acid content with deionized water. Quantification of GNPs was done using Inductively Coupled Plasma–Mass Spectrometry (ICP-MS) (Optima 7300 DV, Perkin Elmer, Waltham, USA).

Confocal fluorescence imaging

Live-cell imaging of GNP uptake and DTX action was performed on a Nikon C2-Plus inverted laser scanning confocal microscope using 60X (CFI Apochromat TIRF, NA 1.49) and 100X (CFI Apochromat TIRF, NA 1.49) objectives. GNPs were functionalized with PEG, RGD, and a PEG-Cy5 complex (excitation 633 nm, emission filter 650 nm LP) as described before. MTs were labeled with a viral transfection stain (CellLight Tubulin-GFP, BacMam 2.0, Thermo-Fisher). The virus contains DNA coding for an α-tubulin/GFP fusion construct. This method was chosen as all other live-cell tubulin stains investigated are taxane-based and would compete with DTX for binding sites.

Cells were cultured in 3 cm coverslip-bottomed dishes (MatTek, Ashland, MA) in FluoroBrite DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). Cells were incubated for >24 h with the viral stain before DTX and fluorescent GNPs were added to the media. Cells were imaged after 24 h of incubation. To investigate the extent of exocytosis, we exchanged the media containing GNPs and DTX with fresh media and the cells were imaged 24 h after the incubation.

When imaging, the same acquisition settings were used between samples within an experiment. Image processing was performed using ImageJ. As the viral stain transfection efficiency was low, and individual cell brightness varied, the tubulin channel (green in images) brightness was selected to allow for the maximum number of visible and unsaturated cells. GNP channel brightness was scaled to show distribution (not quantity) of NPs unless otherwise mentioned.

Radiation treatment

Cells were prepared in 6-well plates and inoculated with GNPs and DTX as similar to uptake studies as described in the section titled “Docetaxel and GNP inoculation.” After inoculation for 24 h, the plates were placed between two 5 cm solid water blocks at the isocenter of a Varian TruBeam medical linear accelerator and given a 2 Gy radiation dose with a 6 MV beam (28 x 28 cm field size, 202 monitor units). Control cells (no GNPs and no DTX) were transported to the linear accelerator, but not irradiated. Both radiated and non-radiated cells were returned to the incubator for 1 h. After the incubation period, cells were washed with PBS three times and added I mL of trypsin and incubated for another 5 min. After trypsinization, cell concentrations were measured using both an automatic cell counter and manually via trypan blue exclusion assay (Gibco) on a hemocytometer. The resulting cell suspensions were used for the clonogenic and proliferation assays to assess the efficacy of each treatment.

Clonogenic assay

The HeLa cell suspensions were seeded into 10 cm dishes at seeding densities of 500/dish for control cells (no DTX, and no GNPs) and cells treated with GNPs (no DTX). The seeding density for cells treated with DTX was 40,000/dish (DTX alone and DTX plus GNPs) due to much lower survival fraction. These plates were incubated for 14 days and colonies were stained with methylene blue (BioShop) for manual counting. The number of colonies vs plated cells was normalized by the plating efficiency of control cells to obtain the survival fraction.^20,21 Two-way analysis of variance of irradiated samples was performed using the CFAssay package for R.^22,23 A clonogenic assay was also performed on MDA-MB-231 as well. Due to its longer doubling time and DTX’s cell cycle arrest, the treated cells did not develop colonies of sufficient size before the control cells grew too confluent.

Proliferation assay

Cells from the radiation experiment were seeded into three black-walled clear-bottom 96-well plates (Costar) (10³ cells/well, 100 µL of fresh media) and covered with a breathable membrane to reduce evaporation (Breathe-Easier Membranes). At the time of the reading, the membrane was removed and the media was aspirated. 100 µL of media containing 10% v/v resazurin dye (PrestoBlue, Thermo-Fisher) was added to each of the well followed by incubation for 1 h. Fluorescence was measured using Biotek Cytation one plate reader (filters at Ex 530/25, Em 590/35 nm). Viable cells reduce the resazurin compound, and the fluorescence of the product correlates linearly to the number of viable cells.

Doubling time assay

MDA-MB-231 and HeLa cells were plated at cell densities ranging from 2500 to 10,000 cells/well in black-walled 96-well plates. After incubation of 14, 48, and 72 h, the media was aspirated and replaced with 10% resazurin dye in media and incubated further for 30 min. The plates were read via fluorescence as described for the proliferation assay. An exponential curve was fit to the fluorescence vs time data for each plating density, and the average was taken to be the doubling time.

Immunofluorescence assay

Cells were grown directly on glass coverslips in 6-well dishes. After 24 h of the treatment, cells were fixed with 2% paraformaldehyde/0.2% Triton-X 100 for 20 min at room temperature (PFA) (Sigma–Aldrich) and washed three times with PBS in 5 min intervals. Cells were then treated with 0.1% Triton-X for 20 min at room temperature and washed with PBS 3 times in 5 min intervals followed by treatment with 3% bovine serum albumin (BSA) for 1 hr. After the incubation, coverslips were placed face down on Para film with a 50 µL drop of primary antibody 1:800 (H2A.X Ser 139; Sigma Aldrich; catalog # 05–636) in 3% BSA/PBS and incubated overnight at 4°C. Coverslips were washed with 0.5% BSA/ 0.175% Tween 20/PBS three times for 5 min. Secondary antibody was diluted 1:500 (Donkey anti mouse IgG; Alexa 647. Sigma Aldrich; catalog # A-31571) in 3% BSA/PBS and the coverslips were incubated for 45 min. Coverslips were again washed with 0.5% BSA/ 0.175% Tween 20/PBS three times in 5 min durations followed by washing again with PBS three times for 5 min time intervals. Finally, the coverslips were dried and mounted on microscope glass slides for imaging.

Monte Carlo simulation

Monte Carlo simulations were performed using the TOPAS/TOPAS-nBio v. 3.1 .p2.^24,25 Therapeutic X-rays from a 6 MV Varian TrueBeam linear accelerator attenuated through 10 cm of water were incident on a single 15 nm GNP and the radial dose was calculated. As in our previous studies,^5,21,26 the radial dose was superimposed on each GNP location in the cell. The cells were represented by elliptical shapes with major/minor diameters of 18.5/8.5 μm. The size of the cell nucleus was assumed to be 8 μm. The GNPs were encapsulated inside vesicles of 50 nm diameter, with 10 GNP per vesicle. The vesicles are distributed in the cytoplasm either uniformly or exponentially for cells without or with DTX, respectively. The radial dose enhancement was scored inside the nucleus.

An adapted local effect model was introduced to predict survival fractions. Radiation response parameters of α = 0.019, β = 0.052 and α = 0.282, β = 0.087 were assumed for MDA-MB-231 and HeLa cells, respectively.^10,27

Results and discussion

Characterization of GNPs

GNPs used for this study were functionalized with PEG and a peptide containing the integrin binding domain RGD (RGD peptide) (see schematic Figure 1d). Having PEG is necessary to improve the circulation lifetime for their optimum entrapment within the tumor using its leaky vasculature.³ However, having PEG on the GNP surface reduces their uptake once they reach the tumor cells. Hence, we added a peptide containing integrin binding domain, RGD to increase the uptake of GNPs. This peptide have been used in a wide variety of nanoparticle systems to induce attachment and endocytosis.^19,28–31 To enhance receptor–ligand interaction, we used smaller 15 nm diameter GNPs as the high curvature of smaller GNPs improves the interaction between RGD-peptide and integrin receptors on the cell membrane.³² The size and shape of GNPs used in this study were measured with scanning electron microscopy (SEM), ultraviolet-visible (UV-VIS) spectroscopy, dynamic light scattering (DLS), and ζ potential measurements as summarized in supplement section S1. Based on the SEM images, the nominally 15 nm GNPs were 17.2$\pm$5.6 nm in diameter (Supplement S1a, b). GNPs have a red color in solution, due to a surface plasmon resonance scattering peak whose wavelength is determined by their diameter. The shape of the UV-VIS spectrum for bare vs RGD/PEG modified GNPs did not change appreciably, though the peak wavelength did shift slightly to the red (Supplement S1c, d) since both RGD-peptide and PEG molecules were considerably smaller than the size of GNP. For example, the molecular weight of RGD-peptide and PEG were 1760 and 2000 Da, respectively. However, the addition of PEG and RGD peptide resulted in a significant change in the surface charge (Supplement S1c, e). This is due to replacement of negatively charged citrate molecules with neutral PEG and positively charged RGD peptide molecules. The peak and shape of the UV/VIS spectrum or the surface charge of GNPs when in solution with DTX was not significantly changed, indicating that DTX did not bind to GNPs or induce aggregation of the GNP complexes. We also measured the change in the hydrodynamic diameter through DLS measurements and the results were consistent with previously discussed data (Supplementary S1c).

Cellular uptake of GNPs in the presence of docetaxel

Before moving to uptake studies, we analyzed the variation of cell cycle and growth in the presence of DTX via proliferation assay (Supplement S2a, b) and cell cycle analysis (Supplement S2c, d). The DNA content of cells exposed to 50 nM DTX was compared with control cells using flow cytometry. With the treatment of DTX, most of the cells were arrested in G2/M phase and this is consistent with previous studies for this dose range and exposure time (see Supplement S2c, d).^10,33–36

Cellular uptake of modified GNP complexes was characterized using two cell lines: HeLa and MDA-MB-231. HeLa (doubling time (T_d) = 31 h) is a human cervical cancer line and MDA-MB-231 (T_d = 40 h) is a triple-negative human breast cancer cell line. The concentration of DTX used for in vitro experiments was 50 nM. Over a 24 h exposure, the area under the concentration-time curve (AUC_0→24) is 1200 nM* hr when 50 nM DTX is used over a 24 h time period. This can be compared to an AUC_0→25 of 1284 nM* hr in plasma for a lower clinical dose of 20–30 mg/m².³⁷ One of the other highlights of our approach is that we use a clinically feasible 0.2 nM concentration of GNPs for this study. For example, our approach would require an intravenous administration of a few mg Au/kg instead of g Au/kg making it a potentially clinically relevant approach in the near future.^2,38

These NPs are internalized by receptor-mediated endocytosis and trapped in endosomes. These are transported to the perinuclear region, where they fuse with lysosomes for processing. The waste products are collected into vesicles which are then brought to the cell periphery for excretion (Figure 2a).^17,39 Studies have shown that microtubules are not involved directly in endocytosis, however, the vesicle transport of NPs depends on the MT network.⁹ Recycling of integrin binding receptors to the cell surface can occur before the endosome begins to move along a MT.^40,41 Hence when the MT network is disturbed in the presence of DTX, the endocytosis process should be largely undisturbed while the transport of GNPs within the cell may be affected (right side of Figure 2a).

Figure 2.

Cellular uptake of GNPs in MDA-MB-231 and HeLa cells. (a) Schematic diagram illustrating the path of GNPs (red dots) within a cell in the absence (left side) and presence of DTX (right side), showing (i) endocytosis, (ii) vesicle transport, (iii) processing near microtubule organizing centre (MTOC, green patch), and (iv) exocytosis. (b) Quantification of GNP uptake in the presence of 50 nM DTX (24 h exposure). Error bars are standard deviations from three replicate measurements. *Represents a statistically significant difference from control (Welch’s unequal variance t-test, p < 0.05). (c–f) Optical images of distribution of GNPs (marked in red) and MTs (marked in green) in HeLa and MDA-MB-231, respectively. (c, e) HeLa and MDA-MB-231 control cells (not treated with DTX), respectively. (d, f) HeLa and MDA-MB-231 cells treated with 50 nM DTX, respectively. The scale bar is 25 µm. DTX, docetaxel; GNP, gold nanoparticle; MTOC, microtubule organizing center.

Nanoparticle uptake with exposure to 50 nM DTX was quantified using ICP-MS (Figure 2b) and imaged on a confocal microscope (Figure 2c–f). Both HeLa and MDA-MB-231 treated with DTX had a ~ 70% increase in uptake after 24 h of incubation (Figure 2b). Both cell lines show at least a two-fold increase in the G2/M-phase population with the treatment of DTX (Supplementary Table 2). The cells in G2/M phase are expected to have higher number of GNPs compared to other phases since they have more time to accumulate GNPs before division. Cell division dilutes the per-cell GNP load between daughter cells.⁴² Hence, having more cells in G2/M could lead to higher average number of GNPs within the whole cell population. Thus, we believe that the observed increase in uptake is likely due to DTX halting cell division at G2/M phase and preventing redistribution (Figure 2b).

We also noticed that there was a change in the cell morphology from elliptical (Figure 2c,e) to circular (Figure 2d,f) with the treatment of DTX for both MDA-MB-231 and HeLa cells. We believe that this change in the shape of the cells is due to the stabilization of MTs with the addition of DTX as described in the introduction. A significant difference in distribution of GNPs was seen in cells treated with DTX. For example, GNPs seem to distribute closer to the nucleus as shown in Figure 2d,f. This pattern of distribution of GNP clusters is consistent across several planes of the cell (Supplement section S3). The presence of GNPs within cells were further verified using both dark field and bright field imaging (Supplement section S4).

Retention of NPs in the presence of DTX

Our results in Figure 3a demonstrate that the proportion of GNPs excreted or redistributed was reduced dramatically in cells treated with DTX. The percent of retention was between ~90 and 100% for both MDA-MB-231 and HeLa cells. Chithrani and Chan showed that exocytosis can occur rapidly after the nanoparticle-containing media is replaced, with up to 40% of internalized NPs being released within 4 h.¹⁷ The observed decrease in GNPs within cells for both MDA-MB-231 and HeLa control samples (without the treatment of DTX) is also consistent with redistribution via division following an exponential with decay constant of ln2/T_d.⁴² If exocytosis moderates the uptake rate in normal cells, blocking that process may lead to an increased presence of NPs within cells. The retention observed in the DTX-treated cell samples is consistent with both a lack of redistribution and a cessation of exocytosis. Figure 3d, e shows that the population of cells remains locked in G2/M even after 24 h in DTX-free media supporting the lack of redistribution hypothesis.

Figure 3.

Quantitative and qualitative measurement of GNP retention 24 h after release from DTX and GNPs. (a) Percent retention of GNPs as compared to uptake. *Represents a statistically significant difference (Welch’s unequal variance t-test, p < 0.05). Error bars are standard deviations from three replicate measurements. (b–c) Confocal images of HeLa cells in the absence and presence of DTX, respectively. GNPs are represented in red and MTs are in green. The scale bar is 25 µm. (d–e) Cell cycle distribution after 24 h with and further 24 h release from 50 nM DTX compared to control exposure for HeLa (d) and MDA-MB-231 (e) cells. DTX, docetaxel; GNP, gold nanoparticle.

In normal cells (Figure 3b), MTs are polarized to allow directed inward or outward motion.⁸ When MTs are stabilized, the processing of vesicles containing GNPs is disturbed.⁴³ It is necessary for NPs in the endosomes to be processed by fusing with lysosomes; however, the azimuthal MT bundles formed in the presence of DTX may not link endosomes with lysosomes efficiently. Even if the vesicles are processed, the lack of radially directed MTs prevents them from moving to the cell surface for exocytosis. This would result in retarded outward motion of GNPs and piling up them closer to the nucleus as shown in Figure 3c. For example, we noticed that some of these vesicles collect at the ends of bundles and in "empty" regions, perhaps through diffusion after reaching the end of a MT (see Supplement section S5). Or it could be that the MTs within these bundles are similarly polarized as well as parallel. Vesicles may encounter only ends that direct them back into the empty regions not allowing them to reach the cell surface. The distribution of GNPs within the cell in the presence of DTX seen in Figure 2d was still observed in Figure 3c. We also captured the distribution of GNPs observed during mitosis in control cells vs cells treated with DTX (Supplement section S6a, b). GNPs were excluded from the mitotic spindle apparatus in control cells, while in DTX-treated cells their distribution was less regular. We also noticed that the presence of GNPs did not significantly affect the action of DTX (Supplement section S6c).

Triple combination of RT, DTX, and GNPs

Linear accelerators are used to treat cancer patients in the clinic. Therefore, we used a model set up as illustrated in Figure 4a. Our samples were placed between solid water blocks to mimic a deep-seated tumor in a patient. Previous studies have shown GNPs can be used as a radiation dose enhancer with MeV photon beams due to beam softening with depth in tissue.² Significant dose enhancement from GNPs is limited to less than 1 µm from the GNP surface for such beams (Figure 4b). A radiation dose of 2 Gy with a 6 MeV photon beam resulted in a further 30% decrease in survival vs radiated control when GNPs were incorporated within cells in the absence of DTX (Figure 4c). Our results are consistent with previously published data.^27,44 Addition of GNPs into the RT/DTX combination resulted in an additional 50% decrease in long term cell survival vs RT/DTX combination (Figure 4d). This extra drop in survival indicates an interaction between DTX and GNPs that heightens radiosensitivity beyond an additive effect (p = 0.0298). Hence, the triple combination of DTX, GNPs, and RT could play a major role in the reduction of normal tissue toxicity. Monitoring of the short-term proliferation of cells showed a similar trend as illustrated in Figure 4e,f. The cell survival and proliferation data were obtained using the HeLa cell line. No toxic interaction was observed between GNPs and DTX without radiation, as shown in Supplement section S65. The addition of GNPs did not significantly alter the action of the drug, DTX.

Figure 4.

Triple combination of RT/GNPs/DTX in HeLa cells. (a) Image of irradiation geometry with clinical linear accelerator. (b) Monte Carlo model of radial dose around a single GNP incident by direct particle showers from 6 MV linear accelerator. (c) GNP-mediated radiation dose enhancement after a radiation dose of 2 Gy as determined by clonogenic assay. (d) Comparison of triple combination of RT/GNPs/DTX vs RT/DTX as determined by clonogenic assay. *Represents aninteraction between DTX and GNP treatment conditions from Two-Way ANOVA, p>0.05. Errorbars are 95% confidence intervals from Poisson model of 3 replicatemeasurements. (e) Comparison of proliferation curves of non-irradiated (0 Gy) for GNP/ No DTX vs GNP/ DTX. (f) Comparison of proliferation curves for triple combination of RT/GNPs/DTX vs RT/DTX. DTX, docetaxel; GNP, gold nanoparticle; RT, radiotherapy.

The presence of DTX changes the distribution of GNPs within cells as discussed in Figure 2. Redistribution of GNPs closer to the nucleus is expected to enhance the DNA damage since the increased cross-section of low energy electrons could lead to more free radicals closer to the nucleus as illustrated in schematic Figure 5a. Double strand breaks (DSBs) are the most lethal type of DNA damages and we evaluated the DNA DSBs under the same conditions discussed in Figure 4.⁴⁵ It is shown that the presence of GNPs could enhance the DNA DSBs both at KeV and MV energies.^20,46 It is evident that the presence of GNPs did not introduce additional DSBs in non-irradiated cells (Figure 5b) indicating their biocompatibility at the concentration used in this study. In radiated cells, there was an enhancement in DNA DSBs in the presence of GNPs (Figure 5c). The presence of GNPs and DTX led to a higher DNA DSBs in radiated cells as compared to the irradiated cells with either GNPs or DTX. Our confocal images in Figure 5d support the quantification data in Figure 5b,c (see also Supplementary section S7). Hence, both quantitative and qualitative results shown in Figure 5 support the outcome illustrated in Figure 4. However, cell death from radiation may be caused by damage in other organelles in the cells that were not considered in this study, e.g. mitochondria.⁴⁷ However, we believe that DNA DSBs are one of the dominant parameters to assess the cell damage due to radiation. We also performed a theoretical analysis using GNP uptake data (Figure 2) to further illustrate how the redistribution of GNPs closer to the nucleus affect cell survival as discussed next.

Figure 5.

Assesment of DNA damage. (a) Diagram showing radiosensitizing effect of GNPs (red sphere). X-rays (black waves) produce free radicals in water (blue circles). GNPs produce several secondary electrons and free radicals (red circles) from a single interaction with an X-ray. These free radicals can do damage to DNA (yellow star), causing single- or double-strand break. (b, c) Extent of DSBs in non-radiated and radiated cells, respectively. (d) Confocal images of nuclei with DSB foci for non-radiated (left panel) and radiated (right panel) cells. Nuclei and DSB foci are represented in blue and green, respectively. The scale bar is 25 µm., double strand break; DTX, docetaxel; GNP, gold nanoparticle.

Monte Carlo simulation

Microdosimetric Monte Carlo simulations were performed to simulate the dose enhancement from a modified intracellular GNP distribution. Radial dose profiles of GNPs under irradiation from a clinical 6 MV beam were calculated and superimposed over GNP locations in two model geometries:

1. Vesicles of GNPs distributed uniformly through the cytoplasm (see schematic Figure 6a). Figure 6b is an image of a control cell (not treated with DTX) showing the distribution of GNPs within the cell cytoplasm.

2. Vesicles distributed exponentially from the nucleus falling off radially (see schematic Figure 6c). This is a close representation of GNP distribution in cells treated with DTX as illustrated in Figure 6d.

Figure 6.

Monte Carlo simulations of GNP dose. (a, b) Schematic and an optical image of GNP distribution in a control cell, respectively. GNPs and MTs are represented in red and green, respectively. (c, d) Schematic and an optical image of GNP distribution in a DTX-treated cell, respectively. (e, f) Radial distribution of deposited dose inside the nucleus with GNP-containing vesicles uniformly (Uni) and exponentially (Exp) distributed in the cytoplasm. (g, h) Relative survival fraction for uniformly (Uni) and exponentially (Exp) distributed GNPs for a radiation dose of 2 Gy for HeLa and MDA-MB-231 cells, respectively.

Total numbers and uncertainties of GNPs per cell were taken from the uptake measurements in Figure 2. The dose was scored inside the nucleus (Figure 6e,f). GNPs caused significant dose enhancement, especially in the outer regions of the nucleus, with an increasing number of GNPs resulting in an increased dose enhancement. For example, 513,739 GNPs distributed exponentially with DTX increased the radiation dose up to 50 Gy in the outer layer of the nucleus of HeLa cells. DTX induced a greater number of GNPs localized in close proximity to the nucleus. Therefore, a combined treatment of docetaxel, GNPs, and radiation was more effective to kill cancer cells compared to radiation alone or GNP plus radiation.

An adapted local effect model was introduced to predict survival fractions based on the experimental GNP uptake data (Figure 6g,h). It should be noted that our simulations do not take into account the effects of the treatment with DTX. The survival fraction is calculated only considering the additional cell kill induced by the presence of GNPs and their distribution. The radiation response parameters (α and β) used in the calculations were the same as for the cells without DTX treatments. However, due to the expected synergistic effects of the two radiation sensitizers/enhancers, the difference in the observed biological effects between GNP and GNP + DTX in the experiments is expected to be higher than predicted by our simulation. It should further be noted, that the simulations only consider the physical dose enhancement. Other effects, e.g. due to additionally created reactive oxygen species or GNP-induced cell stresses, are not modeled and are expected to result in a difference in the absolute scale of the effect between simulation and experiment. Nevertheless, the experimental trends and relative differences consistently follow the simulation results.

Conclusions

The transport processes that move cellular vesicles containing GNPs around are highly dependent on the MT network of the cell.⁹ Based on our quantitative and qualitative data, the change in MT network in the presence of DTX resulted in a significant effect on exocytosis and cause more perinuclear localization of GNP-containing structures.⁴⁸ GNPs are used as radiation dose enhancers and our Monte Carlo simulations (based on our experimental GNP uptake data) showed that redistribution of GNPs closer to the nucleus resulted in a lower cell survival following a RT dose of 2 Gy. DTX can also act as a radiosensitizer, however, the mechanism of action cell damage in the presence of RT was different compared to GNPs. This led to a synergistic therapeutic effect with the triple combination of RT/GNPs/DTX. We used 2 Gy as our single RT dose since it is one of the most commonly used fractionated doses in the clinic. Further, DTX is used as a chemotherapy agent in the clinic and it is typically given on a weekly basis when combined with RT as it remains in tumors longer than in the rest of the body.^36,49 This has the added advantage that it may also allow retention of GNPs during the fractionated RT treatment. GNPs are being successfully tested in Phase I clinical trials.⁵⁰ Hence, we believe that the roadmap to clinical translation of this triple combination of DTX/GNPs/RT is feasible and our study could lay the foundation for novel treatment approaches in the near future.

Supporting information

The supporting information includes SEM images of GNPs, characterization data, cell cycle data, and additional confocal, dark field and bright field images.