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Gold nanoparticle cellular uptake, toxicity and radiosensitisation in hypoxic conditions
Radiotherapy and Oncology 110 (2014) 342–347
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Hypoxia
Gold nanoparticle cellular uptake, toxicity and radiosensitisation in hypoxic conditions Suneil Jain a,⇑,1, Jonathan A. Coulter b,1, Karl T. Butterworth a, Alan R. Hounsell c, Stephen J. McMahon a, Wendy B. Hyland c, Mark F. Muir d, Glenn R. Dickson a, Kevin M. Prise a, Fred J. Currell d, David G. Hirst b, Joe M. O’Sullivan a a Centre for Cancer Research and Cell Biology, School of Medicine, Queen’s University Belfast; b Experimental Therapeutics Research Group, School of Pharmacy, Queen’s University Belfast; c Medical Physics Agency, Northern Ireland Cancer Centre, Belfast; and d Centre for Plasma Physics, School of Mathematics and Physics, Queen’s University Belfast, United Kingdom
a r t i c l e
i n f o
Article history: Received 2 January 2013 Received in revised form 2 November 2013 Accepted 10 December 2013 Available online 17 January 2014 Keywords: Gold nanoparticles Radiosensitisers Toxicity Hypoxia
a b s t r a c t Background and purpose: Gold nanoparticles (GNPs) are novel agents that have been shown to cause radiosensitisation in vitro and in vivo. Tumour hypoxia is associated with radiation resistance and reduced survival in cancer patients. The interaction of GNPs with cells in hypoxia is explored. Materials and methods: GNP uptake, localization, toxicity and radiosensitisation were assessed in vitro under oxic and hypoxic conditions. Results: GNP cellular uptake was significantly lower under hypoxic than oxic conditions. A significant reduction in cell proliferation in hypoxic MDA-MB-231 breast cancer cells exposed to GNPs was observed. In these cells significant radiosensitisation occurred in normoxia and moderate hypoxia. However, in near anoxia no significant sensitisation occurred. Conclusions: GNP uptake occurred in hypoxic conditions, causing radiosensitisation in moderate, but not extreme hypoxia in a breast cancer cell line. These findings may be important for the development of GNPs for cancer therapy. Ó 2014 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 110 (2014) 342–347
There has been intense interest in the use of gold nanoparticles (GNPs) for cancer therapy because of their small size, high contrast, biocompatibility, preferential accumulation in tumours and ability to bind functional moieties [1,2]. Multiple studies have demonstrated GNP radiosensitisation in vitro and in vivo, however, the exact mechanism by which sensitisation occurs remains to be elucidated [3–5]. Sensitisation has commonly been attributed to physical dose enhancement occurring at kilovoltage (kV) photon energies due to increased photoelectric photon absorption by high-Z materials. However, recent in vitro studies have also demonstrated radiosensitisation at megavoltage (MV) energies, commonly used in the treatment of cancer [4,5]. The enhancement at these energies has been accounted for within the framework of the local effect model [6,7]. It is clear that GNPs, which have sizes comparable to many proteins are not inert, and chemical or biological mechanisms of
⇑ Corresponding author. Address: Centre for Cancer Research and Cell Biology, Queen’s University Belfast, 51 Lisburn Road, Belfast BT97AB, United Kingdom. E-mail address: [email protected] (S. Jain). 1 These authors contributed equally to this work. 0167-8140/$ - see front matter Ó 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.radonc.2013.12.013
sensitisation must be considered. Indeed GNPs have been shown to cause cytotoxicity, apoptosis, altered gene expression, cell cycle disruption and reactive oxygen species production [8–10]. GNPs have also been shown to potentiate the effect of the chemotherapeutic agent bleomycin in vitro [4]. Somewhat surprisingly, there is almost no published evidence of GNP interactions with hypoxic cells in vitro or in vivo, either alone or in combination with other treatment modalities, such as radiation or chemotherapy. Tumour hypoxia in human cancers is associated with an aggressive malignant phenotype, increased metastasis, reduced survival, chemoresistance and radioresistance [11,12], overall being a major limiting factor in the effectiveness of external beam radiotherapy in solid tumours. Many questions regarding nanoparticle interactions with hypoxic cells remain to be answered. For example, will hypoxic cells take up GNPs at the same rate as oxic cells? Will GNPs affect cellular proliferation and cytotoxicity of hypoxic cells? Will GNPs cause radiosensitisation in hypoxic cells, in which, indirect free radical mediated effects are reduced or absent? The aim of this work was to assess the uptake, toxicity and radiosensitisation of GNPs in cancer and normal cells in hypoxic conditions as a basis for the design of future nanoparticle strategies targeting tumour hypoxia.
S. Jain et al. / Radiotherapy and Oncology 110 (2014) 342–347
Materials and methods Gold nanoparticles 1.9 nm spherical GNPs (Aurovist™) used in previous radiation studies were purchased from Nanoprobes Inc. (Yaphank, NY) [6,13]. GNPs were suspended in sterile water (Sigma, UK), filtered through a 0.2 lm filter and stored at 20 °C as per manufacturer’s instructions. This stock solution was diluted in culture media immediately prior to use. All cells were exposed to 12 lM GNPs, based on an optimal concentration for radiosensitisation in previous studies [4]. Cell culture DU145 human prostate cancer cells, MDA-MB-231 breast cancer cells and L132 lung epithelial cells were cultured in Roswell Park Memorial Institute (RPMI) 1640, Dulbeccos’ modified eagle medium (DMEM) and minimum essential medium (MEM), respectively. All cells were purchased from the American Type Culture Collection (ATCC). All media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Invitrogen, UK). Cells were maintained in a tissue culture incubator at 37 °C with 5% CO2/95% air. Hypoxic studies Hypoxic conditions were generated in two ways. For GNP cell uptake, localization and toxicity studies, cells were plated in 35 mm2 petri dishes for 24 h to adhere. Cells and 1.9 nm GNPcontaining medium were then incubated at 37 °C in a hypoxic workstation (In Vivo2 400, Ruskinn, Bridgend, UK) with 0.1% oxygen, 5% CO2, 95% nitrogen for 4 h until hypoxic. Hypoxia was confirmed using a resaurzin anaerobic indicator (Oxoid, Hampshire, UK). Hypoxic medium containing GNPs was added to cells in the hypoxic workstation for 24 h, after which uptake and toxicity assays were carried out. Cells remained in hypoxia for the duration of the study. For colony formation and radiation experiments hypoxic cells were exposed to GNPs (1.9 nm; 12 lM) in hypoxic medium for 24 h in a hypoxic workstation and transferred in airtight PMMA chambers (developed in-house) to the 160 X-ray machine. The chamber was gassed under positive pressure with 95% nitrogen/5% CO2/0.1% O2 or 95% nitrogen/5% CO2/1% O2 for 1 h prior to and during radiation. Following irradiation cells were replated and maintained in oxic conditions to form colonies.
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Cell proliferation assay 1 104 cells were plated for 24 h, exposed to 1.9 nm GNPs for 24 h, washed twice in PBS and incubated in fresh culture medium. Cell numbers in control and GNP exposed samples were counted at 0 h and 144 h using a Z1 Beckman Coulter counter (Brea, CA, USA). In hypoxic studies cells remained in hypoxia throughout. Clonogenic survival assays 7.5 104 cells were plated in 35 mm2 dishes for 24 h, exposed to GNPs in culture medium for 24 h, then irradiated with 0–18 Gy X-rays. Cells were washed twice in PBS, trypsinised, counted, replated in 6 well plates and incubated for 9–14 days in oxic conditions to form colonies. Colonies were stained with 0.4% crystal violet and counted using a Colcount automated colony counter (Oxford Optronix, UK). Surviving fractions (SF) were calculated relative to non-irradiated cells and fitted to the linear quadratic (LQ) equation S = exp( aD bD2) using least-squares regression in Prism 5.0 (GraphPad Software, CA, USA). The area under the curve (AUC), which represents the mean inactivation dose (MID), was obtained and the sensitizer enhancement ratio (SER) calculated by dividing the MID of non-exposed cells with gold exposed cells as published [15]. 160 kVp X-rays Irradiations were performed using a Faxitron CP-160 (kVp) X-ray generator (Lincolnshire, IL, USA). Six 35 mm2 dishes were arranged concentrically equidistant from the centre of the radiation field with a focus to surface distance of 30 cm. The X-ray beam was filtered with 0.8 mm beryllium and 0.5 mm copper. The output was measured using a calibrated ionisation chamber traceable to the National Physical Laboratory, Teddington, UK and was 0.69 Gy/min (±2%). The beam uniformity was assessed using Gafchromic film (ISP corp) and the field diameter (90% isodose line) was 16.0 cm. All samples were treated within this field to achieve dose variations of <10%. Statistical analysis All experiments were carried out in triplicate with results expressed as mean ± standard error (SE). Statistically significant differences were calculated using the two-tailed unpaired t-test or one-way analysis of variance with a p value of 60.05 considered significant.
Inductively coupled plasma–atomic emission spectroscopy A total of 7.5 104 cells were plated for 24 h then exposed to GNPs for 24 h, washed three times in PBS, trypsinised, counted, and digested in aqua regia (1 part 100% nitric acid:3 parts 100% hydrochloric acid). Gold content was determined using a Perkin Elmer Optima 4300 DV Inductively Coupled Plasma Optical Emission Spectrometer (Shelton, CT, USA). The number of GNPs per cell was determined as described by Gu et al. [14]. Transmission electron microscopy (TEM) 1.5 105 cells were plated for 24 h then exposed to GNPs for a further 24 h in 21% or 0.1% oxygen. Cells were washed twice in PBS, trypsinised, pelleted and fixed in 4% gluteraldehyde in 0.1 M sodium cocodylate. Cells were post-fixed in 1% osmium tetroxide, dehydrated in ethanol and embedded in agar resin. 60–70 nm sections were cut using an ultramicrotome, placed on copper grids, stained with uranyl acetate and lead citrate and imaged with an FEI Technai F20 TEM (Hillsboro, OR, USA).
Results GNP uptake and cell localization Initial experiments aimed to assess 1.9 nm GNP uptake and intracellular distribution in hypoxic DU145 prostate cancer, MDA-MB-231 breast cancer and L132 normal immortalised lung epithelial cells over a 24 h period. In all three cell lines GNP uptake was linear with time between 1 h and 24 h as shown in Fig. 1a. The rate of GNP uptake was calculated using linear regression analysis for the time period 1 h to 6 h post-GNP exposure in each of the 3 cell lines. There was no significant difference in the mean rate of GNP uptake at 1.70 106, 1.86 106 and 1.29 106 for DU145, MDA-MB-231 and L132 cells/h, respectively. Similarly, there was no significant difference in the mean number of GNPs endocytosed by cells at 24 h in the three cell lines (Fig. 1b). Results in oxic conditions have previously been published [8]. When compared to uptake in equivalent oxic experiments, mean GNP uptake at 24 h was significantly lower in hypoxic DU145 cells than in oxic
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Gold nanoparticle hypoxic radiosensitisation
200 nm
Fig. 2. TEM images of hypoxic (0.1% oxygen) L132 cells after 24 h exposure to 1.9 nm GNPs. Aggregates of dense GNPs are observed in cytoplasmic lysosomes throughout the cell.
200–300 lm diameter lysosomal structures situated throughout the cytoplasm. GNPs appeared aggregated within these structures. No GNPs were visualised freely in the cytosol or in the cell nucleus. GNP toxicity
Fig. 1. (a) 1.9 nm GNP uptake with time in hypoxic cells (0.1% oxygen), DU145, MDA-MB-231 and L132. (b) Comparison of total number of 1.9 nm GNP per cell in hypoxic (0.1% oxygen) and oxic (21% oxygen) DU145, MDA-MB-231 and L132 cells and 1% oxygen (MDA-MB-231 cells) after 24 h exposure. DU145, MDA-MB-231 and L132 cells had a relative uptake of 0.29, 0.53 and 0.56 for hypoxic cells relative to aerobic cells. Results are the mean of three independent experiments ± SE.
DU145 cells (4.71 107 and 1.60 108, respectively, p = 0.03) [8]. While the mean GNP uptake at 24 h was less in hypoxic MDA-MB231 and L132 cells than oxic cells the difference was not significant (p = 0.08, p = 0.12, respectively). DU145, MDA-MB-231 and L132 cells had a relative uptake of 0.29, 0.53 and 0.56 for hypoxic cells compared to aerobic cells. Uptake at 1% O2 in MDA-MB-231 cells was comparable to 21% O2. The rate of GNP uptake 1–6 h post-exposure was significantly greater in all 3 cell lines in oxic compared with hypoxic cell lines with uptake reaching a plateau in oxic, but not hypoxic cells. TEM was used to visualize 1.9 nm GNP uptake and intracellular distribution in hypoxic DU145, MDA-MB-231 and L132 cells. A typical distribution image is shown in Fig. 2. The GNP intracellular distribution was comparable to similar experiments carried out in 21% oxygen [8]. GNPs were observed in large quantities in
With evidence of GNP uptake in hypoxic cells, the effects of GNP exposure on cellular proliferation and colony forming ability were assessed. The number of hypoxic control cells was less at 6 days than cells grown in 21% oxygen, with DU145, MDA-MB-231 and L132 cells reaching 22%, 21% and 27% of the cell numbers in oxic conditions respectively (Fig. 3a–c). There was a significant reduction in cellular proliferation in hypoxic MDA-MB-231 cells exposed to GNPs compared to controls by 6 days (p = 0.0043). There was no significant reduction in cell numbers at day 6 in DU145 and L132 GNP exposed cells compared with control cells. Control and GNP cell numbers were compared after 6 days growth in oxic and hypoxic conditions. Both the DU145 and MDA-MB-231 cell lines had a greater GNP-induced reduction in cell growth in hypoxia than air (1.51 and 1.44 fold respectively), however, the GNPs had similar growth inhibitory effects in L132 cells in hypoxic and oxic conditions. Colony formation of GNP exposed cells was compared to results obtained in oxic conditions in the three cell lines (Fig. 3d–f). There was no significant difference in colony forming ability in any of the cell lines in hypoxic conditions. MDA-MB-231 controls had a plating efficiency (PE) of 0.48 ± 0.03 and gold exposed hypoxic cells a PE of 0.40 ± 0.001 in 0.1% oxygen (p = 0.07). GNP radiosensitisation Radiation clonogenic survival assays were performed in all cell lines at 21% and 0.1% oxygen and MDA-MB-231 cells at 1% oxygen. Results in oxic conditions have previously been published [4]. Fig. 4a demonstrates the effect of hypoxia on the radiation sensitivity of MDA-MB-231 control cells with enhancement ratios of 2.0 and 2.6 at 1% and 21% oxygen concentrations respectively relative to 0.1% oxygen. Fig. 4b–d show survival curves fitted to the linear quadratic model for MDA-MB-231 control cells irradiated with 160 kVp X-rays in 21%, 1% and 0.1% oxygen. The SERs are comparable at 21% and 1% oxygen concentrations at 1.41 and 1.39 (p = 0.01, 0.005), respectively, but at 0.1% oxygen the SER is much lower at 1.1 (p = 0.17) (Table 1). Radiosensitisation occurred with a significant reduction in the MID for GNP exposed MDA-MB-231 cells at 21%, from 3.46 to 2.45, and 1% oxygen concentrations from 4.48
S. Jain et al. / Radiotherapy and Oncology 110 (2014) 342–347
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Fig. 3. Comparison of total cell counts 6 days after plating (a) DU145, (b) MDA-MB-231 or (c) L132 cells exposed to 0 or 12 lM GNP for 24 h in 21% oxygen or 0.1% oxygen. There was a significant reduction in proliferation in oxic DU145 cells and oxic and hypoxic MDA-MB-231 cells. Clonogenic survival of (d) DU145, (e) MDA-MB-231 and (f) L132 cells exposed to 1.9 nm GNP for 24 h at 21%, 1% or 0.1% oxygen. There was a significant reduction in colony formation in oxic MDA-MB-231 cells. Results are the mean of three independent experiments ± SE. PE = plating efficiency.
to 3.21, but not at 0.1% oxygen (p = 0.01, 0.005 and 0.17, respectively). No significant radiosensitisation occurred in DU145 or L132 cells exposed to GNPs for 24 h in 0.1% oxygen or 21% oxygen. Discussion This study represents the first systematic analysis of GNP uptake, localization, toxicity and radiosensitisation in hypoxic conditions. Multiple clinical studies have demonstrated tumour hypoxia correlates with adverse outcome in cancer patients including those treated with radical radiotherapy. For instance, a study of patients with locally advanced cervical cancer treated with surgery alone or radiotherapy alone showed tumour hypoxia was an independent predictor for overall survival [16]. Extreme hypoxia causes a chemical radioresistance due to a lack of oxygen fixation of free radicals at the time of radiation
delivery [12]. Some oxygen enhancement occurs with oxygen levels >0.1% and the effect nears maximum value if oxygen levels are >2.5%. The physiological oxygen levels of normal tissues are commonly in the range 3.5–6%, however, oxygen levels <2.5% often occur in human cancers. For instance, in a large head and neck cancer study of tumour hypoxia the median number of readings per tumour less than 0.33% oxygen was 19% [17]. The reason for reduced GNP uptake in hypoxic cells demonstrated in this study is currently not known. As expected, all cells grew more slowly in 0.1% oxygen than in air under otherwise identical culture conditions, due to a switch to inefficient anaerobic glycolytic metabolism in hypoxia (glycolysis generates 19 fold less ATP per molecule of glucose compared with oxidative phosphorylation) [18]. This reduced energy production may in part explain the reduced GNP uptake in hypoxic cells as receptor mediated endocytosis (RME), the considered mechanism of GNP cellular
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Gold nanoparticle hypoxic radiosensitisation
Fig. 4. Clonogenic radiation survival curves for MDA-MB-231 cells. (a) Control cells demonstrating the change in radiosensitivity with oxygen concentration. (b) Control cells and GNP exposed in 21% oxygen. (c) Control and GNP exposed cells in 1% oxygen. (d) Control and GNP exposed cells in 0.1% oxygen. Enhancement ratios of 1.41, 1.39 and 1.1 were noted for 21%, 1% and 0.1% oxygen, respectively. Survival curves are corrected for the effect of GNP exposure alone. All results are the mean of three independent experiments ± SE.
Table 1 Table of a, b, SF4, SER and values for MDA-MB-231 cells irradiated at three oxygen concentrations in the presence or absence of 1.9 nm GNPs (12 lM). Oxygen%
0.1 1 21
Condition
Control GNP Control GNP Control GNP
MDA-MB-231
p
a [Gy 1]
b [Gy
2
0.021 ± 0.018 0.126 ± 0.053 0.003 ± 0.025 0.107 ± 0.036 0.019 ± 0.025 0.091 ± 0.031
0.006 ± 0.002 0.002 ± 0.005 0.079 ± 0.007 0.098 ± 0.011 0.052 ± 0.007 0.093 ± 0.011
]
SF4
SER
MID
0.833 0.61 0.638 0.344 0.386 0.15
1.1
8.97 8.12 4.48 3.21 3.46 2.45
1.39 1.41
0.17 0.01 0.005
SF4 = surviving fraction at 4 Gy; SER = sensitizer enhancement ratio; MID = mean inactivation dose.
uptake, is known to be energy dependent, occurring at 37 °C but not at 4 °C [19]. Furthermore, it is known that increased levels of lactic acid produced as a product of anaerobic metabolism can alter cellular pH, potentially changing the aggregation potential of GNPs [20]. Hypoxia has been shown to reduce cellular uptake and efficacy of drugs including cisplatin, bleomycin and 5FU [21]. Possible mechanisms of treatment resistance include reduced free radical production leading to reduced DNA damage, reduced cell cycling, reduced apoptosis and clonal selection of resistant cells in hypoxic environments [21]. We observed significant radiosensitisation in MDA-MB-231 cells in moderate hypoxia (1% oxygen) with an SER of 1.39. This level of hypoxia is often observed in human tumours. However, the SER in near anoxia (0.1% oxygen) in this study was much lower
at 1.1, compared with 1.41 in 21% oxygen. It is likely that a reduction in GNP cellular uptake in hypoxia (47% reduction in 0.1% oxygen and 13% in 1% oxygen compared with 21% oxygen in MDA-MB-231 cells) contributes to the observed decrease in sensitisation. If the radiosensitisation occurs primarily due to physical effects then a reduction in GNP uptake would be expected to reduce the SER [6]. Similarly, if biological interactions with GNPs cause the sensitisation, a reduction in cellular uptake is likely to influence the magnitude of the observed effect. The reduced SER could also indicate that sensitisation depends on indirect free radical radiation effects which are responsible for much less radiation damage in near anoxia due to a lack of oxygen fixation. The mechanisms by which GNPs cause radiosensitisation are not fully established at present. Most commentators feel the
S. Jain et al. / Radiotherapy and Oncology 110 (2014) 342–347
primary mechanism is physical and should mainly be observed at kV photon energies due to high contrast material enhancing the photo-electric effect of kV photons. However, the results of biological studies published to date suggest that alternative processes may be important. For instance, GNPs have been shown to cause cytotoxicity, apoptosis, altered gene expression, cell cycle disruption and reactive oxygen species production and to potientiate the effect of radiomimetics such as bleomycin [8–10]. Furthermore, autophagy, is a lysosome-based degradative pathway which is essential for cellular homoeostasis [22,23]. A study demonstrated autophagy concomitant with oxidative stress in lung epithelial cells exposed to 20 nm citrate-coated GNPs in vitro [24]. A separate study reported GNP induced accumulation of autophagosomes and impairment of lysosomal degradation capacity due to alkalinisation of lysosomal pH [22]. Interference with autophagy has been shown to lead to radiosensitisation in many studies, including in MDA-MB-231 breast cancer cells in vitro [23,25]. No studies have examined the role of autophagy in GNPmediated radiosensitisation as yet and this will be the subject of future work. The field of nanomedicine has the potential to provide novel radiosensitisers for clinical use. However, challenges including nanotoxicity, cost, batch-to-batch variability and off-target effects will need to be overcome. Furthermore, this study demonstrates nanoparticle uptake is lower in hypoxic cells. In vivo, it is known that diffusion distances from tumour vasculature to chronically hypoxic cells can be large and it is likely novel mechanisms of drug delivery will be required to target these regions. Two studies have directly investigated nanoparticles in hypoxic cell models. A novel in vitro study utilized the phagocytic actions of blood monocytes to engulf 60 nm diameter gold nanoshells (GNS) and transport them to hypoxic tumour regions in 3D tumour models [26]. Treatment of the hypoxic cells with laser tuned to the resonance frequency of the GNS caused marked cytotoxicity compared to non-nanoparticle exposed cells. This novel approach could be used to target nanoparticles to regions of tumour hypoxia. An alternative approach used laser therapy and GNSs to induce mild hyperthermia in hypoxic tumour xenograft models to enhance the effects of a single 10 Gy dose of 125 kVp X-rays in mice [27].
Conclusions This work provides evidence of 1.9 nm GNP uptake in hypoxic cells in three cell lines. The rate of GNP uptake was lower in hypoxic conditions in all cell lines. GNPs inhibited cellular proliferation in hypoxic MDA-MB-231 cells, but had no significant effect on colony forming ability. Radiosensitisation by GNPs in MDA-MB-231 breast cancer cells was observed under moderate hypoxia, but was of a lower magnitude in near anoxia, and this may relate to reduced uptake of GNPs in hypoxic conditions. In vivo studies of GNPs are required to further assess uptake, toxicity and radiosensitisation.
Role of the funding source This work was supported by Men Against Cancer and Cancer Research UK (grant no. C1278/A990 to DGH and C1513/A7047 to KMP). The funding bodies had no role in the study design or the writing of the manuscript.
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Radiotherapy and Oncology journal homepage: www.thegreenjournal.com
Hypoxia
Gold nanoparticle cellular uptake, toxicity and radiosensitisation in hypoxic conditions Suneil Jain a,⇑,1, Jonathan A. Coulter b,1, Karl T. Butterworth a, Alan R. Hounsell c, Stephen J. McMahon a, Wendy B. Hyland c, Mark F. Muir d, Glenn R. Dickson a, Kevin M. Prise a, Fred J. Currell d, David G. Hirst b, Joe M. O’Sullivan a a Centre for Cancer Research and Cell Biology, School of Medicine, Queen’s University Belfast; b Experimental Therapeutics Research Group, School of Pharmacy, Queen’s University Belfast; c Medical Physics Agency, Northern Ireland Cancer Centre, Belfast; and d Centre for Plasma Physics, School of Mathematics and Physics, Queen’s University Belfast, United Kingdom
a r t i c l e
i n f o
Article history: Received 2 January 2013 Received in revised form 2 November 2013 Accepted 10 December 2013 Available online 17 January 2014 Keywords: Gold nanoparticles Radiosensitisers Toxicity Hypoxia
a b s t r a c t Background and purpose: Gold nanoparticles (GNPs) are novel agents that have been shown to cause radiosensitisation in vitro and in vivo. Tumour hypoxia is associated with radiation resistance and reduced survival in cancer patients. The interaction of GNPs with cells in hypoxia is explored. Materials and methods: GNP uptake, localization, toxicity and radiosensitisation were assessed in vitro under oxic and hypoxic conditions. Results: GNP cellular uptake was significantly lower under hypoxic than oxic conditions. A significant reduction in cell proliferation in hypoxic MDA-MB-231 breast cancer cells exposed to GNPs was observed. In these cells significant radiosensitisation occurred in normoxia and moderate hypoxia. However, in near anoxia no significant sensitisation occurred. Conclusions: GNP uptake occurred in hypoxic conditions, causing radiosensitisation in moderate, but not extreme hypoxia in a breast cancer cell line. These findings may be important for the development of GNPs for cancer therapy. Ó 2014 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 110 (2014) 342–347
There has been intense interest in the use of gold nanoparticles (GNPs) for cancer therapy because of their small size, high contrast, biocompatibility, preferential accumulation in tumours and ability to bind functional moieties [1,2]. Multiple studies have demonstrated GNP radiosensitisation in vitro and in vivo, however, the exact mechanism by which sensitisation occurs remains to be elucidated [3–5]. Sensitisation has commonly been attributed to physical dose enhancement occurring at kilovoltage (kV) photon energies due to increased photoelectric photon absorption by high-Z materials. However, recent in vitro studies have also demonstrated radiosensitisation at megavoltage (MV) energies, commonly used in the treatment of cancer [4,5]. The enhancement at these energies has been accounted for within the framework of the local effect model [6,7]. It is clear that GNPs, which have sizes comparable to many proteins are not inert, and chemical or biological mechanisms of
⇑ Corresponding author. Address: Centre for Cancer Research and Cell Biology, Queen’s University Belfast, 51 Lisburn Road, Belfast BT97AB, United Kingdom. E-mail address: [email protected] (S. Jain). 1 These authors contributed equally to this work. 0167-8140/$ - see front matter Ó 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.radonc.2013.12.013
sensitisation must be considered. Indeed GNPs have been shown to cause cytotoxicity, apoptosis, altered gene expression, cell cycle disruption and reactive oxygen species production [8–10]. GNPs have also been shown to potentiate the effect of the chemotherapeutic agent bleomycin in vitro [4]. Somewhat surprisingly, there is almost no published evidence of GNP interactions with hypoxic cells in vitro or in vivo, either alone or in combination with other treatment modalities, such as radiation or chemotherapy. Tumour hypoxia in human cancers is associated with an aggressive malignant phenotype, increased metastasis, reduced survival, chemoresistance and radioresistance [11,12], overall being a major limiting factor in the effectiveness of external beam radiotherapy in solid tumours. Many questions regarding nanoparticle interactions with hypoxic cells remain to be answered. For example, will hypoxic cells take up GNPs at the same rate as oxic cells? Will GNPs affect cellular proliferation and cytotoxicity of hypoxic cells? Will GNPs cause radiosensitisation in hypoxic cells, in which, indirect free radical mediated effects are reduced or absent? The aim of this work was to assess the uptake, toxicity and radiosensitisation of GNPs in cancer and normal cells in hypoxic conditions as a basis for the design of future nanoparticle strategies targeting tumour hypoxia.
S. Jain et al. / Radiotherapy and Oncology 110 (2014) 342–347
Materials and methods Gold nanoparticles 1.9 nm spherical GNPs (Aurovist™) used in previous radiation studies were purchased from Nanoprobes Inc. (Yaphank, NY) [6,13]. GNPs were suspended in sterile water (Sigma, UK), filtered through a 0.2 lm filter and stored at 20 °C as per manufacturer’s instructions. This stock solution was diluted in culture media immediately prior to use. All cells were exposed to 12 lM GNPs, based on an optimal concentration for radiosensitisation in previous studies [4]. Cell culture DU145 human prostate cancer cells, MDA-MB-231 breast cancer cells and L132 lung epithelial cells were cultured in Roswell Park Memorial Institute (RPMI) 1640, Dulbeccos’ modified eagle medium (DMEM) and minimum essential medium (MEM), respectively. All cells were purchased from the American Type Culture Collection (ATCC). All media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Invitrogen, UK). Cells were maintained in a tissue culture incubator at 37 °C with 5% CO2/95% air. Hypoxic studies Hypoxic conditions were generated in two ways. For GNP cell uptake, localization and toxicity studies, cells were plated in 35 mm2 petri dishes for 24 h to adhere. Cells and 1.9 nm GNPcontaining medium were then incubated at 37 °C in a hypoxic workstation (In Vivo2 400, Ruskinn, Bridgend, UK) with 0.1% oxygen, 5% CO2, 95% nitrogen for 4 h until hypoxic. Hypoxia was confirmed using a resaurzin anaerobic indicator (Oxoid, Hampshire, UK). Hypoxic medium containing GNPs was added to cells in the hypoxic workstation for 24 h, after which uptake and toxicity assays were carried out. Cells remained in hypoxia for the duration of the study. For colony formation and radiation experiments hypoxic cells were exposed to GNPs (1.9 nm; 12 lM) in hypoxic medium for 24 h in a hypoxic workstation and transferred in airtight PMMA chambers (developed in-house) to the 160 X-ray machine. The chamber was gassed under positive pressure with 95% nitrogen/5% CO2/0.1% O2 or 95% nitrogen/5% CO2/1% O2 for 1 h prior to and during radiation. Following irradiation cells were replated and maintained in oxic conditions to form colonies.
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Cell proliferation assay 1 104 cells were plated for 24 h, exposed to 1.9 nm GNPs for 24 h, washed twice in PBS and incubated in fresh culture medium. Cell numbers in control and GNP exposed samples were counted at 0 h and 144 h using a Z1 Beckman Coulter counter (Brea, CA, USA). In hypoxic studies cells remained in hypoxia throughout. Clonogenic survival assays 7.5 104 cells were plated in 35 mm2 dishes for 24 h, exposed to GNPs in culture medium for 24 h, then irradiated with 0–18 Gy X-rays. Cells were washed twice in PBS, trypsinised, counted, replated in 6 well plates and incubated for 9–14 days in oxic conditions to form colonies. Colonies were stained with 0.4% crystal violet and counted using a Colcount automated colony counter (Oxford Optronix, UK). Surviving fractions (SF) were calculated relative to non-irradiated cells and fitted to the linear quadratic (LQ) equation S = exp( aD bD2) using least-squares regression in Prism 5.0 (GraphPad Software, CA, USA). The area under the curve (AUC), which represents the mean inactivation dose (MID), was obtained and the sensitizer enhancement ratio (SER) calculated by dividing the MID of non-exposed cells with gold exposed cells as published [15]. 160 kVp X-rays Irradiations were performed using a Faxitron CP-160 (kVp) X-ray generator (Lincolnshire, IL, USA). Six 35 mm2 dishes were arranged concentrically equidistant from the centre of the radiation field with a focus to surface distance of 30 cm. The X-ray beam was filtered with 0.8 mm beryllium and 0.5 mm copper. The output was measured using a calibrated ionisation chamber traceable to the National Physical Laboratory, Teddington, UK and was 0.69 Gy/min (±2%). The beam uniformity was assessed using Gafchromic film (ISP corp) and the field diameter (90% isodose line) was 16.0 cm. All samples were treated within this field to achieve dose variations of <10%. Statistical analysis All experiments were carried out in triplicate with results expressed as mean ± standard error (SE). Statistically significant differences were calculated using the two-tailed unpaired t-test or one-way analysis of variance with a p value of 60.05 considered significant.
Inductively coupled plasma–atomic emission spectroscopy A total of 7.5 104 cells were plated for 24 h then exposed to GNPs for 24 h, washed three times in PBS, trypsinised, counted, and digested in aqua regia (1 part 100% nitric acid:3 parts 100% hydrochloric acid). Gold content was determined using a Perkin Elmer Optima 4300 DV Inductively Coupled Plasma Optical Emission Spectrometer (Shelton, CT, USA). The number of GNPs per cell was determined as described by Gu et al. [14]. Transmission electron microscopy (TEM) 1.5 105 cells were plated for 24 h then exposed to GNPs for a further 24 h in 21% or 0.1% oxygen. Cells were washed twice in PBS, trypsinised, pelleted and fixed in 4% gluteraldehyde in 0.1 M sodium cocodylate. Cells were post-fixed in 1% osmium tetroxide, dehydrated in ethanol and embedded in agar resin. 60–70 nm sections were cut using an ultramicrotome, placed on copper grids, stained with uranyl acetate and lead citrate and imaged with an FEI Technai F20 TEM (Hillsboro, OR, USA).
Results GNP uptake and cell localization Initial experiments aimed to assess 1.9 nm GNP uptake and intracellular distribution in hypoxic DU145 prostate cancer, MDA-MB-231 breast cancer and L132 normal immortalised lung epithelial cells over a 24 h period. In all three cell lines GNP uptake was linear with time between 1 h and 24 h as shown in Fig. 1a. The rate of GNP uptake was calculated using linear regression analysis for the time period 1 h to 6 h post-GNP exposure in each of the 3 cell lines. There was no significant difference in the mean rate of GNP uptake at 1.70 106, 1.86 106 and 1.29 106 for DU145, MDA-MB-231 and L132 cells/h, respectively. Similarly, there was no significant difference in the mean number of GNPs endocytosed by cells at 24 h in the three cell lines (Fig. 1b). Results in oxic conditions have previously been published [8]. When compared to uptake in equivalent oxic experiments, mean GNP uptake at 24 h was significantly lower in hypoxic DU145 cells than in oxic
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Gold nanoparticle hypoxic radiosensitisation
200 nm
Fig. 2. TEM images of hypoxic (0.1% oxygen) L132 cells after 24 h exposure to 1.9 nm GNPs. Aggregates of dense GNPs are observed in cytoplasmic lysosomes throughout the cell.
200–300 lm diameter lysosomal structures situated throughout the cytoplasm. GNPs appeared aggregated within these structures. No GNPs were visualised freely in the cytosol or in the cell nucleus. GNP toxicity
Fig. 1. (a) 1.9 nm GNP uptake with time in hypoxic cells (0.1% oxygen), DU145, MDA-MB-231 and L132. (b) Comparison of total number of 1.9 nm GNP per cell in hypoxic (0.1% oxygen) and oxic (21% oxygen) DU145, MDA-MB-231 and L132 cells and 1% oxygen (MDA-MB-231 cells) after 24 h exposure. DU145, MDA-MB-231 and L132 cells had a relative uptake of 0.29, 0.53 and 0.56 for hypoxic cells relative to aerobic cells. Results are the mean of three independent experiments ± SE.
DU145 cells (4.71 107 and 1.60 108, respectively, p = 0.03) [8]. While the mean GNP uptake at 24 h was less in hypoxic MDA-MB231 and L132 cells than oxic cells the difference was not significant (p = 0.08, p = 0.12, respectively). DU145, MDA-MB-231 and L132 cells had a relative uptake of 0.29, 0.53 and 0.56 for hypoxic cells compared to aerobic cells. Uptake at 1% O2 in MDA-MB-231 cells was comparable to 21% O2. The rate of GNP uptake 1–6 h post-exposure was significantly greater in all 3 cell lines in oxic compared with hypoxic cell lines with uptake reaching a plateau in oxic, but not hypoxic cells. TEM was used to visualize 1.9 nm GNP uptake and intracellular distribution in hypoxic DU145, MDA-MB-231 and L132 cells. A typical distribution image is shown in Fig. 2. The GNP intracellular distribution was comparable to similar experiments carried out in 21% oxygen [8]. GNPs were observed in large quantities in
With evidence of GNP uptake in hypoxic cells, the effects of GNP exposure on cellular proliferation and colony forming ability were assessed. The number of hypoxic control cells was less at 6 days than cells grown in 21% oxygen, with DU145, MDA-MB-231 and L132 cells reaching 22%, 21% and 27% of the cell numbers in oxic conditions respectively (Fig. 3a–c). There was a significant reduction in cellular proliferation in hypoxic MDA-MB-231 cells exposed to GNPs compared to controls by 6 days (p = 0.0043). There was no significant reduction in cell numbers at day 6 in DU145 and L132 GNP exposed cells compared with control cells. Control and GNP cell numbers were compared after 6 days growth in oxic and hypoxic conditions. Both the DU145 and MDA-MB-231 cell lines had a greater GNP-induced reduction in cell growth in hypoxia than air (1.51 and 1.44 fold respectively), however, the GNPs had similar growth inhibitory effects in L132 cells in hypoxic and oxic conditions. Colony formation of GNP exposed cells was compared to results obtained in oxic conditions in the three cell lines (Fig. 3d–f). There was no significant difference in colony forming ability in any of the cell lines in hypoxic conditions. MDA-MB-231 controls had a plating efficiency (PE) of 0.48 ± 0.03 and gold exposed hypoxic cells a PE of 0.40 ± 0.001 in 0.1% oxygen (p = 0.07). GNP radiosensitisation Radiation clonogenic survival assays were performed in all cell lines at 21% and 0.1% oxygen and MDA-MB-231 cells at 1% oxygen. Results in oxic conditions have previously been published [4]. Fig. 4a demonstrates the effect of hypoxia on the radiation sensitivity of MDA-MB-231 control cells with enhancement ratios of 2.0 and 2.6 at 1% and 21% oxygen concentrations respectively relative to 0.1% oxygen. Fig. 4b–d show survival curves fitted to the linear quadratic model for MDA-MB-231 control cells irradiated with 160 kVp X-rays in 21%, 1% and 0.1% oxygen. The SERs are comparable at 21% and 1% oxygen concentrations at 1.41 and 1.39 (p = 0.01, 0.005), respectively, but at 0.1% oxygen the SER is much lower at 1.1 (p = 0.17) (Table 1). Radiosensitisation occurred with a significant reduction in the MID for GNP exposed MDA-MB-231 cells at 21%, from 3.46 to 2.45, and 1% oxygen concentrations from 4.48
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Fig. 3. Comparison of total cell counts 6 days after plating (a) DU145, (b) MDA-MB-231 or (c) L132 cells exposed to 0 or 12 lM GNP for 24 h in 21% oxygen or 0.1% oxygen. There was a significant reduction in proliferation in oxic DU145 cells and oxic and hypoxic MDA-MB-231 cells. Clonogenic survival of (d) DU145, (e) MDA-MB-231 and (f) L132 cells exposed to 1.9 nm GNP for 24 h at 21%, 1% or 0.1% oxygen. There was a significant reduction in colony formation in oxic MDA-MB-231 cells. Results are the mean of three independent experiments ± SE. PE = plating efficiency.
to 3.21, but not at 0.1% oxygen (p = 0.01, 0.005 and 0.17, respectively). No significant radiosensitisation occurred in DU145 or L132 cells exposed to GNPs for 24 h in 0.1% oxygen or 21% oxygen. Discussion This study represents the first systematic analysis of GNP uptake, localization, toxicity and radiosensitisation in hypoxic conditions. Multiple clinical studies have demonstrated tumour hypoxia correlates with adverse outcome in cancer patients including those treated with radical radiotherapy. For instance, a study of patients with locally advanced cervical cancer treated with surgery alone or radiotherapy alone showed tumour hypoxia was an independent predictor for overall survival [16]. Extreme hypoxia causes a chemical radioresistance due to a lack of oxygen fixation of free radicals at the time of radiation
delivery [12]. Some oxygen enhancement occurs with oxygen levels >0.1% and the effect nears maximum value if oxygen levels are >2.5%. The physiological oxygen levels of normal tissues are commonly in the range 3.5–6%, however, oxygen levels <2.5% often occur in human cancers. For instance, in a large head and neck cancer study of tumour hypoxia the median number of readings per tumour less than 0.33% oxygen was 19% [17]. The reason for reduced GNP uptake in hypoxic cells demonstrated in this study is currently not known. As expected, all cells grew more slowly in 0.1% oxygen than in air under otherwise identical culture conditions, due to a switch to inefficient anaerobic glycolytic metabolism in hypoxia (glycolysis generates 19 fold less ATP per molecule of glucose compared with oxidative phosphorylation) [18]. This reduced energy production may in part explain the reduced GNP uptake in hypoxic cells as receptor mediated endocytosis (RME), the considered mechanism of GNP cellular
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Gold nanoparticle hypoxic radiosensitisation
Fig. 4. Clonogenic radiation survival curves for MDA-MB-231 cells. (a) Control cells demonstrating the change in radiosensitivity with oxygen concentration. (b) Control cells and GNP exposed in 21% oxygen. (c) Control and GNP exposed cells in 1% oxygen. (d) Control and GNP exposed cells in 0.1% oxygen. Enhancement ratios of 1.41, 1.39 and 1.1 were noted for 21%, 1% and 0.1% oxygen, respectively. Survival curves are corrected for the effect of GNP exposure alone. All results are the mean of three independent experiments ± SE.
Table 1 Table of a, b, SF4, SER and values for MDA-MB-231 cells irradiated at three oxygen concentrations in the presence or absence of 1.9 nm GNPs (12 lM). Oxygen%
0.1 1 21
Condition
Control GNP Control GNP Control GNP
MDA-MB-231
p
a [Gy 1]
b [Gy
2
0.021 ± 0.018 0.126 ± 0.053 0.003 ± 0.025 0.107 ± 0.036 0.019 ± 0.025 0.091 ± 0.031
0.006 ± 0.002 0.002 ± 0.005 0.079 ± 0.007 0.098 ± 0.011 0.052 ± 0.007 0.093 ± 0.011
]
SF4
SER
MID
0.833 0.61 0.638 0.344 0.386 0.15
1.1
8.97 8.12 4.48 3.21 3.46 2.45
1.39 1.41
0.17 0.01 0.005
SF4 = surviving fraction at 4 Gy; SER = sensitizer enhancement ratio; MID = mean inactivation dose.
uptake, is known to be energy dependent, occurring at 37 °C but not at 4 °C [19]. Furthermore, it is known that increased levels of lactic acid produced as a product of anaerobic metabolism can alter cellular pH, potentially changing the aggregation potential of GNPs [20]. Hypoxia has been shown to reduce cellular uptake and efficacy of drugs including cisplatin, bleomycin and 5FU [21]. Possible mechanisms of treatment resistance include reduced free radical production leading to reduced DNA damage, reduced cell cycling, reduced apoptosis and clonal selection of resistant cells in hypoxic environments [21]. We observed significant radiosensitisation in MDA-MB-231 cells in moderate hypoxia (1% oxygen) with an SER of 1.39. This level of hypoxia is often observed in human tumours. However, the SER in near anoxia (0.1% oxygen) in this study was much lower
at 1.1, compared with 1.41 in 21% oxygen. It is likely that a reduction in GNP cellular uptake in hypoxia (47% reduction in 0.1% oxygen and 13% in 1% oxygen compared with 21% oxygen in MDA-MB-231 cells) contributes to the observed decrease in sensitisation. If the radiosensitisation occurs primarily due to physical effects then a reduction in GNP uptake would be expected to reduce the SER [6]. Similarly, if biological interactions with GNPs cause the sensitisation, a reduction in cellular uptake is likely to influence the magnitude of the observed effect. The reduced SER could also indicate that sensitisation depends on indirect free radical radiation effects which are responsible for much less radiation damage in near anoxia due to a lack of oxygen fixation. The mechanisms by which GNPs cause radiosensitisation are not fully established at present. Most commentators feel the
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primary mechanism is physical and should mainly be observed at kV photon energies due to high contrast material enhancing the photo-electric effect of kV photons. However, the results of biological studies published to date suggest that alternative processes may be important. For instance, GNPs have been shown to cause cytotoxicity, apoptosis, altered gene expression, cell cycle disruption and reactive oxygen species production and to potientiate the effect of radiomimetics such as bleomycin [8–10]. Furthermore, autophagy, is a lysosome-based degradative pathway which is essential for cellular homoeostasis [22,23]. A study demonstrated autophagy concomitant with oxidative stress in lung epithelial cells exposed to 20 nm citrate-coated GNPs in vitro [24]. A separate study reported GNP induced accumulation of autophagosomes and impairment of lysosomal degradation capacity due to alkalinisation of lysosomal pH [22]. Interference with autophagy has been shown to lead to radiosensitisation in many studies, including in MDA-MB-231 breast cancer cells in vitro [23,25]. No studies have examined the role of autophagy in GNPmediated radiosensitisation as yet and this will be the subject of future work. The field of nanomedicine has the potential to provide novel radiosensitisers for clinical use. However, challenges including nanotoxicity, cost, batch-to-batch variability and off-target effects will need to be overcome. Furthermore, this study demonstrates nanoparticle uptake is lower in hypoxic cells. In vivo, it is known that diffusion distances from tumour vasculature to chronically hypoxic cells can be large and it is likely novel mechanisms of drug delivery will be required to target these regions. Two studies have directly investigated nanoparticles in hypoxic cell models. A novel in vitro study utilized the phagocytic actions of blood monocytes to engulf 60 nm diameter gold nanoshells (GNS) and transport them to hypoxic tumour regions in 3D tumour models [26]. Treatment of the hypoxic cells with laser tuned to the resonance frequency of the GNS caused marked cytotoxicity compared to non-nanoparticle exposed cells. This novel approach could be used to target nanoparticles to regions of tumour hypoxia. An alternative approach used laser therapy and GNSs to induce mild hyperthermia in hypoxic tumour xenograft models to enhance the effects of a single 10 Gy dose of 125 kVp X-rays in mice [27].
Conclusions This work provides evidence of 1.9 nm GNP uptake in hypoxic cells in three cell lines. The rate of GNP uptake was lower in hypoxic conditions in all cell lines. GNPs inhibited cellular proliferation in hypoxic MDA-MB-231 cells, but had no significant effect on colony forming ability. Radiosensitisation by GNPs in MDA-MB-231 breast cancer cells was observed under moderate hypoxia, but was of a lower magnitude in near anoxia, and this may relate to reduced uptake of GNPs in hypoxic conditions. In vivo studies of GNPs are required to further assess uptake, toxicity and radiosensitisation.
Role of the funding source This work was supported by Men Against Cancer and Cancer Research UK (grant no. C1278/A990 to DGH and C1513/A7047 to KMP). The funding bodies had no role in the study design or the writing of the manuscript.
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