Inflicting Controlled Nonthermal Damage to Subcellular Structures by

Oct 5, 2010 - Martin Volk*,⊥. † Centre for Nanoscale Science, Department of Chemistry, Crown Street, University of Liverpool, Liverpool L69 7ZD,...
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Inflicting Controlled Nonthermal Damage to Subcellular Structures by Laser-Activated Gold Nanoparticles Zˇeljka Krpetic´,†,¶ Paola Nativo,†,¶ Violaine Se´e,‡ Ian A. Prior,§ Mathias Brust,† and Martin Volk*,⊥ †

Centre for Nanoscale Science, Department of Chemistry, Crown Street, University of Liverpool, Liverpool L69 7ZD, United Kingdom, ‡ Centre for Cell Imaging, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom, § The Physiological Laboratory, Institute of Cellular and Molecular Physiology, University of Liverpool, Liverpool L69 3BX, United Kingdom, and ⊥ Surface Science Research Centre, Department of Chemistry, University of Liverpool, Liverpool L69 7ZD, United Kingdom ABSTRACT We show that low-intensity laser irradiation of cancer cells containing endosomal gold nanoparticles leads to endosome rupture and escape of the nanoparticles into the cytosol without affecting the cells’ viability. The low light intensity of our experiments allows us to rule out photothermal effects as the underlying mechanism, and we present results that suggest photoinduced radicals as the photogenerated active species. This nonthermal mechanism may also be important in the context of cell death at higher laser intensities, which had been reported previously. KEYWORDS Gold nanoparticles, laser irradiation, photothermal effects, HeLa cells

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anotechnology increasingly contributes to the growing inventory of tools designed to selectively detect and/or kill cancer cells.1-6 Here we introduce a laser method to deliberately damage subcellular structures in the presence of intracellular gold nanoparticles. Depending on the power density of the laser and the exposure time, the extent of damage can be controlled from producing highly localized surgical lesions in healthy cells to indiscriminate widespread obliteration of cellular structure and cell death. This high level of control suggests the possibility of developing metal nanoparticles that can operate as laser-triggered nanoscalpels for intracellular surgery. Notably, it can be demonstrated that over the range of laser power densities employed thermal effects are negligibly small. This does not only imply a new, nanoparticle-mediated nonthermal mechanism of inflicting damage to the cell by laser exposure, but may also necessitate a wider interpretation of a sizable body of previous research conducted with higher laser power densities where the damage was habitually attributed solely to the plasmonic heating that occurs under such conditions. The original motivation for this study was to examine the damage caused to cells by laser exposure in the presence of strongly light absorbing intracellular gold nanoparticles at the level of subcellular structures. This was inspired chiefly by the work of El-Sayed and colleagues on the development

of plasmonic photothermal cancer therapy, which has demonstrated the possibility of adjusting the laser intensity so that cells are only killed if they are closely associated with gold nanoparticles or nanorods.7-11 The precise nature of the damage caused by this treatment has not been studied in microscopic detail. To address this lack of knowledge, we have chosen a well-known human epithelial cervical cancer cell line (HeLa) as an experimental model system for which the endocytosis and subsequent endosomal confinement of citrate-stabilized gold nanoparticles has been established.12 Cell incubation with citrate-coated gold nanoparticles leads to their exclusive uptake into endosomes.12 First, we carried out a set of exploratory experiments to ensure that laser exposure would lead to cell death only in the presence of intracellular gold nanoparticles. For this purpose, we exposed the cells to radiation from a continuous argon ion laser with a wavelength of 514 nm, close to the plasmon absorption band of the gold nanoparticles (λ ) 525 nm), and subsequently tested cell viability by a standard Trypan blue exclusion assay carried out immediately after laser exposure (see Supporting Information for experimental details). The loading of the cells with ca. 15 nm gold nanoparticles was kept constant in all experiments by always adhering to the same conditions during the uptake of the particles that typically produce an average content of about 35 000 particles per cell.12 Figure 1a,b shows that at a laser power density of 55 Wcm-2 and an exposure time of 2 min virtually all cells pretreated with gold nanoparticles are killed as indicated by the blue color, whereas untreated cells survive the exposure. A typical TEM image of dead cells

* To whom correspondence should be addressed. Email: [email protected]. Ph: +44 151 794 3317. Fax: +44 151 794 3870. ¶ These authors contributed equally to this work. Received for review: 07/20/2010 Published on Web: 10/05/2010

© 2010 American Chemical Society

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FIGURE 1. Cell viability. (a,b) Comparative light microscopy images of HeLa cells irradiated at 55 Wcm-2 for 2 min (a) without and (b) with internalized gold nanoparticles. Blue color indicates dead cells (Trypan blue test); (c) TEM image of a representative cell with internalized gold nanoparticles after laser exposure at 55 Wcm-2 for 2 min showing complete obliteration of cellular structure (scale bar is 5 µm). (d-i) Time-lapse imaging of HeLa cells loaded with nanoparticles that were not irradiated (d,e), irradiated with 20 Wcm-2 for 1 min (f,g), or with 55 Wcm-2 for 2 min (h,i). After irradiation, cells were stained with Annexin V (green) and propidium Iodide (red) to monitor both apoptotic and necrotic cell death. Representative images at time 1 h (d,f,h) and 35 h (e,g,i) after irradiation are shown. The scale bar is 100 µm.

after the same laser exposure is shown in Figure 1c, indicating that under such conditions most intracellular structures are completely obliterated; the gold nanoparticles seem well dispersed throughout the cell, and thus are hard to locate and count. These results are representative for a range of different exposure times and intensities. Power densities as low as 30 Wcm-2 gave qualitatively similar results with only a slightly increased number of surviving cells. Under the same irradiation conditions, the cells always remained alive and appeared undamaged in the absence of gold nanoparticles. This indicates that there is a large window of conditions for the selective elimination of cells that contain gold nanoparticles. At lower power densities (20 Wcm-2) practically all cells survive the laser exposure even in the presence of gold nanoparticles. We further demonstrated that no significant delayed cell death was triggered by this low power laser irradiation in the presence of nanoparticles by live cell © 2010 American Chemical Society

imaging for up to 35 h after laser irradiation, using Annexin V and propidium Iodide to monitor both apoptotic and necrotic cell death (see Supporting Information for experimental details). Figure 1d-g shows that no significant apoptosis or necrosis was detected upon low-power laser irradiation compared to control nonirradiated cells (2% apoptosis and 5% necrosis in control versus 5% apoptosis and 4% necrosis after laser irradiation with 20 Wcm-2 for one minute), whereas high-power laser irradiation for 2 min instantaneously killed and fixed the cells, Figure 1h,i. Besides, after low laser irradiation the cells divided throughout the time-lapse experiment (see Movie1 in the Supporting Information), indicating that they were healthy. This is crucial for the future use of the technique in biological applications and proves that there are no long-term effects of the low level irradiation on cell fate. To assess the localized physical damage inflicted by this treatment the cells were subsequently inspected by TEM. In 4550

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FIGURE 2. Laser inflicted subcellular damage. TEM micrographs of gold nanoparticles in HeLa cells before (a) and after (b-f) laser irradiation showing examples of the damage to the endosomes; (b-d) dissolution of the membrane of endosomes filled with fewer particles (red arrows) while endosomes filled with more particles tend to remain intact or suffer only minor damage (blue arrows); (e,f) escape of nanoparticles into the cytosol after surgical rupture of the endosomal membrane. Laser power density and exposure time: (b) 6 Wcm-2, 7 min; (c,f) 20 Wcm-2, 1 min; (d) 20 Wcm-2, 2 min; (e) 6 Wcm-2, 3 min. Scale bars are 500 nm.

the absence of laser irradiation, no endosomal escape occurred and no nanoparticles were found in the cytosol; this was explicitly tested by analyzing nonirradiated control areas on the dishes used for irradiation experiments, see Figure 2a and Figure S7 in the Supporting Information. The images shown in Figure 2b-f are representative for the type of intracellular damage typically observed after low level irradiation. All visible damage is limited to the endosomes, which under the conditions of these experiments are the only organelles that contain gold nanoparticles. Careful inspection reveals in many cases surgical damage to the endosomal membrane, allowing particles to escape into the cytosol, Figure 2e,f. Alternatively, the membrane may disintegrate completely, leaving behind only the formerly enclosed particles and a shadow of debris, Figure 2d. From viewing hundreds of similar images a clear trend emerges © 2010 American Chemical Society

by which the degree of damage to the endosomal membrane increases with decreasing particle loading of the endosome. Multilamellar late endosomes with massive particle loadings, for example, often survive the laser treatment intact, or suffer only minor damage, under conditions where smaller endosomes are fully dissolved. Examples are shown in Figure 2b-d, all of which show these two effects occurring in close vicinity and thus under identical irradiation conditions. In addition to the representative images shown in Figure 2, a gallery of further examples of the different damage features discussed is provided in the Supporting Information. What is the underlying physical basis of the phenomena observed here? A careful look at the recent literature reveals that a number of plasmonic heating experiments, both for photothermal imaging and manipulation, have been re4551

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after turning on a laser beam with a power density of 20 Wcm-2 and a diameter of 1.55 mm; even after 2 min of irradiation the temperature has increased by less than 1.5 °C. The use of a larger beam diameter results in a slightly higher temperature increase (Figure S3 in the Supporting Information), but for all experiments yielding only endosomal rupture, that is, those using irradiation for one minute at a power density of not more than 20 Wcm-2, the temperature increased by at most 3 °C, and endosomal damage and escape of nanoparticles into the cytosol were also observed under conditions yielding less than 1 °C temperature increase. The maximum temperature calculated for experiments leading to cell death (2 min of irradiation with a power density of 55 Wcm-2 and a beam diameter of 1.55 mm) is 4 °C (Figure S3 in the Supporting Information). Irradiation was performed at ambient temperatures (22 °C), so that any temperature increase by less than 15 °C can be safely concluded to not have any thermal effects on the cells, since cells are cultured at 37 °C, allowing us to exclude unequivocally that such effects are responsible for the damage observed in our experiments. These results are in contrast with previous reports on photothermal treatment of cancer cells. In particular, ElSayed and co-workers showed sample heating by up to ∼100 °C for an incident power density of 50 Wcm-2.8 However, the cell cultures used in these experiments contained significantly higher concentrations of nanoparticles than in our experiments, resulting in conditions where up to 25% of the incident light is absorbed in the sample, whereas in our experiments, only ∼0.5% of the incident laser light is absorbed (see Supporting Information for a detailed discussion), which necessarily reduces the temperature increase. It is interesting to note that El-Sayed and coworkers observed cell death only under conditions where the temperature is raised to more than 70 °C by laser irradiation, corresponding to the temperature at which thermal destruction by oven heating occurs, supporting the notion of a photothermal mechanism.8 This is in contrast to our observations, which show cell death under conditions of heating by only a few degrees, ruling out a photothermal mechanism. One may speculate that this difference could arise from the different locations of the nanoparticles in these experiments; whereas in our experiments, the nanoparticles are localized within the cells, El-Sayed and coworkers attached nanoparticles to the cell surface only, and this may have prevented cell death by the mechanism active in our experiments where nanoparticles have direct access to the cell interior. A plausible suggestion for this mechanism may be the involvement of photochemical effects. Free radicals and singlet oxygen are well-known to cause significant damage to subcellular structures, in particular to membranes, which is the basis of photodynamic therapy.27,28 The rupture of the endosomal membrane by photochemically generated reactive oxygen species (ROS) in the presence of an appropriate

FIGURE 3. Heating of sample. (Main) Time dependence of the overall (background) temperature increase in the HeLa cell monolayer at the center of a laser beam with 20 Wcm-2 power density and 1.55 mm diameter after turning on irradiation, calculated from finite element simulations; (inset) local heating above the background temperature around an endosome of 400 µm diameter containing 750 nanoparticles, the dashed line indicates the endosome surface; see Supporting Information for details.

ported that at first sight look similar to our experiments but differ significantly in terms of the conditions of laser exposure.6-11,13-22 A critical parameter that varies over orders of magnitude between different reports is the power density of the laser employed, that is, the amount of energy that the laser delivers per time and area. In particular, pulsed lasers are able to deliver far more energy in a short time frame than continuous ones and are usually employed when the aim is to achieve significant localized heating of the nanoparticles. Other important parameters are size, shape, and concentration of the nanoparticles, which influence significantly the value and wavelength dependence of their absorption coefficient. We carefully investigated the amount of localized heating in the immediate vicinity of irradiated nanoparticles as well as within nanoparticle-containing endosomes and conclude that under our experimental conditions the localized temperature increase above the background temperature is limited to fractions of a degree, see the inset to Figure 3 as well as Figures S1 and S2 in the Supporting Information. This contrasts with reports of local heating by several degrees Celsius near gold nanoparticles under cw-illumination.23-25 It has to be noted that these other studies were performed under conditions where the laser beam was tightly focused, normally close to the diffraction limit, yielding illumination intensities on the order of 105-106 Wcm-2, that is, several orders of magnitude higher than those used here. Our results of negligible local heating at a single NP by cw-illumination are also in full agreement with the results of a recent theoretical investigation.26 We also calculated the rise of the overall background temperature in the cell culture due to the deposition of energy, using finite-element simulations, details of which are given in the Supporting Information. Figure 3 shows the time dependence of the temperature increase in the cell layer © 2010 American Chemical Society

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FIGURE 4. Confocal microscopy: laser exposure causes oxidative stress. Reactive oxygen species (ROS) monitored by the appearance of green fluorescence; (a) HeLa cells incubated with gold nanoparticles after laser irradiation; (b) same as (a) but in the absence of gold nanoparticles; (c) same as (a) but without laser irradiation; and (d) control experiment without gold nanoparticles and without laser irradiation. Laser power density in (a) and (b) was 20 Wcm-2 for 1 min exposure time. Scale bars are 20 µm. (e) Quantitative comparison of ROS generation in panels a-d.

dye has very recently been reported by Febvay et al.29 In our system, we also detected a significantly increased amount of ROS in the cells, even 20-30 min after laser exposure, as shown in Figure 4. Thus, we suggest as one potential mechanism for the observed effects the production of free radicals by laser irradiation of gold nanoparticles, possibly as a consequence of plasmon-assisted photoemission of electrons. For comparison, we repeated some experiments with platinum nanoparticles that do not have a plasmon band near the laser wavelength, and no detectable damage was inflicted on the cells (see Supporting Information). Our interpretation is also consistent with the observation that larger agglomerations of nanoparticles appear to cause less damage than smaller ones. The more particles are present within a volume fraction the higher will be the proportion of photoelectrons that are reabsorbed and radicals that are quenched by the gold. This type of photoemission is quite an unusual phenomenon, albeit not unprecedented,30-32 and there is clearly an arising need for more research. While in vacuum green light could not cause photoemission of electrons from a gold surface, in electrolyte solution the Fermi level of the particles freely varies with charge so that the energy threshold for photoemission can be significantly lowered. In addition, the electron is not emitted to vacuum level but into an energetically lower state such as the conduction band of the electrolyte solution. This has been described first by Brodsky and Pleskov30 for bulk metals in contact with an electrolyte solution and by Henglein and co-workers31,32 for metal colloids. It has to be noted that the ROS detected after the low level laser irradiation are not responsible for delayed apoptosis (Figure 1). Although a high level of ROS has been described to induce cell death in some cellular systems,33 there is now growing evidence that ROS can also mediate intracellular signaling and regulate proliferation or differentiation depending on their subcellular localization and levels.34 © 2010 American Chemical Society

In summary, we have demonstrated that exposure of HeLa cells to continuous green laser light in the presence of suitably light absorbing intracellular gold nanoparticles can cause cell death at higher laser power densities and damage to endosomes at lower power densities, both in the absence of significant thermal effects. By further tuning down the laser power and carefully controlling the exposure time, highly selective localized damage can be inflicted to the endosomal membrane. It should be possible to exploit this effect for more controlled intracellular surgery using single gold nanoparticles associated with the targeted subcellular features of interest. Our observations also have implications for a body of important previous and ongoing work that employs plasmonic heating for a range of purposes. This includes photothermally induced release,18 phase transitions,15 DNA melting16,17 and hyperthermia.6-11 While all these phenomena involve higher laser power densities or absorber concentrations to achieve heating, they may also be affected by the hitherto unconsidered photochemical effects discussed here. The present contribution does not answer the question to what extent previous interpretations of such experiments may have to be revisited, but it highlights the need for more research to elucidate the precise nature and role of photochemical processes that occur either without or alongside heating when intracellular metal nanoparticles are exposed to laser light at or near plasmon resonance conditions. Acknowledgment. The authors thank Professor David Schiffrin, Professor Dr. Carsten So¨nnichsen, Dr. Raphael Le´vy, and Dr. David Spiller for helpful discussions. Z.K., P.N., and M.B. acknowledge financial support from the European Union (NACARDIO). V.S. holds a BBSRC David Phillips Research Fellowship (BB/C520471/1). The argon ion laser was on loan from the EPSRC-funded laser loan pool at the STFC Central Laser Facility. 4553

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Supporting Information Available. Experimental details; estimate of laser-irradiation induced temperature increase; irradiation of HeLa cells incubated with Pt-citrate nanoparticles; representative images showing endosomal gold nanoparticles in unirradiated HeLa cells; gallery of representative images showing three different phenomena that are typically observed in parallel in all samples of HeLa cells containing endosomal gold nanoparticles after irradiation at lower laser power densities [(i) surgical damage (rupture) of endosomal membrane, (ii) complete dissolution of endosomal membrane, and (iii) preferential damage to endosomes filled with smaller amounts of gold nanoparticles]; time-lapse movie of HeLa cells for 35 h after low laser irradiation. This material is available free of charge via the Internet at http://pubs. acs.org.

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