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lOP PUBLISHING NAracrrociiNctoor Nano ethnology 21(20I0) 085102 ( IOpp) doi: I O. IORR/0957-1484/21/8/085102 Tunable plasmonic nanobubbles for cell theranostics E Y Lukianova-HlebI, E Y Hanna', .1 H Harner3 and D 0 Lapotko" Laboratory for Laser Cytotechnologies, A V Lykov Heat and Mass Transfer Institute. 15 Brovka Street, Minsk. 220072. Belarus Department of Head and Neck Surgery. The University of Texas M 13 Anderson Cancer Center. 1515 Holcombe Boulevard, Houston, TX, 77030. USA 3 Department of Physics and Astronomy. Rice University, 6100 Main Street. Houston. TX 77005, USA E-mail: Received 9 December 2009, in final form 28 December 2009 Published 25 January 2010 Online at stacks.iop.org/Nano/21/085102 Abstract Combining diagnostic and therapeutic processes into one (theranostics) and improving their selectivity to the cellular level may offer significant benefits in various research and disease systems and currently is not supported with efficient methods and agents. We have developed a novel method based on the gold nanoparticle-generated transient photothermal vapor nanobubbles, that we refer to as plasmonic nanobubbles (PNB). After delivery and clusterization of the gold nanoparticles (NP) to the target cells the intracellular PNBs were optically generated and controlled through the laser fluence. The PNB action was tuned in individual living cells from non-invasive high-sensitive imaging at lower fluence to disruption of the cellular membrane at higher fluence. We have achieved non-invasive 50-fold amplification of the optical scattering amplitude with the PNBs (relative to that of NPs), selective mechanical and fast damage to specific cells with bigger PNBs, and optical guidance of the damage through the damage-specific signals of the bubbles. Thus the PNBs acted as tunable theranostic agents at the cellular level and in one process that have supported diagnosis, therapy and guidance of the therapy. 1. Introduction functional agents due to their unique properties, such as the plasmon resonances of noble metal NPs, and without chemical Combining diagnosis and therapy in one process is an loads. Plasmon resonances can be activated optically and emerging biomedical method referred to as theranostics [1, 2]. convert incident light into scattered (optical) and absorbed A distinct goal of theranostics is to selectively target specific (thermal) components with the potential for diagnostic and (diseased) tissues or cells to increase diagnostic and therapeutic therapeutic applications. accuracy. The major promise of theranostics is to bring So far plasmon resonant NPs have demonstrated excellent together key stages of a medical treatment, such as the biocompatibility [6], optical diagnostic [7-10] and therapeutic diagnosis and therapy, and thus to make a treatment shorter, potentials [7, 9, 11, 12]. However, background scattering safer and more efficient. However, this goal requires by cells and tissues often dominates the scattering signal, adequate tools with a high multi-functionality and selectivity. resulting in the low sensitivity of NP-based imaging and The initial phase of the development of theranostics has diagnostic methods. This was improved with photothermal already revealed the two general challenges: lack of multi- (PT) techniques [13], but required higher laser-induced functional methods and agents, and the lack of selectivity and temperatures that can be thermally detrimental to cells and specificity of available agents (that ultimately requires cell and molecules. Furthermore, the therapeutic applications of molecular levels). Several theranostic methods have employed plasmon resonant NPs also employ PT effects such as nanoparticles (NPs) as the carriers of diagnostic agents and hypothermia [7, 9, 12] and pressure waves [14] for inactivation drugs [3-5]. However, NPs themselves may also act as multi- of molecular and cellular targets. However, these are macro- 0957-44184!10/085102+10530.00 20I0 top Publishing Ltd hinted in the UK EFTA01113062 Nanotcchnology 21(2010)085102 E Y Lukianova-Hleb a al pump probe law laser 111 rfrfr 4a+ b Figure I. PNB cell theranostic with multi-stage tunable PNB: (a) cell is targeted with NP-antibody conjugates and intracellular NP clusters are formed through the receptor-mediated endocytosis. (b) the 1st (diagnostic) PNB provides the data on a cell and allows one to determine the parameters of the next laser pulse. (c) the 2nd PNB delivers mechanical impact (cell damage though membrane disruption is shown) and this action is guided through the increased optical scattering (red arrows) of the 2nd PNB; the PNB is tuned by varying the fluence of the pump pulse (green arrows). rather than nanoscale effects that cannot be localized and through the mechanisms of antibody—antigen interaction and precisely controlled within a single cell. All this, together with endocytosis (figure 1(a)). Remote (optical) and non-invasive the challenges of NP delivery, poses significant limitations to activation and sensing of PNBs around such intracellular combining accurate diagnosis and targeted therapy in a single clusters was realized in individual living cells with free laser and fast nanometer-scale process. beams. We hypothesized that a combination of the photothermal When activated by a laser pulse, an intracellular plasmonic properties of plasmonic nanoparticles with those of the nanoparticle (figure 1(b)) acts a heat source and generates transient vapor bubbles may be a key solution of the above a transient PNB in the surrounding medium. PNBs of problems through the development of a tunable nanoscale nanometer-scale size and nanosecond-scale duration act as theranostic probe that is not a nanoparticle but a nanoparticle- diagnostic probes by scattering light from the probe laser. generated event—the plasmonic nanobubble (PNB), which Larger micrometer-scale PNBs provide a localized therapeutic combines high optical brightness with localized mechanical action through a mechanical (non-thermal) impact due to impact. In the current work we have studied the optical their rapid expansion and collapse, thus disrupting the cell generation and detection of PNBs around gold nanoparticles membrane(see figure 1(c)). Optical monitoring of the in individual living cells, with the focus on tuning the PNB disruptive PNBs can guide their therapeutic action. Thus properties in one cell and evaluating the multi-functionality of the PNBs may combine diagnostics, therapy, and therapy the PNB. guidance. Despite the extensive studies of the photothermal properties of nanoparticles [7, IS, 16] the generation of photothermal vapor bubbles around them remains an 2. Materials and methods under-recognized phenomenon. Although laser-induced 2.1. Principle ofPNB theranostics macro-bubbles have been studied for various biomedical tasks [17], the studies of bubbles around optically excited Cell theranostics employs a tunable and transient probe, and nanoparticles [18] at the nanometer scale [19] are rather a vapor bubble (figure 1) is generated with a short laser pulse scarce. Furthermore, the PNBs differ appreciably from the around plasmon resonant gold nanoparticles (NP), which we macro-bubbles, where the threshold laser fluence for bubble refer to as a plasmonic nanobubble. The PNB is a system that generation increases with the size of the absorber [20]. results from the interaction of optical radiation with a NP and For nanoparticles the reverse is true: the bigger the size its environment. of the plasmonic nanoparticles, the lower the laser fluence The optical and mechanical properties of PNB depend threshold for bubble generation [21-23]. In addition, PNBs upon its diameter (tunable in the range of from 50 nm to concentrate the laser-induced thermal impact around the 50 µm) and lifetime (tunable in the range of from 10 ns nanoparticles, unlike macro-bubbles [21, 22]. To improve to 10 µs). The short lifetime of PNBs makes them highly the selectivity of the PNB generation we have targeted cells transient phenomena that exist on demand. For target- with relatively small functionalized NPs (which can enter cells specific generation of the PNBs we have selectively formed unlike larger absorbers) that formed intracellular NP clusters clusters of relatively safe gold NPs around molecular targets due to receptor-mediated endocytosis [24, 25]. At a set in cancer cells. Gold NPs, conjugated to diagnosis-specific laser fluence, the nanoparticle cluster will generate a PNB, antibodies have been delivered and aggregated into NP clusters where individual nanoparticles will not due to their smaller EFTA01113063 Nanotechnology 21(2010)085102 E Y Lukianova-Hleb eta! size [21, 24, 251. Thus, PNBs can be selectively generated in specific cells, do not use chemicals, and rely only on Long laser pulse nanometer-scale phenomena of light and heat that are natural 532 nm.10 ns for all living systems. Short laser pulse *OM■ 532 nm.0.5 ns Mi 2.2. Generation ofPNBs Cell PNB generation was experimentally realized by using laser PNB pulse-heated intracellular gold NPs. Evaporation of the Pulsed probe laser medium around a nanoparticle (NP) involves several processes: 690nm.0.5 ns NP laser pulse-induced thermalization of a NP occurs in I ps [16]: thermal diffusion from a NP to adjoining medium forms a Sample Probe laser chamber thin vapor layer around a NP; finally, the PNB begins to 633 nm expand to a maximal diameter and then collapses. The bubble lifetime may be considered as proportional to its maximal r diameter [17, 19, 20, 26, 271. The minimal fluence of a single laser pulse that provides bubble generation is defined as the PNB threshold fluence. PNBs were generated around CCD 50 nm gold spheres and in individual living cells. The pulse wavelength (532 nm) and duration (0.5 ns) were chosen so to provide maximal localization of the released heat and at Photodetector the same time to avoid the generation of shock and pressure waves. If the localization of a photothermal (PT) impact is Figure 2. Experimental setup: single gold NPs in water or individual required, there should be no pressure and shock waves, and cells in the sample chamber were mounted on the stage of an inverted also the thermal diffusion losses should be minimized. When optical microscope: PNB generation was provided by focused single the optical pulse duration exceeds the acoustic relaxation time, pulses (532 nm. 0.5 ns): a pulsed probe laser (690 nm. 0.5 ns) no pressure or shock wave would emerge. Next, when the provided time-resolved optical scattering imaging of the PNB and a optical pulse duration is less than the thermal relaxation time, continuous probe laser (633 nm. I mW) provided monitoring of the optical scattering of PNBs though their time responses. An the losses due to thermal diffusion are negligible, and the entire additional pulsed laser (532 nm. 10 ns, I ml cm-2) was used for heat released is concentrated in a small volume around the heat excitation of fluorescence in the cells. source. In our work we have employed a pulse of length 0.5 ns, 532 nm (STA-01 SH, Standa Ltd, Vilnius). The pump laser beam was directed into the illumination path of an inverted directed to the sample collinearly with the probe pulse and its optical microscope and was focused into the sample. Single axial intensity was monitored by a high-speed photodetector cells or NPs and single events were studied (figure 2). (PDBIIOAC, Thorlabs Inc.). The time response mode allowed measurement of the PNB lifetime that characterizes a maximal 2.3. Detection and imaging of the PNBs diameter of the bubble and thus allows one to quantify its therapeutic impact. Image and response modes were used PNB detection has been realized with two optical methods that simultaneously, thus combining the imaging and measuring of take advantage of the excellent optical scattering properties of the lifetime (figure 2). bubbles [281. These methods were applied earlier by us for the imaging of the photothermal phenomena in living cells with the 2.4. Cells pump-probe laser microscope that we have developed [29, 301. The time-resolved imaging of NPs and PNBs was realized For the in vitro experimental model we have used gold spheres by using side illumination of the sample with a custom made of 50 nm and their conjugates with anti-epidermal growth pulsed probe dye laser beam (0.5 ns) at a wavelength 690 nm factor receptor (EGFR) antibody C225 that were obtained from and with a tunable time delay of 1-10 ns relative to the pump Nanopartz Inc (Salt Lake City, UT). The cells were prepared pulse (figure 2). The scattered by NP or by PNB probe as the monolayers of living EGFR-positive lung carcinoma radiation was imaged with the digital camera (Luka, Andor cells (A549) that were grown into standard 9 mm culture Technologies, Ireland). For quantitative analysis of the optical wells (0024765, Molecular Probes, Inc., Eugene, OR). All amplification by the PNB we have introduced the relative cells were incubated with NPs for 30 min at 37 °C. The scattering amplitude Kx(t) = 11(t) — Mal (0) — 41 that concentration of the NPs during the incubation was adjusted to describes the pixel image amplitude 1(t) of optical scattering 0.9 x 1011 m1-1. NP-C225 conjugates were selectively coupled by a PNB relative to that by a NP, 1(0) (4 is the average with EGFR. This provided a maximal relative concentration of pixel image amplitude of the background). While allowing NPs at the cellular membrane of cancer cells. Secondly, during one to `see' the PNB the pulsed imaging can hardly provide receptor-mediated endocytosis the NPs were internalized and kinetic measurement. The latter was realized in a time response concentrated into clusters of closely packed NPs in endosomal mode. An additional continuous probe beam (633 nm) was compartments. At the end of the incubation procedure large 3 EFTA01113064 Nanotechnology 21 (2010)085102 E Y Lukianova-Hleb eta! V TARGETING \41/0 (a) 0 DIAGNOSTICS N.• (b) TREATMENT and GUIDANCE (c) Figure 3. Targeting the cell with gold NPs (a) and optical generation and detection of the intracellular PNBs: the 1st one non-invasively amplifies optical scattering (b), while increasing the fluence of the pump laser pulse induces the 2nd PNB that mechanically damages the cell (c); I—stages of the PNB theranostic action, II—optical pulsed scattering images of one cell with the membrane border shown with a white line, Ill-o ptical time response of the PNB shows its lifetime, IV—bright field and V—fluorescent (ethidium bromide-specific) images of the cell show it before (a) and after the generation of the 1st (b) and the 2nd (c) PNBs. NP clusters were formed only in those cells with high initial cells were targeted with conjugates of 50 nm gold spheres levels of membrane-bounded NPs, i.e. the target cells, as we to anti-epidermal growth factor receptor antibody C225 and have shown earlier [24, 251. then were exposed in vitro to a single pump laser pulse at Cell viability was evaluated optically with two standard a wavelength near the nanoparticle plasmon resonance peak microscopy techniques. First, a bright field image was obtained (0.5 ns, 532 nm). Optical scattering of the pulsed probe for the cell before and after its exposure to a single pump pulse beam (690 nm) by the gold NPs and by the PNBs in the cells and the difference of these two images was used to detect any was measured as an image pixel amplitude (figure 3-11). Also, PNB-induced changes of the shape of the cell, in particular, the lifetime of the PNB was measured as the duration of a emerging of the blebbing bodies. Blebbing bodies may develop PNB-specific time response that was simultaneously obtained in the cells with damaged cytoskeleton and even with an intact (figure 3-III). We have monitored the damage to the individual membrane. Second, the membrane damage by the PNB was cells after their exposure to the laser pulse by fluorescent detected using a standard fluorescent method by monitoring the imaging of the uptake of the ethidium bromide (that stains cellular uptake of ethidium bromide (EtBr) dye that enters only cells with a disrupted membrane) and the blebbing (that is the cells with a compromised membrane. Fluorescent images associated with the cytoskeleton damage). Scattering by were obtained for each cell before and after PNB generation. gold NPs accumulated by individual A549 cells after 30 min Though these methods did not provide monitoring of the long- incubation at 37 °C (figure 3(a)-II) was found to be quite low term viability, they could be applied on site and to specific and its image amplitudes were close to the scattering image individual cells during the generation of the PNBs and without amplitudes associated with cellular organelles. We have used removing the cells from the sample chamber. Individual cells the NP scattering image as a reference for quantifying the (60 in each population) were irradiated with single laser pulses amplification of optical scattering by the PNBs. The first at 532 nm, which is close to the peak of the maximum of the pump laser pulse was applied to individual cells at a fluence plasmon resonance of the gold NPs. The laser beam diameter of 0.24 .1 cm-2 (above the bubble generation threshold), which was 14 µm in the sample plane to provide the exposure of the induced a PNB within the cell, as was detected with the whole cell. Thus the single events were analyzed in individual probe laser image (figure 3(b)-II). The lifetime of this PNB cells. Each experiment was repeated from 3 to 5 times with a was relatively short, 25 ns, according to its time response newly grown cell population. (figure 3(b)-III). This PNB has amplified the scattering by 9.2 times relative to the scattering by the gold NPs. After the PNB generation bright field (figure 3(b)-IV) and fluorescent 3. Results (figure 3(b)-V) microscopy images of the cell showed no deviation from the pre-pulse conditions shown in figure 3(a)- 3.1. Gold NP-generated PNBs in living cells IV, V, respectively. The absence of fluorescence and blebbing Generation and detection of tunable PNBs in living cells was implies that the cell has survived the laser pulse and the PNB. studied in individual living A549 lung carcinoma cells. The We have detected only one PNB despite the apparent fact that 4 EFTA01113065 Nanotochnology 21(2010)085102 E Y Lukianova-Hleb eta! Table I. Parameters of the PNBs and intracellular photothennal bubbles. Experimental Process and NP-treated Intact cells (cell chromophore-generated parameters cell state cells (PNB) photothermal bubbles) Pump laser (532 nm, 0.5 ns) PNB generation 0.09 ± 0.03 2.72 ± 1.8 threshold fluence (J cm2) Cell damage 1.0± 0.75 2.72± 1.8 PNB lifetime (ns) Surviving cells 44± 17 nia Damaged cells 213± 100 145 th 50 below the PNB generation threshold for the smaller NP clusters 1,0 or single NPs. This result has demonstrated high specificity 0,8 of the PNB generation compared to the specificity of the nanoparticle imaging (figure 3(a)-II). The sensitivity of PNB 0,6 diagnosis versus NP diagnosis is clearly seen by comparing C 0,4 ci figure 3(a)-II with (b)-II: under identical imaging conditions a- the amplitude of the NP scattering was much lower than that for 0,2 the PNB scattering so it did not produce any detectable image. Next, the second laser pulse was shortly applied to the ,0 0,1 1 100 same cell at the increased fluence of 1.76 J cm-2. The second Laser pulse finance (J/cm2) PNB (figure 3(c)-II) was much brighter with its scattering b amplitude being amplified 290 times relative to that of the NPs and was also much longer (figure 3(c)-III) than the 1st PNB. C Within 30-60 s after the PNB generation the fluorescent image has shown the penetration of the dye inside the cell (figure 3(c)- = 100 V) and the bright field image has shown the formation of the blebbing bodies (figure 3(c)-IV). These have indicated the disruption of the cellular membrane and, possibly, of the CO cytoskeleton. This experiment has demonstrated the ability to CO tune the intracellular PNB by varying the laser fluence from 0,1 non-invasive imaging (with an almost 10-fold improvement in Laser pulse fluence (J/cm2) optical scattering signal) to cell disruption. C 75- 3.2. PNB and cell damage We have studied the cell-damaging properties of the PNBs by varying the laser pulse fluence so as to analyze the probability of bubble generation and the probability of cell damage among YS 25- intact (untreated) and NP-treated cells. Each single cell in the population was irradiated with a single laser pulse of 0 specific fluence and then the cell population-averaged values 0 2.5 50 75 100 125 were obtained (figure 4(a)). NP treatment has lowered the Bubble lifetime (ns) threshold laser fluence for the bubble generation by almost 30 Figure 4. Influence of the fluence of a single pump laser pulse (532 times relative to the intact cells (figure 4(a), table 1). As a mu. 0.5 ns) on the PNB parameters and on the damage as measured function of pulse laser fluence, the probabilities of cell damage in individual A549 cells: (a) PNB generation probability (PRB): and of the bubble generation coincided for intact cells, but were (4} —cells incubated with NP-C225 conjugates. (•)—intact cells; significantly separated in the NP-treated cells (figure 4(a)). At cell damage probability (PD): (0)—cells incubated with NP-C225 conjugates. (o)—intact (b) PNB lifetime: (*)—cells incubated pulse fluences of 0.06-0.22 J cm-2, intracellular PNBs were with NP-C225 conjugates. (•)—intact cells; (c) amplification of generated in NP-treated cells without damaging the majority of optical scattering amplitude by the PNB (relatively to gold NPs) in the host cells (figure 4(a)), while the same cells were damaged the NP-treated cells as function of the PNB lifetime (i.e. maximal at 10 times higher fluences (table 1). size of the PNB). We have simultaneously measured the lifetime of the PNB in each irradiated cell as a function of the laser pulse fluence. For relatively small PNBs their lifetime is nearly endocytosis assumes the internalization of many NPs. This proportional to their maximal diameter [17, 19, 20, 26, 271 can be explained with the threshold nature of the PNB: the and was used as its measure. The PNB lifetime has increased fluence level was sufficient for the generation of the PNB only linearly with the laser fluence in the NP-treated and in the around the biggest clusters of NPs, while this fluence was intact cells (figure 4(b)). The lifetime of the non-invasive 5 EFTA01113066 Nanotochnology 21(2010)085102 E Y Lukianova-Hlob era! PNBs was found to be about 5 times shorter than that of the The optical parameters of the damaging PNBs differ damaging ones (table 1). This implies a similar difference in significantly from those of the non-invasive PNB: the lifetime the maximal diameters of non-invasive and damaging PNBs. was several times longer (table I) and the image pixel We consider that the maximal diameter of the PNB plays the amplitude was 10-50 times higher. This may allow direct major role in the cell damage: small PNBs do not damage the and simultaneous guidance of the cell damage due to the cell, while the increase of the PNB size by several times has PNB and without additional techniques. The detection of any induced almost immediate disruption of the cellular membrane intracellular PNB with the image amplitude and/or lifetime and skeleton, as was revealed with fluorescent (membrane being above specific thresholds can be considered as a sign of damage—see figure 3(c)-V) and bright field (appearance of cell damage since these PNB parameters have correlated to the the blebbing bodies—see figure 3(c)-IV) microscopy. The damage-related phenomena observed independently with the assumption about the mechanical damage mechanism is in standard methods. line with our recent data on the thermal insulating effect of PNBs [21, 22] (that have proved that, unlike laser-heated 11 PNBs as optical probes NPs, PNBs do not deliver significant thermal impact to their environment) and with independent data for opto- and Finally, we have evaluated the sensitivity and specificity of sonoporation of the cells [31-38]. The latter studies have the PNBs as imaging probes. We have measured the optical used ultrasound and optical breakdown mechanisms to induce scattering amplification effect of small (non-invasive) PNBs as vapor (cavitation) bubbles at cell membranes and have reported a function of the PNB lifetime, since the lifetime correlates that the vapor bubbles with the diameter above 2 pm caused with the bubble diameter (see figure 4(c)). The data presented irreparable damage to cellular membranes. Based on the were averaged for the cell populations and were obtained at obtained results we have estimated the cell damage threshold specific laser pulse fluences. The amplification coefficient lifetime of the PNB to be about 110 ns. We have found Ksc (measured relative to scattering amplitudes for gold NPs) that intact cells cannot support such small non-lethal PNB linearly increases with the PNB lifetime. This implies the (table I) and the generation of the photothermal laser-induced PNB-based mechanism of the amplification: the vapor—liquid bubbles was always associated with cell damage [39, 40], border of the PNB creates a gradient of the refractive index, suggesting that the endogenous optical absorbers in intact and the scattering efficiency of the PNB is determined by its cells cannot generate small PNBs. As for the NP-treated diameter (that correlates to the PNB lifetime). The PNBs have cells, we may suggest that the PNBs with a maximal diameter yielded 10-50-fold optical amplification without disrupting the smaller than 300 nm would be non-invasive to living cells cell membrane or inducing blebbing. (PNB function: non-invasive imaging), those in the range of Our previously reported modeling of PNB optical 500-1000 nm would produce local reversible disruption (PNB scattering [28] has predicted such amplification. In this work function: intracellular delivery of the drugs or other agents), we have used a Mie simulation code, developed for multilayer and PNBs in the range from I to 10 pm would mechanically concentric spheres, to model the scattering by the vapor destroy individual cells (PNB function: cell damage). bubble (with gold NP inside) relative to the scattering by a We have also found that at laser fluences below the PNB gold NP alone. By applying the developed model for gold generation threshold, the NPs in cells still were significantly spheres and silica-gold shells of the various dimensions we heated by the laser pulse but did not cause detectable damage have found the general trend of increase in scattering against to the cells. Also, the exposure of the cell to 16 pump laser bubble radius. This can be qualitatively understood from pulses (at 15 Hz frequency), instead of a single pulse, did Rayleigh scattering, which states that the scattering intensity not influence the cell viability and the level of the damage is proportional to 46/),.4, where d is the particle diameter and threshold fluence, which suggests that the cell damage results A is the wavelength, provided d is smaller than A. Compared from a single event rather than from an accumulative effect to NPs alone, PNBs could potentially produce 1-3 orders of of the sequence of the PNBs. Thus, the PNB damage magnitude amplification in scattering intensity. The modeling mechanism is mechanical, non-thermal, and rapid: a single results obtained, as well as the above experimental results, laser pulse induces an expanding PNB that disrupts the cellular predict the better scattering efficiency of the bubble compared cytoskeleton and plasma membrane causing the blebbing to the NP, mainly due to the increased diameter of the bubble and cell staining with the membrane-penetrating dye. Also, and to the vapor-liquid interface that it temporary creates. during the generation of the cell-damaging PNBs with sub- A probe of such large diameter (up to 200-500 nm) cannot microsecond lifetime, we did not observe the damage in the be delivered into the cell without compromising its viability, collateral cells where no PNBs were generated. This has though it can be temporally generated there for a short time and demonstrated a cell level selectivity of the PNB mechanism in non-invasive way. Furthermore, our model has predicted an of cell damage. Our ongoing work will include the study of optical attenuating effect around NPs that do not generate the the cell structure after the PNB generation and of the long- bubbles but are also heated with the pump laser pulse above term viability of the surviving cells (including a zebrafish the evaporation threshold for the environment. This attenuating in vivo model). Nevertheless, the NP-generated intracellular effect has recently been confirmed by us in experiments with PNBs have demonstrated a localized, fast, selective and easily gold NPs in water [23]. Therefore, applying the single pulse controllable (through the laser pulse fluence level) mechanism at a specific fluence we may selectively amplify, by several of cell damage. orders of magnitude, the optical scattering around clusters of 6 EFTA01113067 Nanotochnology 21(2010)085102 E Y Lukianova-Hleb eta! gold NPs while suppressing the scattering around single NPs around the NP clusters. These results illustrate the improved or small clusters. The amplification/attenuation effect can be specificity and selectivity of the PNB method relative to those optimized by adjusting the fluence of the pump pulse so as to based on the direct application of NPs as optical or thermal provide maximal specificity of PNB diagnosis. agents: the specificity and selectivity of the NP-based methods Specificity of the diagnosis and selectivity of the therapy (such as optical scattering diagnosis and hypothermia, see the are the top priorities in cancer treatment. These were the Discussion for details) is compromised by the unavoidable objectives for developing the NP cluster mechanism for the contribution from non-specifically coupled (targeted) single generation of plasmonic nanobubbles. The specificity of PNB NPs. In our method, a few non-specifically coupled NPs could diagnosis depends upon NP parameters as of the PNB sources not form a big cluster that requires a considerable amount of and can be significantly increased if NP clusters, not single NPs accumulated at cell membrane. Therefore, the biggest NPs, are used to generate the PNBs. This rule has been clusters can be formed only in target cells, thus providing the verified during our previous studies of PNBs [21, 23-25]: basis for the cell-specific theranostic application of PNBs. the aggregation of NPs into a cluster significantly lowered the PNB generation threshold fluence of the pump laser 4. Discussion pulse. Therefore, at a specific fluence level the PNBs will be generated only around NP clusters and will not emerge around The above experiments have demonstrated several interesting single (e.g. non-specifically coupled) NPs. Thus we have features of intracellular NP-generated PNBs: considered intracellular NP clusterization as the main solution to improving diagnostic specificity and therapeutic selectivity • the PNB probe is a transient event, not an object and so its load and presence in the cell are minimized to of the PNBs. To prove this concept we have experimentally sub-microsecond times, while it uses relatively safe gold varied the parameters that influence NP clusterization through nanoparticles and low levels of laser fluence in a single the mechanism of receptor-mediated endocytosis: diagnosis- pulse mode; associated vectors conjugated to gold NPs (EGFR antibodies versus non-conjugated NPs), targeting conditions and also • the main diagnostic and therapeutic property of the PNB is have compared the PNB generation in aqueous suspensions characterized by its maximal diameter (lifetime) that can of the same gold NPs and their clusters. We have varied be precisely controlled and varied in the sub-micrometer several endocytosis-related parameters. We have decreased the range with the fluence of the laser pulse; also the PNB incubation temperature to 4 °C (which suppresses endocytosis) diameter can be conveniently monitored through the PNB and have used the NPs without a C225 antibody. In both lifetime and scattering amplitude; cases PNB generation thresholds increased from 0.09 ± 0.03 • as the PNB can concentrate, within its volume, the thermal to 0.11 ± 0.06 J cm-2 (for 4 °C incubation with NP-C225) and energy released by the NP [21, 22], its outer action has to 0.33 ± 0.18 J cm2 (for 37 °C incubation with unconjugated a mainly mechanical, non-thermal nature and this may NPs), which implies that NP-C225 conjugates were selectively prevent thermal damage to surrounding molecules and linked to EGFR and then were internalized through the collateral normal cells. receptor-mediated endocytosis. It is interesting to compare the PNB with currently avail- Next, we have independently verified the physical basis able multi-functional probes that have been reported for for the selectivity of the PNB generation around the NP theranostic applications and that can be classified into clusters compared to single NPs. The PNBs were generated several groups (table 2): fluorescent probes [41-44], and detected in water suspensions around the single gold capsule-type probes (liposomes, micelles, polyelectrolyte NPs (50 nm spheres) and around their clusters that were and polymer capsules) [45-51], non-plasmonic nanoparti- intentionally prepared by adding NaCI into the suspension cles [1, 42-44, 52, 53], plasmonic nanoparticles [7, 9, 42, 54] of the NPs. NP clusterization was verified by monitoring and gas-filled or cavitation bubbles [31-34, 55, 56]. Their the extinction spectra of the NP suspensions (showing one theranostic properties were analyzed for the diagnosis, therapy plasmon peak near 532 nm) and their clusters (showing and guidance, and also for cell level selectivity and safety (ta- significant broadening of the peak though still without shifting ble 2). As for the fluorescent probes, the PNB may provide the its maximum). We compared the PNB generation threshold ultimate imaging sensitivity: it was reported previously that fluences in living cells and around isolated NPs and their the optical scattering efficiency of gold nanoparticles is 4-5 clusters. The intracellular PNB generation threshold fluence orders of magnitude higher than the fluorescent efficiency of (0.09 ± 0.03 J cm2 ) was found to be closer to that for the the brightest fluorescent molecules [57], and we have demon- NP clusters in water (0.055 ± 0.02 J cm-2) rather than the strated in our experiments that the optical scattering efficiency threshold for the single NPs in water (0.18 ± 0.06 J cm-2). of PNB is 10-100 times higher than that for gold nanopar- This also indicates that the generation of the intracellular PNBs ticles. Therefore, we may conclude that PNBs will provide occurs around NP clusters and not around single NPs. Also, much better imaging sensitivity than fluorescent probes. The the clusterization of the NPs has lowered the PNB generation specificity demonstrated for PNB cannot be achieved through threshold by almost three times. Therefore, no PNBs could nanoparticle scattering (or through fluorescence) because any have been generated around single NPs (including those non- non-specifically coupled NP (or fluorescent probe) increases specifically coupled to non-target cells) at the laser pulse the optical background and the probability of false-positive di- fluence level that is sufficient for the generation of the PNBs agnosis. The combination of improved brightness of the PNBs 7 EFTA01113068 Nanotochnology 21 (2010)085102 E Y Lukianova-Hlob eta! Table 2. Comparison of the diagnostic, therapeutic and guidance potentials of the common biomedical probes (high—very efficient. low—not efficient or not applied (or low selectivity. specificity)). Function Cell level selectivity Probe Diagnosis Therapy Guidance and specificity Safety Fluorescent probes High Low High (drug High Low (chemical conjugates) toxicity) Micelles. polymers Low (unless loaded Low Low (unless loaded depends upon and liposomes with other probes) other probes) release method Nanoparticles High (magnetic High (thermal Low Low (magnetic resonance Low (chemical (general) resonance imaging) therapy) and photo-acoustic toxicity) imaging) Plasmonic High (optical High (photo- Low High (delivery and optical High for gold nanoparticles scattering and thermal scattering imaging) nanoparticles photo-acoustic therapy) imaging) Gas and cavitation High (acoustic and High Low Low High bubbles optical imaging) with the threshold mechanism of their generation around NP by the size of the bubble. This parameter can be precisely clusters (that can be selectively formed around target molecules controlled through the optical pulse. Furthermore, the optical and cells as we have shown previously in [25, 58]) principally scattering efficiency of the bubble directly correlates to its improves the specificity of PNB diagnosis relative to NP- or diameter and so can be used to guide a biological action fluorescent probe-based methods. that is determined by its diameter: delivery of endocytosed The other example is related to therapeutic properties of molecules by intracellular PNB that disrupt the endosomes plasmonic NPs that are optically heated with continuous 17, 9] (smallest bubbles); delivery of the extra-cellular molecules or pulsed [11, 12] optical radiation. The thermal mechanism through the perforation of the cellula