Article pubs.acs.org/Langmuir
Transient Photothermal Spectra of Plasmonic Nanobubbles Ekaterina Y. Lukianova-Hleb,† Elisabetta Sassaroli,† Alicia Jones,† and Dmitri O. Lapotko*,†,‡ †
Department of Biochemistry and Cell Biology and ‡Department of Physics and Astronomy, Rice University, Houston, Texas 77005, United States S Supporting Information *
ABSTRACT: The photothermal efficacy of near-infrared gold nanoparticles (NP), nanoshells, and nanorods was studied under pulsed high-energy optical excitation in plasmonic nanobubble (PNB) mode as a function of the wavelength and duration of the excitation laser pulse. PNBs, transient vapor nanobubbles, were generated around individual and clustered overheated NPs in water and living cells. Transient PNBs showed two photothermal features not previously observed for NPs: the narrowing of the spectral peaks to 1 nm and the strong dependence of the photothermal efficacy upon the duration of the laser pulse. Narrow red-shifted (relative to those of NPs) near-infrared spectral peaks were observed for 70 ps excitation laser pulses, while longer sub- and nanosecond pulses completely suppressed near-infrared peaks and blue shifted the PNB generation to the visual range. Thus, PNBs can provide superior spectral selectivity over gold NPs under specific optical excitation conditions. therapeutics,30,33,35,36 theranostics,37−39 and surgery,40,41 especially in combination with near-infrared (NIR) plasmon resonances, associated with maximal biosafety and penetration range in tissues.42 However, the conditions of PNB generation correspond to transient overheating, melting, and even destruction of plasmonic NPs during their interaction with the excitation (pump) laser radiation, especially in the case of those with structure-dependent NIR plasmon resonances (rods, shells, and cages).43,44 Under such conditions the plasmonic properties of metal NPs undergo rapid dynamic changes during their interaction with the excitation laser pulse.45−48 Such conditions cannot be described by most of the current models of plasmonic interaction since they do not take into account dynamic changes in the electrical, optical, and thermal properties of NPs. In this work we experimentally and theoretically studied the influence of the wavelength, duration, and fluence of the excitation laser pulse on the photothermal efficacy of PNB generation around gold NPs with NIR resonances, nanorods (NR), and hollow nanoshells (NS) in water and living cells.
1. INTRODUCTION Plasmonic nanoparticles (NP) are the ultimate sources of heat on a nanoscale due to the high efficacy of the conversion of light into heat by plasmon resonance. The photothermal properties of such NPs offer many applications including biomedical ones for both diagnosis and therapy.1−16 These applications also reveal some challenges however: the optical doses required for therapeutic hyperthermia1−18 are quite high (20−26000 J/cm2), and the long duration of the optical treatment (measured in minutes) causes significant thermal losses and collateral biodamage due to thermal diffusion from the heated NPs. To minimize the thermal diffusive losses and nonspecific thermal damage to healthy tissues, short pulsed optical excitation19 and NPs with near-infrared (NIR) plasmon resonance, such as gold nanoshells,20−22 nanorods,23,24 and nanocages,25 were developed. However, the amount of NPs required for the desired diagnostic or therapeutic effect can be so high that it often results in macro- rather than nanoresolution with poor localization of the effect. The limited specificity of the precise targeting of NPs (due to unavoidable nonspecific uptake) further compromises the selectivity of their thermal effect. All these factors impede the major advantage of plasmonic NPs, which is their ability to deliver and manipulate external energy on the nanoscale, including at the cellular level. To address the above limitations, we recently suggested the use of mechanical, nonthermal, localized, and tunable effects of transient vapor nanobubbles generated around superheated plasmonic NPs upon their excitation with short laser pulses.26−33 These nanobubbles were named plasmonic nanobubbles (PNBs) because they receive their energy through the mechanism of plasmon resonance.28,29 In addition to their localized mechanical impact, the optical scattering brightness of PNBs is superior to that of plasmonic NPs.27,28,34 For all these reasons they are promising candidates for cell-level diagnosis,34 © 2012 American Chemical Society
2. EXPERIMENTAL SECTION 2.1. Nanoparticles. We used gold NPs with a structure-dependent and tunable peak of the plasmon resonance in the near-infrared and sizes in the range of 20−70 nm. We used commercially available gold solid nanorods (NR, 25 × 75 nm, optical extinction peak at 710 nm in water suspension under room temperature) (Nanopartz Inc., Loveland, CO) and synthesized gold hollow nanoshells (NS, 50 ± 7 nm diameter, 2.7 ± 0.8 nm thickness, optical extinction peak at 665 nm Received: December 28, 2011 Revised: February 16, 2012 Published: February 16, 2012 4858
dx.doi.org/10.1021/la205132x | Langmuir 2012, 28, 4858−4866
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Article
Figure 1. (a) Experimental scheme for PNB generation and detection around gold NPs. Pulsed pump (excitation) laser used three different sources with pulse durations of 70 and 400 ps and 10 ns and tunable wavelength and fluence. NPs and PNBs were imaged and monitored using optical scattering of the two probe laser beams: cw (633 nm) and pulsed (576 nm). (b) Optical scattering time-resolved image of an individual gold NS cluster in water obtained with a pulsed probe laser at 576 nm. (c) Optical scattering time-resolved image of PNB generated around the NS cluster in water with a single 70 ps pump pulse at a wavelength of 778 nm. (d) Time response of the same PNB as shown in c was obtained with a cw probe laser at 633 nm. under the same conditions) following established protocols22 based on reaction of replacement of the silver core with a gold shell (see Supporting Information for details). The structure and size of the NSs were verified with transmission electron microscopy (TEM) (JEOL 2010, Jeol Ltd., Tokyo, Japan) by measuring the diameter and shell thickness of at least 100 NSs. Solid gold spheres (NSPs) were used for reference spectroscopy measurements and obtained from Ted Pella Inc. (15703, #15706, Redding, CA). Optical spectra for “cold” excitation conditions were obtained for water suspensions of NPs with a spectrophotometer (USB 650 Red Tide spectrometer, Ocean Optics, Inc., Dunedin, FL). Water samples of gold NPs were prepared on standard microscope slides and coverslips of 18 mm diameter. The sample was prepared by adding 5 μL polysterene microspheres of 15 μm diameter (Spherotech Inc., Lake Forest, IL) to 50 μL of the NP suspension. These microspheres were used as spacers between the microscope glass slide and a coverslip to provide a specific height (15 μm) of the liquid. The concentration of gold NPs was adjusted so that their low surface density would exclude exposure of closely located NPs under laser irradiation and thus provide a study of individual NPs. NP clusters (objects consisting of 5−50 aggregated particles) were prepared by adding sodium chloride to the water suspension of NPs at a concentration of NaCl 10 mg/mL and resuspending the NP clusters in water to adjust their concentration of NP clusters to that of the single NPs. Individual NPs or their clusters were visualized with optical scattering (see section 2.4) for their positioning into the center of the laser beam with a motorized microscope stage (8MT167-100, Standa Ltd., Vilnius, Lithuania). To minimize the effect of heterogeneity of the NP cluster size, we irradiated and measured only clusters with similar levels of pixel image amplitudes of optical scattering (see section 2.4 and Figure 1b). 2.2. Cells and Nanoparticle Targeting. Squamous carcinoma cells (HN31) were cultured in DMEM High Glucose medium (Cat. no. 10013 CV) from Mediatech supplemented with MEM Vitamin Solution (Cat. no. 11120) and MEM NEAA (Cat. no. 11140) both from Gibco and penicillin−streptomycin (Cat. no. 30002 CT) from Mediatech. To study plasmonic nanobubble (PNB) generation, the cells were seeded at a density of 700 000 cells/mL in the culture slides (μ Slide VI 0,4, Ibidi LLC, Martinsried, Germany). Cells were grown 24 h in these slides before treatment with NPs. The 50 nm NSs were conjugated with Panitumumab (Amgen Inc., Thousand Oaks, CA), an antibody against the epidermal growth factor receptor that is overexpressed by HN31 cancer cells, by BioAssayWorks LLC (Ijamsville, MD). NS conjugates were incubated with the cells for 24 h under physiological conditions and washed from unbound NPs before PNB generation. The small size of NSs provided efficient targeting through receptor-mediated endocytosis and formation of intracellular NS clusters.30,34,39,49−51
2.3. Optical Excitation of Nanoparticles. We exposed individual NPs and their clusters in water and individual cells to single laser pulses with a specific wavelength, duration, and fluence, the latter being high enough to exceed the PNB generation threshold. We applied single pump laser pulses with a Gaussian intensity profile and tunable wavelengths (532−900 nm) and pulse durations of 70 ps (PL2250, Ekspla, Vilnius, Lithuania), 400 ps (dual-laser system STA-01, Standa Ltd., Vilnius, Lithuania), and 10 ns (Nd:YAG laser LS2132, Lotis TII, Minsk, Belarus) (Figure 1a). The fluence of each single laser pulse was controlled with a polarizing attenuator and measured by registering the image of the laser beam at the sample plane with the image device, EM CCD camera (Luka model, Andor Technology, Northern Ireland), and the pulse energy with the energy meter (Ophir Optronics, Ltd., Israel). The fluence was calculated using the pulse energy and image with the beam diameter measured at the level of 1/e2 relative to the maximaum. The setup was assembled on an optical table (RS 1000TM, Newport Corp., Irvine, CA) and used an inverted optical microscope Zeiss A200 (Carl Zeiss MicroImaging GmbH, Germany) for handling of the samples and all imaging and optical detection. 2.4. Photothermal Response of Plasmonic NPs to Laser Pulses. The photothermal response of plasmonic NPs to laser pulses was measured as (a) optical scattering effects with two techniques and (b) modification of the structure and size of the NP with TEM. PNBs generated around gold NPs were studied through their optical scattering time-resolved images (Figure 1c) and time responses (Figure 1d) using the optical scattering effects of the vapor bubble27,28,52 and two corresponding probe lasers. A continuous (cw) probe laser beam of very low power (633 nm,
