Size Affects the Stability of the Photoacoustic Conversion of Gold

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Size Affects the Stability of the Photoacoustic Conversion of Gold Nanorods Lucia Cavigli, Marella de Angelis, Fulvio Ratto, Paolo Matteini, Francesca Paoletti Rossi, Sonia Centi, Franco Fusi, and Roberto Pini J. Phys. Chem. C, Just Accepted Manuscript • Publication Date (Web): 27 Jun 2014 Downloaded from http://pubs.acs.org on June 28, 2014

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The Journal of Physical Chemistry

Size Affects the Stability of the Photoacoustic Conversion of Gold Nanorods Lucia Cavigli,∗,† Marella de Angelis,† Fulvio Ratto,† Paolo Matteini,† Francesca Rossi,† Sonia Centi,‡ Franco Fusi,‡,† and Roberto Pini† Istituto di Fisica Applicata Nello Carrara, Consiglio Nazionale delle Ricerche, via Madonna del Piano 10, Sesto Fiorentino,50019 (Italy), and Dipartimento di Fisiopatologia Clinica, Università degli Studi di Firenze, Viale G. Pieraccini 6, Firenze, 50139 (Italy) E-mail: [email protected]

∗ To

whom correspondence should be addressed di Fisica Applicata Nello Carrara, Consiglio Nazionale delle Ricerche, via Madonna del Piano 10, Sesto Fiorentino,50019 (Italy) ‡ Dipartimento di Fisiopatologia Clinica, Università degli Studi di Firenze, Viale G. Pieraccini 6, Firenze, 50139 (Italy) † Istituto

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Abstract Gold nanorods exhibit intense optical absorption bands in the near-infrared region of principal interest for applications in biomedical optics, which originate from sharp plasmon resonances. This high absorbance, combined with the biochemical inertness and targetability of gold nanoparticles, makes these materials excellent candidates to provide contrast in photoacoustic imaging and for other applications such as the selective hyperthermia of cancer. One issue demoting the potential of gold nanorods as contrast agents in photoacoustic applications is their limited photostability, which falls below relevant permissible exposure limits. In particular, when gold nanorods are resonantly excited by laser pulses in the nanosecond duration regime, there may occur phenomena like reshaping into rounder nanoparticles as well as fragmentation and sublimation, which modify their optical absorption bands and hinders their efficiency of photoacoustic conversion. Here we investigate the influence of nanoparticle size on the photostability and reproducibility of photoacoustic conversion of gold nanorods embedded in biomimetic phantoms. We compare samples containing gold nanorods with different sizes but the same shapes and overall optical densities. We demonstrate clear size effects as the thresholds of optical fluences for nanoparticle deformation improve from below 2 to above 6 mJ/cm2 with nanoparticle miniaturization from 22 to 5 nm effective radii. We interpret these results in terms of a better thermal coupling and faster heat dissipation from smaller nanoparticles to their environment, originating from their larger specific surface area.

KEYWORDS: Photoacoustic, gold nanorods, photostability, size effects

Introduction Imaging and therapeutic techniques based on photoacoustic (PA) effects are attracting much interest for medical applications due to their profitable combination of optical and acoustic features. 1–5 In the PA process, ultrasounds are generated by the absorption of short light pulses in the nanosecond duration regime, which triggers a cascade of rapid heating and thermoelastic ex2 ACS Paragon Plus Environment

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pansion. 6–8 The combination of optical excitation with acoustic detection allows one to reach far deeper penetration into turbid materials than purely optical imaging techniques, while maintaining the high contrast and spectroscopic-based specificity of optical imaging. 5,9 PA imaging has been used to detect pathologies such as cancer using both its endogenous features 3,5 and the application of exogenous contrast agents with high optical absorbance. 3,5 Higher fluences (energy per surface unit) of the optical excitation may activate other effects such as bubble formation and subsequent cavitation, which has been exploited for new therapies such as to remove malignant cells that were selectively targeted with an exogenous contrast agent. 10–14 Various contrast agents are used to increase the sensitivity and spectroscopic specificity of PA signals, including dyes 15–17 and plasmonic nanoparticles. 18,19 Among all, gold nanorods (GNRs) are a preferred choice for many imaging and therapeutic applications. Their optical absorption features a longitudinal mode of plasmon oscillations (LP) centered in the near infrared (NIR) window of highest interest in biomedical optics, i.e. where the penetration of light into biological tissue is highest, and a rather weaker transverse mode at around 520 nm. The LP peak position is very sensitive to the aspect ratio (AR), i.e. the ratio between longitudinal and transverse size, of the GNRs and can be tuned through the entire NIR region. 20,21 Moreover GNRs are suitable for conjugation with ligands to gain molecular specificity and the possibility to target diseases such as cancer. The modulation of the size, shape and coating of GNRs allows one to tune their optical properties and biological profiles in view of specific applications. The relationships between nanoparticle parameters and functional performances is a subject matter of great current interest. 22–25 In the use of GNRs as contrast agents for PA applications, one of the most critical issues is their instability. 26–32 In particular, when GNRs are irradiated with nanosecond laser pulses in resonance with their plasmon oscillations, overheating may trigger phenomena like their reshaping into more stable and rounder nanoparticles or even thermionic emission and fragmentation at high enough fluences. 26–33 In turn these structural transformations modify the optical absorbance of GNRs, which really jeopardizes their potential to provide contrast at the due wavelength. Much research activity is underway to improve the photostability of GNRs. Several studies have shown that modifying



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the nanoparticle coating may increase their photothermal stability. 32,34 For instance, the addition of silica shells imparts some steric constraint against reshaping and better thermal coupling to the environment. In this context, it is also of fundamental importance to identify threshold fluences Fth before the nanoparticles degrade in the timespan of an imaging or therapeutic procedure. Several values of Fth have been reported in the literature ranging from a few to hundreds of mJ/cm2. This large variability depends on different parameters such as nanoparticle coating 31,32 and dispersion medium as well as the measurement criteria most of all. 29–32,35 In this work, we focus both on the definition of practical measurement criteria and the effect of nanoparticle parameters on Fth . We set Fth at the very onset of nanoparticle reshaping in a biomimetic phantom after a train of 50 laser pulses with the usual repetition rate of 10 Hz. The phantom was composed of a hydrogel of chitosan, 36 which is thermally and mechanically similar to bodily tissue. We note that the number of laser pulses is not a critical parameter, because heat generation and dissipation from individual GNRs occur in a few hundreds of picoseconds 37 and so multiple stimuli are independent. Nanoparticle reshaping was measured combining PA experiments with VIS-IR spectrometry and a model deconvolution. We address the influence of nanoparticle size on their photostability and reproducibility of PA conversion. Suspensions of GNRs were synthesized with different size 38 and comparable AR statistics and then integrated in 50 µ m thick chitosan films 22,39 before optical excitation. Fth was found to exhibit a clear pattern to nanoparticle size. These results provide immediate practical indications for the design of plasmon resonant nanoparticles for specific PA applications such as biomedical imaging and microsurgery.


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Experimental section Synthesis of gold nanorods in chitosan films Chemicals were purchased from Sigma Aldrich and used as received. GNRs with different size statistics were obtained by autocatalytic reduction of chloroauric acid by ascorbic acid in a growth solution containing cetrimonium bromide and silver nitrate, which was supplemented with gold nuclei as it is described in references. 20,38 With the purpose to keep the same AR distribution and modulate the volume statistics, different concentrations of gold nuclei were injected in different batches from the same growth solution. This approach was guided by the idea that the nanoparticle shapes are governed by the composition of the growth solution 20,21 while the nanoparticle sizes may depend on the number density of the gold nuclei. Indeed the autocatalytic nature of nanoparticle growth 20,38,40 ensures that the distribution of the same total amount of gold among different numbers of gold nuclei may bring to either of a smaller number of larger nanoparticles or a larger number of smaller nanoparticles. In a first approximation, the use of the same total amount of gold would entail comparable optical absorbance in the LP bands, 41 i.e. about the same efficiency of photothermal conversion. Figure 1 displays an analysis of TEM micrographs from a series of samples with different concentrations of gold nuclei, such as those shown in the Supporting Information (figures S1 to S6 in Supporting Information). These samples display comparable AR statistics and different volume distributions in decent agreement with our speculation. We note that figures S1 to S6 reveal additional subtle modifications, such as a reduction of quasi spherical byproducts and appearance of so-called dogbone profiles 38,42 at lower concentrations of gold nuclei. The emergence of dogbone profiles leaves its distinctive mark in the plasmonic band in the green window (see also figure S7 in Supporting Information), which undergoes a progressive intensification, red-shift and broadening. 38 Hereafter we label our samples as GNR5, GNR8, GNR11, GNR15 and GNR22, with the numbers denoting their average effective radii (radius of a sphere having the same volume as the rod) in nm, 38 according to an analysis of figure 1a.



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Chitosan films doped with GNRs (GNR-CH) were produced by pouring an acidic suspension of GNRs (Au 800 µ M) containing 3.5 % w/v chitosan into circular polystyrene molds. 43,44 After 24 h drying in an oven at 25◦ C, cross-linking of the sample was induced by alkalinization with a NaOH 1M solution for 5 minutes followed by abundant rinsing with water to restore neutrality. The film thickness and diameter were about 50 µ m and 17 mm respectively.

Figure 1: From the left: a) volume distribution, b) aspect ratio distribution and c) optical extinction spectra of the various samples (from bottom to top: GNR5, GNR8, GNR11, GNR15, GNR22).

Figure 2: Sketch of the set-up used for the photoacoustic experiments (O: objective, L: focusing lens, BS: Beam Splitter, EM: Energy Meter).


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Structural and optical characterization The optical extinction of the GNR-CH phantoms was characterized by a Jasco V-560 spectrophotometer. Local effects over a microscopic lengthscale were addressed with a portable spectrometer (Mod. EPP2000 by Stellarnet Inc., Florida USA) equipped with optical fibers in the excitation and detection arms. In order to represent the experimental data in terms of nanoparticle shapes, we deconvoluted the extinction spectra by an analytical representation of the volumetric distributions of nanoparticle AR and the use of Gans lineshape with the analytical approximation of the dielectric function of gold by Etchegoin et al., 45 as it is described in detail in refs 36,44. Whole agreement with the experimental spectra was achieved with a complex AR distribution parameterized as a linear combination of two Weibull functions. For TEM analysis, droplets of the aqueous suspensions of GNRs before inclusion in chitosan phantoms were left to dry on formvar carbon coated copper TEM grids, which were inspected under a (S)TEM Microscope (Philips CM12, Amsterdam, The Netherlands). Nanoparticle volume and AR distributions were retrieved from a random selection of 500 nanoparticles per sample.

Photoacoustic characterization A photoacoustic set-up was used to test the photostability and PA response of GNR-CH films (a sketch of the set-up is shown in figure 2). Films were kept in a plastic holder and immersed in a petri dish filled with deionized water. An opaque mask with 50 holes (1 mm diameter each) was superimposed on the films in order to pin a set of excitation points. A pre-amplified needle hydrophone (Precision Acoustics, Dorset, UK, sensor diameter 1 mm, frequency range 1-20 MHz) was mounted on a micrometric translation and rotational stage to control the transducer position. The hydrophone distance from the samples was kept constant during all the experiments. The excitation source was an optical parametric oscillator (OPO) pumped with the third harmonic of a Q-switched Nd:YAG laser (Continuum Surelite OPO plus, Santa Clara, USA, wavelength range 400-2500 nm, pulse duration 5 ns, repetition rate 10 Hz). The laser beam was focused perpendicular to the film layer with a spot of 300 µ m diameter. The fluence was adjusted with an 7 ACS Paragon Plus Environment


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attenuator set in front of the laser exit. For each laser pulse, the photoacoustic signal acquired by the hydrophone was recorded with a digital oscilloscope (Mod. RTO1004, Rohde&Schwarz GmbH, Munich, Germany) and the fluence fluctuations were monitored with a pyroelectric detector (Mod. RjP-735, Laser Precision, USA).

Figure 3: a) Ratio R of the intensities of PAref taken after and before irradiation at each Fexc versus Fexc for sample GNR8. b) Extinction spectra from sample GNR8 upon excitation at increasing fluence. c) The distribution of AR from sample GNR8 after irradiation at different fluences.

FEM modeling The photothermal process was evaluated in the GNRs and their surroundings by modeling the light absorption and consequent temperature dynamics. A commercial software (Comsol Multiphysics 4.3a, COMSOL AB, Stockholm, Sweden) was used to solve a bi-dimensional, axisymmetric geometrical model of a gold nanorod immersed in a chitosan medium by a Finite Element Model (FEM). Equation 1 describes the temporal and spatial variations of temperature in the nanoparticle and its environment due to light absorption and was solved both for the GNRs and chitosan:

ρnC pn

∂T − ∇2 (kn T ) = Qext . ∂t

(1)

ρn , C pn , kn are the density (kg/m3 ), heat capacity (J/(kgK)) and thermal conductivity (W/(mK)) of the n-th medium (i.e. gold and chitosan), respectively and were taken from the literature. 44,46,47 Qext (W/m3 ) is the heat source term which is due to the absorption of the laser light by GNRs and 8 ACS Paragon Plus Environment

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is described by equation 2. 37,46,48 Qext =

Cabs I , vp

(2)

where Cabs is the optical absorption cross-section (nm2 ) at the wavelength of interest, v p is the nanoparticle volume (nm3 ), and I is the laser power density (W/m2 ). Cabs was simulated for nanoparticles of representative shapes and sizes by the use of Gans approximation, as described elsewhere. 27,28,36,44,49,50 We note that, according to this approximation, Cabs scales with nanoparticle volume and so Qext remains constant inside nanoparticles with the same shape. The medium surrounding the gold nanoparticles was considered as an infinite element, while heat exchange at the interface between gold and chitosan was modeled as reported in ref. 51: Φ = h (TCH − TGNR ) ,

(3)

where Φ(W/m2 ) is the heat flux at the boundary, h(W/(m2 K)) is the thermal conductance, TCH (◦ C) is the equilibrium temperature in the chitosan matrix and TGNR (◦ C) is the gold temperature at the boundary. As an initial condition the GNR and chitosan matrix were supposed to be in thermal equilibrium at T= 25◦ C. The geometrical model was described with an extremely fine, physics-controlled triangular mesh with 6743 degrees of freedom, which was solved as a time dependent problem. The accuracy of the model was tested against the results of the problem described in ref. 51 (data reported in figure S8 in Supporting Information).

Results and discussion Figure 1c shows the extinction spectra of the five GNR-CH films before irradiation (from bottom to top: GNR5, GNR8, GNR11, GNR15, GNR22). Panels 1a and 1b report the volumetric distributions of nanoparticle volumes and AR. While the trend in size from figure 1 is clear, all the AR distributions remain fairly similar to each other except for sample GNR22, which peaks at smaller



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AR. A comparison of these AR distributions with the extinction spectra in figure 1c suggests a good nanoparticle dispersion 43 except for sample GNR22, where partial nanoparticle aggregation may take place, as it is confirmed by relevant TEM micrographs (see figure S6 in Supporting Information). The influence of size on the photothermal stability and PA response of the GNR-CH films was investigated by means of PA experiments combined with VIS-IR spectroscopy. We fixed the excitation wavelength in PA measurements at 810 nm for all samples, which is close to their LP peak (see figure 1c) and corresponds to an AR around 3.8 according to our solution of the Gans approximation. 27,28,36,44,49,50 Our choice to use the same wavelength for all samples reflects our intent to examine a subset of GNRs with uniform AR and isolate the influence of nanoparticle size on their thermal properties and PA response. Indeed we assume that the average fluence used in this study cannot heat the sample much beyond the timescale of the individual pulse and length scale of the individual nanoparticle. Therefore only that nanoparticle fraction with AR around 3.8 resonating with the optical excitation at 810 nm can undergo modifications, with little involvement of all other nanoparticles. We note that this picture differs much from a photothermal effect under CW irradiation, 36 where sample heating drives a modification of all nanoparticles, i.e. including all those that poorly absorb the laser light.

Threshold measurements A critical condition to exploit the potential of GNRs as contrast agents for PA imaging is the definition of a range of fluences where their PA response is stable and reproducible over time. First of all, we established an experimental probe of stability. We irradiated a point of each sample (corresponding to the first hole in the sample mask) at 1 mJ/cm2 for at least 100 pulses and recorded the corresponding PA signals. We extracted the trend of PA intensity (defined as the maximum of the positive voltage peak from the needle hydrophone) as a function of pulse number and verified that it remained well constant over time. This condition implies that the samples did not undergo transformations, as it also turns out from a comparison of the VIS-IR spectra from the irradiated 10 ACS Paragon Plus Environment

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spots before and after excitation (see figure S9 in Supporting Information). Next, we irradiated different points of the samples with 50 pulses at different nominal fluences with average values Fexc (one nominal fluence per point, selected by a hole in the mask). In order to counterweight the laser intensity fluctuations, the amplitude of the PA signal from each individual pulse was normalized to the ratio of its own fluence and Fexc , which reflects a linear approximation of the PA conversion within these fluctuations. For each Fexc we probed an average PA intensity over 50 pulses at 1 mJ/cm2 (PAref hereafter) both before and after irradiation at Fexc and analyzed its variation. We found that PAref underwent degradation after irradiation above some fluence Fth , which varies from sample to sample. A decrease of the PA signal at PAref following irradiation at a given Fexc is an unequivocal evidence that there has been an irreversible change of the optical and structural properties of the samples. This decrease can be quantified by measuring the ratio R between PAref after and before irradiation at each Fexc . We then define the damage threshold fluence Fth as the value when R begins to differ from one beyond the statistical fluctuations. The advantages of measuring Fth by the variations of R, rather than from the variations of PA signal intensity at Fexc are, first of all, that PAref is acquired in conditions of stability, so we can average over several pulses and improve the quality of the measurements. On the contrary, for excitations close to or above the threshold, it would be necessary to analyze variations of the intensity at the level of the single pulse, because the nanoparticle heating is not cumulative and the sample transforms within a few pulses. In the second place, since PAref is measured at the same point before and after irradiation, we can analyze and compare different spots of the same sample and even different samples without the obstacle of their inhomogeneity. As an example, figure 3a displays the trend of R as a function of Fexc in the case of sample GNR8. The complete measurements for all the samples are reported in figure S10 of the Supporting Information. R remains close to unity up to 4 mJ/cm2. However, at higher fluences, R begins to decrease, indicating a partial transformation of the GNRs. This process is confirmed by the extinction spectra taken after irradiation and shown in figure 3b. From this data, we estimated a Fth = 4 ± 0.5 mJ/cm2 for sample GNR8. At fluences slightly exceeding this threshold (e.g. 5 mJ/cm2, red dashed line in



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figure 3b) the LP peak is still intense but there occurs a slight depletion in resonance with the laser wavelength. In order to describe this modification in terms of volumetric distributions of nanoparticle AR, we deconvoluted the extinction spectra by the use of Gans lineshape. 36,44 We found that when the samples were irradiated at Fth (red dashed line in figure 3c), only the total Au volume in GNRs with AR close to 3.8 did decrease, with the others remaining unaffected except for some increase of nanoparticles with shorter AR, which may result from reshaping of the formers. Thus, at Fth , nanoparticles close to resonance with the laser emission tend to become rounder, which shifts their plasmon oscillations to lower wavelengths and so impairs their potential for PA conversion.

Figure 4: PA signal from sample GNR8 at a) 3.7 mJ/cm2 (< Fth ), b) 5.3 mJ/cm2 (> Fth ) and c) 20 mJ/cm2 (>> Fth ) after 1 (black line), 3 (red line), 10 (green line), 30 (blue line) and 40 (light blue line) laser shots. At higher fluences (e.g. Fexc > 10 mJ/cm2, green and blue lines in figure 3b) we measured an overall damping and blue-shift of the LP peaks with a clear depletion at 810 nm. The corresponding AR distributions (figure 3c, green and blue lines) fell to zero on the high AR side beyond 3.8 (see the blue line in figure 3c). In this range of excitations also those nanoparticles further and further from resonance started to overheat and reshape, likely due to their absorbance tails and the samples became transparent in the NIR region. We note an overall loss of total gold volume, which may result from processes of nanoparticle sublimation and decomposition into ultrasmall fragments with poor plasmon behavior. 33 In figure 4, we show the raw PA signals corresponding to single laser shots generated at fixed nominal fluences (in figure 4a at 3.7 mJ/cm2 < Fth , in 4b at 5.3 mJ/cm2 > Fth , in 4c at 20 mJ/cm2 >> Fth ) for sample GNR8 (the same data for all the samples are reported in figure S11 of the Support12 ACS Paragon Plus Environment

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ing Information). At fluences below the damage threshold, the PA signal is stable and reproducible after 50 pulses, confirming the feasibility of GNRs as PA contrast agents. However, when the fluence becomes larger than Fth , one single laser shot is enough to cause a partial degradation of the GNRs. Depending on Fexc , we observed a decrease by about 25 % for Fexc ≈ Fth (see figure 4b) up to more than 50 % for Fexc >>Fth (see figure 4c). While nanoparticle heating is not cumulative 37 with a repetition rate of 10 Hz, cumulative effects may still arise from incremental transformations from pulse to pulse and statistical jitter. The relative transformation after the second pulse seems to depend on the extent of damage after the first pulse and the probability to trigger additional overheating.

Size effects Figure 5 displays the experimental Fth as a function of nanoparticle effective radius, as obtained from the data of figure S10 in the Supporting Information. The most striking finding is that the value of Fth exhibits a clear trend with nanoparticle size: the smaller the nanoparticles the higher these thresholds. The inset of figure 5 shows the PA response corresponding to a single shot excitation at fluences lower than the damage thresholds for the various samples. The signal to noise ratio (SNR) is always well above unity, even for the sample with the largest nanoparticles (GNR22, light blue in the inset of figure 5) where the damage threshold is the lowest. However the best SNRs are observed in those samples with smaller GNRs thanks to the possibility to use higher fluences (up to 6 mJ/cm2) at comparable optical absorbance. We note that these fluences fall well within the maximal permissible exposure (MPE) for biomedical applications of 30 mJ/cm2 at 800 nm, 52 which emphasizes the importance of our results and calls for additional efforts to improve the nanoparticle stability and enable a full exploitation of the optical parameters.



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Modeling Since a higher surface to volume ratio would suggest a lower intrinsic stability for smaller nanoparticles, which is a common notion in nanoparticle research, 24,41 the origin of our findings may seem counterintuitive and must be found elsewhere. We note that both the absorbance cross section and heat capacity roughly scale with nanoparticle volume, 24,41 i.e. smaller and larger GNRs exhibit the same efficiency of photothermal conversion and would reach the same temperatures in the absence of dissipation. However we conjecture that the larger specific surface area of smaller nanoparticles may translate into much higher thermal coupling to the environment and lesser overheating. 8,53 In order to corroborate this hypothesis, we performed numerical simulations of the photothermal conversion from individual GNRs with the same shape (AR = 3.8) and different effective radii, according to the typical values from figure 1 under individual light pulses of 5 ns at 810 nm and 3 mJ/cm2. Figure 6 displays the evolution of the average nanoparticle temperature with time. Overheating is much higher for larger nanoparticles (dashed dotted dotted light blue line in figure 6), which is believed to trigger their transformation. In the inset, it is shown the 2D temperature map of the smallest GNR at the end of laser exposure. The complete 3D temperature map as a function of time is provided in the Supporting Information. Our interpretation emphasizes the role of thermal coupling to the environment in the photostability of GNRs. A more efficient heat transfer, in this case due to the larger specific surface area of smaller nanoparticles, would lead to a more efficient cooling and so prevent nanoparticle melting or similar effects. Another strategy to enhance the photostability of GNRs consists of the use of rigid shells such as silica. Silanized GNRs seem to benefit from a steric constraint against reshaping. However we note that the higher thermal diffusivity at the interfaces between gold and silica and silica and water, rather than that between gold and water, which was evoked to explain the higher efficiency of photoacoustic conversion from silanized GNRs, 32 may also play a role to enhance their photostability, well in line with our interpretation. Meanwhile, we speculate that the higher local temperatures around larger nanoparticles may enhance their potential to generate nonlinear processes such as bubble formation and cavitation, 14 ACS Paragon Plus Environment

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which will be the subject of future investigations.

Figure 5: Trend of Fth as a function of effective nanoparticle radius. In the inset: comparison of PA response with a single pulse excitation at fluence F

Size Affects the Stability of the Photoacoustic Conversion of Gold

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