Photostability of Gold Nanorods upon Endosomal Confinement in

Article pubs.acs.org/JPCC

Photostability of Gold Nanorods upon Endosomal Confinement in Cultured Cells Lucia Cavigli,† Alberto Cini,‡,§ Sonia Centi,† Claudia Borri,† Sarah Lai,† Fulvio Ratto,*,† Marella de Angelis,† and Roberto Pini† †

Istituto di Fisica Applicata “Nello Carrara”, Consiglio Nazionale delle Ricerche, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze, Italy ‡ Dipartimento di Fisica e Astronomia, Università degli Studi di Firenze, Via G. Sansone 1, 50019 Sesto Fiorentino, Firenze, Italy § ACTISACTIVE SENSORS S.R.L., Via Baldanzese, 17, 50041, Calenzano, Firenze, Italy S Supporting Information *

ABSTRACT: The photoinstability of plasmonic particles remains one remarkable obstacle before their clinical penetration as powerful contrast agents, for instance, in photoacoustic imaging. In particular, gold nanorods easily revert to nanospheres and so lose their best optical features under exposure to few-nanoseconds-long laser pulses. While this issue is attracting much attention and stimulating ad hoc solutions, such as the addition of rigid shells, the biological environment may cause even more instability. For instance, a frequent outcome of the interaction between this type of particles and malignant or immune cells is their tight confinement into endocytic vesicles. In this study, we assess whether this configuration may make an adverse impact on the photostability of gold nanorods, due to the effect of heat confinement. We compare experimental measurements from a limited set of representative samples and verify their relevance by the use of numerical simulations. Under conditions that are typical for photoacoustic microscopy, we estimate the threshold fluence for the onset of photoinstability to remain around 7 mJ·cm−2, independent of the distance among neighboring particles, within accessible limits. Then, we simulate the effect of pulse duration in our model of endocytic confinement. Only in a μs regime of lesser potential for biomedical optics do we predict this configuration to destabilize the gold nanorods, still by as little as 15−20%. Our results span from the femtosecond up to the continuous wave regimes of irradiation and suggest that the biological interface does not pose a major threat on the photostability of plasmonic particles for most biomedical applications, including the photoacoustic imaging and photothermal ablation of cancer.

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of excellent specificity and blood compatibility in vitro.22 However, its translation in vivo is hampered by a complexity of biobarriers,17,24−27 such as the mononuclear phagocyte system, which captures most particles in the liver and spleen. Due to these issues, there are emerging alternative approaches, including the use of cellular vehicles, i.e., immune cells or stem cells that exhibit strong tropism to malignant lesions28−38 and may be isolated from a patient, loaded with plasmonic particles in vitro and then injected back into their host, in order to serve as Trojan horses. Another significant issue is the photoinstability of gold nanorods, as a consequence of overheating and premelting. It is well established that upon absorption of optical power, these particles tend to reshape and become more stable and

INTRODUCTION

The surge of photoacoustic imaging as a hot topic in biomedicine1−5 has gone hand in hand with the development of relevant contrast agents, such as various solutions of plasmonic particles.6−10 In particular, gold nanorods have been proposed as an ideal candidate for photoacoustic applications.11−16 These particles hold a unique combination of intense optical absorbance in the so-called therapeutic window of biotissue, robustness, bioinertness, and ease of modification with biopolymers and molecular probes, which may serve to target malignant tumors by systemic administration. However, the fulfillment of this potential still remains a challenge. Often, the ability of gold nanorods to reach tumors through the bloodstream is delegated to the anomalous permeability and retention of hyperproliferative lesions17−19 and may be enhanced by the use of molecular probes of malignant phenotypes.20−23 This strategy is fostered by the achievement © XXXX American Chemical Society

Received: January 26, 2017 Revised: March 1, 2017 Published: March 1, 2017 A

DOI: 10.1021/acs.jpcc.7b00840 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C rounder,20,39−47 which is accompanied by a blueshift of their plasmonic resonances47 and loss of their optical features in the therapeutic window. Indeed, the plasmonic oscillations of gold nanospheres overlap the optical absorbance of principal biodyes, such as hemoglobin, and exhibit little potential for diagnostics. Reshaping of gold nanorods has been documented in a variety of regimes of irradiation, ranging from femtoseconds-long pulses to continuous waves,20,39−47 yet at optical fluences differing by orders of magnitude, for the most part due to the kinetics of heat dissipation.47 However, even within the same regime of irradiation, there exists a substantial variability from report to report. For instance, in the case of nanosecondslong pulses of principal interest in photoacoustics, the reshaping thresholds that have been reported in the literature range from a few to hundreds of mJ/cm2, which depends on different parameters, such as the size, shape, and coating of the particles, as well as a broad inhomogeneity of measurement criteria.47 The evidence that reshaping may begin well below relevant permissible exposure limits (around 30 mJ/cm2) has motivated efforts to enhance the photostability of gold nanorods by consolidating their shapes and/or accelerating their heat dissipation. For instance, Chen et al. have demonstrated that a shell of silica helps to constrain the shape of prolate particles and maybe also to improve their thermal coupling to an aqueous environment.44,48 Recently, some of us have reported that the size of prolate particles is another key player, with the smaller the particles, the larger their specific surface area and so the better their thermal diffusivity and photostability.47 To our knowledge, none of the previous reports have ever quantified the effect of cellular uptake on the photostability of gold nanorods. Whether it be of malignant cells or cellular vehicles, endocytic capture is a frequent mechanism for the internalization of these particles, which ends in their tight confinement inside intracellular vesicles. This condition holds the risk of causing additional photoinstability due to thermal coupling of nearby particles. Indeed, the effect of heat confinement in endocytic vesicles is exploited to gain more specificity in nonlinear photoacoustic applications, such as cellular microsurgery by photocavitation.49,50 Here, by the interplay of experimental measurements and numerical simulations, we address the effect of endocytic confinement on the photostability of a standard suspension of gold nanorods. More general implications of our work include the relevance of previous measurements of the photostability of homogeneous suspensions of gold nanorods that are intended for biomedical applications.

probably due to Coulombic interactions with their phospholipids.55−57 In our previous work,38 we have documented that the principal mechanism for the internalization of these particles is their endocytic uptake, although the presence of free cytosolic individuals cannot be ruled out.58 More details are given in refs 37, 38, and 59. A macrophagic line was chosen to investigate the effect of the endocytic uptake on the photostability of gold nanorods. J774a.1 murine monocytes/macrophages were purchased from American Type Culture Collection (ATCCTIB-67, Manassas, VA, USA), seeded on plastic culture dishes in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin− streptomycin solution (BioWhittaker, Lonza, Visp, Switzerland) and maintained in an incubator under standard conditions (37 °C, 5% CO2, 100% relative humidity). In brief, 5 × 105 J774a.1 cells were plated in Petri dishes and cultured in complete medium for 24 h and then in serum-free medium, in order to maintain starvation. Appropriate aliquots of polycationic gold nanorods in PBS were diluted in serum-free medium, so as to achieve a final concentration of 400 μM Au, and left in incubation for 24 h. Thereafter, macrophages were harvested, rinsed several times with PBS, and fixed with 3.6% paraformaldehyde in PBS for 10 min at room temperature. Either of polycationic gold nanorods alone or macrophages treated with these particles were embedded into chitosan hydrogels in the shape of thin cylinders with a diameter of 2 cm and a thickness of ∼50 μm.60,61 The volumetric fraction of macrophages in these hybrid samples was around 10%, which is representative of the extent of leukocytic infiltrates in solid tumors.62 Measurement of Photostability Threshold. The photostability threshold from the hybrid hydrogels was investigated by the method that is described in refs 47 and 37. In brief, we used a noninvasive, photoacoustic probe to analyze the transformation of the particles after an optical excitation. In practice, we resorted to a homemade photoacoustic microscope to monitor this transformation by taking the ratio R of the efficiencies of photoacoustic conversion from the various samples after/before irradiation with a train of 50 pulses from an optical parametric oscillator (Continuum Surelite OPO Plus, Santa Clara, USA, wavelength 810 nm, pulse duration 5 ns). A decay of R from unity denotes a photoinstability. In general, a plot of R as a function of the average fluence of the optical excitation starts from 1 and then exhibits a decreasing sigmoid trend, as it is typical for any population with a negative growth rate, which reflects a depletion of particles in resonance with the irradiation. Here, the photostability threshold is set in correspondence to a value of R of 3 , which is significantly lower

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EXPERIMENTAL AND THEORETICAL METHODS Preparation of Samples. Unless it is otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without additional purification. Gold nanorods were prepared by the autocatalytic reduction of chloroauric acid by ascorbic acid in the presence of cetrimonium bromide and silver nitrate, as it is described elsewhere.51,52 In order to gain biocompatibility and to mediate their internalization into cellular vehicles, these particles were modified with mixtures of α-methoxy-ω-mercaptopolyethylene glycol (MW ≈ 5000, Iris Biothech, Marktredwitz, Germany) and (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide. The latter is a quaternary ammonium compound and provides for a polycationic profile that enhances the nonspecific interactions of the particles with plasmatic membranes,38,53,54

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than 1, in statistical meaning, in our setup.37,47 Simulation of Photostability. In an attempt to corroborate our experimental evidence and address its generalizability to more or less plausible configurations of the samples and parameters of their optical excitation, we performed numerical simulations by the use of finite element methods. Overheating over a critical temperature and premelting of the particles were evoked as the principal reason for the onset of photoinstability, as it is widely accepted.45,47,63 An estimate for this critical point was worked out from the interplay of bulk and surface melting,64 which depends on pulse duration, as it is discussed in Supporting Information. In turn, the photostability threshold was defined as the optical fluence that overheats the particles B


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The Journal of Physical Chemistry C

phages were described as arrays of endocytic vesicles with ccp symmetry (see Figure 1b). Also in this case, the ccp arrangement was chosen for the sake of numerical convenience and to respect the symmetry of the relevant interactions. Each vesicle was treated as a heat source, according to Q(t) = σabsNendFf(t), with Nend being the local density by number of the particles (the label “end” denoting samples with endosomes). The entire simulation box was set to the thermal parameters of water, disregarding the transient step at the gold−water interface.72−74 We note that, at variance with its discrete counterpart, this continuous model depends on a larger number of parameters, i.e., the local and global densities of particles and the size and density of the endocytic vesicles. The combination of our discrete and continuous models covers a broad range of length and time scales but fails to describe well the crossover where the interparticle distances, the size of the endocytic vesicles, and the extent of heat diffusion all fall into the same length scale. The principal features of both models are summarized in Table 1.

above their critical point. All simulations were carried out with Comsol Multiphysics (Stockholm, Sweden), which has been used by many authors, in order to describe both the optical features65−70 and the photothermal response47,70−72 from gold nanorods and similar plasmonic constructs. The cross section σabs for the optical absorbance from gold nanorods in resonance with an excitation at ∼800 nm, with their typical aspect ratio (AR) and volume (V), was modeled in the frequency domain, as it is discussed in Supporting Information. The photothermal conversion was solved in the time domain in two different models that may either represent well the heat confinement and thermal coupling among particles or endocytic vesicles. Both models were used to describe both extremes of all particles either confined within endocytic vesicles or free in a homogeneous suspension. We expect that any intermediate configuration, such as the coexistence of particles confined within endocytic vesicles and free in the cytosolic medium, would fall between these extremes. Whenever heat was believed to diffuse over distances in the order of those between particles and much shorter than the size of the endocytic vesicles, we used a discrete model featuring compact arrays of particles with cubic close-packed (ccp) symmetry (see Figure 1a). The ccp arrangement was chosen for

Table 1. Summary of the principal features of both numerical models Discrete nature of particles Finite size of endosomes

Discrete model

Continuous model

Yes No

No Yes

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RESULTS AND DISCUSSION Description of Samples. In this work, we have resorted to the same particles and cellular vehicles that we introduced in ref 38. Elsewhere,37 we reported that the inclusion in chitosan hydrogels is a preservative treatment for the anatomy of macrophagic cells. The inset of Figure 2 displays a transmission

Figure 1. Cartoon of the discrete (a) and continuous (b) models that are used to describe the effects of heat confinement and thermal coupling among particles and endosomes, respectively. The size of the various features and simulation boxes was varied at will.

the sake of numerical convenience and to respect the symmetry of the interactions among particles. As for the values of the interparticle distances, we refer to the discussion in Supporting Information, which takes into account that it is only a minority fraction of all particles to resonate with the optical stimulation. Each particle was treated as a heat source [W m−3], according to Q(t) = σabsFf(t)/V, with F and f(t) being the optical fluence and temporal profile [s−1] of the optical pulse. The temporal profile was assumed to be Gaussian, which is a decent representation of the emission from our oscillator. The medium around the particles was set to the thermal parameters of water, and the gold−water interface was described with the interfacial thermal resistance from refs 73 and 74. We note that whenever this discrete model holds, it is only the local density of particles to matter and additional parameters, such as the size and density of endocytic vesicles, are negligible. Conversely, whenever heat was thought to diffuse over distances much longer than those between particles and, in particular, in the order of the size of the endocytic vesicles, a continuous model was applied, disregarding the discrete nature of the particles. Samples without macrophages were modeled as a homogeneous heat source, according to Q(t) = σabsNw/o endFf(t), with Nw/o end being the global density by number of the particles (the label “w/o end” referring to samples without endosomes). Instead, samples with macro-

Figure 2. Comparison of the spectra of optical extinction of chitosan hydrogels containing gold nanorods alone (broken line) or macrophages loaded with gold nanorods (solid line). Inset: TEM closeup of one macrophage, displaying two endosomes enclosing representative aggregates of gold nanorods (reprinted by permission from Adv. Funct. Mater. 2016, 7178−7185, copyright 2016, Wiley).38

electron micrograph zooming into a representative cell that was cultured with polycationic gold nanorods and confirms that most particles are confined within endocytic vesicles. However, we acknowledge that there may coexist free cytosolic individuals.58 The size of these vesicles is smaller than or on the order of 1 μm and a rough estimate for their mutual distances is shorter than or around 5 μm, as it is discussed in more detail in ref 38. Figure 2 also shows the spectra of optical C


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parameters of the optical excitation, we have run numerical simulations of the photothermal processes that drive the irreversible transformation of gold nanorods. The first set of simulations was intended to understand the generalizability of our findings to other cases of endocytic confinement under the same pulse duration of a few nanoseconds, which is standard in photoacoustics. In a period of 5 ns, the increase of temperature happens to be confined within a few nanometers of the particles. Their discrete nature cannot be disregarded, while the boundaries of the endocytic vesicles hardly play any role because their heat dissipation is irrelevant. Therefore, the photothermal conversion is described well by the discrete model, which implies that the threshold fluence only depends on the local density of particles, while additional parameters, such as the size and density of endocytic vesicles, i.e., the amount of particles per cell or the density of cells, are negligible. Figure 4 displays the dependence of the threshold fluence on the nearest neighbor distance between particles in resonance

extinction of chitosan hydrogels containing a homogeneous dispersion of gold nanorods and macrophagic cells loaded with gold nanorods. We note that the typical plasmonic bands around 520 and 810 nm are well visible in both samples, which suggests that the internalization exerts little effect on the optical features of these particles,38 in spite of their endocytic packing.20,75 In turn, this lack of plasmonic coupling implies that the interparticle distances exceed 10 nm or so,59,76 probably because of the steric hindrance that comes from the hydration of polyethylene glycol. Measurement of Photostability. The shape and the intensity of the photoacoustic signals from chitosan hydrogels containing a homogeneous dispersion of gold nanorods and macrophagic cells loaded with gold nanorods are almost identical. Figure 3a demonstrates the stability of these signals under the fluence that was chosen as a probe for the efficiency of photoacoustic conversion.

Figure 4. (a) Numerical simulation of the threshold fluence for particles in resonance with the optical excitation, as a function of their nearest neighbor distance. As for the precise meaning of these distances, the reader is referred to the discussion in Supporting Information. A square and an asterisk denote the data points that correspond to the cases with and without endosomes, respectively, in our particular experimental conditions. The open black and full blue symbols denote configurations that are sterically possible and impossible, respectively, in the case of a typical suspension of gold nanorods. Insets b and c display examples of thermal maps for a nearest neighbor distance of 130 nm, a fluence of 10 mJ·cm−2, 0.5 and 7.5 ns after the peak of the optical pulse, respectively. The color codes range from RT to 1390 and 105 °C in insets b and c, respectively.

Figure 3. (a) Examples of photoacoustic signals recorded under a regime of photostability for chitosan hydrogels containing gold nanorods alone (broken line) or macrophages loaded with gold nanorods (solid line). (b) Stability of the efficiency of photoacoustic conversion vs optical fluence for chitosan hydrogels containing gold nanorods alone (circles) or macrophages loaded with gold nanorods (squares). Data were fitted to a sigmoid curve (broken and solid lines, respectively) in order to achieve a quantitative definition of a photostability threshold. Error bars were propagated from the fluctuations of the photoacoustic probe.

Figure 3b displays the measurement of the photostability of both samples. The trend is indistinguishable from case to case. For the sample containing gold nanorods alone, a quantitative value for the threshold fluence is (6.9 ± 0.7) mJ·cm−2, which is consistent with our previous findings,47 although in our experience, particles from different batches may exhibit slight discrepancy. For the sample containing macrophagic cells loaded with gold nanorods, the threshold fluence was found to amount to (7.1 ± 0.9) mJ·cm−2. We underline that these values are well below the permissible exposure limits for biomedical applications, which are as high as 30 mJ·cm−2.77 While this observation confirms that the photoinstability of gold nanorods is one limitation for photoacoustic imaging, our data reassure that their integration into cellular vehicles or probably also their uptake from malignant cells does not exacerbate this problem. Simulation of Photostability. In an attempt to corroborate our experimental evidence and to challenge its generalizability for similar configurations of the samples and

with the optical excitation. Below ∼50 nm, particles come into thermal contact and so their melting occurs sooner and sooner as their mutual distances get shorter and shorter. However, above this value, the threshold fluence is found to saturate to a limit of ∼4 mJ·cm−2. The data points that represent our homogeneous dispersion of gold nanorods and our macrophagic cells loaded with gold nanorods are indicated in Figure 4 by the use of an asterisk and a square, respectively. In both cases, our calculation of the interparticle distances takes into account that it is only 1/30 of the gold nanorods in our ensemble to resonate well with the optical excitation, as it is discussed in Supporting Information. The case of the endocytic vesicles corresponds to a density that is 1/10 of a close-packed arrangement of gold nanorods and fits well both with our transmission electron micrographs and with our previous estimate of a number of particles per macrophagic cell of around 105 (each monocyte with a diameter of ∼10 μm D


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The Journal of Physical Chemistry C contains an average of six endocytic vesicles with a mutual distance of 5 μm, and each endocytic vesicle with a diameter of 1 μm may house up to 1.4 × 105 close-packed particles, which is thus about a factor of 10-fold above our previous measurement; see Supporting Information for additional detail).38 Possible particles free in the cytosolic medium would correspond to contributions holding any distance between the asterisk and the square. Since both distances fall in the plateau at ∼4 mJ·cm−2, the confinement of gold nanorods inside endocytic vesicles does not deteriorate their photostability. These results are in qualitative agreement with the experimental evidence that the photostability is the same in both samples. In addition, this conclusion is robust even when the density of the particles in the endocytic vesicles is increased by as much as a factor of 10, which corresponds to the closepacked limit. However, a regime of thermal contact may become accessible with future developments, including a better monodispersity of the gold nanorods, which would downshift the lower limit of sterically possible distances, a higher thermal conductance at the gold−water interface, which would reshape the graph in Figure 4, or the use of unpolarized light, which would change both. Although our simulations neglect relevant effects, such as phase transitions in water,67,71 the absolute value of ∼4 mJ·cm−2 is in reasonable agreement with our experimental measurement as well, especially when it is recalled its derivation from an ideal pattern of particles that are in full resonance with the optical excitation. The second set of simulations was meant to understand whether the lack of effect of the endocytic confinement may apply to more or less similar regimes of optical excitation as well. In this case, we investigated the threshold fluence as a function of pulse duration. We focused on the specific configurations of our two samples containing a homogeneous dispersion of gold nanorods and macrophagic cells loaded with gold nanorods, in terms of typical sizes and distances among particles and endocytic vesicles. We warn the reader that our quantitative estimates become less and less generalizable as more and more steric parameters would enter into play, while moving from the extremes of full heat confinement within the particles, which is independent of any distance, to full heat dissipation over macroscopic length scales, which would depend on the local and global densities of particles and the size and density of the endocytic vesicles. All simulations were performed both in the discrete and in the continuous frameworks. Figure 5 summarizes our calculations. Whenever periodic boundary conditions apply, the threshold fluence saturates at both shorter and longer pulse durations. In the very short limit, heat Q becomes confined within the discrete or continuous heat sources and temperature T becomes independent of pulse Q duration, i.e., T ∼ RT + C with Clocal heat capacity of the

Figure 5. (a) Numerical simulation of the threshold fluence for particles in resonance with the optical excitation as a functions of pulse duration, both for samples containing gold nanorods alone (w/o end.) and for macrophages loaded with gold nanorods (end.), both modeled with the continuous (cont.) and the discrete (discr.) frameworks. The larger black and smaller gray symbols denote our choice of the most and least appropriate frameworks, respectively, with respect to the length scale of heat diffusion. (b) Relative increase of the threshold fluence in samples containing gold nanorods alone with respect to those with macrophages loaded with gold nanorods, as a function of pulse duration. The empty symbol falls in a regime where both the continuous and the discrete frameworks lose accuracy, in the presence of endosomes, and results from a linear interpolation.

these limits, the photostability threshold depends on pulse duration only through the melting temperature, as it is discussed in Supporting Information. Instead, between these limits, the threshold fluence grows with the extent of heat dissipation out of the discrete or continuous heat sources. The best models are the discrete framework at shorter durations, as long as heat remains confined within distances on the order of the size of the endocytic vesicles, and the continuous framework at longer durations, as soon as thermal gradients become negligible between particles. In the case of the homogeneous dispersion of gold nanorods, the crossover between these frameworks is ideal because the discrete model is complete and does not fail to describe any boundary. Conversely, these frameworks do not overlap well in the case of the endocytic vesicles loaded with gold nanorods, when the range of heat diffusion falls between the interparticle distances and the size of the endocytic vesicles. In this window of pulse durations around 1 μs, an estimate of the threshold fluence may be achieved by interpolation, on account of the linearity of all photothermal processes. As a rule of thumb, we note that the threshold fluence displays a sharp dependence on pulse duration but does not change much whether or not particles are clustered in endocytic vesicles. In addition, we postulate that by falling between these two extremes, any intermediate configuration of particles both confined within endocytic vesicles and free in the cytosolic medium or of denser arrays of smaller vesicles would conform to the same trend. Figure 5b shows the relative difference between the photostability threshold in the homogeneous dispersion of gold nanorods and the macrophagic cells loaded

local

heat sources, within the heat sources, and T ∼ RT, elsewhere. In principle, our discrete model in this limit would be generalizable to any configuration of gold nanorods. Conversely, in the very long limit, Q diffuses over length scales that are much longer than those between heat sources and again T Q becomes independent of pulse duration, i.e., T ∼ RT + C global

with Cglobal the heat capacity of the simulation box, everywhere. In this limit, the quantitative relevance of our models is limited to the specific amounts of particles in our samples. Beyond E


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longer, more parameters would enter into play, such as the size and density of the endocytic vesicles. For instance, we have addressed the effect of pulse duration in our representative case of sizes and distances among neighboring vesicles around 1 and 5 μm, respectively, up to the continuous wave regime. In this configuration, we expect the effect of endocytic confinement to remain subtle for any type of irradiation, with a loss of threshold fluence peaking around 20% only in a microsecond regime of lesser interest for biomedical applications. In practice, as for the stability of photoacoustic conversion from plasmonic particles, our findings reassure that the parametrization that is carried out, in most previous work, in standard suspensions and phantoms is likely to remain valid in vitro and in vivo. However, we warn the expert that as progress will enhance the monodispersity of the gold nanorods, say by a factor of 10, or the thermal conductance at the gold−water interface, it will become critical to make sure to assess the photostability of these particles in their biological context.

with gold nanorods. Only for durations between about 100 ns and 100 μs of lesser interest for typical biomedical applications does the endocytic confinement cause a loss of photostability, which still remains within 20% of that of a homogeneous dispersion. The absolute values of the optical thresholds toward the continuous wave regime are compatible with those that we measured in our previous work.45 Also, the remarkable increase of threshold fluence with pulse duration is in qualitative agreement with the variance of previous reports on the transformation of gold nanorods in different regimes of irradiation, ranging from femtoseconds-long pulses to continuous waves.20,39−47 Our prediction insists on the premelting of particles that maintain their optical absorbance until a critical temperature. However, we acknowledge that more subtle and distinct effects may come into play, such as thermal transitions in the aqueous environment,67,71 which may, for example, drive the collapse of the PEG shells59 and trigger plasmonic coupling.20,75 We remind the reader that our quantitative estimates hold universal validity only in the limit of heat confinement in the particles (below about a few nanoseconds) and become less and less generalizable as heat dissipation matches the interparticle distances (probably between a few nanoseconds and 100 ns), where slight variations would occur from case to case of endocytic confinement, and then the size and distances among the endocytic vesicles (say above 100 ns), where more parameters would enter into play, such as the amount of particles per cell, the density of cells, etc. Indeed, in the macroscopic limit of heat dissipation, i.e. in the context of photothermal applications, it would be the overall temperature only rather than the optical fluence to govern the photostability of gold nanorods.45 In an attempt to challenge the validity of our numerical simulations in the continuous wave regime of optical excitation of interest for the photothermal ablation of cancer, we performed the experimental tests that are described in Supporting Information. We show that neither in this case did we observe a loss of photostability upon endocytic confinement of the particles, at least until the thermal decomposition of the chitosan hydrogel60 began.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.7b00840. Detailed description of our simulation of the optical absorbance of a gold nanorod, our calculation for the density of particles in the various samples, our model of the critical temperature for surface melting of a gold nanorod, and our experimental assessment of the photostability of the various samples in a continuous wave regime of optical excitation (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Lucia Cavigli: 0000-0002-6279-8869 Fulvio Ratto: 0000-0001-6721-9486

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Notes

The authors declare no competing financial interest.

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CONCLUSIONS Our results confirm that the photostability of gold nanorods for photoacoustic applications is a limiting factor because its threshold fluence falls much below relevant permissible exposure limits that are around 30 mJ·cm−2. Overheating and reshaping into rounder particles are established as the principal reason for the photoinstability of gold nanorods, as well as similar plasmonic alternatives, including nanoshells, nanocages, etc. Here, we have compared the photostability of PEGylated gold nanorods with a polycationic profile before and after phagocytic uptake from macrophagic cells, with the fear that their tight aggregation within endocytic vesicles may cause additional instability, due to the effect of heat confinement. We have found a loss of efficiency of photoacoustic conversion to begin above ∼7 mJ·cm−2 in both cases, with indistinguishable behavior. We have corroborated this evidence by the use of FEM simulations of the optical and photothermal response from different configurations of gold nanorods. In the context of optical pulses shorter than a few nanoseconds, i.e., in the domain of photoacoustic imaging, the endocytic confinement exerts little effect on the photostability over a broad range of interparticle distances. As the optical pulses get longer and

ACKNOWLEDGMENTS We wish to thank Dr Matthew Tippett-Vannini for proofreading our manuscript. This work was partially supported by Regione Toscana and European Commission within the frame of the ERANET+ Projects LUS BUBBLE and by the Italian National Flagship Project NANOMAX-ENCODER.

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