Regulating Interparticle Proximity in Plasmonic Nanosphere Aggregates to Enhance Photoacoustic Response and Photothermal Stability

Designing plasmonic nanoparticles for biomedical photoacoustic (PA) imaging involves tailoring material properties at the nanometer scale. A key in developing plasmonic PA contrast nanoagents is to engineer their enhanced optical responses in the near‐infrared wavelength range, as well as heat transfer properties and photostability. This study introduces anisotropic plasmonic nanosphere aggregates with close interparticle proximity as photostable and efficient contrast agents for PA imaging. Silver (Ag) is particularly attractive because it has the strongest optical response and highest heat conductivity among plasmonic metals. The results demonstrate that close interparticle proximity in silver nanoaggregates (AgNAs), spatially confined within a polymer shell layer, leads to blackbody‐like optical absorption, resulting in robust PA signals through efficient pulsed heat generation and transfer. Additionally, the AgNAs exhibit a high photodamage threshold highlighting their potential to outperform conventional plasmonic contrast agents for high‐contrast PA imaging over multiple imaging sessions. Furthermore, the capability of the AgNAs are demonstrated for molecular PA cancer imaging in vivo by incorporating a tumor‐targeting peptide moiety.

[16][17][18] Silver (Ag) possesses favorable characteristics for generating enhanced PA signals and contrast, due to its robust surface plasmon resonance effect in the 300-1200 nm wavelength range, along with the highest thermal conductivity among plasmonic metals. [19]Various potential forms of AgNPs exist, and anisotropic nanostructures characterized by a high aspect ratio, such as nanoplates or nanorods, are frequently utilized to produce high PA signals due to their large absorption cross-sections within the NIR window. [18,20,21][23][24] PA imaging typically uses high-intensity laser pulses over 10 mJ cm −2 to increase PA signals by maximizing fluence of laser light.[27][28] Therefore, developing AgNPs with a high photodamage threshold and absorption cross-section in the NIR window is crucial for high-contrast PA imaging over multiple imaging sessions.
[31] When separate plasmonic NSs come into close interparticle proximity with one another, plasmon mode hybridization occurs, resulting in a redshift of the peak optical absorption towards longer wavelengths. [30,31]he intensity of the optical absorption for the coupled NSs is determined by the number of NSs and the interparticle spacing. [30,31]Therefore, the creation of anisotropic AgNS aggregates (AgNAs) that resemble porous anisotropic nanostructures can achieve strong NIR-light absorption via interparticle plasmon coupling along the long axis, i.e., anisotropic plasmon coupling.[37][38][39] Nevertheless, controlling the structural homogeneity, aggregate size, and interparticle spacing between adjacent plasmonic NSs within plasmonic NAs remains challenging.
Here, we introduce a strategy for creating anisotropic Ag-NAs, composed of small, photostable AgNSs for multi-session PA imaging with high imaging contrast.Our approach leverages polymer template-mediated growth to consistently control the size and interparticle spacing of AgNSs in AgNAs.[42] Our results indicated that interparticle proximity between adjacent AgNSs in AgNAs can control absorption responses at NIR wavelengths, regulating PA signal generation.We also showed that AgNAs exhibited higher PA signals than counterpart AuNAs by promoting heat transfer into the local environment.Furthermore, AgNAs had excellent photostability, showing the potential of the AgNAs to outperform existing plasmonic contrast agents for high-contrast PA imaging across multiple imaging sessions.We finally validate the utility of the AgNAs for in vitro and in vivo PA imaging.

Development of AgNAs with Varying Interparticle Proximity
We hypothesized that the development of anisotropic AgNAs, composed of small AgNSs, will enhance PA signal amplitude, and therefore enable high-contrast PA imaging for two main reasons: 1) interparticle plasmon coupling between proximate AgNSs along the long axis of AgNAs will maximize the absorption cross-section in the NIR optical window, and 2) Ag has the highest heat conductivity among plasmonic metals to promote heat transfer into the surrounding medium.
To create anisotropic AgNAs and to control interparticle proximity between neighboring AgNSs, we developed a seedmediated growth approach based on in situ template-mediated growth utilized to achieve nanoaggregated plasmonic NPs. [40,43]eed-mediated Ag growth of 2 nm-sized Au seeds within an empty ellipsoid nanospace (40 nm × 200 nm) covered by a resorcinol-formaldehyde polymer layer was carried out to form AgNAs (Figure S1, Supporting Information).The AgNAs were intentionally designed to have a size of less than 300 nm to enhance their ability to circulate in the bloodstream for an extended period of time. [44,45]The Au seeds within the ellipsoid nanospace were utilized to initiate conformal Ag growth with negligible influence on optical properties of AgNAs (Figure S2, Supporting Information).Since optical responses of AgNAs in the NIR wavelength range are affected by interparticle proximity between neighboring AgNSs in AgNAs, it is essential to create closely packed AgNSs within the ellipsoid space during the Ag growth process, while preventing self-nucleation of free Ag-NPs outside the space.For the uniform Ag growth of the seeds within the space, polyvinylpyrrolidone (PVP) and sodium hydroxide (NaOH) were utilized as a capping agent and reagent, respectively, to control the reduction rate of Ag ions [46][47][48] (Figure 1a; Figure S3, Supporting Information).
To understand the role of the Ag reduction rate on the Ag growth of the seeds within the ellipsoid space covered by the polymer layer, the concentration of NaOH in the growth process was altered. [48]A fast reduction rate of Ag ions in the growth step with a high NaOH concentration (69 mM) induced free nucleation of AgNPs outside the ellipsoid space, while a slow reduction rate of Ag ions with a low NaOH concentration (18 mM) led to a uniform seed growth of the seeds without generating any free AgNPs (Figure 1b).The AgNS diameter of AgNAs obtained using a low NaOH concentration in the growth process was larger than AgNS diameters obtained from moderate and high NaOH concentrations (36 mM and 69 mM, respectively), due to the uniform growth of AgNSs (Figure 1c; Figure S4, Supporting Information).Furthermore, the larger diameter AgNSs in AgNAs exhibited strong absorption at NIR wavelengths compared to other AgNAs due to the enhanced interparticle plasmon coupling based on closer interparticle proximity between nearby AgNSs (Figure 1d).
To fabricate AgNAs with adjustable interparticle proximity between nearby AgNSs without the formation of free AgNPs, we modulated the diameter and interparticle distances of AgNSs by modulating the added volume of Ag precursors (from 100 μL to 500 μL) in the growth process to synthesize different AgNA formulations, designated as AgNA-1, AgNA-2, AgNA-3, and AgNA-4, respectively (Figure 1e; Figure S5, Supporting Information).By increasing the added Ag precursors in the growth process, the average AgNS diameter inside the ellipsoid space increased from 6.6 nm to 10.6 nm, decreasing the interparticle distances from 14.3 nm to 10.6 nm (Figure 1f,g; Figure S5, Supporting Information).Specifically, AgNA-3 and AgNA-4 displayed a similar diameter and interparticle spacing of AgNSs (Figure 1f,g).The inability of AgNA-4 to achieve closer interparticle distances than AgNA-3 could be attributed to strong repulsive forces between proximate AgNSs, imposed by electrostatic interaction and steric hindrance. [49,50]A low magnification TEM image confirmed the structural homogeneity of AgNAs (Figure S6, Supporting Information).UV-vis-NIR spectroscopy indicated that as the interparticle proximity became closer in AgNAs, the NIR light absorption increased due to enhanced interparticle plasmon coupling, which was reflected through colorimetric observation of AgNAs (Figure 1h,i).Overall, our seeded growth strategy can control the sphere diameter and interparticle spacing in anisotropic AgNAs, providing us with fine control over their optical characteristics.

Influence of The Interparticle Proximity between Neighboring AgNSs on PA Signal Generation
Due to the different proximities between AgNSs, all AgNAs exhibited different optical absorption characteristics (Figure 1).We expected that PA responses from AgNAs would also differ because PA signal amplitude is proportional to the optical absorption intensity of the NPs. [3,14]To validate this relationship, we prepared each AgNA solution with the same solvent (water), solution volume, and Ag mass concentration (adjusted to the added Ag ions in the growth process) and compared PA signals generated by each AgNA (Figure 2a).Results showed that the close interparticle proximity between adjacent AgNSs (AgNA-3 and AgNA-4) exhibited stronger PA signal amplitudes compared to AgNA-1 and AgNA-2.Moreover, AgNA-3 exhibited slightly higher PA signal amplitudes compared to AgNA-4 although the difference was not statistically significant (Figure 2b; Figure S7, Supporting Information).
In theory, PA signal amplitude from NP solutions is the product of the Grüneisen parameter, laser fluence, and absorption coefficient.14] In our PA experiment, because we kept the NP solvent and laser fluence same, the only different variable between each AgNA sample (AgNA-1, AgNA-2, AgNA-3, and AgNA-4) is their optical absorption coefficient, which is directly associated with pulsed heat generation from AgNAs under pulsed laser illumination. [9,14,15]To estimate optical absorption coefficients of the AgNAs, we computationally calculated their optical extinction, absorption, and scattering crosssections within the 680-900 nm spectral range using the finitedifference time-domain (FDTD) numerical method (Figure 2c-f).Our simulation results showed that, as the mean diameter of AgNSs increased with decreasing interparticle distances in Ag-NAs, the absorption cross-section within the 680-900 nm wave-length range was significantly increased due to more efficient interparticle plasmon coupling (Figure 2c-f).Furthermore, AgNA-3 and AgNA-4 exhibited comparable optical absorption crosssections within the NIR spectral range (Figure 2e,f), attributed to their similar diameter and interparticle spacing of AgNSs in AgNAs (Figure 1f,g).
Next, the absorption coefficient for each AgNA was calculated in the 680-900 nm spectral range, based on the calculated absorption cross-section for all AgNAs.The absorption coefficient for each AgNA is directly proportional to the absorption cross-section per unit Ag mass because the Ag mass concentration in all AgNA solutions was maintained (see details in Note S2, Supporting Information).Results showed that AgNA-3 had the highest absorption cross-sections per unit Ag mass at 680-900 nm wavelengths among all AgNAs (Figure 2g).Our computational analysis indicates that when Ag mass (or atomic) concentration of the AgNA solutions is identical, AgNA-3 can exhibit stronger optical absorption via more efficient interparticle plasmon coupling in the NIR optical window, resulting in more efficient pulsed heat generation, compared to the other AgNAs (Figure 2h).This computational result matches our PA result (Figure 2b).These results confirm that AgNA-3 has desirable interparticle proximity between adjacent AgNSs to produce higher PA signal amplitude, compared to the other AgNAs.

Influence of The Heat Transfer Properties of Plasmonic NAs on PA Signal Amplitude
Next, we hypothesized that PA signal generation from plasmonic NAs will be impacted by the heat transfer properties of building block NSs. [9,14,17]To test this hypothesis, we fabricated plasmonic NAs composed of materials with different heat conductivities (Figure 3a).For this experiment, the counterpart NA, AuNA-3, was created using Au spheres for two reasons: 1) Au has the second highest heat conductivity among plasmonic metals; and 2) in the presence of PVP, the reduction rate of Ag ions is similar to that of Au ions [46,47] to create plasmonic NAs with similar sphere diameter and interparticle spacing in the growth process.TEM analysis confirmed that the plasmonic NAs, including AgNA-3 and AuNA-3, had nearly the same sphere diameter and interparticle spacing, i.e., similar interparticle proximity between neighboring NSs within a single NA (Figure S8, Supporting Information).The optical spectra of AgNA-3 and AuNA-3 were determined to be in the 680-900 nm wavelength range via UVvis-NIR spectroscopy and FDTD simulations (Figure 3b,c).Results showed that AuNA-3 had a slightly larger absorption crosssections than AgNA-3 (Figure S9, Supporting Information).We then analyzed PA signals from the two different NAs within the 680-900 nm spectral range while keeping the same solvent (water), NA concentration, laser fluence, and solution volume.Despite the lower optical absorption cross-section of AgNA-3 compared to AuNA-3, AgNA-3 exhibited approximately 30% higher PA signals than AuNA-3, which seemed to be contradictory to the conventional PA theory [1][2][3][4] (Figure 3d; Figure S10, Supporting Information).
Under pulsed illumination of an NP solution, laser photons are primarily absorbed by the NPs rather than the solvent, resulting in pulsed heat dissipation. [9,51]Due to time-dependent thermal leakage, PA signal from the NP solution is influenced by the rate of heat transfer from the NPs to the surrounding medium. [14,52]In Fourier's law of thermal conduction from a construct to the surrounding environment, the rate of heat transfer is determined by the heat conductivity of the NPs, heat transfer area, and temperature variation of the construct within a medium.In our PA experiment involving AgNA-3 and AuNA-3, the only difference between those NAs lies in their construct composition.Therefore, the factors governing the rate of pulsed heat transfer are their heat conductivity and temperature variation of the NAs within the medium under pulsed laser illumination [53][54][55] (see details and equations in Note S2, Supporting Information).While AgNA-3 exhibits an ≈ 10% lower temperature increase under pulsed laser illumination than AuNA-3 due to the lower absorption cross-section of AgNA-3, [55,56] the heat conductivity of Ag is 40% higher than the heat conductivity of Au.Thus, due to the high heat conductivity of Ag, AgNA-3 can exhibit an ≈ 30% higher rate of heat transfer than AuNA-3.This difference in the heat transfer properties between AgNA-3 and AuNA-3 agreed with our PA results (Figure 3e).Our results indicate the importance of components in the design of plasmonic NAs for enhancing PA signal amplitudes by modulating the NAs' heat transfer properties.

High Photodamage Threshold of AgNAs for PA Imaging Over Multiple Imaging Sessions
Compared to conventional plasmonic contrast agents, such as AuNRs, AgNAs have two promising characteristics that reduce their susceptibility to photodamage under pulsed laser illumination.First, an aggregate comprised of photostable, small-sized NSs will have a higher photodamage threshold than anisotropic NPs. [22,57]Second, the porous structure of AgNA-3 will decrease the diffusion coefficient, [32][33][34] which results in a slow diffusion of Ag atoms and a high photodamage threshold under pulsed laser illumination.
To estimate the photodamage threshold of AgNAs, the solution of AgNA-3 (hereafter, denoted as AgNA) was illuminated for 1000 laser pulses at different laser fluences (5 mJ cm −2 , 10 mJ cm −2 , 20 mJ cm −2 , and 30 mJ cm −2 ).Results showed that Ag-NAs exhibited no changes in structural and optical characteristics and generated stable PA signals linearly increased with laser fluence up to 20 mJ cm −2 (Figure 4a-d; Figure S11, Supporting Information).At the laser fluence of 30 mJ cm −2 , a particle coalescence between proximate AgNSs was observed, resulting in optical absorption decreases and PA signal decay of ≈ 40% in the NIR window after 1000 pulses (Figure 4a-c; Figure S11, Supporting Information).Nonetheless, the photostability of AgNAs up to 20 mJ cm −2 was superior compared to other existing plasmonic PA contrast agents, including AuNRs, [58] Au nanostars, [59] Au nanoprisms, [26] and silica-coated AuNRs, [28] which are typically photodamaged around 10 mJ cm −2 , meaning that AgNAs had a 100% higher photodamage threshold (Table S1, Supporting Information).Our results demonstrate the potential of AgNAs for high-contrast PA imaging over multiple imaging sessions in the NIR optical window.

In Vitro and In Vivo PA Cancer Imaging by Employing RGD-Functionalized AgNAs
We tested the capability of AgNAs for biomedical PA imaging of cancer both in vitro and in vivo.We first functionalized AgNAs with arginine-glycine-aspartic acid (RGD) tripeptides to recognize the integrin  v  3 receptor overexpressed in various cancer cell lines (Figure S12a, Supporting Information). [60,61]Serial changes of the surface charges of AgNAs validated successful RGD functionalization on the surface of AgNAs (Figure S12b, Supporting Information).Furthermore, AgNAs did not show any changes in their morphological characteristics, including the diameters and interparticle distances of AgNSs, under low pH conditions.This suggests that AgNAs can maintain their structural stability in an acidic tumor environment [62] (Figure S13, Supporting Information).Next, we carried out in vitro PA imaging of MDA-MB 231 breast cancer cells labeled with RGDfunctionalized AgNAs (Figure 5a).AgNAs did not cause any significant cell death up to a concentration of 50 μg mL −1 (Figure S14, Supporting Information).Labeled cells were embedded in a gelatin-based tissue-mimicking dome-shaped phantom for PA imaging.Labeled cells displayed considerably higher PA signal amplitudes than unlabeled cells (Figure 5b,c).Labeled cells were even detected at low concentrations of approximately 20 cells μL −1 in vitro (Figure 5d,e).PA signals from the labeled cells did not show any signal decay over 1400 laser pulses (Figure S15, Supporting Information).Moreover, AgNA-labeled cancer cells exhibited significantly stronger PA signal generation compared to AgNS-labeled cancer cells (Figure S16, Supporting Information).These in vitro results demonstrate the capability of AgNAs for PA cell imaging with high imaging contrast.
Finally, we demonstrated in vivo PA imaging of the AgNAs.As a preliminary study, AgNAs were injected via the tail vein and the accumulation of AgNAs was monitored in the spleen where NPs accumulate following systemic delivery (Figure S17, Supporting Information).After AgNA injection (24 h), PA signal amplitude at 700 nm wavelength increased, indicating that AgNAs could be detected with high imaging contrast (Figure S17a-c, Supporting Information).In vivo results from spleen imaging showcased the feasibility of monitoring the passive accumulation of AgNAs using US/PA imaging.Moreover, the biodistribution of Cy5-conjugated AgNAs was evaluated.Following systemic injection in vivo, major organs were harvested and analyzed by fluorescence imaging 24 hours later (Figure S18, Supporting Information).In addition, a histopathological analysis of the organs was carried out to assess potential tissue inflammation, damage, or lesions 24 hours after in vivo administration of AgNAs.Histology showed no signs of toxicity in the major organs treated with AgNAs compared to the saline-treated control (Figure S19, Supporting Information).Subsequently, we conducted a more intricate in vivo demonstration by monitoring the accumulation of targeted, RGD-coupled AgNAs for PA cancer imaging in MDA-MB 231 tumor-bearing mice (Figure 5f).In vivo US/PA imaging showed a 10-fold increase in PA signal at the tumor site for the mice that received RGD-coupled AgNAs compared to the background control (Figure 5g,h).Collectively, these results demonstrated the potential of AgNAs to serve as an exogenous contrast agent for high-contrast PA imaging in vivo.

Conclusion
We created plasmonic contrast nanoagents that exhibit strong optical absorption in the NIR wavelength range and high heat conductivity, thus allowing for prolonged PA imaging with high contrast.Specifically, anisotropic AgNAs consisting of small-sized AgNSs with tunable interparticle proximities were developed to control the optical properties of the AgNAs in the NIR wavelength range based on different interparticle plasmon coupling.Results showed that AgNAs with close interparticle proximities produced high PA signals due to the enhanced interparticle plasmon coupling between proximate AgNSs and the highest heat conductivity of Ag among plasmonic metals.We also investigated material composition of the NAs and showed the enhanced PA signal generation from AgNAs compared to AuNAs.We further validated the photodamage threshold of our AgNAs.The photostability of our AgNAs surpassed conventional exogenous contrast agents for longitudinal PA imaging by showing a higher photodamage threshold of the AgNAs.Finally, using RGD-coupled AgNAs as a targeted imaging contrast agent, in vivo PA imaging visualized the accumulation of RGD-coupled AgNAs at the tumor site with high imaging contrast, demonstrating the potential for molecular imaging of targeted AgNAs.In the future, the structural and physical parameters, such as size, shape, polymer, and composition, of our plasmonic NAs can be further controlled to create a PA contrast agent with desired optical and thermal characteristics.Furthermore, this flexibility in manipulating structural and physical parameters of nano-sized aggregates offers the potential to customize the physicochemical characteristics of nano-sized aggregates for biomedical applications to ensure optimal biocompatibility.Our future research will extend to assessing the longterm systemic toxicity and clearance pathway of diverse NA compositions.

Figure 1 .
Figure 1.Synthesis of AgNAs with tunable interparticle proximity via a seed-mediated growth approach within an ellipsoid space.a) A seed-mediated growth step to modulate interparticle proximity between neighboring AgNSs in AgNAs.b) TEM images of AgNAs synthesized at different concentrations of the reducing agent in the seed-mediated growth process.c) Corresponding schematic of the effect of the reduction rate on the Ag growth within the ellipsoid space in AgNAs.d) Corresponding UV-vis-NIR spectra of AgNAs fabricated at different reduction rates.e) TEM images of AgNAs with tunable interparticle proximities by modulating the added Ag ions in the growth process.f, g) Corresponding quantifications of the diameters of AgNSs and interparticle distances in AgNAs.h) UV-vis-NIR spectra of AgNAs.i) Colorimetric images of AgNAs.The scale bars are 200 nm.Data represents the mean ± standard deviation (n = 32).

Figure 2 .
Figure 2. Effect of interparticle proximity of AgNAs on PA signal generation.a) A schematic to illustrate the experimental design to investigate PA responses from AgNAs with different interparticle proximities.b) PA signal amplitudes from AgNAs with different interparticle proximities in the 680-900 nm spectral range.c-f) Extinction, absorption, and scattering cross-sections of different AgNAs in the 680-900 nm spectra range calculated via a finite-difference time-domain solution.g) Calculated absorption cross-sections per unit Ag mass for different AgNAs in the 680-900 nm spectral region.h) Proposed mechanism of PA signal generation from AgNAs with different interparticle proximities.Data represents the mean ± standard error (n = 8).

Figure 3 .
Figure 3.Effect of the composition of plasmonic NAs on PA signal generation.a) A schematic to illustrate the experimental design to investigate PA responses from plasmonic NAs with different compositions.b, c) Experimental and theoretical extinction spectra of the plasmonic NAs in the 680-900 nm spectral range, corresponding to PA imaging wavelengths.d) PA signals from AgNA-3 and AuNA-3 in the 680-900 nm spectral range.e) Proposed mechanism of PA signal generation from plasmonic NAs with different material compositions.Data represents the mean ± standard error (n = 8).

Figure 4 .
Figure 4. Estimation of photothermal stability of AgNAs (AgNA-3) under pulsed laser illumination at different laser fluences.a) TEM images of AgNAs before and after 700 nm pulsed laser illumination for 1000 pulses at different laser fluences.Scale bars are 200 nm.b) Schematic of the structural transformation of AgNAs under pulsed laser illumination.c) PA signals from AgNA-3 under pulsed laser illumination (700 nm) for 1000 pulses at 5 mJ cm −2 , 10 mJ cm −2 , 20 mJ cm −2 , and 30 mJ cm −2 .d) PA amplitudes of AgNAs as a function of laser fluence.Data represents the mean ± standard deviation (n = 3).

Figure 5 .
Figure 5.In vitro and in vivo US/PA imaging using AgNAs (AgNA-3).a) Schematic of in vitro US/PA cell imaging using a dome-shaped tissue-mimicking phantom containing MDA-MB 231 cells labeled with RGD-coupled AgNAs.b, c) US/PA images (700 nm) and PA spectra of the dome phantoms containing the unlabeled and labeled cells.d, e) US/PA images and PA amplitudes (700 nm) from the dome phantoms containing labeled cells with different cell densities.f) Schematic of in vivo US/PA cancer imaging using RGD-coupled AgNAs.g, h) in vivo US/PA imaging (700 nm) and PA spectra of the tumor region before and after the intravenous injection of RGD-coupled AgNAs.The scale bars are 4 mm.Data represents the mean ± standard deviation (n = 3) for in vitro and in vivo experiments.