Nanoparticle-Augmented Photoacoustic Imaging Enables Calibration-Free Temperature Monitoring in Tissue

Introduction: : Thermal therapies hold great promise for cancer treatment by enabling tumor ablation, tumor microenvironment modulation, and controlled drug delivery. However, their clinical efficacy depends critically on accurate, real-time temperature monitoring. While techniques such as magnetic resonance (MR), ultrasound (US), and photoacoustic (PA) imaging have been employed, their reliance on tissue-specific properties limits their ability to provide calibration-free, reliable temperature measurements across heterogeneous tumor environments. We demonstrated that nanoparticle (NP)-mediated PA thermometry can achieve calibration-free temperature sensing by stabilizing the local aqueous environment around the NPs. Building on this foundation, we aim to extend NP-enhanced PA thermometry to complex tumor models. Thus, this approach could enable accurate, calibration-free temperature mapping across tumor tissues, advancing the precision and effectiveness of thermal therapies.

Materials and

Methods: : Gold nanospheres (AuNSs) were synthesized via sodium citrate reduction and functionalized with mPEG-SH. The PEGylated AuNSs (PEG-AuNSs) were imaged using transmission electron microscopy (TEM; HT7700, Hitachi). To assess their PA thermal response in different environments, a tube phantom was prepared using polyethylene tubes filled with PEG-AuNSs at an optical density (OD) of 2 and olive oil concentrations of 0% (water only), 10% (low-fat), or 50% (high-fat). Tubes were sealed, mounted in a custom holder, and submerged in a water-filled container heated by a temperature-controlled circulator (Isotemp 3013H, Thermo Fisher). Temperature near the imaging plane was monitored with a thermocouple. PA images were acquired using the Vevo® LAZR system (Fujifilm VisualSonics) at three cross-sections, with 75 frames per temperature. To model NP-augmented PA thermometry in tissue, RAW 264.7 macrophages were labeled via overnight incubation in AuNS-containing media (OD = 2) and fixed in 10% neutral buffered formalin. Labeled cells were embedded in tissue-mimicking phantoms consisting of a polyacrylamide base with rectangular inclusions. Each inclusion contained AuNS-labeled cells (1k cells/μL) and olive oil at 0%, 10%, 30%, or 50% (v/v). After degassing and mixing, mixtures were cast into phantom voids and refrigerated until solidified. Imaging followed the same protocol as for tube phantoms, with 100 PA frames acquired per temperature at three cross-sections. Image analysis was performed in MATLAB (MathWorks). PA signal amplitude was averaged within manually defined regions of interest (ROIs), normalized to baseline, and fitted to temperature via linear regression to extract the thermal response slope.

Results, Conclusions, and Discussions:: AuNSs were synthesized and PEGylated to enhance colloidal stability in physiological environments, such as aqueous and lipid-rich media. TEM imaging confirmed the morphology of PEG-AuNSs (Figure 1A). To assess the temperature dependence of their PA signal, PEG-AuNSs were suspended in water, low-fat (10% olive oil), and high-fat (50% olive oil) solutions. Across 33–43 °C, all samples exhibited similar linear increases in PA signal with temperature (Figure 1B). Linear regression slopes of normalized PA signal versus temperature showed no significant difference among groups (n=3, Figure 1C). These results indicate that PEG-AuNSs retain a water-based local environment, resulting in similar PA thermal response regardless of the surrounding bulk composition.
To evaluate this behavior in a more biologically relevant context, AuNSs were internalized by macrophages and embedded in tissue-mimicking phantoms containing 0%, 10%, 30%, or 50% olive oil (Figure 1D). PA signals from AuNS-labeled cells also increased linearly with temperature across all inclusions (Figure 1E), and slopes of PA signal change were not significantly different from the water-only control (n=3, Figure 1F). These results support that appropriate nanoparticle surface chemistry enables NPs to retain aqueous surrounding layers, preserving their PA thermal response even in lipid-rich tissue background.
These findings highlight the potential of NP-mediated, calibration-free PA thermometry for guiding thermal therapies, particularly in heterogeneous tissues where composition is unknown. However, in deep tissues, challenges arise from light scattering and acoustic attenuation that can significantly reduce PA signal strength and signal-to-noise ratio (SNR), limiting temperature sensing accuracy. Therefore, further investigation including in vivo demonstration is required for proving the reliability of the method for guiding the thermal therapies of solid tumor with complex biological tissue properties.

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