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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation Undergraduate Fire Talks

Assessing the size-dependent uptake of gold nanoparticles to develop custom, cell-specific photoacoustic imaging strategies

Saturday, October 11, 2025

8:55 AM - 9:00 AM PDT

Location: Room 30D

Presenting Author(s)

Sheryl A. Loden

Undergraduate Researcher
The University of Virginia
McGregor, Texas, United States

Co-Author(s)

Caroline R. Fox

Graduate Student
University of Virginia
Charlottesville, Virginia, United States

Primary Investigator(s)

Kelsey Kubelick

Assistant Professor
University of Virginia
Charlottesville, Virginia, United States

Introduction: : Photoacoustic imaging (PAI) is a hybrid imaging modality that combines the advantages of light and sound. PA signals are generated by irradiating an imaging target with pulsed laser light to induce local thermoelastic expansion which, in turn, releases sound waves quantifiable by ultrasound transducers. Thus, PAI generates high resolution, high contrast images based on optical absorption at ultrasound imaging depths. Exogenous contrast agents are frequently used to generate enhanced PA signals. Gold nanospheres (AuNSs) are commonly employed due to their strong optical absorption, biocompatibility, and size tunability. AuNS size is an important design parameter with implications for cellular uptake, targeting, and clearance that directly impacts PA properties and imageability. Current PAI research leverages cellular uptake of exogenous absorbers for developing specialized cancer theranostics. For example, AuNSs can be used to label cancer cells for in vivo longitudinal monitoring, image-guided drug delivery, and localized thermal ablation of tumors. In all cases, AuNS size impacts particle uptake or targeting, and hence, downstream PA detection for image-guided therapies. To design optimal cancer cell-specific imaging strategies, there is a need to deeply investigate the effect of AuNS size on cellular uptake and subsequent PAI signal. This study examines the effect of AuNS size on breast, cervical, and colon cancer cell uptake and PA signal generation in order to establish trends describing cellular preferences for AuNSs.

Materials and

Methods: : AuNSs were synthesized using established seed-mediated growth protocols [1]. The volume of seed solution was adjusted to yield AuNSs with different diameters, where 1 mL, 200 uL, 50 uL, and 20 uL of a seed solution yielded 26 nm, 44 nm, 69 nm, and 84 nm diameter spheres, respectively. AuNSs were characterized with dynamic light scattering (DLS), transmission electron microscopy (TEM), and UV-Vis spectroscopy. EO771 breast, HeLa cervical, and MC-38 colorectal cancer cell lines were cultured according to standard protocols. Upon reaching 80 percent confluency, cells were labeled with AuNSs. Prior to labeling, the AuNSs were sterilized under UV light for thirty minutes, and then cells were labeled via 24-hour incubation with AuNSs at an optical density of 1. Next, UV-Vis measurements were performed to confirm cell labeling. After thoroughly washing cells to remove residual nanoparticles, AuNS-labeled cell samples were harvested, fixed in neutral buffered formalin, and resuspended at a constant cell concentration. A tissue-mimicking phantom was prepared for PA imaging. Fixed cells were embedded in gelatin inclusions, with three inclusion replicates for each experimental group. Combined ultrasound and photoacoustic (US/PA) images were acquired with the Vevo LAZR-X system (10.0-22.0 MHz; 680–970 nm). Imaging data was exported for post-processing via Vevo Lab software (version 5.9.0). GraphPad Prism (version 10.4.1) was used to analyze data and generate figures.

Results, Conclusions, and Discussions:: AuNSs were synthesized with diameters of 26 nm, 44 nm, 69 nm, and 84 nm, as confirmed by DLS, UV-vis, and TEM (Fig A, B). Peak optical absorption shifted according to AuNS diameter, as expected. Microscope images confirmed that EO771, HeLa, and MC-38 cells were successfully labeled with AuNSs, indicated by a darker cell coloration in brightfield photomicrographs (Fig C; bottom panel). In PAI experiments in tissue-mimicking phantoms, EO771, HeLa, and MC-38 cells all exhibited a red-shifted PA spectrum (Fig D) due to surface plasmon resonance (SPR), resulting from AuNS coupling due to their close proximity when localized to endosomes after cell uptake [2]. However, each cell line exhibited different PA signatures according to AuNS diameter. At 700 nm wavelength, EO771 cells generated the greatest PA signal when labeled with the smallest diameter AuNSs (26 nm). HeLa and MC-38 cells display the opposite trend, as both cell lines exhibited the greatest PA signal when labeled with the largest diameter AuNSs (84 nm), followed by the 69 nm diameter spheres. PA signals were markedly decreased in HeLa and MC-38 cells when labeled with 44 nm or 26 nm AuNSs. Variation in PA intensity trends among EO771, HeLa, and MC-38 groups suggests AuNS size preferences for uptake unique to each cell line. Enhanced SPR coupling may also explain the increased PA intensity of the 84 nm and 69 nm AuNSs within HeLa and MC-38 cells. For all three cell lines, these data suggest that PA intensity is a function of AuNS diameter, uptake levels, and coupling, which were shown to vary among cell lines. Characterization of these trends allows PAI parameters to be tuned to the unique preferences of cancer cells, ultimately laying the foundation for customizable theranostic imaging solutions.

Acknowledgements and/or References (Optional): : 1) Gao, C., Vuong, J., Zhang, Q., Liu, Y., & Yin, Y. (2012). One-step seeded growth of Au nanoparticles with widely tunable sizes. Nanoscale, 4(9), 2875. https://doi.org/10.1039/c2nr30300k

2) Jain, P. K., & El-Sayed, M. A. (2010). Plasmonic coupling in noble metal nanostructures. Chemical Physics Letters, 487(4–6), 153–164. https://doi.org/10.1016/j.cplett.2010.01.062