Tracking Virus-Like-Particles using Ultrasound-Guided Photoacoustic Imaging

Introduction: : Virus-like particles (VLPs) are increasingly recognized as versatile vaccine and drug delivery platforms due to their biocompatibility, uniform nanoscale architecture, and ability to be chemically modified. VLPs are self-assembling protein-based structures that can be functionalized with imaging agents, antigens, or therapeutic molecules. Efficient accumulation of VLPs in lymph nodes is particularly important for eliciting strong immune responses, as lymph nodes serve as critical sites for antigen presentation and immune cell activation. Despite their clinical potential, the dynamics of VLP trafficking and localization within lymphatic tissues remain poorly understood. Conventional imaging methods, such as in vivo imaging systems (IVIS) and magnetic resonance imaging have been used to evaluate VLP distribution, but they are limited by sensitivity, spatial resolution, and imaging depth. Imaging limitations hinder further efforts to improve delivery strategies and evaluate therapeutic outcomes. Ultrasound-guided photoacoustic (US/PA) imaging offers a powerful alternative by combining the deep tissue penetration and real-time resolution of ultrasound with contrast generated by light-absorbing agents. In this study, we labeled VLPs with a near-infrared dye (NIR) and tracked them to the lymph nodes using US/PA imaging. Data gathered from tracking experiments provide information for the rational design of VLPs with specific physical and chemical properties tailored for different applications.

Materials and

Methods: : T93AHA VLPs were genetically engineered to incorporate azidohomoalanine residues at position 93, allowing site-specific attachment of functional groups. The NIR Cyanine 7 (Cy7) was conjugated to the VLPs using copper(I)-catalyzed azide–alkyne cycloaddition. [1,2] Dye conjugation efficiency, measured as the number of dyes per VLP, was calculated from Cy7 absorbance (UV-Vis spectroscopy) and protein concentration estimated by the Bradford assay. Particle size was determined using dynamic light scattering. All US/PA imaging was conducted using the Vevo LAZR 2100 system (20 MHz transducer, 680–950 nm). For in vitro phantom studies, Cy7-labeled and unlabeled VLPs were embedded in a tube phantom at optical densities (OD = 0, 1, 2, and 4). For ex vivo imaging, Cy7-VLPs were injected subcutaneously near the right hind limb of BALB/c mice, and VLP photostability was evaluated using US/PA imaging. Cy7-VLPs were injected subcutaneously near the right hind limb of BALB/c mice, and imaging was performed at 0 and 24 hours post-injection. Three-dimensional datasets were acquired using a motorized stage (step size: 0.152 mm), with regions of interest including the injection site and the inguinal lymph node. At 24 hours post-injection, lymph nodes were collected and imaged using the IVIS.

Results, Conclusions, and Discussions:: Results and

Discussion:
Dye conjugation was quantified using UV-Vis absorbance and Bradford assay, yielding an average of approximately 20 Cy7 molecules per VLP (Fig. A). Cy7-VLPs had a hydrodynamic radius of 19.1 ± 0.3 nm (Fig. B). In phantom imaging, Cy7-VLPs showed significantly higher PA signal compared to unlabeled controls, confirming the ability to detect Cy-VLPs (Figs. C–D). The particles were photostable, with minimal signal loss after 1,500 laser pulses (Fig. E, F, G). Cy7-VLPs showed clear changes in PA signal intensity within the lymph node over time. By 24 hours post-injection, a substantial increase in PA signal intensity was observed in the lymph node, indicating accumulation of the labeled particles (Fig. H). To further validate the US/PA findings, ex-vivo IVIS imaging was performed on excised lymph nodes. A strong Cy7-VLPs fluorescence signal was detected in lymph nodes from injected animals, while control lymph nodes exhibited negligible fluorescence. (Fig. E).

Conclusion:
This study demonstrates the successful use of US/PA imaging to track Cy7-VLPs to lymph nodes, providing a non-invasive, high-resolution method for monitoring VLP migration and accumulation. The results confirm that Cy7-VLPs are stable, efficiently conjugated, and detectable using US/PA imaging, with significant accumulation in lymph nodes observed 24 hours post-injection. This approach offers a promising strategy for optimizing lymphatic-targeted delivery of therapeutic particles, with potential applications in vaccine and drug delivery systems.

Acknowledgements and/or References (Optional):: [1] Strable. E, Prasuhn.D.E, Udit, A. K, Brown. S, Link. A. J, Ngo. J. T, Lander. G, Quispe. J, Potter. C. S, Carragher. B, Tirrell. D. A, Finn. M. G, Unnatural Amino Acid Incorporation into Virus-Like Particles. Bioconjugate Chem. 2008, 19, 866–875.
[2] Hovlid. ML, Lau. JL, Breitenkamp. K, Higginson. CJ, Laufer. B, Manchester. M, Finn. MG. Encapsulated atom-transfer radical polymerization in Qβ virus-like nanoparticles. ACS Nano. 2014 Aug 26;8(8):8003-14.