Info
Background:
Dendritic cell (DC) immunotherapy is a promising strategy in cancer treatment due to its ability to initiate and regulate tumor-specific immune responses. By presenting tumor antigens and activating T cells, DCs can induce durable antitumor immunity. However, clinical efficacy remains limited by three major challenges [1-3]. First, there is a lack of non-invasive methods to monitor DC migration and biodistribution in vivo, limiting verification of DC delivery to target sites. In addition, administered DCs exhibit inefficient trafficking to lymph nodes, where antigen presentation and T cell priming occur, substantially reducing therapeutic impact. Overcoming these barriers is essential to realize the full therapeutic potential of DC-based immunotherapy.

Hypothesis/Objective:
We propose a nanotechnology-enabled platform for image-guided delivery of DCs. To enable image-guidance, we employ longitudinal, noninvasive ultrasound and photoacoustic imaging to enable dynamic assessment of DC biodistribution. To improve delivery and address the limitation of poor DC trafficking, a magnetic homing strategy actively encourages DC migration and retention toward target tissues, i.e. lymph nodes or primary tumor, upon exposure to an external magnetic field.

Methods:
Gold nanorods (AuNR) and iron nanospheres (FeNS) were synthesized in-house to enable PA imaging and magnetic delivery, respectively. DC 2.4 cells were co-incubated with both nanoparticles at varying optical density ratios of AuNR/FeNS. Following overnight incubation, residual particles were removed by washing with phosphate buffered saline. The remaining dual-labeled DCs (Fig. A) were harvested for downstream analysis and experiments. In vitro cell viability was assessed using an MTT assay. Magnetic responsiveness was quantified using a cell‑sorting based magnetic pulling assay. Dual-labeled DCs were suspended in solution in a microcentrifuge tube. An external magnetic field was applied to the tube (Miltenyi MACs separator) for ~5 mins, followed by counting of trapped cells. Finally, dual-labeled DCs were prepared in a gelatin phantom for assessments of in vitro image-guided magnetic delivery. Dual-labeled DCs were embedded in inclusions, where % gelatin varied to alter viscosity. An external magnet (NdFeB, ~5 lbs pull force) was placed near the inclusion. US/PA images were acquired with the FUJIFILM Visualsonics Vevo LAZR-X (UHF 29X transducer) at λ=680-970 nm. Data was exported for post-processing using the built-in VevoLAB software and MATLAB.

Results:
Transmission electron microscopy (TEM) confirmed successful synthesis or AuNRs and FeNSs. Dual-labeling was verified by bright field microscopy. The MTT assay study showed high cell viability, confirming cytocompatibility of the dual labeling approach. The magnetic separation assay demonstrated increasing accumulation of magnetically responsive cells with increasing labeling density of FeNS. The result demonstrates feasibility of magnetic trapping of DCs in a simple, fluid environment (Fig. B). To more rigorously demonstrate potential of image-guided magnetic delivery, dual-labeled DCs were embedded in a viscous, tissue-mimicking phantom, followed by application of an external magnet. Dual-labeled DCs generated strong PA signals at 900 nm wavelength (Fig. C). Furthermore, US/PA imaging results conveyed cellular migration toward the magnet, confirming photomagnetic properties. Differences in migration and localization at the magnet were observed according to inclusion viscosity.

Conclusion:
These in vitro results demonstrate feasibility of a first‑in‑kind theranostic platform. Results confirm cytocompatibility of the proposed dual-labeling strategy and support future in vivo studies. This all-in-one strategy, combining image-guided targeted delivery with enhanced cellular function, has potential to coordinate and enhance DC activation towards improved therapeutic efficacy.

Image/Figure:
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Image/Figure Caption:
Dual-labeled DCs (A) demonstrate photomagnetic properties for magnetic delivery (B) and US/PA imaging (C).

Author
Kelsey Kubelick
University of Virginia