Design and characterization of in situ cell-penetrating multi-modal gadolinium-gold nanoparticles for MR and CT imaging

Contrast agents capable of labeling cells in situ are essential for tracking individual cells as they migrate through tissues during dynamic biological processes. Gold nanoparticles (AuNPs) conjugated with gadolinium (Gd) and fluorochromes offer multimodal detection via magnetic resonance imaging (MRI), computed tomography (CT), and fluorescence microscopy. In this study, a systematic strategy was employed to incrementally increase the complexity of Gd-labeled AuNPs (GdAuNPs) and evaluate four distinct surface chemistries for in situ cell labeling. Comprehensive characterization of GdAuNP synthesis and stability-using inductively coupled plasma mass spectrometry, UV/visible spectroscopy, transmission electron microscopy, and MR relaxometry-demonstrated high reproducibility and a long shelf-life. Following intracerebroventricular or intrastriatal injection, histological analyses revealed that GdAuNPs labeled over 80 % of neurons in the striatum and approximately 20 % of neural stem cells (NSCs) in the subventricular zone. Only GdAuNPs functionalized with single-stranded DNA (ssDNA) were efficiently internalized by cells; GdAuNPs lacking ssDNA remained extracellular and were removed during immunohistochemical processing. ssDNA-labeled GdAuNPs localized peri-nuclearly following endocytosis. In microglia, GdAuNP also accumulated near the nucleus, whereas in macrophages, all GdAuNP formulations-including those with ssDNA-were mostly sequestered within phagosomes, indicating uptake via phagocytosis. The most effective design, termed type D GdAuNP, featured Gd chelates conjugated both to ssDNA and directly to the AuNP surface. These nanoparticles exhibited the highest MR sensitivity and contrast-to-noise ratio in MRI after in situ labeling and were also robustly detected by μCT. This stepwise approach to nanoparticle optimization demonstrates the potential to enhance multimodal imaging sensitivity, supporting the feasibility of a noninvasive visualization of in situ labeled neurons and NSCs.