Transneuronal Transport of Ligand-Conjugated Upconversion Nanoparticles from the Peripheral to the Central Nervous System

Abstract Despite significant advancements in neuro-nanotechnology, the movement of nanomaterials between peripheral and central neurons for applications such as cargo delivery, imaging, and tracking remains a formidable challenge. This limitation highlights the critical necessity for developing engineered materials that can facilitate efficient intra-axonal transport and trans-synaptic translocation. In this study, we report the design and synthesis of core–shell–shell upconversion nanoparticles (CSS-UCNPs) featuring a NaYF4:YbEr core sequentially coated with active NaYF4:YbNd and inert NaYF4 shells. To achieve neuron-specific targeting, the architecture was surface-functionalized with poly(ethylene glycol) (PEG)-maleimide and conjugated to the RVG29 peptide (CSS-PEG-RVG29). Characterization by transmission electron microscopy, dynamic light scattering, and spectroscopic analyses confirmed uniform particle size (42.9 nm radius) and efficient upconversion luminescence (λex = 808 nm, λem = 500−575 nm). In vitro assays demonstrated low cytotoxicity in Neuro2a, NSC34, and C6 cell lines at concentrations up to 500 μg/mL, enhanced cellular uptake mediated by RVG29, and no detectable alterations in spinal neuronal action potential firing, as assessed by patch-clamp electrophysiology. In vivo, intrafascicular injection into the mouse sciatic nerve (2 mg/mL, 2 μL) resulted in nanoparticle transport to the dorsal part of the spinal cord, with localization in higher regions of the central nervous system (CNS) and cortices, as visualized by multiphoton microscopy. In contrast, control experiments utilizing scrambled RVG29-conjugated nanoparticles demonstrated negligible accumulation within the CNS. These findings establish CSS-PEG-RVG29 as a promising platform for peripheral-to-CNS cargo delivery, with broad potential applications.