In vitro Uptake Analysis of Cholera Toxin B- and Wheat Germ Agglutinin- Functionalized Nanoparticles

Cholera Toxin B (CTB) binds large-diameter myelinated dorsal root ganglion (DRG) neurons (Ab- and Ad-fibers), targeting mechanoreceptors and proprioceptors; Wheat Germ Agglutinin (WGA) binds small-diameter unmyelinated DRG neurons (both peptidergic and non-peptidergic C-fibers), targeting nociceptors. Both WGA and CTB are used for neuroanatomical tract tracing. The objective of this study was to functionalize natural nanoparticles, e.g., Extracellular Vesicles (EVs), and synthetic nanoparticles, e.g., Liposomes, with CTB or WGA to determine whether they accumulate specifically in neurons. Our hypothesis was that CTB- or WGA-tagged nanoparticles would be taken up by a neuronal cell line (ACS 5004) more than by a HEK 293 kidney cell line. EVs were isolated from conditioned media using Size Exclusion Chromatography, and liposomes were synthesized using the thin-film hydration method. Both types of nanoparticles were tagged using lipid linkers. Labeled (with PKH26, a lipophilic dye) nanoparticles were added to ACS 5004 and HEK 293 cell lines for 24 hours, and uptake was assessed by confocal microscopy with quantitative analysis using an arbitrary unit ratio relative to DAPI staining. The results showed that only CTB-EVs were specifically taken up by ACS 5004 in a dose-response manner, whereas other nanoparticles did not show increased uptake compared to naïve nanoparticles. In conclusion, CTB-EVs represent a possible strategy for uptake and retrograde transport to DRG neurons in vivo. Disclosure of funding: Midwest Institute of Comparative Stem Cell Biotechnology This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.