Presentation Information
[P04-477]Development of Membrane-Inserted Extracellular Vesicle Labeling Probes Based on Organic Semiconductor backbones
○Yuta Kido1, Riku Kawasaki1, Yoshihiro Sasaki2, Ryosuke Mizuta2, Hikaru Takaya3, Keita Yamana1, Atsushi Ikeda1 (1. Grad. Sch. Adv. Sci. Eng., Hirosihma Univ (Japan), 2. Grad. Sch. Eng., Kyoto Univ (Japan), 3. Fac. Life Environ. Sci., Teikyo Univ. Sci. (Japan))
Keywords:
Extracellular vesicles,Drug delivery system,π-conjugate molecule
Purpose: The purpose of this study is to develop a stable and quantitative labeling strategy for extracellular vesicles (EVs).EVs are promising carriers for drug delivery systems due to their biocompatibility and intrinsic targeting ability; however, their in vivo and intracellular behavior remains difficult to analyze because it is challenging to distinguish exogenous EVs from endogenous populations, and conventional dyes have several limitations. Establishing a reliable labeling method will enable accurate tracking of EVs, facilitate mechanistic understanding of EV-mediated communication, and improve the design and evaluation of EV-based DDS.
Methods To address these limitations, we designed and synthesized fluorescent transmembrane molecules with an organic semiconductor backbone. The structure was engineered so that the π-conjugated backbone length matches the lipid bilayer thickness, enabling stable membrane insertion. Polar terminal groups were introduced to suppress intermolecular aggregation and improve water dispersibility. Furthermore, the molecules were complexed with β-cyclodextrin nanoparticles cross-linked with PEG chains to enhance dispersibility. These supramolecular complexes were introduced into EVs membranes via a noninvasive exchange reaction. Incorporation into EVs was evaluated using fluorescence spectroscopy, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Additionally, labeling stability was examined under physiological conditions to assess suitability for long-term tracking. The influence of labeling on EV physicochemical properties was also investigated.
Results Fluorescence spectroscopy, NTA, and TEM confirmed incorporation of the molecules into EVs membranes while preserving vesicle integrity. Quantitative analysis revealed approximately one hundred molecules were incorporated into each EV. In addition, confocal laser scanning microscopy of Colon-26 cells incubated with labeled EVs showed intracellular fluorescence signals, indicating effective uptake and stable labeling. Furthermore, negligible fluorescence leakage and minimal photobleaching were observed, demonstrating superiority of this labeling strategy over conventional dyes, particularly for prolonged observation and quantitative analysis. Importantly, no significant changes in particle size distribution or morphology were detected after labeling.
Conclusion Organic semiconductor-based transmembrane molecules function as stable fluorescent anchors for EVs membranes and provide a platform for EV tracking and quantitative analysis in nanomedicine. This approach is expected to contribute to deeper understanding of EV behavior in biological systems and accelerate development of next-generation EV-based therapeutic technologies. Furthermore, this strategy may offer opportunities for integrating functional imaging and therapeutic delivery within a single EV-based system. This method enables more reliable comparisons.
Methods To address these limitations, we designed and synthesized fluorescent transmembrane molecules with an organic semiconductor backbone. The structure was engineered so that the π-conjugated backbone length matches the lipid bilayer thickness, enabling stable membrane insertion. Polar terminal groups were introduced to suppress intermolecular aggregation and improve water dispersibility. Furthermore, the molecules were complexed with β-cyclodextrin nanoparticles cross-linked with PEG chains to enhance dispersibility. These supramolecular complexes were introduced into EVs membranes via a noninvasive exchange reaction. Incorporation into EVs was evaluated using fluorescence spectroscopy, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Additionally, labeling stability was examined under physiological conditions to assess suitability for long-term tracking. The influence of labeling on EV physicochemical properties was also investigated.
Results Fluorescence spectroscopy, NTA, and TEM confirmed incorporation of the molecules into EVs membranes while preserving vesicle integrity. Quantitative analysis revealed approximately one hundred molecules were incorporated into each EV. In addition, confocal laser scanning microscopy of Colon-26 cells incubated with labeled EVs showed intracellular fluorescence signals, indicating effective uptake and stable labeling. Furthermore, negligible fluorescence leakage and minimal photobleaching were observed, demonstrating superiority of this labeling strategy over conventional dyes, particularly for prolonged observation and quantitative analysis. Importantly, no significant changes in particle size distribution or morphology were detected after labeling.
Conclusion Organic semiconductor-based transmembrane molecules function as stable fluorescent anchors for EVs membranes and provide a platform for EV tracking and quantitative analysis in nanomedicine. This approach is expected to contribute to deeper understanding of EV behavior in biological systems and accelerate development of next-generation EV-based therapeutic technologies. Furthermore, this strategy may offer opportunities for integrating functional imaging and therapeutic delivery within a single EV-based system. This method enables more reliable comparisons.
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