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<p>Current extracellular vesicle (EV) isolation workflows are dominated by size- and density-based approaches, which provide limited insight into surface chemical properties of vesicular particles. Here, we report a hydrophobic interaction chromatography (HIC) workflow for resolving EV fractions along differences in membrane interfacial hydrophobicity. Using a commercially available HIC column, reproducible fractionation of EV samples was achieved, yielding discrete fractions that differed in retention behaviour and lipid composition. Transmission electron microscopy (TEM) showed vesicle-like nanoparticles with EV-consistent morphology, indicating preservation of vesicle integrity during HIC. Lipidomic profiling by reversed-phase liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (RP-LC-Q-TOF-MS) revealed fraction-specific differences in lipid composition. Importantly, we demonstrate the feasibility of direct injection of pre-cleaned biofluids onto the HIC column, enabling fractionation of nanoparticle populations containing EVs without prior ultracentrifugation. Furthermore, an operational interfacial hydrophobicity index derived from lipidomic data showed a clear correlation with HIC retention, providing an orthogonal compositional descriptor consistent with the proposed fractionation mechanism. Together, this hydrophobicity-based strategy introduces a previously unexplored physicochemical dimension to EV analysis, revealing chemically structured heterogeneity that is not accessible using conventional separation strategies. The workflow provides a practical framework for fractionating EVs in a manner directly relevant to lipidomic profiling and studies of EV membrane chemistry.</p>

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Hydrophobic interaction chromatography resolves extracellular vesicle fractions with distinct lipidomic signatures

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月20日

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  • Michał Młynarczyk,
  • Wiktoria Więckowska,
  • Mariusz Belka,
  • Raphael Ewonde Ewonde,
  • Jagoda Mantej,
  • Mikołaj Klimczuk,
  • Felicja Gajdowska,
  • Jorge Matinha-Cardoso,
  • Paula Tamagnini,
  • Danuta Gutowska-Owsiak,
  • Paulo Oliveira,
  • Sebastiaan Eeltink,
  • Weronika Hewelt-Belka

摘要

Current extracellular vesicle (EV) isolation workflows are dominated by size- and density-based approaches, which provide limited insight into surface chemical properties of vesicular particles. Here, we report a hydrophobic interaction chromatography (HIC) workflow for resolving EV fractions along differences in membrane interfacial hydrophobicity. Using a commercially available HIC column, reproducible fractionation of EV samples was achieved, yielding discrete fractions that differed in retention behaviour and lipid composition. Transmission electron microscopy (TEM) showed vesicle-like nanoparticles with EV-consistent morphology, indicating preservation of vesicle integrity during HIC. Lipidomic profiling by reversed-phase liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (RP-LC-Q-TOF-MS) revealed fraction-specific differences in lipid composition. Importantly, we demonstrate the feasibility of direct injection of pre-cleaned biofluids onto the HIC column, enabling fractionation of nanoparticle populations containing EVs without prior ultracentrifugation. Furthermore, an operational interfacial hydrophobicity index derived from lipidomic data showed a clear correlation with HIC retention, providing an orthogonal compositional descriptor consistent with the proposed fractionation mechanism. Together, this hydrophobicity-based strategy introduces a previously unexplored physicochemical dimension to EV analysis, revealing chemically structured heterogeneity that is not accessible using conventional separation strategies. The workflow provides a practical framework for fractionating EVs in a manner directly relevant to lipidomic profiling and studies of EV membrane chemistry.