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Standardized Method to Functionalize Plasma-Extracellular Vesicles via Copper-Free Click Chemistry for Targeted Drug

Maria Chiara Ciferri1, Silvia Bruno1, Nicole Rosenwasser1

  • 1Department of Experimental Medicine, University of Genova, Largo Rosanna Benzi 10, Genova 16132, Italy.

ACS Applied Bio Materials
|January 16, 2024
PubMed
Summary

We developed a copper-free click chemistry method to functionalize extracellular vesicles (EVs) from plasma. This novel technique enables reliable EV surface modification for improved drug delivery and diagnostics without cytotoxicity.

Keywords:
click chemistryextracellular vesiclessurface functionalizationtumor cells

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Area of Science:

  • Biotechnology
  • Nanomedicine
  • Cell Biology

Background:

  • Extracellular vesicles (EVs) show promise for targeted drug delivery and diagnostics.
  • Current EV surface modification methods, like copper-catalyzed click chemistry, face challenges with cytotoxicity and biological interference.
  • Functionalizing EVs derived from complex biofluids like plasma is particularly difficult.

Purpose of the Study:

  • To develop a standardized, copper-free click chemistry method for extracellular vesicle (EV) functionalization.
  • To optimize EV isolation from plasma for purity and yield.
  • To validate the functionalization process and characterize the resulting modified EVs.

Main Methods:

  • Compared three EV isolation techniques, selecting sucrose cushion ultracentrifugation (sUC) for optimal purity/yield.
  • Optimized copper-free click chemistry conditions for EV surface functionalization using a fluorescently labeled azide.
  • Characterized functionalized EVs (click-EVs) for identity, size, cellular uptake, and intracellular trafficking.

Main Results:

  • Sucrose cushion ultracentrifugation (sUC) provided the best purity/yield ratio for plasma-derived EVs.
  • Successfully functionalized plasma-EVs using copper-free click chemistry, creating 'click-EVs'.
  • Click-EVs retained their identity and size, were efficiently internalized by tumor cells, and partially utilized endosomal recycling pathways.

Conclusions:

  • A standardized, copper-free click chemistry approach enables reliable functionalization of plasma-derived EVs.
  • This method overcomes limitations of previous techniques, offering a safer alternative for EV modification.
  • The developed protocol provides a foundation for advancing EV-based applications in drug delivery, diagnostics, and therapeutics.