Effects of Lyophilization of Arginine-rich Cell-penetrating Peptide-modified Extracellular Vesicles on Intracellular Delivery

Anticancer Res. 2019 Dec;39(12):6701-6709. doi: 10.21873/anticanres.13885.


Background/aim: Extracellular vesicles (exosomes, EVs) (30-200 nm in diameter) are secreted by various cells in the body. Owing to the pharmaceutical advantages of EVs, an EV-based drug delivery system (DDS) for cancer therapy is expected to be the next-generation therapeutic system. However, preservation methods for functional and therapeutic EVs should be developed. Here, we developed the method of lyophilization of arginine-rich cell penetrating peptide (CPP)-modified EVs and investigated the effects of lyophilization on the characteristics of EVs.

Materials and methods: Particle size, structure, zeta-potential, and cellular uptake efficacy of the arginine-rich CPP-modified EVs were analyzed. The model protein saporin (SAP), having anti-cancer effects, was encapsulated inside the EVs to assess the cytosolic release of EV content after cellular uptake.

Results: Lyophilization of the EVs did not affect their particle size, structure, zeta-potential, and cellular uptake efficacy; however, the biological activity of the encapsulated SAP was inhibited by lyophilization.

Conclusion: Lyophilization of EVs may affect SAP structures and/or reduce the cytosolic release efficacy of EV's content after cellular uptake and needs attention in EV-based DDSs.

Keywords: Extracellular vesicles; cell-penetrating peptides; exosomes; lyophilization; saporin.

MeSH terms

  • Animals
  • Arginine*
  • CHO Cells
  • Cell Survival
  • Cell-Penetrating Peptides / administration & dosage
  • Cell-Penetrating Peptides / chemistry
  • Cell-Penetrating Peptides / pharmacokinetics*
  • Cricetulus
  • Extracellular Vesicles / metabolism*
  • Freeze Drying
  • Humans
  • Particle Size
  • Pharmaceutical Vehicles*
  • Pinocytosis
  • Preservation, Biological / methods
  • Saporins / administration & dosage
  • Saporins / pharmacokinetics*
  • Tetraspanin 30 / metabolism


  • CD63 protein, human
  • Cell-Penetrating Peptides
  • Pharmaceutical Vehicles
  • Tetraspanin 30
  • Arginine
  • Saporins