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Page 44 Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10

production in Mtb by analyzing Mtb cells under conditions of high vesicle production, such as iron
restriction and cells lacking VirR. Transcriptomic analysis showed common upregulation of the iniBAC
operon in association with high vesicle production in Mtb. Genetic and vesicle production analysis
demonstrated that the dynamin-like proteins (DLPs) encoded by this operon, IniA and IniC, are necessary
for release of extracellular vesicles by Mtb in culture and in infected macrophages. The first line antibiotic,
isoniazid was found to stimulate vesicle release in a DLP-dependent manner. Extracellular vesicles purified
from WT Mtb cultures or concentrated from the extracellular medium of infected macrophages stimulated
release of pro- inflammatory cytokines in uninfected bone marrow-derived macrophages, whereas an
iniAC mutant showed poor immunostimulatory activity. Our results provide a new understanding of the
function of mycobacterial DLPs and mechanistic insights into vesicle biogenesis and enable studies that
address the role of extracellular vesicles in TB pathogenesis.

32. Intracellular delivery methods using biofunctional peptide-modified extracellular vesicles

Authors: Ikuhiko Nakase
E-mail: i-nakase@21c.osakafu-u.ac.jp
Affiliations: Graduate School of Science, Osaka Prefecture University, Naka-ku, Sakai-shi, Japan.
Abstracts: Extracellular vesicles (exosomes, EVs) with encapsulation of biofunctional molecules (e.g.,
enzymes and genes) are highly expected to be next-generation therapeutic carriers because of their
pharmaceutical advantages including effective usage of cell-to-cell communication routes, controlled
immunogenicity, absence of cytotoxicity, brain targeting through BBB. However, methods for increasing
the cellular EVs uptake efficacy must be developed to achieve effective intracellular delivery of EV contents.
In this presentation, I will introduce novel techniques to enhance cellular EV uptake by modification of
[1-3]
functional peptides on EV membranes especially using an antimicrobial protein, CAP18 derived cell-
[4]
penetrating peptides for macropinocytosis induction and effective cellular uptake. We previously found
that macropinocytosis (accompanied by actin reorganization, ruffling of the plasma membrane, and
engulfment of large volumes of extracellular fluid) is important route for enhanced cellular EV uptake .
[5]
CAP18- derived sC18 peptide has high abilities for cellular uptake, and dimer-type structure of sC18
[4]
peptides, (sC18)2, shows further cell membrane penetration . In this study, we found that (sC18)2 peptides
induced macropinocytosis via glycosaminoglycan on plasma membranes of targeted cells. Furthermore,
modification of the sC18 peptides or (sC18)2 peptides with stearyl-moiety on EV membranes significantly
enhanced cellular EV uptake via macropinocytosis induction. In addition, ribosome-inactivating protein,
saporin-artificially encapsulated EVs with modification of the (sC18)2 peptides showed glycosaminoglycan-
dependent cell-killing activity. Our experimental techniques and findings are considered to contribute to
the development for EV-based intracellular delivery system via macropinocytosis.

REFERENCES
1. Nakase I, Noguchi K, Fujii I, Futaki S. Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced
internalization induced by macropinocytosis. Sci Rep 2016 6:34937.
2. Nakase I, Noguchi K, Aoki A, Takatani-Nakase T, Fujii I, Futaki S. Arginine-rich cell-penetrating peptide-modified extracellular vesicles
for active macropinocytosis induction and efficient intracellular delivery. Sci Rep 2017;7:1991.
3. Nakase I, Ueno N, Katayama M, et al. Receptor clustering and activation by multivalent interaction through recognition peptides
presented on exosomes. Chem Commun (Camb) 2016;53:317-20.
4. Gronewold A, Horn M, Ranđelović I, et al. Characterization of a cell-penetrating peptide with potential anticancer activity.
ChemMedChem 2017;12:42-9.
5. Nakase I, Kobayashi NB, Takatani-Nakase T, Yoshida T. Active macropinocytosis induction by stimulation of epidermal growth factor
receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes. Sci Rep 2015;5:10300.