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The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye
Published on: August 16, 2014
Small Extracellular Vesicle-Mediated Peptide Delivery to the Mouse Corneal Endothelium In Vivo
JeongGoo Lee1, Sun Young Lee1, Dimitrios Pollalis1
1Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California.
Purpose:
To investigate the utility of human corneal endothelial cell (hCEC)-derived small extracellular vesicles (sEVs) as a delivery system to the mouse corneal endothelium (CE) in vivo.
Design:
Laboratory study.
Subjects:
C57BL/6 mice.
Methods:
hCEC-derived sEV were isolated from culture media using differential ultracentrifugation. Size, number, and morphology were characterized using nanoparticle tracking analysis and transmission electron microscopy (TEM). sEV markers were confirmed using an sEV detection antibody panel. PKH26-labeled sEV were injected into the anterior chamber of wild-type mice, and corneal endothelial uptake of sEV was assessed by colocalization of 4',6-diamidino-2-phenylindole and PKH26 fluorescence on corneal whole mounts. Saponin permeabilization and sonication were used to load FITC-conjugated peptide into sEV. Delivery to mouse CE in vivo following intracameral injection was assessed by colocalization analysis of PKH26-labeled sEV with FITC-conjugated peptide. An average of 5 × 108 to 1 × 109 particles/plate with a peak size of 120 nm sEV were isolated.
Main Outcome Measures:
sEV-mediated endothelial targeting.
Results:
Morphology and size of sEVs were confirmed by TEM, and the presence of sEV markers, flotillin-1, ICAM, programmed cell death 6 interacting protein (ALIX), immunoglobulin superfamily member 8 (CD81), lysosome-associated membrane protein 3 (CD63), annexin A5, and tumor susceptibility gene 101 (TSG101), was confirmed by immunoblotting. Confocal microscopy of whole-mounted mouse corneas, performed 2 days after intracameral injection of PKH26-labeled sEVs containing FITC-conjugated peptide, showed localization of sEVs within the CE. Colocalization of PKH26 and FITC signals in the mouse CE confirmed delivery of the peptide cargo via hCEC-derived sEVs in the mouse CE in vivo.
Conclusions:
We demonstrate proof of principle of using hCEC-derived sEVs for delivery of potential therapeutic agents to the mouse CE in vivo via intracameral injection.
Financial Disclosures:
Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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