Atomic force microscopy nano-characterization of bee-derived extracellular vesicles

Session

Poster Session Two

Authors

Dr. Sebastian Aguayo (2, 3), Dr. Christina Schuh (1)

Affiliations

1. Centro de Medicina Regenerativa, Facultad de Medicina, Clínica Alemana-Universidad del Desarrollo
2. School of Dentistry, Faculty of Medicine, Pontificia Universidad Católica de Chile,
3. Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Católica de Chile

Keywords

Honeybee EVs, Atomic Force Microscopy, EV characterization

Abstract text

Introduction: Since ancient times, bee-derived products like honey or royal jelly have been used in medicine. Among their applications are wound healing, antimicrobial treatments as well as treatment of dental-derived conditions. However, in modern medicine, difficulties remain to transfer these approaches into clinics due to complex standardization and quality control. In recent years, extracellular vesicles (EVs) have been identified as one of the active components of bee products with both anti-bacterial and anti-inflammatory effects, and therefore are a promising opportunity for clinical translation. In this study, we highlight the development of an atomic force microscopy (AFM) based approach for the morphological and adhesive characterization of isolated bee-product-derived EVs. 

Methods: Bee-derived products were dissolved in particle-free PBS and debris was removed by serial centrifugation. Subsequently, supernatants were subjected to ultracentrifugation at 1x105xg for 70 min. Pellets were washed and ultracentrifuged again, and finally stored at -80°C. For basic characterization particles were quantified with nanoparticle tracking analysis and vesicle shape was verified with transmission electron microscopy. For the non-destructive immobilization of EVs, freshly cleaved mica discs were coated with a 50ul droplet of 0.1M poly-L-lysine (PLL) for 10 mins, washed twice with deionized water (dH2O), and dried with a gentle stream of N2. Once fully air-dried, a droplet of EV suspension was incubated on the PLL-treated mica substrates for 15min at room temperature and subsequently rinsed with dH2O to remove unattached EVs. Imaging of PLL and EVs was carried out in tapping mode in air employing an Asylum MFP 3D-SA (Asylum Research, US) with TAP300GD-G cantilevers (calibrated k~24 N/m; BudgetSensors, Bulgaria). Then, force-distance curves were obtained on the surface of individualized EVs with a 10nN setpoint and constant speed. Finally, morphological parameters and adhesion forces of EVs were extracted with Gwyddion v2.62 and the proprietary Asylum Research AFM software (v. 16.10.211).

Results: Basic EV characterization revealed presence of intact EVs within the expected parameters. NTA showed particles within 100-150nm and TEM verified the expected vesicle shape. The use of PLL-coated mica was an effective approach to immobilize bee-derived EVs in a non-destructive manner. AFM characterization confirmed that individualized EVs were mostly in the size range previously observed with NTA, and presented a nanoscale morphology consistent with previous reports. Also, reduced EV adhesion and clumping were correlated with sample purity following ultracentrifugation steps. 

Conclusion: AFM is an effective tool to determine the morphological and adhesive properties of bee-derived product EVs at the single vesicle level. This nanoscale approach could potentially be used as an indicator for quality control regarding the purity, concentration, and mechanical properties of non-mammalian derived EVs. 

Acknowledgements: This project was funded by ANID Fondecyt #1220803.