DEVELOPMENT OF BIOINSPIRED PROTEOLIPOSOMES AND CELL-DERIVED NANOVESICLES AS OSTEOGENIC SYNTHETIC EXTRACELLULAR VESICLES FOR BONE REGENERATION

Jun 29, 2022, 2:40 PM
10m
Room: S3 A

Room: S3 A

Speaker

Brunet, Mathieu Y. (School of Chemical Engineering, University of Birmingham)

Description

In regenerative medicine, extracellular vesicles (EVs) have been increasingly studied as alternative acellular therapies overcoming the limitations of cell-based strategies. Derived from mineralising osteoblasts, EVs demonstrated their osteogenic potency suggesting their potential as a novel bone regenerative therapy. However, the clinical translation of EVs remains limited by issues associated with the scalability, reproducibility and purity of these naturally-derived nanoparticles. In this study, the characterisation of mineralising-osteoblast-derived EVs (MO-EVs) was performed to inspire the development of osteogenic synthetic EVs.
EVs were collected from cultures of mineralising osteoblasts over a 2-week period and the EV-isolation was performed by ultracentrifugation. Subsequently, the size, ζ-potential, morphology and particle concentration of these nanovesicles was characterised and the presence of tetraspanin markers (CD9, CD63 and CD81) was confirmed using nano-flow cytometry. Furthermore, the pro-osteogenic capacity of MO-EVs was assessed in vitro via quantifying alkaline phosphatase (ALP) activity and calcium deposition. From the composition analysis of MO-EVs, bio-inspired proteoliposomes harboring ALP and/or annexin VI were formulated via the thin-film hydration method followed by extrusion. Both the activity of the proteins post-insertion and their resulting incorporation efficiency in proteoliposomes were then determined. Additionally, cell-derived nanovesicles (CDNs) were produced by the serial extrusion of mineralising osteoblasts and the resulting synthetic EVs were similarly characterised.
The isolation of MO-EVs was validated as positivity for all tetraspanin markers was reported for these sub-100 nm vesicles. Notably, their osteogenic potency was confirmed in vitro on osteoblasts as MO-EVs increased significantly ALP activity, calcium deposition and collagen production after a 2-week treatment. MO-EVs were found enriched in several annexin proteins which guided the formulation of bio-inspired proteoliposomes. The insertion of both ALP and annexin VI was successful with >30% incorporation efficiency obtained for all formulations. Importantly, EV-inspired liposomes harboring annexin VI or ALP were found to be functional with the validation of the mediation of Ca2+-influx by annexin VI inside proteoliposomes and the confirmation of ALP enzymatic activity. Moreover, CDNs were also successfully produced as a nanoparticle population with an EV-size was obtained after serial extrusion. Both EV-inspired proteoliposomes and CDNs’ osteogenic potencies were then compared to MO-EVs after 14 days in osteogenic conditions.
Taken together, these results shows the potential of the development of synthetic EVs as biomimetic nanocarriers to accelerate the clinical translation of EV-based therapies for bone regeneration.

20941834084

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