title: Bioderived therapeutics for chronic liver diseases
Creators: name: Ganguin, Aymar Abel
Publisher: name: Universität Bern
Year: 2023
Subjects: subject: 540 Chemistry
subject: 570 Life sciences; biology

description: The increasing prevalence of fibrosis and the lack of specific treatments calls for novel anti-fibrotic therapeutics. We set out to establish a protocol for the formation and purification of artificial EVs, termed cell-derived nanovesicles (cdNVs) from differently pre-treated HSCs, with fibrosis-resolving properties. We have previously shown the release of EVs with different compositions and activities, depending on the HSC cell status, and their fibrosis-resolving properties[1]. However, due to limitations in EV usage they are not suitable as anti-fibrotic therapeutics. We sought to overcome those drawbacks by creating cdNVs, while preserving the HSC fibrosis resolution potential of EVs, to create potential bioderived therapeutics for chronic liver diseases. 
In Chapter 2 different cdNV formation methods were tested to establish an upstream process starting from quiescent, naïve and TGF-β1 activated LX-2 cells. Therefore, several methods were evaluated based on their lysis efficiency, as well as on the quality (size, PDI, yield) of the formed cdNVs. Additionally, fluorescent microscopy experiments were performed to assess the ability of the formed cdNVs to induce a phenotypical shift of cdNV-treated activated LX-2 cells into a quiescent-like state. 
Building upon the previous chapter, in Chapter 3 we established methodological practices for the purification, enrichment, and sterilization of cdNVs derived from differently pre-treated LX-2 cells. Additionally, soluble factors were collected during the downstream process acting as a control to investigate activity loss and activity specificity. 
Chapter 4 covers all the activity studies performed on LX-2 cells treated with cdNVs formed through the established upstream and downstream processes. Genotypical and phenotypical deactivation in naïve, as well as TGF-β1 activated LX-2 cells upon cdNV treatment was assessed via fluorescent microscopy and real-time polymerase chain reaction (qPCR) on several markers involved in HSC activation. Surface corona and lipids were investigated for their role in cell deactivation. Further, homing of cdNVs was studied and compared between naïve and TGF-β1 activated LX-2 cells via flow cytometry.
We believe that our work lays the foundation for LX-2 cdNVs, which confirmed to be a promising candidate for the development of anti-fibrotic therapies.
In Chapter 5, we describe a novel approach that employs additive manufacturing to develop bioorthogonal and biodegradable hydrogel microbeads. These microbeads can be loaded with various liposomal formulations, creating a platform that enables sustained release of multiple small drug candidates. These drug candidates, both hydrophilic and hydrophobic, are encapsulated in different liposomal formulations leading to a sustained release in the peritoneum. As proof-of-concept gefitinib was used, which is a small hydrophobic drug against peritoneal adhesion, as well as tumours epidermal growth factor receptor mutations.
[1] Zivko C, Fuhrmann K, Fuhrmann G, Luciani P. Following the SPARC: tracking Secreted Protein Acidicand Rich in Cysteine associated with LX-2 ́s extracellular vesicles as a non-destructive modality to evaluate lipid-based antifibrotic treatments. Commun Biol 2021; 5(1):1155.