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Methyl Donating artificial organelles to support liver cells in Non-alcoholic fatty liver disease

Periodic Reporting for period 1 - MetD-AO (Methyl Donating artificial organelles to support liver cells in Non-alcoholic fatty liver disease)

Reporting period: 2019-09-01 to 2021-08-31

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world, encompassing a spectrum of liver damage. Multiple issues are involved on the cellular level in failing liver often involve enzyme deficiencies, and the accumulation of reactive oxygen species (ROS) and reduced levels of glutathione (GSH). In healthy individuals, GSH is present in high levels in liver cells, playing a key role in removing ROS. In fatty liver cells, the missing antioxidant together with high ROS levels presents a toxic combination further pressuring the injured organ.
NAFLD is recognized as an emerging medical challenge, and alternative concepts as potential medical interventions are highly sought after. Therefore, the findings of my fellowship contribute to the fundamental understanding how bottom-up synthetic biology can contribute to address biomedical challenges with the aim to provide a solution for affected individuals in the society.
The goal of my fellowship was to explore a bottom-up synthetic biology approach eventually aiming at efficiently replenishing deficiencies in the liver. The overall objective included the design of polymeric GSH reductase activity mimicking polymer nanoparticles as artificial organelles (AO). AOs are typically nano-sized single compartment reactors, aimed to perform a specific encapsulated biocatalytic reaction within a cell to substitute for missing or lost function. AO design involves several challenges such as to have an appropriate carrier system, an adequate enzymatic reaction and the ability to be transported into the cytosol of the cell.
The first aspect involved the synthesis of the bioactive polymer with methyl donation. A small library of various amphiphilic block copolymers were synthesized by addition−fragmentation chain-transfer (RAFT) polymerization. As a main monomer cholesteryl methacrylate was used to form the hydrophobic block due to its capabilities to promote self-assemble as part of an ABC. In order to tune the self-assembly of the future ABC other small monomers were copolymerised. 2-carboxyethyl acrylate (CEA) was utilized as part of the hydrophilic block and fluorescently labelled. PCEA was expected to act as a membranolytic polymer guiding the cytosolic placement.
Glutathione is a tripeptide that plays a key role in reducing reactive oxygen spices, which is often associated with enhancement of the liver damage, making this concept an equally impactful approach than the initially planned methyl donation. This peptide is active in its reduced thiol form, however is readily oxidized forming a disulfide bond upon removal of oxidizing agents. Glutathione could be successfully installed on any carboxyl group containing polymer such as PCEA.
The synthesized polymers were assembled either into hybrid polymer lipid vesicles (HV) or micelles. Assembling HVs is an obvious choice since cholesterol is naturally found in lipid membranes in mammalian cells, therefore expected that the polymer chains can easily insert into the lipid bilayer. Both type of assemblies were successfully made, showing stability up to several weeks. HVs with various ratios of polymer and soyPC was systematically evaluated for the membrane properties. Their ability to be associated with a model enzyme -galactosidase was studied and the enzymatic reaction was visualized with confocal laser scanning microscopy inside of the giant vesicles. The type of polymers had a large influence on the lifetime of the enzyme, inserted in the lipid membrane. The micelles’ and HVs’ interaction with giant liposomes was investigated to gain a basic understanding of polymer-lipid interactions towards mimicking lysosomal escape, and assemblies with pH sensitive lipids were found to be the most interactive.
The activity of the bioactive polymers was evaluated by the capability of glutathione reductase to break the disulfide bond between the tripeptide and the polymer. All polymers we able to serve as a substrate for the enzyme. The polymers were processed slower than the same amount of oxidized glutathione, which was attributed to the harder accessibility due to the larger polymer chain. Cell lysate from hepatocytes were prepared the GR present in this environmet was also able to utilize the polymers.
The assembled carrier systems were evaluated on two different cell lines. All types of assemblies as well as glutathione modified polymers were found to be non-toxic for both types of cells, and the uptake efficacy could be enhanced with a longer time of exposure. In addition to regular HepG2 cells, we also used fatty cells and showed comparable uptake efficacy and no inherent short-term cytotoxicity.
The cellular faith of the assemblies were followed by their fluorescent tag and the localization of the carriers in the cellular environment was visualize. For HVs we found that the polymer and the lipid were processed differently and the integrity of the assemblies were lost. The polymers were found to be co-localized with the liposomes while the lipids were not visible after prolonged waiting time following exposure to the carriers. This indicated that PCEA alone might not be sufficient to aid cytosolic placement of the carriers. Even so, the assemblies in this form has not yielded lysosomal escape insights of the process has been gained, and contributed to the understanding of the design criteria for the AOs.
We also started to explore the intracellular glutathione donation in regular and fatty hepatic cells. With the aim to assess the efficacy of the glutathione donation, we stressed the cells with acetaminophen. The glutathione modified polymers were non-toxic to the cells and could be dosed in high amounts, but the benefit of the glutathione donation remains inconclusive and would need further investigations such as using alternative stressers and more specialize read-out assay.
Taken all together, the chemistry challenges were successfully addressed and the basic biological evaluation was completed. The work already resulted in two peer-reviewed original articles and further three original articles are at different stages in the publication process.
The results I obtained were exploited by other researcher illustrated by the citations of my published articles. My results have also contributed ongoing projects in my host group, already leading to further peer reviewed articles.
Therapeutic cell mimicry provides radically new solutions such as synthesis of artificial enzymes or assembly of synthetic organelles and cells. However, the general design criteria is not very well understood to obtain functional nanoreactors that can be placed in the cytosol of mammalian cells. The research carried out in my fellowship has contributed to several aspect of this challenge. First, a bioactive unit was synthesized with biologically relevant function. Second, we showed that a broad range of properties of hybrid lipid polymer vesicles, which are promising candidates to be used as carriers, can be influenced by their building blocks.. Lastly, the fundamental understanding about the cell are processing hybrid vesicles was improved due to my findings.
Taken all together the findings of the research further advanced the state of the art of the field, and the results were disseminated to the highest degree it was possible in the current situation.
Scheme of the project