Impact of four common hydrogels on amyloid-β (Aβ) aggregation and cytotoxicity: Implications for 3D models of Alzheimer’s disease

Abstract

The properties of a hydrogel utilized in 3D culture can influence cell phenotype and morphology, yielding striking similarities to cellular processes that occur in vivo. Indeed, research areas including regenerative medicine, tissue engineering, cancer models, and stem cell cultures have readily utilized 3D biomaterials to investigate cell biological questions. However, cells are only one component of this milieu. Macromolecules play roles as bioactive factors and physical structures. Yet, investigations of macromolecular biophysics largely focus on pure molecules in dilute solution. Biophysical processes such as protein aggregation underlie diseases including Alzheimer’s disease, which is hallmarked by accumulated neurotoxic amyloid-β (Aβ) aggregates. Previously, we demonstrated that Aβ cytotoxicity is attenuated when cells are cultured within type I collagen hydrogels vs. on 2D substrates. Here, we investigated whether this phenomenon is conserved when Aβ is confined within hydrogels of varying physiochemical properties, notably mesh size and bioactivity. We investigated Aβ structure and aggregation kinetics in solution and in hydrogels (collagen, agarose, hyaluronic acid and polyethylene glycol) using fluorescence correlation spectroscopy and thioflavin T assays. Our results reveal that all hydrogels tested were associated with Aβ cytotoxicity attenuation. We suggest that confinement itself imparts a profound effect, possibly by stabilizing Aβ structures and shifting the aggregate equilibrium toward larger species. It is likely that the milieu that exist within cells and tissues also influences protein-protein interactions; thus, we suggest that it is critical to evaluate whether protein structure, function, and stability are altered in 3D systems vs. ideal solutions and 2D culture.

Collagen hydrogel confinement of Amyloid-β (Aβ) accelerates aggregation and reduces cytotoxic effects

“…6 may be skewed to favor the more hydrophilic species that exist within the collagen gel. In another work, we tested a series of hydrogel types with varying hydrophilicity, charge content, and mesh size [97] . All gel types attenuated A β toxicity, but the nucleation and aggregation kinetics that were measured in collagen gels were slower than in either agarose or crosslinked PEG, suggesting that confinement may be more important than a specific collagen-A β interaction.…”

Stability of proteins encapsulated in Michael‐type addition polyethylene glycol hydrogels

“…However, the tertiary structure of lysozyme responds rapidly to changes in denaturant concentration (van den Berg et al, 1999), and we suggest that differences in exposure time had minimal impact on lysozyme unfolding as the experiment was conducted for 120 min while it should have taken approximately 100 min for an equilibrium concentration of GuSCN or acrylamide to be reached between the surrounding solution and the gel. Ghosh et al, 2020;Wang & Akcora, 2017) but few have focused on PEG gels (Simpson et al, 2020). Our data indicated that PEG gels did not destabilize lysozyme, although no clear conclusion could be drawn whether lysozyme was stabilized.…”

“…Others have shown that crowding and confinement could preserve protein structure, stability, and activity (Eggers & Valentine, 2001; Ghosh et al, 2020; Wang & Akcora, 2017) but few have focused on PEG gels (Simpson et al, 2020). Our data indicated that PEG gels did not destabilize lysozyme, although no clear conclusion could be drawn whether lysozyme was stabilized.…”

“…The effects of confinement on protein folding have been investigated by theoretical (Minton, 1992(Minton, , 2006Zhou, Rivas, et al, 2008), computational (Cheng et al, 2018;Chu et al, 2020;Xu et al, 2017), and experimental studies (Asuri & Kunkel, 2014;Simpson et al, 2020;Wang & Akcora, 2017), but significant disagreement exists between the results of these studies. For PEG hydrogels in particular, much research has been focused on controlling protein release (Bhattarai et al, 2005;Kharkar et al, 2014;Lin & Anseth, 2009;Van de Wetering et al, 2005), but few have focused on protein stability and structure long-term or under stress conditions (Simpson et al, 2020). Such detailed investigations will enable the translation of PEG hydrogels as biotherapeutic delivery devices.…”

Single-Molecule Nanotechnologies: An Evolution in Biological Dynamics Detection

“…Dynamic parameters of the fluorescently labeled molecules can be discerned by fitting the autocorrelation curve with a suitable model function . In contrast to smFRET measurements, smFCS is not limited to a detection size of 1–10 nm and is sensitive to distances shorter than 1 nm, , making it applicable to a wide range of investigations in the biological sciences such as obtaining reaction rates, diffusion coefficients, and affinity constants. − …”