CHEMISTRY

Understanding and manipulating the interactions between foreign bodies and cell membranes during endo- and phagocytosis is of paramount importance, not only for the fate of living cells, but also for numerous biomedical applications. This study aims to elucidate the role of variables such as particle shape, curvature, orientation, membrane tension, and adhesive strength in this essential process, using a minimal experimental biomimetic system comprising giant unilamellar vesicles and rod-like particles. We find that the particle wrapping process is dictated by the balance between the elastic energy penalty and adhesion energy gain, leading to two distinct engulfment pathways, tip-first and side-first, emphasizing the particle orientation's significance in determining the pathway. Our study contributes to a comprehensive understanding of the mechanistic intricacies of endocytosis by highlighting how the interplay between the particle's shape, curvature, orientation, membrane tension, and adhesive strength can influence the engulfment pathway. Moreover, our findings could help in better design of future targeted drug delivery applications.

α-synuclein (αS) is a dynamic intrinsically disordered protein (IDP) with a wide variety of interaction partners, but also the propensity to assemble into amyloid fibrils, a condition characterizing Parkinson’s disease. To investigate the origin of αS’s intra- and inter-molecular interactions at the amino acid level, we combined single-molecule fluorescence resonance energy transfer (smFRET) experiments with coarse-grained molecular dynamics (CG-MD) simulations. We find excellent agreement between experiments and simulations. Our results show that αS is highly dynamic throughout its entire chain with global good solvent scaling behaviour under physiological salt conditions. Nonetheless, we observe local intra- and inter-regional interactions in αS to cause deviations. Specifically, compaction in the NAC region, caused by hydrophobic aliphatic contacts, mostly between valine and alanine residues, and cation-π interactions between lysine and tyrosine, which also contribute to a slightly shorter than expected distance between the C- and N-termini. Also, hydrogen bonds seem to compact the NAC region. In addition, we find expansion of the C-terminal region, explained by intraregional electrostatic repulsion and increased chain stiffness from several prolines. Overall, our study demonstrates the effectiveness of combining smFRET experiments with CG-MD simulations to investigate the key interactions in highly dynamic IDPs at the amino acid level.

Dynamic covalent chemistry (DCC) provides a versatile toolbox of reactions, consisting of diverse bonds that are strong yet dynamic. Implementing dynamic covalent bonds in materials enables interesting properties such as self-healing behavior and stress relaxation over a wide timescale. Accordingly, DCC has also attracted interest for extracellular matrix (ECM) mimics, enabling better resemblance to the natural dynamicity of the ECM than static bonds and being stronger than physical bonds.

This work presents a novel type of thiol-based dynamic covalent chemistry used as a crosslinking strategy to form tuneable, dynamic hydrogels for cell encapsulation. The system consists of two precursors, whereby the functional groups are attached to a polyethylene glycol (PEG) backbone. A bifunctional linear precursor, synthesized with good yield (60%) and excellent substitution (100%), and a, commercially available, 4-arm precursor with four functional groups. By mixing the two precursors, under mild ambient conditions (aqueous buffer solution pH 6.6-8.0, room temperature), hydrogels are formed rapidly in a few seconds to minutes. The resulting hydrogels have tunable mechanical properties, show self-healing and -recovery properties, and exhibit moderate stress-relaxation. This showcases the applicability of this novel thiol-mediated dynamic covalent chemistry to make adaptable hydrogels, that will be further investigated for use as ECM mimics.

To mimic the extracellular matrix (ECM) we need a dynamic network, with biochemical cues, that can relax and dissipate stress. Furthermore, we need to match the physiological characteristics of cardiac tissue.

Alginate hydrogels can be formed through different crosslinking methods. Covalent methods, such as photo-crosslinking, produce stable but brittle hydrogels. To make the network dynamic, we propose the introduction of non-covalent host-guest chemistry. These interactions are more stable and controllable in comparison to ionic crosslinking.

First, we functionalize alginate with a methacrylate group, this allows UV-induced crosslinking with a photo-initiator. Next, we use the FGGC peptide because it can react with the methacrylate group through the thiol-click chemistry and the aromatic group of phenylalanine can form a 2:1 complex with cucurbit[8]uril (CB[8]). CB[8] is a macrocycle with a  large cavity that can accommodate two planar, hydrophobic guests.

The thiol-click chemistry allows to graft peptides to the alginate backbone that enhance cell-ECM communication. The reversible bond strength is dependent on the guest; therefore, the stress relaxation profile of the hydrogels can be adapted to the stage of the developing heart. We intend to use this system to culture stem cell-derived cardiomyocytes and observe their communication with other cells and the ECM.