Collagen type I mimicking peptide additives to functionalize synthetic supramolecular hydrogels.

Small bioactive peptide sequences derived from extracellular matrix proteins possess the ability to interact with cell receptors. As such, these peptide additives are excellent mimics to develop materials for 3D cell culture. Two types of supramolecular modified collagen type I mimicking peptide additives are presented; UPy-GFOGER (39 amino acids), with a novel superstructure, and the more simplistic UPy-DGEA (7 amino acids). Here, we studied the impact of the conformational differences between both peptide additives, on their biological performance. Various analyzing techniques demonstrated the ability of the supramolecular UPy-GFOGER to self-assemble into short nanofibers with brush-like outer features, suggesting trimerization into a triple helix. UPy-DGEA is a short additive without a complex structure. Since, collagen type I is a major component of the human corneal stroma, primary keratocytes (PKs) are encapsulated within the functionalized hydrogels to provide insights in the induced bioactivity of both additives. Incorporation of UPy-GFOGER supported an elongated morphology and (re-)differentiation of the encapsulated PKs, while tiny round-shaped cells were observed within the hydrogels functionalized with UPy-DGEA. This difference in biological success between UPy-GFOGER and UPy-DGEA indicates the difficulty of using short peptide additives without a complex structure to mimic the complex structure of natural collagen.

Artificial cells with viscoadaptive behavior based on hydrogel-loaded giant unilamellar vesicles.

Viscoadaptation is an essential process in natural cells, where supramolecular interactions between cytosolic components drive adaptation of the cellular mechanical features to regulate metabolic function. This important relationship between mechanical properties and function has until now been underexplored in artificial cell research. Here, we have created an artificial cell platform that exploits internal supramolecular interactions to display viscoadaptive behavior. As supramolecular material to mimic the cytosolic component of these artificial cells, we employed a pH-switchable hydrogelator based on poly(ethylene glycol) coupled to ureido-pyrimidinone units. The hydrogelator was membranized in its sol state in giant unilamellar lipid vesicles to include a cell-membrane mimetic component. The resulting hydrogelator-loaded giant unilamellar vesicles (designated as HL-GUVs) displayed reversible pH-switchable sol-gel behavior through multiple cycles. Furthermore, incorporation of the regulatory enzyme urease enabled us to increase the cytosolic pH upon conversion of its substrate urea. The system was able to switch between a high viscosity (at neutral pH) and a low viscosity (at basic pH) state upon addition of substrate. Finally, viscoadaptation was achieved via the incorporation of a second enzyme of which the activity was governed by the viscosity of the artificial cell. This work represents a new approach to install functional self-regulation in artificial cells, and opens new possibilities for the creation of complex artificial cells that mimic the structural and functional interplay found in biological systems.

Hepatic Spheroid Formation on Carbohydrate-Functionalized Supramolecular Hydrogels.

Two synthetic supramolecular hydrogels, formed from bis-urea amphiphiles containing lactobionic acid (LBA) and maltobionic acid (MBA) bioactive ligands, are applied as cell culture matrices in vitro. Their fibrillary and dynamic nature mimics essential features of the extracellular matrix (ECM). The carbohydrate amphiphiles self-assemble into long supramolecular fibers in water, and hydrogels are formed by physical entanglement of fibers through bundling. Gels of both amphiphiles exhibit good self-healing behavior, but remarkably different stiffnesses. They display excellent bioactive properties in hepatic cell cultures. Both carbohydrate ligands used are proposed to bind to asialoglycoprotein receptors (ASGPRs) in hepatic cells, thus inducing spheroid formation when seeding hepatic HepG2 cells on both supramolecular hydrogels. Ligand nature, ligand density, and hydrogel stiffness influence cell migration and spheroid size and number. The results illustrate the potential of self-assembled, carbohydrate-functionalized hydrogels as matrices for liver tissue engineering.

Photo-Crosslinked Coumarin-Containing Bis-Urea Amphiphile Hydrogels.

The design of photo-responsive supramolecular hydrogels based on coumarin dimerization and de-dimerization is described. The photo-responsive coumarin unit is chemically incorporated into an oligo(ethylene glycol) (OEG) bis-urea amphiphile that is capable of co-assembling with non-functionalized OEG amphiphile, to form supramolecular fibers. UV light with two different wavelengths (365 nm and 254 nm) is employed to induce a photo-reversible dimerization and de-dimerization process of coumarin units, respectively. The co-assembled solutions could be photo-crosslinked to induce a sol-to-gel transition through dimerization of coumarin with 365 nm UV light, and de-dimerization occurs with 254 nm UV light, to provide a weaker gel. In this system, the mechanical strength of supramolecular hydrogels can be tuned using the irradiation time, providing precise control of gelation in a supramolecular hydrogelator.

Tuning the affinity of amphiphilic guest molecules in a supramolecular polymer transient network.

Dynamicity plays a central role in biological systems such as in the cellular microenvironment. Here, the affinity and dynamics of different guest molecules in a transient supramolecular polymer hydrogel system, i.e. the host network, are investigated. The hydrogel system consists of bifunctional ureido-pyrimidinone (UPy) poly(ethylene glycol) polymers. A monofunctional complementary UPy guest is introduced, designed to interact with the host network based on UPy-UPy interactions. Furthermore, two other guest molecules are synthesized, being cholesterol and dodecyl (c12) guests; both designed to interact with the host network via hydrophobic interactions. At the nanoscale in solution, differences in morphology of the guest molecules were observed. The UPy-guest molecule formed fibers, and the cholesterol and c12 guests formed aggregates. Furthermore, cellular internalization of fluorescent guest molecules was studied. No cellular uptake of the UPy-cy5 guest was observed, whereas the cholesterol-cy5 guest showed membrane binding and cellular uptake. Also the c12-cy5 guest showed cellular uptake. Formulation of the guest molecules into the UPy hydrogel system was done to study the guest-host affinity. No changes in mechanical properties as measured with rheology were found upon guest-hydrogel formulation. Fluorescence recovery after photobleaching showed the diffusive properties of the cy5-functionalized guests throughout the host network. The c12 guest displayed a relatively fast mobility, the UPy guest displayed a decrease in mobility, and the cholesterol-guest remained relatively stable in the host network with little mobility. This demonstrates the tunable dynamic differences of affinity-based interaction between guest molecules and the host network. Interestingly, the cholesterol guest is internalized in cells and is robustly incorporated in the hydrogel network, while the UPy guest is not taken up by cells but shows an affinity to the hydrogel network. These results show the importance of guest-hydrogel affinity for future drug release. However, if modified with cholesterol these guests, or future drugs, will be taken up by cells; if modified with a UPy unit this does not occur. In this way both the drug-hydrogel interaction and the cell internalization behavior can be tuned. Regulating the host-guest dynamics in transient hydrogels opens the door to various drug delivery purposes and tissue engineering.

Engineering the Dynamics of Cell Adhesion Cues in Supramolecular Hydrogels for Facile Control over Cell Encapsulation and Behavior.

The extracellular matrix (ECM) forms through hierarchical assembly of small and larger polymeric molecules into a transient, hydrogel-like fibrous network that provides mechanical support and biochemical cues to cells. Synthetic, fibrous supramolecular networks formed via non-covalent assembly of various molecules are therefore potential candidates as synthetic mimics of the natural ECM, provided that functionalization with biochemical cues is effective. Here, combinations of slow and fast exchanging molecules that self-assemble into supramolecular fibers are employed to form transient hydrogel networks with tunable dynamic behavior. Obtained results prove that modulating the ratio between these molecules dictates the extent of dynamic behavior of the hydrogels at both the molecular and the network level, which is proposed to enable effective incorporation of cell-adhesive functionalities in these materials. Excitingly, the dynamic nature of the supramolecular components in this system can be conveniently employed to formulate multicomponent supramolecular hydrogels for easy culturing and encapsulation of single cells, spheroids, and organoids. Importantly, these findings highlight the significance of molecular design and exchange dynamics for the application of supramolecular hydrogels as synthetic ECM mimics.

Porphyrin-Based Supramolecular Flags in the Thermal Gradients' Wind: What Breaks the Symmetry, How and Why.

The Spontaneous Symmetry Breaking (SSB) phenomenon is a natural event in which a system changes its symmetric state, apparently reasonless, in an asymmetrical one. Nevertheless, this occurrence could be hiding unknown inductive forces. An intriguing investigation pathway uses supramolecular aggregates of suitable achiral porphyrins, useful to mimic the natural light-harvesting systems (as chlorophyll). Using as SSB probe supramolecular aggregates of 5,10,15,20-tetrakis[p(ω-methoxypolyethyleneoxy)phenyl]porphyrin (StarP), a non-ionic achiral PEGylated porphyrin, we explore here its interaction with weak asymmetric thermal gradients fields. The cross-correlation of the experimental data (circular dichroism, confocal microscopy, atomic force microscopy, and cryo-transmission electron microscopy) revealed that the used building blocks aggregate spontaneously, organizing in flag-like structures whose thermally-induced circular dichroism depends on their features. Finally, thermal gradient-induced enantioselectivity of the supramolecular flag-like aggregates has been shown and linked to their size-dependence mesoscopic deformation, which could be visualized as waving flags in the wind.

In Vivo Retention Quantification of Supramolecular Hydrogels Engineered for Cardiac Delivery.

Recent advances in the field of cardiac regeneration show great potential in the use of injectable hydrogels to reduce immediate flush-out of injected factors, thereby increasing the effectiveness of the encapsulated drugs. To establish a relation between cardiac function and retention of the drug-encapsulating hydrogel, a quantitative in vivo imaging method is required. Here, the supramolecular ureido-pyrimidinone modified poly(ethylene glycol) (UPy-PEG) material is developed into a bioactive hydrogel for radioactive imaging in a large animal model. A radioactive label is synthesized, being a ureido-pyrimidinone moiety functionalized with a chelator (UPy-DOTA) complexed with the radioactive isotope indium-111 (UPy-DOTA-111 In) that is mixed with the hydrogel. Additionally, bioactive and adhesive properties of the UPy-PEG hydrogel are increased by supramolecular introduction of a UPy-functionalized recombinant collagen type 1-based material (UPy-PEG-RCPhC1). This method enables in vivo tracking of the nonbioactive and bioactive supramolecular hydrogels and quantification of hydrogel retention in a porcine heart. In a small pilot, cardiac retention values of 8% for UPy-PEG and 16% for UPy-PEG-RCPhC1 hydrogel are observed 4 h postinjection. This work highlights the importance of retention quantification of hydrogels in vivo, where elucidation of hydrogel quantity at the target site is proposed to strongly influence efficacy of the intended therapy.

Influence of the Assembly State on the Functionality of a Supramolecular Jagged1-Mimicking Peptide Additive.

Expanding the bioactivation toolbox of supramolecular materials is of utmost relevance for their broad applicability in regenerative medicines. This study explores the functionality of a peptide mimic of the Notch ligand Jagged1 in a supramolecular system that is based on hydrogen bonding ureido-pyrimidinone (UPy) units. The functionality of the peptide is studied when formulated as an additive in a supramolecular solid material and as a self-assembled system in solution. UPy conjugation of the DSLJAG1 peptide sequence allows for the supramolecular functionalization of UPy-modified polycaprolactone, an elastomeric material, with UPy-DSLJAG1. Surface presentation of the UPy-DSLJAG1 peptide was confirmed by atomic force microscopy and X-ray photoelectron spectroscopy analyses, but no enhancement of Notch activity was detected in cells presenting Notch1 and Notch3 receptors. Nevertheless, a significant increase in Notch-signaling activity was observed when DSLJAG1 peptides were administered in the soluble form, indicating that the activity of DSLJAG1 is preserved after UPy functionalization but not after immobilization on a supramolecular solid material. Interestingly, an enhanced activity in solution of the UPy conjugate was detected compared with the unconjugated DSLJAG1 peptide, suggesting that the self-assembly of supramolecular aggregates in solution ameliorates the functionality of the molecules in a biological context.

Supramolecular Modification of a Sequence-Controlled Collagen-Mimicking Polymer.

Structurally and functionally well-defined recombinant proteins are an interesting class of sequence-controlled macromolecules to which different crosslinking chemistries can be applied to tune their biological properties. Herein, we take advantage of a 571-residue recombinant peptide based on human collagen type I (RCPhC1), which we functionalized with supramolecular 4-fold hydrogen bonding ureido-pyrimidinone (UPy) moieties. By grafting supramolecular UPy moieties onto the backbone of RCPhC1 (UPy-RCPhC1), increased control over the polymer structure, assembly, gelation, and mechanical properties was achieved. In addition, by increasing the degree of UPy functionalization on RCPhC1, cardiomyocyte progenitor cells were cultured on "soft" (∼26 kPa) versus "stiff" (∼68-190 kPa) UPy-RCPhC1 hydrogels. Interestingly, increased stress fiber formation, focal adhesions, and proliferation were observed on stiffer compared to softer substrates, owing to the formation of stronger cell-material interactions. In conclusion, a bioinspired hydrogel material was designed by a combination of two well-known natural components, i.e., a protein as sequence-controlled polymer and UPy units inspired on nucleobases.