Controlled, supramolecular polymer formulation to engineer hydrogels with tunable mechanical and dynamic properties

Nature uses combined covalent (chemical bonds) and non-covalent (physical bonds) synthesis in a highly, controlled multi-step fashion to create functional materials with different mechanical and dynamic properties out of similar building blocks. Surprisingly, this control in fully synthetic systems remains elusive, even though the effects of formulation pathways on the assembly processes have been emphasized—highlighting the importance of and relationship between energy landscapes and function in synthetic systems. Here, we control multiple, coherent supramolecular assembly processes (fiber formation and crosslinking) to formulate hydrogels with tunable mechanical and dynamic properties. Hydrogels are prepared via two different formulation methods using similar building blocks (monofunctional and bifunctional supramolecular monomers), including (1) the mixing of the supramolecular monomers under basic conditions (Fbasic) and (2) the mixing of the supramolecular monomers under neutral conditions (Fneutral). In Fbasic, network formation is induced via simultaneous fiber formation and crosslinking, yielding homogeneously mixed networks which are dense, stiff (~10 kPa) and robust. In Fneutral, network formation is induced through sequential fiber formation and crosslinking, yielding heterogenous, soft (~2 kPa) and dynamic networks. With these results, we advance towards the controlled, multistep, non-covalent synthesis to create larger hierarchical, functional structures, similar to nature.</p

Development of a Fully Synthetic Corneal Stromal Construct via Supramolecular Hydrogel Engineering

Recent advances in the field of ophthalmology show great potential in the design of bioengineered constructs to mimic the corneal stroma. Hydrogels based on synthetic supramolecular polymers, are attractive synthetic mimics of the natural highly hydrated corneal stroma. Here, a fully synthetic corneal stromal construct is developed via engineering of an injectable supramolecular hydrogel based on ureido-pyrimidinone (UPy) moieties. The hydrogel displays a dynamic and tunable behavior, which allows for control of biochemical and mechanical cues. Two hydrogels are developed, a fully synthetic hydrogel functionalized with a bioactive cyclic arginine-glycine-aspartate UPy (UPy-cRGD) additive, and a hybrid hydrogel based on UPy-moieties mixed with collagen type I fibers. Both hydrogels supported cell encapsulation and associated cellular deposition of extracellular matrix (ECM) proteins after 21 days. Excitingly, the hydrogels support the activation of isolated primary keratocytes into stromal fibroblasts as well as the differentiation toward more quiescent corneal stromal keratocytes, demonstrated by their characteristic long dendritic protrusions and a substantially diminished cytokine secretion. Furthermore, cells survive shear stresses during an injectability test. Together, these findings highlight the development of an injectable supramolecular hydrogel as a synthetic corneal stromal microenvironment able to host primary keratocytes.</p

Antimicrobial peptide modification of biomaterials using supramolecular additives

Biomaterials based on non-active polymers functionalized with antimicrobial agents by covalent modification or mixing are currently regarded as high potential solutions to prevent biomaterial associated infections that are major causes of biomedical device failure. Herewith a strategy is proposed in which antimicrobial materials are prepared by simply mixing-and-matching of ureido-pyrimidinone (UPy) based supramolecular polymers with antimicrobial peptides (AMPs) modified with the same UPy-moiety. The N-terminus of the AMPs was coupled in solution to an UPy-carboxylic acid synthon resulting in formation of a new amidic bond. The UPy-functionalization of the AMPs did not affect their secondary structure, as proved by circular dichroism spectroscopy. The antimicrobial activity of the UPy-AMPs in solution was also retained. In addition, the incorporation of UPy-AMPs into an UPy-polymer was stable and the final material was biocompatible. The addition of 4 mol % of UPy-AMPs in the UPy-polymer material protected against colonization by Escherichia coli, and methicillin-sensitive and -resistant strains of Staphylococcus aureus. This modular approach enables a stable but dynamic incorporation of the antimicrobial agents, allowing at the same time for the possibility to change the nature of the polymer, as well as the use of AMPs with different activity spectra.</p

Engineering antimicrobial supramolecular polymer assemblies

Antibacterial resistance against conventional antibiotics has emerged as a global health problem. To address this problem, antimicrobial peptides (AMPs) have been recognized as alternatives due to their fast-killing activity and less propensity to induce resistance. Here, the AMPs are engineered via a supramolecular fashion to control and increase their biological performance. The AMPs are modified with ureido-pyrimidinone (UPy) to obtain UPy-AMP monomers, followed by modular self-assembling to realize antibacterial UPy-AMP supramolecular polymers. These positively charged assemblies are illustrated as stable, short fibrous or rod-like UPy-AMP nanostructures with enhanced antibacterial activity and modulable cytotoxicity. Moreover, these antibacterial UPy-AMP assemblies can be internalized by both THP-1 derived macrophages and human kidney cells, which would be an effective potential therapy to deliver the AMPs into mammalian cells to address intracellular infections. Overall, the results present here demonstrate that supramolecular engineering of AMPs provides a powerful tool to enhance the antibacterial activity, modulate cytotoxicity and accelerate the clinical application of AMPs.</p

Engineering antimicrobial supramolecular polymer assemblies

Antibacterial resistance against conventional antibiotics has emerged as a global health problem. To address this problem, antimicrobial peptides (AMPs) have been recognized as alternatives due to their fast-killing activity and less propensity to induce resistance. Here, the AMPs are engineered via a supramolecular fashion to control and increase their biological performance. The AMPs are modified with ureido-pyrimidinone (UPy) to obtain UPy-AMP monomers, followed by modular self-assembling to realize antibacterial UPy-AMP supramolecular polymers. These positively charged assemblies are illustrated as stable, short fibrous or rod-like UPy-AMP nanostructures with enhanced antibacterial activity and modulable cytotoxicity. Moreover, these antibacterial UPy-AMP assemblies can be internalized by both THP-1 derived macrophages and human kidney cells, which would be an effective potential therapy to deliver the AMPs into mammalian cells to address intracellular infections. Overall, the results present here demonstrate that supramolecular engineering of AMPs provides a powerful tool to enhance the antibacterial activity, modulate cytotoxicity and accelerate the clinical application of AMPs.</p

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.</p

Development of an Antimicrobial Peptide SAAP-148-Functionalized Supramolecular Coating on Titanium to Prevent Biomaterial-Associated Infections

Titanium implants are widely used in medicine but have a risk of biomaterial-associated infection (BAI), of which traditional antibiotic-based treatment is affected by resistance. Antimicrobial peptides (AMPs) are used to successfully kill antibiotic-resistant bacteria. Herein, a supramolecular coating for titanium implants is developed which presents the synthetic antimicrobial and antibiofilm peptide SAAP-148 via supramolecular interactions using ureido-pyrimidinone supramolecular units (UPy-SAAP-148GG). Material characterization of dropcast coatings shows the presence of UPy-SAAP-148GG at the surface. The supramolecular immobilized peptide remains antimicrobially active in dropcast polymer films and can successfully kill (antibiotic-resistant) Staphylococcus aureus, Acinetobacter baumannii, and Escherichia coli. Minor toxicity for human dermal fibroblasts is observed, with a reduced cell attachment after 24 h. Subsequently, a dipcoat coating on titanium implants is developed and tested in vivo in a subcutaneous implant infection mouse model with S. aureus administered locally on the implant before implantation to mimic contamination during surgery. The supramolecular coating containing 5 mol% of UPy-SAAP-148GG significantly prevents colonization of the implant surface as well as of the surrounding tissue, with no signs of toxicity. This shows that supramolecular AMP coatings on titanium are eminently suitable to prevent BAI.</p

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.</p

Heparin-guided binding of vascular endothelial growth factor to supramolecular biomaterial surfaces

Growth factors can steer the biological response to a biomaterial post implantation. Heparin is a growth factor binding molecule that can coordinate growth factor presentation to cells and therefore is able to regulate many biological processes. One way to functionalize biomaterials with heparin and growth factors is via a supramolecular approach. Here, we show a proof-of-concept study in which a supramolecular approach based on ureido-pyrimidinone (UPy) was used, which allows for modular functionalization. PCLdiUPy was functionalized with a UPy-modified heparin binding peptide (UPy-HBP) to facilitates binding of heparin, which in turn can bind vascular endothelial growth factor (VEGF) via its heparin binding domain. The adsorption of both heparin and VEGF were studied in two different functionalization approaches (pre-complex and two-step) and at different molecular ratios. Quartz crystal microbalance with dissipation energy adsorption data showed that VEGF and pre-complexed heparin:VEGF adsorbed non-specifically, with no distinguish between non-specific adsorption and heparin guided-adsorption. On the biological side, heparin guided-adsorption of Heparin:VEGF enhanced HUVECs surface coverage as compared to non-specific adsorption. These results provide a detailed insight on the molecular sandwich which is useful for new design strategies of supramolecular biomaterials with well-controlled immobilization of different growth factors.</p