Bioorthogonal Tetrazine Carbamate Cleavage by Highly Reactive trans-Cyclooctene.

The high rate of the 'click-to-release' reaction between an allylic substituted trans-cyclooctene linker and a tetrazine activator has enabled exceptional control over chemical and biological processes. Here we report the development of a new bioorthogonal cleavage reaction based on trans-cyclooctene and tetrazine, which allows the use of highly reactive trans-cyclooctenes, leading to 3 orders of magnitude higher click rates compared to the parent reaction, and 4 to 6 orders higher than other cleavage reactions. In this new pyridazine elimination mechanism, wherein the roles are reversed, a trans-cyclooctene activator reacts with a tetrazine linker that is substituted with a methylene-linked carbamate, leading to a 1,4-elimination of the carbamate and liberation of a secondary amine. Through a series of mechanistic studies, we identified the 2,5-dihydropyridazine tautomer as the releasing species and found factors that govern its formation and subsequent fragmentation. The bioorthogonal utility was demonstrated by the selective cleavage of a tetrazine-linked antibody-drug conjugate by trans-cyclooctenes, affording efficient drug liberation in plasma and cell culture. Finally, the parent and the new reaction were compared at low concentration, showing that the use of a highly reactive trans-cyclooctene as the activator leads to a complete cycloaddition reaction with the antibody-drug conjugate in seconds vs hours for the parent system. Although the subsequent release from the IEDDA adduct is slower, we believe that this new reaction may allow markedly reduced click-to-release reagent doses in vitro and in vivo and could expand the application scope to conditions wherein the trans-cyclooctene has limited stability.

Sequence of Polyurethane Ionomers Determinative for Core Structure of Surfactant-Copolymer Complexes.

The core of micelles self-assembled from amphiphiles is hydrophobic and contains little water, whereas complex coacervate core micelles co-assembled from oppositely charged hydrophilic polymers have a hydrophilic core with a high water content. Co-assembly of ionic surfactants with ionic-neutral copolymers yields surfactant-copolymer complexes known to be capable of solubilizing both hydrophilic and hydrophobic cargo within the mixed core composed of a coacervate phase with polyelectrolyte-decorated surfactant micelles. Here we formed such complexes from asymmetric (PUI-A2) and symmetric (PUI-S2), sequence-controlled polyurethane ionomers and poly(N-methyl-2-vinylpyridinium iodide)29-b-poly(ethylene oxide)204 copolymers. The complexes with PUI-S2 were 1.3-fold larger in mass and 1.8-fold larger in radius of gyration than the PUI-A2 complexes. Small-angle X-ray scattering revealed differences in the packing of the similarly sized PUI micelles within the core of the complexes. The PUI-A2 micelles were arranged in a more ordered fashion and were spaced further apart from each other (10 nm vs. 6 nm) than the PUI-S2 micelles. Hence, this work shows that the monomer sequence of amphiphiles can be varied to alter the internal structure of surfactant-copolymer complexes. Since the structure of the micellar core may affect both the cargo loading and release, our findings suggest that these properties may be tuned through control of the monomer sequence of the micellar constituents.

Synthesis and Self-Assembly of Bay-Substituted Perylene Diimide Gemini-Type Surfactants as Off-On Fluorescent Probes for Lipid Bilayers.

Interest in bay-substituted perylene-3,4:9,10-tetracarboxylic diimides (PDIs) for solution-based applications is growing due to their improved solubility and altered optical and electronic properties compared to unsubstituted PDIs. Synthetic routes to 1,12-bay-substituted PDIs have been very demanding due to issues with steric hindrance and poor regioselectivity. Here we report a simple one-step regioselective and high yielding synthesis of a 1,12-dihydroxylated PDI derivative that can subsequently be alkylated in a straightforward fashion to produce nonplanar 1,12-dialkoxy PDIs. These PDIs show a large Stokes shift, which is specifically useful for bioimaging applications. A particular cationic PDI gemini-type surfactant has been developed that forms nonfluorescent self-assembled particles in water ("off state"), which exerts a high fluorescence upon incorporation into lipophilic bilayers ("on state"). Therefore, this probe is appealing as a highly sensitive fluorescent labelling marker with a low background signal for imaging artificial and cellular membranes.

Heterogeneity of Network Structures and Water Dynamics in κ-Carrageenan Gels Probed by Nanoparticle Diffusometry.

A set of functionalized nanoparticles (PEGylated dendrimers, d = 2.8-11 nm) was used to probe the structural heterogeneity in Na+/K+ induced κ-carrageenan gels. The self-diffusion behavior of these nanoparticles as observed by 1H pulsed-field gradient NMR, fluorescence recovery after photobleaching, and raster image correlation spectroscopy revealed a fast and a slow component, pointing toward microstructural heterogeneity in the gel network. The self-diffusion behavior of the faster nanoparticles could be modeled with obstruction by a coarse network (average mesh size <100 nm), while the slower-diffusing nanoparticles are trapped in a dense network (lower mesh size limit of 4.6 nm). Overhauser dynamic nuclear polarization-enhanced NMR relaxometry revealed a reduced local solvent water diffusivity near 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO)-labeled nanoparticles trapped in the dense network, showing that heterogeneity in the physical network is also reflected in heterogeneous self-diffusivity of water. The observed heterogeneity in mesh sizes and in water self-diffusivity is of interest for understanding and modeling of transport through and release of solutes from heterogeneous biopolymer gels.

Click-to-Release from trans-Cyclooctenes: Mechanistic Insights and Expansion of Scope from Established Carbamate to Remarkable Ether Cleavage.

The bioorthogonal cleavage of allylic carbamates from trans-cyclooctene (TCO) upon reaction with tetrazine is widely used to release amines. We disclose herein that this reaction can also cleave TCO esters, carbonates, and surprisingly, ethers. Mechanistic studies demonstrated that the elimination is mainly governed by the formation of the rapidly eliminating 1,4-dihydropyridazine tautomer, and less by the nature of the leaving group. In contrast to the widely used p-aminobenzyloxy linker, which affords cleavage of aromatic but not of aliphatic ethers, the aromatic, benzylic, and aliphatic TCO ethers were cleaved as efficiently as the carbamate, carbonate, and esters. Bioorthogonal ether release was demonstrated by the rapid uncaging of TCO-masked tyrosine in serum, followed by oxidation by tyrosinase. Finally, tyrosine uncaging was used to chemically control cell growth in tyrosine-free medium.

Investigating the Cellular Specificity in Tumors of a Surface-Converting Nanoparticle by Multimodal Imaging.

Active targeting of nanoparticles through surface functionalization is a common strategy to enhance tumor delivery specificity. However, active targeting strategies tend to work against long polyethylene glycol's shielding effectiveness and associated favorable pharmacokinetics. To overcome these limitations, we developed a matrix metalloproteinase-2 sensitive surface-converting polyethylene glycol coating. This coating prevents nanoparticle-cell interaction in the bloodstream, but, once exposed to matrix metalloproteinase-2, i.e., when the nanoparticles accumulate within the tumor interstitium, the converting polyethylene glycol coating is cleaved, and targeting ligands become available for binding to tumor cells. In this study, we applied a comprehensive multimodal imaging strategy involving optical, nuclear, and magnetic resonance imaging methods to evaluate this coating approach in a breast tumor mouse model. The data obtained revealed that this surface-converting coating enhances the nanoparticle's blood half-life and tumor accumulation and ultimately results in improved tumor-cell targeting. Our results show that this enzyme-specific surface-converting coating ensures a high cell-targeting specificity without compromising favorable nanoparticle pharmacokinetics.

A novel approach to tracer-kinetic modeling for (macromolecular) dynamic contrast-enhanced MRI.

PURPOSE: To develop a novel tracer-kinetic modeling approach for multi-agent dynamic contrast-enhanced MRI (DCE-MRI) that facilitates separate estimation of parameters characterizing blood flow and microvascular permeability within one individual. METHODS: Monte Carlo simulations were performed to investigate the performance of the constrained multi-agent model. Subsequently, multi-agent DCE-MRI was performed on tumor-bearing mice (n = 5) on a 7T Bruker scanner on three measurement days, in which two dendrimer-based contrast agents having high and intermediate molecular weight, respectively, along with gadoterate meglumine, were sequentially injected within one imaging session. Multi-agent data were simultaneously fit with the gamma capillary transit time model. Blood flow, mean capillary transit time, and bolus arrival time were constrained to be identical between the boluses, while extraction fractions and washout rate constants were separately determined for each agent. RESULTS: Simulations showed that constrained multi-agent model regressions led to less uncertainty and bias in estimated tracer-kinetic parameters compared with single-bolus modeling. The approach was successfully applied in vivo, and significant differences in the extraction fraction and washout rate constant between the agents, dependent on their molecular weight, were consistently observed. CONCLUSION: A novel multi-agent tracer-kinetic modeling approach that enforces self-consistency of model parameters and can robustly characterize tumor vascular status was demonstrated.

Triggered Drug Release from an Antibody-Drug Conjugate Using Fast "Click-to-Release" Chemistry in Mice.

The use of a bioorthogonal reaction for the selective cleavage of tumor-bound antibody-drug conjugates (ADCs) would represent a powerful new tool for ADC therapy, as it would not rely on the currently used intracellular biological activation mechanisms, thereby expanding the scope to noninternalizing cancer targets. Here we report that the recently developed inverse-electron-demand Diels-Alder pyridazine elimination reaction can provoke rapid and self-immolative release of doxorubicin from an ADC in vitro and in tumor-bearing mice.

Complex Coacervate Core Micelles with Spectroscopic Labels for Diffusometric Probing of Biopolymer Networks.

We present the design, preparation, and characterization of two types of complex coacervate core micelles (C3Ms) with cross-linked cores and spectroscopic labels and demonstrate their use as diffusional probes to investigate the microstructure of percolating biopolymer networks. The first type consists of poly(allylamine hydrochloride) (PAH) and poly(ethylene oxide)-poly(methacrylic acid) (PEO-b-PMAA), labeled with ATTO 488 fluorescent dyes. We show that the size of these probes can be tuned by choosing the length of the PEO-PMAA chains. ATTO 488-labeled PEO113-PMAA15 micelles are very bright with 18 dye molecules incorporated into their cores. The second type is a (19)F-labeled micelle, for which we used PAH and a (19)F-labeled diblock copolymer tailor-made from poly(ethylene oxide)-poly(acrylic acid) (mPEO79-b-PAA14). These micelles contain approximately 4 wt % of (19)F and can be detected by (19)F NMR. The (19)F labels are placed at the end of a small spacer to allow for the necessary rotational mobility. We used these ATTO- and (19)F-labeled micelles to probe the microstructures of a transient gel (xanthan gum) and a cross-linked, heterogeneous gel (κ-carrageenan). For the transient gel, sensitive optical diffusometry methods, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, and super-resolution single nanoparticle tracking, allowed us to measure the diffusion coefficient in networks with increasing density. From these measurements, we determined the diameters of the constituent xanthan fibers. In the heterogeneous κ-carrageenan gels, bimodal nanoparticle diffusion was observed, which is a signpost of microstructural heterogeneity of the network.

NMR nanoparticle diffusometry in hydrogels: enhancing sensitivity and selectivity.

From the diffusional behavior of nanoparticles in heterogeneous hydrogels, quantitative information about submicron structural features of the polymer matrix can be derived. Pulsed-gradient spin-echo NMR is often the method of choice because it measures diffusion of the whole ensemble of nanoparticles. However, in (1)H diffusion-ordered spectroscopy (DOSY), low-intensity nanoparticle signals have to be separated from a highly protonated background. To circumvent this, we prepared (19)F labeled, PEGylated, water-soluble dendritic nanoparticles with a (19)F loading of ~7 wt % to enable background free (19)F DOSY experiments. (19)F nanoparticle diffusometry was benchmarked against (1)H diffusion-T2 correlation spectroscopy (DRCOSY), which has a stronger signal separation potential than the commonly used (1)H DOSY experiment. We used bootstrap data resampling to estimate confidence intervals and stabilize 2D-Laplace inversion of DRCOSY data with high noise levels and artifacts, allowing quantitative diffusometry even at low magnetic field strengths (30 MHz). The employed methods offer significant advantages in terms of sensitivity and selectivity.

Balancing Scaffold Degradation and Neo-Tissue Formation in In Situ Tissue Engineered Vascular Grafts.

An essential aspect of cardiovascular in situ tissue engineering (TE) is to ensure balance between scaffold degradation and neo-tissue formation. We evaluated the rate of degradation and neo-tissue formation of three electrospun supramolecular bisurea-based biodegradable scaffolds that differ in their soft-block backbone compositions only. Scaffolds were implanted as interposition grafts in the abdominal aorta in rats, and evaluated at different time points (t = 1, 6, 12, 24, and 40 weeks) on function, tissue formation, strength, and scaffold degradation. The fully carbonate-based biomaterial showed minor degradation after 40 weeks in vivo, whereas the other two ester-containing biomaterials showed (near) complete degradation within 6-12 weeks. Local dilatation was only observed in these faster degrading scaffolds. All materials showed to some extent mineralization, at early as well as late time points. Histological evaluation showed equal and non-native-like neo-tissue formation after total degradation. The fully carbonate-based scaffolds lagged in neo-tissue formation, presumably as its degradation was (far from) complete at 40 weeks. A significant difference in vessel wall contrast enhancement was observed by magnetic resonance imaging between grafts with total compared with minimal-degraded scaffolds.

Off-the-Shelf Synthetic Biodegradable Grafts Transform In Situ into a Living Arteriovenous Fistula in a Large Animal Model.

Current vascular access options require frequent interventions. In situ tissue engineering (TE) may overcome these limitations by combining the initial success of synthetic grafts with long-term advantages of autologous vessels by using biodegradable grafts that transform into autologous vascular tissue at the site of implantation. Scaffolds (6 mm-Ø) made of supramolecular polycarbonate-bisurea (PC-BU), with a polycaprolactone (PCL) anti-kinking-coil, are implanted between the carotid artery and jugular vein in goats. A subset is bio-functionalized using bisurea-modified-Stromal cell-derived factor-1α (SDF1α) derived peptides and ePTFE grafts as controls. Grafts are explanted after 1 and 3 months, and evaluated for material degradation, tissue formation, compliance, and patency. At 3 months, the scaffold is resorbed and replaced by vascular neo-tissue, including elastin, contractile markers, and endothelial lining. No dilations, ruptures, or aneurysms are observed and grafts are successfully cannulated at termination. SDF-1α-peptide-biofunctionalization does not influence outcomes. Patency is lower in TE grafts (50%) compared to controls (100% patency), predominantly caused by intimal hyperplasia. Rapid remodeling of a synthetic, biodegradable vascular scaffold into a living, compliant arteriovenous fistula is demonstrated in a large animal model. Despite lower patency compared to ePTFE, transformation into autologous and compliant living tissue with self-healing capacity may have long-term advantages.

Controlling the growth and shape of chiral supramolecular polymers in water.

A challenging target in the noncovalent synthesis of nanostructured functional materials is the formation of uniform features that exhibit well-defined properties, e.g., precise control over the aggregate shape, size, and stability. In particular, for aqueous-based one-dimensional supramolecular polymers, this is a daunting task. Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing out attractive noncovalent forces within the hydrophobic core of the polymerizing building blocks with electrostatic repulsive interactions on the hydrophilic rim we managed to switch from elongated, rod-like assemblies to small and discrete objects. Intriguingly this rod-to-sphere transition is expressed in a loss of cooperativity in the temperature-dependent self-assembly mechanism. The aggregates were characterized using circular dichroism, UV and 1H-NMR spectroscopy, small angle X-ray scattering, and cryotransmission electron microscopy. In analogy to many systems found in biology, mechanistic details of the self-assembly pathways emphasize the importance of cooperativity as a key feature that dictates the physical properties of the produced supramolecular polymers.

Semi-synthetic degradable notochordal cell-derived matrix hydrogel for use in degenerated intervertebral discs: Initial in vitro characterization.

Low back pain is the leading cause of disability worldwide, but current therapeutic interventions are palliative or surgical in nature. Loss of notochordal cells (NCs) and degradation of the healthy matrix in the nucleus pulposus (NP), the central tissue of intervertebral discs (IVDs), has been associated with onset of degenerative disc changes. Recently, we established a protocol for decellularization of notochordal cell derived matrix (NCM) and found that it can provide regenerative cues to nucleus pulposus cells of the IVD. Here, we combined the biologically regenerative properties of decellularized NCM with the mechanical tunability of a poly(ethylene glycol) hydrogel to additionally address biomechanics in the degenerate IVD. We further introduced a hydrolysable PEG-diurethane crosslinker for slow degradation of the gels in vivo. The resulting hydrogels were tunable over a broad range of stiffness's (0.2 to 4.5 kPa), matching that of NC-rich and -poor NP tissues, respectively. Gels formed within 30 min, giving ample time for handling, and remained shear-thinning post-polymerization. Gels also slowly released dNCM over 28 days as measured by GAG effusion. Viability of encapsulated bone marrow stromal cells after extrusion through a needle remained high. Although encapsulated NCs stayed viable over two weeks, their metabolic activity decreased, and their phenotype was lost in physiological medium conditions in vitro. Overall, the obtained gels hold promise for application in degenerated IVDs but require further tuning for combined use with NCs.

Evaluation of pliable bioresorbable, elastomeric aortic valve prostheses in sheep during 12 months post implantation.

Pliable microfibrous, bioresorbable elastomeric heart valve prostheses are investigated in search of sustainable heart valve replacement. These cell-free implants recruit cells and trigger tissue formation on the valves in situ. Our aim is to investigate the behaviour of these heart valve prostheses when exposed to the high-pressure circulation. We conducted a 12-month follow-up study in sheep to evaluate the in vivo functionality and neo-tissue formation of these valves in the aortic position. All valves remained free from endocarditis, thrombotic complications and macroscopic calcifications. Cell colonisation in the leaflets was mainly restricted to the hinge area, while resorption of synthetic fibers was limited. Most valves were pliable and structurally intact (10/15), however, other valves (5/15) showed cusp thickening, retraction or holes in the leaflets. Further research is needed to assess whether in-situ heart valve tissue engineering in the aortic position is possible or whether non-resorbable synthetic pliable prostheses are preferred.

Disentangle a Complex MALDI TOF Mass Spectrum of Polyethylene Glycols into Three Separate Spectra via Selective Formation of Protonated Ions and Sodium or Potassium Adducts.

In MALDI TOF MS analysis, complicated mass spectra can usually be recorded for polymers with high affinities to protons and alkali metal ions. For these polymers, protonated ions and sodium and potassium adducts can often be formed concomitantly. By distributing these ions into three separate spectra of protonated ions, sodium adducts, and potassium adducts, significantly simplified spectra can be acquired. Mass spectra consisting of only sodium or potassium adducts can often be obtained by simply adding sodium salt and potassium salt, respectively. We report here a method to selectively generate protonated ions. A polyethylene glycol (PEG) sample with amino end groups was selected as the model polymer and α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix. Octadecylamine (ODA) or a mixture of a tetrabutylammonium (TBA) salt and an ammonium salt was used as the co-matrix to inhibit the release of sodium and potassium ions and their related adducts into the MALDI gas phase plume. By depositing the polymer sample on top of a preloaded layer of CHCA with a co-matrix, the generation of Na+ and K+ adducts is suppressed, while [ODA + H]+ and NH4+ released from the preloaded matrix layer can serve as protonation reagents to protonate the polymer molecules via proton transfer reactions. It is clearly demonstrated that disentangling a complex mass spectrum filled densely with various series of ions into three separate spectra, with each one consisting of only one type of ions, allows unambiguous identification of mass peaks and greatly helps the interpretation of MS results.

Micellization of Sequence-Controlled Polyurethane Ionomers in Mixed Aqueous Solvents.

While the impact of compositional parameters such as block length and ionic content on the micellization of (polymeric) amphiphiles is widely investigated, the influence of monomer sequence has received far less attention until recently. Here, we report the synthesis of two sequence-controlled polyurethane ionomers (PUIs) prepared via a stepwise coupling-deprotection strategy, and compare their solution association in aqueous-organic mixtures. The two PUIs are highly similar in mass and overall composition, yet differ markedly in the sequence of building blocks. PUI-A2 comprises a polytetrahydrofuran (pTHF) block connected to an alternation of isophorone diamine (IPDA) and dimethylolpropionic acid (DMPA) units that together are also arranged in a blockwise manner. The result is a macromolecular structure with a comparatively hydrophobic tail (pTHF) and a hydrophilic headgroup, which structure is reminiscent of those of traditional surfactants, albeit much larger in size. PUI-S2 instead resembles a bolaamphiphilic architecture with a pTHF midblock connected on either end to a singly charged segment comprising DMPA and IPDA. We detect micellization below a threshold cosolvent volume fraction (φsolv) of 0.4 in aqueous-organic mixtures with tetrahydrofuran (THF), ethanol, and isopropyl alcohol. We use scattering tools to compare the aggregation number (N agg) and hydrodynamic radius (R h) of PUI-S2 and PUI-A2 micelles. Irrespective of the solvent composition, we observe in the micellar window of φsolv < 0.4, lower N agg for PUI-S2 micelles compared to PUI-A2, which we attribute to packing restraints associated with its bolaamphiphilic architecture. The increase in micellar size with increasing φsolv is much more pronounced for PUI-S2 than for PUI-A2. The micellar mass decreases with increasing φsolv for both PUIs; the effect is modest for PUI-S2 compared to PUI-A2 and is not observed in the most apolar cosolvent studied (THF). Upon the approach of the micellization boundary φsolv ≈ 0.4, both types of PUI micelles become less compact in structure, as (in most cases) PUIs are released and as micellar dimensions increase.

Inconsistency in Graft Outcome of Bilayered Bioresorbable Supramolecular Arterial Scaffolds in Rats.

There is a continuous search for the ideal bioresorbable material to develop scaffolds for in situ vascular tissue engineering. As these scaffolds are exposed to the harsh hemodynamic environment during the entire transformation process from scaffold to neotissue, it is of crucial importance to maintain mechanical integrity and stability at all times. Bilayered scaffolds made of supramolecular polycarbonate-ester-bisurea were manufactured using dual electrospinning. These scaffolds contained a porous inner layer to allow for cellular infiltration and a dense outer layer to provide strength. Scaffolds (n = 21) were implanted as an interposition graft into the abdominal aorta of male Lewis rats and explanted after 1, 3, and 5 months in vivo to assess mechanical functionality and neotissue formation upon scaffold resorption. Results demonstrated conflicting graft outcomes despite homogeneity in the experimental group and scaffold production. Most grafts exhibited adverse remodeling, resulting in aneurysmal dilatation and calcification. However, a few grafts did not demonstrate such features, but instead were characterized by graft extension and smooth muscle cell proliferation in the absence of endothelium, while remaining patent throughout the study. We conclude that it remains extremely difficult to anticipate graft development and performance in vivo. Next to rational mechanical design and good performance in vitro, a thorough understanding of the mechanobiological mechanisms governing scaffold-driven arterial regeneration as well as potential influences of surgical procedures is warranted to further optimize scaffold designs. Careful analysis of the differences between preclinical successes and failures, as is done in this study, may provide initial handles for scaffold optimization and standardized surgical procedures to improve graft performance in vivo. Impact statement In situ vascular tissue engineering using cell-free bioresorbable scaffolds is investigated as an off-the-shelf option to grow small caliber arteries inside the body. In this study, we developed a bilayered electrospun supramolecular scaffold with a dense outer layer to provide mechanical integrity and a porous inner layer for cell recruitment and tissue formation. Despite homogenous scaffold properties and mechanical performance in vitro, in vivo testing as rat aorta interposition grafts revealed distinct graft outcomes, ranging from aneurysms to functional arteries. Careful analysis of this variability provided valuable insights into materials-driven in situ artery formation relevant for scaffold design and implantation procedures.

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.

Co-assembly of precision polyurethane ionomers reveals role of and interplay between individual components.

Industrial and household products, such as paints, inks and cosmetics usually consist of mixtures of macromolecules that are disperse in composition, in size and in monomer sequence. Identifying structure-function relationships for these systems is complicated, as particular macromolecular components cannot be investigated individually. For this study, we have addressed this issue, and have synthesized a series of five sequence-defined polyurethanes (PUs): one neutral-hydrophobic, one single-charged hydrophilic, one single-charged hydrophobic and two double-charged amphiphilic PUs (one symmetric and one asymmetric). These novel precision PUs - that were prepared by using stepwise coupling-deprotection synthetic protocols - have a defined composition, size and monomer sequence, where the chosen sequences were inspired by those that are abundantly formed in the production of industrial waterborne PU dispersions. By performing dynamic light scattering experiments (DLS), self-consistent field (SCF) computations and cryogenic transmission electron microscopy (cryo-TEM), we have elucidated the behavior in aqueous solution of the individual precision PUs, as well as of binary and ternary mixtures of the PU sequences. The double-charged PU sequences ('hosts') were sufficiently amphiphilic to yield single-component micellar solutions, whereas the two more hydrophobic sequences did not micellize on their own, and gave precipitates or ill-defined larger aggregates. Both the neutral-hydrophobic PU and the hydrophilic single-charged PU were successfully incorporated in the host micelles as guests, respectively increasing and reducing the micelle radius upon incorporation. SCF computations indicated that double-charged symmetric PUs stretch whilst double-charged asymmetric PUs are expelled from the core to accommodate hydrophobic PU guests within the micelles. For the ternary mixture of the double-charged symmetric and asymmetric hosts and the neutral-hydrophobic guest we have found an improved colloidal stability, as compared to those for binary mixtures of either host and hydrophobic guest. In another ternary mixture of precision PUs, with all three components not capable of forming micelles on their own, we see that the ensemble of molecules produces stable micellar solutions. Taken together, we find that the interplay between PU-molecules in aqueous dispersions promotes the formation of stable micellar hydrocolloids.