Torsten Rossow

Controlled Synthesis of Cell-Laden Microgels by Radical-Free Gelation in Droplet Microfluidics

Micrometer-sized hydrogel particles that contain living cells can be fabricated with exquisite control through the use of droplet-based microfluidics and bioinert polymers such as polyethyleneglycol (PEG) and hyperbranched polyglycerol (hPG). However, in existing techniques, the microgel gelation is often achieved through harmful reactions with free radicals. This is detrimental for the viability of the encapsulated cells. To overcome this limitation, we present a technique that combines droplet microfluidic templating with bio-orthogonal thiol-ene click reactions to fabricate monodisperse, cell-laden microgel particles. The gelation of these microgels is achieved via the nucleophilic Michael addition of dithiolated PEG macro-cross-linkers to acrylated hPG building blocks and does not require any initiator. We systematically vary the microgel properties through the use of PEG linkers with different molecular weights along with different concentrations of macromonomers to investigate the influence of these parameters on the viability and proliferation of encapsulated yeast cells. We also demonstrate the encapsulation of mammalian cells including fibroblasts and lymphoblasts.

A microgel construction kit for bioorthogonal encapsulation and pH-controlled release of living cells.

pH-Cleavable cell-laden microgels with excellent long-term viabilities were fabricated by combining bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) and droplet-based microfluidics. Poly(ethylene glycol)dicyclooctyne and dendritic poly(glycerol azide) served as bioinert hydrogel precursors. Azide conjugation was performed using different substituted acid-labile benzacetal linkers that allowed precise control of the microgel degradation kinetics in the interesting pH range between 4.5 and 7.4. By this means, a pH-controlled release of the encapsulated cells was achieved upon demand with no effect on cell viability and spreading. As a result, the microgel particles can be used for temporary cell encapsulation, allowing the cells to be studied and manipulated during the encapsulation and then be isolated and harvested by decomposition of the microgel scaffolds.

A modular construction kit for supramolecular polymer gels

Supramolecular polymer gels are swollen networks of macromolecules interconnected by transient, non-covalent bonds; they form an extraordinarily useful class of soft, stimuli-sensitive materials. To optimize the use of supramolecular polymer gels in applications, their physical and chemical properties must be understood. This understanding is ideally achieved using model systems that allow the type and strength of supramolecular chain crosslinking to be varied to a great extent without concurrent alteration of the properties of the covalent polymer backbones. We introduce a system that provides these requirements. We use linear chains of electrophilic methacryl-succinimidyl (MASI) modified poly(N-isopropylacrylamide) (pNIPAAm). These polymers can be modified in a modular fashion by replacing their electrophilic MASI units by nucleophilic amine-functionalized derivatives of custom, supramolecular crosslinkable functionalities. We follow this approach and prepare a set of pNIPAAm polymers that consist of exactly the same polymer backbone functionalized with different types of crosslinkable sidegroups. These polymers are then crosslinked by addition of low molecular weight linkers that are complementary to the motifs on the polymer. We use multiple hydrogen bonding based on diaminotriazine and maleimide, cyanuric acid and Hamilton wedges, and diaminotriazine and cyanuric acid; we also use metal complexation based on terpyridine and different metal salts. This approach creates supramolecular networks of greatly varying rheological properties, from low viscous liquids to elastic gels, each showing consistent and quantitative correlation between the gel mechanical properties and the binding strength of the respective constituent supramolecular crosslinking motifs. Exploiting the good solubility of the common pNIPAAm backbone polymer in a variety of solvents allows these networks to be prepared and studied in different media with unprecedented consistency and flexibility.

Supramolecular polymer gels with potential model-network structure

Supramolecular polymer gels are swollen networks of non-covalently interconnected macromolecules with a variety of potential applications as soft, stimuli-sensitive materials. The utility of these materials is based on their mechanical properties, which are determined on two levels. On a molecular scale, the strength of transient chain crosslinking is a main contributor; whereas on above-molecular scales, an additional contributor is the polymer network topology. In this paper, we present a modular toolkit to form supramolecular polymer networks that allows both these contributors to be controlled. Our approach is based on transition-metal mediated linking of star-shaped poly(ethylene glycol) building blocks that are end-capped with terpyridine moieties. This allows supramolecular networks of greatly varying strengths of transient interlinkage to be prepared by a modular choice of the linking metal ion and the surrounding solvent. We follow this approach and prepare a set of different supramolecular polymer gel networks with mechanical properties that are quantitatively related to the strength of their constituent crosslinking complexes. Static light scattering reveals just minor nanometer-scale polymer network inhomogeneity in some of the gels, whereas others exhibit non-negligible nanostructural heterogeneity. In the latter gels, we find the mechanical strength and resistance to relaxation to be greater than expected, indicating clustering of supramolecular crosslinks to be a mechanism of enforcement.

Supramolecular Hydrogel Capsules Based on PEG: A Step Toward Degradable Biomaterials with Rational Design

Supramolecular microgel capsules based on polyethylene glycol (PEG) are a promising class of soft particulate scaffolds with tailored properties. An approach to fabricate such particles with exquisite control by droplet-based microfluidics is presented. Linear PEG precursor polymers that carry bipyridine moieties on both chain termini are gelled by complexation to iron(II) ions. To investigate the biocompatibility of the microgels, living mammalian cells are encapsulated within them. The microgel elasticity is controlled by using PEG precursors of different molecular weights at different concentrations and the influence of these parameters on the cell viabilities, which can be optimized to exceed 90% is studied. Reversion of the supramolecular polymer cross-linking allows the microcapsules to be degraded at mild conditions with no effect on the viability of the encapsulated and released cells.

Hybrid Microgels with Thermo-Tunable Elasticity for Controllable Cell Confinement

Stimuli-responsive hydrogels are able to change their physical properties such as their elastic moduli in response to changes in their environment. If biocompatible polymers are used to prepare such materials and if living cells are encapsulated within these networks, their switchability allows the cell-matrix interactions to be investigated with unprecedented consistency. In this paper, thermo-responsive macro- and microscopic hydrogels are presented based on azide-functionalized copolymers of poly(N-(2-hydroxypropyl)-methacrylamide) and poly(hydroxyethyl methacrylate) grafted with poly(N-isopropylacrylamide) side chains. Crosslinking of these comb polymers is realized by bio-orthogonal strain-promoted azide-alkyne cycloaddition with cyclooctyne-functionalized poly(ethylene glycol). The resulting hybrid hydrogels exhibit thermo-tunable elasticity tailored by the polymer chain length and grafting density. This bio-orthogonal polymer crosslinking strategy is combined with droplet-based microfluidics to encapsulate living cells into stimuli-responsive microgels, proving them to be a suitable platform for future systematic stem-cell research.

Supramolecular Polymer Networks: Preparation, Properties, and Potential

Supramolecular polymer networks consist of macromolecules interconnected by transient, noncovalent bonds such as those through hydrogen bonding, transition metal complexation, hydrophobic interaction, ionic attraction, or π–π stacking. These networks form an extraordinarily useful class of soft, stimuli-sensitive materials. Although they assemble to strong materials under favorable conditions, they are easily disassembled under other conditions. This ambivalent nature renders supramolecular polymer networks useful for applications in drug delivery, tissue engineering, self-healing, and shape-memory materials. These applications require a deep and comprehensive understanding of the physical chemistry of supramolecular networks, with a particular view to the complex interplay between their structure, dynamics, and properties. Approaches that have attempted to derive such knowledge are often based on investigations of supramolecular polymer networks in the melt or of supramolecular polymer networks swollen in organic media. These approaches are reviewed in the first part of this chapter. In the second part, we focus on the preparation and characterization of supramolecular hydrogels based on synthetic and natural precursors and reveal their utility and potential in life science applications.

Tin(IV) oxide coatings from hybrid organotin/polymer nanoparticles.

Tin dioxide coatings are widely applied in glasses and ceramics to improve not only optical, but also mechanical properties. In this work, we report a new method to prepare SnO(2) coatings from aqueous dispersions of polymer/organotin hybrid nanoparticles. Various liquid organotin compounds were encapsulated in polymeric nanoparticles synthesized by miniemulsion polymerization. Large amounts of tetrabutyltin and bis(tributyltin) could be successfully incorporated in cross-linked and noncross-linked polystyrene nanoparticles that served as sacrificial templates for the formation of tin oxide coatings after etching with oxygen plasma or calcination. Cross-linked polystyrene particles containing bis(tributyltin)--selected for having a high boiling point--were found to be especially suited for the oxide coating formation. The content of metal in the particles was up to 12 wt %, and estimations by thermogravimetrical indicated that at least 96% of the total organotin compound was converted to SnO(2). The resulting coatings were mainly identified as tetragonal SnO(2) (cassiterite) by X-ray diffraction, although a coexistence of this phase with orthorhombic SnO(2) was observed for samples prepared with bis(tributyltin).

Hybrid Polymer-Network Hydrogels with Tunable Mechanical Response

Hybrid polymer-network gels built by both physical and covalent polymer crosslinking combine the advantages of both these crosslinking types: they exhibit high mechanical strength along with excellent fracture toughness and extensibility. If these materials are extensively deformed, their physical crosslinks can break such that strain energy is dissipated and irreversible fracturing is restricted to high strain only. This mechanism of energy dissipation is determined by the kinetics and thermodynamics of the physical crosslinking contribution. In this paper, we present a poly(ethylene glycol) (PEG) based material toolkit to control these contributions in a rational and custom fashion. We form well-defined covalent polymer-network gels with regularly distributed additional supramolecular mechanical fuse links, whose strength of connectivity can be tuned without affecting the primary polymer-network composition. This is possible because the supramolecular fuse links are based on terpyridine–metal complexation, such that the mere choice of the fuse-linking metal ion adjusts their kinetics and thermodynamics of complexation–decomplexation, which directly affects the mechanical properties of the hybrid gels. We use oscillatory shear rheology to demonstrate this rational control and enhancement of the mechanical properties of the hybrid gels. In addition, static light scattering reveals their highly regular and well-defined polymer-network structures. As a result of both, the present approach provides an easy and reliable concept for preparing hybrid polymer-network gels with rationally designed properties.

Microfluidic Synthesis of Pharmacologically Responsive Supramolecular Biohybrid Microgels

Biohybrid hydrogels that change their mechanical properties in response to pharmacological cues hold high promises as externally controlled drug depots for biomedical applications. In this study, we devise a generically applicable method for the synthesis of micrometer-scale, injection-ready biohybrid materials. We use droplet-based microfluidics to generate monodisperse pre-microgel fluid droplets, wherein which we react fluorescein-modified 8-arm poly(ethylene glycol) with a thiol-functionalized humanized anti-fluorescein single chain antibody fragment and vinylsulfone-functionalized 8-arm poly(ethylene glycol), resulting in the formation of stable, narrowly dispersed supramolecular microgels (30 and 150 μm diameter). We demonstrate that the addition of free fluorescein to these microgels results in a weakening of their hydrogel structure, eventually leading to its disintegration. This method of formation of pharmacologically responsive biohybrid hydrogels in an injection-ready formulation is a pioneering example of a general approach for the synthesis of biohybrid hydrogel-based drug depots for biomedical applications.