Sebastian Seiffert

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.

Smart Microgel Capsules from Macromolecular Precursors

Microgel particles and capsules which consist of multiple layers can be fabricated using droplet microfluidics, but in existing methods, emulsion templating forms layers of dissimilar polarity. In this paper, we fabricate functional microgel capsules that consist of two miscible yet distinct layers. We use microfluidic devices to template micrometer-sized drops that are loaded with prepolymerized precursors and solidify them through a polymer-analogous reaction. This allows the particle morphology to be controlled and prevents pronounced interpenetration of the different layers despite their miscibility. We use polyacrylamide and poly(N-isopropylacrylamide) precursors to form thermoresponsive core-shell microparticles and demonstrate their utility for encapsulation and controlled release applications.

Hyperbranched polyglycerols on the nanometer and micrometer scale

We report the preparation of polyglycerol particles on different length scales by extending the size of hyperbranched polyglycerols (3 nm) to nanogels (32 nm) and microgels (140 and 220 μm). We use miniemulsion templating for the preparation of nanogels and microfluidic templating for the preparation of microgels, which we obtain through a free-radical polymerization of hyperbranched polyglycerol decaacrylate and polyethylene glycol-diacrylate. The use of mild polymerization conditions allows yeast cells to be encapsulated into the resultant microgels with cell viabilities of approximately 30%.

Dynamic supramolecular poly(isobutylene)s for self-healing materials

Mono- and bifunctional supramolecular poly(isobutylene)s (PIBs) bearing hydrogen-bonding motifs (barbituric acid or a Hamilton wedge) are prepared by a combination of living carbocationic polymerization (LCCP) and azide–alkyne “click” reactions to investigate their dynamics and self-healing behaviour. Barbituric acid (7) or Hamilton wedge (8) functionalized polymers (3a–c, 4a–d, 5a–c, 6a) with molecular weights of ∼3000 up to 30 000 g mol−1 exhibit complete end group transformation as proven by NMR and MALDI methods. Temperature-dependent rheology in the melt reveals thermoreversible formation of supramolecular clusters. Stoichiometric mixing of the polymers by solution blending affects the extent of clustering by specifically interacting barbituric acid/Hamilton wedge moieties. Frequency-dependent measurements on bifunctional barbituric acid functionalized PIBs reveal a strong rubbery plateau and terminal flow, caused by the formation of dynamically bridged clusters. In addition, fluorescence recovery after photobleaching (FRAP) measurements on the same supramolecular polymers reveal a multitude of different chain dynamics. Small discs of these polymers show self-healing at room temperature after being cut and brought into contact at the fractured surface.

Systematic evaluation of FRAP experiments performed in a confocal laser scanning microscope

The diffusion coefficient as well as the dimensionality of the diffusion process can be determined by straightforward and facile data analysis, when fluorescence recovery after photobleaching (FRAP) is measured as a function of time and space by means of confocal laser scanning microscopy. Experiments representing one‐dimensional diffusion from a plane source or two‐dimensional diffusion from a line source are readily realized. In the data analysis, the deviations of the actual initial conditions from ideal models are consistently taken into account, so that no calibration measurements are needed. The method is applied to FRAP experiments on solutions of Rhodamine B in glycerol and aqueous suspensions of polymethyl methacrylate microspheres.

Small but Smart: Sensitive Microgel Capsules

Microgel capsules are micrometer-sized particles that consist of a cross-linked and swollen polymer network complexed with additives. These capsules can be actuated by external stimulation if they are formed from sensitive or supramolecular polymer networks. To make this truly useful, it is crucial to control the microgel size, shape, and loading; this can be achieved by droplet-based microfluidic templating.

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.

Janus Microgels Produced from Functional Precursor Polymers

Micrometer-sized Janus particles of many kinds can be formed using droplet microfluidics, but in existing methods, the microfluidic templating is strongly coupled to the material synthesis, since droplet solidification occurs through rapid polymerization right after droplet formation. This circumstance limits independent control of the material properties and the morphology of the resultant particles. In this paper, we demonstrate a microfluidic technique to produce functional Janus microgels from prefabricated, cross-linkable precursor polymers. This approach separates the polymer synthesis from the particle gelation, thus allowing the microfluidic droplet templating and the functionalization of the matrix polymer to be performed and controlled in two independent steps. We use microfluidic devices to emulsify semidilute solutions of cross-linkable, chemically modified or unmodified poly(N-isopropylacrylamide) precursors and solidify the drops via polymer-analogous gelation. The resultant microgel particles exhibit two distinguishable halves which contain most of the modified precursors, and the unmodified matrix polymer separates these materials. The spatial distribution of the modified precursors across the particles can be controlled by the flow rates during the microfluidic experiments. We also form hollow microcapsules with two different sides (Janus shells) using double emulsion droplets as templates, and we produce Janus microgels that are loaded with a ferromagnetic additive which allows remote actuation of the microgels.

Synthesis of Monodisperse Microparticles from Non-Newtonian Polymer Solutions with Microfluidic Devices

Microfluidic devices can form emulsions that are highly uniform in size;[1–3] they can also form compound emulsions, in which each supradroplet contains exactly the same number of internal droplets, packed in exactly the same configuration.[4–6] Because the drops can be formed with a highly controlled structure and uniformity, they are useful as templates to synthesize monodisperse particles. In such a process, microfluidic devices are used to form droplets with the desired structure, which are then solidified to produce particles. This allows synthesis of particles with a variety of shapes, including Janus particles, nonspherical dimers, and core–shell capsules.[7–10] However, the fluid precursors must be compatible with the formation of drops in microfluidic devices: this precondition limits the applicability of this technique. For example, this circumstance requires fluids with a low viscosity, negligible viscoelastic response, and moderate interfacial tension. If even one of these constraints is not met, it is difficult to achieve drop formation in the stable dripping regime. Instead, jetting occurs, resulting in the production of polydisperse particles.[11] This represents a significant limitation, as the most useful materials for making particles typically have properties that do not meet these constraints. For example, lipid melts, due to their amphiphilic chemical properties, tend to be viscous and have low interfacial tension with oil or aqueous carrier phases. Solutions of long-chain polymers like poly(N-isopropylacrylamide) (pNIPAM)[12] or polyurethane–polybutadienediol (pU–pBDO),[13] form excellent particles, but tend to be viscous and have significant viscoelastic response at the shear rates needed for controlled drop formation.[1,11,14] As a consequence, these fluids, and many others like them, cannot be used to synthesize particles in microfluidic devices, greatly limiting the applicability of this technique. To overcome the limitations with such fluids, a more robust drop formation mechanism is needed.

Controlled fabrication of polymer microgels by polymer-analogous gelation in droplet microfluidics

We fabricate thermo-responsive polymer microgels by combining microfluidic pre-gel emulsification with polymer-analogous gelation. This separates the microgel formation from the polymer synthesis; it combines highly controlled microfluidic templating with the great flexibility of preparative polymer chemistry, allowing each to be controlled independently. We produce monodisperse pre-gel droplets from semidilute solutions of photocrosslinkable poly(N-isopropylacrylamide) precursors. The size and morphology of these droplets can be precisely controlled by the microfluidic emulsification, provided the molecular weight of the precursor is limited. Using polymer-analogous gelation rather than monomer chain-growth gelation yields gels with a higher efficiency of crosslinking and a greater homogeneity on nano- and micrometre scales, as determined by oscillatory shear rheology, static light scattering, and optical microscopy. We also demonstrate the applicability of our method to fabricate microgel particles with well-defined concentrations of functional sites.