Albumin Microspheres as “Trans-ferry-beads” for Easy Cell Passaging in Cell Culture Technology

Protein hydrogels represent ideal materials for advanced cell culture applications, including 3D-cultivation of even fastidious cells. Key properties of fully functional and, at the same time, economically successful cell culture materials are excellent biocompatibility and advanced fabrication processes allowing their easy production even on a large scale based on affordable compounds. Chemical crosslinking of bovine serum albumin (BSA) with N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) in a water-in-oil emulsion with isoparaffinic oil as the continuous phase and sorbitan monooleate as surfactant generates micro-meter-scale spherical particles. They allow a significant simplification of an indispensable and laborious step in traditional cell culture workflows. This cell passaging (or splitting) to fresh culture vessels/flasks conventionally requires harsh trypsinization, which can be omitted by using the “trans-ferry-beads” presented here. When added to different pre-cultivated adherent cell lines, the beads are efficiently boarded by cells as passengers and can be easily transferred afterward for the embarkment of novel flasks. After this procedure, cells are perfectly viable and show normal growth behavior. Thus, the trans-ferry-beads not only may become extremely affordable as a final product but also may generally replace trypsinization in conventional cell culture, thereby opening new routes for the establishment of optimized and resource-efficient workflows in biological and medical cell culture laboratories

In situ preparation of stabilizing units for Pickering emulsions and some applications

Self-assembly is a very versatile tool for the fabrication of materials on the nanoscale and plays a predominant role in the formation and stabilization of both classical (surfactant stabilized) and Pickering (particle stabilized) emulsions which for their part find numerous applications in e. g. biomedicine, cosmetics and food industry. Furthermore, emulsion droplets can serve as templates for different materials as well as compartments for reactants or chemical reactions. One of the most important tasks with respect to both the preparation and the application of emulsions is to understand the underlying self-assembly process and gain control over it as this enables the rational design of emulsions with specific physical and chemical properties. The different projects covered within this thesis focus on the in situ preparation of stabilizing units for Pickering emulsions by different self-assembly processes at the oil-water interface. Subsequently, the as-prepared emulsion droplets are modified either physically or chemically. Although in any case the miniemulsion technique is applied in order to obtain emulsions with droplet sizes in the sub-μm range, two fundamentally different systems are investigated: on the one hand, commercially available hydrophilic silica nanoparticles are hydrophobized in situ by the self-assembly with neutral polymeric hydrophobizing agents and consequently used to stabilize aqueous droplets with a diameter between 180 and 450 nm in an oily continuous phase. In the first step, the adsorption behaviour of the hydrophobizing agent on silica nanoparticles as well as the influence of size and charge of the particles, kind and amount of hydrophobizing agent and the composition of the aqueous dispersed phase on emulsion characteristics is evaluated. In the second step, the initially milky-white emulsion is heated to evaporate water from the inside of the droplets and subsequently change the composition of the aqueous phase. As the stabilizing particles are small compared to the wavelength of visible light, one emulsion droplet and the corresponding stabilizing nanoparticles act as one single scattering object. This enables the fine-tuning of the refractive index (RI) of the emulsion droplets by evaporation of water and consequently the preparation of Pickering emulsions with a remarkable transmittance of up to 86 % across the visible spectrum without adjusting the RI of the particles. This property is unique in the field of Pickering emulsions as the only reports on highly transparent particle stabilized emulsions deal with polymeric particles whose RI is carefully matched with the one of both the continuous and the dispersed phase before preparing the emulsion. The second emulsion system is used to establish a completely new approach of stabilizing emulsions. Water-soluble organic dyes are shown to self-assemble into aggregates in situ at the oil-water interface and these dye aggregates act as very efficient stabilizers for oil-in-water emulsions. Thus, in contrast to conventional Pickering emulsions, the stabilizing particular units are formed in situ instead of being already employed in the form of nanoparticles. By comparing the stabilization behaviour of fluorescein as an example for water-soluble dyes with the one of the classical amphiphile sodium dodecyl sulfate (SDS) exemplarily, similarities and differences between surfactants and dye aggregates as stabilizers for direct miniemulsions are revealed. Different parameters such as interfacial tension, concentration of the stabilizer, salinity of the aqueous phase, pH-value and polarity of the organic oil are taken into account and discussed. With respect to potential applications, not only the stabilization but also the controlled destabilization of emulsions might be of special interest. For Pickering emulsions stabilized by dye aggregates, different methods of demulsification including heating, addition of electrolyte, change in pH-value and addition of solid adsorbents are investigated and show the great potential of this new kind of stabilizer regarding the controlled phase separation of emulsions which are typically stable for years when letting unaffected. Since it could be shown that styrene can be employed as the organic liquid in dye stabilized emulsions, the follow-up work focusses on the free radical polymerization of styrene in emulsion droplets stabilized by fluorescein and alizarin yellow. The system containing alizarin yellow is investigated in detail with respect to the influence of dye concentration and ultrasonication time on the resulting latex as well as the nucleation mechanism of the polymerization. In the last step, oil droplets containing the silica precursor tetraethyl orthosilicate (TEOS) are stabilized by different anionic and cationic dyes and the hydrolysis and condensation reaction at the oil-water interface is investigated. By the choice of the proper dye, either one silica capsule or many monodisperse sub-20 nm silica particles are obtained from one single emulsion droplet. The formation of small nanoparticles in emulsions stabilized by negatively charged dyes is consequently investigated as this reaction is unexpected while the formation of silica capsules has already been described in literature for emulsions stabilized by e. g. cetyltrimethylammonium bromide (CTAB). As the oil-water interface in miniemulsions is much bigger than macroscopic interfaces, the kinetics of hydrolysis and condensation reaction is increased significantly which enables the synthesis of silica particles at ambient temperature and pH-value under static conditions. As organic solvents can be abandoned completely and the stabilizing dye can be removed with the help of solid adsorbents easily, particle dispersions comprising only water at neutral pH, particles and traces of ethanol can be prepared in accordance with the idea of “green chemistry” which is in contrast to literature known synthesis routes to sub-20 nm silica nanoparticles. In order to get a better understanding of the reaction mechanism, several parameters such as concentration of the dye, salinity, pH-value, reaction temperature, reaction time and kind of the stabilizing dye are taken into account and varied systematically. In summary, different self-assembly processes were investigated in order to understand their role in the formation of stabilizing units for Pickering emulsions with droplet sizes in the sub- μm range. The respective findings contribute to the subsequent rational physical or chemical modification of emulsion droplets. Hence, the preparation of highly transparent Pickering emulsions stabilized by commercially available silica nanoparticles in a very simple two-step process without adjusting the RI of the stabilizing particles could be implemented. On the other hand, water-soluble organic dyes which are frequently applied for colouring applications were introduced as new building blocks for the in situ preparation of dye aggregates which act as a kind of molecular scale Pickering stabilizer in direct miniemulsion systems. These emulsion droplets were successfully used as templates for i) the free radical polymerization of styrene inside the droplets which enables the preparation of surfactant free latex and ii) the interfacial hydrolysis and condensation reaction of TEOS which results in either silica capsules if positively charged dyes are employed as stabilizers or sub-20 nm silica particles in the case of anionic dyes. As the dyes are easily removable from the emulsion system by the addition of an appropriate solid adsorbent, these emulsions can be separated in a stabilizer-free oil and a stabilizer-free aqueous phase in a very controlled manner which is unique in the field of both Pickering and classical emulsions. Thus, dye stabilized emulsions offer a great potential for several applications including the compartmentalization of chemical reactions as they enable a very simple work-up

Dye Aggregates as New Stabilizers for (Mini)emulsions

Water-soluble organic dyes such as fluorescein are widely used, mainly for coloration of, e.g., biological samples and groundwater tracing, and they are not obviously amphiphilic by molecular structure like surfactants. Here, we show that the dyes alone stabilize oil-in-water emulsions. Exemplarily, fluorescein is compared with the classical surfactant sodium dodecyl sulfate (SDS) by means of surface/interfacial tension, concentration of stabilizer and electrolyte, as well as pH. The principle can be extended to further classes of water-soluble dyes, which keep up with or exceed SDS by efficiency. Various organic liquids of different polarities can be employed and be polymerized in the case of styrene as disperse phase. Thus, surfactant free latex solely stabilized by water-soluble dyes is accessible. The emulsions can be destabilized by absorption of the dyes to hydrogels, and their complete removal is easily followed visually. The stabilization mechanisms are different for SDS and the dyes: The latter stabilize droplets not as monomers but by their aggregates as molecular scale Pickering stabilizers, which is a new concept of stabilization

Investigation of the Mechanism of SiO2_2 Particle and Capsule Formation at the Oil–Water Interface of Dye-Stabilized Emulsions

In a previous contribution we described the formation of silica nanostructures in dye-stabilized nanoemulsions from tetraethyl orthosilicate droplets in water. Depending on the type of dye, either capsules (crystal violet, CV) or nanoparticles (congo red, CR) are formed. The thorough study of the sol-gel process uses a combination of time- and/or temperature-resolved small-angle X-ray scattering, transmission electron microscopy, and $^1$H NMR spectroscopy to elucidate the detailed kinetics and mechanism of structure formation. In both cases, small nuclei of 1.5-2 nm are formed, followed by either a fast cluster-cluster (CV) or a much slower monomer-cluster aggregation (CR). The former leads to a cross-linked network and finally to patchy capsules, while the latter leads to individual nanoparticles (SNPs). From an Avrami plot it can be deduced that the SNPs are formed by an interface-controlled one-dimensional growth process. The mechanisms are based on the different local environments at the oil-water interface, which is either slightly acidic (CV) or fairly basic (CR). The kinetics differ by a factor between 3 and 20 and are presumably caused by the different mobility of the catalyzing species H$^+$ or OH$^-$

Inverse Pickering Emulsions with Droplet Sizes below 500 nm

Inverse Pickering emulsions with droplet diameters between 180 and 450 nm, a narrow droplet size distribution, and an outstanding stability were prepared using a miniemulsion technique. Commercially available hydrophilic silica nanoparticles were used to stabilize the emulsions. They were hydrophobized in situ by the adsorption of various neutral polymeric surfactants. The influence of different parameters, such as kind and amount of surfactant as hydrophobizing agent, size and charge of the silica particles, and amount of water in the dispersed phase, as well as the kind of osmotic agent (sodium chloride and phosphate-buffered saline), on the emulsion characteristics was investigated. The systems were characterized by dynamic light scattering, transmission electron microscopy, cryo-scanning electron microscopy (cryo-SEM), thermogravimetric analysis, and semiquantitative attenuated total reflection infrared spectroscopy. Cryo-SEM shows that some silica particles are obviously rendered hydrophilic and form a three-dimensional network inside the droplets

Green Chemistry in Red Emulsion: Interface of Dye Stabilized Emulsions as a Powerful Platform for the Formation of sub-20-nm SiO₂ Nanoparticles

Dye stabilized nanoemulsions offer the unique possibility of creating both silica capsules and sub-20-nm particles with precise control of particle size and narrow dispersity from the same system by the choice of the proper dye. The large o/w interface enhances the kinetics of particle formation significantly over macroscopic interfaces which enables the synthesis of silica nanoparticles without any catalyst or elevated temperatures under static conditions. This is in contrast to syntheses for sub-20-nm silica nanoparticles described until now which can normally not be conducted at neutral pH and/or room temperature without stirring. Furthermore, the synthesis can be run without any additional organic solvent and the dyes can be easily removed from the dispersion which opens the pathway to silica dispersions containing only particles, traces of ethanol and water at neutral pH without centrifugation, washing, or redispersion in accordance with the idea of “green chemistry”