Robust Protocol for the Synthesis of BSA Nanohydrogels by Inverse Nanoemulsion for Drug Delivery.

In a highly efficient and reproducible process, bovine serum albumin (BSA) nanogels are prepared from inverse nanoemulsions. The concept of independent nanoreactors of the individual droplets in the nanoemulsions allows high protein concentrations of up to 0.6% in the inverse total system. The BSA gel networks are generated by the 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride coupling strategy widely used in protein chemistry. In a robust work-up protocol, the hydrophobic continuous phase of the inverse emulsion is stepwise replaced by water without compromising the colloidal stability and non-toxicity of the nanogel particles. Further, the simple process allows the loading of the nanogels with various cargos like a dye (Dy-495), a drug (ibuprofen), another protein [FMN-binding fluorescent protein (EcFbFP)], and oligonucleotides [plasmid DNA for enhanced GFP expression in mammalian cells (pEGFP c3) and a synthetic anti-Pseudomonas aeruginosa aptamer library]. These charged nanoobjects work efficiently as carriers for staining and transfection of cells. This is exemplarily shown for a phalloidin dye and a plasmid DNA as cargo with adenocarcinomic human alveolar basal epithelial cells (A549), a cell revertant of the SV-40 cancer rat cell line SV-52 (Rev2), and human breast carcinoma cells (MDA-MB-231), respectively.

Investigation of the Mechanism of SiO2 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 1H 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-.

BSA Hydrogel Beads Functionalized with a Specific Aptamer Library for Capturing Pseudomonas aeruginosa in Serum and Blood.

Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide. Pseudomonas aeruginosa is a WHO-alerted multi-resistant pathogen of extreme importance as a cause of sepsis. Septicemia patients have significantly increased survival chances if sepsis is diagnosed in the early stages. Affinity materials can not only represent attractive tools for specific diagnostics of pathogens in the blood but can prospectively also serve as the technical foundation of therapeutic filtration devices. Based on the recently developed aptamers directed against P. aeruginosa, we here present aptamer-functionalized beads for specific binding of this pathogen in blood samples. These aptamer capture beads (ACBs) are manufactured by crosslinking bovine serum albumin (BSA) in an emulsion and subsequent functionalization with the amino-modified aptamers on the bead surface using the thiol- and amino-reactive bispecific crosslinker PEG4-SPDP. Specific and quantitative binding of P. aeruginosa as the dedicated target of the ACBs was demonstrated in serum and blood. These initial but promising results may open new routes for the development of ACBs as a platform technology for fast and reliable diagnosis of bloodstream infections and, in the long term, blood filtration techniques in the fight against sepsis.

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.

Double in Situ Preparation of Raspberry-like Polymer Particles.

Well-defined raspberry-like poly(styrene- co-4-vinylpyridine)-SiO2 nanocomposite particles with a diameter of around 200 nm were easily prepared by a double in situ process in nanoemulsion with the water-soluble dye Eosin Y as the stabilizer. During radical polymerization of the nanodroplets comprising styrene, 4-vinylpyridine (4-VP), and tetraethoxysilane (TEOS), the silane phase is expelled from the polymer phase to the oil/water (o/w) interface. In the later polymerization stage, SiO2 nanoparticles with a size of around 25 nm were produced via the in situ sol-gel reaction of TEOS at the o/w interface promoted by the negatively charged dye. The pyridine moieties in the copolymer fix the SiO2 nanoparticles on the surface of the polymer particles by electrostatic interactions without any sign of free unbound silica particles as proven by transmission electron microscopy.

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".

Directing intracellular supramolecular assembly with N-heteroaromatic quaterthiophene analogues.

Self-assembly in situ, where synthetic molecules are programmed to organize in a specific and complex environment i.e., within living cells, can be a unique strategy to influence cellular functions. Here we present a small series of rationally designed oligothiophene analogues that specifically target, locate and dynamically self-report their supramolecular behavior within the confinement of a cell. Through the recognition of the terminal alkyl substituent and the amphiphilic pyridine motif, we show that the cell provides different complementary pathways for self-assembly that can be traced easily with fluorescence microscopy as their molecular organization emits in distinct fluorescent bands. Importantly, the control and induction of both forms are achieved by time, temperature and the use of the intracellular transport inhibitor, bafilomycin A1. We showcase the importance of both intrinsic (cell) and extrinsic (stimulus) factors for self-organization and the potential of such a platform toward developing synthetic functional components within living cells.

Highly Transparent w/o Pickering Emulsions without Adjusting the Refractive Index of the Stabilizing Particles.

Pickering emulsions with a remarkable transmittance of up to 86% across the visible spectrum have been prepared without adjusting the refractive index (RI) of the stabilizing particles to those of the aqueous and oil phases. Commercially available hydrophilic silica particles with a diameter of 20 nm, which are hydrophobized partially in situ, were used to stabilize water droplets with diameters below 400 nm in IsoparM. In this system, the stabilizing particles and the emulsion droplets act as one single scattering object, which renders RI-matching of the particles unnecessary. By either evaporation of some water from the droplets or addition of an appropriate organic liquid to the oil phase, it is possible to match the RI of the droplets (aqueous phase + particles) with that of the continuous phase, which minimizes scattering and results in highly transparent emulsions.

Alizarin Yellow R (AYR) as compatible stabilizer for miniemulsion polymerization.

HYPOTHESIS: Many solid particles have been used in Pickering stabilized (mini)emulsions. Stabilizing "particles" can be also formed in situ e.g. by aggregation of dye molecules as reported recently. Among the dyes sodium 2-hydroxy-5-[(E)-(4-nitrophenyl)diazenyl]benzoate (Alizarin Yellow R, (AYR)) is one of the best stabilizers. It is assumed to act as sole stabilizer also in heterophase polymerizations and offers a great potential for applications. EXPERIMENTS: Aqueous solutions of AYR in varying concentrations (0.3, 0.5, 1.0, 1.5, 2.0mg/mL (dye/water)) were employed as continuous phase in direct miniemulsions. The oil phase comprised ethenylbenzene (styrene) and hexadecane. The effects of AYR concentration and ultrasonication time on size and distribution of the droplets were investigated. The miniemulsions were polymerized with a water-soluble azo-initiator (2,2'-azobis[n-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate, VA-057) and conversion and kinetics were determined. FINDINGS: The AYR is successfully employed as stabilizer in Pickering-like miniemulsion polymerizations of styrene. The higher the AYR concentrations the more stable the miniemulsions, the smaller the droplet sizes and the narrower the distributions are, ranging from ca. 450 to 180nm and 0.38 to 0.15, respectively. The nucleation mechanism of the polymer particles could be revealed by the number ratio of droplets and particles and follows droplet nucleation. This is confirmed by polymerization kinetics, which is in accordance with classical miniemulsion polymerization, too.

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.

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.