Rapid Particle Patterning in Surface Deposited Micro-Droplets of Low Ionic Content via Low-Voltage Electrochemistry and Electrokinetics.

Electrokinetic phenomena are a powerful tool used in various scientific and technological applications for the manipulation of aqueous solutions and the chemical entities within them. However, the use of DC-induced electrokinetics in miniaturized devices is highly limited. This is mainly due to unavoidable electrochemical reactions at the electrodes, which hinder successful manipulation. Here we present experimental evidence that on-chip DC manipulation of particles between closely positioned electrodes inside micro-droplets can be successfully achieved, and at low voltages. We show that such manipulation, which is considered practically impossible, can be used to rapidly concentrate and pattern particles in 2D shapes in inter-electrode locations. We show that this is made possible in low ion content dispersions, which enable low-voltage electrokinetics and an anomalous bubble-free water electrolysis. This phenomenon can serve as a powerful tool in both microflow devices and digital microfluidics for rapid pre-concentration and particle patterning.

Solvent-free liquid crystals and liquids based on genetically engineered supercharged polypeptides with high elasticity.

A series of solvent-free elastin-like polypeptide liquid crystals and liquids are developed by electrostatic complexation of supercharged elastin-like polypeptides with surfactants. The smectic mesophases exhibit a high elasticity and the values can be easily tuned by varying the alkyl chain lengths of the surfactants or the lengths of the elastin-like polypeptides.

Thermotropic liquid crystals from biomacromolecules.

Complexation of biomacromolecules (e.g., nucleic acids, proteins, or viruses) with surfactants containing flexible alkyl tails, followed by dehydration, is shown to be a simple generic method for the production of thermotropic liquid crystals. The anhydrous smectic phases that result exhibit biomacromolecular sublayers intercalated between aliphatic hydrocarbon sublayers at or near room temperature. Both this and low transition temperatures to other phases enable the study and application of thermotropic liquid crystal phase behavior without thermal degradation of the biomolecular components.

Recombinant supercharged polypeptides restore and improve biolubrication.

Recombinant supercharged polypeptides (SUPs) with low cytotoxicity are developed and applied to rejuvenate the lubrication of naturally occurring salivary conditioning films (SCFs). SUPs with 72 positive charges adsorbed and rigidified the SCFs and recruited mucins to form a hydrated layer. These SCFs with SUPs have higher mechanical strength and sustain lubricating effect for longer duration compared with only SCFs.

Enhancing cellular uptake of GFP via unfolded supercharged protein tags.

One of the barriers to the development of protein therapeutics is effective delivery to mammalian cells. The proteins must maintain a careful balance of polar moieties to enable administration and distribution and hydrophobic character to minimize cell toxicity. Numerous strategies have been applied to this end, from appending additional cationic peptides to supercharging the protein itself, sometimes with limited success. Here we present a strategy that combines these methods, by equipping a protein with supercharged elastin-like polypeptide (ELP) tags. We monitored cellular uptake and cell viability for GFP reporter proteins outfitted with a range of ELP tags and demonstrated enhanced uptake that correlates with the number of positive charges, while maintaining remarkably low cytotoxicity and resistance to degradation in the cell. GFP uptake proceeded mainly through caveolae-mediated endocytosis and we observed GFP emission inside the cells over extended time (up to 48 h). Low toxicity combined with high molecular weights of the tag opens the way to simultaneously optimize cell uptake and pharmacokinetic parameters. Thus, cationic supercharged ELP tags show great potential to improve the therapeutic profile of protein drugs leading to more efficient and safer biotherapeutics.

De novo design of supercharged, unfolded protein polymers, and their assembly into supramolecular aggregates.

Here we report for the first time the design and expression of highly charged, unfolded protein polymers based on elastin-like peptides (ELPs). Positively and negatively charged variants were achieved by introducing lysine and glutamic acid residues, respectively, within the repetitive pentapeptide units. Subsequently it was demonstrated that the monodisperse protein polyelectrolytes with precisely defined amino acid compositions, sequences, and stereochemistries can be transferred into superstructures exploiting their electrostatic interactions. Hollow capsules were assembled from oppositely charged protein chains by using the layer-by-layer technique. The structures of the capsules were analyzed by various microscopy techniques revealing the fabrication of multilayer containers. Due to their low toxicity in comparison to other polyelectrolytes, supercharged ELPs are appealing candidates for the construction of electrostatically induced scaffolds in biomedicine.

Novel single-chain Fv' formats for the generation of immunoliposomes by site-directed coupling.

Immunoliposomes generated by coupling of antibodies to the liposomal surface allow for an active targeting of entrapped compounds to diseased areas. Single-chain Fv fragments (scFv) represent the smallest part of an antibody containing the entire antigen-binding site. They can be coupled in a defined and site-directed manner through genetically engineered cysteine residues, for example, those added at the C-terminus. Here, we have performed a comparative analysis of various scFv' variants with cysteine residues present at the end of a C-terminal extension of varying length and composition (HC variants) or introduced in the linker sequence connecting the variable heavy and light chain domain (LC variants). Using a scFv fragment directed against fibroblast activation protein (FAP) as a model antibody, we could show that all variants can be employed for the generation of active immunoliposomes, although the presence of three additional cysteine residues in one scFv' molecule resulted in decreased binding of immunoliposomes compared to that of immunoliposomes generated with scFv' molecules containing only one additional cysteine residue. In order to further improve the scFv' format by reducing the number of additional amino acid residues, we also generated molecules with the hexahistidyl-tag incorporated into the linker sequence together with a cysteine residue either at position 1 or 3 of the linker sequence (LCH variants). These newly designed scFv' molecules may be particularly suitable for the generation of immunoliposomes and other antibody conjugates, limiting the number of additional residues in these antibody molecules to a minimum.