Glycerol monooleate-based nanocarriers for siRNA delivery in vitro.

We present studies of the delivery of short interfering ribonucleic acid (siRNA) into a green fluorescent protein (GFP) expressing cell line, using lipid nanocarriers in cubic lyotropic liquid crystal form. These carriers are based on glycerol monooleate (GMO) and employ the use of varying concentrations of cationic siRNA binding lipids. The essential physicochemical parameters of the cationic lipid/GMO/siRNA complexes such as particle size, ζ otential, siRNA uptake stability, lyotropic mesophase behavior, cytotoxicity,and gene silencing efficiency were systematically assessed. We find that the lipid nanocarriers were effectively taken up by mammalian cells and that their siRNA payload was able to induce gene silencing in vitro. More importantly, it was found that the nonlamellar structure of some of the lipid nanocarrier formulations were more effective at gene silencing than their lamellar structured counterparts. The development of cationic lipid functionalized nonlamellar GMO-based nanostructured nanoparticles may lead to improved siRNA delivery vehicles.

Salt induced lamellar to bicontinuous cubic phase transitions in cationic nanoparticles.

The development of improved methods to allow the low energy production of cubic phase forming nanoparticles (cubosomes) is highly desired. The lamellar to hexagonal and cubic phase change of these lipid nanoparticles has previously been induced via the lowering of pH and the addition of calcium ions to anionic lipid nanoparticles. We have developed a method to produce low polydispersity cubosomes without the requirement of high energy input such as shear, sonication or homogenization under physiological conditions. We have found that the simple addition of phosphate buffered saline solution to aqueous dispersions of cationic liposome vesicles made with phytantriol results in the spontaneous formation of cubosomes after vortex mixing. This finding demonstrates the potential of utilizing this technique to incorporate shear and temperature sensitive compounds into cubosomes under extremely mild conditions for biomedical and nanotechnological applications.

Surface plasmon optical detection of beta-lactamase binding to different interfacial matrices combined with fiber optic absorbance spectroscopy for enzymatic activity assays.

In this study, we describe the attachment of biotin-functionalized beta-lactamase to different types of interfacial architectures. Generic biotin-NeutrAvidin binding matrices were assembled using biotin-terminated alkanethiol and poly (L-lysine)-g-poly (ethylene glycol) polymer. Quantitative comparisons were made between different matrices and binding strategies. In addition, the feasibility of regeneration was tested. Our results show that in general all matrices were well suited for the binding of the protein, although quantitative differences were observed and will be discussed. Furthermore, the results obtained by surface plasmon resonance spectrometer and optical waveguide measurements show excellent correlation. For all five matrices investigated, real time enzymatic activity assays of beta-lactamase were performed by a detection scheme that combines an affinity and a catalytic sensor. The results show that the surface-immobilized enzymes are stable and sufficiently active for highly sensitive catalytic activity measurements. The effect of surface immobilization on the catalytic activity of the enzyme is discussed.

Combined affinity and catalytic biosensor: in situ enzymatic activity monitoring of surface-bound enzymes.

Surface Plasmon Resonance Spectroscopy (SPR) and miniature Fiber Optic Absorbance Spectroscopy (FOAS) were combined to monitor in situ and quantitatively an enzymatic model reaction catalyzed by beta-lactamase. The enzyme was covalently immobilized to the gold surface of a SPR chip, which was functionalized with NeutrAvidin through a biotinylated alkanethiol self-assembled monolayer, thus serving as a highly sensitive affinity biosensor. SPR was used to control the density of the surface-bound enzyme. Nitrocefin as the enzymatic substrate was allowed to react with the immobilized enzyme in the SPR flow cell, and its turnover was detected with the FOAS system acting as the catalytic biosensor. The coupling of the two techniques has a substantial potential for highly controlled on-line monitoring of surface-bound enzyme activity. The FOAS technique may also be easily employed as an add-on device to other types of affinity sensing instruments.

NTA-Functionalized Poly(L-lysine)-g-Poly(Ethylene Glycol): A Polymeric Interface for Binding and Studying 6 His-tagged Proteins.

In this paper, a novel graft copolymer, poly-(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) with part of the PEG chains carrying a terminal nitrilotriacetic acid group (NTA) was synthesized. Through electrostatic interactions, these polycationic graft co-polymers assemble spontaneously from aqueous solution onto negatively charged surfaces, forming polymeric monolayers that present NTA groups at controlled surface densities on a highly PEGylated background. The NTA-functionalized PLL-g-PEG surfaces proved to be highly resistant to nonspecific adsorption in contact with human serum while allowing the specific and reversible surface binding of GFPuv-6His and ß-lactamase-6His in native conformation. Micropatterns consisting of NTA-functionalized PLL-g-PEG in a background of PLL-g-PEG were produced using the "molecular assembly patterning by lift-off" technique. Exposure to Ni2+and GFPuv-6His resulted in a protein pattern of excellent contrast as judged by fluorescence microscopy. Furthermore, optical waveguide lightmode spectroscopy (OWLS) and a miniature fiber optic absorbance spectrometer (FOAS) were combined as affinity and catalytic biosensor to monitor in situ and quantitatively the amount of immobilized ß-lactamase-6His and to determine the activity of the immobilized enzyme. The NTA-functionalized PLL-g-PEG surface is considered to be a promising sensor platform for binding 6 His-tagged proteins thanks to the simplicity and cost-effectiveness of the surface modification protocol, high specificity and nearly quantitative reversibility of the protein binding, and the potential to fabricate microarrays of multiple capture molecules.

Immobilization of the enzyme beta-lactamase on biotin-derivatized poly(L-lysine)-g-poly(ethylene glycol)-coated sensor chips: a study on oriented attachment and surface activity by enzyme kinetics and in situ optical sensing.

Understanding the conformation, orientation, and specific activity of proteins bound to surfaces is crucial for the development and optimization of highly specific and sensitive biosensors. In this study, the very efficient enzyme beta-lactamase is used as a model protein. The wild-type form was genetically engineered by site-directed mutagenesis to introduce single cysteine residues on the surface of the enzyme. The cysteine thiol group is subsequently biotinylated with a dithiothreitol (DTT)-cleavable biotinylation reagent. beta-Lactamase is then immobilized site-specifically via the biotin group on neutral avidin-covered surfaces with the aim to control the orientation of the enzyme molecule at the surface and study its effect on enzymatic activity using Nitrocefin as the substrate. The DTT-cleavable spacer allows the release of the specifically bound enzyme from the surface. Immobilization of the enzyme is performed on a monolayer of the polycationic, biotinylated polymer PLL-g-PEG/PEG-biotin assembled on niobium oxide (Nb2O5) surfaces via neutral avidin as the docking site. Two different assembly protocols, the sequential adsorption of avidin and biotinylated beta-lactamase and the immobilization of preformed complexes of beta-lactamase and avidin, are compared in terms of immobilization efficiency. In situ optical waveguide lightmode spectroscopy and colorimetric analysis of enzymatic activity were used to distinguish between specific and unspecific enzyme adsorption, to sense quantitatively the amount of immobilized enzyme, and to determine Michaelis-Menten kinetics. All tested enzyme variants turned out to be active upon immobilization at the polymeric surface. However, the efficiency of immobilized enzymes relative to the soluble enzymes was reduced about sevenfold, mainly because of impaired substrate (Nitrocefin) diffusion or restricted accessibility of the active site. No significant effect of different enzyme orientations could be detected, probably because the enzymes were attached to the surface through long, flexible PEG chain linkers.