Fabian J. Eber

The Impact of Aspect Ratio on the Biodistribution and Tumor Homing of Rigid Soft-Matter Nanorods

The size and shape of nanocarriers can affect their fate in vivo, but little is known about the effect of nanocarrier aspect ratio on biodistribution in the setting of cancer imaging and drug delivery. The production of nanoscale anisotropic materials is a technical challenge. A unique biotemplating approach based on of rod-shaped nucleoprotein nanoparticles with predetermined aspect ratios (AR 3.5, 7, and 16.5) is used. These rigid, soft-matter nanoassemblies are derived from tobacco mosaic virus (TMV) components. The role of nanoparticle aspect ratio is investigated, while keeping the surface chemistries constant, using either PEGylated stealth nanoparticles or receptor-targeted RGD-displaying formulations. Aspect ratio has a profound impact on the behavior of the nanoparticles in vivo and in vitro. PEGylated nanorods with the lowest aspect ratio (AR 3.5) achieve the most efficient passive tumor-homing behavior because they can diffuse most easily, whereas RGD-labeled particles with a medium aspect ratio (AR 7) are more efficient at tumor targeting because this requires a balance between infusibility and ligand-receptor interactions. The in vivo behavior of nanoparticles can therefore be tailored to control biodistribution, longevity, and tumor penetration by modulating a single parameter: the aspect ratio of the nanocarrier.

Inducible site-selective bottom-up assembly of virus-derived nanotube arrays on RNA-equipped wafers.

Tobacco mosaic virus (TMV) is a tube-shaped, exceptionally stable plant virus, which is among the biomolecule complexes offering most promising perspectives for nanotechnology applications. Every viral nanotube self-assembles from a single RNA strand and numerous identical coat protein (CP) subunits. Here we demonstrate that biotechnologically engineered RNA species containing the TMV origin of assembly can be selectively attached to solid surfaces via one end and govern the bottom-up growth of surface-linked TMV-like nanotubes in situ on demand. SiO(2) wafers patterned by polymer blend lithography were modified in a chemically selective manner, which allowed positioning of in vitro produced RNA scaffolds into predefined patches on the 100-500 nm scale. The RNA operated as guiding strands for the self-assembly of spatially ordered nanotube 3D arrays on the micrometer scale. This novel approach may promote technically applicable production routes toward a controlled integration of multivalent biotemplates into miniaturized devices to functionalize poorly accessible components prior to use. Furthermore, the results mark a milestone in the experimental verification of viral nucleoprotein complex self-assembly mechanisms.

New Approaches for Bottom-Up Assembly of Tobacco Mosaic Virus- Derived Nucleoprotein Tubes on Defined Patterns on Silica- and Polymer-Based Substrates

The capability of some natural molecular building blocks to self-organize into defined supramolecular architectures is a versatile tool for nanotechnological applications. Their site-selective integration into a technical context, however, still poses a major challenge. RNA-directed self-assembly of tobacco mosaic virus-derived coat protein on immobilized RNA scaffolds presents a possibility to grow nucleoprotein nanotubes in place. Two new methods for their site-selective, bottom-up assembly are introduced. For this purpose, isothiocyanate alkoxysilane was used to activate oxidic surfaces for the covalent immobilization of DNA oligomers, which served as linkers for assembly-directing RNA. Patterned silanization of surfaces was achieved (1) on oxidic surfaces via dip-pen nanolithography and (2) on polymer surfaces (poly(dimethylsiloxane)) via selective oxidization by UV-light irradiation in air. Atomic force microscopy and X-ray photoelectron spectroscopy were used to characterize the surfaces. It is shown for the first time that the combination of the mentioned structuring methods and the isothiocyanate-based chemistry is appropriate (1) for the site-selective immobilization of nucleic acids and, thus, (2) for the formation of viral nanoparticles by bottom-up self-assembly after adding the corresponding coat proteins.

Modified TMV Particles as Beneficial Scaffolds to Present Sensor Enzymes

Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes, and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50% of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional “high-binding” microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins.

Tailoring the surface properties of tobacco mosaic virions by the integration of bacterially expressed mutant coat protein

Due to its small dimensions and high stability, tobacco mosaic virus (TMV) is used as nano-scaffold frequently. Its surface can be engineered to meet specific needs for technical, medical or materials applications. However, not all technically desirable TMV coat protein (CP) mutants can be propagated in plants successfully, if they change the efficiency of virion assembly. In order to circumvent this problem, a novel wild type (wt) CP-assisted and RNA-directed assembly procedure was designed for a recalcitrant CP mutant: Although pure hexahistidine-tagged CP cannot form particles on its own with TMV RNA in vitro, it was integrated into full-length particles if blended with wt CP in different proportions. The resulting rods formed dense monolayers with short range alignment on silicon substrates, substantially different from the largely wavy patterns obtained with wt TMV. Since they also mediated efficient ZnO deposition under mild conditions, the approach has yielded a new class of biotemplates which are amenable to the formation of nanostructured hybrid materials with adjustable texture for various applications.