Free steroli and sterylglycosides (SG) from oat leaves and seeds were isolated by conventional thin layer chromatography (TLC) and subjected to high performance liquid chromatography (HPLC) for resolution of molecular species. Acylsterylglycolides, isolated by TLC, were converted to SG by mild alkaline hydrolysis and determined as SG. Sterols and SG were injected onto the column without any chemical treatment and the separated species were detected at 200 nm. The separation of SG-species follows exactly the separation of free sterols.Though gas liquid chromatography still is the method of choice, advantages of HPLC is to analyse directly the SG-specia without hydrolysis and derivatization as compared to GLC. After TLC the sterol-and the SG-fraction are injected directly onto the column. This is extremely important for labile sterylglycosides or sterols, as demonstrated for the avenasterols.

Intrinsically disordered proteins (IDPs) play an important role in molecular biology and medicine because their induced folding can lead to so-called conformational diseases, where β-amyloids play an important role. Still, the molecular folding process into the different substructures, such as parallel/ antiparallel or extended β-sheet/crossed β-sheet is not fully understood. The recombinant spider silk protein eADF4(Cx) consisting of repeating modules C, which are composed of a crystalline (pep-c) and an amorphous peptide sequence (pep-a), can be used as a model system for IDP since it can assemble into similar structures. In this work, blend films of the pep-c and pep-a sequences were investigated to modulate the β-sheet formation by varying the molar fraction of pep-c and pep-a. Dichroic Fouriertransform infrared spectroscopy (FTIR), circular dichroism, spectroscopic ellipsometry, atomic force microscopy, and IR nanospectroscopy were used to examine the secondary structure, the formation of parallel and antiparallel β-sheets, their orientation, and the microscopic roughness and phase formation within peptide blend films upon methanol post-treatment. New insights into the formation of filament-like structures in these silk blend films were obtained. Filament-like structures could be locally assigned to βsheet-rich structures. Further, the antiparallel or parallel character and the orientation of the formed β-sheets could be clearly determined. Finally, the ideal ratio of pep-a and pep-c sequences found in the fibroin 4 of the major ampullate silk of spiders could also be rationalized by comparing the blend and spider silk protein systems.

Orientation analysis of the β-sheet structure within films of the established recombinant spider silk protein eADF4­(C16) was performed using a concept based on dichroic transmission– and attenuated total reflection–Fourier transform infrared spectroscopy, lineshape analysis, assignment of amide I components to specific vibration modes, and transition dipole moment directions of β-sheet structures. Based on the experimental dichroic ratio R, the order parameter S of β-sheet structures was calculated with respect to uniaxial orientation. Films of eADF4­(C16) were deposited on untexturized (Si) and unidirectionally scratched silicon substrates (Si-sc) and post-treated with MeOH vapor. Freshly cast thin and thick eADF4­(C16) films out of hexafluoroisopropanol featured β-sheet contents of ≈6%, which increased to >30% after MeOH post-treatment in dependence of time. Pseudo-first order folding kinetics were obtained, suggesting a transition from an unfolded to a folded state. In MeOH post-treated thin films with diameters in the nanometer range, a significant orientation of β-sheets was obtained regardless of the texturization of the silicon substrate (Si, Si-sc). This was rationalized by dichroic ratios of the amide I component at 1696 cm–1 assigned to the (0, π) mode of antiparallel β-sheet structures, whose transition dipole moment M is located in parallel to both β-sheet plane and chain direction. The calculated high molecular order parameter S ≈ 0.40 suggested vertically (out-of-plane) oriented antiparallel β-sheet stacks with tilt angles of γ ≈ 39° to the surface normal. Microscale (thick) films, in contrast, revealed low order parameters S ≈ 0. Scanning force microscopy on thin eADF4 films at silicon substrates showed dewetted polymer film structures rather at the micro-scale. These findings give new insights in the role of the β-sheet crystallite orientation for the mechanical properties of spider silk materials.

In this conceptual contribution, thin functional coatings consisting of either pure polyelectrolytes (PELs) or complexes between oppositely charged PELs at model and applied substrates are outlined. Latter PEL/PEL complexes were deposited by two concepts. In a first well-known concept, PEL multilayers (PEM) were consecutively deposited according to the layer-by-layer (LbL) technique. In a second less known concept, PEL complex (PEC) nanoparticles (NPs) preformed by mixing polycation (PC) and polyanion (PA) solutions were deposited in one step. Both concepts based on binary oppositely charged PELs are compared to one based on a single polycation system. Examples shall be given on adhesiveness, nanostructure, and biomedical applications of PEM and PEC NP coatings. In situ attenuated total reflection (ATR) infrared (IR) spectroscopy, circular dichroism (CD), and scanning force microscopy (SFM) were used for molecular, optical, and microscopic characterization. At first, results on the adsorbed amount and wet adhesiveness of pure (single-component) PEL coatings as a function of charge density are given to motivate coatings of mixed oppositely charged PELs. Second, the wet adhesiveness of PEM and PEC NP coatings of identical PEL compounds in aqueous media varying the molar charge ratio ( n-/ n+) and the deposition step z, respectively, is compared. Upon comparing the three PEL deposition concepts, it is suggested that the lack or absence of excess charge at the PEL/surface interface is one of the main factors for the wet adhesiveness of all pure PEL, PEM, and PEC NP coatings. Finally, the potential of PEM and PEC NP coatings for biomedical applications is outlined. Concerning biopassivation, PEM coatings excessed or terminated by PA repel proteins with low isoelectric points. Concerning bioactivation, PEM coatings loaded with antibiotics as well as PEC NP coatings loaded with therapeutic bisphosphonates showed retarded, optionally temperature responsive drug release for applications in acute surgery and bone healing, and immunoglobulin/PEL complex coatings might open theranostic applications.

The bone therapeutic drug zoledronate (ZOL) was loaded at and released by polyelectrolyte complex (PEC) particle films composed of either pure poly(ethyleneimine) (PEI) or maltose-modified poly(ethyleneimine) (PEI-M) and oppositely charged cellulose sulfate attached to model germanium (Ge) substrates by solution casting. Dispersions of colloidally stable polyelectrolyte complex (PEC) particles in the size range 11–141 nm were obtained by mixing PEI or PEI-M, CS and ZOL in defined stoichiometric ratios. TRANS-FTIR spectroscopy was used to determine the stability of the PEC films against detachment, in-situ-ATR-FTIR spectroscopy for the ZOL loss in the PEC film and UV–VIS spectroscopy for the ZOL enrichment of the release medium. Films of casted ZOL/CS/PEI-M or ZOL/CS/PEI particles were stable in contact to water, while films of the pure drug (ZOL) and of the binary systems ZOL/PEI-M or ZOL/PEI were not stable against detachment. Retarded releases of ZOL from various PEC films compared to the pure drug film were observed. The molecular weight of PEI showed a considerable effect on the initial burst (IB) of ZOL. No significant effect of the maltose modification of PEI-25 K on IB could be found. Generally, after one day the ZOL release process was finished for all measured ZOL/PEC samples and residual amounts of 0-30% were obtained. Surface adhesive drug loaded PEC particles are promising drug delivery systems to supply and release a defined amount of bone therapeutics and to functionalize bone substitution materials.