Harald Plank

Development of an Itraconazole-loaded nanostructured lipid carrier (NLC) formulation for pulmonary application

Itraconazole-loaded NLC for pulmonary application were developed. In Precirol ATO 5 and oleic acid Itraconazole had the highest solubility. The solid lipid and the oil were mixable in a ratio 9:1 possessing a melting point above body temperature. 0.4% Itraconazole was dissolved in this lipid blend. Eumulgin SLM 20 was the stabilizer with the highest affinity to the lipid blend used as particle matrix. 2.5% Eumulgin SLM 20 was sufficient to obtain NLC with a narrow particle size distribution and sufficient stability. The tonicity of the formulation was adjusted with glycerol. Sterility was obtained by autoclaving. Neither the addition of glycerol nor autoclaving had an influence on the particle size and the zeta potential of Itraconazole-loaded NLC. SEM images showed spherical particles confirming the particle size measured by light scattering techniques. An entrapment efficiency of 98.78% was achieved. Burst release of Itraconazole from the developed carrier system was found. Itraconazole-loaded NLC possessed good storage stability. Nebulizing Itraconazole-loaded NLC with a jet stream and an ultrasonic nebulizer had no influence on the particle size and the entrapment efficiency of Itraconazole in the particle matrix, being a precondition for pulmonary application.

Cellulose surface degradation by a lytic polysaccharide monooxygenase and its effect on cellulase hydrolytic efficiency

Background: Lytic polysaccharide monooxygenase (LPMO) has recently been discovered to depolymerize cellulose. Results: Dynamic imaging was applied to reveal the effects of LPMO and cellulase activity on solid cellulose surface. Conclusion: Critical features of surface morphology for LPMO synergy with cellulases are recognized. Significance: Direct insights into cellulose deconstruction by LPMO alone and in synergy with cellulases are obtained. Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.

Dissecting and reconstructing synergism - in situ visualization of cooperativity among cellulases

Background: Synergistic interplay of cellulases is key for efficiency of cellulose hydrolysis. Results: In situ observation of individual and synergistic action of endo- and exo-cellulases on a polymorphic cellulose substrate reveals specificity of individual enzyme components for crystalline or amorphous regions. Conclusion: Cellulase synergism is governed by mesoscopic morphological characteristics of the cellulose substrate. Significance: Advanced knowledge basis for rational optimization of cellulose saccharification. Cellulose is the most abundant biopolymer and a major reservoir of fixed carbon on earth. Comprehension of the elusive mechanism of its enzymatic degradation represents a fundamental problem at the interface of biology, biotechnology, and materials science. The interdependence of cellulose disintegration and hydrolysis and the synergistic interplay among cellulases is yet poorly understood. Here we report evidence from in situ atomic force microscopy (AFM) that delineates degradation of a polymorphic cellulose substrate as a dynamic cycle of alternating exposure and removal of crystalline fibers. Direct observation shows that chain-end-cleaving cellobiohydrolases (CBH I, CBH II) and an internally chain-cleaving endoglucanase (EG), the major components of cellulase systems, take on distinct roles: EG and CBH II make the cellulose surface accessible for CBH I by removing amorphous-unordered substrate areas, thus exposing otherwise embedded crystalline-ordered nanofibrils of the cellulose. Subsequently, these fibrils are degraded efficiently by CBH I, thereby uncovering new amorphous areas. Without prior action of EG and CBH II, CBH I was poorly active on the cellulosic substrate. This leads to the conclusion that synergism among cellulases is morphology-dependent and governed by the cooperativity between enzymes degrading amorphous regions and those targeting primarily crystalline regions. The surface-disrupting activity of cellulases therefore strongly depends on mesoscopic structural features of the substrate: size and packing of crystalline fibers are key determinants of the overall efficiency of cellulose degradation.

Molecular alignments in sexiphenyl thin films epitaxially grown on muscovite

Abstract The epitaxial orientations of highly crystalline para-sexiphenyl (C 36 H 26 ) films on mica (001) surfaces are investigated by selected area electron diffraction (SAED) and transmission electron microscopy (TEM). Films at the early growth stage (growth time 26 s) and at an advanced growth stage (growth time 10 min) are studied. Films at the early growth stage exhibit only three-dimensional islands with an average size of 60×30×10 nm 3 , whereas films at an advanced growth stage consist of long oriented nano-fibres with a needle-like morphology. We identified three different types of epitaxial relations between the mica (001) substrate and the sexiphenyl crystallites, which are the same in both growth stages. Moreover, within a single island as well as within a single fibre crystalline domains with these three epitaxial orientations are observed. At the advanced growth stage, these domains are aligned antiparallel or perpendicular to the fibre axes; the typical size of the domains is 20 nm.

Visualizing cellulase activity

Commercial exploitation of lignocellulose for biotechnological production of fuels and commodity chemicals requires efficient—usually enzymatic—saccharification of the highly recalcitrant insoluble substrate. A key characteristic of cellulose conversion is that the actual hydrolysis of the polysaccharide chains is intrinsically entangled with physical disruption of substrate morphology and structure. This “substrate deconstruction” by cellulase activity is a slow, yet markedly dynamic process that occurs at different length scales from and above the nanometer range. Little is currently known about the role of progressive substrate deconstruction on hydrolysis efficiency. Application of advanced visualization techniques to the characterization of enzymatic degradation of different celluloses has provided important new insights, at the requisite nano‐scale resolution and down to the level of single enzyme molecules, into cellulase activity on the cellulose surface. Using true in situ imaging, dynamic features of enzyme action and substrate deconstruction were portrayed at different morphological levels of the cellulose, thus providing new suggestions and interpretations of rate‐determining factors. Here, we review the milestones achieved through visualization, the methods which significantly promoted the field, compare suitable (model) substrates, and identify limiting factors, challenges and future tasks. Biotechnol. Bioeng. 2013; 110: 1529–1549.

Cellulases Dig Deep IN SITU OBSERVATION OF THE MESOSCOPIC STRUCTURAL DYNAMICS OF ENZYMATIC CELLULOSE DEGRADATION

Background: The exact mechanism by which cellulases degrade cellulose is still elusive. Results: An empirical model of the structural dynamics of cellulose degradation is shown. Conclusion: Enzymatic cellulose hydrolysis is subjected to deceleration and acceleration caused by periodically emerging internal limitations and overcoming them. Significance: Understanding structural dynamics of enzymatic cellulose disintegration is pivotal for making biofuel production from lignocellulosic feedstock economic. Enzymatic hydrolysis of cellulose is key for the production of second generation biofuels, which represent a long-standing leading area in the field of sustainable energy. Despite the wealth of knowledge about cellulase structure and function, the elusive mechanism by which these enzymes disintegrate the complex structure of their insoluble substrate, which is the gist of cellulose saccharification, is still unclear. We herein present a time-resolved structural characterization of the action of cellulases on a nano-flat cellulose preparation, which enabled us to overcome previous limitations, using atomic force microscopy (AFM). As a first step in substrate disintegration, elongated fissures emerge which develop into coniform cracks as disintegration continues. Detailed data analysis allowed tracing the surface evolution back to the dynamics of crack morphology. This, in turn, reflects the interplay between surface degradation inside and outside of the crack. We observed how small cracks evolved and initially increased in size. At a certain point, the crack diameter stagnated and then started decreasing again. Stagnation corresponds with a decrease in the total amount of surface which is fissured and thus leads to the conclusion that the surface hydrolysis “around” the cracks is proceeding more rapidly than inside the cracks. The mesoscopic view presented here is in good agreement with various mechanistic proposals from the past and allows a novel insight into the structural dynamics occurring on the cellulosic substrate through cellulase action.

Fundamental Proximity Effects in Focused electron Beam Induced Deposition

Fundamental proximity effects for electron beam induced deposition processes on nonflat surfaces were studied experimentally and via simulation. Two specific effects were elucidated and exploited to considerably increase the volumetric growth rate of this nanoscale direct write method: (1) increasing the scanning electron pitch to the scale of the lateral electron straggle increased the volumetric growth rate by 250% by enhancing the effective forward scattered, backscattered, and secondary electron coefficients as well as by strong recollection effects of adjacent features; and (2) strategic patterning sequences are introduced to reduce precursor depletion effects which increase volumetric growth rates by more than 90%, demonstrating the strong influence of patterning parameters on the final performance of this powerful direct write technique.

Simulation-Guided 3D Nanomanufacturing via Focused Electron Beam Induced Deposition

Focused electron beam induced deposition (FEBID) is one of the few techniques that enables direct-write synthesis of free-standing 3D nanostructures. While the fabrication of simple architectures such as vertical or curving nanowires has been achieved by simple trial and error, processing complex 3D structures is not tractable with this approach. In part, this is due to the dynamic interplay between electron-solid interactions and the transient spatial distribution of absorbed precursor molecules on the solid surface. Here, we demonstrate the ability to controllably deposit 3D lattice structures at the micro/nanoscale, which have received recent interest owing to superior mechanical and optical properties. A hybrid Monte Carlo-continuum simulation is briefly overviewed, and subsequently FEBID experiments and simulations are directly compared. Finally, a 3D computer-aided design (CAD) program is introduced, which generates the beam parameters necessary for FEBID by both simulation and experiment. Using this approach, we demonstrate the fabrication of various 3D lattice structures using Pt-, Au-, and W-based precursors.

Electron-beam-assisted oxygen purification at low temperatures for electron-beam-induced pt deposits: towards pure and high-fidelity nanostructures.

Nanoscale metal deposits written directly by electron-beam-induced deposition, or EBID, are typically contaminated because of the incomplete removal of the original organometallic precursor. This has greatly limited the applicability of EBID materials synthesis, constraining the otherwise powerful direct-write synthesis paradigm. We demonstrate a low-temperature purification method in which platinum-carbon nanostructures deposited from MeCpPtIVMe3 are purified by the presence of oxygen gas during a post-electron exposure treatment. Deposit thickness, oxygen pressure, and oxygen temperature studies suggest that the dominant mechanism is the electron-stimulated reaction of oxygen molecules adsorbed at the defective deposit surface. Notably, pure platinum deposits with low resistivity and retain the original deposit fidelity were accomplished at an oxygen temperature of only 50 °C.

The influence of beam defocus on volume growth rates for electron beam induced platinum deposition.

Electron beam induced deposition (EBID) is a versatile method for the controlled fabrication of conducting, semi-conducting and non-conducting structures down to the nanometer scale. In contrast to ion beam induced deposition, EBID processes are free of sputter effects, ion implantation and massive heat generation; however, they have much lower deposition rates. To push the deposition efficiency further towards its intrinsic limits, the individual influences of the process parameters have to be explored. In this work a platinum pre-cursor is used for the deposition of conducting nanorods on highly oriented pyrolytic graphite. The study shows the influence of a beam defocus during deposition on the volume growth rates. The temporal evolution of volume growth rates reveals a distinct maximum which is dependent on the defocus introduced, leading to an increase of deposited volumes by a factor 2.5 after the same deposition times. The observed maximum is explained by an increasing and saturating electron yield contributing to the final deposition process and constantly decreasing diffusion abilities of the pre-cursor molecules toward the tip of the nanorods, which is further supported by dwell time experiments.