Rapid assessment of homogeneity and stability of amorphous solid dispersions by atomic force microscopy--from bench to batch.

PURPOSE: To verify the robustness and fundamental value of Atomic Force Microscopy (AFM) and AFM-based assays to rapidly examine the molecular homogeneity and physical stability of amorphous solid dispersions on Hot-Melt-Extrudates. METHODS: Amorphous solid dispersions were prepared with a Hot-Melt Extruder (HME) and profiled by Raman Microscopy and AFM following a sequential analytical routine (Multi-Scale-Imaging-of-Miscibiliy (MIMix)). Extrudates were analyzed before and after incubation at elevated temperature and humidity. The data were compared with published results as collected on miniaturized melt models. The value of molecular phase separation rates for long term stability prediction was assessed. RESULTS: Data recorded on the extrudates are consistent with those published, and they can be compared side by side. Such direct data comparisons allow the identification of possible sources of extrudate heterogeneities. The surface roughness analysis of fracture-exposed interfaces is a novel quantitative way to trace on the nanometer scale the efficiencies of differently conducted HME-processes. Molecular phase separation rates are shown to be relevant for long term stability predictions. CONCLUSIONS: The AFM-based assessment of API:excipient combinations is a robust method to rapidly identify miscible and stable solid dispersions in a routine manner. It provides a novel analytical tool for the optimization of HME processes.

Formation and healing of micrometer-sized channel networks on highly mobile Au(111) surfaces.

Practical protocols are presented to reproducibly prepare micrometer-sized Au(111) substrates. Au(111) terraces of micrometer dimensions and atomic smoothness were crystallized by flame-annealing vacuum-deposited gold films on glass and on millimetric amorphous gold shots. Gold films and shots that were slowly cooled in a moderately applied stream of nitrogen gas exhibited large and stable crystal surfaces with Au(111) morphologies. Similarly, flame-annealed gold samples cooled with another protocol--in much rougher streams of nitrogen gas--produced morphologically unstable and highly mobile Au(111) layers. Within the first hour after preparation, however, rapid microscale restructuring in the layers produced complex morphologies of hexagonal channel networks and islands that were predominantly triangular. These channeled gold layers fused slowly in the following hours, with velocities of 0.01-0.2 A/s, as quantified by digital image correlation (DIC). Atomically smooth, stable, and predominantly triangular Au(111) terraces on the scale of micrometers were observed approximately 24 h after the sample preparations.

Components for high speed atomic force microscopy.

Many applications in materials science, life science and process control would benefit from atomic force microscopes (AFM) with higher scan speeds. To achieve this, the performance of many of the AFM components has to be increased. In this work, we focus on the cantilever sensor, the scanning unit and the data acquisition. We manufactured 10 microm wide cantilevers which combine high resonance frequencies with low spring constants (160-360 kHz with spring constants of 1-5 pN/nm). For the scanning unit, we developed a new scanner principle, based on stack piezos, which allows the construction of a scanner with 15 microm scan range while retaining high resonance frequencies (>10 kHz). To drive the AFM at high scan speeds and record the height and error signal, we implemented a fast Data Acquisition (DAQ) system based on a commercial DAQ card and a LabView user interface capable of recording 30 frames per second at 150 x 150 pixels.

Sacrificial bonds and hidden length dissipate energy as mineralized fibrils separate during bone fracture.

Properties of the organic matrix of bone as well as its function in the microstructure could be the key to the remarkable mechanical properties of bone. Previously, it was found that on the molecular level, calcium-mediated sacrificial bonds increased stiffness and enhanced energy dissipation in bone constituent molecules. Here we present evidence for how this sacrificial bond and hidden length mechanism contributes to the mechanical properties of the bone composite, by investigating the nanoscale arrangement of the bone constituents and their interactions. We find evidence that bone consists of mineralized collagen fibrils and a non-fibrillar organic matrix, which acts as a 'glue' that holds the mineralized fibrils together. We believe that this glue may resist the separation of mineralized collagen fibrils. As in the case of the sacrificial bonds in single molecules, the effectiveness of this mechanism increases with the presence of Ca2+ ions.

Influence of the degradation of the organic matrix on the microscopic fracture behavior of trabecular bone.

In recent years, the important role of the organic matrix for the mechanical properties of bone has become increasingly apparent. It is therefore of great interest to understand the interactions between the organic and inorganic constituents of bone and learn the mechanisms by which the organic matrix contributes to the remarkable properties of this complex biomaterial. In this paper, we present a multifaceted view of the changes of bone's properties due to heat-induced degradation of the organic matrix. We compare the microscopic fracture behavior (scanning electron microscopy; SEM), the topography of the surfaces (atomic force microscopy; AFM), the condition of bone constituents [X-ray diffraction (XRD), thermogravimetric analysis (TGA), and gel electrophoresis], and the macromechanical properties of healthy bovine trabecular bone with trabecular bone that has a heat-degraded organic matrix. We show that heat treatment changes the microfracture behavior of trabecular bone. The primary failure mode of untreated trabecular bone is fibril-guided delamination, with mineralized collagen filaments bridging the gap of the microcrack. In contrast, bone that has been baked at 200 degrees C fractures nondirectionally like a brittle material, with no fibers spanning the microcracks. Finally, bone that has been boiled for 2 h in PBS solution fractures by delamination with many small filaments spanning the microcracks, so that the edges of the microcracks become difficult to distinguish. Of the methods we used, baking most effectively weakens the mechanical strength of bone, creating the most brittle material. Boiled bone is stronger than baked bone, but weaker than untreated bone. Boiled bone is more elastic than untreated bone, which is in turn more elastic than baked bone. These studies clearly emphasize the importance of the organic matrix in affecting the fracture mechanics of bone.

Rigid design of fast scanning probe microscopes using finite element analysis.

To improve the performance of atomic force microscopes regarding speed and noise sensitivity, it is important to consider the mechanical rigidity of the actuator (scanner), and the overall mechanical structure. Using finite element analysis in the design process, it was possible to increase the first resonance frequency from 950 Hz for the whole system to 23.4 kHz for the whole system. This constitutes a factor of approximately 25 in resonance frequency and a factor of 625 in stiffness and, hence, noise immunity.

Force spectroscopy of collagen fibers to investigate their mechanical properties and structural organization.

Tendons are composed of collagen and other molecules in a highly organized hierarchical assembly, leading to extraordinary mechanical properties. To probe the cross-links on the lower level of organization, we used a cantilever to pull substructures out of the assembly. Advanced force probe technology, using small cantilevers (length <20 microm), improved the force resolution into the sub-10 pN range. In the force versus extension curves, we found an exponential increase in force and two different periodic rupture events, one with strong bonds (jumps in force of several hundred pN) with a periodicity of 78 nm and one with weak bonds (jumps in force of <7 pN) with a periodicity of 22 nm. We demonstrate a good correlation between the measured mechanical behavior of collagen fibers and their appearance in the micrographs taken with the atomic force microscope.

Evidence that collagen fibrils in tendons are inhomogeneously structured in a tubelike manner.

The standard model for the structure of collagen in tendon is an ascending hierarchy of bundling. Collagen triple helices bundle into microfibrils, microfibrils bundle into subfibrils, and subfibrils bundle into fibrils, the basic structural unit of tendon. This model, developed primarily on the basis of x-ray diffraction results, is necessarily vague about the cross-sectional organization of fibrils and has led to the widespread assumption of laterally homogeneous closepacking. This assumption is inconsistent with data presented here. Using atomic force microscopy and micromanipulation, we observe how collagen fibrils from tendons behave mechanically as tubes. We conclude that the collagen fibril is an inhomogeneous structure composed of a relatively hard shell and a softer, less dense core.

Investigations into the polymorphism of rat tail tendon fibrils using atomic force microscopy.

Collagen type I displays a typical banding periodicity of 67 nm when visualized by atomic force or transmission electron microscopy imaging. We have investigated collagen fibers extracted from rat tail tendons using atomic force microscopy, under different ionic and pH conditions. The majority of the fibers reproduce the typical wavy structure with 67 nm spacing and a height difference between the peak and the grooves of at least 5 nm. However, we were also able to individuate two other banding patterns with 23+/-2 nm and 210+/-15 nm periodicities. The small pattern showed height differences of about 2 nm, whereas the large pattern seems to be a superposition of the 67 nm periodicity showing height differences of about 20 nm. Furthermore, we could show that at pH values of 3 and below the fibril structure gets dissolved whereas high concentrations of NaCl and CaCl(2) could prevent this effect.