Garth J. Simpson

An electrochemical fabrication process for the assembly of anisotropically oriented collagen bundles

Controlled assembly of collagen molecules in vitro remains a major challenge for fabricating the next generation of engineered tissues. Here we present a novel electrochemical alignment technique to control the assembly of type-I collagen molecules into highly oriented and densely packed elongated bundles at the macroscale. The process involves application of electric currents to collagen solutions which in turn generate a pH gradient. Through an isoelectric focusing process, the molecules migrate and congregate within a plane. It was possible to fabricate collagen bundles with 50-400 microm diameter and several inches length via this process. The current study assessed the orientational order, and the presence of fibrillar assembly in such electrochemically oriented constructs by polarized optical microscopy, small angle X-ray scattering, second harmonic generation, and electron microscopy. The mechanical strength of the aligned crosslinked collagen bundles was 30-fold greater than its randomly oriented-crosslinked counterpart. Aligned crosslinked collagen bundles had about half the strength of the native tendon. Tendon-derived fibroblast cells were able to migrate and populate multiple macroscopic bundles at a rate of 0.5mm/day. The anisotropic order within biocompatible collagenous constructs was conferred upon the nuclear morphology of cells as well. These results indicate that the electrochemically oriented collagen scaffolds carry baseline characteristics to be considered for tendon/ligament repair.

Chirality in Nonlinear Optics

The past decade has witnessed the emergence of new measurement approaches and applications for chiral thin films and materials enabled by the observations of the high sensitivity of second-order nonlinear optical measurements to chirality. In thin films, the chiral response to second harmonic generation and sum frequency generation (SFG) from a single molecular monolayer is often comparable with the achiral response. The chiral specificity also allows for symmetry-allowed SFG in isotropic chiral media, confirming predictions made approximately 50 years ago. With these experimental demonstrations in hand, an important challenge is the construction of intuitive predictive models that allow the measured chiral response to be meaningfully related back to molecular and macromolecular structure. This review defines and considers three distinct mechanisms for chiral effects in uniaxially oriented assemblies: orientational chirality, intrinsic chirality, and isotropic chirality. The role of each is discussed in experimental and computational studies of bacteriorhodopsin films, binaphthol, and collagen. Collectively, these three model systems support a remarkably simple framework for quantitatively recovering the measured chiral-specific activity.

Selective Detection of Protein Crystals by Second Harmonic Microscopy

The unique symmetry properties of second harmonic generation (SHG) microscopy enabled sensitive and selective imaging of protein microcrystals with negligible contributions from solvated proteins or amorphous protein aggregates. In studies of microcrystallites of green fluorescent protein (GFP) prepared in 500 pL droplets, the SHG intensities rivaled those of fluorescence, but with superb selectivity for crystalline regions. GFP in amorphous aggregates and in solution produced substantial background fluorescence, but no detectable SHG. The ratio of the forward-to-backward detected SHG provides a measure of the particle size, suggesting detection limits down to crystallites 100 nm in diameter under low magnification (10x). In addition to being sensitive and highly selective, second-order nonlinear optical imaging of chiral crystals (SONICC) is directly compatibility with virtually all common protein crystallization platforms.

Nonlinear Optical Imaging of Integral Membrane Protein Crystals in Lipidic Mesophases

Second-order nonlinear optical imaging of chiral crystals (SONICC) is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments. High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and/or lipids that often form various mesophases. Detection of crystals in such media by conventional optical methods (e.g., intrinsic UV fluorescence, birefringence, bright-field image analysis, etc.) is often complicated by optical scattering and by the small sizes of the crystals that routinely form. SONICC is shown to be well-suited for this application, by nature of its compatibility with imaging in scattering media and its high selectivity for protein crystals. Bright second harmonic generation (SHG) (up to 18 million counts/s) was observed from even relatively small crystals (5 mum) with a minimal background due to the surrounding lipid mesophase ( approximately 1 thousand counts/s). The low background nature of the resulting protein crystal images permitted the use of a relatively simple, particle counting analysis for preliminary scoring. Comparisons between a particle counting analysis of SONICC images and protocols based on the human expert analysis of conventional bright-field and birefringence images were performed.

Surface Roughness by Contact versus Tapping Mode Atomic Force Microscopy

To evaluate and compare tapping mode and contact mode AFM measurements of surface roughness, images of quartz and mica were acquired by both methods and the height distributions and variance correlation functions analyzed. Significant deviation from the expected Gaussian profiles for the height distributions were observed for contact mode images of quartz but not for tapping mode images. Additionally, variance correlation functions were found to be highly scan size dependent for contact mode images and scan size invariant for tapping mode images. One possible explanation for the observed differences is that the scan speed limit is exceeded in contact mode for linear scan velocities as low as 0.5 μm/s.

Selective Detection and Quantitation of Organic Molecule Crystallization by Second Harmonic Generation Microscopy

Second order nonlinear optical imaging of chiral crystals (SONICC) was applied to selectively detect crystal formation at early stages and characterize the kinetics of nucleation and growth. SONICC relies on second harmonic generation (SHG), a nonlinear optical effect that only arises from noncentosymmetric ordered domain structures, which include crystals of chiral molecules. The model systems studied include pharmaceutically relevant compounds: griseofulvin and chlorpropamide. SONICC demonstrates low detection limits producing an 8 order of magnitude improvement relative to macroscopic average techniques and 5 order of magnitude improvement relative to optical microscopy. SONICC was also applied to examine the kinetics of crystallization in amorphous griseofulvin. The results show that SONICC enables simultaneous monitoring of individual crystal growth, nucleation rate, and macroscopic crystallization kinetics.

Assembly of Dithiocarbamate-Anchored Monolayers on Gold Surfaces in Aqueous Solutions

Dithiocarbamates (DTCs) can be formed by the in situ condensation of polar alkylamines with CS 2, and assembled into dithiocarbamate-anchored monolayers (DAMs) on Au substrates in aqueous solutions. Primary and secondary amines can both be used to prepare DTCs, but have significant differences in their reactivities and product stabilities. Ultraviolet absorption spectroscopy provides a convenient method for monitoring in situ DTC formation as well as the formation of potential byproducts. The kinetics of DAM assembly on Au substrates as measured by second harmonic generation (SHG) indicated first-order rate processes and saturation coverages similar to those of alkanethiols on Au. However, the rate of adsorption did not change with DTC concentration in a manner expected of Langmuir kinetics, and is attributed to the competitive adsorption of alkylammonium counterions to the freshly oxidized Au substrate. These analyses establish a practical range of conditions for preparing DAMs from polar amines using in situ DTC formation.

Effect of Substrates on Naproxen-Polyvinylpyrrolidone Solid Dispersions Formed via the Drop Printing Technique

Solid dispersions have been used to improve the bioavailability of poorly water-soluble drugs. However, drug solid-state phase, compositional uniformity, and scale-up problems are issues that need to be addressed. To allow for highly controllable products, the drop printing (DP) technique can provide precise dosages and predictable compositional uniformity of active pharmaceutical ingredients in two-/three-dimensional structures when integrated with edible substrates. With different preparation conditions, DP was conducted to fabricate naproxen (NAP)-polyvinylpyrrolidone solid dispersions with chitosan and hydroxypropyl methylcellulose films as the substrate. Scanning electron microscopy, X-ray diffraction, second harmonic generation microscopy, and atomic force microscopy analyses were performed to characterize the microstructure and spatial distribution of NAP in the solid dispersions. The results identified that composition, temperature, and substrate type all had an impact on morphology and crystallization of samples. The surface energy approach was combined with classical nucleation theory to evaluate the affinity between the nucleus of NAP and substrates. Finally, the collective results of the drug were correlated to the release profile of NAP within each sample.

Structural origins of circular dichroism in surface second harmonic generation

Circular dichroic (CD) ratios often exceeding 100% have been reported in previous surface second harmonic generation (SHG) measurements of chiral surface films, offering promise for the development of unique characterization methods to study biologically interesting surface systems. In this work, the molecular and surface structural origins of these large dichroic differences were explored in theoretical treatments and modeling calculations. Several new conclusions were formed regarding chirality in electric dipole allowed SHG measurements; (1) SHG-CD is allowed even in uniaxial films of achiral chromophores, provided they assemble with asymmetry in the twist angle, (2) for systems dominated by one or more chiral tensor element, this same asymmetry in surface packing is required for SHG-CD to be observed, and (3) large SHG-CD ratios are predicted in standard reflection and transmission measurements of ultrathin films in systems with spectral overlap between multiple excited states. The methodology describ...

Impact of Polymers on the Precipitation Behavior of Highly Supersaturated Aqueous Danazol Solutions

The phase behavior of supersaturated solutions of a relatively hydrophobic drug, danazol, was studied in the absence and presence of polymeric additives. To differentiate between phase separation to a noncrystalline phase and phase separation to a crystalline phase, an environmentally sensitive fluorescent probe was employed. Induction times for crystallization in the presence and absence of polymeric additives were studied using a combination of ultraviolet and fluorescence spectroscopy. It was found that, when danazol was added to aqueous media at concentrations above the amorphous solubility, liquid-liquid phase separation was briefly observed prior to crystallization, resulting in a short-lived, drug-rich noncrystalline danazol phase with an initial size of around 500 nm. The addition of polymers was found to greatly extend the lifetime of the supersaturated two phase system, delaying the onset of crystallization from a few minutes to a few hours. Below a certain threshold danazol concentration, found to represent the amorphous solubility, only crystallization was observed. Thus, although the addition of polymers was unable to prevent danazol from precipitating once a threshold concentration was exceeded, they did inhibit crystallization, leading to a solution with prolonged supersaturation. This observation highlights the need to determine the structure of the precipitating phase, since it is linked to the resultant solution concentration time profile.