Distinguishing between COOH, COO- , and hydrogen disordered COOH sites in solids with 13 C chemical shift anisotropy and T1 measurements.

Since 1993, it has been known that 13 C chemical shift tensor (i.e., δ11 , δ22 , and δ33 ) provides information sufficient to distinguish between COOH and COO- sites. Herein, four previously unreported metrics are proposed for differentiating COOH/COO- moieties. A new relationship is also introduced that correlates the asymmetry (i.e., δ11 -δ22 ) of COOH sites to the proximity of hydrogen bond donating partners within 2.6 Å with high accuracy (±0.05 Å). Conversely, a limitation to all proposed metrics is that they fail to distinguish between COO- and hydrogen disordered COOH sites. To reconcile this omission, a new approach is proposed based on T1 measurements of both 1 H and 13 C. The 13 C T1 values are particularly sensitive with the T1 for hydrogen disordered COOH moieties found to be nearly six times smaller than T1 's from COO- sites.

Laser machined micropatterns as corrosion protection of both hydrophobic and hydrophilic magnesium.

Magnesium and its alloys are promising candidate materials for medical implants because they possess excellent biocompatibility and mechanical properties comparable to bone. Furthermore, secondary surgical operations for removal could be eliminated due to magnesium's biodegradability. However, magnesium's degradation rate in aqueous environments is too high for most applications. It has been reported that hydrophobic textured surfaces can trap a surface gas layer which acts as a protective barrier against corrosion. However, prior studies have not investigated separately the role of the texture and hydrophobic treatments on magnesium corrosion rates. In this study, pillar-shaped microstructure patterns were fabricated on polished high purity magnesium surfaces by ablation with a picosecond laser. Some micropatterned samples were further processed by stearic acid modification (SAM). Micropatterned surfaces with SAM had hydrophobic properties with water droplet contact angles greater than 130°, while the micropatterned surfaces without SAM remained hydrophilic. The corrosion properties of textured and smooth magnesium surfaces in saline solution were investigated using electrochemical impedance spectroscopy (EIS) and optical microscopy. Corrosion rates on both hydrophobic and hydrophilic laser machined surfaces were reduced ∼90% relative to polished surfaces. Surprisingly, corrosion rates were similar for both hydrophobic and hydrophilic surfaces. Indirect evidence of local alkalization near microstructures was found and was hypothesized to stabilize the Mg(OH)2 layer, thereby inhibiting corrosion on hydrophilic surfaces. This is different than the corrosion resistance mechanism for superhydrophobic surfaces which makes use of gas adhesion at the liquid solid interface. These results suggest additional processing to render the magnesium hydrophobic is not necessary since it does not significantly enhance the corrosion resistance beyond what is conferred by micropatterned textures.

NMR structural characterization from one-bond 13 C13 C couplings: Complete assignment of a hydrogen-poor depsidone.

The connectivity, conformation, tautomeric form, and dynamics of a new depsidone (perisalazinic acid) were characterized using one-bond 13 C13 C NMR scalar couplings (1 JCC ) obtained from the INADEQUATE experiment. Characterization of perisalazinic acid using more conventional NMR techniques is problematic due to the extremely limited number of CH protons present. In the present study, 81 candidate structures were considered and a best fit structure was selected by comparing computed 1 JCC values for each candidate to 15 experimental values. Of the six flexible moieties in perisalazinic acid, three are adequately represented by a single orientation stabilized by intramolecular hydrogen bonding. The three remaining groups are present as mixtures of conformers with two sites consisting of a pair of conformations and another disordered over six orientations. This study demonstrates the feasibility of complete three-dimensional structural characterization of an unknown using only theoretical and experimental 1 JCC values.

Adhesion and Particle Removal from Surface-Tethered Poly(N-Isopropylacrylamide) Coatings Using Hydrodynamic Shear Forces.

Thermally responsive coatings of poly(N-isopropylacrylamide), or poly(NIPAAm), have a volume phase transition temperature (VPTT) near 32 °C. Below this temperature, the coating imbibes water and swells. Above this temperature, the coating rejects water and collapses. Herein, a spinning disk method is used to determine the hydrodynamic shear stress necessary to remove 10 µm polystyrene (PS) microspheres capped with either carboxylic acid (COOH) functionality or immunoglobulin (IgG) proteins from the coatings as a function of coating thickness and temperature. In the case of the PS-COOH, the hydrodynamic shear stress necessary to remove the microspheres was consistently larger below the VPTT than above the VPTT of the poly(NIPAAm) coating. In the case of PS-IgG, the trend was reversed, in which the hydrodynamic shear stress necessary to remove the microspheres was consistently smaller below the VPTT than above the VPTT. Simple scaling relationships were developed to explain the findings within the Johnson-Kendall-Roberts (JKR) model of contact mechanics, which illustrates the delicate interplay between the pull-off force and contact radius (as determined by the coating shear modulus) in governing particle removal from soft surfaces with hydrodynamic forces.

Interfacial Phenomena of Natural Dispersants for Crude Oil Spills.

A natural surfactant was studied to simulate the dispersion process of crude oil in water. The interfacial phenomena of this natural dispersant was compared with a commercially available chemical dispersant, COREXIT EC9500A. This functional surfactant was extracted from the mucilage of the Opuntia ficus-indica cactus species. The evaluation to determine the efficacy to disperse crude oil of the cactus-based mucilage extract (nongelling extract, NE) was based on characterizing surface and interfacial tension, dispersion efficiency, mixing effects, salinity effects, stability, and droplets size distributions. We found that surface tension values follow a linear relationship with respect to the natural logarithm of the concentrations of NE. The application of NE in the water phase led to decreasing oil/water interfacial tensions. Surface tension tests were also used to quantify the effect of oil-in-water (O/W) emulsion ratios once either natural or commercialized dispersants were added. A key finding of our work is that the surface tension between typical 6% and 3% v/v O/W emulsions was significantly reduced with the addition of discrete amounts of NE. This result indicated that the dynamic balance between O/W and water-in-oil (W/O) emulsions was thermodynamically more stable toward O/W emulsion states with NE. We also found that O/W emulsions with higher dispersion effectiveness were formed for both 10 and 35 practical salinity units, as the dispersant to oil ratios increased, with a significant correlation to the mixing energy. We observed that the O/W emulsions with natural dispersants had a significantly smaller weighted average diameter compared to those with COREXIT EC9500A. Such a phenomenon can be explained by understanding intermolecular interactions due to the structure and type of dispersant. In conclusion, cactus-based mucilage extracts could be used as environmentally benign dispersants and, therefore, reduce negative social perceptions of the application of dispersants to clean up spilled oil.

Responsive coatings from naturally occurring pectin polysaccharides.

Pectin polysaccharides have significant potential as all-natural, non-toxic "green" coatings that exhibit thermally-cued swelling behavior. Herein, ultra-thin coatings of highly-esterified pectin polysaccharides were cross-linked with calcium chloride (CaCl2) and their swelling in water was investigated with ellipsometry. At low temperatures, the coatings swell to 2-3 times their dry layer thickness. As the temperature is increased, the coatings show a pronounced decrease in swollen thickness, reminiscent of the hydrophilic-hydrophobic transition observed in lower critical solution temperature (LCST) polymers. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy establishes that this transition is driven by dehydration of the esterified galacturonic acid residues along the pectin backbone. By adjusting both the CaCl2 concentration used to crosslink the pectin coatings as well as pH of swelling medium, the pectin coatings could be judiciously tuned for a desired swelling response as a function of temperature. Due to their non-toxic and responsive nature, it was further demonstrated that such coatings could be used in applications to control cell adhesion.

Remotely Controlled Micromanipulation by Buckling Instabilities in Fe3O4 Nanoparticle Embedded Poly(N-isopropylacrylamide) Surface Arrays.

The micromanipulation of biological samples is important for microbiology, pharmaceutical science, and related bioengineering fields. In this work, we report the fabrication and characterization of surface-attached microbeam arrays of 20 µm width and 25 µm height made of poly(N-isopropylacrylamide), a thermoresponsive polymer, with embedded spherical or octopod Fe3O4 nanoparticles. Below 32 °C, the microbeams imbibe water and buckle with an amplitude of approximately 20 µm. Turning on an AC-magnetic field induces the microbeam array to expel water due to the heating effect of the nanoparticles (magnetic hyperthermia), leading to a reversible transition from a buckled to nonbuckled state. It is observed that the octopod nanoparticles have a heating rate 30% greater (specific absorption rate, SAR) than that of the spherical nanoparticles, which shortens the time scale of the transition from the buckled and nonbuckled state. The return of the microbeams to the buckled state is accomplished by turning off the AC magnetic field, the rate of which is dictated by dissipation of heat and is independent of the type of nanoparticle. It is further demonstrated that this transition can be used to propel 50 µm spherical objects along a surface. While the motion is random, this study shows the promise of harnessing shape-shifting patterns in microfluidics for object manipulation.

Protein-surface interactions on stimuli-responsive polymeric biomaterials.

Responsive surfaces: a review of the dependence of protein adsorption on the reversible volume phase transition in stimuli-responsive polymers. Specifically addressed are a widely studied subset: thermoresponsive polymers. Findings are also generalizable to other materials which undergo a similarly reversible volume phase transition. As of 2015, over 100,000 articles have been published on stimuli-responsive polymers and many more on protein-biomaterial interactions. Significantly, fewer than 100 of these have focused specifically on protein interactions with stimuli-responsive polymers. These report a clear trend of increased protein adsorption in the collapsed state compared to the swollen state. This control over protein interactions makes stimuli-responsive polymers highly useful in biomedical applications such as wound repair scaffolds, on-demand drug delivery, and antifouling surfaces. Outstanding questions are whether the protein adsorption is reversible with the volume phase transition and whether there is a time-dependence. A clear understanding of protein interactions with stimuli-responsive polymers will advance theoretical models, experimental results, and biomedical applications.

A Cell ELISA for the quantification of MUC1 mucin (CD227) expressed by cancer cells of epithelial and neuroectodermal origin.

Quantitative analysis of MUC1, a cell membrane associated mucin, expressed by intact cells of epithelial origin previously has been limited to flow cytometry, which requires using large quantities of cells and antibodies. Here, for the first time, we report the development of a novel Cellular-based Enzyme Linked Immunosorbent Assay (Cell ELISA) to quantify the expression of MUC1 by cell lines of epithelial and neuroectodermal origin using an antibody recognizing a specific tandem repeat found in the extracellular domain of MUC1. In contrast to flow cytometry, this method requires a much lower number of cells. We report here the results obtained from two variants of this Cell ELISA in live and fixed cells. We found that the Cell ELISA in live cells was not sensitive enough to detect a difference in MUC1 levels between the normal cells and tumor cells. However, we found that Cell ELISA in fixed cells followed by whole cell staining was a dependable method of MUC1 level detection in the normal and tumor cells showing significantly higher levels of MUC1 receptor in the tumor cells when compared to the normal controls. Therefore, we conclude that the Cell ELISA in fixed cells is an efficient method for quantifying the expression of MUC1 by epithelial and neuroectodermal cancer cell lines.

Thermoresponsive PNIPAM Coatings on Nanostructured Gratings for Cell Alignment and Release.

Thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) has been widely used as a surface coating to thermally control the detachment of adsorbed cells without the need for extreme stimuli such as enzyme treatment. Recently, the use of 2D and 3D scaffolds in controlling cell positioning, growth, spreading, and migration has been of a great interest in tissue engineering and cell biology. Here, we use a PNIPAM polymer surface coating atop a nanostructured linear diffraction grating to controllably change the surface topography of 2D linear structures using temperature stimuli. Neutron reflectometry and surface diffraction are utilized to examine the conformity of the polymer coating to the grating surface, its hydration profile, and its evolution in response to temperature variations. The results show that, in the collapsed state, the PNIPAM coating conforms to the grating structures and retains a uniform hydration of 63%. In the swollen state, the polymer expands beyond the grating channels and absorbs up to 87% water. Such properties are particularly desirable for 2D cell growth scaffolds with a built-in nonextreme tissue-release mechanism. Indeed, the current system demonstrates advanced performance in the effective alignment of cultured fibroblast cells and the easy release of the cells upon temperature change.

Effects of fluid shear stress on polyelectrolyte multilayers by neutron scattering studies.

The structure of layer-by-layer (LbL) deposited nanofilm coatings consists of alternating polyethylenimine (PEI) and polystyrenesulfonate (PSS) films deposited on a single crystal quartz substrate. LbL-deposited nanofilms were investigated by neutron reflectomery (NR) in contact with water in the static and fluid shear stress conditions. The fluid shear stress was applied through a laminar flow of the liquid parallel to the quartz/polymer interface in a custom-built solid-liquid interface cell. The scattering length density profiles obtained from NR results of these polyelectrolyte multilayers (PEM), measured under different shear conditions, showed proportional decrease of volume fraction of water hydrating the polymers. For the highest shear rate applied (ca. 6800 s(-1)) the water volume fraction decreased by approximately 7%. The decrease of the volume fraction of water was homogeneous through the thickness of the film. Since there were not any significant changes in the total polymer thickness, it resulted in negative osmotic pressures in the film. The PEM films were compared with the behavior of thin films of thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) deposited via spin-coating. The PEM and pNIPAM differ in their interactions with water molecules, and they showed opposite behaviors under the fluid shear stress. In both cases the polymer hydration was reversible upon the restoration of static conditions. A theoretical explanation is given to explain this difference in the effect of shear on hydration of polymeric thin films.

Combining Nonadhesive Materials into Microstructured Composite Surfaces Induces Cell Adhesion and Spreading.

The synergistic effects between surface topography and chemical functionality were investigated in order to enhance the adhesion of cells to poorly adhesive materials. It has been established that many cell types are weakly adhesive to thin films of poly(N-isopropylacrylamide), or PNIPAAm, and self-assembled triethylene glycol-terminated alkanethiols (EG3SAM). To enhance adhesion, a topographical cue in the form of electrospun micron-width fibers of PNIPAAm was deposited to each coating. Both coatings, without fibers, supported less than 5% cell adhesion. With the deposition of the PNIPAAm fibers to the PNIPAAm coating, cell adhesion increased to 60% of the theoretical maximum at intermediate fiber coverage before decreasing again; however, cell spreading remained minimal. With the deposition of the PNIPAAm fibers onto the EG3SAM surface, cell adhesion increased to almost 100% at 60% fiber coverage, and unlike the PNIPAAm surface, cell spreading was significantly enhanced. This difference in spreading coincided with the assembly of fiber-associated focal adhesions when the underlying surface was EG3SAM, but not PNIPAAm. These findings indicate that the presence of a sparse topographical feature can stimulate cell adhesion on a typically nonadhesive material, but a chemical dissimilarity between the topographic features and the background is necessary for cell spreading. This work demonstrates for the first time that combining nonadhesive materials into microtextured composites can lead to cell adhesion and spreading on par with strongly adhesive surfaces.

Shape-changing hydrogel surfaces trigger rapid release of patterned tissue modules.

The formation and assembly of diverse tissue building blocks is considered a promising bottom-up approach for the construction of complex three-dimensional tissues. Patterned shape-changing materials were investigated as an innovative method to form and harvest free-standing tissue modules with preserved spatial organization and cell-cell connections. Arrays of micro-scale surface-attached hydrogels made of a thermoresponsive polymer were used as cell culture supports to fabricate tissue modules of defined geometric shape. Upon stimulation, these hydrogels swelled anisotropically, resulting in significant expansion of the culture surface and subsequent expulsion of the intact tissue modules. By varying the network crosslink density, the surface strain was modulated and a strain threshold for tissue module release was identified. This mechanical mechanism for rapid tissue module harvest was found to require inter- and intra-cellular tension. These results suggest that the cell-matrix adhesions are disrupted by the incompatibility of surface expansion with tissue module cohesion and stiffness, thus providing a novel method of forming and harvesting tissue building blocks by a mechanism independent of the thermal stimulus that induces the biomaterial shape change.

Poly(N-isopropylacrylamide) cross-linked coatings with phototunable swelling.

A series of terpolymers were synthesized comprising the following monomers: N-isopropylacrylamide (NIPAAm), a stimuli-responsive structural unit that swells and collapses in response to temperature; methacryloxybenzophenone (MaBP), a photo-cross-linking unit that is activated at a wavelength of 365 nm; and phenacyl methacrylate (PHEm), a photolabile protected carboxyl group that can be deprotected at a wavelength of 254 nm. It is shown that the terpolymers can be photo-cross-linked at long UV wavelength light (λ = 365 nm) to establish surface-attached, cross-linked coatings and subsequently photochemically cleaved at short UV wavelength light (λ = 254 nm), which is found to be consistent with first-order kinetics. The photocleavage reaction produces free carboxylic groups, which can be used to locally tune the swelling characteristics and transition temperature of the coating, which depends on both the irradiation exposure and the overall PHEm content. For instance, for a terpolymer with 7.1 mol % PHEm, the transition temperature between the swollen and collapsed states increased from 20 to 50 °C at a pH of 8.5 with an exposure dose of 0.52 J/cm(2) at 254 nm. Finally, photocleavage can be used to create chemically patterned regions to provide a basis by which to conjugate cationic markers, proteins, or nanoparticles to the terpolymer coating.

Controlled release niosome embedded chitosan system: effect of crosslink mesh dimensions on drug release.

We report on a model chemotherapy drug delivery system comprising nonionic surfactant vesicles (niosomes) packaged within a temperature-sensitive chitosan network. This smart packaging, or package-within-a package system, provides two distinct advantages. First, the gel prevents circulation of the niosomes and maintains delivery in the vicinity of a tumor. Second, the chitosan network protects the niosomes against fluctuations in tonicity, which affects delivery rates. Tonicity is the sum of the concentrations of the solutes which have the capacity to exert an osmotic force across the membrane. All release rate experiments were conducted with 5,6-carboxyfluorescein, a fluorescent dye. Release rates were monitored from both bare niosomes alone and niosome-embedded, chitosan networks. It was observed that chitosan networks prolonged delivery from 100 h to 55 days in low ionic strength environment and pH conditions similar to a tumor site. The primary effect of chitosan is to add control on release time and dosage, and stabilize the niosomes through a high ionic strength surrounding that prevents uncontrolled bursting of the niosomes. Secondary factors include crosslink density of the chitosan network, molecular weight of the individual chitosan polymers, dye concentration within the niosomes, and the number density of niosomes packaged within the chitosan network. Each of these factors can be altered to fine-tune release rates.

Influence of lipid membrane rigidity on properties of supporting polymer.

Temperature-sensitive hydrogel polymers are utilized as responsive layers in various applications. Although the polymer's native characteristics have been studied extensively, details concerning its properties during interaction with biorelated structures are lacking. This work investigates the interaction between a thermoresponsive polymer cushion and different lipid membrane capping layers probed by neutron reflectometry. N-isopropylacrylamide copolymerized with methacroylbenzophenone first supported a lipid bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and subsequently 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The polymer-membrane systems were investigated above and below the polymer transition temperature (37 and 25°C). Although the same cushion supported each lipid membrane, the polymer hydration profile and thickness were markedly different for DPPE and DPPC systems. Because DPPE and DPPC have different bending rigidities, these results establish that the polymer-membrane interaction is critically mediated by the mechanics of the membrane, providing better insight into cell-hydrogel interactions.

In-plane correlations in a polymer-supported lipid membrane measured by off-specular neutron scattering.

Polymer-supported single lipid bilayers are models to study configurations of cell membranes. We used off-specular neutron scattering to quantify in-plane height-height correlations of interfacial fluctuations of such a lipid bilayer. As temperature decreased from 37 °C to 25 °C, the polymer swells and the polymer-supported lipid membrane deviates from its initially nearly planar structure. A correlation length characteristic of capillary waves changes from 30 µm at 37 °C to 11 µm at 25 °C, while the membrane bending rigidity remains roughly constant in this temperature range.

Surface instabilities in ultrathin, cross-linked poly(N-isopropylacrylamide) coatings.

Near-the-surface instabilities with a cusplike morphology were observed in ultrathin photo-cross-linked poly(N-isopropylacrylamide) coatings upon swelling in water. The characteristic wavelength of the instability was approximately 25 times the dry thickness and scaled linearly with coating thickness between 30 and 1200 nm. Above 1200 nm, slippage of the coating along the confining substrate led to reticulated patterns with a much larger wavelength. To help interpret the origin of the instability, the coatings were also exposed to a solvent slightly worse than water (acetone) and a solvent slightly better than water (isopropanol). In all cases, the characteristic wavelength scaled linearly with respect to the swelling induced by each solvent. Both water and isopropanol produced well-defined cusps or folds in the gel surface, while acetone produced semiordered blisters that grew into one another. The features produced in acetone may be a consequence of swelling being close to the threshold value for the loss of planar stability. Through the use of a first-order linear perturbation of the Flory-Rehner model, it is shown that the emergence of a characteristic wavelength is consistent with an inhomogeneous distribution of solvent that results from diffusion of solvent into a dry coating.

Viscoelastic response of photo-cross-linked poly(N-isopropylacrylamide) coatings by QCM-D.

The viscoelastic behavior of surface-tethered poly(N-isopropylacrylamide), or poly(NIPAAm), networks in contact with aqueous solutions was characterized by the quartz crystal microbalance with dissipation (QCM-D). To avoid ambiguities in the data analysis, four integer multiples of the dry thickness (h = k x 36 nm, where k = 1, 2, 3, 4) were analyzed at the third through ninth overtones of the QCM-D signal. At the third overtone, the networks resembled rigid films and the viscoelastic behavior could not be ascertained. With increasing overtones, however, the films showed deviation from the rigid film limit, allowing for analysis of the viscoelastic parameters. As the network collapsed over the temperature range of 15-40 degrees C, the shear modulus of the network increased by a factor of 24 (from 5 to 120 MPa); the shear viscosity, on the other hand, increased only by a factor of 2 (from 40 to 80 cP). The high values of the shear modulus suggest the QCM-D probes a regime where the polymer mesh does not adequately relax, and the high values of the shear viscosity suggest significant polymer-polymer coil overlap above and below the demixing temperature. Finally, the influence of NaCl on the viscoelastic response was measured. Interestingly, NaCl affects the shear modulus of the networks but not the dynamics.

Model lipid membranes on a tunable polymer cushion.

A hydrated, surface-tethered polymer network capable of fivefold change in thickness over a 25-37 degrees C temperature range has been demonstrated via neutron reflectivity and fluorescence microscopy to be a novel support for single lipid bilayers in a liquid environment. As the polymer swells from 170 to 900 A, it promotes both in- and out-of-plane fluctuations of the supported membrane. The cushioned bilayer proved to be very robust, remaining structurally intact for 16 days and many temperature cycles. The promotion of membrane fluctuations offers far-reaching applications for this system as a surrogate biomembrane.