ABSTRACT:The adsorption and conformation of bovine serum albumin (BSA) on gold nanoparticles (AuNPs) were interrogated both qualitatively and quantitatively via complementary physicochemical characterization methods. Dynamic light scattering (DLS), asymmetric-flow field flow fractionation (AFFF), fluorescence spectrometry, and attenuated total reflectanceFourier transform infrared (ATR-FTIR) spectroscopy were combined to characterize BSA-AuNP conjugates under fluid conditions, while conjugates in the aerosol state were characterized by electrospray-differential mobility analysis (ES-DMA). The presence of unbound BSA molecules interferes with DLS analysis of the conjugates, particularly as the AuNP size decreases (i.e., below 30 nm in diameter). Under conditions where the γ value is high, where γ is defined as the ratio of scattering intensity by AuNPs to the scattering intensity by unbound BSA, DLS size results are consistent with results obtained after fractionation by AFFF. Additionally, the AuNP hydrodynamic size exhibits a greater proportional increase due to BSA conjugation at pH values below 2.5 compared with less acidic pH values (3.4-7.3), corresponding with the reversibly denatured (E or F form) conformation of BSA below pH 2.5. Over the pH range from 3.4 to 7.3, the hydrodynamic size of the conjugate is nearly constant, suggesting conformational stability over this range. Because of the difference in the measurement environment, a larger increase of AuNP size is observed following BSA conjugation when measured in the wet state (i.e., by DLS and AFFF) compared to the dry state (by ES-DMA). Molecular surface density for BSA is estimated based on ES-DMA and fluorescence measurements. Results from the two techniques are consistent and similar, but slightly higher for ES-DMA, with an average adsorbate density of 0.015 nm -2 . Moreover, from the change of particle size, we determine the extent of adsorption for BSA on AuNPs using DLS and ES-DMA at 21°C, which show that increasing the concentration of BSA increases the measured change in AuNP size. Using ES-DMA, we observe that the BSA surface density reaches 90% of saturation at a solution phase concentration between 10 and 30 μmol/L, which is roughly consistent with fluorescence and ATR-FTIR results. The equilibrium binding constant for BSA on AuNPs is calculated by applying the Langmuir equation, with resulting values ranging from 0.51 Â 10 6 to 1.65 Â 10 6 L/mol, suggesting a strong affinity due to bonding between the single free exterior thiol on N-form BSA (associated with a cysteine residue) and the AuNP surface. Moreover, the adsorption interaction induces a conformational change in BSA secondary structure, resulting in less R-helix content and more open structures (β-sheet, random, or expanded).

Interfacial adhesion and friction are important factors in determining the performance and reliability of microelectromechanical systems. We demonstrate that the adhesion of micromachined surfaces is in a regime not considered by standard rough surface adhesion models. At small roughness values, our experiments and models show unambiguously that the adhesion is mainly due to van der Waals dispersion forces acting across extensive non-contacting areas and that it is related to 1/Dave2, where Dave is the average surface separation. These contributions must be considered because of the close proximity of the surfaces, which is a result of the planar deposition technology. At large roughness values, van der Waals forces at contacting asperities become the dominating contributor to the adhesion. In this regime our model calculations converge with standard models in which the real contact area determines the adhesion. We further suggest that topographic correlations between the upper and lower surfaces must be considered to understand adhesion completely.

The relationship between ECM mechanics and cell behavior is dynamic, as cells remodel and respond to changes in their local environment. Most in vitro substrates are static and supraphysiologically stiff; thus, platforms with dynamic and reversible mechanical changes are needed. Here, we developed hyaluronic acid-based substrates capable of sequential photodegradation and photoinitiated crosslinking reactions to “soften” and then “stiffen” the hydrogels over a physiologically-relevant range of moduli. Reversible mechanical signaling to adhered cells was demonstrated with human mesenchymal stem cells. In situ hydrogel softening (from ~14 to 3.5 kPa) led to a decrease in cell area and nuclear localization of YAP/TAZ, and subsequent stiffening (from ~3.5 to 28 kPa) increased cell area and nuclear localization of YAP/TAZ. Each photoreaction was cytocompatible and tunable, rendering this platform amenable to studies of dynamic mechanics on cell behavior across many cell types and contexts.

Surface-sensitive quantitative studies of competitive molecular adsorption on nanoparticles were conducted using a modified attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy method. Adsorption isotherms for thiolated poly(ethylene glycol) (SH-PEG) on gold nanoparticles (AuNPs) as a function of molecular mass (1, 5, and 20 kDa) were characterized. We find that surface density of SH-PEG on AuNPs is inversely proportional to the molecular mass (M(m)). Equilibrium binding constants for SH-PEG, obtained using the Langmuir adsorption model, show the binding affinity for SH-PEG is proportional to M(m). Simultaneous competitive adsorption between mercaptopropionic acid (MPA) and 5 kDa SH-PEG (SH-PEG5K) was investigated, and we find that MPA concentration is the dominant factor influencing the surface density of both SH-PEG5K and MPA, whereas the concentration of SH-PEG5K affects only SH-PEG5K surface density. Electrospray differential mobility analysis (ES-DMA) was employed as an orthogonal characterization technique. ES-DMA results are consistent with the results obtained by ATR-FTIR, confirming our conclusions about the adsorption process in this system. Ligand displacement competitive adsorption, where the displacing molecular species is added after completion of the ligand surface binding, was also interrogated by ATR-FTIR. Results indicate that for SH-PEG increasing M(m) yields greater stability on AuNPs when measured against displacement by bovine serum albumin (BSA) as a model serum protein. In addition, the binding affinity of BSA to AuNPs is inhibited for SH-PEG conjugated AuNPs, an effect that is enhanced at higher SH-PEG M(m) values.

There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness.photodegradable hydrogel | human mesenchymal stem cell | spatial matrix stiffness

Competitive adsorption kinetics between thiolated polyethylene glycol (SH-PEG) and mercaptopropionic acid (MPA) on gold nanoparticles (Au-NPs) were studied using a prototype physical characterization approach that combines dynamic light scattering (DLS) and electrospray differential mobility analysis (ES-DMA). The change in hydrodynamic particle size (intensityaverage) due to the formation of SH-PEG coatings on Au-NPs were measured by DLS in both two component (Au-NP + MPA or Au-NP + SH-PEG) and three component (Au-NP +MPA + SH-PEG) systems. ES-DMA was employed to quantify the surface coverage of SH-PEG and establish a correlation between surface coverage and the change in particle size measured by DLS. A change in equilibrium binding constant for SH-PEG on Au-NPs at various concentrations of SH-PEG and MPA showed that the presence of MPA reduced the binding affinity of SH-PEG to the Au-NP surface. Kinetic studies showed SH-PEG was desorbed from the Au-NP surface following a second-order desorption model after subsequently introducing MPA. The desorption rate constant of SH-PEG from the Au-NP surface by MPA displacement was strongly affected by the concentration of MPA and the excess SH-PEG in solution.

Spatial and temporal organoid control Stem cell–derived organoids form through self-organization and serve as models for organ development, function, and disease, with potential applications in drug development and personalized medicine. However, in the absence of external guidance, developmental processes are stochastic, resulting in variable end products that differ significantly from the native organ. Gjorevski et al . developed approaches for specifying the initial organoid geometry to build intestinal organoids of defined shape, size, and cell distributions, forming structures that are predictable, more similar to normal organs, and reproducible (see the Perspective by Huycke and Gartner). These methods identify symmetry-breaking mechanisms in intestinal morphogenesis and have potential for standardizing organoid-based therapies and facilitating the refinement of mechanistic studies. —BAP

We demonstrate a high-resolution in situ experimental method for performing simultaneous size classification and characterization of functional gold nanoparticle clusters (GNCs) based on asymmetric-flow field flow fractionation (AFFF). Field emission scanning electron microscopy, atomic force microscopy, multi-angle light scattering (MALS), and in situ ultraviolet-visible optical spectroscopy provide complementary data and imagery confirming the cluster state (e.g., dimer, trimer, tetramer), packing structure, and purity of fractionated populations. An orthogonal analysis of GNC size distributions is obtained using electrospray-differential mobility analysis (ES-DMA). We find a linear correlation between the normalized MALS intensity (measured during AFFF elution) and the corresponding number concentration (measured by ES-DMA), establishing the capacity for AFFF to quantify the absolute number concentration of GNCs. The results and corresponding methodology summarized here provide the proof of concept for general applications involving the formation, isolation, and in situ analysis of both functional and adventitious nanoparticle clusters of finite size.