Determination of Peptide<b>–</b>Surface Adsorption Free Energy for Material Surfaces Not Conducive to SPR or QCM using AFM

The interactions between peptides and proteins with material surfaces are of primary importance in many areas of biotechnology. While surface plasmon resonance spectroscopy (SPR) and quartz crystal microbalance (QCM) methods have proven to be very useful in measuring fundamental properties characterizing adsorption behavior, such as the free energy of adsorption for peptide–surface interactions, these methods are largely restricted to use for materials that can readily form nanoscale-thick films over the respective sensor surfaces. Many materials including most polymers, ceramics, and inorganic glasses, however, are not readily suitable for use with SPR or QCM methods. To overcome these limitations, we recently showed that desorption forces (<i>F</i>_des) obtained using a standardized AFM method linearly correlate to standard-state adsorption free energy values (Δ<i>G°</i>^<i></i>_ads) measured from SPR in phosphate buffered saline (PBS: phosphate buffered 140 mM NaCl, pH 7.4). This approach thus provides a means to determine Δ<i>G°</i>^<i></i>_ads for peptide adsorption using AFM that can be applied to any flat material surface. In this present study, we investigated the <i>F</i>_des–Δ<i>G°</i>^<i></i>_ads correlation between AFM and SPR data in PBS for a much broader range of systems including eight different types of peptides on a set of eight different alkanethiol self-assembled monolayer (SAM) surfaces. The resulting correlation was then used to estimate Δ<i>G°</i>^<i></i>_ads from <i>F</i>_des determined by AFM for selected bulk polymer and glass/ceramic materials such as poly­(methyl methacrylate) (PMMA), high-density polyethylene (HDPE), fused silica glass, and a quartz (100) surface. The results of these studies support our previous findings regarding the strong correlation between <i>F</i>_des measured by AFM and Δ<i>G°</i>^<i></i>_ads determined by SPR, and provides a means to estimate Δ<i>G°</i>^<i></i>_ads for peptide adsorption on macroscopically thick samples of materials that are not conducive for use with SPR or QCM

Structure of Hydrated Poly(d,l-lactic acid) Studied with X-ray Diffraction and Molecular Simulation Methods

The effect of hydration on the molecular structure of amorphous poly­(d,l-lactic acid) (PDLLA) with 50:50 L-to-D ratio has been studied by combining experiments with molecular simulations. X-ray diffraction measurements revealed significant changes upon hydration in the structure functions of the copolymer. Large changes in the structure functions at ∼10 days of incubation coincided with the large increase in the water uptake from ∼1 to ∼40% and the formation of voids in the film. Computer modeling based on the recently developed TIGER2/TIGER3 mixed sampling scheme was used to interpret these changes by efficiently equilibrating both dry and hydrated models of PDLLA. Realistic models of bulk amorphous PDLLA structure were generated as demonstrated by close agreement between the calculated and the experimental structure functions. These molecular simulations were used to identify the interactions between water and the polymer at the atomic level including the change of positional order between atoms in the polymer due to hydration. Changes in the partial O–O structure functions, about 95% of which were due to water–polymer interactions, were apparent in the radial distribution functions. These changes, and somewhat smaller changes in the C–C and C–O partial structure functions, clearly demonstrated the ability of the model to capture the hydrogen-bonding interactions between water and the polymer, with the probability of water forming hydrogen bonds with the carbonyl oxygen of the ester group being about 4 times higher than with its ether oxygen

Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces

Ribonuclease A (RNase A) is a small globular enzyme that lyses RNA. The remarkable solution stability of its structure and enzymatic activity has led to its investigation to develop a new class of drugs for cancer chemotherapeutics. However, the successful clinical application of RNase A has been reported to be limited by insufficient stability and loss of enzymatic activity when it was coupled with a biomaterial carrier for drug delivery. The objective of this study was to characterize the structural stability and enzymatic activity of RNase A when it was adsorbed on different surface chemistries (represented by fused silica glass, high-density polyethylene, and poly­(methyl-methacrylate)). Changes in protein structure were measured by circular dichroism, amino acid labeling with mass spectrometry, and in vitro assays of its enzymatic activity. Our results indicated that the process of adsorption caused RNase A to undergo a substantial degree of unfolding with significant differences in its adsorbed structure on each material surface. Adsorption caused RNase A to lose about 60% of its native-state enzymatic activity independent of the material on which it was adsorbed. These results indicate that the native-state structure of RNase A is greatly altered when it is adsorbed on a wide range of surface chemistries, especially at the catalytic site. Therefore, drug delivery systems must focus on retaining the native structure of RNase A in order to maintain a high level of enzymatic activity for applications such as antitumor chemotherapy