Influence of surface curvature on unfolding of proteins on nanoparticles

Background.

Protein adsorption plays a crucial role in various fields in biotechnology and biomedical engineering. Adsorptive methods are commonly applied for separation of biomolecules by chromatography and membrane absorbers (Jungbauer, 2005). On the other hand, for certain unit operations, but also in the biomedical field, protein adsorption is not desired and should be avoided. Examples are fouling of filters in bioseparation, automated sampling devices in bioprocessing, in-situ analytics, or medical devices implanted into the body. A deeper understanding of protein adsorption and the underlying processes is required. This will also help to develop new materials for bioseparation, bioprocessing or biomedical applications, but will also contribute to the field of biocatalysis.

One promising possibility to intensify adsorptive processes is the use of non-porous nanoparticles for protein adsorption in a batch contactor, followed by a counter-current extraction as desorption step (Vorauer-Uhl et al., 1992, Tarmann et al., 2008). Nanoparticles have been manufactured at various sizes from 20-200 nm with different technologies and materials. Metastable states such as gels and glasses have found application in a wide range of products including toothpaste, hand cream, paints, and car tires (Shen et al., 2006). The equilibrium and metastable state behaviour of nanoparticle suspensions are often described by simple fluid models, where particles are treated as hard cores and interacting with short-range attractions. So it seems to be possible to use nanoparticles also for bioseparation purposes. Dry-gel conversion methods have been successfully applied for the synthesis of mesostructured silica materials with the particle size in submicron range. Duration of self-assembly and dry-gel conversion influences the structure and surface morphology of such silica particles. Submicron particles with uniform mesoporous structure can be formed that can be further modified by grafting with cationic or anionic groups. This leads to further stabilization of the suspension and allows electrostatic interaction between proteins and the nanoparticles.

In case of nanoparticles, it is not clear how proteins adsorb on their surface. In case of macroporous beads the protein adsorbs on the fibres, which form the porous structure (Jungbauer et al., 2005). Silica nanoparticles may have only a micropore-structure and it is not clear how the curvature of the particle affects the adsorbing protein. So one has to distinguish between the effects on protein deformation caused by the curvature and the effects caused by the surface energy. Surfaces have been either described on a molecular level or attempts have been made to calculate the surface energy. The contact angle has been determined using different solvents and several approaches have been applied to extract the surface energy from such measurements using the VanOss-Good-Chaudhury model. Recently, it was possible to fabricate nanoparticles with different charge densities, but also with the same charge density but different size. These particles are excellent models for the present project.

Aims and methods.

The aim is to elucidate the effect of surface curvature of nanoparticles on the unfolding of large adsorbing proteins. This needs elucidation of the surface energy of nanoparticles and the development of a proper method for its measurement. Silica nanoparticles will be fabricated at a size ranging from 20-200 nm and grafted with positively and negatively charged ligands at various densities. As the size of the particles roughly corresponds to that of large biomolecules, the adsorption step is likely to be accompanied by a partial unfolding of the protein which may lead to a loss of its activity or functionality. We hypothesise that the curvature of the particle, determined by its radius, has a dominant influence on this denaturing effect. Thus, we intend to investigate the extent of protein unfolding in correlation with the particle size and the surface energy. The extent of partial unfolding will be assessed by attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy that monitors changes in the secondary structure. From these experiments it should be possible to identify a critical lower particle diameter. Furthermore it will be investigated if this behaviour depends on the extent of saturation of the particle. The equilibrium binding capacity will be determined for particles of different size and a correlation with the theoretical surface area will be established.

A method to measure the surface energy will be developed using a similar approach as previously described with inverse gas chromatography. The nanoparticles will be packed into a capillary and pulses of solvents with different polarity will be injected. Using the retention of different solvents, the surface energy can be calculated by the VanOss-Good-Chaudhury model. The effect of protein unfolding mediated by nanoparticles of the same size but different surface energy can be measured. This will help to elucidate the contribution of the surface energy. Furthermore, unfolding will be studied with different adiabatic compressibility. Nanoparticles can be also coagulated in the presence of proteins. Either the charge repulsion is shielded by the adsorbed protein or the protein acts as a bridging agent and the nanoparticles will flocculate. Varying the salt concentration and pH of the environment can induce this effect and protein denaturation can be investigated. To bridge the gap to bioseparation desorption experiments must be conducted to see how efficiently the protein can be removed from such nanoparticles.

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