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Ordered mesoporous silica (OMS) materials are receiving great attention as possible carriers for valuable but unstable drugs as, for example, therapeutic proteins. A key issue is to prove that the therapeutic protein is effectively able to penetrate the pores of OMS during the adsorption step. Here, we immobilized an antibody fragment [F(ab')GAMIgG] conjugated with ultrasmall gold nanoparticles (GNPs) onto amino-functionalized SBA-15 (SBA-NH2) mesoporous silica. The aim of this work is the visualization of the location of the conjugates adsorbed onto SBA-NH2 with transmission electron microscopy (TEM). Because of the ultrasmall size of GNPs (<1 nm), we use the silver enhancement procedure to amplify their size. In this procedure, ultrathin sections of conjugate-loaded SBA-NH2 particles are prepared by a ultramicrotome sectioning technique. The ultrasmall GNPs located on the top side of the 70-90 nm thick slices act as microcrystallization nucleation sites for the deposition of reduced metallic silver. Consequently, the ultrasmall GNPs increase their size. This allows for the direct imaging of the conjugates adsorbed. We clearly localize the F(ab')GAMIgG-GNPs conjugates either on the external surface of the particles or inside the mesopores of SBA-NH2 through TEM.

Silica-based ordered mesoporous materials are very attractive matrices to prepare smart depot systems for several kinds of therapeutic agents. This work focuses on the well-known SBA-15 mesoporous silica and lysozyme, an antimicrobial protein. In order to improve the bioadhesion properties of SBA-15 particles, the effect of hyaluronic acid (HA) functionalization on lysozyme adsorption was investigated. SBA-15 samples having high (H-SBA) and low (L-SBA) levels of functionalization were analyzed during the three steps of the preparations: (1) introduction of the -NH2 groups to obtain the SBA-NH2 samples; (2) functionalization with HA to obtain the SBA-HA matrices; (3) adsorption of lysozyme. All silica matrices were characterized through N2-adsorption/desorption isotherms, small-angle X-ray scattering, transmission electron microscopy, thermogravimetric analysis, and Fourier transform infrared spectroscopy. The whole of the experimental data suggests that a high level of functionalization of the silica surface allows for a negligible lysozyme adsorption mainly due to unfavorable electrostatic interactions (H-SBA-NH2) or steric hindrance (H-SBA-HA). A low degree of functionalization of the silica surface brings about a very good performance toward lysozyme adsorption, being 71% (L-SBA-NH2) and 63% (L-SBA-HA) respectively, compared to that observed for original SBA-15. Finally, two different kinetic models--a "pseudo-second order" and a "intraparticle diffusion"--were compared to fit lysozyme adsorption data, the latter being more reliable than the former.

Turbidity titrations are used to study the ion specific aggregation of hemoglobin (Hb) below and physiological salt concentration in the pH range 4.5-9.5. At a salt concentration 50 mM cations promote Hb aggregation according to the order Rb(+) > K(+) ~ Na(+) > Cs(+) > Li(+). The cation series changes if concentration is increased, becoming K(+) > Rb(+) > Na(+) > Li(+) > Cs(+) at 150 mM. We interpret the puzzling series by assuming that the kosmotropic Li(+) will bind to kosmotropic carboxylates groups-according to the law of matching water affinities (LMWA)-whereas the chaotropic Cs(+) will bind to uncharged protein patches due to its high polarizability. In fact, both mechanisms can be rationalized by invoking previously neglected ionic nonelectrostatic forces. This explains both adsorption to uncharged patches and the LMWA as a consequence of the simultaneous action of electrostatic and dispersion forces. The same interpretation applies to anions (with chaotropic anions binding to chaotropic amine groups). The implications extend beyond hemoglobin to other, still unexplained, ion specific effects in biological systems.

Experiments on bovine serum albumin (BSA) via potentiometric titration (PT) and electrophoretic light scattering (ELS) are used to study specific-ion binding. The effect is appreciable at a physiological concentration of 0.1 M. We found that anions bind to the protein surface at an acidic pH, where the protein carries a positive charge (Z(p) > 0), according to a Hofmeister series (Cl(-) < Br(-) < NO(3)(-) < I(-) < SCN(-)), as well as at the isoionic point (Z(p) = 0). The results obtained require critical interpretation. The measurements performed depend on electrostatic theories that ignore the very specific effects they are supposed to reveal. Notwithstanding this difficulty, we can still infer that different 1:1 sodium salts affect the BSA surface charge/pH curve because anions bind to the BSA surface with an efficiency which follows a Hofmeister series.

The points of zero charge/potential of proteins depend not only on pH but also on how they are measured. They depend also on background salt solution type and concentration. The protein isoelectric point (IEP) is determined by electrokinetical measurements, whereas the isoionic point (IIP) is determined by potentiometric titrations. Here we use potentiometric titration and zeta potential (ζ) measurements at different NaCl concentrations to study systematically the effect of ionic strength on the IEP and IIP of bovine serum albumin (BSA) aqueous solutions. It is found that high ionic strengths produce a shift of both points toward lower (IEP) and higher (IIP) pH values. This result was already reported more than 60 years ago. At that time, the only available theory was the purely electrostatic Debye-Hückel theory. It was not able to predict the opposite trends of IIP and IEP with ionic strength increase. Here, we extend that theory to admit both electrostatic and nonelectrostatic (NES) dispersion interactions. The use of a modified Poisson-Boltzmann equation for a simple model system (a charge regulated spherical colloidal particle in NaCl salt solutions), that includes these ion specific interactions, allows us to explain the opposite trends observed for isoelectric point (zero zeta potential) and isoionic point (zero protein charge) of BSA. At higher concentrations, an excess of the anion (with stronger NES interactions than the cation) is adsorbed at the surface due to an attractive ionic NES potential. This makes the potential relatively more negative. Consequently, the IEP is pushed toward lower pH. But the charge regulation condition means that the surface charge becomes relatively more positive as the surface potential becomes more negative. Consequently, the IIP (measuring charge) shifts toward higher pH as concentration increases, in the opposite direction from the IEP (measuring potential)., (© 2011 American Chemical Society)

Potentiometric titrations were used to estimate the surface charge density of SBA-15 mesoporous silica in different salt solutions. It was found that surface charge depends both on cation type, following a Hofmeister series (Cs(+) < Guanidinium(+) < K(+) < Na(+) < Li(+)), and on salt concentration (in the range 0.05-1 M). The surface charge series is reproduced by theoretical calculations performed using a modified Poisson-Boltzmann equation that includes ionic dispersion forces with ab initio ion polarizabilities and hydrated ions. The hydration model assigns an explicit hydration shell to kosmotropic (strong hydrated) ions only. The Hofmeister series appears to be due to the combination of ion-surface dispersion interactions and ion hydration.