- Mineralized Bionanoparticle Pickering Emulsions (2008)
- Various aspects of mineralized, bionanoparticle-stabilized Pickering emulsions were investigated in this work. Proteins (HSA, BSA, fetuin-A), protein cages (ferritin) and plant viruses (CPMV, TYMV, TMV) were employed to produce functional nanopatterned surfaces and interfaces by means of Pickering emulsions which were employed in biomimetic hydroxy apatite mineralization to produce inorganic biocompatible surfaces and hollow mineral spheres. The work accounts for the emerging interest and significant technological implications of protein and particle stabilized emulsions as well as bio-inorganic nanostructured composite materials. First, the bionanoparticles were characterized with SEC, UV-Vis, SLS, DLS, TEM and SDS-PAGE. Solutions of BSA, fetuin-A and ferritin contained aggregates and had to be fractionated with SEC for the following studies. Structural characterization of the viruses was accomplished by TEM with negative staining. Further, CPMV was labelled with fluorescein and tetramethylrhodamine. The preparation of CPMV-solutions from frozen leaves of cowpea plants is shortly described, too. Next, kinetic investigations of the adsorption process of the bionanoparticles at the decane/buffer solution interface were performed with pendant drop tensiometry. Interfacial pressure isotherms were recorded for BSA, HSA, fetuin-A, ferritin, CPMV and TYMV. A detailed explanation of the previously developed model of variable surface states of adsorbed proteins and various aspects of protein adsorption experiments were necessary as basis for the discussion and the modelling of the isotherms and the dynamic surface tension. The surface pressure isotherms of BSA and ferritin as well as the dynamic surface tension of BSA could be modelled successfully and were in excellent agreement with previous reports. Further, oil-in-water Pickering emulsions with perfluorooctane or an UV-crosslinkable PDMS precursor oil were prepared. The liquid-liquid interface could be imaged by confocal laser scanning fluorescence microscopy while the solid interfaces could be accessed with SEM and AFM. The bionanoparticles at the interface formed a dense assembly but the pattern did not show any long-range order. Interestingly, the rods of TMV formed no liquid-crystalline array but an irregular, sparse assembly at the interface. Height determinations of the adsorbed virus with AFM and TEM of cross-sections from embedded interfaces showed that TMV was not significantly immersed into the oil phase. The adsorption of BSA and ferritin could be further proven by fluorescence microscopy of solid Pickering emulsions that have reacted with fluorescently labelled antibodies. Finally, mineralization with hydroxy apatite (HAP) was conducted with various solutions under different conditions. The use of a simulated body fluid (SBF) allowed a biomimetic mineralization. A pre-incubation step on bioactive glass particles was necessary to induce HAP growth with SBF at physiological concentrations. Further, 1.5-fold concentrated SBF solution (1.5 SBF/cit) and an oversaturated calcium phosphate solution (Ca/P/cit), both containing citrate, were employed. The development of hydroxy apatite coatings on PET as model substrate was followed with time. PDMS-surfaces with immobilized proteins were used to mimic bio-related interfaces. Extensive structural and compositional characterization was performed with SEM and EDX. The induction and growth of hydroxy apatite was discussed and compared in detail for surfaces with different immobilized bionanoparticles and for different mineralization conditions. Dense coatings were achieved with SBF and Ca/P/cit. Surfaces prepared with ferritin and SDS yielded generally the smoothest and most compact HAP layers. Finally, true bionanoparticle Pickering emulsions (i.e. liquid-liquid interfaces) were used to produce mineral capsule shells. The development of the interfacial mineralization was investigated after various incubation times and exchanges of the mineralization solutions. The observed surface morphologies were explained by different mechanisms of nuclei formation and HAP growth. Particularly, the use of 1.5 SBF/cit or Ca/P/cit led to a dense pattern of single separated nuclei at the interface which tended to grow together to form compact shells. Ferritin-based Pickering emulsions were usually mineralized more weakly than BSA- and fetuin-A-based ones. In the case of Ca/P/cit, the slow mineralization of ferritin-coated oil droplets led to a controlled and very homogeneous formation of thin HAP composite shells. The combination of different mineralization mechanisms allowed the preparation of capsules with various controlled shell thicknesses and surface morphologies. Finally, the work contributes to the basic understanding of protein adsorption at liquid-liquid interfaces and their interfacial structures and shows new ways for the production of composite biomaterials through interfacial templating.