Characterization of Amyloid Fibril Networks by Atomic Force Microscopy.

Dense networks of amyloid nanofibrils fabricated from common globular proteins adsorbed to solid supports can improve cell adhesion, spreading and differentiation compared to traditional flat, stiff 2D cell culture substrates like Tissue Culture Polystyrene (TCPS). This is due to the fibrous, nanotopographic nature of the amyloid fibril networks and the fact that they closely mimic the mechanical properties and architecture of the extracellular matrix (ECM). However, precise cell responses are strongly dependent on the nanostructure of the network at the cell culture interface, thus accurate characterization of the immobilized network is important. Due to its exquisite lateral resolution and simple sample preparation techniques, Atomic Force Microscopy (AFM) is an ideal technique to characterize the fibril network morphology. Thus, here we describe a detailed protocol, for the characterization of amyloid fibril networks by tapping mode AFM.

Preparation of Amyloid Fibril Networks.

Networks of amyloid nanofibrils fabricated from common globular proteins such as lysozyme and ß-lactoglobulin have material properties that mimic the extracellular microenvironment of many cell types. Cells cultured on such amyloid fibril networks show improved attachment, spreading and in the case of mesenchymal stem cells improved differentiation. Here we describe a detailed protocol for fabricating amyloid fibril networks suitable for eukaryotic cell culture applications.

Quantifying Young's moduli of protein fibrils and particles with bimodal force spectroscopy.

Force spectroscopy is a means of obtaining mechanical information of individual nanometer-scale structures in composite materials, such as protein assemblies for use in consumer films or gels. As a recently developed force spectroscopy technique, bimodal force spectroscopy relates frequency shifts in cantilevers simultaneously excited at multiple frequencies to the elastic properties of the contacted material, yet its utility for quantitative characterization of biopolymer assemblies has been limited. In this study, a linear correlation between experimental frequency shift and Young's modulus of polymer films was used to calibrate bimodal force spectroscopy and quantify Young's modulus of two protein nanostructures: ß-lactoglobulin fibrils and zein nanoparticles. Cross-sectional Young's modulus of protein fibrils was determined to be 1.6 GPa while the modulus of zein nanoparticles was determined as 854 MPa. Parallel measurement of ß-lactoglobulin fibril by a competing pulsed-force technique found a higher cross-sectional Young's modulus, highlighting the importance of comparative calibration against known standards in both pulsed and bimodal force spectroscopies. These findings demonstrate a successful procedure for measuring mechanical properties of individual protein assemblies with potential use in biological or packaging applications using bimodal force spectroscopy.

Chitosan-coated amyloid fibrils increase adipogenesis of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have the potential to revolutionize medicine due to their ability to differentiate into specific lineages for targeted tissue repair. Development of materials and cell culture platforms that improve differentiation of either autologous or allogenic stem cell sources into specific lineages would enhance clinical utilization of MCSs. In this study, nanoscale amyloid fibrils were evaluated as substrate materials to encourage viability, proliferation, multipotency, and differentiation of MSCs. Fibrils assembled from the proteins lysozyme or ß-lactoglobulin, with and without chitosan coatings, were deposited on planar mica surfaces. MSCs were cultured and differentiated on fibril-covered surfaces, as well as on unstructured controls and tissue culture plastic. Expression of CD44 and CD90 proteins indicated that multipotency was maintained for all fibrils, and osteogenic differentiation was similarly comparable among all tested materials. MSCs grown for 7days on fibril-covered surfaces favored multicellular spheroid formation and demonstrated a >75% increase in adipogenesis compared to tissue culture plastic controls, although this benefit could only be achieved if MSCs were transferred to TCP for the final differentiation step. The largest spheroids and greatest tendency to undergo adipogenesis was evidenced among MSCs grown on fibrils coated with the positively-charged polysaccharide chitosan, suggesting that spheroid formation is prompted by both topography and cell-surface interactivity and that there is a connection between multicellular spheroid formation and adipogenesis.

Influence of sulphate, chloride, and thiocyanate salts on formation of ß-lactoglobulin-pectin microgels.

Effects of sulphate, chloride, and thiocyanate salts on the heat-induced formation of protein-based microgels from ß-lactoglobulin-pectin complexes were determined as a function of pH and protein-to-polysaccharide ratio. Aggregation temperatures were initially decreased at low ionic strength due to shielding of electrostatic interactions between ß-lactoglobulin and pectin but increased with further increases in ionic strength. Turbidity of heated mixtures and associated sizes of formed microgels were increased with up to 75 mmol kg(-1) ionic strength. Aggregation and microgel formation were relatively increased in the presence of thiocyanate salts compared to chloride salts and relatively decreased in the presence of sulphate salts, indicating that the inverse Hofmeister series was relevant in this system. Topographical analysis of dried microgels by atomic force microscopy verified that microgels were smallest in the presence of sulphate salts and showed that added ions, particularly thiocyanate, increased the deformability of microgels during drying.

Electrostatic stabilization of ß-lactoglobulin fibrils at increased pH with cationic polymers.

In order to improve the stability of ß-lactoglobulin fibrils formed in acidic conditions to increased pH values (pH 3-7), formation of electrostatic complexes between fibrils and cationic polymers chitosan (CH), amine-terminated poly(ethylene glycol) (APEG), low molecular weight poly(ethylenimine) (LPEI), and high molecular weight poly(ethylenimine) (HPEI) was investigated by electrophoretic mobility, turbidimetry, and atomic force microscopy. Except for suspensions with APEG, addition of polycations increased ζ-potential values of the fibrils at pH 5, 6, and 7, verifying their interactions with fibrils. Maximal increase in ζ-potential at pH 7, indicating optimal electrostatic interactivity, occurred at concentrations (w/w) of 0.05, 0.01, and 0.01% (corresponding to 6.9, 50, and 4 µmol·kg(-1)) for CH, LPEI, and HPEI, respectively. Turbidity of fibril solutions at pH 5, indicating isoelectric instability, was decreased significantly with increasing concentration of CH, LPEI, and BPEI, but not with added APEG. Turbidity was increased at pH 7 with added polycation, except for suspensions containing ≥0.02% HPEI. Fibril length and resistance to aggregation, as observed by atomic force microscopy, were increased at pH 5 with increasing concentration of CH and LPEI, yet only HPEI was capable of maintaining the morphology of fibrils at pH 7. Calculated persistence lengths of the fibrils, as compared to pure fibrils at pH 3 (∼4 µm), were only slightly reduced at pH 5 with CH and at pH 7 with HPEI, but increased at pH 5 with LPEI and HPEI. Improvement in the stability of ß-lactoglobulin fibrils at higher pH conditions with the addition of polycations will contribute to their potential utilization in packaging, food, and pharmaceutical applications.