Surfaces covered and functionalized with biomolecules play a key role in nano-biotechnology. At NANOTEC we study the biomolecules at interfaces with high resolution techniques to elucidate the mechanisms of self-assembly and aggregation, investigate the molecular-scale mechanisms of surface force generation and determine the mechanical and viscoelastic properties of biomaterials for their applications in tissue regeneration and biosensing.
Proteins at interfaces
Protein and peptide monolayers/multilayers are created at a liquid/vapour interface by Langmuir techniques. Studies on the aggregation process are performed in situ by the analysis of the compression isotherm curves, obtained under different environmental conditions (T, pH, compression speed), using models derived for classical 3D aggregation, adapted to the 2D world. The structural properties are investigated on mono-layers and multi-layers after the transfer of the film on silicon or glass substrates by Langmuir-Blodgett/Langmuir-Shaefer techniques, by means of scanning probe Microscopy Nanomechanics and Force spectroscopy. Of particular interest are Hydrophobins, small proteins produced by filamentous fungi, as they are biological surfactants whose films and aggregates can be used as models to study the mechanism of protein aggregation, for instancein amyloidosis. Moreover hydrophobins exhibit singular properties that can be exploited for bio-technological application.
Protein aggregation is a process triggered by physical and chemical changes in the surrounding molecular environment that lead to several neurodegenerative human disorders known as amyloid diseases, including Alzheimer’s and Parkinson’s diseases and spongiform encephalopathies. On the other hand, protein aggregation can also be utilized for creating nanostructured materials as protein can self-assemble in fibrils and other organized orphologies with defined nanoscale morphologies.
Protein structures can be modelled by regulating a number of experimental conditions that include temperature, pH, protein concentration, presence of other compounds and substrate of deposition. The structures formed can be characterized by a combination of experimental and theoretical methods. In our case, we have studied the self-assembly features of model proteins such as ß-lactoglobulin and human serum albumin. Their aggregation properties have been investigated in the presence of metals such as copper, zinc and iron, as well as in interaction with solid substrates.
Molecular-scale mechanisms of surface force generation by proteins and biomimetic polymers
There are many examples of biological surfaces that show outstanding lubrication properties (e.g. removal of surface adsorption/adhesion, low friction coefficient and/or high resistance to wear) while exposed to aqueous fluids, e.g. articular cartilage, cornea, teeth, gastro-intestinal and reproductive tracts. Most synthetic lubricants and surface coatings are oil-based and immiscible with water, and fail to provide an efficient or durable lubrication of wet surfaces, which is particularly inconvenient in bioengineering, e.g. for joint replacements, contact lenses, hearth valves, catheters and medical probes. Likewise, synthetic adhesives generally fail to provide efficient surface adhesion in wet conditions whereas many organisms, particularly sea shells, mussel and algae, are able to firmly attach to underwater surfaces. Our project is aimed at clarifying the molecular-scale mechanisms of biological adhesion and lubrication, relating molecular composition to conformation and function, for two classes of proteins: mucin glycoproteins that lubricate many surfaces of the human body, and mussel foot proteins that provide underwater surface adhesion. In both cases, the key molecular feature is the presence of specific functional groups: strongly hydrophilic sugar groups in mucins and surface-binding L-3,4-dihydroxyphenylalanine (DOPA) in mussels.
The study is conducted mainly using the Surface Force Apparatus (SFA) and Atomic Force Microscope (AFM) on protein layers adsorbed on solid surfaces and functional coatings with known physical-chemical properties (surface charge, polarity, roughness, etc.). Insights into the molecular-scale mechanism of surface adhesion and lubrication can be translated into the molecular structure of biomimetic synthetic polymer, which are also considered in this project.
Adhesive and viscoelastic properties of biomolecular thin films and biomaterials
This project is focused on the quantitative analysis of the mechanical properties of polymers, protein layers and biological tissues for biomedical applications.
The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber and provides 2/3 of the eye’s focusing power. The cornea has the structure of a thin shell with the external and internal surfaces having an ellipsoidal geometry. In the last three years our research has been focused mainly on the study of the mechanical properties of the corneal stroma using atomic force microscopy. A part of the research was devoted to the study of the effects induced in corneal tissues by the riboflavin/UV-A cross-linking technique, used for the treatment of the corneal disease known as keratoconus. Another part of the research was aimed to study the depth-dependent mechanical anisotropy of the human corneal stroma at the micro- and nano-level and to determine whether the biomechanics of the stroma involves any relationships between different scales of measurement. The research is carried out using Atomic Force Microscopy and Force Spectroscopy.
Biofunctionalization of materials and devices
Biomimetic and bioinspired materials present an emerging field in the areas of biomedicine, bioengineering, and biological science. Of particular interest is the current trend toward the production of biofunctional materials that are able to interact with the surrounding biological environment thereby enabling applications in tissue engineering, therapy, biosensing and bioimaging. In the case of tissue engineering, biomimetically inspired biomaterials include hydrogels, calcium phosphates (CaP) like hydroxyapatite (HA), magnesium containing coatings and materials containing proteins or peptides of the extracellular matrix (ECM). Low pressure and atmospheric pressure, plasma-assisted approaches can be used in order to improve the attachment of biomolecules onto materials, promote the adsorption of microcarriers to deliver a specific biomolecule (i.e. growth factors or bioactive agents) or plasma deposit in a single step coatings containing the molecules of interest that mimick the ECM environment or coatings containing magnesium and/or hydroxyapatite.
- L. Petrone, A. Kumar, C. N. Sutanto, N. J. Patil, S. Kannan, A. Palaniappan, S. Amini, B. Zappone, C. Verma and A. Miserez, Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins, Nature Communications 6, 8737 (2015) ISSN: 2041-1723; doi: 10.1038/ncomms9737
- B. Zappone, N. Patil, J. Madsen, K. Pakkanen, S. Lee, Molecular Structure and Equilibrium Forces of Bovine Submaxillary Mucin Adsorbed at a Solid-Liquid Interface Langmuir, 31 (15), 4524-4533 (2015) ISSN: 0743-7463; doi: 10.1021/acs.langmuir.5b00548
- R. Guzzi, B. Rizzuti, C. Labate, B. Zappone, M.P. De Santo, Ferric ions inhibit the amyloid fibrillation of ß-lactoglobulin at high temperature, Biomacromolecules, 16, 1794-1801 (2015). ISSN: 1525-7797; doi: 10.1021/acs.biomac.5b00371
- C. Labate, M.P. De Santo, G. Lombardo, M. Lombardo, Understanding Of The Viscoelastic Response Of The Human Corneal Stroma Induced By Riboflavin/Uv-A Cross-Linking At The Nano Level, Plos One, 10, 4 pag UNSP e0122868 (2015), ISSN: 1932-6203 doi: 10.1371/Journal.Pone.0122868
- C. Labate, M. Lombardo, M. P. De Santo, J. Dias. N.M. Ziebarth, G. Lombardo, Multiscale Investigation Of The Depth-Dependent Mechanical Anisotropy Of The Human Corneal Stroma, Investigative Ophthalmology & Visual Science 56 (6), 4053-60 (2015) ISSN: 0146-0404;doi: 10.1167/Iovs.15-16875.
- T. Røn, I. Javakhishvili, N. J. Patil; K. Jankova Atanasova, B. Zappone, S. Hvilsted, S. Lee, Aqueous lubricating properties of charged (ABC) and neutral (ABA) triblock copolymer chains Polymer 55, 4873–4883 (2014). ISSN: 0032-3861; doi: 10.1016/j.polymer.2014.07.049
- G. Da Ponte, E. Sardella, F. Fanelli, S. Paulussen, P. Favia. Atmospheric pressure plasma deposition of poly lactic acid-like coatings with embedded elastin. Plasma processes and polymers 11-4 (2014) 342-352 ISSN: 1612-8850; doi: 10.1002/ppap.201300130
Other selected publications
- Houmadi, R.D. Rodriguez, S. Longobardi, P. Giardina, M. C. Fauré, M. Giocondo, E. Lacaze, Self-Assembly of Hydrophobin Protein Rodlets Studied with Atomic Force Spectroscopy in Dynamic Mode Langmuir 28(5), 2551-2557 (2012). ISSN: 0743-7463; doi: 10.1021/la2028093
- De Stefano, I. Rea, E. De Tommasi, I. Rendina, L. Rotiroti, M. Giocondo, S. Longobardi, A. Armenante, and P. Giardina. Bioactive Modification of Silicon Surface using Self-assembled Hydrophobins from Pleurotus ostreatus. EPJ E-Soft Matter & Biological Physics, 30(2), 181-185 (2009) ISSN: 1292-8941; doi: 10.1140/epje/i2009-10481-y
- Houmadi, F. Ciuchi, M.P. De Santo, L. De Stefano, I. Rea, P. Giardina, A. Armenante, E. Lacaze and M. Giocondo. Langmuir-Blodgett film of hydrophobin protein from Pleurotus ostreatus at the air-water interface, Langmuir, 24(22), 12953–12957, (2008). ISSN: 0743-7463; doi: 10.1021/la802306r
- De Stefano, I. Rea, A. Armenante, I. Rendina, P. Giardina and M. Giocondo. Self-Assembled Biofilm of Hydrophobins Protect the Silicon Surface in the KOH Wet Etch Process, Langmuir 23(15) 7920 (2007) ISSN: 0743-7463; doi: 10.1021/la701189b
- Das; X. Banquy; B. Zappone; G. W. Greene; G. Jay; J. Israelachvili , Synergistic interactions between grafted Hyaluronic acid and Lubricin provide enhanced wear protection and lubrication, Biomacromolecules 14, 1669–1677 (2013). ISSN: 1525-7797; doi: 10.1021/bm400327a
- Zappone, P. J. Thurner, J. Adams, G. E. Fantner, P. K. Hansma, Effect of Ca2+ ions on the adhesion and mechanical properties of adsorbed layers of human Osteopontin, Biophysical Journal, 95, 1-12 (2008). ISSN: 0006-3495; doi: 10.1529/biophysj.108.135889
- Zappone, G. W. Greene, E. Ouroudjev, G. D. Jay, J. N. Israelachvili, Molecular aspects of the boundary lubrication by human Lubricin: effect of disulphide bonds and enzymatic digestion, Langmuir 24, 1495-1508 (2008). ISSN: 0743-7463; doi: 10.1021/la702383n
- Zappone, M. Ruths, G. W. Greene, G. D. Jay, J. N. Israelachvili, Adsorption, lubrication and wear of Lubricin on model surfaces: Polymer brush-like behavior of a glycoprotein, Biophysical Journal 92, 1693-1708 (2007). ISSN: 0006-3495; doi: 10.1529/biophysj.106.088799
- Lombardo M, Lombardo G, Carbone G, De Santo Mp, Barberi R, Serrao S, Biomechanics Of The Anterior Human Corneal Tissue Investigated With Atomic Force Microscopy. Investigative Ophthalmology & Visual Science, 53, 1050-1057 (2012) ISSN: 0146-0404; doi: 10.1167/iovs.11-8720
- Lombardo M, Carbone G, Lombardo G, De Santo Mp, Barberi R, Analysis Of Intraocular Lens Surface Adhesiveness By Atomic Force Microscopy, Journal Of Cataract And Refractive Surgery, 35, 1266-1272 (2009) ISSN: 0886-335; doi: 10.1016/j.jcrs.2009.02.0290