Interactions between Biomolecules and Phospholipid Membranes

The interactions between biomolecules and phospholipid membranes are a key topic to understand biological complexity since many cellular processes occur at specific sites of the cell membrane: not only receptor molecules are localized on its surface but the mechanism of action of different classes of biomolecules, including peptides, enzyme, nucleic acids, cholesterol derivatives and proteins, depends on the way they interact with the cell membrane. We investigated different models of cell membrane with different radii of curvature and composition with special emphasis on phenomena of lateral phase separation that produce transient microdomains, known as lipid raft. Lipid rafts are implicated in processes such as endocytosis, exocytosis, and vesicular trafficking (transport of vesicles across the cell) and are associated to regions of enhanced membrane curvature. Localized changes to membrane curvature in cells are essential for inter- and intracellular communication, in model systems, externally induced curvature changes are expected to drive the lateral organization of the membrane components. Recently, one of the recent reasons of interest for lipid rafts is the increasing experimental evidence of the implication of these domains in pathologically relevant phenomena, which involve the spatiotemporal regulated distribution of membrane-associated proteins, in terms of accumulation and segregation and eventually misfolding and aggregation. Lipid rafts were reproduced in different membrane models in order to investigate both their structure and dynamics in different model systems and to discriminate any preferential interactions of proteins with these ordered microdomains. We chose lysozyme as model protein since previous work reported that lysozyme aggregates into amyloid-like assemblies under conditions, such as acidic pH, high temperatures, presence of organic solvents or under physiological conditions in the presence of lipid membranes. This makes the protein an ideal model to study the effect of membranes on the unfolding and aggregation of pathologically relevant proteins. In parallel, we explored the behavior of a series of inhibitor of FKBP12, a protein of the family of immunophilins, and their interaction with the biological membrane. Due to its central role in immunosuppression and cell proliferation and due to its specific peptidyl-prolyl-isomerase (PPI) function, the FKBP protein family is at the crossroad of several important metabolic pathways. Members of this family, and notably FK506 binding protein (FKBP12), are thought to be involved in neurodegenerative diseases such as Alzheimer disease, Parkinson disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, as well as in proliferation disorders and cancer. Unravelling the mechanism of interaction and inclusion of effective inhibitors in biomimetic membrane models pave the way to drug-delivery strategies and biomimetic nanosensors for the FKBP12 protein. We successfully tested the inhibition of FKBP12 in solution in presence of natural ligands, as FK506 and Rifaximin and with a class of nanomolar ligands newly synthesized, ELTEX compounds. The binding process of the different ligands has been studied by means of photophysical measurements investigating the fluorescence quenching of the tryptophan residue in the binding pocket of FKBP12 by addition of the ligand in solution. At the same time we screened the possibility to include ligands of FKBP12 in planar and curved membrane models to investigate drug-membrane interaction, this study is of importance to understand the mechanism of passage through the membrane, for development of nanosystems, and for delivery of drugs. A biomimetic strategy was followed for the immobilization of the ligands: we selected different phospholipid nanoarchitectures differing in lipid composition, fluidity, number of layers and method of production (incubation versus co-spreading). We studied the incorporation of ligands of FKBP12 in mono and supported lipid bilayers prepared both with the Langmuir-Blodgett technique and for fusion of vesicles as a function of the ligand concentration. The effective incorporation of the ligands in LB film has been verified with UV-Vis absorption and fluorescence measurements which were compared with the respective samples prepared in the absence of binders. More importantly, the experiments demonstrated that the ligands in the LB scaffolds efficiently quench FKBP12 fluorescence in solution as a consequence of ligand-binding to the protein. Among ELTEX compounds, ElteN378, a new low atomic weight ligand, showed activity comparable to that of the macrolide Rapamycin, a compound with high affinity for FKBP12 used as standard. These results open the way for design of a sensor for FKBP12, a possible biomarker for early diagnosis in AD or PD. A FKBP sensor device can be envisaged by judiciously attaching to ElteN378 a suitable polymeric chain ending with an anchoring group for biochemical sensing in Self-Assembled Monolayers or Supported Lipid Bilayers deposited on Gold surfaces. We also faced the problem of efficient delivery and transfer of the drug, generally nanoparticles or liposomal systems are used as carriers of release. We explored also micellar systems because previous in vitro and in vivo experimental results show that micellar polymeric systems incorporate effectively FK506, thus suggesting a wider application as vehicles of release of other biologically active and poorly soluble compounds, such as ELTEX compounds. We investigated micelles and nanocomposite sponges, containing different fluorescent hydrophobic compounds as drug-like molecules that mimic the potential drug in order to monitor the stimulated release. We characterized a biocompatible device for on-demand chemical release in the form of a light-activatable sponge-like nanocomposite scaffold, which assures an excellent control over the principal parameters of the chemical release and dosage in order to sustain effective therapeutic action. The sponge consists of a porous biopolymer scaffold containing a dispersion of gold nanorods, which acts as an absorber of the incoming laser light, and of thermosensitive micelles, which serve as a reservoir for the drug molecules to be released. The photothermal response of the nanoparticles contained inside the sponge triggers a contraction in proximal micelles, thus promoting the expulsion of the drug that in turn is released from the sponge to the external environment. The peculiar physiochemical and structural properties of the nanocomposite sponges impart a number of interesting features to the proposed drug release system, including the possibility of spatially-confining the therapeutic treatment as well as of precisely controlling the amount of released drug as a function of duration and power of the excitation light.