Caratterizzazione delle prorietà strutturali e fotofisiche di biomateriali tramite EPR, FT-IR, spettroscopie Raman confocale ed NMR

Responsabili di progetto

Henriette Molinari, Oktay Gasimov

Accordo

AZERBAIJAN - ANAS-not in force - Azerbaijan National Academy of Sciences

Bando

CNR-ANAS 2016-2017

Dipartimento

Scienze chimiche e tecnologie dei materiali

Area tematica

Scienze chimiche e tecnologie dei materiali

Stato del progetto

Nuovo

Proposta di ricerca

Fabrication of photo-stable rhodamine (RHD) dyes incorporated into polymer matrices, in the solid state, improves the poor photostability of the dye (1-3) observed in solution. A novel alternative is represented by the encapsulation of RHD in bio-host systems, which can confer excellent photophysical properties to the dye. In a recent published example from Milano Lab, a lipid binding protein (LBP) was employed as bio-host system providing high quality homogeneous films, with excellent morphological properties, suitable to use in optoelectronic devices (4).
In the present project different bio-host systems, namely fibroin and proteins belonging to the calycin family, such as beta-lactoglobulin (BLG) and LBP, will be tested. Fibroin is an excellent biocompatible material widely applied in various areas of technology (5). Fibroin solution is easily processable to produce high quality films structure, which can be controlled by solvent treatment. Preliminary results from Baku Lab indicated that accessibility of hydrophobic areas of fibroin can be also modulated by the pH of the solution (6). BLG and LBP represent interesting bio-hosts as a consequence of their promiscuous hydrophobic cavities that can accommodate a wide variety of different ligands (7,8).
Baku Lab has great expertise in physical characterization of fibroin and protein films (9). Milano Lab made significant contribution to BLG structure-function relationship studies (7,8,10) and is involved in the structural characterization of new bio host-guest complexes.
Three main aims characterize the present project and are briefly described in the following.
1. Characterisation of RHD dye derivatives bio-incorporated in fibroin.
Bio-incorporation of RHD derivatives into fibroin will be performed by feeding silkworms with mulberry powder containing various dyes. The mechanism of dye-host interaction together with dye photophysics will be investigated. Free radical formation induced by UV and/or visible light will reveal photostability of the dye. Identity of the free radicals, other than RHD, will indicate the side chains that donates electron to excited RHD molecules. T1, T2, and 3pulse ESEEM (electron spin echo envelop modulation) measurements will be performed for RHD radicals. ESEEM measurements will also reveal whether H-bonds or pi-hydrogen bonds with protein side-chains, contribute to dye stabilisation in the host systems. Fibroin structures, modified by using different organic solvents, will be also characterized. CW-EPR and ESEEM measurements will be performed in solid state. NMR solution spectra of fibroin, at natural abundance, were obtained in aqueous solutions directly from "liquid silk" extracted from B. mori larvae silk glands and from regenerated silk fibroin from B.mori cocoons (11,12,13). Solution NMR spectra at natural abundance will be assayed on samples obtained from silkworms fed with and without RHD, to check whether the dye preferentially interacts with a specific repeat. Solution NMR studies will complement solid state investigations.
2. Charactersation of RHD-biomacromolecules complexes obtained in vitro.
Photophysics and free radical formation will be studied on the samples obtained in vitro by non covalent complexation of dyes with proteins. The obtained results will be compared to those obtained on bio-incorporated RHD samples. Time-resolved ANS fluorescence will be employed to detect fibroin hydrophobic clusters and will guide the choice of the experimental conditions allowing RHD incorporation in various regions of silk fibroin.
Non-covalent complexes between RHD and calycins will be characterized by solution NMR. The fundamental elements contributing to complex stabilization and spectral tuning of the chromophore inside the proteins cavities will be investigated. These complexes, easily processable into thin films, will be characterized exploiting the joint expertise of Milano (NMR) and Baku Laboratories (Surface Enhanced Raman and FTIR spectroscopies of films).
3. Kinetics studies of free radical recombination.
Recombination kinetics of free radicals, generated by UV irradiation in hydrated fibroin, will be measured together with the effects of solvent viscosity. Recombination kinetics (activation energy) of these radicals, activated by high-frequency low amplitude backbone motions of the protein, will be characterised for crystalline and amorphous parts of fibroin. It is expected that backbone dynamics of amorphous part in hydrated forms will be enhanced significantly compared to that of the crystalline part.
References
1) F.Amat-Guerri, A.Costela, J.M.Figueva, F.Florido, R.Sastre, Chem. Phys. Lett. 209, 352, 1993. 2) Costela, I. Garcia-Moreno, J.M.Figuera, F.Amat-Guerti, R.Mallavia, M.D. Santa-Maria, R. Sastre, J. Appl. Phys. 80, 3167, 1996. 3) Costela, I. Garc1a-Moreno, J.M. Figuera, F. Amat-Guerri, R. Sastre, Appl. Phys. Lett. 68, 593, 1996. 4) S. Tomaselli, U. Giovanella, K. Pagano, G. Leone, S. Zanzoni, M. Assfalg, F. Meinardi, H. Molinari, C. Botta, L. Ragona, Biomacromolecules, 14, 3549_3556, 2013. 5) N. Kasoju and U. Bora, Advanced healthcare materials 1, 393-412, 2012. 6) O.K. Gasymov, A. Aydemirova, O. Alekperov, R.B. Aslanov, et al, Phys. Status Solidi C 12, No. 6, 628- 630, 2015. 7) L. Ragona, F. Fogolari, M. Catalano, R. Ugolini, L. Zetta and H. Molinari, J. Biol. Chem. 278, 38840-6, 2003. 8) M. Collini, L. D'Alfonso, H. Molinari, L. Ragona, M. Catalano and G. Baldini, Protein Sci., 12, 1596-603, 2003. 9) O.K. Gasymov, A.R. Abduragimov and B.I.J. Glasgow, Phys. Chem. B. 119, 3962-8, 2015. 10) I. Eberini, C. Sensi, M. Bovi, H. Molinari, M. Galliano, F. Bonomi, S. Iametti and E. Gianazza, Amino acids, 43, 2457-68, 2012. 11) Sagnella, C. Chieco, V. Benfenati, N. Di Virgilio, S. Toffanin, et al, Composites: Part B 68, 281-287, 2015. 12) Suzuki et al., Biomacromolecules, 15, 104-112, 2014. 13)Asakura, T.,Okushita, K and M.P. Williamson, Macromolecules, 48, 2345-2357, 2015.

Obiettivi della ricerca

Specific objectives of the present research are:
o Investigation of the photophysical properties and free radical formation of RHD derivatives/protein complexes in solution and film samples (Baku Lab)
o Solution NMR characterization of newly prepared (employing BLG, LBP and fibroin) RHD host-guest systems (Milano Lab).
o Determination by time-resolved ANS fluorescence of the appropriate solution condition for a selective incorporation of RHD in different fibroin regions (Baku Lab)
o Role of pH and solvent in determining interactions with specific fibroin repeat (Milano and Baku Lab)
o Comparison of structural and photophysical properties of RHD derivatives: i) incorporated in fibroin through silkworms supply (bio-incorporation) and ii) incorporated in proteins via in vitro formation of non covalent host-gest complexes (Milano and Baku Lab)
o Comparison of recombination kinetics in crystalline and amorphous fibroins (Baku Lab)

The objectives described can be reached thank to the complementary available instrumentations in the two laboratories:
EPR, Confocal Raman, and FTIR (Baku Lab) and 600 MHz, 500MHz and 400 MHz NMR spectrometers (Milano Lab).

Ultimo aggiornamento: 24/04/2024