Applicazione della tecnologia sol-gel: sintesi di un materiale ibrido biocompatibile e studio delle proprietà spettroscopiche ed elettrochimiche del citocromo c incapsulato in un bio sol-gel

Aim of the study is the structural characterization of a new hybrid silica biomaterial and the spectroscopic amd electrochemical behaviour of cyt c encapsulated in a bio sol-gel. In the first part of the project we investigated a new silica biomaterial synthesized through the sol-gel method. The inorganic/organic hybrids were synthesized combining an acid chitosan solution with an inorganic phase (TMOS), previously modified by the addition of calcium and phosphate ions. The structural test of the hybrids, containing a different chitosan -to silica ratios (BK1, BK2, BK3) were performed by employing Fourier-transform infrared spectroscopy. Hybrids spectra were compared with the bioactive sol-gel. In vivo, material bioactivity is associated to the ability to form an hydroxycarbonate apatite layer on its surface. To investigate the effect of chitosan amount on sol-gel biocompatibility, bioactivity essays were performed in vitro soaking the samples with static immersion for different time intervals (30 min, 6h e 24h) in a 0.01 M di Tris- HCl solution (pH 7.0). Ions release (Ca2+ e PO43- ) was determinated using couple induced emission spectroscopy (ICP). On BK3 surface, after sample immersion in a 0.01 M Tris-HCl for 48h, confocal microscopy has underlined the formation of crystalline aggregates. Although we can not state unequivocally that they are inequivocally composed by hydroxycarbonate apatite, we observed that the amounts of crystals increases with chitosan amount. Matrix structural changes, induced by chitosan, were investigated by swelling and permeability measurements. Swelling process was investigated to analyze hybrids stability in water environment. Permeability study, tense to clarify chitosan effect on solvent diffusion across the silica matrix, was performed using a fluorescent probe, named RubPy. Swelling measurements, demonstrate that samples weight increase, due to solvent diffusion across the matrix, is strongly correlated with chitosan concentration. Samples with high chitosan amount need longer times to reach an equilibrium state. More structural information are obtained comparing infrared spectrum of the bioactive gel with that of the hybrid material. An interesting result is the absorption band observed around 1400 cm-1, that can be ascribed to the organic modification of the silica matrix and, in particular, to the formation of Si-C bond. The ternary system, CaO- SiO2-P2O5, even if shows interesting properties, cannot be used as support for protein encapsulation; the major impediment is represented by the low pH (< 2.5); we synthesized a binary CaO-SiO2 bioactive system (bio sol-gel) which, although missing the phosphates, contains calcium, an element considered fundamental for biocompatibility. In the present work, we used the horse heart cytocrome c as a protein model. Structural properties of bio sol-gel encapsulated cyt c were investigated comparing the electronic absorption spectra and dicrioic spectra with those of the protein in solution (pH 7; 25° C) .We also investigated i) the acid-induced protein denaturation, (ii) the protein stability and (iii) the electrochemical behaviour of the embedded protein. Our data demonstrate that immobilization process do not alter protein functionality. System protein/ modified electrode show a quasi- reversible voltammetric behaviour in the Fe(III)/Fe(II) redox process, for all the scan rates investigated. The experimentally formal potential, E1/2 = 0.244 V vs NHE, is very close to the formal redox potential determined for soluble cyt c at the same pH (E1/2 = 0.255 V). To investigate system stability cyclic voltammogram of the samples was run for one week; surprisingly the protein preserves its redox activity. The electronic absorption and CD spectra demonstrate that bio sol-gel encapsulated protein do not present structural changes. The acid - induced denaturation process, achieved adding an HCl solution, was investigated by following the shift of the Soret absorption band from 408nm (native state) to 395nm (denaturated protein) as a function of time. Biogel entrapped cyt c clearly requires longer time to unfold than the soluble protein.The protein undergoes denaturation more promptly when entrapped in the salt-doped sol gel (i.e., in the presence of calcium nitrate) with respect to the case it is in pure (i.e., salt missing) sol-gel. Probably, this may be ascribed to the salt that prevent a correct sol gel reticulation and induces formation of larger pores. Also, different samples unfolding kinetics can be correlated with the a different solvent diffusion The refolding process, proved that unfolding-refolding process of cyt c entrapped in the bio sol-gel is fully reversible, under the conditions investigated.