Theranostic applications of fluorescent liquid crystalline nanoparticles

Lipid liquid crystalline nanoparticles can find application as nanocarriers in several fields of the daily life but, very likely, the pharmaceutical arena is the most relevant. Indeed, several problems encountered in drugs administration (e.g. critical sideeffects from antitumor drugs) require alternative, less invasive, but simultaneously efficient therapeutic routes to be explored. Novel fields of personalized nanomedicine are developing in this direction. One of the most interesting is theranostic, which calls for the design of platforms capable of combining therapeutic and diagnostic functionalities. In this optic, we explored the potential of monoolein-based cubosomes and hexosomes as nanocarriers for theranostic purposes. Our work focussed on the design of lipid nanoparticles able to deliver antineoplastic drugs and imaging probes for fluorescent optical in vitro and in vivo imaging. We developed cubosome formulations loaded with antineoplastic drugs and useful for the fluorescence imaging of cells. Such formulations were also actively targeted to cancer cells and coupled with a NIR-emitting fluorophore, which was the promise for in vivo applications. We also investigated hexosomes with encouraging results encapsulating in their lipid matrix a BODIPY derivative with solvatochromic properties, helpful for the understanding of the dye localization. Importantly, we reported (manuscript submitted) the first proof-of-principle for in vivo fluorescence optical imaging application using monoolein-based cubosomes in a healthy mouse animal model. Finally, since relatively little is known about the interaction of cubosomes with biological systems, their effects on lipid droplets, mitochondria and lipid profile of HeLa cells were deeply studied. This thesis is divided in two main parts. The introduction section reports on the essential background of the research field, and it is followed by the publications (published or submitted) resulting from these three years of work.

Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes.

The quantification of protein-ligand interactions is essential for systems biology, drug discovery, and bioengineering. Ligand-induced changes in protein thermal stability provide a general, quantifiable signature of binding and may be monitored with dyes such as Sypro Orange (SO), which increase their fluorescence emission intensities upon interaction with the unfolded protein. This method is an experimentally straightforward, economical, and high-throughput approach for observing thermal melts using commonly available real-time polymerase chain reaction instrumentation. However, quantitative analysis requires careful consideration of the dye-mediated reporting mechanism and the underlying thermodynamic model. We determine affinity constants by analysis of ligand-mediated shifts in melting-temperature midpoint values. Ligand affinity is determined in a ligand titration series from shifts in free energies of stability at a common reference temperature. Thermodynamic parameters are obtained by fitting the inverse first derivative of the experimental signal reporting on thermal denaturation with equations that incorporate linear or nonlinear baseline models. We apply these methods to fit protein melts monitored with SO that exhibit prominent nonlinear post-transition baselines. SO can perturb the equilibria on which it is reporting. We analyze cases in which the ligand binds to both the native and denatured state or to the native state only and cases in which protein:ligand stoichiometry needs to treated explicitly.

Electrosprayed core-shell microspheres for protein delivery.

This communication describes a single-step electrospraying technique that generates core-shell microspheres (CSMs) with encapsulated protein as the core and an amphiphilic biodegradable polymer as the shell. The protein release profiles of the electrosprayed CSMs showed steady release kinetics over 3 weeks without a significant initial burst.

Detection of a single DNA base-pair mismatch using an anthracene-tagged fluorescent probe

A novel anthracene-tagged oligonucleotide can discriminate between a fully-matched DNA target sequence and one with a single mismatching base-pair through a remarkable difference in fluorescence emission intensity upon duplex formation.