Francesca Di Maria

Microwave-assisted synthesis of thiophene fluorophores, labeling and multilabeling of monoclonal antibodies, and long lasting staining of fixed cells.

We report the expedient microwave-assisted synthesis of thiophene based 4-sulfo-2,3,5,6,-tetrafluorophenyl esters whose molecular structure was engineered to achieve blue to red bright fluorescence. The reactivity toward monoclonal antibodies of the newly synthesized fluorophores was analyzed in comparison with that of the corresponding N-succinimidyl esters. Single-fluorophore and multiple-fluorophore labeled antibodies were easily prepared with both types of esters. Multiple-fluorophore labeling with blue and orange emitting fluorophores resulted in white fluorescent antibodies. Thiophene based fluorophores displayed unprecedented fluorescence stability in immunostaining experiments. First-principles TD-DFT theoretical calculations helped us to interpret the behavior of fluorescence emission in different environments.

Live-Cell-Permeant Thiophene Fluorophores and Cell-Mediated Formation of Fluorescent Fibrils

In our search for thiophene fluorophores that can overcome the limits of currently available organic dyes in live-cell staining, we synthesized biocompatible dithienothiophene-S,S-dioxide derivatives (DTTOs) that were spontaneously taken up by live mouse embryonic fibroblasts and HeLa cells. Upon treatment with DTTOs, the cells secreted nanostructured fluorescent fibrils, while cell viability remained unaltered. Comparison with the behavior of other cell-permeant, newly synthesized thiophene fluorophores showed that the formation of fluorescent fibrils was peculiar to DTTO dyes. Laser scanning confocal microscopy of the fluorescent fibrils showed that most of them were characterized by helical supramolecular organization. Electrophoretic analysis and theoretical calculations suggested that the DTTOs were selectively recognized by the HyPro component of procollagen polypeptide chains and incorporated through the formation of multiple H-bondings.

Microwave-assisted synthesis of oligothiophene semiconductors in aqueous media using silica and chitosan supported Pd catalysts

We report an innovative heterogeneous procedure for the preparation of highly pure thiophene oligomers via microwave-assisted Pd catalysis by using silica- and chitosan-supported Pd complexes. This approach is very efficient and greener than the existing homogeneous methodology as it combines a very efficient reaction with improved catalyst separation. Our new, efficient and cleaner microwave approach smoothly afforded the preparation of coupled products in high yields (up to 87% isolated yield, 30–100 min). Thienyl iodides or activated bromides were employed as starting materials and KF as base. The microwave reaction was carried out in aqueous ethanol. The heterogeneous catalyst can be easily removed from the reaction mixture by filtration and reused in consecutive reactions (up to 4 times).

Targeting ordered oligothiophene fibers with enhanced functional properties by interplay of self-assembly and wet lithography

Reproducible spatial control of a self-assembly process of fiber-forming oligothiophenes was achieved by using confinement effects. This strategy allowed the direct integration with a precise control over density, orientation, and size of supramolecular semiconducting fibers in OFET devices, demonstrating that well-aligned fibers exhibit a substantial enhancement of electrical performances.

Poly(3-hexylthiophene) nanoparticles for biophotonics: study of the mutual interaction with living cells

We report on the mutual interaction between poly(3-hexylthiophene) nanoparticles (P3HT-NPs) and human embryonic kidney (HEK-293) cells. P3HT-NPs, prepared in sterile conditions and efficiently uptaken within the live cells cytosol, show well-ordered morphology, high colloidal stability and excellent biocompatibility. Electrophysiology and calcium imaging experiments demonstrate that physiological functions of live cells are fully preserved in the presence of P3HT-NPs. From a complementary point of view, the photophysical properties of P3HT-NPs are also mainly maintained within the cellular environment, as proven by in situ time-resolved photoluminescence. Interestingly, we detect slight modifications in emission spectra and dynamics, which we ascribe to the contribution from the P3HT-NPs surface, possibly due to conformational changes as the result of the interaction with intracellular proteins or the formation of NPs aggregates. This work demonstrates that P3HT-NPs are excellent candidates for use as light sensitive actuators, due to their remarkable physical properties, optimal biocompatibility and capability of interaction with living cells.

Poly(3-hexylthiophene) Nanoparticles Containing Thiophene-S,S-dioxide: Tuning of Dimensions, Optical and Redox Properties, and Charge Separation under Illumination

We describe the preparation of poly(3-hexylthiophene-S,S-dioxide) nanoparticles using Rozens reagent, HOF·CH3CN, either on poly(3-hexylthiophene) (P3HT) or on preformed P3HT nanoparticles (P3HT-NPs). In the latter case, core-shell nanoparticles (P3HT@PTDO-NPs) are formed, as confirmed by X-ray photoelectron spectroscopy measurements, indicating the presence of oxygen on the outer shell. The different preparation modalities lead to a fine-tuning of the chemical-physical properties of the nanoparticles. We show that absorption and photoluminescence features, electrochemical properties, size, and stability of colloidal solutions can be finely modulated by controlling the amount of oxygen present. Atomic force microscopy measurements on the nanoparticles obtained by a nanoprecipitation method from preoxidized P3HT (PTDO-NPs) display spherical morphology and dimensions down to 5 nm. Finally, Kelvin probe measurements show that the coexistence of p- and n-type charge carriers in all types of oxygenated nanoparticles makes them capable of generating and separating charge under illumination. Furthermore, in core-shell nanoparticles, the nanosegregation of the two materials, in different regions of the nanoparticles, allows a more efficient charge separation.

Biocompatible and biodegradable fluorescent microfibers physiologically secreted by live cells upon spontaneous uptake of thiophene fluorophore

Live cells can form multifunctional and environmentally responsive multiscale assemblies of living and non-living components. We recently reported the results of a unique approach to introduce supplementary properties, fluorescence in particular, into fibrillar proteins produced by live fibroblasts and extruded into the ECM. In this work, we demonstrate that the physiological secretion of fluorescent nanostructured microfibers upon the spontaneous uptake of the appropriate fluorophore extends to living cells derived by different tissue contexts. We also show that live cells seeded on fluorescent microfibers have a different fate in terms of the cellular morphology, cytoskeleton rearrangement and viability. These results suggest that the microfibers, which are biocompatible and biodegradable, can be used as multiscale biomaterials to direct the cell behaviour.

Physiological formation of fluorescent and conductive protein microfibers in live fibroblasts upon spontaneous uptake of biocompatible fluorophores.

We have recently reported initial results concerning an original approach to introduce additional properties into fibrillar proteins produced by live fibroblasts and extruded into the ECM. The key to such an approach was biocompatible, fluorescent and semiconducting synthetic molecules which penetrated spontaneously the cells and were progressively encompassed via non-bonding interactions during the self-assembly process of the proteins, without altering cell viability and reproducibility. In this paper we demonstrate that the intracellular secretion of fluorescent microfibers can be generalized to living primary and immortalized human/mouse fibroblasts. By means of real-time single-cell confocal microscopy we show that the fluorescent microfibers, most of which display helical morphology, are generated by intracellular coding of the synthetic molecules. We also describe co-localization experiments on the fluorescent microfibers isolated from the cell milieu demonstrating that they are mainly made of type-I collagen. Finally, we report experimental data indicating that the embedded synthetic molecules cause the proteins not only to be fluorescent but also capable of electrical conductivity.

Photocatalytic Activity of Polymer Nanoparticles Modulates Intracellular Calcium Dynamics and Reactive Oxygen Species in HEK-293 Cells

Optical modulation of living cells activity by light-absorbing exogenous materials is gaining increasing interest, due to the possibility both to achieve high spatial and temporal resolution with a minimally invasive and reversible technique and to avoid the need of viral transfection with light-sensitive proteins. In this context, conjugated polymers represent ideal candidates for photo-transduction, due to their excellent optoelectronic and biocompatibility properties. In this work, we demonstrate that organic polymer nanoparticles, based on poly(3-hexylthiophene) conjugated polymer, establish a functional interaction with an in vitro cell model (Human Embryonic Kidney cells, HEK-293). They display photocatalytic activity in aqueous environment and, once internalized within the cell cytosol, efficiently generate reactive oxygen species (ROS) upon visible light excitation, without affecting cell viability. Interestingly, light-activated ROS generation deterministically triggers modulation of intracellular calcium ion flux, successfully controlled at the single cell level. In perspective, the capability of polymer NPs to produce ROS and to modulate Ca2+ dynamics by illumination on-demand, at non-toxic levels, may open the path to the study of biological processes with a gene-less approach and unprecedented spatio-temporal resolution, as well as to the development of new biotechnology tools for cell optical modulation.

Controlling the functional properties of oligothiophene crystalline nano/microfibers via tailoring of the self-assembling molecular precursors

Oligothiophenes are π‐conjugated semiconducting and fluorescent molecules whose self‐assembly properties are widely investigated for application in organic electronics, optoelectronics, biophotonics, and sensing. Here an approach to the preparation of crystalline oligothiophene nano/microfibers is reported based on the use of a “sulfur overrich” quaterthiophene building block, T4S4 , containing in its covalent network all the information needed to promote the directional, π–π stacking‐driven, self‐assembly of Y‐T4S4‐Y oligomers into fibers with hierarchical supramolecular arrangement from nano‐ to microscale. It is shown that when Y varies from unsubstituted thiophene to thiophene substituted with electron‐withdrawing groups, a wide redistribution of the molecular electronic charge takes place without substantially affecting the aggregation modalities of the oligomer. In this way, a structurally comparable series of fibers is obtained having progressively varying optical properties, redox potentials, photoconductivity, and type of prevailing charge carriers (from p‐ to n‐type). With the aid of density functional theory (DFT) calculations, combined with powder X‐ray diffraction data, a model accounting for the growth of the fibers from molecular to nano‐ and microscale is proposed.