Supramolecular oligothiophene microfibers spontaneously assembled on surfaces or coassembled with proteins inside live cells.

During the last few decades, multifunctional nano- and microfibers made of semiconducting π-conjugated oligomers and polymers have generated much interest because of a broad range of applications extending from sensing to bioelectronic devices and (opto)electronics. The simplest technique for the fabrication of these anisotropic supramolecular structures is to let the molecules do the work by spontaneous organization driven by the information encoded in their molecular structure. Oligothiophenes-semiconducting and fluorescent compounds that have been extensively investigated for applications in thin-film field-effect transistors and solar cells and to a lesser extent as dyes for fluorescent labeling of proteins, DNA, and live cells-are particularly suited as building blocks for supramolecular architectures because of the peculiar properties of the thiophene ring. Because of the great polarizability of sulfur outer-shell electrons and the consequent facile geometric deformability and adaptability of the ring to the environment, thiophene can generate multiple nonbonding interactions to promote non-covalent connections between blocks. Furthermore, sulfur can be hypervalent, i.e., it can accommodate more than the eight electrons normally associated with s and p shells. Hypervalent oligothiophene-S,S-dioxides whose oxygen atoms can be involved in hydrogen bonding have been synthesized. These compounds are amphiphilic, and some of them are able to spontaneously cross the membrane of live cells. Hypervalent nonbonding interactions of divalent sulfur, defined as weak coordination to a proximate nitrogen or oxygen, have also been invoked in the solid-state packing of many organic molecules and in the architecture of proteins. In this Account, we describe two different types of thiophene-based building blocks that can induce the spontaneous formation of nanostructured microfibers in very different environments. The first, based on the synthesis of "sulfur-overrich" hexamers and octamers, leads to surface-independent self-assembly of microfibers-helical or rodlike depending on the groups attached to the same identical inner core-that are crystalline, fluorescent, and conductive and display chirality despite the lack of chiral carbon atoms on the building blocks. Supramolecular polymorphic microfibers are also formed, and they are characterized by very different functional properties. The second, based on a rigid oligothiophene-S,S-dioxide, leads to coassembled protein-oligothiophene microfibers that are physiologically formed inside live cells. The oligothiophene-S,S-dioxide can indeed spontaneously cross the membrane of live cells and be directed toward the perinuclear region, where it is recognized and incorporated by specific peptides during the formation of fibrillar proteins without being harmful to the cells. Coassembled oligothiophene-protein microfibers are progressively formed through a cell-mediated physiological process. Thanks to the oligothiophene blocks, the microfibers possess fluorescence and charge-conduction properties. By means of fluorescence imaging, we demonstrated that various types of live cells seeded on these microfibers were able to internalize and degrade them, experiencing in turn different effects on their morphology and viability, suggesting a possible use of the microfibers as multiscale biomaterials to direct cell behavior. On the whole, our results show the great versatility of oligothiophene building blocks and allow us to foresee that their capabilities of spontaneous assembly in the most different environments could be exploited in much more exciting research fields than those explored to date.

Oligothiophenes as fluorescent markers for biological applications.

This paper summarizes some of our results on the application of oligothiophenes as fluorescent markers for biological studies. The oligomers of thiophene, widely known for their semiconductor properties in organic electronics, are also fluorescent compounds characterized by chemical and optical stability, high absorbance and quantum yield. Their fluorescent emission can be easily modulated via organic synthesis by changing the number of thiophene rings and the nature of side-chains. This review shows how oligothiophenes can be derivatized with active groups such as phosphoramidite, N-hydroxysuccinimidyl and 4-sulfotetrafluorophenyl esters, isothiocyanate and azide by which the (bio)molecules of interest can be covalently bound. This paper also describes how molecules such as oligonucleotides, proteins and even nanoparticles, tagged with oligothiophenes, can be used in experiments ranging from hybridization studies to imaging of fixed and living cells. Finally, a few multilabeling experiments are described.

Spontaneous Coassembly of the Protein Terthiophene into Fluorescent Electroactive Microfibers in 2D and 3D Cell Cultures.

Protein-based microfibers are biomaterials of paramount importance in materials science, nanotechnology, and medicine. Here we describe the spontaneous in situ formation and secretion of nanostructured protein microfibers in 2D and 3D cell cultures of 3T3 fibroblasts and B104 neuroblastoma cells upon treatment with a micromolar solution of either unmodified terthiophene or terthiophene modified by mono-oxygenation (thiophene → thiophene S-oxide) or dioxygenation (thiophene → thiophene S,S-dioxide) of the inner ring. We demonstrate via metabolic cytotoxicity tests that modification to the S-oxide leads to a severe drop in cell viability. By contrast, unmodified terthiophene and the respective S,S-dioxide cause no harm to the cells and lead to the formation and secretion of fluorescent and electroactive protein-fluorophore coassembled microfibers with a large aspect ratio, a micrometer-sized length and width, and a nanometer-sized thickness, as monitored in real-time by laser scanning confocal microscopy (LSCM). With respect to the microfibers formed by unmodified terthiophene, those formed by the S,S-dioxide display markedly red-shifted fluorescence and an increased n-type character of the material, as shown by macroscopic Kelvin probe in agreement with cyclovoltammetry data. Electrophoretic analyses and Q-TOF mass spectrometry of the isolated microfibers indicate that in all cases the prevalent proteins present are vimentin and histone H4, thus revealing the capability of these fluorophores to selectively coassemble with these proteins. Finally, DFT calculations help to illuminate the fluorophore-fluorophore intermolecular interactions contributing to the formation of the microfibers.

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.

A successful chemical strategy to induce oligothiophene self-assembly into fibers with tunable shape and function.

Functional supramolecular architectures for bottom-up organic nano- and microtechnology are a high priority research topic. We discovered a new recognition algorithm, resulting from the combination of thioalkyl substituents and head-to-head regiochemistry of substitution, to induce the spontaneous self-assembly of sulfur overrich octathiophenes into supramolecular crystalline fibers combining high charge mobility and intense fluorescence. The fibers were grown on various types of surfaces either as superhelices or straight rods depending on molecular structure. Helical fibers directly grown on a field effect transistor displayed efficient charge mobility and intrinsic 'memory effect'. Despite the fact that the oligomers did not have chirality centers, one type of hand-helicity was always predominant in helical fibers, due to the interplay of molecular atropisomerism and supramolecular helicity induced by terminal substituents. Finally, we found that the new sulfur overrich oligothiophenes can easily be prepared in high yields through ultrasound and microwave assistance in green conditions.

Human Serum Albumin-Oligothiophene Bioconjugate: A Phototheranostic Platform for Localized Killing of Cancer Cells by Precise Light Activation.

The electronic, optical, and redox properties of thiophene-based materials have made them pivotal in nanoscience and nanotechnology. However, the exploitation of oligothiophenes in photodynamic therapy is hindered by their intrinsic hydrophobicity that lowers their biocompatibility and availability in water environments. Here, we developed human serum albumin (HSA)-oligothiophene bioconjugates that afford the use of insoluble oligothiophenes in physiological environments. UV-vis and electrophoresis proved the conjugation of the oligothiophene sensitizers to the protein. The bioconjugate is water-soluble and biocompatible, does not have any "dark toxicity", and preserves HSA in the physiological monomeric form, as confirmed by dynamic light scattering and circular dichroism measurements. In contrast, upon irradiation with ultralow light doses, the bioconjugate efficiently produces reactive oxygen species (ROS) and leads to the complete eradication of cancer cells. Real-time monitoring of the photokilling activity of the HSA-oligothiophene bioconjugate shows that living cells "explode" upon irradiation. Photodependent and dose-dependent apoptosis is one of the primary mechanisms of cell death activated by bioconjugate irradiation. The bioconjugate is a novel theranostic platform able to generate ROS intracellularly and provide imaging through the fluorescence of the oligothiophene. It is also a real-time self-reporting system able to monitor the apoptotic process. The induced phototoxicity is strongly confined to the irradiated region, showing localized killing of cancer cells by precise light activation of the bioconjugate.

Self-complementary nucleoside-thiophene hybrid systems: synthesis and supramolecular organization.

Versatile synthetic methods towards a variety of thiophene-nucleobase hybrid systems are reported. Adenine- and thymine-based modified nucleosides characterized by a bithiophene unit linked to the C5' or C8 position through an ethylenamino or an ethylensulfanyl bridge were synthesized and successfully polymerized in the presence of FeCl(3) . The self-organization properties of the pure polymers as well as their mixtures - with complementary nucleobases - were investigated by means of optical microscopy and AFM in cast film showing complex supramolecular structures resulting from the interplay of multiple intermolecular interactions.

Supramolecular Assembly of Thiophene-Based Oligomers into Nanostructured Fluorescent Conductive and Chiral Microfibers.

The implementation of nano/microelectronic devices requires efficient strategies for the realization of supramolecular structures with desired function and supported on appropriate substrates. This article illustrates a strategy based on the synthesis of thiophene oligomers having the same "sulfur-overrich" quaterthiophene inner core (non bonding interactional algorithm) and different terminal groups. Nano/microfibers are formed on surfaces having a morphology independent of the nature of the deposition substrate and displaying a wide tuning of properties that make the fibers optoelectronically suitable for application in devices.

In Vivo Bioengineering of Fluorescent Conductive Protein-Dye Microfibers.

Engineering protein-based biomaterials is extremely challenging in bioelectronics, medicine, and materials science, as mechanical, electrical, and optical properties need to be merged to biocompatibility and resistance to biodegradation. An effective strategy is the engineering of physiological processes in situ, by addition of new properties to endogenous components. Here we show that a green fluorescent semiconducting thiophene dye, DTTO, promotes, in vivo, the biogenesis of fluorescent conductive protein microfibers via metabolic pathways. By challenging the simple freshwater polyp Hydra vulgaris with DTTO, we demonstrate the stable incorporation of the dye into supramolecular protein-dye co-assembled microfibers without signs of toxicity. An integrated multilevel analysis including morphological, optical, spectroscopical, and electrical characterization shows electrical conductivity of biofibers, opening the door to new opportunities for augmenting electronic functionalities within living tissue, which may be exploited for the regulation of cell and animal physiology, or in pathological contexts to enhance bioelectrical signaling.

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.

Self-organization, optical, and electrical properties of alpha-quinquethiophene-dinucleotide conjugates.

The synthesis and properties of (5')TA(3')-t5 (8a) and (5')CG(3')-t5 (8b) conjugates, in which the self-complementary dinucleotides TA and CG are covalently bound to the central ring of alpha-quinquethiophene (t5), are described. According to molecular mechanics calculations, the preferred conformation of both 8a and 8b is that with the dinucleotide folded over the planar t5 backbone, with the nucleobases facing t5 at stacking distance. The calculations show that the aggregation process of 8a and 8b is driven by a mix of nucleobase-thiophene interactions, hydrogen bonding between nucleobases (non Watson-Crick (W&C) in TA, and W&C in CG), van der Waals, and electrostatic interactions. While 8b is scarcely soluble in any solvents, 8a is soluble in water, indicating that the aggregates of the former are more stable than those of the latter. Microfluidic-induced self-assembly studies of 8a showed the formation of lamellar, spherulitic, and dendritic supramolecular structures, depending on the concentration and solvent evaporation time. The self-assembled structures displayed micrometer dimensions in the xy plane of the substrate and nanometer dimensions in the z direction. Spatially resolved confocal microscopy and spectroscopy showed that the aggregates were characterized by intense fluorescence emission. Cast films of 8a from water solutions showed chirality transfer from the dinucleotide to t5. The hole mobility of the cast films of 8a was estimated using a two-electrode device under high vacuum and found to be up to two orders of magnitude greater than those previously measured for dinucleotide-quarterthiophene conjugates under the same experimental conditions.

Enantiopure polythiophene nanoparticles. Chirality dependence of cellular uptake, intracellular distribution and antimicrobial activity.

The use of intrinsic chiral molecules opens the door to bio-imaging specific tools and to the development of target-therapy. In this work the synthesis and characterization of polythiophenes with alkyl side chains containing one R or S chiral carbon is reported. Enantiopure chiral nanoparticles (R or S NPs) were prepared from the polymers by a reprecipitation method. UV-vis, photoluminescence and circular dichroism spectroscopy of the NPs are described. In vitro analysis and metabolic assays show that both R and S NPs are efficiently taken-up by fibroblast cells without signs of toxicity. SDS-PAGE experiments show that formation of hard protein 'corona' enhances the chirality difference between nanoparticles. Co-localization experiments demonstrate that the cells are able to discriminate between the enantiomeric R and S nanoparticles. Finally, experiments carried out on Gram negative and Gram positive bacteria show that the enantiomeric NPs display different antibacterial activity.

Multifunctional nanostructures based on inorganic nanoparticles and oligothiophenes and their exploitation for cellular studies.

The combination of materials that possess different properties (such as, for instance, fluorescence and magnetism) into one single object of nanoscale size represents an attractive challenge for biotechnology, especially for their potential relevance in biomedical applications. We report here the preparation of novel bifunctional conjugates based on the linkage of inorganic nanoparticles to organic oligothiophene fluorophores (OTFs). In comparison to the organic dyes commonly used in bioimaging and more similarly to colloidal quantum dots, OTFs have broad optical absorption spectra, and therefore OTF fluorophores emitting at different colors can be excited with a single excitation source, allowing for easier multiplexing analysis. In this work we show the preparation of OTF-nanoparticle conjugates based on gold and iron oxide nanoparticles and their characterization using different techniques such as gel electrophoresis, photoluminescence spectroscopy, dynamic light scattering, and so on. In addition, by performing an in vitro study on human tumor cells we show that OTF-nanoparticle conjugates emitting at different colors can be used for multiplexing detection. Also, in the case of iron oxide-OTF conjugates, once uptaken by the cells, we show that they preserve both their fluorescent and their magnetic properties.

Polyvinyl-locked versus free quaterthiophene: effect of spatial constraints on the electronic properties of n-hexylquaterthiophene.

A soluble, low-weight fraction of poly(alpha-vinyl,omega-n-hexyl-quaterthiophene), PT4Hex, having n-hexylquaterthiophenes as side-chain groups, is prepared by free-radical polymerization of alpha-vinyl,omega-n-hexyl-quaterthiophene and the corresponding properties compared to those of free di-n-hexylquaterthiophene (T4Hex). Optical analysis (absorption and emission) and X-ray diffraction data indicate that in the polyvinyl-locked architecture the quaterthiophene pendants adopt a cofacial arrangement with a mutual distance close enough for pi-pi orbitals to overlap ( approximately 4 A). As a consequence of the close chain packing, a shift of the reduction potential of about 0.5 V toward less negative values with respect to free T4Hex, is found for PT4Hex films. Due to its enhanced electron affinity, PT4Hex displays an electron-acceptor behavior when blended with alkylated and silylated quaterthiophenes acting as donors.

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.

Ordered assembly of alpha-quinquethiophene on a copper oxide nanotemplate.

The organic semiconductor alpha-quinquethiophene (T5) is used as the active layer in organic field-effect transistors. We have investigated the adsorption of T5 on the (110) surface of copper and on the CuO nanotemplate formed by the high-temperature exposure of Cu(110) to molecular oxygen. The results were obtained with high-resolution scanning tunneling microscopy (STM) under ultra-high-vacuum (UHV) conditions. The adsorption of T5 on copper is an important model system because it mimics the active-layer-electrode interface in organic devices. The molecules were observed to adsorb onto both the pristine Cu(110) surface and the CuO nanotemplate, showing a greater affinity for the pristine copper surface. Surprisingly, however, the T5 molecules assembled with a much higher degree of long-range order on the oxygen-passivated portion of the surface.

Influence of the side functionalization of quinquethiophene-S,S-dioxides on the morphology of blends with poly(3-hexylthiophene): scanning force microscopy reveals.

Blends of an electron donor, i.e. a regioregular poly(3-hexylthiophene) (P3HT), with electron acceptors, a series of soluble quinquethiophene-S,S-dioxides (T5Os) bearing different alkyl side groups were self-assembled at surfaces. Scanning Force Microscopy (SFM) studies revealed that while the T5O symmetrically functionalized with two hexyl groups in the central thiophene (1) self-organizes into micrometer sized crystals embedded in a grainy matrix of P3HT, by substituting the central thiophene of 1 with one hexyl and one methyl unit (2) smaller and less anisotropic crystals of the acceptor having a sub-micrometer scale size were formed. The generation of these crystals is due to the joint effect of different non-covalent intermolecular interactions between the T5Os that self-segregate from the P3HT. By derivatizing the compound 1 with cyclo-hexyl moieties in the four external thiophenes molecule 3 was obtained. Such system was found to assemble into grainy disordered structures when co-deposited with P3HT, providing evidence for the absence of a phase segregation between the two components. Generally, the self-assembly at surfaces is governed by the interplay of intramolecular as well as intermolecular and interfacial interactions. In the present case, the cyclo-hexyl side groups in 3 both induce an intramolecular loss of planarity of the thiophene rings and hinders intermolecular interactions, reducing the tendency of the molecules to self-associate forming large crystals, whereas the symmetrical functionalization of the two central thiophenes with hexyl chains favours the crystallization of the T5O. The reported results demonstrate that subtle differences in the chemical functionalization can lead to different types of molecular architectures at surfaces. This is of importance since controlling the self-organization of pi-conjugated molecules at surfaces towards pre-programmed assemblies is a viable approach to enhance their electronic and luminescent properties, which should help to improve the performance of organic devices.

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.

New biocompatible polymeric micelles designed for efficient intracellular uptake and delivery.

New amphiphilic block copolymers are easily synthesised by post-polymerisation modifications of poly(glycidyl methacrylate) chain derivatives. The obtained material, upon dispersion in water, is capable of self-assembling into robust micelles. These nanoparticles, which are also characterised by adaptable stability, were loaded with different thiophene based fluorophores. The photoluminescent micelles were administered to cultured cells revealing a high and rapid internalisation of structurally different fluorescent molecules by the same internalisation pathway. Appropriate pairs of chromophores were selected and loaded into the micelles to induce Förster resonance energy transfer (FRET). The disappearing of the FRET phenomenon, after cell uptaking, demonstrated the intracellular release of the nanoparticle contents. The studied nanomaterial and the loaded chromophores have also shown to be biocompatible and non toxic towards the tested cells.

Regioselective Oligomerization of 3-(Alkylsulfanyl)thiophenes with Ferric Chloride.

The action of FeCl(3) on 3-(alkylsulfanyl)thiophenes (3-(alkylthio)thiophenes) leads to the one-step formation of regioregular alpha-conjugated oligothiophenes, from trimer to octamer, depending on the solvent used and on the length of the alkyl chain. The regiochemistry of these oligomers is characterized by one inner head-to-head linkage between adjacent rings and by a variable number of lateral head-to-tail junctions. The reaction of ferric chloride with the head-to-head and head-to-tail bis(methylsulfanyl)-2,2'-bithiophenes gives the corresponding tetramers, while the reaction with the tail-to-tail counterpart affords a high molecular weight insoluble material. With the aid of theoretical calculations, these results are interpreted on the basis of the joint effects of the orienting power of the substituents and of the stability of the radical cations formed during the oxidative process.