Multifunctional Photoelectroactive Materials for Optoelectronic Applications Based on Thieno[3,4-b]pyrazines and Thieno[1,2,5]thiadiazoles.

In this study, we introduce a novel family of symmetrical thiophene-based small molecules with a Donor-Acceptor-Donor structure. These compounds feature three different acceptor units: benzo[c][1,2,5]thiadiazole (Bz), thieno[3,4-b]pyrazine (Pz), and thieno[1,2,5]thiadiazole (Tz), coupled with electron donor units based on a carbazole-thiophene derivative. Using Density Functional Theory (DFT), we investigate how the molecular geometry and strength of the central acceptor unit impact the redox and spectroscopic properties. Notably, the incorporation of Pz and Tz moieties induces a significant redshift in the absorption and emission spectra, which extend into the near-infrared (NIR) region, simultaneously reducing their energy gaps (~1.4-1.6 eV). This shift is attributed to the increased coplanarity of the oligomeric inner core, both in the ground (S0 ) and excited (S1 ) states, due to the enhanced quinoidal character as supported by bond-length alternation (BLA) analysis. These structural changes promote better π-electron delocalization and facilitate photoinduced charge transfer processes in optoelectronic devices. Notably, we show that Pz- and Tz-containing molecules exhibit NIR electrochromic behavior and present ambivalent character in bulk heterojunction (BHJ) solar cells. Finally, theoretical calculations suggest that these molecules could serve as effective two-photon absorption (2PA) probes, further expanding their potential in optoelectronic applications.

Insight on the Intracellular Supramolecular Assembly of DTTO: A Peculiar Example of Cell-Driven Polymorphism.

The assembly of supramolecular structures within living systems is an innovative approach for introducing artificial constructs and developing biomaterials capable of influencing and/or regulating the biological responses of living organisms. By integrating chemical, photophysical, morphological, and structural characterizations, it is shown that the cell-driven assembly of 2,6-diphenyl-3,5-dimethyl-dithieno[3,2-b:2',3'-d]thiophene-4,4-dioxide (DTTO) molecules into fibers results in the formation of a "biologically assisted" polymorphic form, hence the term bio-polymorph. Indeed, X-ray diffraction reveals that cell-grown DTTO fibers present a unique molecular packing leading to specific morphological, optical, and electrical properties. Monitoring the process of fiber formation in cells with time-resolved photoluminescence, it is established that cellular machinery is necessary for fiber production and a non-classical nucleation mechanism for their growth is postulated. These biomaterials may have disruptive applications in the stimulation and sense of living cells, but more crucially, the study of their genesis and properties broadens the understanding of life beyond the native components of cells.

Core-Shell Architecture in Poly(3-hexylthiophene) Nanoparticles: Tuning of the Photophysical Properties for Enhanced Neuronal Photostimulation.

This study shows that entirely thiophene-based core@shell nanoparticles, in which the shell is made of the oxidized form of the core polymer (P3HT@PTDOx NPs), result in a type II interface at the particle surface. This enables the development of advanced photon nanotransducers with unique chemical-physical and biofunctional properties due to the core@shell nanoarchitecture. We demonstrate that P3HT@PTDOx NPs present a different spatial localization of the excitation energy with respect to the nonoxidized NPs, showing a prevalence of surface states as a result of a different alignment of the HOMO/LUMO energy levels between the core and shell. This allows for the efficient photostimulation of retinal neurons. Indeed, thanks to the stronger and longer-lived charge separation, P3HT@PTDOx NPs, administered subretinally in degenerate retinas from the blind Royal College of Surgeons rats, are more effective in photostimulation of inner retinal neurons than the gold standard P3HT NPs.

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.

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.

Sterilization of Semiconductive Nanomaterials: The Case of Water-Suspended Poly-3-Hexylthiophene Nanoparticles.

In this work, the feasibility of sterilizing a water suspension of poly-3-hexylthiophene nanoparticles (P3HT-NPs) is investigated using ionizing radiation, either γ-rays or high-energy electrons (e-beam). It is found that regardless of the irradiation source, the size, polydispersity, aggregation stability, and morphology of the NPs are not affected by the treatment. Furthermore, the impact of ionizing radiation on the physicochemical properties of NPs at different absorbed radiation doses (10-25 kGy) and dose rates (kGy time-1 ) is evaluated through different spectroscopic techniques. The results indicate that delivering a high dose of radiations (25 kGy) at a high dose rate, that is, kGy s-1 , as achieved by e-beam irradiation, preserves the characteristics of the polymeric NPs. Differently, the same radiation dose but delivered at a lower dose rate, that is, kGy h-1 , as attained by using a γ-source, can modify the physicochemical properties of the polymer. Sterility tests indicate that an absorbed dose of 10 kGy, delivered either with γ-rays or e-beam, is already sufficient for effective sterilization of the colloidal suspension and for reducing the endotoxin content. Finally, NPs irradiated at different doses, exhibit the same cytocompatibility and cell internalization characteristics in human neuroblastoma SH-SY5Y cells of NPs prepared under aseptic conditions.

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.

Thermodynamically versus Kinetically Controlled Self-Assembly of a Naphthalenediimide-Thiophene Derivative: From Crystalline, Fluorescent, n-Type Semiconducting 1D Needles to Nanofibers.

The control over aggregation pathways is a key requirement for present and future technologies, as it can provide access to a variety of sophisticated structures with unique functional properties. In this work, we demonstrate an unprecedented control over the supramolecular self-assembly of a semiconductive material, based on a naphthalenediimide core functionalized with phenyl-thiophene moieties at the imide termini, by trapping the molecules into different arrangements depending on the crystallization conditions. The control of the solvent evaporation rate enables the growth of highly elaborated hierarchical self-assembled structures: either in an energy-minimum thermodynamic state when the solvent is slowly evaporated forming needle-shaped crystals (polymorph α) or in a local energy-minimum state when the solvent is rapidly evaporated leading to the formation of nanofibers (polymorph ß). The exceptional persistence of the kinetically trapped ß form allowed the study and comparison of its characteristics with that of the stable α form, revealing the importance of molecular aggregation geometry in functional properties. Intriguingly, we found that compared to the thermodynamically stable α phase, characterized by a J-type aggregation, the ß phase exhibits (i) an unusual strong blue shift of the emission from the charge-transfer state responsible for the solid-state luminescent enhancement, (ii) a higher work function with a "rigid shift" of the electronic levels, as shown by Kelvin probe force microscopy and cyclic voltammetry measurements, and (iii) a superior field-effect transistor mobility in agreement with an H-type aggregation as indicated by X-ray analysis and theoretical calculations.

One- and two-photon absorption properties of quadrupolar thiophene-based dyes with acceptors of varying strengths.

The one-photon (1P) and two-photon (2P) absorption properties of three quadrupolar dyes, featuring thiophene as a donor and acceptors of varying strengths, are determined by a combination of experimental and computational methods employing the density functional theory (DFT). The emission shifts in different solvents are well reproduced by time-dependent DFT calculations with the linear response and state specific approaches in the framework of the polarizable continuum model. The calculations show that the energies of both 1P- and 2P-active states decrease with an increase of the strength of the acceptor. The 2P absorption cross-sections predicted by the response theory are accounted for by considering just one intermediate state (S1) in the sum-over-states formulation. For the chromophore featuring the stronger acceptor, the energetic positions of the 1P- and 2P-active states prevent the exploitation of the theoretically predicted very high 2P activity due to the competing 1P absorption into the S1 state.

Nanostructuring Iridium Complexes into Crystalline Phosphorescent Nanoparticles: Structural Characterization, Photophysics, and Biological Applications.

One of the key challenges in materials science is to control the properties of a material by directing its supramolecular arrangement. Here we show that iridium complexes, such as FIrpic, Ir-PPY, and Ir-MDQ, can be organized into crystalline and phosphorescent nanoparticles through the nanoprecipitation method, which allows thorough modification of their functional properties. Moreover, we found that it is possible to combine different iridium complexes into a single multicomponent nanostructure, thus creating nanoparticles whose photonic properties derive from the close spatial proximity of the electronic excited states of the different Ir complexes. The morphology of all nanoparticles was fully characterized by microscopic and spectroscopic techniques, and their ordered arrangement was assessed by X-ray diffraction (XRD) and high-resolution transmission electron microscopy (TEM) measurements. We demonstrate that the nanostructuring of the complexes influences their optical and redox properties-by promoting a fine-tuning of emission, photoluminescence quantum yield, excited state lifetime, HOMO/LUMO energy levels, and energy-transfer processes-as well as their interaction with living cells. Investigations on glioblastoma U-251 MG cells demonstrate that nanostructuring represents an effective tool to regulate the efficiency of cell loading, cell viability, colocalization, and penetration in 3D spheroids.

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.

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.

Semiconducting polymers are light nanotransducers in eyeless animals.

Current implant technology uses electrical signals at the electrode-neural interface. This rather invasive approach presents important issues in terms of performance, tolerability, and overall safety of the implants. Inducing light sensitivity in living organisms is an alternative method that provides groundbreaking opportunities in neuroscience. Optogenetics is a spectacular demonstration of this, yet is limited by the viral transfection of exogenous genetic material. We propose a nongenetic approach toward light control of biological functions in living animals. We show that nanoparticles based on poly(3-hexylthiophene) can be internalized in eyeless freshwater polyps and are fully biocompatible. Under light, the nanoparticles modify the light response of the animals, at two different levels: (i) they enhance the contraction events of the animal body, and (ii) they change the transcriptional activation of the opsin3-like gene. This suggests the establishment of a seamless and biomimetic interface between the living organism and the polymer nanoparticles that behave as light nanotransducers, coping with or amplifying the function of primitive photoreceptors.

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.