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Combined photodynamic and photothermal therapy (PDT and PTT) can achieve more superior therapeutic effects than the sole mode by maximizing the photon utilization, but there remains a significant challenge in the development of related single-molecule photosensitizers (PSs), particularly those with type I photosensitization. In this study, self-assembly of squaraine dyes (SQs) is shown to be a promising strategy for designing PSs for combined type I PDT and PTT, and a supramolecular PS (TPE-SQ7) has been successfully developed through subtle molecular design of an indolenine SQ, which can self-assemble into highly ordered H-aggregates in aqueous solution as well as nanoparticles (NPs). In contrast to the typical quenching effect of H-aggregates on reactive oxygen species (ROS) generation, our results encouragingly manifest that H-aggregates can enhance type I ROS ( • OH) generation by facilitating the intersystem crossing process while maintaining a high PTT performance. Consequently, TPE-SQ7 NPs with ordered H-aggregates not only exhibit superior combined therapeutic efficacy than the well-known PS (Ce6) under both normoxic and hypoxic conditions but also have excellent biosafety, making them have important application prospects in tumor phototherapy and antibacterial fields. This study not only proves that the supramolecular self-assembly of SQs is an effective strategy toward high-performance PSs for combined type I PDT and PTT but also provides a different understanding of the effect of H-aggregates on the PDT performance.

The viscosity that ensures the controlled diffusion of biomolecules in cells is a crucial biophysical parameter. Consequently, fluorescent probes capable of reporting viscosity variations are valuable tools in bioimaging. In this field, red-shifted probes are essential, as the widely used and gold standard probe remains green-emitting molecular rotors based on BODIPY. Here, we demonstrate that pyrrolyl squaraines, red-emissive fluorophores, exhibit high sensitivity over a wide viscosity range from 30 to 4890 mPa·s. Upon alkylation of the pyrrole moieties, the probes improve their sensitivity to viscosity through an enhanced twisted intramolecular charge transfer phenomenon. We utilized this scaffold to develop a plasma membrane probe, pSQ-PM, that efficiently stains the plasma membrane in a fluorogenic manner. Using fluorescence lifetime imaging, pSQ-PM enabled efficient sensing of viscosity variations in the plasma membrane under various conditions and in different cell lines (HeLa, U2OS, and NIH/3T3). Moreover, upon incubation, pSQ-PM stained the membrane of intracellular vesicles and suggested that the lysosomal membranes displayed enhanced fluidity.

Due to the wide application of reporter gene-related visible/NIR-I bioluminescent imaging, multiplexed fluorescence imaging across visible/NIR-I/NIR-II has excellent potential in biomedical research. However, in vivo multiplexed imaging applications across those regions have rarely been reported due to the lack of proper fluorophores. Herein, nine squaraine dyes, which exhibit diverse adsorption and emission wavelengths, were synthesized. Among them, water-soluble SQ 710-5k and SQ 905 were found to have significant absorption differences, which allowed the tumor and lymph nodes to be identified. Then, for the first time, six-channel multiplexed fluorescence imaging across visible/NIR-I/II was achieved by coordination with reporter gene-related bioluminescent phosphors. Additional research revealed that SQ 710-5k exhibited higher-quality blood vessels and tumor imaging in NIR-II. H-aggregates SQ 905 demonstrated a high photothermal conversion efficiency for photothermal therapy. This study proposed an approach to creating small molecular dyes that coordinate with reporter gene-related bioluminescent phosphors for six-color fluorescence imaging.

An organelle-selective vision provides insights into the physiological response of plants and crops to environmental stresses in sustainable agriculture ecosystems. Biological applications often require two-photon excited fluorophores with low phototoxicity, high brightness, deep penetration, and tuneable cell entry. We obtained three aniline-based squaraines (SQs) tuned from hydrophobic to hydrophilic characteristics by modifying terminal pendant groups and substituents, and investigated their steady-state absorption and far-red-emitting fluorescence properties. The SQs exhibited two-photon absorption (2PA) ranging from 750 to 870 nm within the first biological spectral window; their structure-property relationships, corresponding to the 2PA cross sections ( δ 2PA ), and structure differences were demonstrated. The maximum δ 2PA value was ∼1220 GM at 800 nm for hydrophilic SQ3. Distinct biological staining efficiency and selective SQ bioimaging were evaluated utilizing the onion epidermal cell model. Contrary to the hydrophobic SQ1 results in the onion epidermal cell wall, amphiphilic SQ2 tagged the vacuole and nucleus and SQ3 tagged the vacuole. Distinguishable staining profiles in the roots and leaves were achieved. We believe that this study is the first to demonstrate distinct visualisation efficiency induced by the structure differences of two-photon excited SQs. Our results can help establish the versatile roles of novel near-infrared-emitting SQs in biological applications.

Squaraine dyes are a family of compounds known for their relevant photophysical and photochemical properties potentially useful as photosensitizing agents. Since pyridines have been introduced into the skeleton of several families of compounds to enhance their pharmacological activity, and this approach had not yet been performed on squaraines, novel dyes derived from benz[e]indole functionalized with picolyl- and dipicolylamine and N-ethyl and -hexyl chains were designed and synthesized. After being fully characterized, their interaction with human albumin was in vitro and in silico evaluated. Dyes were further assessed for their phototoxicity activity, and the most interesting ones were studied regarding cell localization and induction of morphological cell changes, genotoxicity, apoptosis and cell cycle arrest. The molecules with N-ethyl chains showed the greatest in vitro light-dependent cytotoxic effects, particularly the zwitterionic squaraine dye and the one bearing a single pyridine unit, which also exhibited a more significant interaction with human albumin. Phenotypically, the cells incubated with these squaraines became smaller and rounded after irradiation, the effects varying with the tested concentration. Genotoxic effects were observed even without irradiation, being more evident for the N-ethyl picolylamine-derived dye. The fluorescence emitted by Rhodamine 123 largely coincided with that emitted by the dyes, suggesting that they are found preferentially in mitochondria. After irradiation, an increase in the subG1 population was verified by propidium iodide-staining analysis by flow cytometry, indicative of cell death by apoptosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Three barbiturate squaraine dyes derived from indolenine or benzothiazole, with different barbituric acid derivatives were prepared, characterized and photophysically evaluated by standard spectroscopic methods. As expectable for squaraines, these dyes showed narrow and intense absorption and emission bands in the Vis/NIR region. The interaction of synthesized dyes with bovine and human serum albumins (BSA and HSA) was also evaluated in phosphate buffer (PB). The results revealed that upon the addition of BSA or HSA the complex dye-protein emit more fluorescence, and the emission intensity is directly proportional to the concentration of protein used (0-3.5 µM). The titration tests allowed to calculate the binding constants, in an order of magnitude of 10 4 -10 6 M, as well as the limits of detection and quantification in the nanomolar tens range. All dyes showed a good response to the interaction with both proteins, but the most pronounced envisioning their use as protein labeling was observed for the squaraine dye derived from the indolenine with a 1,3-dimethylbarbituric acid moiety. The molecular docking studies revealed the existence of a binding between the compounds and four sites on the HSA molecule, where one of these four locations is a new binding site with which this series of dye interacts., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Dual-modular imaging approaches combining near-infrared (NIR) fluorescence (FLI) and photoacoustic imaging (PAI) require suitable contrast agents to produce dual-modular signals. Although nanoparticles have been used to develop PAI agents, small molecule-based imaging agents have not been extensively studied, highlighting the need to design new fluorophores with an enhanced multifunctional ability. Thus, in this study, we designed a novel squaraine (SQ)-based dye and reported its rational preparation and conjugation with a cancer targeting peptide. Specifically, benzoindole-derived SQ (BSQ) showed strong absorption and fluorescence properties at above 650 nm under aqueous conditions, with a maximum absorption and emission at 665 and 680 nm, respectively. Moreover, PA signal scanning experiments revealed a maximum signal intensity in the range 680-700 nm. BSQ was also conjugated with cyclic arginine-glycine-aspartic acid (cRGD) to improve its active targeting ability for the α v β 3 integrin, which is overexpressed in various cancer and angiogenic cells. A series of in vitro , in vivo , and ex vivo FLI studies showed that the cRGD conjugated BSQ (BSQ-RGD 2 ) successfully stained and targeted α v β 3 integrin-overexpressing tumor cells and xenografts, which were clearly visualized by FLI and PAI. Therefore, BSQ-RGD 2 can successfully be applied to dual-modular imaging of the specific biomarker in living animals.

Highly stable symmetric and asymmetric squaraine fluorophores have been synthesized featuring an internal salt bridge between a quaternary ammonium cation and the central oxycyclobutenolate ring of the chromophore. Some of our newly synthesized symmetric and asymmetric compounds display increased molar absorptivity, quantum yield in serum, and thermal/photochemical stability over previously reported squaraine-based dyes. Consequently, both classes show great promise in resurfacing the normal environment-labile squaraine dyes as novel imaging agents and scaffolds for fluorescence sensing. Furthermore, incorporating a covalent attachment point away from the conjugated system allows for biological tagging applications without disturbing the optimum optical characteristics of the newly designed fluorophore.

Mucin-type glycoproteins are the principal components of mucus which cover all the mucosal surfaces of the human body. The mucus and mucins are essential mediators of the innate immune system, however in the last decades mucins have been identified even as an important class of cancer biomarkers. Luminogenic materials with fluorescence turn-on behavior are becoming promising materials because of their advantages of label free, relatively inexpensive and simple to use properties for biological detection and imaging. Squaraines are luminogens characterized by high fluorescence in organic media but poor emission in aqueous environments due to their tendency to self-aggregate. Herein we investigate the interaction between porcine gastric mucin (PGM) and several squaraines in aqueous media. While squaraine dyes showed low fluorescence intensity and quantum yield in water, as a result of the formation of aggregates, an enhancement of fluorescence up to 45-fold was achieved when PGM was added. PGM was detected in a linear range of 10-300 μg/mL with a limit of detection of 800 ng/mL. The assay was used to quantify mucin in diluted human serum samples and recoveries of 94.9-116.2% were achieved. To the best of our knowledge, this is the easiest and convenient method for mucin detection in the reported literature., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A novel and simple squaraine-based fluorescent chemosensor SQ-BH bearing the benzoylhydrazine moiety was developed for the highly sensitive and selective detection of Al 3+ in methanol-water mixture. The chemosensing behaviors of SQ-BH and its binding interaction with Al 3+ were explored by various spectroscopic analyses. The reversibility of Al 3+ recognition process was investigated using EDTA. The results of experiments and DFT/TDDFT calculations revealed that the chemosensor SQ-BH obeyed a turn on mechanism which was associated with the inhibited photoinduced electron transfer (PET), the enhanced intramolecular charge transfer (ICT) and the activated chelation enhanced fluorescence (CHEF). Furthermore, the fluorescent chemosensor SQ-BH whose excellent biocompatibility was confirmed by a standard MTT assay could be used to detect Al 3+ in living cells, indicating its potential application value in biological fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier B.V.)

Squaraine dyes are a class of organic dyes with strong and narrow absorption bands in the near-infrared. Despite high molar absorptivities and fluorescence quantum yields, these dyes have been less explored than other dye scaffolds due to their susceptibility to nucleophilic attack. Recent strategies in probe design including encapsulation, conjugation to biomolecules, and new synthetic modifications have seen squaraine dyes emerging into the forefront of biomedical imaging and other applications. Herein, we provide a concise overview of (1) the synthesis of symmetrical and unsymmetrical squaraine dyes, (2) the relationship between structure and photophysical properties of squaraine dyes, and (3) current applications of squaraine dyes in the literature. Given the recent successes at overcoming the limitations of squaraine dyes, they show high potential in biological imaging, in photodynamic and photothermal therapies, and as molecular sensors.

Various squaraine dyes have been developed for biological imaging. Nevertheless, squaraine dyes with emission in the second window (NIR-II, 1000-1700 nm) have few reports largely due to the short of a simple and universal design strategy. In this contribution, molecular engineering strategy is explored to develop squaraine dyes with NIR-II emission. First, NIR-I squaraine dye SQ2 is constructed by the ethyl-grafted 1,8-naphtholactam as donor units and square acid as acceptor unit in a donor-acceptor-donor (D-A-D) structure. To red-shift the fluorescence emission into NIR-II window, malonitrile, as a forceful electron-withdrawing group, is introduced to strengthen square acid acceptor. As a result, the fluorescence spectrum of acceptor-engineered squaraine dye SQ1 exhibits a significant red-shift into NIR-II window. To translate NIR-II fluorophores SQ1 into effective theranostic agents, fibronectin-targeting SQ1 nanoprobe was constructed and showed excellent NIR-II imaging performance in angiography and tumor imaging, including lung metastatic foci in deep tissue. Furthermore, SQ1 nanoprobe can be used for photoacoustic imaging and photothermal ablation of tumors. This research demonstrates that the donor-acceptor engineering strategy is feasible and effective to develop NIR-II squaraine dyes.

Bovine serum albumin (BSA)-squaraine supramolecular adducts with aggregation-induced emission (AIE) properties were prepared and characterized by spectroscopic methods. While squaraine dyes showed a very low fluorescence quantum yield in water, a great enhancement in the fluorescence of the aggregated BSA adducts was achieved due to the abnormal aggregation-induced emission properties of squaraines. The adducts formation was studied from a kinetic point of view showing unexpected structure-properties relationships., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Squaraine dyes have recently attracted interest as potential second generation photosensitizers for photodynamic therapy. Several cationic aminosquaraine dyes bearing benzoselenazole terminal nuclei were synthezised and their cytotoxic activity was tested against four different human tumor cell lines - breast (MCF-7), non-small cell lung (NCI-H460), cervical (HeLa) and hepatocellular (HepG2) carcinomas - and against a non-tumor porcine liver primary cell line (PLP2), both in the absence of light and under irradiation. All dyes, which displayed strong absorption within the phototherapeutic window, were found to exhibit photodynamic activity and were shown to be, in most cases, more cytotoxic, both in the dark and upon irradiation, than their benzothiazole analogues.

Abnormal level of proteins is proved to be associated with diseases. Thus, protein sensing is helpful for clinical diagnosis and therapy. However, there is a great variety of protein species and relatively low concentration of each protein in complicated biological systems including other nonprotein biomolecules. Therefore, it remains challenging to develop an effective method for detecting protein with high selectivity and sensitivity. Herein, a new self-assembly method based on a robust dye SQSS of which two squaraine molecules were conjugated through disulfide bond was developed for highly selective and sensitive detection of serum albumin (SA) in aqueous solution and live cells. SQSS can self-assemble into "compact" aggregates, offering "inert" disulfide group and very low background fluorescence through the combination of aggregation quenching and homogeneous fluorescence resonance energy transfer (homoFRET) quenching. The response of SQSS to SA undergoes two cascade stages. At the first stage, SA drives the compact assemblies of SQSS to form loose ones with fast speed (30 s) through noncovalent interaction, resulting in the enhancement of fluorescence to some extent. In this loose assembly state, the disulfide bond in SQSS is reactive. At the second stage, the Cys34 in SA slowly induced further disassembly through covalent binding with reactive disulfide bond, resulting in fluorescence further increasing and SQSS labeling to SA that cannot be displaced by site binding ligands of SA. The self-assemblies of SQSS can selectively detect SA with continuous near-infrared (NIR) turn-on fluorescence response in 100% aqueous buffer solution. In addition, SQSS showed the potential application of imaging SA in living cells. On the other hand, the loose assembly state of SQSS was also achieved in aqueous solution with 20% CH 3 CN. In this media, thiol-containing glutathione (GSH) caused the disassembly of SQSS with turn-on fluorescence response through interaction with disulfide bond. SQSS can selectively recognize GSH over other amino acids even in the presence of other sulfhydryl amino acids. As a proof-of-concept method, the molecular self-assembly through multisteps interactions would provide an ideal strategy for detection and live-cell imaging of biorelated molecules with high selectivity and signal-to-noise ratio.

The photophysical properties of a deep-red fluorescent squaraine dye can be improved by encapsulating it within a tetralactam macrocycle. Three new tetralactams are described with different substituents (methyl, methoxy, methylenedioxy) on the macrocycle aromatic sidewalls. The capability of each tetralactam to encapsulate a squaraine dye in chloroform solution was determined experimentally using absorption, fluorescence, and NMR spectroscopy. Two of the tetralactams were found to thread a squaraine dye with association constants on the order of 106 M-1, while a third macrocycle exhibited no squaraine affinity. An X-ray crystal structure of the third tetralactam showed that the substituents sterically blocked squaraine association. Of the two tetralactams that encapsulate a squaraine, one induces an increase in squaraine fluorescence quantum yield, while the other quenches the squaraine fluorescence. The results suggest that these new squaraine binding systems will be useful for biological imaging and diagnostics applications.

We introduce a novel strategy to enhance the fluorescence brightness of organic-molecule-based nanoparticles in the second near-infrared window (NIR-II, 1000-1700 nm) by fabricating J-aggregate nanoparticles SQP-NPs(J). Our prepared J-aggregate nanoparticles SQP-NPs(J) show an emission maximum near 1100 nm, and the emission intensity is 4.8-fold higher than that of H-aggregate SQP-NPs(H). In addition, SQP-NPs(J) can be used for NIR-II imaging guided photothermal therapy on MCF-7 tumor-bearing mice due to the fact that SQP-NPs(J) have highly effective photothermal properties, which are significant for precise tumor diagnostics and treatments.

Chloroquine was among the first of several effective drug treatments against malaria until the onset of chloroquine resistance. In light of diminished clinical efficacy of chloroquine as an antimalarial therapeutic, there is potential in efforts to adapt chloroquine for other clinical applications, such as in combination therapies and in diagnostics. In this context, we designed and synthesized a novel asymmetrical squaraine dye coupled with chloroquine (SQR1-CQ). In this study, SQR1-CQ was used to label live Plasmodium falciparum ( P. falciparum ) parasite cultures of varying sensitivities towards chloroquine. SQR1-CQ positively stained ring, mature trophozoite and schizont stages of both chloroquine⁻sensitive and chloroquine⁻resistant P. falciparum strains. In addition, SQR1-CQ exhibited significantly higher fluorescence, when compared to the commercial chloroquine-BODIPY (borondipyrromethene) conjugate CQ-BODIPY. We also achieved successful SQR1-CQ labelling of P. falciparum directly on thin blood smear preparations. Drug efficacy experiments measuring half-maximal inhibitory concentration (IC50) showed lower concentration of effective inhibition against resistant strain K1 by SQR1-CQ compared to conventional chloroquine. Taken together, the versatile and highly fluorescent labelling capability of SQR1-CQ and promising preliminary IC50 findings makes it a great candidate for further development as diagnostic tool with drug efficacy against chloroquine-resistant P. falciparum .

New fluorescent molecular probes, which can selectively target specific cell surface receptors, are needed for microscopy, in vivo imaging, and image guided surgery. The preparation of multivalent probes using standard synthetic chemistry can be a laborious process due to low reaction yields caused by steric effects. In this study, fluorescent molecular probes were prepared by a programmed non-covalent pre-assembly process that used a near-infrared fluorescent squaraine dye to thread a macrocycle bearing a cyclic arginine-glycine-aspartate peptide antagonist (cRGDfK) as a cancer targeting unit. Cell microscopy studies using OVCAR-4 (ovarian cancer) and A549 (lung cancer) cells that express high levels of the integrin αvβ3 or αvβ5 receptors, respectively, revealed a multivalent cell targeting effect. That is, there was comparatively more cell uptake of a pre-assembled probe equipped with two copies of the cRGDfK antagonist than a pre-assembled probe with only one appended cRGDfK antagonist. The remarkably high photostability and low phototoxicity of these near-infrared probes allowed for acquisition of long-term fluorescence movies showing endosome trafficking in living cells. In vivo near-infrared fluorescence imaging experiments compared the biodistribution of a targeted and untargeted probe in a xenograft mouse tumor model. The average tumor-to-muscle ratio for the pre-assembled targeted probe was 3.6 which matches the tumor targeting performance reported for analogous cRGDfK-based probes that were prepared entirely by covalent synthesis. The capability to excite these pre-assembled near-infrared fluorescent probes with blue or deep-red excitation light makes it possible to determine if a target site is located superficially or buried in tissue, a probe performance feature that is likely to be very helpful for eventual applications such as fluorescence guided surgery., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new family of fluorescent thiophene and thienothiophene-containing squaraine dyes is described with tunable wavelengths that cover the absorption/emission range of 600⁻800 nm. The deep-red and near-infrared fluorescent compounds were easily prepared by simple synthesis and purification methods. Spectral studies showed that each squaraine was rapidly encapsulated by a tetralactam macrocycle, with nanomolar affinity in water, to produce a threaded supramolecular complex with high chemical stability, increased fluorescence quantum yield, and decreased fluorescence quenching upon dye self-aggregation. Energy transfer within the supramolecular complex permitted multiplex emission. That is, two separate dyes with fluorescence emission bands that match the popular Cy5 and Cy7 channels, could be simultaneously excited with a beam of 375 nm light. A broad range of practical applications is envisioned in healthcare diagnostics, microscopy, molecular imaging, and fluorescence-guided surgery.

Squaraine dyes have excellent photostability with intensive absorption and strong fluorescence in the near-infrared (NIR) region. However, they display a strong tendency to aggregate in aqueous media because of their poor water solubility, often causing fluorescence quenching that severely limits their in vivo applications, especially for detecting or imaging diagnostic enzymes. In this work, an oligo(ethylene glycol)-functionalized squaraine fluorophore has been developed as an NIR-fluorescent probe that can detect and image the activities of a diagnostic enzyme (leucine aminopeptidase) both in vitro and in vivo. The probe shows near-infrared absorption and emission, a low detection limit (0.61 ng/mL), relatively good aqueous solubility, high selectivity, and little toxicity. In addition, the probe herein was successfully used to track endogenous leucine aminopeptidase both in vitro and in vivo with a nude-mouse model.

A highly sensitive and selective probe for Vancomycin (Van) in aqueous and serum samples is developed in this study. The probe is based on a triad consisting of a near-infrared squaraine dye (Seta-640) conjugated to two anthraquinone molecules via Lys-d-Ala-d-Ala peptides. In the absence of Van, the close proximity and efficient electron-transfer from the excited Seta-640 dye to anthraquinone result in significant fluorescence quenching of the dye ("off"-state). When Van is added, the antibiotic molecules bind with high affinity to the -d-Ala-d-Ala ligands in a 2:1 stoichiometry (Van:triad), resulting in fluorescence recovery that is as high as 30 times ("on"-state). Even though bound Van enhances the fluorescence by reducing the rate of (intrinsic) polarity-induced nonradiative decay process, this effect plays only a minor role. Instead, the main reason behind the observed fluorescence recovery after drug binding is the effective inhibition of electron-transfer; plausibly arising from a steric-induced lengthening of the spatial separation between electron donor and acceptor. The probe has detection limits of 7.0 and 96.9 nM in buffer and human serum, respectively, operates in the clinically relevant range, is insensitive to Van crystalline degradation product (CDP-1), and is easy to operate by using a commonly available fluorescence spectrometer.

The design of fluorescent molecular platforms capable of responding to multiple analytes is a topic of great interest. Herein, the use of a Zn 2+ -complexed unsymmetrical squaraine dye, Sq-Zn 2+ , as a chemical platform for recognizing structurally distinct analytes is reported. The squaraine ring is substituted on one side with a dipicolylamine unit, which acts as the metal ion receptor, whereas the other part of the molecule carries a dibutylaniline moiety, which is an electron donor. The molecular system is unique because it can respond specifically to different types of analytes, namely, atmospheric carbon dioxide, cyclic phosphates, and picric acid. Moreover, the interaction of these analytes can be monitored colorimetrically and fluorimetrically, which favors both qualitative and quantitative analyses. The distinct response towards cyclic and linear phosphates, as well as the selective response towards picric acid, among the various nitroaromatic compounds was achieved with sensitivity at the ppm level. The flexible coordination offered by Zn 2+ plays a significant role in the discrimination of these analytes with high specificity. Dye Sq-Zn 2+ introduced herein is a single-molecule construct that can be used for the selective and sensitive response towards analytes of environmental and biological relevance., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The synthesis of several aminosquaraine cationic dyes displaying strong absorption within the so-called phototherapeutic window (650-850nm) is described. Their cytotoxicity, under dark and illuminated conditions, was tested against several human tumor cell lines (breast, lung, cervical and hepatocellular carcinomas) and non-tumor porcine liver primary cells. All compounds showed to inhibit the growth of the tumor cells upon irradiation more than in the absence of light, in more or less extension, clearly exhibiting photodynamic activity. The photosensitizing ability against some cell lines, together with the low toxicity for the non-tumor primary PLP2 cells displayed by some of the compounds synthetized, turns them into potential candidates as photosensitizers for PDT., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

New unsymmetrical aminosquarylium cyanine dyes were synthesized and their potential as photosensitizers evaluated. New dyes, derived from benzothiazole and quinoline, were prepared by nucleophilic substitution of the corresponding O-methylated, the key intermediate that was obtained by methylation with CF 3 SO 3 CH 3 of the related zwitterionic unsymmetrical dye, with ammonia and methylamine, respectively. All three news dyes herein described displayed intense and narrow bands in the Vis/NIR region (693-714nm) and their singlet oxygen formation quantum yields ranged from 0.03 to 0.05. In vitro toxicity, in Caco-2 and HepG2 cells, indicated that dark toxicity was absent for concentrations up to 5µM (for the less active dye) or up to 1µM (for the two more active dyes). The three dyes present potential as photosensitizers, differing in irradiation conditions and period of incubation in the presence of irradiated dye. The less active dye needs a longer irradiation period to exhibit phototoxicity which is only evident after longer period of contact with cells (24h). However, the remaining two more active dyes produce higher phototoxicity, even at shorter incubation periods (1h), with shorter irradiation time (7min). Although in different extents, these dyes show promising in vitro results as photosensitizers., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

G-quadruplexes (G4s) are peculiar DNA or RNA tertiary structures that are involved in the regulation of many biological events within mammalian cells, bacteria, and viruses. Although their role as versatile therapeutic targets has been emphasized for 35 years, G4 selectivity over ubiquitous double-stranded DNA/RNA, as well as G4 differentiation by small molecules, still remains challenging. Here, a new amphiphilic dicyanovinyl-substituted squaraine, SQgl, is reported to act as an NIR fluorescent light-up probe discriminating an extensive panel of parallel G4s while it is non-fluorescent in the aggregated state. The squaraine can form an unconventional sandwich π-complex binding two quadruplexes, which leads to a strongly fluorescent (Φ F =0.61) supramolecular architecture. SQgl is highly selective against non-quadruplex and non-parallel G4 sequences without altering their topology, as desired for applications in selective in vivo high-resolution imaging and theranostics., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Direct monitoring of apoptotic progression is a major step forward for the early assessment of therapeutic efficacy of certain treatments and the accurate evaluation of the spread of a disease. Here, the regulatory role of glutathione (GSH) is explored as a potential biomarker for tracking apoptosis. For this purpose, a near- infrared (NIR) squaraine dye is introduced that is capable of sensing GSH in a ratiometric manner by switching its emission from NIR (690 nm) to visible region (560 nm). The favorable biocompatible attributes of the probe facilitated the real-time monitoring of apoptotic process in line with the conventional apoptotic assay. Furthermore, the robust nature of the probe was utilized for the quantitative estimation of GSH during different stages of apoptosis. Through this study, an easy and reliable method of assaying apoptosis is demonstrated, which can provide valuable insights in translational clinical research., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Novel glycoconjugated squaraine dyes were synthesized, which exhibited excellent internalization in tumor cells and are potent fluorogenic imaging probes for tumor selective optical imaging.

A conceptually new type of enzymatic cleavage assay is reported that utilizes in situ supramolecular capture of the fluorescent product. A squaraine-derived substrate with large blocking groups at each end of its structure cannot be threaded by a tetralactam macrocycle until the blocking groups are removed by enzyme cleavage. A prototype design responds to viral neuraminidase, an indicator of influenza infection, and also measures susceptibility of the sample to neuraminidase inhibitor drugs. The substrate structure incorporates three key features: (a) a bis(4-amino-3-hydroxyphenyl)squaraine core with bright deep-red fluorescence and excellent photostability, (b) an N-methyl group at each end of the squaraine core that ensures fast macrocycle threading kinetics, and (c) sialic acid blocking groups that prevent macrocycle threading until they are removed by viral neuraminidase. The enzyme assay can be conducted in aqueous solution where dramatic colorimetric and fluorescence changes are easily observed by the naked eye. Alternatively, affinity capture beads coated with macrocycle can be used to immobilize the liberated squaraine and enable a range of heterogeneous analysis options. With further optimization, this new type of neuraminidase assay may be useful in a point of care clinic to rapidly diagnose influenza infection and also determine which of the approved antiviral inhibitor drugs is likely to be the most effective treatment for an individual patient. The assay design is generalizable and can be readily modified to monitor virtually any type of enzyme-catalyzed cleavage reaction.

Efficient and site-specific delivery of anticancer drugs to tumors is important in the development of effective cancer chemotherapy. As an undecapeptide of the tachykinin neuropeptide family, the substance P (SP)/neurokinin-1 receptor (NK1R) system has been identified as a promising ligand-receptor pair in tumor-specific drug delivery. However, the rational design of suitable theranostic agents with high drug loading capacity and tumor targeting for cancer patients remains a great challenge. Herein, we report a dendritic strategy that utilizes the two amine functionalities of lysine to create branch points that allow conjugation of the anticancer drug 5-fluorouracil (5-FU) to the tumor-targeting ligand substance P, along with an additional near-infrared (NIR) squaraine dye, to construct a theranostic dendritic agent, P-FU 4. This cytotoxic theranostic agent, containing four carboxyl-modified 5-FU molecules, has several desirable advantages: i) the ability to self-assemble into nanoparticles; ii) enhanced cytotoxicity with high drug loading capacity (16%) and a specific receptor-targeted interaction with NK1R through the SP moiety; and iii) a high NIR squaraine fluorescence efficiency due to the specific dendron isolation, avoiding aggregation-mediated quenching. As demonstrated in this report, the cytotoxic activity of P-FU 4 is dose-dependent against the tested cancer cells. The improved drug loading capacity with dendritic branching distinctly enhanced cytotoxicity to tumor cells but had little effect on the viability of normal cells. P-FU 4 was preferentially taken up by tumor cells through a receptor-mediated interaction, which was monitored by effective NIR fluorescence with high tissue penetration. Studies using a mouse model revealed that P-FU 4 can significantly inhibit tumor progression, with a tumor-inhibition rate of 60.2%. The receptor-targeted cytotoxic dendritic theranostic agent is highly preferable to standard chemotherapeutic treatments and decreases the negative side effects of medications on healthy cells, which establishes its utility in drug delivery and cancer chemotherapy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Optical activity of hierarchical supramolecular assemblies based on organic dyes would create multiple functional architectures. In this work, three kinds of silica nanoparticles with or without functional groups were synthesized. For the first time, silica nanoparticles can induce positively charged squaraine (SQ) to aggregate to form supramolecular assemblies. Adenosine-5'-triphosphate (ATP) as building blocks was absorbed on the surface of silica nanoparticles through metal-anion coordination and electrostatic interactions, in which the aggregates of SQ was transferred to monomer. The thickness being composed of ATP and SQ on the outside of nanoparticles is about 5 nm. These supramolecular assemblies showed selective turn-on fluorescence response to ATP in near infrared (NIR) region over other ions through metal-anion coordination and electrostatic interactions. These functional silica nanoparticles possessing many advantages provide proof-of-principle "seed crystals" for construction of supramolecular assemblies and platforms for sensing with facile performance.

The cyanine platforms including cyanine, hemicyanine, and squaraine are good candidates for developing chemosensors because of their excellent photophysical properties, outstanding biocompatibility, and low toxicity to living systems. A huge amount of research work involving chemosensors based on the cyanine platforms has emerged in recent years. This review focuses on the development from 2000 to 2015, in which cyanine, hemicyanine, and squaraine sensors will be separately summarized. In each section, a systematization according to the type of detection mechanism is established. The basic principles about the design of the chemosensors and their applications as bioimaging agents are clearly discussed. In addition, we emphasize the advances that have been made in improving the detection performance through incorporation of the chemosensors into nanoparticles.

Using multiple interactions, a simple self-assembly based on a Zn(ii) coordination compound and squaraine () demonstrated a selective turn-on fluorescence response to ATP in the near infrared (NIR) region. More importantly, the self-assembly has been successfully applied to ATP imaging in the mitochondria of the gastric cancer cell line SGC-7901 and monitoring of level fluctuation of ATP during the mitotic period.

One of the major challenges in current cancer therapy is to maximize therapeutic effect and evaluate tumor progression under the scheduled treatment protocol. To address these challenges, we synthesized the cytotoxic peptide (KLAKLAK)2 (named KLAK) conjugated amphiphilic poly(β-thioester)s copolymers (H-P-K) composed of reactive oxygen species (ROS) sensitive backbones and hydrophilic polyethylene glycol (PEG) side chains. H-P-K could self-assemble into micelle-like nanoparticles by hydrophobic interaction with copolymer backbones as cores and PEG and KLAK as shells. The assembled polymer-peptide nanoparticles remarkably improved cellular internalization and accumulation of therapeutic KLAK in cells. Compared to free KLAK peptide, the antitumor activity of H-P-K was significantly enhanced up to ∼400 times, suggesting the effectiveness of the nanoscaled polymer-peptide conjugation as biopharmaceuticals. The higher antitumor activity of nanoparticles was attributed to the efficient disruption of mitochondrial membranes and subsequent excessive ROS production in cells. To realize the ROS monitoring and treatment evaluation, we encapsulated squaraine (SQ) dyes as built-in reporters in ROS-sensitive H-P-K micelles. The overgenerated ROS around mitochondria stimulated the swelling of nanoparticles and subsequent release of SQ, which formed H-aggregates and significantly increased the photoacoustic (PA) signal. We believed that this self-assembled polymer-peptide nanotherapeutics incorporating built-in reporters has great potential for high antitumor performance and in situ treatment evaluation.

We report the synthesis and characterization of a series of symmetrical indolenine-based squaraine dyes along with the evaluation of their singlet oxygen generation efficiency. The photodynamic activity of these new photosensitizers has been evaluated on a human tumor fibrosarcoma (HT-1080) cell line. The cytotoxicity increased over time and is induced by the photoactivation of bromo (Br-C4) and iodio (I-C4) long carbon chain squaraine dyes and the consequent increase in reactive oxygen species (ROS) production (p < 0.001), which leads to necrosis 6 h after treatment. Induction of cytochrome c release, DNA damage and up-regulation of GPX1, NQO1 and SOD2 mRNA gene expression after PDT were investigated., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The electronic absorption spectra, ground-state geometries and electronic structures of symmetric and asymmetric squaraine dyes (SQD1-SQD4) were investigated using density functional theory (DFT) and time-dependent (TD-DFT) density functional theory at the B3LYP/6-311++G** level. The calculated ground-state geometries reveal pronounced conjugation in these dyes. Long-range corrected time dependent density functionals Perdew, Burke and Ernzerhof (PBE, PBE1PBE (PBE0)), and the exchange functional of Tao, Perdew, Staroverov, and Scuseria (TPSSh) with 6-311++G** basis set were employed to examine optical absorption properties. In an extensive comparison between the optical data and DFT benchmark calculations, the BEP functional with 6-311++G** basis set was found to be the most appropriate in describing the electronic absorption spectra. The calculated energy values of lowest unoccupied molecular orbitals (LUMO) were 3.41, 3.19, 3.38 and 3.23 eV for SQD1, SQD2, SQD3, and SQD4, respectively. These values lie above the LUMO energy (-4.26 eV) of the conduction band of TiO₂ nanoparticles indicating possible electron injection from the excited dyes to the conduction band of the TiO₂ in dye-sensitized solar cells (DSSCs). Also, aromaticity computation for these dyes are in good agreement with the data obtained optically and geometrically with SQD4 as the highest aromatic structure. Based on the optimized molecular geometries, relative positions of the frontier orbitals, and the absorption maxima, we propose that these dyes are suitable components of photovoltaic DSSC devices.

In this work, a squaraine dye CS5 was characterized and evaluated for its potential in photodynamic therapy. The studies were performed in ethanol and also in a powdered biopolymer, in this case chitosan. Ground state absorption, absolute fluorescence quantum yields, fluorescence lifetimes, and transient absorption were determined in order to evaluate the advantage of adsorbing the dye onto a biopolymer. Several concentrations of the dye, adsorbed onto chitosan, were prepared in order to evaluate the concentration effect on the photophysical parameters under study. A remarkable increase in the fluorescence quantum yield and lifetimes was detected when compared with the dye in solution. Also, a very clear dependence of the fluorescence quantum yield on the concentration range was found. A lifetime distribution analysis of these systems fluorescence evidenced the entrapment of the dye onto the chitosan environment with a monoexponential decay which corresponds to the monomer emission in slightly different environments. The transient absorption spectrum was obtained without sensitization indicating the existence of a triplet state which takes special importance in the generation of phototoxic species namely singlet oxygen. The subcellular localization of a photosensitizer is critical for efficient photoinduced cell death, in this way, colocalization studies were performed within HeLa cell line (human cervical carcinoma) through confocal microscopy. Toxicity in the dark and phototoxicity of CS5 were also evaluated for the same cellular model.

A regio-selectively squaraine (SQ)-bounded cellulose derivative (4) with a degree of substitution of SQ (DSSQ) of 0.55 was prepared from 6-O-(4-methoxytrityl) cellulose (1) by three reaction steps in 77% total yield. Lauryl SQ carboxylate (8) was also prepared as a reference sample. The photostability of SQ moieties of compound 4 in CHCl3 was not improved when compared with SQ-COOH (7), but that of SQ moiety of compound 8 was improved unexpectedly. The Langmuir-Blodgett monolayer films 4B and 8B on an indium tin oxide (ITO) electrode were successfully prepared from compounds 4 and 8 by a vertical dipping method, respectively. The films 4B and 8B showed photocurrent generation performances in the region of 550-680 nm. The quantum yield at 650 nm of film 4B was higher than that of film 8B. These results showed that the cellulose backbone of compound 4 acts as an effective scaffold for the good photocurrent generation performances and that compound 4 was a promising complementary material of the porphyrin-bounded cellulose derivatives (for photocurrent generation at 400-420 nm) for effective utilization of solar light, as well as the phthalocyanine-bounded cellulose derivatives (for photocurrent generation at 650-720 nm)., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Using dimethylsulfoxonium methylide as the methylene transfer reagent, 2a,8b-dihydrobenzo[b]cyclobute[d]pyran-3-ones were converted into 2,2'-biphenol derivatives as major products and dihydrodibenzofurans as minor products. The reaction mechanism was extrapolated from a deuteration experiment with CD2=S(O)(CD3)2.

A rational design of squaraine dyes with lipophilic and zwitterionic groups tunes cell entry, allowing for selective far-red/near-infrared imaging of plasma membrane vs. endoplasmic reticulum. They exhibit up to 110-fold fluorescence enhancement in biomembranes and enable cellular imaging at 1 nM concentration, which make them the brightest membrane probes to date.

With an objective to develop β-amyloid destabilizing agents, we have investigated the interactions of a few water-soluble near-infrared (NIR)-absorbing squaraine dyes 1-3 with lysozyme and its amyloid aggregates through photophysical and biophysical techniques. These dyes exhibited strong interactions with lysozyme and β-amyloids in addition to serum albumins as evidenced by the absorption and emission changes. The interactions were found to be spontaneous with association constant values in the range of approximately 10(4)-10(5)  m(-1), as confirmed through half-reciprocal analysis and isothermal calorimetric measurements. Uniquely, such effective interactions of the dyes have led to the complete disassembly of the β-amyloid fibrillar structures to form spherical particles approximately 350 nm in size, as confirmed through photophysical, thioflavin assay, circular dichroism (CD), atomic force microscopy (AFM), TEM, and selected-area electron diffraction (SAED) techniques. These results demonstrate that the squaraine dyes 1-3 under investigation act as effective protein-labelling and destabilizing agents of the protein amyloidogenesis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A macrocyclic tetralactam is threaded by a complementary squaraine dye that is flanked by two polyethylene glycol chains to produce a pseudorotaxane complex with favorable near-infrared fluorescence properties. The association thermodynamics and kinetics were measured for a homologous series of squaraines with different N-alkyl substituents at both ends of the dye. The results show that subtle changes in substituent steric size have profound effects on threading kinetics without greatly altering the very high association constant.

The color of squaraine solution shows a "fingerprint" change upon addition of different surfactants. A cross-responsive sensing array based on a small molecular probe was applied to discriminate surfactants with 100% confidence limits. Furthermore, the probe can selectively detect sodium dodecyl sulfate (SDS) with turn-on fluorescence response.

A macrocyclic tetralactam host is threaded by a highly fluorescent squaraine dye that is flanked by two polyethylene glycol (PEG) chains with nanomolar dissociation constants in water. Furthermore, the rates of bimolecular association are very fast with k(on) ≈ 10(6)-10(7) M(-1) s(-1). The association is effective under cell culture conditions and produces large changes in dye optical properties including turn-on near-infrared fluorescence that can be imaged using cell microscopy. Association constants in water are ∼1000 times higher than those in organic solvents and strongly enthalpically favored at 27 °C. The threading rate is hardly affected by the length of the PEG chains that flank the squaraine dye. For example, macrocycle threading by a dye conjugate with two appended PEG2000 chains is only three times slower than threading by a conjugate with triethylene glycol chains that are 20 times shorter. The results are a promising advance toward synthetic mimics of streptavidin/biotin.

Combined near-infrared (NIR) fluorescence and photoacoustic imaging techniques present promising capabilities for noninvasive visualization of biological structures. Development of bimodal noninvasive optical imaging approaches by combining NIR fluorescence and photoacoustic tomography demands suitable NIR-active exogenous contrast agents. If the aggregation and photobleaching are prevented, squaraine dyes are ideal candidates for fluorescence and photoacoustic imaging. Herein, we report rational selection, preparation, and micelle encapsulation of an NIR-absorbing squaraine dye (D1) for in vivo fluorescence and photoacoustic bimodal imaging. D1 was encapsulated inside micelles constructed from a biocompatible nonionic surfactant (Pluoronic F-127) to obtain D1-encapsulated micelles (D1(micelle)) in aqueous conditions. The micelle encapsulation retains both the photophysical features and chemical stability of D1. D1(micelle) exhibits high photostability and low cytotoxicity in biological conditions. Unique properties of D1(micelle) in the NIR window of 800-900 nm enable the development of a squaraine-based exogenous contrast agent for fluorescence and photoacoustic bimodal imaging above 820 nm. In vivo imaging using D1(micelle), as demonstrated by fluorescence and photoacoustic tomography experiments in live mice, shows contrast-enhanced deep tissue imaging capability. The usage of D1(micelle) proven by preclinical experiments in rodents reveals its excellent applicability for NIR fluorescence and photoacoustic bimodal imaging.

Combining a squaraine (S) and a BODIPY (B) chromophore in a heterodimer (SB) and two heterotrimers (BSB and SBS) by alkyne bridges leads to the formation of coupled oscillators whose fluorescence properties are superior compared to the parent squaraine chromophore. The lowest energy absorption and emission properties of these superchromophores are mainly governed by the squaraine part and are shifted by more than 1000 cm(-1) to the red by excitonic interaction between the squaraine and the BODIPY dye. Employing polarization-dependent transient absorption and fluorescence upconversion measurements, we could prove that the lowest energy absorption in SB and BSB is caused by a single excitonic state but by two for SBS. Despite the spectral red-shift of their lowest absorption band, the fluorescence quantum yields increase for SB and BSB compared to the parent squaraine chromophore SQA. This is caused by intensity borrowing from the BODIPY states, which increases the squared transition moments of the lowest energy band dramatically by 29% for SB and 63% for BSB compared to SQA. Thereby, exciton coupling leads to a substantial enhancement of fluorescence quantum yield by 26% for SB and by 46% for BSB and shifts the emission from the red into the near-infrared. In this way, the BODIPY-squaraine conjugates combine the best properties of each class of dye. Thus, exciton coupling in heterodimers and -trimers is a valuable alternative to tuning fluorescence properties by, e.g., attaching substituents to chromophores.

Herein, we show that a far-red arylidene-squaraine dye is stable against nucleophiles, in contrast to arene-squaraines. Owing to the fluorescence enhancement in apolar media together with high brightness and photostability, this dye was successfully applied to detect the oxytocin G protein-coupled receptor and monitor its internalization in living cells.

This article describes the design and development of squaraine-based semiconducting polymer dots (Pdots) that show large Stokes shifts and narrow-band emissions in the near-infrared (NIR) region. Fluorescent copolymers containing fluorene and squaraine units were synthesized and used as precursors for preparing the Pdots, where exciton diffusion and likely through-bond energy transfer led to highly bright and narrow-band NIR emissions. The resulting Pdots exhibit the emission full width at half-maximum of ∼36 nm, which is ∼2 times narrower than those of inorganic quantum dots in the same wavelength region (∼66 nm for Qdot705). The squaraine-based Pdots show a high fluorescence quantum yield (QY) of 0.30 and a large Stokes shift of ∼340 nm. Single-particle analysis indicates that the average per-particle brightness of the Pdots is ∼6 times higher than that of Qdot705. We demonstrate bioconjugation of the squaraine Pdots and employ the Pdot bioconjugates in flow cytometry and cellular imaging applications. Our results suggest that the narrow bandwidth, high QY, and large Stokes shift are promising for multiplexed biological detections.

Polarity-sensitive fluorogenic dyes raised considerable attention because they can turn on their fluorescence after binding to biological targets, allowing background-free imaging. However, their brightness is limited, and they do not operate in the far-red region. Here, we present a new concept of fluorogenic dye based on a squaraine dimer that unfolds on changing environment from aqueous to organic and thus turns on its fluorescence. In aqueous media, all three newly synthesized dimers displayed a short wavelength band characteristic of an H-aggregate that was practically nonfluorescent, whereas in organic media, they displayed a strong fluorescence similar to that of the squaraine monomer. For the best dimer, which contained a pegylated squaraine core, we obtained a very high turn-on response (organic vs aqueous) up to 82-fold. Time-resolved studies confirmed the presence of nonfluorescent intramolecular H-aggregates that increased with the water content. To apply these fluorogenic dimers for targeted imaging, we grafted them to carbetocin, a ligand of the oxytocin G protein-coupled receptor. A strong receptor-specific signal was observed for all three conjugates at nanomolar concentrations. The probe derived from the core-pegylated squaraine showed the highest specificity to the target receptor together with minimal nonspecific binding to serum and lipid membranes. The obtained dimers can be considered as the brightest polarity-sensitive fluorogenic molecules reported to date, having ∼660,000 M(-1) cm(-1) extinction coefficient and up to 40% quantum yield, whereas far-red operation region enables both in vitro and in vivo applications. The proposed concept can be extended to other dye families and other membrane receptors, opening the route to new ultrabright fluorogenic dyes.

Squaraine dyes have attracted significant attention in many areas of daily life from biomedical imaging to semiconducting materials. Moreover, these dyes are used as photoactive materials in the field of solar cells. In the present study, we investigated the structural, electronic, photophysical, and charge transport properties of six benzothiazole-based squaraine dyes (Cis-SQ1-Cis-SQ3 and Trans-SQ1-Trans-SQ3). The effect of electron donating (-OCH3) and electron withdrawing (-COOH) groups was investigated intensively. Ground state geometry and frequency calculations were performed by applying density functional theory (DFT) at B3LYP/6-31G** level of theory. Absorption spectra were computed in chloroform at the time-dependent DFT/B3LYP/6-31G** level of theory. The driving force of electron injection (ΔG (inject)), relative driving force of electron injection (ΔG r (inject)), electronic coupling constants (|VRP|) and light harvesting efficiency (LHE) of all six compounds were calculated and compared with previously studied sensitizers. The ΔG (inject), ΔG r (inject) and |VRP| of all six compounds revealed that these sensitizers would be efficient dye-sensitized solar cell materials. Cis/Trans-SQ3 exhibited superior LHE as compared to other derivatives. The Cis/Trans geometric effect was studied and discussed with regard to electro-optical and charge transport properties.