Cyanine Research Articles

Synthesis of Heptamethine Cyanines from Furfural Derivatives

Despite the widespread theranostic utilization of cyanine dyes (Cy7), their synthetic method is still limited with pyridine or cyclohexanone derivatives as starting materials. Herein, we report the synthesis of Cy7 from furfural derivatives. First, a one-pot reaction strategy is developed to solve the unstable problem of the Stenhouse salts. Second, a stepwise condensation strategy is exploited to regioselectively synthesize asymmetrical Cy7. The methodology possesses advantages, such as easy handling, high yield, wide substrate scopes, and good functional group tolerance.

Features of cyanine dyes aggregation on differently charged TiO2 matrices

Changing the polycondensation reaction route, positively and negatively charged TiO2 nanoparticles have been synthesized. On the base of the synthesized TiO2 nanoparticles, porous TiO2 films have been formed with different surface charges. J-aggregates of two anionic dyes were formed on positively charged TiO2 films, while J-aggregates of cationic dye was formed on negatively charged one. Combining J-aggregates with different J-band spectral positions, we have obtained two composites consisting of J-aggregates pairs with an effective excitation energy transfer between them. In both cases, simple mixing of J-aggregate in solution did not result in the composite formation and only sequential deposition of J-aggregate from corresponding solutions was successful. Interesting, not only charge alternating composite of cationic pseudoisocyanine J-aggregates and anionic thiacarbocyanine J-aggregates was realized, but also the same was done in the case of the composite of two anionic carbocyanine J-aggregates.

General Strategy To Improve the Photon Budget of Thiol-Conjugated Cyanine Dyes.

Maleimide-cysteine chemistry has been a routine practice for the site-specific labeling of fluorophores to proteins since the 1950s. This approach, however, cannot bring out the best photon budget of fluorophores. Here, we systematically measured the Cyanine3/5 dye conjugates via maleimide-thiol and amide linkages by counting the total emitted photons at the single-molecule level. While brightness and signal-to-noise ratios do not change significantly, dyes with thioether linkages exhibit more severe photobleaching than amide linkers. We then screened modern arylation-type bioconjugation strategies to alleviate this damage. Labeling thiols with phenyloxadiazole (POD) methyl sulfone, p-chloronitrobenzene, and fluorobenzene probes gave rise to electron-deficient aryl thioethers, effectively increasing the total emitted photons by 1.5-3 fold. Among the linkers, POD maintains labeling efficiency and specificity that are comparable to maleimide. Such an increase has proved to be universal among bulk and single-molecule assays, with or without triplet-state quenchers and oxygen scavengers, and on conformationally unrestricted or restricted cyanines. We demonstrated that cyanine-POD conjugates are general and superior fluorophores for thiol labeling in single-molecule FRET measurements of biomolecular conformational dynamics and in two-color STED nanoscopy using site-selectively labeled nanobodies. This work sheds light on the photobleaching mechanism of cyanines under single-molecule imaging while highlighting the interplay between the protein microenvironment, bioconjugation chemistry, and fluorophore photochemistry.

Mueller Matrix Polarimetry on Cyanine Dye J-Aggregates.

Cyanine dyes are known to form H- and J-aggregates in aqueous solutions. Here we show that the cyanine dye, S0271, assembles in water into vortex induced chiral J-aggregates. The chirality of the J-aggregates depends on the directionality of the vortex. This study utilised both conventional benchtop CD spectropolarimeters and Mueller matrix polarimetry. It was found that J-aggregates have real chirality alongside linear dichroism and linear and circular birefringence. We identify the factors that are key to the formation of metastable chiral J-aggregates and propose a mechanism for their assembly.

In Vivo Evaluation of a Gallium-68-Labeled Tumor-Tracking Cyanine Dye for Positron Emission Tomography/Near-Infrared Fluorescence Carcinoma Imaging, Image-Guided Surgery, and Photothermal Therapy.

Positron emission tomography (PET)/near-infrared fluorescence (NIRF) dual-modal imaging presents an enticing prospect for tumor diagnosis and surgical navigation. In this study, we developed a novel probe IR808-DOTA for tumor-targeted PET/NIRF imaging, image-guided surgery, and photothermal therapy. This construct had better water solubility and pharmacokinetics than IR808 and had similar photophysical properties, tumor targeting ability, and photothermal anticancer effect to IR808. By a simple labeling process, IR808-DOTA was labeled with gallium-68 and applied as a PET probe for tumor imaging in MCF-7 tumor xenografted mice. IR808-DOTA itself acted as an NIRF imaging agent in the following surgery for intraoperative navigation to aid surgeons in the delineation of tumor margins and visualizing sentinel lymph nodes to facilitate a more thorough tumor resection. Irradiation by laser, IR808-DOTA could prominently inhibit tumor growth in MCF-7 subcutaneous tumor model mice by directly ablating tumor cells, inhibiting tumor proliferation, and promoting tumor cell apoptosis. In summary, 68Ga-DOTA-IR808 could enable a convenient and user-friendly workflow for tumor imaging and guided surgery, and therefore, it may have great prospects for clinical translation as a PET/NIRF dual-modal probe.

Fluorinated Merophosphinine and Phosphinine Dyes: Synthesis and Evaluation of UV-Visible Light Absorption Properties

Merophosphinine and phosphinine dyes were reported as phosphorus atom equivalents of merocyanine and cyanine dyes in the 1960s. Although these dyes are excellent sensitizers for enhancing photographic performance, their development has been considerably retarded because of difficulties in the synthetic process. Previously, while investigating the reactivity of fluoroalkenes with various nucleophiles, we developed fluorinated merophosphinine and phosphinine scaffolds in a single reaction step; however, we did not investigate their ultraviolet-visible (UV-vis) light absorption properties further. Therefore, in this study, we synthesized fluorinated merophosphinines and phosphinine derivatives using triphenylphosphonium ylides with various R substituents [R = H, C6H5, 4-MeOC6H4, 4-PhC6H4, or C6F5] and octafluorocyclopentene to study their absorption characteristics. Thermogravimetric analyses (TGA) indicated that several synthesized derivatives were thermally stable at temperatures above 241 °C. Additionally, when studied in various solvents, the fluorinated merophosphinine and phosphinine dyes exhibited well-defined maximum absorption wavelengths (λabs) in the 364–375 and 420–474 nm ranges, respectively. Thus, fluorinated merophosphinine and phosphinine derivatives may serve as promising thermally stable functional dyes for future technical applications.

Spectral-fluorescent and photochemical study of 6,6′-di(benzoylamino)trimethine cyanine dyes in solutions as possible probes for DNA

Spectral-fluorescent and photochemical properties of trimethine cyanine dyes T-304, T-306, and T-307, having substituents in 6,6′-positions, in various organic solvents, in aqueous buffer solutions, in the presence of surfactants and ethanol additives, and the effect on these properties of addition of DNA have been studied. Strong aggregation of the dyes in aqueous and aqueous buffer solutions has been shown. This is due to increased hydrophobicity of the dyes, which makes it difficult to use them as spectral-fluorescent probes for DNA. In the presence of DNA, trimethine cyanines partially form highly fluorescent complexes of dye monomers with the biomolecule, with slight decomposition of the initial aggregates and the formation of aggregates on DNA molecules. The formation of different types of dye–DNA complexes, i.e., intercalation and binding in the DNA grooves, was modeled by molecular docking. Dye–DNA complexes were also studied by circular dichroism spectroscopy and by thermal dissociation of DNA. To reveal selectivity of the dyes, their interaction with human serum albumin was briefly studied. The presence of moderate concentrations of nonionic surfactants does not lead to a significant decomposition of aggregates, but leads to a biphasic dependence of the fluorescence intensity on the DNA concentration. At the same time, ethanol additives (15%) lead to a more or less linear concentration dependence of the fluorescence intensity, which makes it possible to use these dyes as fluorescent probes for DNA. The effective binding constants of the dyes to DNA and the limits of DNA detection using the dyes in the presence of 15% ethanol were estimated. Photoisomerization and generation of the triplet states of T-304, T-306, and T-307 have been also studied. Along with the fluorescence growth, complexation with DNA leads to an increase in the yield of the triplet states of the dyes. This creates a prerequisite for using the dyes in targeted PDT. In the presence of DNA, the decay kinetics of the triplet states are biexponential, which indicates different types of dye complexes with DNA. The rate constants of oxygen quenching of the triplet states of the dyes bound to DNA are significantly lower than the diffusion-controlled values (taking into account the spin-statistical factor), which is explained by the shielding effect on the triplet molecules in complexes with DNA. The data obtained show that dyes T-304, T-306 and T-307, with addition of 15% ethanol, can be used as possible fluorescent probes for DNA.

Interaction of Gold Nanoparticles with Cyanine Dyes in Cholesteric DNA Submicroparticles: Impact of the Way of Their Introduction into the System

Interaction of Gold Nanoparticles with Cyanine Dyes in Cholesteric DNA Submicroparticles: Impact of the Way of Their Introduction into the System

Low-Dose NIR-II Preclinical Bioimaging Using Liposome-Encapsulated Cyanine Dyes.

Fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700nm) provides a powerful tool for in vivo structural and functional imaging in deep tissue. However, the lack of biocompatible contrast agents with bright NIR-II emission has hindered its application in fundamental research and clinical trials. Herein, a liposome encapsulation strategy for generating ultrabright liposome-cyanine dyes by restricting dyes in the hydrophobic pockets of lipids and inhibiting the aggregation, as corroborated by computational modeling, is reported. Compared with free indocyanine green (ICG, an US Food and Drug Administration-approved cyanine dye), liposome-encapsulated ICG (S-Lipo-ICG) shows a 38.7-fold increase in NIR-II brightness and enables cerebrovascular imaging at only one-tenth dose over a long period (30min). By adjusting the excitation wavelength, two liposome-encapsulated cyanine dyes (S-Lipo-ICG and S-Lipo-FD1080) enable NIR-II dual-color imaging. Moreover, small tumor nodules (2-5mm) canbe successfully distinguished and removed with S-Lipo-ICG image-guided tumor surgery in rabbit models. This liposome encapsulation maintains the metabolic pathway of ICG, promising for clinical implementation.

Optimization Stability in Excited-State-Specific Variational Monte Carlo.

We investigate the issue of optimization stability in variance-based state-specific variational Monte Carlo, discussing the roles of the objective function, the complexity of wave function ansatz, the amount of sampling effort, and the choice of minimization algorithm. Using a small cyanine dye molecule as a test case, we systematically perform minimizations using variants of the linear method as both a standalone algorithm and in a hybrid combination with accelerated descent. We demonstrate that adaptive step control is crucial for maintaining the linear method's stability when optimizing complicated wave functions and that the hybrid method enjoys both greater stability and minimization performance.

Oxygen supplementation liposomes for rheumatoid arthritis treatment via synergistic phototherapy and repolarization of M1-to-M2 macrophages

Rheumatoid arthritis (RA) is one of the most common systemic autoimmune diseases, with no current, clinically effective treatment. Activation of pro-inflammatory M1 macrophages is known to play a vital role in the pathogenesis of RA. Accumulating evidence suggests that phototherapy, especially photodynamic therapy (PDT), could apply as a new therapeutic method for the treatment of RA. However, due to the anoxic environment of RA synovial tissues and the oxygen consumption required for PDT process, the phototherapy efficiency was always reduced. Moreover, surviving and newly invading macrophages will be induced to polarize towards the pro-inflammatory M1-type, resulting in a relapse of the disease. To compensate for these deficits, we designed and developed novel phototherapeutic liposomal nanoparticles (CyI&Hb/FA-LPs) by incorporating heavy atom modified, cyanine dyes (CyI) as photosensitizers, and loading hemoglobin (Hb) into the inner core for relieving the hypoxic microenvironments and repolarizing M1-to-M2 macrophages. Meanwhile, folic acid was attached to the liposome surface to achieve active targeting of M1 macrophages. In vitro and in vivo studies demonstrate that near infrared light irradiation of CyI&Hb/FA-LPs reduces M1 macrophage populations and reduces the expression of hypoxia-inducible factor-1α that drives the remaining M1 macrophages to transition to the anti-inflammatory M2 phenotype. Moreover, intra-articular treatment of CyI&Hb/FA-LPs in a RA mouse model inhibited synovial cell infiltration, and alleviated inflammation and pathological features in the affected joint. This study highlights the potential of CyI-based oxygenated liposomes as a new and versatile strategy for implementing RA targeting phototherapy and M1-to-M2 repolarization. The dual mode phototherapy and O2 supplementation agent appears to function synergistically by reducing M1 macrophage populations through direct killing and induced apoptosis, as well as by inducing M2 phenotype proliferation.

Probing DNA structural heterogeneity by identifying conformational subensembles of a bicovalently bound cyanine dye.

DNA is a re-configurable, biological information-storage unit, and much remains to be learned about its heterogeneous structural dynamics. For example, while it is known that molecular dyes templated onto DNA exhibit increased photostability, the mechanism by which the structural dynamics of DNA affect the dye photophysics remains unknown. Here, we use femtosecond, two-dimensional electronic spectroscopy measurements of a cyanine dye, Cy5, to probe local conformations in samples of single-stranded DNA (ssDNA-Cy5), double-stranded DNA (dsDNA-Cy5), and Holliday junction DNA (HJ-DNA-Cy5). A line shape analysis of the 2D spectra reveals a strong excitation-emission correlation present in only the dsDNA-Cy5 complex, which is a signature of inhomogeneous broadening. Molecular dynamics simulations support the conclusion that this inhomogeneous broadening arises from a nearly degenerate conformer found only in the dsDNA-Cy5 complex. These insights will support future studies on DNA's structural heterogeneity.

Trans-cis isomerization kinetics of cyanine dyes reports on the folding states of exogeneous RNA G-quadruplexes in live cells.

Guanine (G)-rich nucleic acids are prone to assemble into four-stranded structures, so-called G-quadruplexes. Abnormal GGGGCC repeat elongations, and in particular their folding states, are associated with amyotrophic lateral sclerosis and frontotemporal dementia. Due to methodological constraints however, most studies of G quadruplex structures are restricted to in vitro conditions. Evidence of how GGGGCC repeats form into G-quadruplexes in vivo is sparse. We devised a readout strategy, exploiting the sensitivity of trans-cis isomerization of cyanine dyes to local viscosity and sterical constraints. Thereby, folding states of cyanine-labeled RNA, and in particular G-quadruplexes, can be identified in a sensitive manner. The isomerization kinetics, monitored via fluorescence blinking generated upon transitions between a fluorescent trans isomer and a non-fluorescent cis isomer, was first characterized for RNA with GGGGCC repeats in aqueous solution using fluorescence correlation spectroscopy and transient state (TRAST) monitoring. With TRAST, monitoring the isomerization kinetics from how the average fluorescence intensity varies with laser excitation modulation characteristics, we could then detect folding states of fluorescently tagged RNA introduced into live cells.

Albumin-based nanosystem for dual-modality imaging-guided chem-phototherapy against immune-cold triple-negative breast cancer.

Triple-negative breast cancer is a highly aggressive and metastatic tumor; diagnosing it in the early stages is still difficult, and the prognosis for conventional radio-chemotherapy and immunotreatment is not promising due to cancer's immunosuppressive microenvironment. The utilization of protein-based nanosystem has proven to be effective in delivering agents with limited adverse effects, yet the combination of diagnosis and treatment remains a difficult challenge. This research took advantage of natural albumin and organic molecules to construct a self-assemble core-shell nanostructure combining with superparamagnetic iron oxide nanocrystals and heptamethine cyanine dye IR780 through non-covalent interactions. This nanocomposite successfully decreased the transverse relaxation time of the magnetic resonance hydrogen nucleus, resulting in outstanding T2 imaging, as well as emitting near-infrared II fluorescence, thereby the resulting dual-modality imaging tool was applied to improve diagnostic competency. It is noteworthy that the nanocomposites exhibited impressive enzyme-like catalytic and photothermal capabilities, resulting in a successful activation of the immune system to efficiently suppress distant metastatic lesions in vivo. Consequently, this nano-drug-based therapy could be an advantageous asset in reinforcing the immune system and hindering the growth and reappearance of the immune-cold breast cancer.

Usefulness of 4K-resolution Indocyanine Green Endoscope for the Removal of Spontaneous Intracerebral Hematomas.

Indocyanine green (ICG) is a cyanine dye useful for visualizing blood vessels; it has been developed for endoscopy and is used in skull base surgery. Endoscopy is widely used for hematoma removal after an intracerebral hemorrhage since it is minimally invasive and has a shorter operation time than craniotomy. However, with this technique the surgical field is limited and it is difficult to obtain an adequate orientation; thus, it is challenging to locate the bleeding point, and postoperative rebleeding has been reported. We performed intraoperative ICG near-infrared fluorescence imaging to locate the bleeding point. This purpose of this study was to evaluate the usefulness of ICG angiography during endoscopic hematoma removal in two patients, using two endoscope types and comparing their visualization of perforating branches during the procedure. ICG angiography was performed in two different cases of putaminal hemorrhage, using the SPIES NIR/ICG-System and IMAGE1 S Rubina (both KARL STORZ, Tuttlingen, Germany) at the intraoperative bleeding site. The intraoperative use of ICG allowed the clear visualization of the perforating branches and real-time confirmation of active bleeding. We could also distinguish an old hematoma from the active bleeding point. The IMAGE1 S Rubina has adequate brightness for contrast enhancement, allowing surgical manipulation simultaneously to the enhancement phase.ICG fluorescence angiography is useful to identify the damaged vessel and perform hemostasis. We expect other similar devices to be developed in the future, accompanied by flexible and thin rigid endoscopes.

Antibody-guided iodinated cyanine for near-IR photoimmunotherapy

Photoimmunotherapy (PIT) is a cancer treatment modality that synergistically combines immunotherapy and photodynamic treatment (PDT). Many current photosensitizers used in PDT and PIT applications such as porphyrins suffer from insufficient light absorption in the near-IR spectra region, which reduces their phototoxicity. Cyanine dyes exhibit much more pronounced absorption in the near-IR region but they are less explored for PDT and their antibody-guided conjugates have not yet been studied for PIT applications. In this work, for the first time a “cyanine dye — antibody” PIT conjugate is developed and studied. We synthesized novel, highly water soluble, near-IR, diiodinated heptamethine cyanine dye, 2ICy7, conjugated this dye with the breast cancer-specific monoclonal antibody trastuzumab, and investigated the obtained PIT conjugate in the mouse xenograft model to suppress tumor growth versus the parent, non-iodinated Cy7–Ab conjugate. The quantum yields of the singlet oxygen generation in aqueous media for the iodinated 2ICy7 dye and 2ICy7–Ab conjugate were found to be about 3-fold higher compared to the non-iodinated counterparts, Cy7 and Cy7–Ab. As a result, the 2ICy7–Ab conjugate suppressed tumor growth upon PIT four-fold more efficiently than Cy7–Ab, while the immunotherapeutic effect resulted only in 1.4-fold suppression.

Effect of Solubilizing Group on the Antibacterial Activity of Heptamethine Cyanine Photosensitizers.

Antibiotic resistance of pathogenic bacteria dictates the development of novel treatment modalities such as antimicrobial photodynamic therapy (APDT) utilizing organic dyes termed photosensitizers that exhibit a high cytotoxicity upon light irradiation. Most of the clinically approved photosensitizers are porphyrins that are poorly excitable in the therapeutic near-IR spectral range. In contrast, cyanine dyes function well in the near-IR region, but their phototoxicity, in general, is very low. The introduction of iodine atoms in the cyanine molecules was recently demonstrated to greatly increase their phototoxicity. Herein, we synthesized a series of the new iodinated heptamethine cyanine dyes (ICy7) containing various solubilizing moieties, i.e., negatively charged carboxylic (ICy7COOH) and sulfonic (ICy7SO3H) groups, positively charged triphenylphosphonium (ICy7PPh3), triethylammonium (ICy7NEt3) and amino (ICy7NH2) groups, and neutral amide (ICy7CONHPr) group. The effect of these substituents on the photodynamic eradication of Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) pathogens was studied. Cyanine dyes containing the amide and triphenylphosphonium groups were found to be the most efficient for eradication of the investigated bacteria. These dyes are effective at low concentrations of 0.05 µM (33 J/cm2) for S. aureus, 50 µM (200 J/cm2) for E. coli, and 5 µM (100 J/cm2) for P. aeruginosa and considered, therefore, promising photosensitizers for APDT applications. The innovation of the new photosensitizers consisted of a combination of the heavy-atom effect that increases singlet oxygen generation with the solubilizing group's effect improving cell uptake, and with effective near-IR excitation. Such a combination helped to noticeably increase the APDT efficacy and should pave the way for the development of more advanced photosensitizers for clinical use.

Selenium-Substituted Monomethine Cyanine Dyes as Selective G-Quadruplex Spectroscopic Probes with Theranostic Potential.

The binding interactions of six ligands, neutral and monocationic asymmetric monomethine cyanine dyes comprising benzoselenazolyl moiety with duplex DNA and RNA and G-quadruplex structures were evaluated using fluorescence, UV/Vis (thermal melting) and circular dichroism (CD) spectroscopy. The main objective was to assess the impact of different substituents (methyl vs. sulfopropyl vs. thiopropyl/thioethyl) on the nitrogen atom of the benzothiazolyl chromophore on various nucleic acid structures. The monomethine cyanine dyes with methyl substituents showed a 100-fold selectivity for G-quadruplex versus duplex DNA. Study results indicate that cyanines bind with G-quadruplex via end π-π stacking interactions and possible additional interactions with nucleobases/phosphate backbone of grooves or loop bases. Cyanine with thioethyl substituent distinguishes duplex DNA and RNA and G-quadruplex structures by distinctly varying ICD signals. Furthermore, cell viability assay reveals the submicromolar activity of cyanines with methyl substituents against all tested human cancer cell lines. Confocal microscopy analysis shows preferential accumulation of cyanines with sulfopropyl and thioethyl substituents in mitochondria and indicates localization of cyanines with methyl in nucleus, particularly nucleolus. This confirms the potential of examined cyanines as theranostic agents, possessing both fluorescent properties and cell viability inhibitory effect.

Near-Infrared-Emitting Meso-Substituted Heptamethine Cyanine Dyes: From the Synthesis and Photophysics to Their Use in Bioimaging

Heptamethine cyanine dyes were synthesized in good yields by the reaction between quaternary indoles and a pentamethinic salt, under mild reaction conditions minimizing photooxidation. These compounds were used as precursors to prepare meso-substituted derivatives. The cyanine dye precursors presented UV-Vis absorption, related to fully allowed electronic transitions and fluorescence emission in the NIR region, without any evidence of aggregation in both ground and excited states. The substitution at the meso position showed a fundamental role in their photophysics, with the main absorption in the green-orange region related to the monomeric species. Moreover, the excited state photophysics presented emission profiles dependent on the excitation wavelengths, complicating the correlation of spectroscopy and structure. Density Functional Theory and OO-SCS-MP2 calculations under different solvation conditions revealed the heavy impact of conjugation effects on ground and excited states’ geometries and electronic configurations of these compounds. Finally, the observed photophysical features of the meso-substituted heptamethine cyanine dyes were successfully used to explore their application as fluorescent probes in biological media, allowing stable staining in live and fixed cells.

Enhanced Tumor Uptake and Retention of Cyanine Dye-Albumin Complex for Tumor-Targeted Imaging and Phototherapy.

Heptamethine cyanine dyes are widely used for in vivo near-infrared (NIR) fluorescence imaging and NIR laser-induced cancer phototherapy due to their good optical properties. Since most of heptamethine cyanine dyes available commercially are highly hydrophobic, they can usually be used for in vivo applications after formation of complexes with blood plasma proteins, especially serum albumin, to increase aqueous solubility. The complex formation between cyanine dyes and albumin improves the chemical stability and optical property of the hydrophobic cyanine dyes, which is the bottom of their practical use. In this study, the complexes between three different heptamethine cyanine dyes, namely clinically available indocyanine green (ICG), commercially available IR-786 and zwitterionic ZW800-Cl, and bovine serum albumin (BSA), were prepared to explore the effect of cyanine dyes on their tumor uptake and retention. Among the three complexes, IR-786©BSA exhibited increased tumor accumulation with prolonged tumor retention, compared to other complexes. Moreover, IR-786 bound to BSA played an important role in tumor growth suppression due to its cytotoxicity. To achieve complete tumor ablation, the tumor targeted by IR-786©BSA was further exposed to 808 nm laser irradiation for effective photothermal cancer treatment.