Pentamethine Cyanine Dyes Research Articles

Azaindole-based asymmetric pentamethine cyanine dye for mitochondrial pH detection and near-infrared ratiometric fluorescence imaging of mitophagy

Azaindole-based asymmetric pentamethine cyanine dye for mitochondrial pH detection and near-infrared ratiometric fluorescence imaging of mitophagy

An antioxidative-enhanced endoplasmic reticulum-targeted cyanine dye for efficient tumor immunotherapy

Excessive generation of reactive oxygen species (ROS) triggers intense endoplasmic reticulum (ER) stress, emerging as a key mechanism for arousing autoimmune therapy. Therefore, the development of an ER-targeted ROS generator is crucial for advancing self-immunotherapy. However, despite a clear demand for ER-targeting ROS generators, the high-efficiency agents are still limited. In this study, the pentamethine cyanine dye (Cy5) is used as a parent structure to explore the efficient ER-targeted immune activator. Three variants (PCy5-H, PCy5-Cl, and PCy5-2Cl) were synthesized by introducing benzene, chlorobenzene, and dichlorobenzene structures at the meso-position of the Cy5 scaffold. The electron-withdrawing groups (EWGs) effectively reduce the electron density on the conjugate chain, facilitating the intersystem crossing (ISC) process and enhancing the antioxidant capability. Consequently, the antioxidant properties and ROS generation capacity of PCy5-2Cl were enhanced. Furthermore, encapsulating PCy5-2Cl into liposomes significantly improves its retention at the tumor site, inducing robust ER stress. Upon accumulation at the ER sites in tumor cells, PCy5-2Cl nanoparticles induced strong ER stress under near-infrared (NIR) light irradiation. Exposure to necrotic fragments reverses the immunosuppressive microenvironment within the tumor, ultimately promoting dendritic cell maturation and infiltration of cytotoxic T lymphocytes (CTLs). As a result, significant inhibition is observed in both primary and distant tumors, underscoring the efficacy of this EWGs modification strategy for enhancing tumor immunotherapy.

Simultaneous Docking of Antiviral Drugs and Cyanine Dyes with Proteins Using Multiple Ligand Approach

The protein-based nanosystems for targeted drug delivery of a wide array of substances, ranging from small drugs and therapeutic proteins to nucleic acids and genes, attract increasing attention due to their biocompatibility and biodegradability, extraordinary binding capacity for different ligands, accessibility from natural sources, effective drug protection and gentle encapsulation conditions. Due to the multitude of binding pockets and functional groups on the protein surface, these nanocarriers seem to be highly efficient multifunctional nanotheranostic systems that could incorporate both a therapeutic drug and a visualizing agent. This integration serves multiple purposes, including the regulation of drug release, monitoring the alterations at the target site in response to treatment, and offering crucial insights into the efficacy of the intervention in its early stages. The development of these advanced nanosystems necessitates a thorough comprehension of the potential interactions within these intricate systems. In the present study we assessed the potential of six trimethine and seven pentamethine cyanine dyes to serve as visualizing agents in the drug-protein-dye systems which include functionally significant proteins (cytochrome c, serum albumin, lysozyme and insulin and four antiviral drugs, viz. favipiravir, molnupiravir, nirmatrelvir and ritonavir. The ternary systems with the highest dye-protein surface shape complementarity were established for all groups of the examined cyanine dyes. The influence of the cyanine dye structure on the stability of the drug-protein-dye complexes was assessed. The obtained results indicate that the dye-protein affinity is not solely dependent on the length of the polymethine chain. It was found that the most prospective drug delivery systems containing the trimethines and pentamethines as visualizing agents are AK5-6-, AK5-8- and AK3-11-drug-albumin complexes.

Efficient synthesis and characterization of meso-substituted bis(benzothiazolium) pentamethine cyanine dyes and investigation of the impact of meso-substitution on water solubility and the structural rigidity to improve photophysical properties and fluorescent brightness

To achieve structurally rigid pentamethine cyanine dyes with worthful photophysical properties for biological applications, five series of symmetrical meso-substituted bis(benzothiazolium) pentamethine cyanine dyes were synthesized via an efficient and simple triethylamine-promoted approach involved condensation of various N-substituted benzothiazolium salts with preformed β-substituted vinamidinium salts in high yield and purity without the nontrivial chromatographic separation. The chemical structures of the synthesized dyes were confirmed by HPLC-MS, 1H NMR, 13C NMR, and FTIR techniques. The photophysical properties of the new dyes were investigated in methanol, recognizing that the dyes demonstrate excellent fluorescent brightness. The solutions of the dyes exhibited maximum absorption and fluorescence emission in the range of 600–650 nm with high molar absorption coefficients (around 1-3 × 105 dm3 mol−1 cm−1) and 610–670 nm with remarkable fluorescence quantum yield, respectively. In addition, the photostabilities and photoluminescent stabilities of the synthetic dyes were studied in methanol, revealing their outstanding stability and resistance to photodecomposition. Examining the photochemical behavior (UV light) of the dyes in organic solvents with different polarities verified the main role of various meso substitutions on the rigidity of molecular structures and cis-trans isomerization to improve the photophysical properties. The presence of the pyridinium substituent rings into the polymethine chain makes the dyes rigid and more water-soluble because of their bulky form and positive charge, respectively.

Synthesis and biological evaluation of theranostic Trastuzumab–SN38 conjugate for Near-IR fluorescence imaging and targeted therapy of HER2+ breast cancer

Here, we report on the design, synthesis, and biological evaluation of a new theranostic antibody drug conjugate (ADC), Cy5-Ab-SS-SN38, that consists of the HER2-specific antibody trastuzumab (Ab) connected to the near infrared (NIR) pentamethine cyanine dye Cy5 and SN38, which is a bioactive metabolite of the anticancer drug irinotecan. SN38 is bound to an antibody through a glutathione-responsive self-immolative disulfide carbamate linker. For the first time, we explored this linker in ADC and found that it to reduce the drug release rate, which is important for safe drug delivery. The developed ADC exhibited specific accumulation and nanomolar anti-breast cancer activity on HER2-positive (HER2+) cell lines but no effect on HER2-. Animals treated with this ADC exhibited good tolerance. In vivo studies have shown that the ADC had good targeting ability for HER2+ tumors with much higher anticancer potency than trastuzumab itself or a mixture of trastuzumab with SN38. Side-by-side HER2+/HER2-xenograft at the 10 mg/kg dose exhibited specific accumulation and reduction of HER2+ tumor but not accumulation or growth inhibition of HER2-counterpart. The self-immolative disulfide linker implemented in this study was proven to be successful, broadening its utilization with other antibodies for targeted anticancer therapy in general. We believe that the theranostic ADCs comprising the glutathione-responsive self-immolative disulfide carbamate linker are applicable for the treatment and fluorescent monitoring of malignancies and anticancer drug delivery.

Single-molecule characterization of a bright and photostable deep-red fluorescent squaraine-figure-eight (SF8) dye

Squaraine Figure Eight (SF8) dyes are a unique class of deep-red fluorescent dyes with self-threaded molecular architecture that provides structural rigidity while simultaneously encapsulating and protecting the emissive fluorochrome. Previous cell microscopy and bulk phase studies of SF8 dyes indicated order of magnitude enhancements in photostability over conventional pentamethine cyanine dyes such as Cy5. Studies conducted at the single molecule level now reveal that these ensemble level enhancements carry over to the single molecule level in terms of enhanced emission quantum yields, longer times to photobleaching, and enhanced total photon yields. When compared to Cy5, the SF8-based dye SF8(D4)2 possesses a three-fold larger single molecule emission quantum yield, exhibits order of magnitude longer average times before photobleaching, and exhibits twenty times larger photon yields. Additional features such as water solubility, fluorochrome encapsulation to protect it against nucleophilic attack, and selective biomarker targeting capability make SF8-based dyes promising candidates for biological labeling and microscopy applications and single molecule tracking.

Ratiometric Fluorescence Monitoring of Antibody-Guided Drug Delivery to Cancer Cells.

Ratiometric measurements utilizing two independent fluorescence signals from a dual-dye molecular system help to improve the detection sensitivity and quantification of many analytical, bioanalytical, and pharmaceutical assays, including drug delivery monitoring. Nevertheless, these dual-dye conjugates have never been utilized for ratiometric monitoring of antibody (Ab)-guided targeted drug delivery (TDD). Here, we report for the first time on the new, dual-dye TDD system, Cy5s-Ab-Flu-Aza, comprising the switchable fluorescein-based dye (Flu) linked to the anticancer drug azatoxin (Aza), reference pentamethine cyanine dye (Cy5s), and Her2-specific humanized monoclonal Trastuzumab (Herceptin) antibody. The ability of ratiometric fluorescence monitoring of drug release was demonstrated with this model system in vitro in the example of the human breast cancer SKBR3 cell line overexpressing Her2 receptors. The proposed approach for designing ratiometric, antibody-guided TDD systems, where a "drug-switchable dye" conjugate and a reference dye are independently linked to an antibody, can be expanded to other drugs, dyes, and antibodies. Replacement of the green-emitting dye Flu, which was found not detectable in vivo, with a longer-wavelength (red or near-IR) switchable fluorophore should enable quantification of drug release in the body.

Excited state relaxation in cationic pentamethine cyanines studied by time-resolved spectroscopy

In this work, we examined the nature and pathways of electronic excitation relaxation at low temperature in cationic pentamethine pyrylocyanine dye, which in the ground state is characterized by symmetric and non-symmetric localization of cationic charge on the carbon chain between the terminal groups. The carbon chain in such symmetric and non-symmetric charge localizations is close to polymethine and polyene conformations, respectively. The effect of symmetry breaking during the two-way transition polymethine – polyene in the optically excited state on the electronic properties of dyes is considered. Combined quantum-chemical calculations and time-resolved spectral measurements allowed us to characterize the symmetric and non-symmetric structures in the ground and excited states with specific peaks of transitions and lifetimes. The features of the polymethine structure of the dye are attributed to large-radius polaron formation, whereas for the polyene structures the model of excitons formed on the double bonds of the carbon chain can be used to analyze the relaxation pathways. The energy diagram model for relaxation pathways of optically excited electronic states has been proposed. The proposed model can be characteristic for pentamethine cyanine dyes with the symmetric and non-symmetric distribution of cationic charge at the carbon chain between the terminal groups in the ground and excited states.

Synthesis of pH-sensitive benzothiazole cyanine dye derivatives containing a pyridine moiety at the meso position

Four benzothiazole-derived pentamethine cyanine dyes were synthesized with a pyridine moiety at the meso-position of the methine chain with a reaction yield of 89–93%. These dyes were prepared with high purity in a three-step synthesis as prospective pH-responsive probes. Each compound absorbed and fluoresced between 630 and 670 nm within the far-red range of the visible spectrum with high molar absorptivity values calculated between 134,910 L mol −1 cm−1 and 163,816 L mol −1 cm−1 and quantum yields between 9% and 17%. When a 20 μM solution of dye derivatives are exposed to hydrochloric acid in the range of pH 5–6 in ethanol, all the dyes experienced a blueshift in absorbance and emission as well as a decrease in spectral intensity that enables them to operate as pH probes. These properties were further validated via computational studies showing pyridine's influence on each molecules' spectral properties, and pathways for electron delocalization in the presence and absence of acid were proposed. © 2021 Elsevier Science. All rights reserved.

Dual-dye systems comprising activatable fluorescein dye and hydrophobic or hydrophilic Cy5 reference fluorophore for ratiometric drug delivery monitoring

Ratiometric fluorescence detection in biological samples in vitro and in vivo mitigates several issues in conventional single-signal measurements such as light absorption and scattering, dye photobleaching, and instrumental parameters. Herein, two new systems for ratiometric monitoring of drug delivery are synthesized and investigated. These systems comprise a non-fluorescent fluorescein-based dye, Flu, bound to the anticancer drug, azatoxin (Aza), through a hydrolytically cleavable acrylate ester linker and either hydrophobic (Cy5h) or hydrophilic (Cy5s) pentamethine cyanine dye providing a red-light reference signal. Upon drug release, the green Flu fluorescence notably increases enabling ratiometric tracking of drug release events. The spectral properties of the developed conjugates and fluorescently monitored drug release rates are investigated in biologically relevant media with respect of the Cy5 dye hydrophilicity.

Fluorous Soluble Cyanine Dyes for Visualizing Perfluorocarbons in Living Systems.

The bioorthogonal nature of perfluorocarbons provides a unique platform for introducing dynamic nano- and microdroplets into cells and organisms. To monitor the localization and deformation of the droplets, fluorous soluble fluorophores that are compatible with standard fluorescent protein markers and applicable to cells, tissues, and small organisms are necessary. Here, we introduce fluorous cyanine dyes that represent the most red-shifted fluorous soluble fluorophores to date. We study the effect of covalently appended fluorous tags on the cyanine scaffold and evaluate the changes in photophysical properties imparted by the fluorous phase. Ultimately, we showcase the utility of the fluorous soluble pentamethine cyanine dye for tracking the localization of perfluorocarbon nanoemulsions in macrophage cells and for measurements of mechanical forces in multicellular spheroids and zebrafish embryonic tissues. These studies demonstrate that the red-shifted cyanine dyes offer spectral flexibility in multiplexed imaging experiments and enhanced precision in force measurements.

High-Yielding Water-Soluble Asymmetric Cyanine Dyes for Labeling Applications

A simple and efficient microwave-assisted synthesis of asymmetric pentamethine cyanine dyes with various functional groups was developed, which allows high-yielding results. The synthesized dyes are modifiable and suitable for single-molecule imaging in biological and medical sciences by application of click chemistry or classic esterification and amidation.

Far-red pentamethine cyanine dyes as fluorescent probes for the detection of serum albumins.

Benzothiazole based cyanine dyes with bridged groups in the pentamethine chain were studied as potential far-red fluorescent probes for protein detection. Spectral-luminescent properties were characterized for unbound dyes and in the presence of serum albumins (bovine (BSA), human (HSA), equine (ESA)), and globular proteins (β-lactoglobulin, ovalbumin). We have observed that the addition of albumins leads to a significant increase in dyes fluorescence intensity. However, the fluorescent response of dyes in the presence of other globular proteins was notably lower. The value of fluorescence quantum yield for dye bearing a sulfonate group complexed with HSA amounted to 42% compared with 0.2% for the free dye. The detection limit of HSA by this dye was greater than 0.004 mg ml−1 which indicates the high sensitivity of dye to low HSA concentrations. Modelling of structure of the dyes complexes with albumin molecules was performed by molecular docking. According to these data, dyes could bind to up to five sites on the HSA molecule; the most preferable are the haemin-binding site in subdomain IB and the dye-binding site in the pocket between subdomains IA, IIA and IIIA. This work confirms that pentamethine cyanine dyes could be proposed as powerful far-red fluorescent probes applicable for highly sensitive detection of albumins.

DNA Photocleavage in the Near-Infrared Wavelength Range by 2-Quinolinium Dicarbocyanine Dyes.

Here, we report the syntheses of two pentamethine cyanine dyes containing quinolinium rings and substituted with either hydrogen (3) or bromine (4) at the meso carbon. The electron withdrawing bromine atom stabilizes dye 4 in aqueous buffer, allowing complex formation to occur between the dye and double-helical DNA. UV–visible, CD, and fluorescence spectra recorded at low DNA concentrations suggest that dye 4 initially binds to the DNA as a high-order aggregate. As the ratio of DNA to dye is increased, the aggregate is converted to monomeric and other low-order dye forms that interact with DNA in a non-intercalative fashion. The brominated dye 4 is relatively unreactive in the dark, but, under 707–759 nm illumination, generates hydroxyl radicals that cleave DNA in high yield (pH 7.0, 22 °C). Dye 4 is also taken up by ES2 ovarian carcinoma cells, where it is non-toxic under dark conditions. Upon irradiation of the ES2 cells at 694 nm, the brominated cyanine reduces cell viability from 100 ± 10% to 14 ± 1%. Our results suggest that 2-quinolinium-based carbocyanine dyes equipped with stabilizing electron withdrawing groups may have the potential to serve as sensitizing agents in long-wavelength phototherapeutic applications.

Optimizing the Biodistribution of Radiofluorinated Barbiturate Tracers for Matrix Metalloproteinase Imaging by Introduction of Fluorescent Dyes as Pharmacokinetic Modulators.

Dysregulated expression or activation of matrix metalloproteinases (MMPs) is observed in many kinds of life-threatening diseases. Therefore, MMP imaging-for example, with radiolabeled MMP inhibitors (MMPIs)-potentially represents a valuable tool for clinical diagnostics using noninvasive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. Despite numerous preclinical imaging approaches, translation to a clinical setting has not yet been successful. We introduce and oppose three potential radiofluorinated MMP-targeted imaging probes, modified by the introduction of pentamethine cyanine (Cy5) dyes and therefore containing both radio- as well as fluorescent label with respect to their capability to assess MMP activity in vivo by means of scintigraphic (PET) and/or fluorescent (NIRF) imaging. New hybrid MMPI tracer candidates, structurally based on radiofluorinated pyrimidine-2,4,6-triones (barbiturates) from previous approaches, were synthesized by convenient two-step syntheses. In the first step, Cy5 dyes, varying in the number of sulfonate groups (nSO3- = 1, 2, or 4) and bearing an additional "clickable" alkyne moiety, were coupled to the barbiturate MMPI by amide formation. In the second step, the [18F]fluoride radiolabel was introduced into the resulting Cy5 dye conjugates by "radio-click" chemistry. Biodistribution studies of these hybrid tracer candidates were assessed and compared in C57BL/6 mice by PET as well as fluorescence imaging. MMP activity was imaged in a MMP-positive mouse model of irritant contact dermatitis (ICD) by PET and sequential fluorescence reflectance imaging (FRI), respectively. In vivo data were validated by scintillation counting, gelatin zymography, and MMP-histology. Three new potential hybrid MMP imaging probes were prepared, differing essentially in the number of sulfonate groups, introduced by Cy5 dye components. Although the hydrophilicity of these compounds was substantially increased, 10a (nSO3- = 1) and 10b (nSO3- = 2) were still rapidly eliminated via unfavorable hepatobiliary pathways, as observed in earlier approaches. Only 11 (nSO3- = 4) showed delayed in vivo clearance and a shift towards higher renal elimination. In the chosen mouse model of ICD, only 11 (nSO3- = 4) significantly accumulated in the inflamed mouse ear, which could be precisely visualized by means of PET and FRI.

Bifunctional reactive pentamethine cyanine dyes for biomedical applications

New unsymmetrical pentamethine cyanine dyes containing two non-equivalent reactive groups selected from carboxyl, amino and hydroxyl in different combinations were synthesized and their spectral properties investigated. Amino and hydroxy groups were used for binding the dyes to the anticancer drug, chlorambucil (CLB) via biodegradable amide or ester linker while the second reactive functionality could be utilized for attachment at mild conditions to a targeting carrier such as an antibody or peptide. These conjugates can thereby potentially be suitable for fluorescence-based monitoring of drug delivery. The CLB release profiles triggered by biodegradable linker were investigated in buffer solutions and cell medium using LC-MS and spectrofluorimetric methods.

Design and synthesis of symmetrical pentamethine cyanine dyes as NIR photosensitizers for PDT

Herein, we report the synthesis and spectroscopic characterization of novel Near Infra-Red (NIR) pentamethine cyanine dyes, as potential photosensitizers for Photodynamic Therapy (PDT) characterized by a strong absorption in the tissue transparency window (600–800 nm). The heteroaromatic benzoindolenine ring of various symmetrical cyanine dyes has been differently functionalized and quaternarized as a result of a structure-activity study and to determine the substituent effect on the cellular uptake, ROS production and photodynamic activity. These probes present low cytotoxicity in dark, but promote phototoxic effect, upon irradiation, in human fibrosarcoma cell line (HT-1080) with interesting and unexpected structure to property activity.

Template‐Free Self‐Assembly of Dimeric Dicarbocyanine Dyes

AbstractSymmetrical dicarbocyanine dyes are known to cooperatively assemble on DNA templates. With the goal of developing dyes that are capable of template‐free self‐assembly, two N‐linked dimeric benzothiazole containing pentamethine cyanine dyes have been synthesized. UV‐visible spectroscopy of the dimeric dyes reveals very strong propensities for self‐assembly unlike their monomeric dye counterparts. The N‐propyl linked dye exhibits pronounced H‐aggregation at micromolar dye concentrations with the absorption maxima blue‐shifted by nearly 200 nm. In contrast, an N‐hexyl linked dye preferentially folds into H‐dimers. Similar aggregates of the monomeric dicarbocyanine dyes are known to be observed at much higher dye concentrations or in presence of suitable templates such as DNA molecules. The aggregate species of the dimeric dyes are identified based on shifts in their absorbance maxima and quenched fluorescence. The calculated electronic structure of the dimeric dyes supports their observed aggregation behaviour. Further, introduction of DNA molecules with AT‐tracts leads to transformation of the spontaneously formed dimeric dye H‐aggregates and H‐dimers into DNA‐bound dye species.

Synthetic route to novel zwitterionic pentamethine indocyanine fluorophores with various substitutions

Two series of novel zwitterionic water-soluble pentamethine cyanine dyes with geometrically balanced structures were synthesized and characterized. One series of cyanine dyes is the «zwitterionic» fluorophores, which contain positively charged trimethylammonioalkyl and negatively charged sulfonate groups. The second series of cyanine dyes contains a sulfonate group on one indolenine nucleus and a carboxyl group linked through an N-acylsulfonamide bond on the other indolenine nucleus. Dye-modified deoxyuridine triphosphates were synthesized and evaluated as reagents for nucleic acid labeling using PCR with Taq DNA-polymerase. The efficiency of the incorporation of labeled nucleotides was investigated via real-time PCR and using a «KRAS-Biochip» commercial testing system.

Synthesis and Optical Properties of Pentamethine Cyanine Dyes With Carboxylic Acid Moieties.

Cyanine dyes possessing carboxylic acid groups have been used in many different fields of study. The acid groups can act as handles for bioconjugation or as metal chelators. Several pentamethine cyanine dyes with propionic acid handles were synthesized and their optical properties were studied to determine their usefulness as fluorescent probes. The optical properties studies performed include the absorbance and emission maxima values as well as the calculation of quantum yield and molecular brightness levels. Molecular models were also calculated to help analyze the dyes’ behavior and were compared with similar dyes with varying alkyl chain lengths replacing the acid moieties.