Organic dyes in contemporary medicinal chemistry and biomedicine. I. From the chromophore to the bioimaging/bioassay agent

Abstract The present review was provoked by the demand of a comprehensive overview on the recent scientific achievements revealing new horizons for advanced applications of organic dyes in service of contemporary medicinal chemistry, pharmacy and biomedicine. The review outlines the basic structural characteristics, physicochemical properties and biological activity of various dye families and suggests modified classifications of dyes according to their structural moieties and bioorganic functionalities serving the necessities of modern analytical chemistry and biochemistry. A major part of the review focuses on the pros and cons of the use of dyes as vectors in bioanalytic assays. The latter is based on comparative analyses of the limitations of some widely applied classical methodologies vs. the advantages and outcomes of the application of newly-designed dye molecules in modern dye-based bioassay techniques.


History of dyes: a brief overview
Historically, the use of dyes in the textile industry dated more than 4,000 years ago. Until the middle of the nineteenth century, only dyes of natural origin were used [1]. The revolution in the synthesis of synthetic dyes is associated with the research of William Henry Perkin who synthesized quinine in 1856, which in turn led to the discovery of the dye Mauveine (aniline purple or Perkin purple) [2,3]. Although Perkin was not actually the first scientist who synthesized an aniline dye, his perception helped him to realise the significant role of the purple dye in a commercial aspect. Initially he gave it the name 'Tyrian purple' as a reference to the ancient Murex barrel. The new colour was also supported by the French Empress Eugenie, who was famous for her affinity for fashion. Later on, Perkin renamed the dye mauveine due to the association of the term with Parisian haute couture. The supervisor of Perkin Hoffman later synthesized the aniline dye rosaniline [4].
The discovery of mauveine played an important role in the development of immunology and chemotherapy. In 1891, the German chemist, immunologist and physician Paul Ehrlich discovered that certain cells or organisms have the ability to selectively absorb specific types of dyes. He then found that it was possible to inject a large enough dose of dye to kill pathogenic microorganisms if the dye did not affect other cells. Ehrlich continued to use dyes against syphilis. These are the first experiments using a chemical compound to selectively kill bacteria in the body. The German chemist used methylene blue to attack Plasmodium, which is responsible for malaria [5].
Following the chronological development of dyes, it should be outlined that at the end of the nineteenth century another dye family -the acridine dyes -first extracted from coal tar, began their life in the fabric industry. During the first decade of the twentieth century, Erlich and Beneda initiated their application as antimalarial agents. After Browning's proposition of the use of acridines as antimicrobials, the dyes were widely applied in antimicrobial therapy during World War I and II, prior to the discovery of penicillin [6].

Chemical structure insights
Almost all dyes are aromatic organic compounds that can be considered as benzene derivatives. Benzene, naphthalene and other aromatic hydrocarbons absorb light in the ultraviolet but not in the visible spectral range, so they are colourless. In order to absorb visible light, aromatic nuclei must form larger molecules known as chromogens, which do not always have an aromatic nature. This leads to an uneven distribution of the electron density within the molecule. To be a dye, an organic compound must be able to impart its colour to another substance. This property is achieved by adding to the organic molecule one or more substituent groups called auxochromes, which are colour modifiers of the chromogen [8]. The auxochrome is a functional group containing unshared electron pairs. When linked to a chromophore, it causes changes in the absorption wavelength and intensity. The direct relatation of the functional groups to the π-system of the chromophore could cause a wavelength increase and in turn, intensification of absorption. . The general structure of a dye is presented in Figure 1.
Auxochromes structure has a characteristic presence of at least one unshared electron pair, responsible for the presence of resonance structures. The folllowing functional groups are included in the auxochromes class: the hydroxyl (−OH) group, the amino (−NH 2 ) group (protonated in acidic medium), quaternary N-atoms (positively charged in each medium), the sulphone group (-SO 3 H) (negatively charged in aqueous solution regardless of pH), the aldehyde group (−CHO), methyl mecraptan group (−SCH 3 ), carboxyl group (-COOH) (ionized to anions in neutral and acidic medium), etc. [9][10][11]

Structural and functional classifications of dyes
The chemical composition and structure of dyes determine their colour, properties and use and provides one of the main rational criteria for their classification.
The dyes are classified as per the chemical structure of their chromophores according to the numbering and ordering systems used in the Colour Index and in Conn's Biological Stains. Table 1 in this review presents a  • dyeing of polyamide fibres; • staining of erythrocytes and cytoplasm in trichrome staining methods in human and veterinary histology; • -fixing tissue agents; [12,13] azo -N = N-
• -for polychrome staining of animal tissue [43]  comprehensive classification according to the latter criteria additionally covering major dye uses and bioactivities [44].
To understand and reveal the chemical structure of dyes and the associated specific properties of individual dyes and dye families in general, it is of utmost importance to differentiate two of their structural units: the chromophore and the luminophore (fluorophore). The chromophore is that part of the organic dye molecule which is responsible for its colour or the group of atoms in a molecule in which the electronic transition is responsible for a given spectral band. While the luminophore is the part of the organic molecule that is responsible for a given emission band when it undergoes luminescence. In this respect, according to the type of the fluorophores, the structural functions and biomedical applications new classes of dyes have been recently extensively designed, developed and synthesized. The conditional classification of these dye classes and subclasses is summarized in Table 2.

Dyes in fluorescence imaging
Fluorescence imaging has been identified as one of the most powerful techniques for the efficient visualization of temporal and spatial changes and mechanisms of biological phenomena in living cells, biological tissues and organs [22,55]. The major advantage of this analytical approach over other widely applied techniques such as magnetic resonance imaging, ultrasound and positron emission tomography, lies in its ability to reveal detailed spatiotemporal information regarding living cells and ex vivo tissue sections [56].
Various dyes classes have been assessed as suitable candidates for efficient fluorescent imaging. The basic criteria taken into consideration are high water solubility, photo and thermal stability, high molar extinction coefficients, high fluorescence quantum yield, high sensitivity and short response times [22]. Fluorescein and rhodamine xanthene dyes have been successfully utilized as efficient fluorescent cores for fluorescent probes acting in the green and red wavelength regions [57], as well as in non-destructive testing and bio-imaging [58].
However, the complete understanding of the biochemical mechanisms requires imaging exploration at deeper or intact tissue of living organisms. The extreme heterogeneity of tissues associated with strong scattering of visible light restrains the penetration capacity of conventional fluorescence imaging, thus presenting the major challenge of this method [57]. Most of these limitations have been overcome by recently elaborated near-infrared (NIR) fluorescence imaging techniques, operating in the NIR window between 700 and 1700 nm and even in the NIR-II range within 1000-1700 nm, that have been extensively developed and applied for deep-tissue biological explorations [58][59][60]. These methodologies have the characteristics of suppressed photon scattering and diminished tissue autofluorescence and have found application for the dynamic and high-contrast exploration of neural activity, cerebral blood flow, tumour, microenvironment, in immunoassays, etc. [57] Though, NIR fluorescence has emerged as a cheap, safe and real-time navigation modality, it requires the development of novel molecular fluorophores with specific chemical and photophysical properties associated with longer wavelengths, intensified brightness, improved photostability, chemical stability, strong penetrability and negligible damage to biological tissues and living cells [58].

NIR fluorophores in bioimaging
Newly-developed classes of near-infrared fluorescence probes constitute of conventional fluorescent dyes such as pentamethine and heptamethine cyanine, rhodamine, xanthene, tetrapyrrole, thiazine and oxazine dyes [61]. They have been successfully applied for the labelling of antibodies, proteins, nucleic acids, peptides and other biomolecules, and the dye-biomolecules conjugates are featured with more intensive brightness and higher photo stability as compared to fluorescein [22]. Kushida et al. (2015) reviewed procedures for the development of silicon-substituted xanthene dyes (2,7-N,N,N′,N′-tetramethyl-9-dimethyl-10-hydro-9silaanthracene, Si-rhodamines and Tokyo Magentas) acting as novel near-infrared fluorescent cores, based on the substitution of the O-atom at the 10-position of xanthene by a Si-atom. The characteristic features of silicon-substituted xanthene dyes have been utilized for in vivo tumour imaging in mice, triple-colour imaging of neuronal activity in mouse brain slices, and for highly sensitive live cell protein labelling for super-resolution microscopy [53]. Novel heptamethine cyanine dyes with increased water solubility, high quantum yields and large Stokes's shift were synthesized by various methods such as: changing the central chlorocyclohexenyl group, introducing a carboxylic group and iodination [20]. Porphyrins and Phthalocyanines belong to the near-infrared fluorescence dyes containing a tetrapyrrole group. There are reports on the application of porphyrin dyes to macromolecules (DNA, proteins) for the modification of their structure, biological properties and functions [60]. The fluorescent properties of rhodamine dyes were modified by processes associated with opening/ closing of aromatic rings or photo-induced effects [60]. Although thiazide and oxazine-based near-infrared fluorescent probes are easily synthesized smaller molecules, their application is limited due to their low fluorescence quantum yield [61]. Croconaine dyes are defined as donor-acceptor-donor type zwitterionic compounds which have extended π-conjugation. Their bioimaging and theranostics applications were ascertained by a straightforward condensation reaction synthesis procedure, tailored structures and improved near-infrared photophysical properties [62]. Scientific studies have proved the valuable properties of BODIPY dyes: large molecular absorption coefficients, well-resolved sharp spectral features, high fluorescent quantum yields, insensitivity to the polarity of the solvent, narrow fluorescence peaks, long fluorescence lifetime, photostability [61,63]. Thus, BODIPY dyes have been identified as one of the main design targets for the development of novel near-infrared fluorescent dyes. The specificity of the BODIPY core structure allows an infinite array of possible chemical modifications including π-conjugated system rings extension, alkenyl and alkynyl α-substitutions, directed towards physicochemical properties tuning [61,63]. According to Berraud-Pache et al. [62] despite the state-of-the-art investigations, the cogent design of near-infrared fluorescent dyes with beneficial properties encounters strains and difficulties.

Classical vs. contemporary dye-based metabolic activity assays
The indication of cell physiology changes by means of dye-based metabiotic activity assays is based either on fluorescence intensity changes or emission spectral alterations [64]. The wide array of cytotoxicity and cell viability assays applied in biomedicine, toxicology and pharmacology is related to the great variety of cell functions enzyme activity, cell membrane permeability, cell adherence, adenosine triphosphate (ATP) production, co-enzyme production and nucleotide uptake activity, as well as to the specific physicochemical and biochemical properties of numerous classes of conventional and newly synthesized organic dyes [65][66][67]. According to Aslantürk [64] metabolic assays could be classified as: dye exclusion, colourimetric, fluorometric assays and luminometric assays [64][65][66][67] • high dye-to-protein ratio without causing precipitation of conjugates. • biomolecule, cell and tissue labels for fluorescence microscopy, cell biology, molecular biology.
covering the types of dyes applied the general principle of the assay, as well as the basic pros and cons of the corresponding metabolic assay. Thus, the available up-to-date scientific data on dye-based assays according to the stated criteria was summarized and structured in Table 3.
Recently, automated benchtop image-based cell counters for rapid cell concentration and viability • preliminary tests to determine the optimal time of staining; • data analysis performed in a highly manual and subjective manner using limited image analysis techniques and complex software. [67,[77][78][79] measurements have been designed. Chan et al. [77] demonstrated the applicability of image-based cytometry for cell viability detection by using single-, dual-, or multi-stain nucleic acid and enzymatic stain techniques. Based on their investigations, the scholars concluded that fluorescent viability staining allows the exclusion of cellular debris and non-nucleated cells from the analysis. The latter results outlined the possibility to eliminate the preliminary purification step during sample preparation, which in turn would improve the efficiency of the method [77]. Datki et al. [78] developed an innovative protein assay based on the fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulphonic acid dipotassium salt (BisANS) and proved its applicability for protein quantification of various biological and chemical samples. The dye molecules characterized with: satisfactory water solubility, significant photostability at adequate pH, high kinetics rate of interaction with proteins. Besides, the dye did not display exclusive sensitivity to the chelator molecules, the detergent agents and the inhibitor ingredients [78].
Zlatskiy et al. [79] established significant differences in the effectiveness of three biological dyes: FDA (fluorescein diacetate), PI (propidium iodide) and trypan blue, for cell viability assessment in vitro of cancer and normal cell lines at different deuterium/protium ratios by a combination of laser diffraction and viability dye staining. The experimental data revealed no sensitivity of trypan blue, partial sensitivity of FDA and PI, and high sensitivity of Alamar blue. The authors suppose that the deviations in the sensitivity of the methodology could be attributed to deuterium/protium dependent chemical interactions of dyes which provoke alterations in the chirality of the active substances [15,79,80].
Scientific literature reports various methods of phagocytosis investigations. A common flow cytometry one implies bacteria labelling with fluorescein isothiocyanate (FITC) dye. The disadvantages of the method include: necessity of preliminary quenching of cell surface-bound bacteria before the quantitative analyses, as well as indistinguishable bacterial invasion and phagocytosis. The technique was compromised also due to the decreased fluorescence of the dye in acidic medium [87][88][89]. Other flow phagocytosis assays, for analysing the activity of macrophages apply carboxyfluorescein succinimidyl ester (CFSE) or CellTracker dye. Although the latter highly fluorescent at physiological pH dye penetrates cell membranes , overestimation of the actual potential of the phagocytosis of cancer cells is possible. It could derive from inappropriate detection of intercellular binding [89].
Recent studies prove that the fluorescence intensity of pHrodo dyes at neutral pH is low, while acidification in the lysosome and cancer cells provokes the emission of significantly higher fluorescence. pHrodo dyes utilized for cancer cells labelling interact with the primary amines on cancer cells forming covalently bonded pH probes, which display increased fluorescence at acidic pH [90]. As a result, internalized particles would be detected due to their intensive fluorescence, while nonphagocytosed particles adhered to the outer membrane will not be assayed. pHrodo dyes have been also applied for labelling dead neural stem cells, heat-killed bacteria, dead neural stem cells, lipids, proteins, nanoparticles, etc. [86,89,90].
Lenzo et al. [89] studied the application of a pH-sensitive pHrodo dye for measuring the active phagocytosis of P. gingivalis by the application of phagocytic particles -initially inactivated dye-labelled Escherichia coli. The method allowed the determination of phagocytosed unopsonized bacteria within an active phagosome under acidic conditions. Besides, bacteria stuck on the surface or in compromised endosomes could be excluded from the analysis due to lack of fluorescence [89].
Lindner et al. [81] conducted a comparative study of the efficiency and applicability of pHrodo red-and pHrodo green-labelled particles in combination with different phagocytic cell types in phagocytosis assays by flow cytometry and high-content imaging. The results outlined that both applied pHrodo dyes facilitated the specific detection of phagocytosed particles [81].
To overcome the limitations of previously applied classical flow cytometry-based phagocytosis assays, Nam et al. [91] developed and applied an optimized fluorescence microscopy protocol for determination of cancer cells phagocytosis using pHrodo-succinimidyl ester (pHrodo-SE) dye. Due to the low pH of the phagolysosome, engulfed cancer cells can be visualized and distinguished from the cancer cells sticked to the outer phagocyte surface [91].
Richey et al. [92] investigated the macrophagemediated phagocytosis and dissolution of amyloid-like fibrils in mice using Dylight80021 and pHrodo red30 fluorophores. Based on the experimental results, the scientific team concluded that the applied methodology could be successfully used to study phagocytosis and cell mediated dissolution of amyloid materials [92].
The performance of the dyes alamarBlue®, neutral red (NR), Viral ToxGlo™ (VTG) and WST-1 in the assessment of the antiviral activities of biologically active compounds against RNA viruses was studied by Smee et al. [93]. It was established that the 50% virus-inhibitory (EC50) concentrations for each inhibitor/virus combination were not significantly dependent on the dye characteristics. But, the efficiency of the dyes in distinguishing the vitality of virus-infected cultures varied in the presence of cells not killed by the virus infection [93].
A number of these assays have been widely applied, improved and optimized using novel dye classes and instruments. Still, numerous studies continue their improvement and adaptation [80]. Continued advances in fluorophore design, dual-laser scanning, multispectral imaging, and spinning disk applications are expected to provide significant improvements in the near future.

Challenges in the development of novel chromophores
The considerable recent research interest in the development of design approaches and synthesis methods associated with the photophysical properties of organic chromophores has been provoked by their benefits for various analytical, biochemical and biomedical applications [21].
The classical assessment of RNA, DNA and proteins in clinical research applies in situ immuoenzymatic methods utilizing peroxidase and alkaline phosphatase catalysing chromogenic stains (3,3′-diaminobenzidine, Fast Red, Fast Blue), conversion into insoluble products. From another aspect the basic features of conventional chromogens comprise of: broad absorbance spectra, provision of sufficient biomarker morphology and signal range. However, the urge for high contrast restricts the number of visually distinguished chromogens applied to the same sample. The latter effect represses dyes efficacy for guaranteeing multiplexed detection in biological samples, which in turn certainly claims the design of new generation of chromogens [94].
Scientific literature reports the utilization of dyes that strongly absorb relatively narrow bands of light, as a possible technique to achieve better colour discrimination in multiplexed chromogenic assays. The methodology is based on the conception of the removal of a small fraction of the visible spectrum by an individual dye.
Day et al. [94] generated a new class of chromogens by covalent conjugation of tyramine to dyes. The new chromogens were remarkably concordant, in both signal morphology and dynamic range, with broad absorbing chromogen, able to produce a number of spectrally distinct chromogens, and these chromogens can be effectively integrated into multiplexed brightfield in situ assays [94]. Holzapfel et al. [95] established an innovative approach for fluorescence multiplexing using spectral imaging and combinatorics. The methodology proposed consisted of the design of new independent probes from covalently-linked combinations of individual fluorophores [95].
In general, the basic advantages of multiplexed biotechnologies are associated with improved quality of diagnosis, providing greater association with clinical outcome and reducing the consumption of limited clinical material.
Wang et al. [21] synthesized two novel π-extended red-emitting hybrid xanthene dyes. Each of the novel molecules carries two spirolactone rings and combines a seminaphthorhodafluor (SNARF) moiety in a single molecule. The results established that the dyes existed in double-ring-opened forms in aqueous solution which were able to emit deep-red fluorescence and were mitochondria-targetable. Due to the fact that the absorption and fluorescence properties of the newly-designed fluorophores can be regulated, the authors propose the applicability of the hybrid xanthene dyes as fluorescence tracers for various biological and analytical applications [21,96].

Conclusions
The emerging requirements of modern sciences in the fields of chemistry, biology, biochemistry, biophysics, medicine, pharmacy and drug discovery imposes the establishment of highly specific and efficient dye-based assays for the identification of cell structural components and observation of cellular responses applying natural and synthetic organic dyes with various biofunctionalities, specific photosensitivity and nontoxicity. The innovative dye-based assays have to provide sensitivity, specificity and reliability from the detection of simple cell organelles to more complex monitoring of cell signalling, death pathways and toxicity. To overcome these challenges, however, the 'new era' scientists have to be aware of the bioactivities, specific dye-biomatrix interactions and biological dyes toxicology profiles.

Author contributions
Conceptualization, Z.Y. and D.I.; writing-original draft preparation, Z.Y., D.I., N.N and M.T.; writing-review and editing, Z.Y., D.I.; visualization, Z.Y. and N.N.; supervision, Z.Y. All authors have read and agreed to the published version of the manuscript.

Data availability
The data that support the findings of this study are available on request from the corresponding author.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The APC was funded by the Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria.