Hydrocyanines: a versatile family of probes for imaging radical oxidants in vitro and in vivo

Abstract

The hydrocyanines are a class of dyes that can detect reactive oxygen species (ROS) in cell culture, tissue explants, and in vivo. The hydrocyanines selectively react with radical oxidants, such as superoxide via an amine oxidation mechanism to generate cyanine dyes, thereby imaging ROS. The hydrocyanines can detect nanomolar levels of cellular ROS with tunable emission wavelengths between 560–830 nm. The hydrocyanine dyes have excellent stability against auto-oxidation and can be easily synthesized, and this enabled their rapid commercialization. Herein, we discuss in detail the properties of the hydrocyanine dyes and their current applications across biology and medicine.

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Corinne Sadlowski

Dr. Corinne Sadlowski received her undergraduate degree from the University of New Hampshire in 2008. She then completed her Ph.D. in organic chemistry from the University of Vermont in 2014, where she worked on the total synthesis of indole alkaloids. Dr. Sadlowski joined Professor Murthy's lab as a postdoctoral fellow in the Department of Bioengineering at the University of California, Berkeley. Her research focuses on the synthesis of new fluorescent probes for imaging and the design of drug delivery vehicles.

Santanu Maity

Santanu Maity was born in Kolkata, India. He finished his Ph.D. from the University of Geneva under the supervision of Prof. Stefan Matile in the field of supramolecular photovoltaics. Followingly, he joined Biocon in India as a research scientist and he joined Murthy lab in the year 2012. Since then he has been working in various research topic like endosomal hydrolysable polymer, on demand drug delivery and development of a new family of a fluorescent probe to image ROS, aldehyde dehydrogenase (ALDH) and other important analytes.

Kousik Kundu

Kousik Kundu studied chemistry at the University of Burdwan, (BSc Hon's) and the Indian Institute of Technology, Kharagpur (MSc). He received his PhD in Organic Chemistry from the University of Maryland, College Park. Upon graduation, he joined Dr. Niren Murthy's group as a Postdoctoral Research Fellow in the Biomedical Engineering Department at the Georgia Institute of Technology. He is one of the co-inventors of Hydrocyanine Technology. He co-founded a start-up in Atlanta before joining LI-COR Biosciences in 2011. Since 2014, he has been working at Nalco-Champion, an ECOLAB company. His current research focuses on solving flow assurance challenges in upstream oil production.

Niren Murthy

Dr. Niren Murthy is a professor in the Department of Bioengineering at the University of California at Berkeley. Dr. Murthy's laboratory is an interdisciplinary laboratory that focuses on the development of new materials for drug delivery and molecular imaging. Dr. Murthy received the NSF CAREER award in 2006, and the 2009 Society for Biomaterials Young Investigator Award. The Murthy laboratory has developed several new biomaterials for drug delivery and molecular imaging, such as the hydrocyanines.

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Design, System, Application

Reactive oxygen species (ROS) play an important role in biology and medicine. In this review, we have summarized the use of hydrocyanine or fused hydrocyanines as imaging probes for superoxide and hydroxyl radicals in a variety of in vitro and in vivo models. Hydrocyanines can be synthesized from commercially available cyanine probes in a single step reduction. In addition, they have a fast response rate, fluorescence enhancement in the presence of ROS, and a detection sensitivity in the nanomolar range. In biology, endogenous ROS are produced in nanomolar concentration and therefore hydrocyanines are a versatile probe for cellular imaging applications.

1. Introduction

Radical oxidants play a pivotal role in biology¹ and therefore technologies that image them have the potential to have a significant impact across science and medicine.² Reactive oxygen species (ROS) refer to the collective metabolites of oxygen that play an essential role in the biology of aerobic organisms and include peroxides, superoxide, hydroxyl radical, and singlet oxygen. They are classified as either endogenous ROS, involved in physiological functions from cell signaling to oxidative stress, or exogenous ROS, produced from ionizing radiation or environmental pollutants. During aerobic oxidation, the shuttling of electrons leads to the generation of ATP in which molecular oxygen is the final electron acceptor to produce water. However, this process can easily malfunction and create partially reduced oxygen intermediates that can be damaging to the surrounding environment. Though a proportion of ROS is necessary for cellular viability, a non-homeostatic surge of ROS is indicative of dysfunction and often leads to a diseased state, commonly referred to as oxidative stress. It is therefore essential for the diagnosis of disease that ROS can be detected and monitored for therapeutic treatment.

ROS have been implicated in a broad variety of diseases and over the past decade, technologies that have accurately detected cellular ROS have positively impacted both science and medicine. Radical oxidants are implicated in cancer and atherosclerosis, and are also involved in a variety of fundamental physiological and pathophysiological processes, such as aging and inflammation. In addition, ROS are a powerful biomarker for measuring tumorigenesis and for estimating the activity of atherosclerotic plaques, which is critical for determining disease progression.³ Moreover, ROS play a critical role in the efficacy of both antibiotic and anticancer therapies,⁴ influencing the process of DNA mutagenesis that consequently drive mutations responsible for drug resistance.⁵ Across the disciplines, radical oxidants have been implicated in over 150 diseases such as inflammation, aging, toxicity, cell growth, cell differentiation, cellular damage, tumor survival, and their drastic biological consequences have stimulated tremendous interest among the scientific community.⁶

Considerable efforts have therefore been made to develop tools that image ROS accurately, however, imaging radical oxidants has been difficult because of their small size, short lived half-life, and low concentration, and therefore cannot be imaged by conventional methods such as antibody strategies.⁷ In addition, developing chemical probes to image ROS has also been challenging because of the difficulty in identifying a fluorescent probe that has the necessary reactivity and kinetics required to compete with cellular ROS scavengers such as glutathione or superoxide dismutase (SOD), yet also retains the stability and photophysical properties needed to prevent high levels of background fluorescence.⁸ Although several ROS probes such 2′,7′-dichlorodihydrofluorescein diacetate (H₂-DCFDA), dihydroethidium (DHE), and dihydrorhodamine (DHR) have been developed, they all suffer from instability, which leads to spontaneous autoxidation and rapid photobleaching.⁹ Despite immense interest, currently there are no probes available for ROS detection that have the sensitivity, versatility, and reliability needed to simultaneously detect ROS in cell culture, ex vivo, and in vivo, and this has hindered progress across multiple areas of science and medicine.⁹

2. Advantages of the hydrocyanines: a new family of fluorescent ROS probes

2.1 Overview

The hydrocyanines are a new family of fluorescent probes, discovered in 2009 by Kundu et al.,¹⁰ which can image radical oxidant ROS in cell culture, in tissue sections, and in vivo, and have the potential to significantly impact multiple areas of biology and medicine (see Fig. 1 and 2). Since their inception, approximately 100 papers have been published using hydrocyanine dyes. They are currently commercially available from 2 sources and over 7 hydrocyanine-based products have been developed.¹¹ The hydrocyanines have several features that have made them one of the most commonly used probes in ROS biology; they have tunable emission wavelengths, tunable physical properties, high quantum yields, and photophysical stability, and can therefore be easily engineered for a wide variety of biological applications. Furthermore, the synthesis of the hydrocyanines is extremely straightforward and can be manufactured via a one-step reduction of cyanine dyes with sodium borohydride, making their synthesis both economical and easy to commercialize.

Fig. 1 The hydrocyanines are a new family of probes for imaging radical oxidants. They are synthesized by reduction of fluorescent cyanine dyes to non-fluorescent and membrane permeable hydrocyanines. Reactive oxygen species (ROS) such as superoxide or hydroxyl radical can selectively turn on fluorescence via oxidation of hydrocyanines to cyanine dyes and thereby image ROS.¹⁰

Fig. 2 Hydrocyanines have multiple applications. Any cyanine dye can be reduced into a hydrocyanine via a simple 1-step reduction process. Consequently, hydrocyanines with different optical and physical properties can be synthesized for various biological applications.

2.2 Hydrocyanine synthesis and radical oxidant sensing mechanism

The chemical structure of the hydrocyanines and the mechanism by which they detect radical oxidants is shown in Fig. 1. Hydrocyanines are synthesized through a 1-step reduction of cyanine dyes with sodium borohydride via a 1,2-hydride addition to the iminium cation of the cyanine scaffold. The hydride addition disrupts the extended π-conjugation and consequently turns off the fluorescence of the parent cyanine dye.¹⁰ Hydrocyanines are therefore non-fluorescent, however, in the presence of radical oxidants (HO˙ or O₂˙⁻), they undergo oxidation, resulting in hydride ion elimination on the heterocycle. This oxidation regenerates the iminium cation and thus turns on fluorescence.^10,12 The first step in the radical-mediated oxidation is loss of an electron from the reduced amine, resulting in hydrogen abstraction and elimination. Deuterium labelling studies have demonstrated that the rate-limiting step is hydrogen abstraction, similar to other amine oxidation reactions.¹² In addition, deuterium labeling studies have also demonstrated that the sodium borohydride is delivered to the iminium cation with excellent regioselectivity.¹² Finally, the reduction of cyanine dyes provides nearly quantitative yields of the corresponding hydrocyanines without producing any major by-products, and therefore allows for straightforward purification.¹⁰

2.3 Photophysical properties of the hydrocyanines

The hydrocyanines have outstanding photophysical properties because they generate cyanine dyes after oxidation by radical oxidants. The cyanine dyes are one of the largest classes of dyes used in molecular imaging and have a wide range of attractive photophysical and biophysical properties, such as emission wavelengths ranging from 480 nm to 850 nm and log P values ranging from +5 to −4.^10,12 Almost any cyanine dye can be converted into a hydrocyanine via sodium borohydride reduction, enabling tunable physical and photophysical properties for ROS imaging among a wide range of biomedical applications.^10,12 In addition, the excitation and emission properties of the hydrocyanines are compatible with commonly used laboratory equipment since they require the same fluorescence settings as cyanines dyes, which are widely used for imaging in confocal microscopes, FACS machines, and in vivo imagers.

A number of diverse hydrocyanines have been synthesized which span the visible and near-IR region and are suitable for imaging in microscopy, flow cytometry, and in vivo imaging applications. In addition, hydrocyanines have been synthesized that can image intracellular and extracellular ROS via manipulation of their hydrophobic/hydrophilic balance. For example, the hydrocyanines hydro-Cy3, hydro-Cy5, hydro-IR-676, and hydro-Cy7 (Table 1) can detect intracellular ROS as they are initially membrane-permeable molecules; however, upon oxidation by intracellular ROS, they are converted into charged and membrane-impermeable molecules and consequently accumulate within cells that are overproducing ROS. In contrast, the hydrocyanines hydro-ICG, hydro-IR783, hydro-IRDye 800CW are charged and membrane-impermeable molecules and therefore are suitable for measuring extracellular ROS production.¹⁰ Finally, cyanine dyes with targeting moieties such as folate, arginine–glycine–aspartate (RGD), hyaluronic acid (HA), epidermal growth factor (EGF), polyethylene glycol (PEG), chitosan, and antibodies can be easily transformed into targeted ROS probes, and can potentially facilitate new insights in redox biology and disease progression.¹⁰ Thus, reduced cyanine dyes represent a powerful synthetic methodology for generating fluorescent ROS sensors.

Table 1 Structure, excitation/emission wavelengths, and advantages of hydrocyanine probes for biological applications

Structure	Probe name	Molecular weight (g mol^−1)	λ Ex/Em (nm)	Membrane permeable	Cell culture	Live animal imaging
	Hydro-Cy3	414.63	535/560	+	+	−
	Hydro-Cy5	384.56	635/660	+	+	+
	Hydro-Cy7	410.59	735/760	+	−	+
	Hydro-ICG	798.96	535/560	−	−	+
	Hydro-IR783	873.47	535/560	−	−	+

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2.4 Specificity of hydrocyanines to radical oxidants

Cells produce several different types of reactive oxygen and nitrogen species and the specificity of the hydrocyanines towards different reactive oxygen and nitrogen species has therefore been measured. The specificity of the hydrocyanines hydro-Cy3 and hydro-Cy7 toward 12 different reactive oxygen and nitrogen species has been reported. For example, Fig. 3a demonstrates that hydro-Cy3 has high selectivity towards radical oxidants (superoxide and hydroxyl radical) and is non-responsive towards Fe²⁺, singlet oxygen, hydrogen peroxide, nitric oxide, peroxynitrates, and various other oxidizing agents.¹⁰ The selectivity of the hydrocyanines towards radical oxidants arises from their reaction mechanism involving two consecutive one-electron oxidations, similar to the oxidation of DHE.¹¹

Fig. 3 (a) Hydrocyanines have high specificity for superoxide and radical oxidants over other reactive oxygen and nitrogen species. (b) Hydrocyanines have nanomolar sensitivity towards the hydroxyl radical and are significantly more sensitive than DHE (see ref. 10 for details).

2.5 Sensitivity of the hydrocyanines toward radical oxidant detection

The ability of the hydrocyanines to detect hydroxyl radical in vitro has been studied and compared against DHE. Fig. 3b demonstrates that both hydro-Cy3 and hydro-Cy7 have nanomolar sensitivity towards radical oxidants. Both exhibited linear relationships (r² = 0.99) between fluorescence intensity and hydrogen peroxide concentration (1–30 nm range) with Fenton's reagent. Hydrocyanines have significantly better sensitivity compared to DHE, which had an r² value of only 0.36 in the same nanomolar concentration range of radical oxidants.

2.6 Kinetics of the hydrocyanines toward auto-oxidation and reaction with radical oxidants

A major problem with traditional fluorescent ROS probes is their autoxidation in aqueous solutions, which creates high levels of background fluorescence and interferes with ROS measurements. This can be attributed to the lower thermodynamic oxidation driving force of hydrocyanines resulting from a lower resonance stabilization of oxidation intermediates and products compared to those of DHE, which has a polycyclic aromatic backbone structure. Fig. 4a demonstrates the significantly higher stability of hydrocyanines against autoxidation compared to ROS probes such as DHE, although the resulting cyanine dyes are sensitive to degradation from ROS. Hydro-Cy7 and hydro-Cy3 both have half-lives of approximately 3 days in aqueous pH 7.4 buffer at 37 °C and have two orders of magnitude more stability to autoxidation compared to DHE, which had a half-life of only 30 minutes. The greater stability of hydrocyanines therefore makes them ideally suited to detect ROS in biological samples with higher accuracy.

Fig. 4 (a) Hydrocyanines have significantly higher stability than DHE. (b) Hydrocyanines reacts with radicals (i.e. hydroxyl radical) with a half-life of a few minutes. (c) Hydrocyanines have minimal toxicity even at a 1 mM concentration.¹⁰

The kinetics of the amine oxidation of hydrocyanines by radical oxidants are critical for their efficacy as ROS detection probes. Fig. 4b demonstrates that the half-life of hydrocyanine oxidation by radicals (i.e. hydroxyl radical) is a few minutes (see ref. 10, ESI).

2.7 Hydrocyanine toxicity

Chemical probes need to have minimal toxicity in order to avoid any secondary biological effects. The toxicity of hydrocyanines has been measured in cell culture on rat aortic smooth muscle cells (RASMs) to detect plaque activity via ROS production when stimulated with angiotensin II, which is implicated in the development of atherosclerosis and hypertension. Cell viability assay on RASMs showed that a 1 mM concentration of hydro-Cy3 caused negligible levels of toxicity and demonstrated that the hydrocyanines have the biocompatibility needed for cell culture and other biological studies (see ref. 10, ESI).

3. Applications of the hydrocyanines

The hydrocyanines have found numerous applications in biotechnology due to their ability to measure ROS in vitro and in vivo. In the last five years, hydrocyanines have been used to measure ROS in a variety of cell culture experiments; in vivo for live animal imaging and in organ culture via histological analysis. Collectively, these experiments have significantly advanced our understanding of the role of ROS in biology. The hydrocyanines are now commercially available from LICOR Biosciences under the trade name ROSstar, and modified hydrocyanines from Life Technologies.¹¹

3.1 Microbiome imaging

A central question in microbiome biology is how do intestinal epithelial cells distinguish between pathogenic bacteria and commensal bacteria.¹³ This problem was solved by the Neish laboratory and collaborators from 2009–2014, and they have demonstrated commensal bacteria induce intestinal epithelial cells to generate ROS via a NOx-mediated process and that this ROS is critical for a variety epithelial functions such as barrier integrity, angiogenesis, and cell division.¹³ The hydrocyanines were a key reagent used to elucidate the role of ROS signaling pathways in orchestrating homeostasis between commensal bacteria and intestinal epithelial cells.¹³ For example, in 2011 the Neish laboratory was able to demonstrate that the commensal bacteria Lactose rhamnose were able to stimulate intestinal epithelial cells to make ROS via a NADPH-mediated process.¹³ Their initial experiments were done using Caco2 cells, which were then treated with Lactobacillus rhamnosus and hydro-Cy3 that demonstrated in vitro commensal bacteria can induce ROS production in intestinal epithelial cells. Their hydro-Cy3 experiment is described in Fig. 6, and demonstrates that hydro-Cy3 can measure ROS in cell culture.¹³

This initial finding was later followed up by in vivo experiments with hydrocyanines, which demonstrated that intestinal epithelial cells produce ROS in response to commensal bacteria.¹³ In these studies, the sterile mice were fed with commensal bacteria followed by an intraperitoneal injection (IP) of hydro-Cy3. As a control, mice were given PBS and an IP injection of hydro-Cy3. After 4 hours, the mice were sacrificed and histological sections of the intestines were made and analyzed for ROS. Fig. 5 demonstrated that mice treated with commensal bacteria had a very high level of ROS production, as demonstrated by the fluorescent red staining from hydro-Cy3, which was significantly higher than the control mice. Furthermore, the red staining is predominantly in the epithelial cells facing the lumen and is significantly higher than other traditional cell types that make ROS, such as macrophages or neutrophils. This experiment demonstrated that commensal bacteria induced intestinal epithelial cells to make ROS. In addition, these experiments demonstrated that hydrocyanines could be used in vivo to identify cells that make ROS and we anticipate that this property of the hydrocyanines will have numerous applications in biology.

Fig. 5 Commensal bacteria induce production of ROS in intestinal epithelia. a) Fluorescent images of ROS generation in scratch-wounded Caco-2 monolayers treated with media, LGGlo, LGGhi or insulin, and then loaded with 100 μm hydro-Cy3. b) Fluorescently labelled whole mount preparations of proximal small intestine taken from mice injected with hydro-Cy3 followed by oral gavage with HBSS or LGG for 1 h. (Upper) 10x magnification; (lower) 40× magnification. Data are representative of two independent experiments with n = 3 mice per group.¹³

Extensive studies have been conducted using hydrocyanines to elucidate the mechanism by which ROS helps orchestrate gut homeostasis. For example, they were able to demonstrate that microbiota-induced ROS is essential for wound repair and functions via the induction of MAP kinases.¹³ In addition, ROS generated from the microbiota is essential for stem cell proliferation and also for cell motility via inactivation of focal adhesion kinase phosphatases. Finally, the Neish group was able to demonstrate that commensal bacteria in drosophila also generate ROS and that non-commensal bacteria do not have this ability, suggesting that this is an evolutionarily conserved process used by multicellular organisms to identify bacteria, which can be beneficial to the gut.¹³

3.2 ROS signaling pathways in immunology elucidated with the hydrocyanines

ROS play a key role in numerous immunological processes and hydrocyanines have been used by the Pulendran laboratory to investigate the role of ROS in driving a Th2 response.¹⁴ Tang et al. used hydrocyanines to demonstrate that the Th2 response to cysteine proteases requires ROS-mediated signaling, which is generated by epithelial cells. Tang et al. treated mice with papain via an intramuscular injection and 6 hours later injected them with hydro-Cy5 at the same injection site, excised the skin from the injection site, co-stained, and analysed for ROS production via oxidation of hydro-Cy5. Robust ROS production was detected mainly in epithelial cells with a weak signal in CD11c+ DCs in the dermis (Fig. 6).

Fig. 6 ROS production in skin in response to immunization with papain is dependent on ROS. Immunofluorescence confocal microscopy of the site of immunization with OVA plus papain, stained for DAPI (blue), hydro-Cy5 (red), and CD11c (green) to assess ROS activity. Far right, enlargement of area outlined at left; arrows indicate some hydro-Cy5 staining in DCs. Original magnification, ×20 (main images).¹⁴

Hydrocyanines can measure ROS in a plate reader format and in live cells, which enables quantitative analysis of ROS. Several laboratories have now used hydrocyanines to measure ROS in a plate reader format and this has contributed significantly to the elucidation of numerous cell signaling pathways. For example, the Kemp laboratory was able to demonstrate that doxorubicin generated superoxide in CD4+ T cells via a NADPH-dependent process. This exemplified how ROS play a protective role against cell death and can therefore be exploited to lower toxicity in off-target organs (Fig. 7).¹⁴

Fig. 7 A novel murine aortic coarctation model to acutely create a region of low magnitude oscillatory WSS in vivo to test the hypothesis that acute changes of WSS in vivo induce upregulation of inflammatory proteins (VCAM-1) mediated by ROS and imaged by hydro-Cy3 (A). The lower images show representative confocal images (63x) from en face mounted aortas stained for either VCAM-1 (B and C) or superoxide (D and E). Images B and D were taken from the upstream region while the images C and E were taken from the downstream region.¹⁵

3.3 Hydrocyanines reveal ROS plays a central role in atherosclerosis and a variety of pulmonary diseases

ROS plays a central role in both atherosclerosis and a variety of pulmonary diseases. For example, ROS has been implicated in the development of atherosclerosis and vascular inflammation. However, the biological mechanisms that cause inflammation and ROS production in the vasculature have been difficult to identify in vivo. Low shear stress in the lumen of the blood vessels has been implicated as an important contributor in the pathogenesis of atherosclerosis, however, their role in generating vascular inflammation and ROS has never been clearly understood. Willett et al. developed a murine aortic coarctation model to create regions of low magnitude oscillatory wall shear stress in vivo and used hydro-Cy3 to analyze if regions of low shear stress generate high levels of ROS and inflammation.¹⁵ To determine ROS production in vivo, Willet et al. injected hydro-Cy3 into the aorta of these mice before sacrificing them. The mice were then analyzed via histology to determine ROS production and vascular inflammation. Willet et al. observed that regions of low shear stress had high levels of ROS production and vascular markers of inflammation, such as VCAM1. Thus, the hydrocyanines can measure ROS at a cellular level in diverse tissues.¹⁵

ROS also plays a critical role in a variety of pulmonary diseases. Although the role of ROS in generating pulmonary inflammation and tissue damage is well known, ROS are also involved in a variety of physiologic processes that are essential for healthy tissue function. For example, the Helms laboratory was able to use hydro-Cy7 to demonstrate that ROS generated by NADPH oxidases regulated alveolar epithelial sodium channel activity and lung fluid balance in vivo and thus plays a critical role in protecting the lung against edema (Fig. 8).¹⁶

Fig. 8 (a) Longitudinal in vivo imaging of ROS as a surrogate for implant-associated inflammation using Hydro-ICG. (b) Immunohistochemical staining for macrophages and neutrophils along with imaging of ROS in implant-associated inflammation using hydro-Cy5. (c) Longitudinal ROS imaging in response to controlled release of dexamethasone from PLGA microparticles after intravenous delivery of hydro-Cy7.¹⁷

3.4 In vivo imaging of inflammation

ROS plays a pivotal role in driving inflammation and imaging ROS therefore has the potential to serve as a diagnostic for early stage inflammation.¹⁷ Selvam et al. used hydrocyanines to investigate if ROS can be used as a surrogate marker of inflammation in the context of implant-associated inflammation.¹⁷ For these experiments, they used the near-IR hydrocyanine dye, hydro-ICG, delivered either locally or intravenously in mice to measure ROS in vivo in the vicinity of an implant. They were able to demonstrate that hydro-ICG can measure ROS production and thus inflammation in vivo in response to implanted poly(ethylene terephthalate) (PET) disks or injected poly (lactic-co-glycolic acid) (PLGA) microparticles in a longitudinal manner. In vivo ROS imaging with the hydrocyanines correlated with conventional analysis of inflammation and also enabled non-invasive monitoring of modulation of the inflammatory responses via release of the anti-inflammatory agent dexamethasone. In vivo imaging of ROS by hydrocyanines can therefore serve as a surrogate measure for monitoring implant-associated inflammation as well as for evaluating the efficacy of therapeutic approaches to modulate host responses to implanted medical devices.¹⁷

ROS has also been implicated in the pathogenesis of osteoarthritis (OA) and imaging ROS therefore has the potential to serve as a biomarker for osteoarthritis development. Xie et al. demonstrated that in vivo fluorescence imaging of ROS with hydrocyanines can be used to noninvasively monitor OA progression and to assess the efficacy of pharmacologic interventions in small animals (Fig. 9).¹⁸ This study also used a combination of contrast-enhanced micro computed tomography (mCT) and fluorescence imaging to characterize inflammation in articular cartilage, subchondral bone, and vascularization. They were able to correlate these parameters with ROS production, thus providing significant insight into the role of ROS in OA progression.

Fig. 9 In vivo imaging of ROS was used as an indicator of inflammation in rat joints with osteoarthritis induced by monosodium iodoacetate (MIA). Hydro-indocyanine green ROS images were obtained at 1, 11, and 21 days post-injection.¹⁸

3.5 ROS imaging in cancer

Several researchers have also used hydrocyanines to investigate the role of ROS in cancer biology. For example, Burton et al. used hydrocyanines to investigate the molecular mechanisms by which the Snail transcription factor promoted epithelial mesenchymal transition (EMT) in prostate cancer cells, and determined that it is an ROS-dependent process that can be inhibited by muscadine grape skin extract (MSKE).¹⁹ This was accomplished by generating Snail overexpressing prostate cancer cell lines and examining their ROS production with hydro-Cy3. In addition, the Heintz laboratory was able to use hydrocyanines to demonstrate that the anticancer drug thiostrepton functions by modifying mitochondrial thioredoxin, which causes an increase in ROS.²⁰ Finally, the Tae laboratory has used near-IR emitting hydrocyanines to image ROS in tumors. In their studies, the hydrocyanines were encapsulated in pluronic micelles and were able to image cancers at an earlier stage more effectively than with cyanine dyes alone due to selective ROS-mediated activation in the tumor.²¹

3.6 Neurological applications of the hydrocyanines

ROS plays a central role in a variety of neurological pathologies ranging from ophthalmic diseases to various types of chronic or acute brain injury, and the hydrocyanines consequently have the potential to make a significant contribution to these areas of research. For example, oxidative stress is an important contributor to macular degeneration and diabetic retinopathy, and imaging it has the potential to enable early diagnosis. Toward this goal, the Pardue lab has demonstrated that the hydrocyanine hydro-800CW can detect light-induced oxidative stress in the retina by scanning laser ophthalmoscopy in living mice, and can be used as a surrogate marker to non-invasively assess inflammation in the eye.²² Hydro-800CW therefore has the potential to detect retinal ROS in vivo and improve the screening and monitoring of numerous causes of blindness such as diabetic retinopathy, glaucoma, and age-related macular degeneration, all of which have oxidative stress as a significant component. Hydrocyanines have also been used by Schoknecht et al. in the context of brain ischemia to measure the dynamics of cortical perfusion, blood brain barrier permeability, and cell damage in a rat photothrombosis vascular occlusion model.²³ The commercially available hydrocyanine ROSstar 650 (LI-COR Biosciences, Lincoln, NE, USA), was used for these studies. Lastly, the Berezin lab used a near-IR hydrocyanine probe for the in vivo detection of ROS as a marker of tissue response to ischemia and a precursor to angiogenesis and remodeling.²⁴

Conclusions

In summary, the hydrocyanines are a new class of fluorescent probes that image ROS and have numerous applications across biology and medicine.

Notes and references

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