Advances in Imaging Reactive Oxygen Species

Hydrocyanines

Since their discovery in 2009 (4), hydrocyanine probes have been used for detection of superoxide and hydroxyl ROS both in vitro and in vivo (4,5), with detection limits down to nanomolar concentrations. A wide range of hydrocyanines with desired properties can be prepared in a single synthetic step, making them accessible even to labs with limited synthetic infrastructures.

Hydrocyanines were first discovered by Kundu et al. (4) in 2009 by reducing commercially available cyanine dyes with NaBH₄. The hydrocyanine hydro-Cy3 successfully imaged ROS in rat aortic smooth muscle cells after treatment with the angiotensin II peptide, a system that mimics the development of atherosclerosis and hypertension. In addition, hydro-Cy7, a near-infrared (NIR) dye, was used to image ROS in mice during a lipopolysaccharide-mediated inflammatory response. In the years following, hydrocyanines were extensively investigated for ROS imaging, most of which were covered in a previous review (5). We will therefore focus on recent developments in the field of hydrocyanines in the past 3 years.

Although hydrocyanines have many uses, they do have some limiting factors. These limitations include high autooxidation, low Stokes shifts, and low solubility, as well as their product cyanine dyes’ lability to ROS (6). A strategy for reducing some of these shortcomings was published by Maity et al. (6), who introduced a new class of thiophene-bridged hydrocyanines (Fig. 1A). Thiophene-bridged hydrocyanines showed superior stability to autooxidation, with 89% of the probe remaining after a 48-h incubation in phosphate-buffered saline, compared with 42% for hydro-Cy5 (Fig. 1B). After oxidation, THBC generated a fluorescent product with significant stability toward ROS-mediated degradation (Fig. 1C). However, despite their promise, THBC derivatives have not been thoroughly studied, partly because of their relatively complex synthesis compared with regular hydrocyanines. Regardless, the increase in chemical stability and the improved photophysical properties yield a promising scaffold that should be further investigated.

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FIGURE 1.

Modification of hydrocyanines for increased stability and in vivo sensitivity. (A) Structure of recently reported thiophene-bridged hydrocyanines (THBC). (B) Increase in stability of THBC toward autooxidation in phosphate-buffered saline. (C) Increase in stability of active thiophene-bridged cyanines (gray) toward ROS-dependent degradation, compared with Cy3 (orange). Graph represents percentage of probe intact after incubation with 250 μM of indicated ROS. (Reprinted from Maity et al. (6).)

Because hydrocyanines are well established in their use for ROS detection, recent advances in the field have focused on building multifunctional systems to increase selectivity and introduce additional functions. For example, Al-Karmi et al. (7) introduced an ¹⁸F label on IR780, allowing for PET-based biodistribution studies of hydrocyanine probes. Andina et al. (8) used a modular design of a reported dye connected to hydro-Cy5 via 2 complementary peptide nucleic acid moieties for measuring extracellular ROS. The use of a reporter dye allowed them to account for distribution differences by using a ratio-based readout. In a study by Zhang et al. (9), glioblastomas were selectively imaged by conjugating hydro-Cy5 to the integrin α_vβ₃-targeting peptide Arg-Trp-(d-Arg)-Asn-Arg. Using this strategy, tumors could be imaged selectively in the presence of other inflammatory tissues.

Xanthene Probes

Xanthene dyes are some of the most frequently used probes for ROS detection, with reduced derivatives of fluorescein and rhodamine being the most abundant. They can easily be prepared through direct reduction of their oxidized derivatives. Xanthene dyes were initially used as H₂O₂-selective probes but have been shown to react readily with hydroxyl and peroxyl radicals and with several RNS (10). This characteristic has reduced their practicality in favor of more selective probes, although they are still frequently used.

Although fluorescein and rhodamine-based dyes are heavily used for ROS detection, they do not possess any specificity toward the ROS source. One way to achieve organelle specificity was explored by Zhang et al. (11), who used silicon-rhodamine-based NIR fluorescent probes to target lysosomal ROS. These probes showed high specificity toward the highly reactive ROS, HClO, HO⋅ and ONOO⋅ allowing for imaging of ROS in the lysosomes of cancer cells. This strategy was further explored by Wang et al., when they created a NIR probe for in vivo imaging of HClO/ONOO^⋅ in an idiopathic pulmonary fibrosis mouse model (12).

Sulfonyls

Sulfonyl-protected dyes were developed to battle shortcomings associated with other protecting groups such as acetyls, which show significant deprotection from hydrolysis in addition to perhydrolysis (13). The superoxide-selective sulfonyl probes were introduced when Maeda et al. (14) developed a diprotected fluorescein analog, although the observed selectivity depended on the sulfonyl substituent. Further studies were done to eliminate the impact of side reactions by modifying the leaving group. In particular, the triflate-protected probe HKSOX-1 showed excellent resistance to thiol-mediated cleavage, thus allowing for selective detection of superoxide in zebrafish embryos (15). Sulfonyls are still being developed for use in selective superoxide detection, with recent applications including superoxide detection in mitochondria (16) and the use of 2-photon excitation microscopy (TPEF) probes (17).

Arylboronates

Arylboronic acids have long been known for their reactivity toward H₂O₂, in which addition of peroxide to the boron leads to an aryl-group migration and subsequent phenol formation (18). Boronic acids mask the fluorescence of fluorophores, yielding H₂O₂-responsive probes. This method was pioneered by the Chang lab (19) when it synthesized a diboronic acid-modified fluorescein that selectively reacted with H₂O₂ in the presence of other ROS. After this seminal study, aryl boronic acid–capped fluorophores remain a prevalent strategy in the selective detection of H₂O₂. Recent advances include detection of H₂O₂ in ischemia–reperfusion injury (20), chemiluminescent detection through a capped luciferin substrate (21), and the use of boronate-capped probes that trigger 1,6-elimination reactions on phenol generation (20).

Phosphinates

The nucleophilic property of superoxide can also be exploited to cleave phosphinate groups. This strategy was spearheaded by Xu et al. (22) when they synthesized diphenylphosphinate-capped fluorescein and naphthofluorescein analogs, which were able to detect superoxide down to 0.1 nM. These probes are also useful for in vitro imaging, as was illustrated by imaging superoxide formation in macrophages after stimulation with phorbol 12-myristate 13-acetate, an oxidative burst stimulant. This strategy was further expanded on by Zhang et al. (23), who created a NIR dye that allowed for in vivo imaging of superoxide. Recently, this strategy was also expanded to include bioluminescence by Liu et al. (24), who used a phosphinate-capped luciferin to detect polystyrene-induced superoxide formation in cells. Furthermore, a mitochondria-targeting probe (NA-T) that saw a shift in fluorescence after phosphinate deprotection and subsequent 1,6-elimination to release an unmodified diketopyrrolopyrrole dye has been developed (25). NA-T showed high specificity toward superoxide-mediated cleavage in the presence of a wide range of ROS and was used for imaging of phorbol 12-myristate 13-acetate/lipopolysaccharide–triggered superoxide formation in Daphnia magna.

Microbubbles

Microbubbles vary in size from 1 to 10 μm in diameter. Conventional microbubbles are composed of lipids, polymers, and surfactants, among others, which encapsulate a gas. However, by chemically modifying hydrazine, N₂ encapsulation as an organic compound is possible. The Murthy lab has previously demonstrated the use of chemically generated microbubbles to image in vivo oxidative stress via ultrasound (28). Allylhydrazine encapsulated within a liposome proved efficient in detecting 10 mM ROS in vitro and in vivo (28). In the presence of ROS, the amine from allylhydrazine oxidizes into 2-propenyl-diazene and thus allows for a retro-ene reaction that results in the release of nitrogen and propylene gas. This increase in gas concentration creates acoustic impedance that can be detected acoustically.

Photoacoustic Imaging Using Nanoparticles

When tissue absorbs light and converts it to heat via vibrational relaxation, thermoelastic expansion may result and then generate ultrasound waves. This is known as the photoacoustic effect. Previously, researchers have used polymers and nanoparticles as a nanoplatform for developing photoacoustic probes (29). In vivo ratiometric photoacoustic imaging is a relatively new method that has been used to monitor ROS and RNS (29,30). When a nanoparticle is coupled with an ROS-sensitive dye, the nanoparticle emits an optoacoustic signal. In the presence of ROS, absorption or fluorescence of the chromophore is blue-shifted because of degradation whereas absorption or fluorescence of the nanoparticle remains unaffected. Overlaying the images when monitoring at NIR versus visible wavelength generates a representative image.

Using nanoparticles to image and generate ROS in cancer cells is of great importance for theranostics. By incorporating multifunctional groups on semiconducting perylene diimide, Chen and team developed a theranostic nanoparticle, perylene diimide-IR790s-iron/cisplatin (31), for ratiometric photoacoustic imaging. IR790s, a cyanine derivative, absorbs light at 790 nm but decomposes in the presence of ROS, losing its absorptivity. The change in absorption ratio between perylene diimide and IR790s allows for ratiometric imaging. The cisplatin prodrug activates nicotinamide adenine dinucleotide phosphate oxidase, thus forming superoxides followed by H₂O₂ generation by superoxide dismutase. Fe³⁺ then converts H₂O₂ into hydroxyl radicals via the Fenton reaction, which then reacts with IR790s to quench absorption. This technique to induce ROS for cancer therapy also allows for real-time monitoring during treatment.

Photoacoustic Imaging Using Upconversion Nanocrystals

Detecting multiple radical species independently is invaluable in ROS imaging. By incorporating 2 cyanine dyes—hydro-Cy5 and Cy7, which are sensitive to 2 specific reactive species—onto the surface of an upconversion nanoprobe, Xing and team were able to screen the presence of ROS and RNS via multispectral optoacoustic tomography by ratiometrically monitoring multiple radicals (32). Upconversion nanocrystals have been used because of their anti–Stokes-shift properties. Unlike TPEF, upconversion nanocrystals rely on sequential, rather than simultaneous, absorption of photons. Therefore, it is possible to excite the nanocrystal with NIR light and observe emission in the visible spectra (33). Because emission of the nanocrystal overlaps the absorption of the cyanine dye, individual monitoring of the cyanine dye is possible. In the presence of ROS there is a fluorescence increase and, thus, an increase in optoacoustic signal. Conversely, in the presence of RNS there is a decrease in fluorescence and, thus, a decrease in the optoacoustic signal (Fig. 2).

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FIGURE 2.

(i) Illustration of HCy5 (ROS) and Cy7 (RNS) on upconversion nanocrystal (UCN) surface. In presence of ROS, nonfluorescent HCy5 is converted into fluorescent Cy5, whereas in presence of RNS, fluorescent Cy7 decomposes, causing signal reduction at 800 nm. (ii) Schematic representation of UCN in live mice for bioimaging. MSQT = multispectral optoacoustic tomography; UCL = upconverted luminescence. (Reprinted from Ai et al. (32).)

Chemiluminescence

Unlike fluorophores, chemiluminescent probes do not autofluoresce, thus making them desirable for bioimaging. However, some drawbacks are a short emission time and short wavelengths. To address the issue of short emission time, Ren and team synthesized a chemiluminescent polymer dot probe (hemin-Pdots) that relies on chemiluminescence resonance energy transfer to enhance the chemiluminescence lifetime to about 10 h in the presence of an L-012, a luminol analog, and H₂O₂ (34). In the presence of H₂O₂, L-012 oxidizes, and chemiluminescence that is 100 times greater than that of luminol is observed (34). Slow diffusion of the oxidized L-012 through the hemin-Pdots nanoparticles increases the chemiluminescence lifetime via chemiluminescence resonance energy transfer, where the luminol is the energy donor and the hemin-Pdot is the acceptor. Indeed, the slow diffusion dynamics of such chemiluminescent systems show a 700-fold enhanced chemiluminescence over 10 h.

One of the problems in ROS imaging via luminescence is correlating the readout to ROS concentration. Contag and team have previously described use of a reporter probe, coelenterazine, to measure superoxide dynamics (35). Interestingly, they were able to observe a change in chemiluminescence during cellular respiration, as well as in response to variant glucose concentrations. By generating an in vitro baseline using coelenterazine, their system shows promise not only for in vivo imaging but also for quantification of superoxide concentrations.