Single Atom Stabilization of Phosphinate Ester-Containing Rhodamines Yields Cell Permeable Probes for Turn-On Photoacoustic Imaging

Abstract

Photoacoustic imaging (PAI) is an emerging imaging technique that uses pulsed laser excitation with near-infrared (NIR) light to elicit local temperature increases through non-radiative relaxation events, ultimately leading to the production of ultrasound waves. The classical xanthene dye scaffold has found numerous applications in fluorescence imaging, however, xanthenes are rarely utilized for PAI since they do not typically display NIR absorbance. Herein, we report the ability of Nebraska Red (NR) xanthene dyes to produce photoacoustic (PA) signal and provide a rational design approach to reduce the hydrolysis rate of ester containing dyes, affording cell permeable probes.

To demonstrate the utility of this approach, we construct the first cell permeable rhodamine-based, turn-on PAI imaging probe for hypochlorous acid (HOCl) with maximal absorbance within the range of commercial PA instrumentation. This probe, termed SNR₇₀₀-HOCl, is capable of detecting exogenous HOCl in mice. This work provides a new set of rhodamine-based PAI agents as well as a rational design approach to stabilize esterified versions of NR dyes with desirable properties for PAI. In the long term, the reagents described herein could be utilized to enable non-invasive imaging of HOCl in disease-relevant model systems.

Introduction

PAI is a promising imaging modality for preclinical research and point-of-care applications.^[1,2,3] Commercial PAI instruments utilize pulsed NIR lasers, capable of exciting dyes with absorbance in the 680–980 nm range, in order to increase the penetration depth of excitation light by minimizing interference from hemoglobin and decrease phototoxicity.^[4,5,6] PA signal is generated through non-radiative relaxation leading to cycles of thermoelastic expansion events that give rise to pressure waves which can be detected by ultrasound transducers.^[7] This photoacoustic effect was first described by Alexander Graham Bell in 1880.^[8] Given that biological tissues are 1000-fold more transparent to sound compared to light, imaging depths in the cm range can be achieved by PAI.^[7,9] Furthermore, PAI is an attractive alternative to deep-tissue imaging techniques such as magnetic resonance imaging (MRI), X-ray computed tomography (CT), and positron emission tomography (PET) due to its relatively low cost and the absence of ionizing radiation. Nonetheless, PAI is currently limited by the lack of diversity of small molecule scaffolds capable of producing PA readouts, which in turn, limits the ability to generate PA probes for biologically relevant analytes.

The BODIPY,^{[10,11,12,13,14]} hemicyanine,^[3,15,16,17] and cyanine scaffolds^[18] have been the primary small-molecule dyes employed for PAI due to compatibility of their maximal absorbance with commercial PAI instrumentation. Recent chemistry-focused efforts have leveraged activity-based sensing approaches^{[19,20,21,22,23]} to develop turn-on or acoustogenic small molecule PA reporters from these scaffolds.^{[4,5,6,16,17]} Such reporters operate through judicious choice of a functional group that selectively reacts with a target analyte and subsequent installation of that functional group on a dye to render it nonabsorbent in the NIR. Upon reaction with the target analyte, a chemical transformation occurs such that the dye becomes NIR absorbent and PA active. This approach has resulted in acoustogenic probes for nitric oxide,^[18,24,25] glutathione,^[16] copper,^[2] and alkaline phosphatase.^[3] However, greater chemical diversity in PA active small molecule dyes is necessary to expand the toolbox of acoustogenic probes across a broader range of biologically relevant analytes.

Xanthene dyes, typified by rhodamine, rhodol, and fluorescein are historically among the most utilized optical probes and have been extensively studied.^{[26,27,28,29,30]} In particular, spirocyclization at the electrophilic C-9’ position of xanthenes with a functional group capable of selectively reacting with a target analyte is a well-established approach for constructing absorbance off-on probes (Figure 1).^[31,32] Indeed, this spiroring-opening strategy has proven to be generalizable across a wide range of analytes including reactive oxygen and nitrogen species,^[19,33] metal ions,^[34] and enzymes.^[28] Unfortunately, the ability to generate acoustogenic probes has been hindered by the inability to utilize the highly generalizable xanthene spiroring-opening strategy for analyte sensing since the maximal absorbance of these dyes does not typically fall within the 680–980 nm range of commercial PAI instrumentation. Nonetheless, there are a limited number of examples of xanthene dyes capable of producing PA signal.^[35,36,37] For example, previous work has described a Si-rhodamine-based turn-on PA probe (termed PA-MMSiNQ, see Figure S14a for structure) for HOCl,^[37] a potential intracellular marker of acute myeloid leukemia (AML),^[38] that relied on a spirocyclic thioether for detection. While this work represents an elegant use of fluorescence quenching groups to design PA probes, this reporter is not cell permeable and does not display maximal absorbance in the wavelength range of commercial PAI instrumentation. Alternatively, a fluorescent phosphine-oxide-containing rhodamine probe for HOCl with maximal absorbance at 710 nm has been described.^[39] However, the ability of this probe to produce PA signal has not been reported. Thus, to the best of our knowledge, the probe described in this work represents the first cell permeable rhodamine-based, turn-on PAI imaging probe for HOCl with maximal absorbance in the range of commercial instrumentation.

Figure 1.

Spiroring-opening at the C-9’ position of rhodamine dyes has afforded visible turn-on absorbance/fluorescence probes for a variety of analytes (top). Stabilization of the phosphinate ester in NR dyes provides acoustogenic, rhodamine-based probes that are compatible with commercial PAI instrumentation (bottom).

Efforts to red-shift the absorbance of xanthenes have demonstrated that introduction of heteroatom functionalities at the bridging position of xanthenes, such as B,^[40,41,42] C,^[43] S,^[44,45] Si,^[46] Ge,^[47] SO₂,^[48] and C═O,^[49] can lead to a significant red-shift in absorbance. Using this strategy, our lab has recently developed phosphinate-containing rhodamine dyes which we termed Nebraska Red (NR) dyes.^[50,51] These dyes display ~115 nm red-shifts in absorbance compared to the parent dyes containing oxygen at the bridging position, and can be used for a variety of applications in chemical biology, including construction of absorbance off-on probes.^{[50,51,52,53,54]} We hypothesized that the relatively high extinction coefficient (ε > 10⁴ M⁻¹ cm⁻¹) and NIR absorbance of these dyes would make them excellent candidates for PAI. Herein, we screen a series of four rhodamine-based NR dyes for their ability to generate PA signal. From this initial screen, a phosphinate ester-containing NR dye, NR₇₀₀, that displays relatively rapid hydrolysis under physiological conditions (pH ~7.4), was identified as a promising lead. In order to stabilize this dye to hydrolysis and extend its lifetime in aqueous solutions, we employed rational design to implement a one atom change. This produced a cell permeable thiophosphinate NR derivative, SNR₇₀₀, with a 3.6-fold increase in stability in aqueous media, while retaining desirable photophysical properties for PAI. We further leveraged this stabilized dye to construct an acoustogenic probe for intracellular HOCl, the enzymatic product of myeloperoxidase (MPO), which is a clinically relevant diagnostic marker of AML.^[55,56] This turn-on PA probe, termed SNR₇₀₀-HOCl, is capable of producing a 12.5-fold increase in PA signal in the presence of HOCl, detecting as little as 500 nM HOCl, and providing contrast at imaging depths up to 2.9 cm in tissue. Moreover, SNR₇₀₀-HOCl was capable of detecting exogenous HOCl in mice. In the long term, cell permeable xanthene-based dyes that are compatible with commercial PAI instrumentation could provide valuable tools for construction of acoustogenic reporters for disease relevant analytes.

Results and Discussion

Screening NR Dyes for PA Signal

To determine whether NR dyes were capable of producing PA signal, we screened a set of four previously published, rhodamine-based dyes with maximal absorbance ≥666 nm (Figure 2a).^[50] This set of dyes contains pairs of phosphinate and phosphinate ethyl esters with varying molar extinction coefficients and quantum yields. Gratifyingly, we observed PA signal that overlaid well with the absorbance spectra of each dye within this panel (Figures S1 and S2). Within this panel, we observed consistently higher relative PA signal for phosphinate esters (NR₇₀₀ and NR₇₄₄) compared to their phosphinate analogues (NR₆₆₆ and NR₆₉₈, Figure 2b and c), even accounting for the 66.3% absorbance of NR₆₆₆ at 680 nm (Figure S2). Given that NR₇₀₀ produced the maximal PA signal in our panel, 45% greater than NR₇₄₄ (Figure 2c), we chose this dye as a scaffold for creation of an acoustogenic probe. While the phosphinate ethyl ester of NR₇₄₄ is resistant to hydrolysis, even when incubated at 37°C for 48 h in the presence of 50% FBS in cell culture media,^[52] we have previously shown that NR₇₀₀ hydrolyzes on the minutes timescale under physiological conditions to produce NR₆₆₆.^[50] In the context of a fluorescent probe, the hydrolysis of NR₇₀₀ can be an advantage for facilitating cellular uptake followed by hydrolysis to the brighter cell impermeable NR₆₆₆ or by providing a means for gated delivery of cargo in cells with subsequent hydrolysis to NR₆₆₆ as a readout of delivery.^[50] Unfortunately, as a probe for PAI, hydrolysis of NR₇₀₀ to NR₆₆₆ is less desirable since this would result in a significant decrease in observable PA signal (Figure S1, NR₆₆₆ versus NR₇₀₀). Accordingly, we set out to identify a rational design approach that could stabilize the phosphinate ester of NR₇₀₀ to hydrolysis while simultaneously maintaining PA signal.

Figure 2.

Photoacoustic signal of previously published NR dyes. a) Structures of NR₆₆₆, NR₆₉₈, NR₇₀₀, and NR₇₄₄ along with their photophysical properties. b) Photoacoustic images of the respective dyes (50 μM) in 1.2 cm thick tissue phantoms. c) Comparison of maximal PA intensities from each dye (see Figure S1) (background corrected mean±SD, n=6).

Rational Design of a Stabilized NR Phosphinate Ester-Containing Dye

Based on the work of Haake and colleagues,^[57] we hypothesize that hydrolysis of NR₇₀₀ to NR₆₆₆ occurs through nucleophilic attack of an hydroxide anion at phosphorus (Figure S3). Although NR₇₄₄ displays dramatically reduced hydrolysis compared to NR₇₀₀, we chose to pursue an approach that would minimally alter the spectroscopic properties of NR₇₀₀ to preserve PA signal. Inspired by the reduced hydrolysis of ATP-γ-S compared to ATP,^[58,59] we hypothesized that exchanging the P═O group for a P═S group would significantly decrease hydrolysis of the resulting thiophosphinate without appreciably impacting PA signal generation. To test this hypothesis, we synthesized a thiophosphinate analog of NR₇₀₀, termed SNR₇₀₀, starting from a previously reported NR₇₀₀ intermediate (1, Scheme 1).^[50] Treatment with Lawesson’s reagent followed by oxidation, yielded the target dye in a concise synthetic route. As hypothesized, SNR₇₀₀ retained a strikingly similar absorbance and emission profile (Figure S4) as well as photophysical properties (Figure 3a) compared to NR₇₀₀. However, installation of the thiophosphinate in SNR₇₀₀ led to a dramatic 3.6-fold decrease in the rate of ester hydrolysis compared to NR₇₀₀ (Figure 3b). Next, we assessed the ability of SNR₇₀₀ to produce PA signal. Indeed, SNR₇₀₀ retained 82% of the PA intensity of NR₇₀₀ (Figure 4), making it more intense than NR₆₆₆, NR₆₉₈, and NR₇₄₄. Initial confocal imaging studies also demonstrated that SNR₇₀₀ was more cell permeable than the parent NR₇₀₀ dye (Figure S5), an important feature for detection of intracellular analytes. These results confirm the ability to reduce hydrolysis of esters in NR dyes with a single atom modification, while maintaining desirable photophysical properties for imaging applications.

Scheme 1.

Synthesis of SNR₇₀₀.

Figure 3.

Incorporation of a thiophosphinate ester significantly reduces ester hydrolysis in NR dyes. a) Comparison of SNR₇₀₀ and NR₇₀₀ photophysical properties. b) Pseudo first-order rates of hydrolysis of NR₇₀₀ or SNR₇₀₀ at varying concentrations of hydroxide with 100 nM dye (mean±SD, n=3).

Figure 4.

SNR₇₀₀ retains PA signal intensity. a) PA images of 50 μM NR₇₀₀ and SNR₇₀₀ in 1.2 cm thick tissue phantoms. b) Quantified PA intensity from panel a (background corrected mean±SD, n=6).

Design and Synthesis of an Acoustogenic Probe for HOCl

Having identified a robust, PA active NR dye, we set out to leverage the classic spiroring-opening approach^[31,32] to generate a cell permeable, acoustogenic probe with maximal absorbance within the range of commercial PAI instrumentation. As an initial target analyte, we chose HOCl, the enzymatic product of MPO. Importantly, intracellular expression of MPO is used as a marker for diagnosis of AML in the clinic^[55,56] and previous studies have shown that patient-derived myeloblasts produce intracellular HOCl at concentrations of 2.4–5.6 μM.^[38] Given that expression of MPO and its subsequent production of HOCl is highly restricted to myeloid cells, aberrant intracellular HOCl production could represent a potentially underutilized diagnostic marker for AML. Thus, we envisioned the development of a cell permeable, acoustogenic SNR₇₀₀-based probe for HOCl. Ultimately, such a probe may provide a non-invasive approach to detecting AML in the clinic relative to bone marrow biopsy. To obtain an SNR₇₀₀-based acoustogenic probe for HOCl, we chose our previously reported NR-HOCl sensor, which contains a spirocyclic thioether^[60] that selectively reacts with HOCl, as a starting point.^[50] Using Lawesson’s reagent we converted NR-HOCl into SNR₇₀₀-HOCl in one step (Scheme 2). In the presence of HOCl, we envisioned that SNR₇₀₀-HOCl would oxidize to the corresponding sulfonate, yielding a species displaying NIR absorbance/fluorescence and PA signal. Gratifyingly, the increased hydrolytic stability afforded by the thiophosphinate motif was conserved in SNR₇₀₀-HOCl, which displayed a 3.3-fold slower fluorescence decrease upon reaction with HOCl compared to NR-HOCl, indicating that the P═S group remains intact after reaction with HOCl (Figure S6).

Scheme 2.

Synthesis of SNR₇₀₀-HOCl.

In Vitro and Cellular Selectivity for HOCl

Before testing the ability of SNR₇₀₀-HOCl to produce PA signal, we first evaluated the selectivity of SNR₇₀₀-HOCl using fluorescence. In vitro examination showed that SNR₇₀₀-HOCl is stable and non-fluorescent in aqueous media, while exhibiting a 280-fold turn-on fluorescence signal in response to HOCl, with peak emission at 734 nm (Figure 5 and S7). In addition, SNR₇₀₀-HOCl showed excellent selectivity towards HOCl compared to a panel of reactive oxygen (ROS) and nitrogen (RNS) species consisting of H₂O₂, tert-butyl hydroperoxide, superoxide, ·OH, ·OtBu, peroxynitrite, and nitric oxide (Figure S8), while displaying virtually no cellular toxicity at concentrations relevant to imaging experiments in a luciferase-expressing AML cell line (HL-60-Luc2, Figure S9). Encouraged by these findings we evaluated the ability of SNR₇₀₀-HOCl to detect HOCl within living cells. As an initial test, separate samples of RAW264.7 macrophage cells were dosed with increasing concentrations of HOCl or left untreated and incubated with SNR₇₀₀-HOCl. As expected, no NIR fluorescence was observed in untreated RAW264.7 cells, however upon addition of exogenous HOCl, a reproducible dose-dependent response to exogenous HOCl was observed across biological replicates (Figure S10). To assess the selectivity of SNR₇₀₀-HOCl for endogenously produced HOCl, we chose the RAW264.7 macrophage cell line which is known to produce HOCl upon stimulation with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA). Upon overnight incubation with LPS (1 μg/mL) followed by 2 hr stimulation with PMA (0.1 μg/mL), we observed a 63% increase in fluorescence relative to untreated cells (Figure S11). Lastly, we investigated the ability of SNR₇₀₀-HOCl to detect endogenous HOCl in HL-60-Luc2 cells which have been shown to produce steady-state levels of 1 μM HOCl.^[38] Indeed, a clear turn-on fluorescence signal was observed in this AML cell line which decreased by 52% upon addition of an MPO inhibitor, 4-aminobenzoic acid hydrazide (ABAH, Figure 6 and Figure S12). Taken together, these experiments demonstrate the ability to selectively detect HOCl both in vitro and within living cells.

Figure 5.

SNR₇₀₀-HOCl (5 μM) displays a clear absorbance and fluorescence turn-on signal in the presence of 5 μM HOCl. Assays were performed in DPBS (1% DMF). The inset shows absorbance and NIR fluorescence images of samples.

Figure 6.

Selective detection of endogenous HOCl in living cells with SNR₇₀₀-HOCl. Confocal images of HL-60-Luc2 cells incubated with SNR₇₀₀-HOCl (10 μM) in the absence or presence of the MPO inhibitor ABAH (200 μM).

Acoustogenic Detection of HOCl

Having established the ability of SNR₇₀₀-HOCl to selectively detect endogenous HOCl within living cells, we next evaluated SNR₇₀₀-HOCl as an acoustogenic probe. Within tissue phantoms, virtually no PA signal was observed from SNR₇₀₀-HOCl prior to addition of HOCl. However, addition of HOCl induced the formation of a robust PA signal with a peak at 690 nm in PBS and a 12.5-fold signal increase over background (Figure 7a). Encouraged by these results, we next investigated the limit of detection (LOD) of SNR₇₀₀-HOCl for HOCl using PAI in tissue phantoms. By varying the concentration of HOCl, we observed an LOD by PAI of 500 nM for HOCl (Figure 7b). While this LOD was ~100-fold higher compared to the fluorescence LOD of SNR₇₀₀-HOCl for HOCl (Figure S13), the PAI LOD is below the range of intracellular HOCl measured in patient-derived myeloblasts (2.4–15.6 μM).^[38]

Figure 7.

Acoustogenic detection of HOCl using SNR₇₀₀-HOCl. a) SNR₇₀₀-HOCl (25 μM) displays a clear photoacoustic turn-on signal in the presence of 50 μM HOCl (background corrected mean±SD, n=6). The inset shows PA images of the indicated samples. b) Photoacoustic signal from SNR₇₀₀-HOCl (50 μM) in the presence of the indicated concentration of HOCl (mean±SD, n=3). All experiments were performed in DPBS (1% DMF) in 1.2 cm thick tissue phantoms.

Since the maximal absorbance of SNR₇₀₀-HOCl lies within the range of commercial instrumentation, we hypothesized that this probe would produce increased signal relative to a previously reported cell impermeant Si-rhodamine-based probe, PA-MMSiNQ with maximal absorbance at 660 nm.^[37] Indeed, under identical conditions we observed a 2.2-fold increase in PA signal for SNR₇₀₀-HOCl compared to PA-MMSiNQ (Figure S14). These results highlight the utility of NR-based dyes for the development of turn-on PA probes for intracellular analytes.

Acoustogenic Imaging of HOCl in Tissues

As a proof-of-concept for deep-tissue imaging with SNR₇₀₀-HOCl, we cut chicken breast cylinders of varying diameters (Figure S15) and encased them in a tissue mimicking phantom to facilitate loading onto PAI instrumentation (Figure S16). Using this experimental setup, we were able to observe signal from SNR₇₀₀-HOCl stimulated with 100 μM HOCl up to a depth of 2.9 cm in tissue (Figure 8 and S17). These results further highlight the ability to leverage the spiroring-opening strategy in NR dyes to produce acoustogenic probes that are compatible with commercial PAI instrumentation.

Figure 8.

Evaluation of PA imaging depth for SNR₇₀₀-HOCl in tissues. a) PA images of a DPBS blank and 100 μM SNR₇₀₀-HOCl with 100 μM HOCl in chicken breast tissue of the indicated depth. b) Quantified PA signal from panel a across 11 positions, 0.5 mm apart for each indicated depth of tissue, scanned three times (mean±SD, n=33).

Acoustogenic Imaging of Exogenous HOCl in Mice

Lastly, we asked whether SNR₇₀₀-HOCl was capable of detecting exogenous HOCl in mice using PAI. For these experiments, nude mice (NU/J, Jackson Laboratory) were subcutaneously injected in the right flank with either saline, SNR₇₀₀-HOCl alone, or SNR₇₀₀-HOCl immediately followed by 2.5 equivalents of HOCl. Mice were subsequently imaged and multispectral unmixing algorithms were used to separate the photoacoustic signal from activated SNR₇₀₀-HOCl and endogenous chromophores, namely hemoglobin and oxyhemoglobin. Mice receiving SNR₇₀₀-HOCl followed by HOCl displayed a clear increase in PA signal compared to both saline and SNR₇₀₀-HOCl without HOCl at the injection site (Figure 9, Figure S18). These results underscore the potential of SNR₇₀₀-HOCl as a promising tool for the intracellular detection of HOCl in living animals. Ongoing research in our laboratory is focused on utilizing SNR₇₀₀-HOCl to detect AML in live animal models.

Figure 9.

PAI of SNR₇₀₀-HOCl in mice. Spectrally unmixed PA images were acquired from nude mice following subcutaneous injection on the right flank. Representative cross-sectional images (top) are shown from animals injected with 100 μL of 100 μM SNR₇₀₀-HOCl (a), 100 μL of 100 μM SNR₇₀₀-HOCl immediately followed by 100 μL of 250 μM HOCl (b), and saline (c). The bottom images in each panel represent maximal intensity projections of cross-sectional images from the mouse chest (left) to its hips (right). Arrows indicate the injection site. d) Quantified PA signal after multispectral unmixing within regions of interest from three subsequent cross-sectional images, spaced 1 mm apart, in each mouse from panel a (n=3 mice), b (n=4 mice), and c (n=3 mice). *** indicates a p-value of <0.001. The color scheme used in the PA images is as follows: blue represents hemoglobin, red represents oxyhemoglobin, and green represents activated SNR₇₀₀-HOCl.

Conclusions

In summary, we have demonstrated the ability of NR dyes to produce PA signal. Using rational design, we employed a single atom change to reduce the hydrolysis of the phosphinate ester in SNR₇₀₀ by 3.6-fold compared to the parent dye. This stabilized dye retains peak absorbance in the NIR for hours as well as cell permeability, enabling PAI using commercial instrumentation. Further building on this scaffold, we repurposed the well-established spiroring-opening absorbance off-on analyte detection strategy of xanthenes dyes to afford an acoustogenic probe for HOCl, a potential intracellular diagnostic marker for AML.^[55,56] PA turn-on signal from SNR₇₀₀-HOCl was capable of detecting clinically relevant concentrations of HOCl in tissue phantoms and provided imaging depths up to 2.9 cm in tissues. Lastly, we demonstrated the ability of SNR₇₀₀-HOCl to detect exogenous HOCl in mice. To the best of our knowledge, SNR₇₀₀-HOCl is the first cell permeable xanthene-based, acoustogenic probe for HOCl with maximal absorbance within the range of commercial PAI instrumentation. Future efforts in our lab are focused on increasing the signal-to-noise of NR-based acoustogenic probes for HOCl, which can be accomplished using modifications that decrease Φ,^[10,11,37] and their evaluation in animal models of AML. Such agents would reduce the amount of probe required to achieve observable signal and provide more sensitive detection of HOCl. In a larger context, this work provides design principles for stabilizing phosphinate esters in NR dyes to hydrolysis, providing the community with cell permeable NIR absorbing xanthenes that can be used for PAI with commercial instrumentation. We expect that the ability to leverage the lexicon of spiroring-opening absorbance off-on strategies for xanthene dyes,^[31,32] in the context of heteroatom xanthenes with NIR absorbance,^{[35,39,48,49,50,54,61]} will significantly expand the toolbox of acoustogenic probes for biologically relevant analytes.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.