Azaphosphinate Dyes: A Low Molecular Weight Near-Infrared Scaffold for Development of Photoacoustic or Fluorescence Imaging Probes

Abstract

Near-infrared (NIR) dyes are desirable for biological imaging applications including photoacoustic (PA) and fluorescence imaging. Nonetheless, current NIR dyes are often plagued by relatively large molecular weights, poor water solubility, and limited photostability. Herein, we provide the first examples of azaphosphinate dyes which display desirable properties such as low molecular weight, absorption/emission above 750 nm, and remarkable water solubility. In PA imaging, an azaphosphinate dye exhibited a 4.1-fold enhancement in intensity compared to commonly used standards, the ability to multiplex with existing dyes in whole blood, imaging depths of 2.75 cm in a tissue model, and contrast in mice. An improved derivative for fluorescence imaging displayed a > 10-fold reduction in photobleaching in water compared to the FDA-approved indocyanine green dye and could be visualized in mice. This new dye class provides a robust scaffold for the development of photoacoustic or NIR fluorescence imaging agents.

Graphical Abstract

We present a new class of NIR dyes termed azaphosphinate NR dyes with desirable properties including low molecular weight, absorption/ emission above 750 nm, and remarkable water solubility. Derivatives of these dyes can be used for photoacoustic or fluorescence imaging, providing a new NIR scaffold for the design of imaging probes.

Introduction

Small molecule dyes with absorption and emission maxima in the 680–980 nm range of the NIR are highly desirable for bioimaging applications due to decreased background from endogenous chromophores and increased tissue penetration in this wavelength range.^[1,2,3,4] Although several efforts have identified promising new NIR scaffolds,^{[5,6,7,8,9,10,11,12,13]} current dyes within the 680–980 nm range often suffer from relatively high molecular weights, poor water solubility, and decreased chemical and photostability.^[14,15] Indeed, the classic FDA-approved dyes within this wavelength range, indocyanine green (ICG) and methylene blue (MB, Figure S1), suffer from these issues. For example, sulfonation is required to increase the water solubility of ICG. However, this modification does not entirely suppress aggregation of this dye. Moreover, the flexible nature of the polymethine chromophore within this dye renders it susceptible to excited state photochemistry that limits dye photostability.^[16] On the other hand, MB is a relatively low molecular weight thiazine dye scaffold with emission tailing into the NIR region. Although this dye has been widely used for imaging purposes,^{[17,18,19,20,21,22]} its tendency to form H-type dimers in aqueous solution^{[23,24,25,26,27]} limits its utility for imaging applications. Thus, there is a need for continued development of next generation NIR dyes with improved properties. The availability of such dyes will ultimately improve capabilities in emerging areas of biomedical imaging.

Photoacoustic (PA) and NIR fluorescence imaging represent two such emerging imaging techniques. In the case of PA imaging, pulsed NIR excitation of a dye results in the production of pulsed sound waves through the PA effect.^[28] As opposed to fluorescence imaging, PA provides increased imaging depth due to reduced scattering of sound in tissues.^[1,3,29] Although recent efforts have identified elegant approaches to enable turn-on (or acoustogentic) signal generation from small molecule PA probes,^{[30,31,32,33]} PA imaging is still limited by the relative lack of diversity in robust scaffolds that can be used to design PA probes. Alternatively, NIR fluorescence imaging relies on monitoring the emission of NIR absorbing and emitting fluorophores, providing increased tissue penetration relative to dyes in the visible spectral region. Since both PA and NIR fluorescence imaging rely on the availability of robust NIR absorbing dyes, both techniques are limited by the previously mentioned issues of aggregation, relatively large molecular weight, and reduced chemical and photostability of NIR dyes.^[14,15] Consequently, chemistry-focused efforts to expand the toolbox of low molecular weight, robust NIR dyes are needed to ultimately advance PA as well as fluorescence imaging.

While ICG and MB have been FDA-approved for over 50-years and extensively utilized in both PA and NIR fluorescence imaging, probe development has been hindered by the inherent photophysical and chemical limitations of these dyes. Notably, the structure of MB aggregates have been extensively studied and indicates that an intermolecular sulfur-nitrogen dipole-dipole interaction and π-π stacking of the chromophore leads to dimer formation (Figure 1a).^[34,35,36] Given the low molecular weight and desirable chemical and photostability of MB, we were intrigued by the idea of suppressing aggregation of this scaffold while simultaneously red-shifting excitation and emission into the NIR range.

Figure 1.

a) A previously reported structure of MB dimers in aqueous solution. b) The core azaphosphinate dye from this work. Phosphinate substitution inhibits dye aggregation.

Substitution of the bridging oxygen atom in xanthene scaffolds with relatively electron-withdrawing functionalities such as silicon^[37,38,39] and phosphorus^[40,41,42] substituents results in a decrease in HOMO-LUMO energy gaps and a substantial red-shift in absorbance and emission maxima of the resulting dyes. Our lab has previously developed a series of phosphinate-containing xanthenes, termed Nebraska Red (NR) dyes.^[40,41] These dyes display > 100 nm red-shifts in absorbance and emission compared to the parent dyes, and can be used for various applications in chemical biology including self-reporting cargo delivery, targeted toxicity in cancer cells, and imaging.^[41,43,44] More recently, the Miller group has extended the approach of heteroatom substitution to oxazine dyes, which are structurally related to MB, yielding dimethylsilyl-containing azasiline dyes.^[45] The resulting azasiline dyes are red-shifted by 57–83 nm relative to the parent oxazine. This seminal work clearly demonstrates the potential to red-shift thiazine-type scaffolds using the bridging atom replacement strategy. However, an increase in the quantum yield of these dyes in aqueous solutions was observed upon detergent addition, indicating that the dimethylsilyl group does not fully inhibit aggregation of this scaffold. Inspired by this work, we hypothesized that the sp³ hybridized and negatively charged phosphinate functionality may result in more potent disruption of dye aggregation in this family (Figure 1b). Herein, we show that phosphinate substitution in the MB scaffold virtually abolishes aggregation in aqueous samples. Furthermore, the resulting azaphosphinate dyes display absorbance and emission above 750 nm and improved photostability compared to ICG. Derivatives of this new scaffold provide either PA or fluorescence readouts in living cells and mice. Thus, this robust, low molecular weight scaffold provides a new template for construction of improved PA and fluorescence imaging probes.

Results and Discussion

Design and Synthesis of Azaphosphinate Dyes.

Work from the Miller group indicated that the inclusion of methyl groups at the 4 and 6 positions of azasiline dyes increased their stability.^[45] Accordingly, we envisioned the construction of a tetramethyl azaphosphinate NR derivative (Scheme 1). Before embarking on a synthetic campaign, we utilized recently published trendlines from the calibration of computational estimations of absorbance and emission to experimental parameters for xanthene-based NR dyes.^[46] This calibrated computational data enables rapid prediction of NIR dye absorbance and emission with mean percent errors of 2.2 and 2.8%, respectively. Using this approach, we predicted absorbance and emission maxima of 715 and 732 nm, respectively, for NR₇₅₁ (see Supporting Information). Confident that phosphinate substitution would induce a substantial red-shift in excitation and emission, we began the synthesis of this new dye by reductive amination with 3-bromo-5-methylaniline yielding the dimethylaniline intermediate 1. Lithiation of dimethylaniline 1 and subsequent treatment with ethyl dichlorophosphate furnished the phosphinate ester 2. Lastly, nitrosation-cyclization^[47] and simultaneous ester cleavage yielded the final dye.

Scheme 1.

Synthesis of NR₇₅₁.

Photophysical Characterization of NR₇₅₁.

Next, we measured the photophysical properties of our new NR dye. Gratifyingly, in PBS the dye showed an absorbance and emission maxima of 751 and 779 nm, which is 87 and 94 nm red-shifted compared to MB, respectively (Figure 2a and b). Moreover, these experimental values correspond to 4.8 and 6% errors in computationally predicted absorbance and emission, respectively, and indicate the ability to utilize our experimentally calibrated computational dataset^[46] to predict absorbance and emission of structurally diverse dyes. Based on the absorbance maxima of this dye, we named it NR₇₅₁ to be consistent with previous NR dyes.^[40,41] Next, we measured the molar extinction coefficient (ε), quantum yield (Φ), and fluorescence lifetime (τ) of NR₇₅₁ in PBS (Figure 2b). NR₇₅₁ exhibited a low quantum yield of 0.4%, indicating significant non-radiative relaxation from the excited state. Since PA signal generation relies on non-radiative relaxation to induce local pressure increases through thermal expansion events,^[30,33,48] we surmised that NR₇₅₁ may be a useful PA agent. In support of this notion, we calculated the nonradiative decay rate (k_nr) for NR₇₅₁ using the observed Φ and τ.^[49] Indeed, NR₇₅₁ exhibits a rapid nonradiative decay rate of 1.42×10¹⁰ s⁻¹ which is 5.4-fold faster than MB (2.63×10⁹ s⁻¹). These results indicated that NR₇₅₁ may show promise as a PA agent.

Figure 2.

a) Normalized absorption and emission spectra of NR₇₅₁ in PBS. b) Photophysical properties of NR₇₅₁ in PBS.

Water Solubility of NR₇₅₁.

Prior to the analysis of PA signal generation by NR₇₅₁, we investigated the ability of the phosphinate functionality to suppress aggregation in NR₇₅₁. Gratifyingly, the absorbance of increasing concentrations of NR₇₅₁ in PBS yielded a linear relationship up to at least 200 μM (Figure 3a). Alternatively, the absorbance of MB showed a clear deviation from linearity with increasing concentration (Figure 3a), a characteristic signature of aggregation in MB and other small molecule dyes.^{[23,24,50,51]} Moreover, solutions containing increasing concentrations of MB displayed increased intensity of a blue-shifted absorbance band at 605 nm, which has been assigned to H-aggregates of MB (Figure 3b).^[50] Addition of 1% Tween-20 suppressed the 605 nm absorbance peak of MB while the absorption spectra of NR₇₅₁ remained virtually unchanged under identical conditions (Figure S2). These experiments demonstrate that the phosphinate functionality can red-shift the absorbance and emission of azaphosphinate NR dyes, and effectively abolish aggregation in relevant concentration regimes used for imaging.

Figure 3.

a) The relationship between concentration and absorbance for MB and NR₇₅₁ in PBS. b) Normalized absorbance spectra for increasing concentrations of NR₇₅₁ and MB in PBS.

Solvatochromic Nature of NR₇₅₁.

To investigate the solvatochromic properties of NR₇₅₁, we measured its photophysical properties in a panel of solvents (Figure S3). Interestingly, a solvent-dependent shift in absorbance and emission maxima was observed (Figure 4a and b). Peak absorbance and emission ranged from 727–751 nm and 743–772 nm, respectively, and correlated with the solvent polarity parameter E_T(30)^[52] (Figure 4c). The brightest fluorescence emission (defined as ε x Φ) was observed in dichloromethane (35-fold increase compared to water, Figure 4d and e). These results indicate the potential of NR₇₅₁ as a solvatochromic indicator.

Figure 4.

Normalized absorbance (a) and emission (b) spectra of NR₇₅₁ in the indicated solvents. c) The correlation between either NR₇₅₁ absorbance (red) or emission (blue) maxima in wavenumber versus E_T(30) for each solvent. d) Fluorescence brightness of NR₇₅₁ in the indicated solvent. e) Photophysical properties of NR₇₅₁ in the indicated solvent.

PA Signal Generation from NR₇₅₁.

Having fully characterized the spectral and physical properties of NR₇₅₁, we evaluated the ability of this new dye to produce PA signal. PA signal from PBS solutions containing increasing concentrations of NR₇₅₁ in a tissue-mimicking phantom consisting of 5% agarose and 2.5% milk were linear to at least 50 μM (Figure S4). As anticipated, the PA signal from NR₇₅₁ overlaid with the absorbance profile for the dye, producing maximal signal at 750 nm (Figure 5a and b). Next, we compared the PA signal from NR₇₅₁ to PA-HD (Figure S5a), a hemicyanine based PA scaffold with similar absorbance profile to NR₇₅₁,^[53] in aqueous solutions containing BSA or FBS. NR₇₅₁ displayed significantly increased PA intensity in PBS compared to PA-HD (4.8-fold, Figure S5b). This trend of increased PA intensity of NR₇₅₁ compared to PA-HD was maintained in 5% BSA (1.7-fold, Figure S5c) and FBS (1.4-fold, Figure S5d), although the presence of protein slightly reduced PA signal from NR₇₅₁ while enhancing signal from PA-HD. Additionally, NR₇₅₁ displayed enhanced PA photostability in aqueous solutions containing protein (13.1-fold in PBS with 5% BSA and 3.5-fold in FBS) compared to PA-HD (Figure S6).

Figure 5.

a) PA images of PBS solutions containing 50 μM NR₇₅₁ (left) or no dye (right) in tissue-mimicking phantoms consisting of 5% agarose and 2.5% milk. b) Overlay of normalized PA and absorbance spectra of NR₇₅₁. c) PA spectra from the indicated dyes (50 μM each) in tissue-mimicking phantoms.

Although MB is a commonly used standard in PA imaging its absorbance maximum lies outside of the range of commercial instrumentation (Figures S1 and S2). PA signal intensity of equivalent concentrations of NR₇₅₁ and MB demonstrated that NR₇₅₁ produced a 4.1-fold higher PA signal compared to MB using commerical PA imaging instrumentation (Figure 5c). Additionally, we observed a 10% increase in PA signal from NR₇₅₁ relative to ICG and a more defined PA spectra for NR₇₅₁ (potentially due to aggregation of ICG, Figure 5c). Broad absorbance spectra can complicate spectral unmixing during multiplexed imaging. Taken together, these data demonstrate that the increased solubility and structural rigidity of azaphosphinate dyes can be leveraged to afford probes with improved PA signal output and photostability in aqueous media compared to conventional dyes.

Multiplexed PA Imaging with NR₇₅₁.

Encouraged by the ability of NR₇₅₁ to produce relatively intense PA signal with well-defined features, due to increased solubility, we asked whether MB and NR₇₅₁ PA signals could be spectrally unmixed. As a proof-of-principle for multiplexed PA imaging with NR₇₅₁ and MB, we prepared a two-channel tissue-mimicking phantom for simultaneously imaging samples of each dye. First, we investigated the ability to distinguish between NR₇₅₁ or MB and PBS. Indeed, spectral unmixing yielded images in which the signal from each dye could be clearly distinguished (Figure S7a and b). To provide a more physiologically relevant test, we also asked whether solutions of NR₇₅₁ or MB could be imaged in defibrinated sheep blood. Here too, the PA signal from each dye could be resolved from oxyhemoglobin using spectral unmixing (Figure S7c and d). Lastly, we investigated the ability to simultaneously distinguish between PA signal from NR₇₅₁ or MB in defibrinated sheep blood. Indeed, we were able to resolve the PA signal from each dye (Figure 6). These experiments provide a proof-of-principle for multiplexed PA imaging with NR₇₅₁ and MB.

Figure 6.

a) Spectral unmixing of 100 μM NR₇₅₁ (green) or MB (magenta) PA signal in defibrinated sheep’s blood using a two-channel tissue mimicking phantom consisting of 5% agarose and 2.5% milk. b) Mean pixel intensity of NR₇₅₁ and MB within the indicated region of interest (red circle) from panel a.

PA Imaging in Tissues.

Tissue imaging is an established approach to evaluate the performance of PA reagents since tissues simulate optical and acoustic parameters found in living animals (optical absorption and scattering, speed of sound, acoustic attenuation, acoustic backscatter coefficient, etc.).^[54,55,56] To determine the maximal imaging depth of NR₇₅₁ in tissues, solutions of 50 μM dye in PBS were placed at the center of chicken breast cylinders of increasing thickness (Figure S8). PA imaging of these samples resulted in a gradual decrease in PA signal with increasing tissue depth, yielding an imaging depth of 2.75 cm for NR₇₅₁ under these conditions (Figure 7). This data demonstrates that azaphosphinate NR dyes provide a robust scaffold for the development of PA imaging reagents.

Figure 7.

a) PA images of PBS solutions with or without 50 μM NR₇₅₁ in chicken breast cylinders of increasing thickness. b) Average PA signal intensity from 7 positions, scanned in triplicate within the indicated region of interest (red circle) from panel a. **** indicates p<0.0001 (two sample independent t-test). c) PA signal-to-noise ratios (SNR) at the indicated imaging depths.

PA Imaging in Living Mice.

Encouraged by the ability to image NR₇₅₁ in the above tissue model, we investigated the ability to resolve signal from NR₇₅₁ in living mice. First, we assessed the toxicity of NR₇₅₁ in cell culture (Figure S9) as well as compared singlet oxygen genration from NR₇₅₁ to MB (Figure S10). We did not observe cellualr toxicity at concentrations used in this study up to 24 hrs in cell culture nor did we observe evidence for singlet oxygen genration from NR₇₅₁ under the conditions used herein. Accordingly, we administered subcutaneous injections of NR₇₅₁ (1 mg/kg) in the right abdomen. The mice were then scanned from the chest to the abdomen. The PA signal from NR₇₅₁ could be clearly distinguished from endogenous absorbers such as hemoglobin and oxyhemoglobin by employing a linear-regression, spectral unmixing approach (Figure 8a and b). Additionally, quantified PA signal from NR₇₅₁ in the injection region, for n = 3 mice in each group, indicated a 93.3-fold increase in PA signal in mice injected with NR₇₅₁ compared to saline alone (Figure 8c). These experiments demonstrate the ability to resolve PA signal from NR₇₅₁ in living mice.

Figure 8.

Representative cross-sectional (a) or maximal intensity projections of cross-sectional images from the mouse chest (top) to abdomen (bottom, b) PA images of nude mice following subcutaneous injection of 100 μL saline without (left) or with 1 mg/kg NR₇₅₁ (right). c) Average PA signal intensity for NR₇₅₁ after spectral unmixing within the indicated region of interest (red circle) from panel a (n = 3). *** indicates p<0.001 (two-tailed independent t-test).

Enhancing Azaphosphinate Fluorescence.

Having demonstrated the utility of NR₇₅₁ as a PA imaging probe, we turned our attention toward improving the fluorescence properties of this dye. Given the remarkable water solubility of NR₇₅₁, we hypothesized that twisted intramolecular charge transfer (TICT) may be suppressing the Φ of this scaffold.^{[57,58,59,60]}

Therefore, we set out to inhibit TICT using the well-established azetidine auxochrome.^[60] An azetidine-containing derivative, termed NR₇₅₁-Az, was synthesized by modifying published procedures for azasiline dyes (Scheme 2).^[45] Briefly, the starting material 1-(3-bromo-5-methylphenyl) azetidine was prepared via Buchwald-Hartwig amination. Phosphinate amide 3 was furnished by lithiation followed by addition to diethylphosphoramidous dichloride and subsequent oxidation with hydrogen peroxide in situ. After dibromination of phosphinate amide 3 with 1,3-dibromo-5,5-dimethylhydantoin (DDH), the resulting intermediate 4 was subjected to lithiation followed by addition to isopentyl nitrite and iron (II) chloride catalyzed reduction in situ. Finally, NR₇₅₁-Az was obtained by dehydrogenation of the precursor followed by phosphine amide hydrolysis under acidic conditions. We then measured the spectroscopic properties of this new dye. Similar to NR₇₅₁, NR₇₅₁-Az exhibited an absorption maximum of 751 nm and an emission maximum of 775 nm (Figure 9a and b). However, the Φ increased by 2-fold to 0.8%, leading to a 1.8-fold increase in fluorescence brightness compared to NR₇₅₁ (Figures 2b and 9b). This increase in quantum yield resulted in a 2.8-fold decrease in PA signal from NR₇₅₁-Az compared to NR₇₅₁ (Figure S11), presumably due to decreased vibrational relaxation of NR₇₅₁-Az.^[30] Thus, in addition to acting as PA probes, azaphosphinate NR dyes also provide a novel scaffold for the development of robust NIR fluorescent imaging probes.

Scheme 2.

Synthesis of NR₇₅₁-AZ.

Figure 9.

a) Normalized absorbance and emission spectra of NR₇₅₁-Az in PBS. b) Photophysical properties of NR₇₅₁-Az in PBS.

Chemical- and Photo-Stability of Azaphosphinate Dyes.

Based on the structural rigidity of the azaphosphinate NR scaffold and the ability of azetidine auxochromes to increase photostability,^[60] we hypothesized that both NR₇₅₁ and, to a greater extent, NR₇₅₁-Az would display significantly enhanced chemical and photostability. Therefore, we first evaluated the chemical stability of azaphosphinate NR dyes. Both dyes exhibited virtually identical absorbance spectra across a range of biologically relevant pH values (4–9) over 4 hours (Figure S12). To investigate the stability of these dyes in the presence of biologically relevant reactive oxygen/nitrogen species (ROS/RNS), we conducted stability assays in the presence of HOCl, H₂O₂, TBHP, ONOO⁻, ·OH, · OtBu, and NO. The results revealed that NR₇₅₁ and NR₇₅₁-Az maintained their absorbance for a minimum of 12 hours in the presence of the majority of ROS/RNS in our panel (Figure S13). However, in the presence of ONOO⁻, we observed a 22 and 58% decrease in absorbance for NR₇₅₁ and NR₇₅₁-Az, respectively (Figure S13). Next, we examined the stability of NR₇₅₁ and NR₇₅₁-Az in commonly used cell culture components. Within 4 hours of exposure to DMEM or FBS, NR₇₅₁ displayed a 54 or 32% decrease in absorbance in the respective media (Figure S14). Conversely, NR₇₅₁-Az displayed relatively enhanced stability in both DMEM and FBS. Finally, we also investigated the photostability of azaphosphinate NR dyes. As a comparison, we chose the FDA-approved dye ICG. Indeed, NR₇₅₁ displayed a 10.0-fold longer photobleaching half-life compared to ICG in PBS, while virtually no fluorescence decay was observed for NR₇₅₁-Az (Figure S15). However, we observed that the half-life of NR₇₅₁ photobleaching was accelerated in samples containing protein (FBS or BSA) presumably due to protein binding (Figure S15b and c). The azetidine auxochromes in NR₇₅₁-Az virtually abolished photobleaching in BSA and increased the photobleaching half-life relative to NR₇₅₁ by 2.8-fold in FBS (Figure S15b and c). Overall, these experiments demonstrate that azaphosphinate NR dyes are well suited for biological imaging applications.

NIR Fluorescence Microscopy with NR₇₅₁-Az.

As a preliminary assessment of the utility of azaphosphinate NR dyes for fluorescence imaging, we investigated the ability to stain cellular organelles using confocal microscopy. For this purpose, we chose NR₇₅₁-Az due to its optimized fluorescence brightness. Previous work from our lab has shown that phosphinate dyes are typically membrane impermeable,^[40,41,43] likely due to the negatively charged phosphinate. Indeed, treatment of HeLa cells with NR₇₅₁-Az resulted in no observable cellular fluorescence from NR₇₅₁-Az (Figure 10a), further highlighting the membrane impermeable nature of phosphinate dyes. We have previously shown that phosphinate esterification can dramatically increase the membrane permeability of NR dyes,^{[40,41,43,44]} however initial studies of a phosphinate ethyl ester-containing derivative of NR₇₅₁ demonstrated virtually instantaneous phosphinate ester hydrolysis in water (Figure S16). We are currently investigating approaches to further stabilize phosphinate esters in azaphosphinate NR dyes using structural modifications, in a similar fashion to stabilized esters in rhodamine-based NR dyes.^[41,44] As an alternative approach, we were inspired by the formation of complexes between SDS and MB,^[61] and hypothesized that a cationic lipid such as cetyltrimethylammonium bromide (CTAB) could form a complex with NR₇₅₁-Az similarly to other dyes.^[62,63,64] We envisioned that the resulting CTAB/dye complex would mask the negative charge on NR₇₅₁-Az, thereby increasing its permeability to cellular membranes. Indeed, the NR₇₅₁-Az/CTAB complex, termed NR₇₅₁-Az-CTAB, significantly enhanced the membrane permeability of NR₇₅₁-Az (Figure 10b). Importantly, the concentration of CTAB used in this study was not toxic to HeLa cells (Figure S17). Co-treatment of HeLa cells with NR₇₅₁-Az-CTAB and organelle trackers indicated that NR₇₅₁-Az fluorescence was mainly localized in mitochondria (Figure 10c and Figure S18). While the structure and mechanism of cellular uptake of the CTAB/dye complex is currently under investigation, these data demonstrate the ability to deliver phosphinate-containing dyes to living cells and the potential of NR₇₅₁-Az for organelle labeling and NIR fluorescence imaging applications.

Figure 10.

Confocal microscopy images of HeLa cells incubated with 20 μM NR₇₅₁-Az (a) or NR₇₅₁-Az-CTAB (b). c) Confocal microscopy images of the indicated organelle tracker and 50 μM NR₇₅₁-Az-CTAB. Merged images show predominant localization to the mitochondria. Scale bars = 20 μm.

NIR Fluorescence Imaging with NR₇₅₁-Az in Living Mice.

To demonstrate the ability to visualize NR₇₅₁-Az fluorescence in a more complex system, we injected saline solutions without or with 50 μM NR₇₅₁-Az intramuscularly in the hindlimbs of live mice and imaged immediately following injection. Indeed, a clear 5.0-fold increase in fluorescence from hindlimb sites injected with NR₇₅₁-Az was observed compared to saline (Figure 11 and Figure S19). These results indicate that the quantum yield enhancement in NR₇₅₁-Az can be leveraged for fluorescence imaging in mice.

Figure 11.

a) A representative fluorescence image of a nude mice with intramuscular injection sites for saline (left hindlimb) or 50 μM NR₇₅₁-Az in saline containing 0.5% DMSO (right hindlimb) indicated. b) Average fluorescence intensity within the indicated region of interest (red circle) from panel a (n = 3 mice in each group). Mice were imaged immediately following injection. *** indicates p<0.001 (two-tailed independent t-test).

Conclusions

We have rationally designed a low molecular weight dye with NIR absorption and emission in the 750 nm range. Our design strategy, which employed the incorporation of a phosphinate functionality into the oxazine scaffold, not only red-shifted the absorbance and emission of the resulting dye but also dramatically suppressed aggregation in aqueous solutions. To the best of our knowledge, NR₇₅₁ is the lowest molecular weight, water soluble dye with absorbance and emission maxima in this spectral range.

Leveraging the improved water solubility of NR₇₅₁, we demonstrated that this dye scaffold can produce a robust PA signal. Indeed, NR₇₅₁ produces a 4.1- and 1.1-fold stronger PA signal on commercial instrumentation than currently used standards such as MB and ICG. Given the well-defined PA signal profile of this dye, multiplexing with currently available PA probes, such as MB, in blood samples is possible. Moreover, in its current form, NR₇₅₁ can achieve 2.75 cm imaging depths in tissue samples and can be visualized in live mice. Our lab is currently pursuing the use of NR₇₅₁ as a scaffold for developing in vivo PA imaging probes, activatable PA probes, and more intense PA reagents.

Lastly, we demonstrated the ability to increase the fluorescence brightness of NR₇₅₁ through the incorporation of azetidine auxochromes, yielding NR₇₅₁-Az. This improved fluorescent dye displays reduced photobleaching compared to ICG in water. We further developed a protocol for the delivery of NR₇₅₁-Az into living cells for fluorescence microscopy applications and demonstrated the ability to resolve this dye in live mice. Our laboratory is currently pursuing targeted NR₇₅₁-Az probes for animal imaging, activatable probes for analyte sensing, and approaches to further increase the fluorescence brightness of this scaffold.

In summary, we report a new class of azaphosphinate NR dyes with desirable properties that can be leveraged for either PA or NIR fluorescence imaging. Ultimately, the choice of a water soluble dye, such as the azaphosphinates described herein, or a more hydrophobic dye such as ICG will depend on the imaging application. In certain cases, the enhanced fluorescence and photostability of ICG^[65,66] upon protein binding may be useful for imaging structures that are readily accessible in the blood stream. However, protein binding may complicate the design of probes for intracellular imaging applications, limit visualization of spatially restricted areas, or hinder reaction with handles for analyte sensing or in vivo labeling. We anticipate that the availability of this new, highly water soluble dye class will stimulate the design of robust imaging probes to illuminate biology. We suspect that other dye scaffolds may also benefit from the coarse tuning and solubility-enhancing effects of the phosphinate functionality.

Data Availability Statement

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