Near-Infrared Photoacoustic Imaging Probe Responsive to Calcium

Oct 25, 2016 - (15, 16) However, with respect to responsive imaging probes for PAI, very few reports are available. For detecting specific enzymatic a...
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Near-infrared photoacoustic imaging probe responsive to calcium Anurag Mishra, Yuanyuan Jiang, Sheryl Roberts, Vasilis Ntziachristos, and Gil Gregor Westmeyer Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03039 • Publication Date (Web): 25 Oct 2016 Downloaded from http://pubs.acs.org on October 27, 2016

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Analytical Chemistry

Near-infrared photoacoustic imaging probe responsive to calcium Anurag Mishra*,†,‡, Yuanyuan Jiang†,‡,‫ال‬, Sheryl Roberts†,‡, Vasilis Ntziachristos †,ξ, Gil G. Westmeyer*, †,‡, ψ †

Institute for Biological and Medical Imaging, Helmholtz Zentrum München, 85764 Neuherberg, Germany. Institute of Developmental Genetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany. ξ Chair for Biological Imaging, Technical University of Munich, 80333 Munich, Germany. ψ Department of Nuclear Medicine, Technical University of Munich, 81675 Munich, Germany. ‡

‫ال‬

Present address: Max Planck Institute for Biological Cybernetics, Spemannstrasse 41, 72076 Tuebingen, Germany.

ABSTRACT: Photoacoustic imaging (PAI) is an attractive imaging modality that can volumetrically map the distribution of photoabsorbing molecules with deeper tissue penetration than multiphoton microscopy. To enable dynamic sensing of divalent cations via PAI, we have engineered a new reversible near infrared probe that is more sensitive to calcium as compared to other biologically relevant cations. The metallochromic compound showed a strong reduction of its peak absorbance at 765 nm upon addition of calcium ions that was translated into robust signal changes in photoacoustic images. Therefore, the heptamethine cyanine dye will be an attractive scaffold to create a series of metallochromic sensors for molecular PAI.

Divalent cations have several fundamental influences in many critical biological functions, including muscle contraction, vascular tone, nerve transmission, and many enzyme-mediated processes.1 Calcium (Ca2+) is prominently involved in synaptic activity,2 signalling of immune and neuroendocrine cells as well as in biomechanics of contractile tissue.3 Magnesium (Mg2+) ions play a critical role as enzyme cofactors in DNA synthesis4 and they also modulate signal transduction,5 and ion channels.6 Zinc (Zn2+) is a vital cofactor in metalloproteins required for the normal functioning of nervous tissue with unique importance in myelin synthesis. It is furthermore colocalized with glutamate in neurons7 and copackaged with insulin in vesicles that are released from beta cells in a Ca2+-dependent manner.8 Copper (Cu2+) is also involved in brain specific functions9 as well as an essential cofactor of important enzymes which are involved in redox reactions.10 Currently, fluorescence imaging is the preferred modality for measuring intracellular cations fluxes and various selective cation indicators exist that have led to valuable insights into cell signaling.11 However, the absorbance and emission spectra of most fluorescent indicators are in the visible wavelength range (350 to 650 nm) where substantial tissue absorption and scattering limits the penetration depth for optical imaging. In multiphoton techniques, (near)-infrared light is used to focally excite fluorophores and enable cellular resolution. However, due to scattering and absorption, the maximum imaging depth is still limited to ~1 millimeter.12,13 Fluorophores with (one-

photon) absorption and emission bands in the near-infrared (NIR) range enable imaging of their tissue distribution by tomographic techniques such as fluorescence molecular tomography (FMT). However, despite the use of sophisticated models for light propagation, the resolution of FMT is severely affected by light scattering.14 Thus, molecular imaging of cell signalling dynamics with high spatiotemporal resolution in tissue deeper than a few mm is still a formidable challenge. Photoacoustic Imaging (PAI) can overcome these constraints because its resolution is insensitive to light scattering and it can operate with NIR light to enable deep tissue penetration.15,16 This is possible as PAI maps the distribution of photoabsorbers in tissue by detecting ultrasound waves that are generated when absorbed photons are converted into local heat and cause thermoelastic expansion (photoacoustic effect).15,16 Photoacoustic images can be acquired at multiple wavelengths such that specific photoabsorbers can be identified based on their photoacoustic spectra. Imaging probes for PAI ideally have the absorbance peak in the near-infrared wavelength region where absorption of endogenous molecules such as hemoglobin is relatively weak. Since imaging volumes can furthermore be obtained without scanning, PAI can also achieve a higher temporal resolution than scanning or projection methods.16 For the practice of preclinical neuroimaging in rodents, this set of features allows for capturing a mouse brain in its entire dorsoventral extent (~7 mm) at up to tens of micrometers resolution and with each frame or volume acquired at tens of Hertz depending on the imaging geometry.17,18

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Scheme 1. Schematic representation of NIR Ca2+ sensing PAI probe, L, in its unbound and Ca2+-bound form.

To date, PAI of brain function has been mainly concerned with measuring changes in blood flow and oxygenation (hemodynamic changes) that are indirectly linked to neuronal activation via neurovascular coupling.17-20 However, photoacoustic imaging of molecular signals of brain activity has hardly been achieved. This is mainly due to the absence of dynamic imaging probes for PAI that change their photoacoustic signal as a function of an analyte of interest, preferably in a reversible manner. Based on the photoacoustic effect, such an analyte-dependent photoacoustic signal change can be obtained from a chromophore that alters its absorbance spectrum (chromism) in response to an analyte. Hence, an ideal chromophore for photoacoustic imaging should have high molar extinction coefficient, low quantum yield (to maximize nonradiative decay) and high photobleaching resistance. It has been shown that to obtain static contrast in PAI, a NIR dye such as indocyanine green (ICG) can be detected at nanomolar concentrations in tissue.15,16 However, with respect to responsive imaging probes for PAI, only very few reports are available. For detecting specific enzymatic activity, activatable probes were developed based on proteolyticallyinduced cellular uptake 21 or agglomeration of chromophorebearing peptides.22 Recently, we introduced a probe that was targeted to cell surface N-methyl-D-aspartate (NMDA) receptors from where it was outcompeted by addition of glutamate and imaged by PAI.23 In addition, pH-dependent photoacoustic signal changes were observed from a liposomal preparation of a croconaine dye 24 and from semiconducting polymer nanoparticles in response to reactive oxygen species.25 For metal detection, a Cu2+-responsive probe was reported in which Cu2+ activated a 2-picolinic ester bond cleavage resulting in an irreversible change of the photoacoustic spectra.26 Further-

Scheme 2. Synthesis and characterisation of L. Reagents and conditions: (a) K2CO3, anhydrous MeCN, 68%; (b) Fe, HCl, EtOH:H2O (9:1), 55%; (c) Ethyl bromoacetate, proton sponge, anhydrous DMF, 53%; (d) i) 1M BBr3 in CH2Cl2, ii) NaOH, THF:H2O (9:1), 25%; (e) NaH, anhydrous DMF:DMSO (9:1), 5%.

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more, a porphyrin-based probe and an optode-based nanosensor were used for detection of uranium and lithium in vivo via PAI, respectively.27,28 Herein, we bring forward a new NIR metallochromicimaging probe that changes its photoacoustic signal upon binding to Ca2+ and can be used to reversibly measure calcium fluctuation via PAI. We set out to create the NIR probe L, where the known Ca2+-chelating moiety [4 = APTRAmorpholinoamide (carboxymethyl) ({4‐hydroxy‐2‐[2‐ (morpholin‐4‐yl)‐2‐oxoethoxy]phenyl})amino]acetic acid)] is positioned onto the cyclohexenyl ring of a heptamethine cyanine dye (Scheme 1). The commercial NIR dye core based on heptamethine cyanine dye precursor (IR-780®) is well suited for PAI due to a high molar extinction coefficient and a low quantum yield.15,29 It has also been shown that the cyclicheptacyanine dye series are more photostable as compared to its indocarbocyanine counterpart (Cy-7).29 L was synthesized starting with the construction of the Ca2+-chelating moiety in 5 steps (Scheme 2). Stepwise alkylation of 5-methoxy‐2‐nitrophenol with 2-bromo‐1‐(morpholin ‐ 4‐yl)ethan‐1‐one in anhydrous MeCN afforded compound 1, where nitro was reduced by using carbonyl-Fe and catalytic amount of conc. HCl, to give 2 in 55% yield. This amine 2 was alkylated with ethyl bromoacetate in anhydrous DMF to give compound 3, from which the hydroxy 4 was obtained by subsequent deprotections using base hydrolysis of the ethyl groups and demethylation of aryl methyl ether by BBr3. The final PAI probe (L) was synthesised by O-alkylation of 4 onto IR-780 [λmax 780 nm] scaffold using NaH as base in anhydrous DMF:DMSO (9:1) with 5% yield and subsequently purified by reverse-phase HPLC. The negatively charged Ca2+-sensing unit enhances water solubility and generally reduces its tendency to bind nonspecifically to endogenous proteins.30 Therefore, the photophysical characterisation of L could be carried out in aqueous solution [pH 7.20, 294K]. The absorption spectrum of L showed a broad absorption band centred at 765 nm, with a secondary band around 695 nm (Fig. 1A). Upon exchanging Cl on IR-780® to -O (from 4), λmax hypsochromically shifted from 780 nm to 765 nm. The extinction coefficient (ε) of L at 765 nm was measured as 194,000 M-1 cm-1 (Fig. S2, ESI) which is comparable to the ε of cyclic-heptacyanine based NIR dyes with an intense π→π∗ (0→0 vibronic) transition. The shoulder at 695 nm is also π→π∗ mode, relating to the 0→1 or 0→2 vibronic transitions.31 The emission spectra of L was measured following excitation at 680 nm and a weak emission band was obtained that centered at 790 nm, giving a Stokes’ shift of ~25 nm (Fig. 1B). Similar dye structures reported previously were shown to have quantum yields that fall in between 5-10%.32 The high extinction coefficient in the NIR makes L an excellent PAI sensor because the background absorption (of oxygen-dependent photoacoustic changes due to hemoglobin) is relatively low in this wavelength range. In order to determine the Ca2+ responsive behaviour of L, we conducted a spectroscopic characterisation of L with various concentrations of free [Ca2+] in 30 mM 3-(N-morpholino) propane sulphonic acid (MOPS) buffer containing 100 mM KCl and different ratios of K2EGTA/CaEGTA (pH 7.2, 20 µM [L] ) at 310K. The absorbance peak amplitude of L decreased to half with increasing [Ca2+] (0 → 39 µM; Fig. 1A). Similarly, the fluorescence emission dropped to half upon excitation at 680 nm (Fig. 1B). The IC50 values were determined from

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the Ca2+-dependent change in the peak absorbance (765 nm) and fluorescence (790 nm) and calculated as 11.3 µM and 12.4 µM respectively [Fig. 1C (blue line) and Fig. S4, ESI). These values are lower than that of other APTRA (o-aminophenolN,N,O-triacetic acid) based molecules (22 µM for Ca2+),33 probably because we positioned the APTRAmorpholinoamide (donor) via a strong electron donating group (-O, present on para position to –N(CH2COOH)2) at the bulky chromophore (acceptor). We also observed a reduction of the molar extinction coefficient of L from 194,000 to 100,000 M1 cm-1 upon binding of one equivalent amount of Ca2+ (Fig. S2, ESI). We additionally monitored the stability of the absorbance readings from L and its Ca2+-adduct in buffer and observed only ~5% signal decrease over 2 h (Fig. S3 ESI). It is known that lone pair of electron in aniline derivative

Figure 1. Spectroscopic characterisation of L to determine Ca2+ sensitivity and selectivity over other biologically relevant metals. Changes in absorbance (A) and fluorescence spectra (B) (normalised) induced by various concentrations of free [Ca2+] (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30 and 39 µM) in 30 mM MOPS buffer containing 100 mM KCl (pH 7.2, 20 µM L) at 310K. The excitation wavelength was 680 nm. (C) Peak absorbance maxima (at 765 nm) measured from L (20 µM) upon addition of 50 µM [Ca2+], [Zn2+] and [Cu2+] in 30 mM MOPS, 100 mM KCl (pH 7.2, 310 K). (D) IC50 values for Ca2+ and Mg2+ were determined from the peak absorbance value at 765 nm in 100 mM KCl, 30 mM MOPS (20 µM L, pH 7.2, 310 K). The Ca2+ binding curve is shown in blue and the black curve shows the binding curve for Mg2+. (E) Absorbance at 765 nm obtained from L (20 µM) in 30 mM MOPS, 100 mM KCl (pH 7.2, 310 K), mixing with 20 µM (1 equi.) each of [Mg2+]/[Zn2+]/[Cu2+], after subsequent addition of 30 µM [Ca2+] and 60 µM EDTA. (F) L (20 µM) was added to Human Serum (HS) (pH 7.2, 310 K) and the absorbance was sequentially measured before and after mixing with 4 mM EDTA, after subsequent addition of 1 mM [Ca2+] and 1 mM EDTA.

are normally delocalised onto its aromatic ring upon protonation.34 Therefore, the lone pair of electron on -N atom in APTRA is expected to localise upon Ca2+ coordination which leads to a decrease in absorbance. Tethering an APTRAmorpholinoamide (donor) on IR-780 (acceptor) is thus expected to give similar delocalisation from the electron-rich ion-chelating moiety to the chromophoric dye moiety.35 According to this model, coordination of [Ca2+] reduces the absorbance of L because the electron-movement of the APTRAmorpholinoamide moiety is prevented, i.e. the -N lone pair of electron localises (Scheme 1). Furthermore, we have also examined the effect of H+ on the observed absorbance of L. There is only a marginal change in the absorbance spectrum of L (10 µM) upon a pH change from 5-8 ([Fig. S5, ESI). Therefore, within the physiological pH range, H+ should not confound the Ca2+-dependent absorbance changes. In addition, we examined the effect of the standard protein Bovine serum albumin (BSA) on the measured absorbance of L. Upon addition of 5 mM of BSA in 30 mM MOPS (pH 7.2, 294K), a similar halving of the absorbance (as without BSA) was observed in the absence and presence of 2 equivalents of [Ca2+] with L (10 µM) (Fig. S6, ESI). To evaluate the selectivity of L with Ca2+ over other biologically relevant divalent cations, we performed selectivity studies with individual divalent cations in buffer. Absorbance measurements of L (20 µM) were taken in solutions containing Zn2+ or Cu2+ (50 µM each) (Fig. 1C). As expected, due to the six-coordination site in the chelating unit of L, neither Cu2+ nor Zn2+ exhibit changes in the absorbance spectra, as both metals prefer 4-coordinate square-planar geometry, and are unable to bind cooperatively to the lone pair of electrons of -N and -O atoms on the phenyl. We also did not observe any significant signal change in the peak absorbance upon addition of biologically relevant mono- (Na+ and K+) and trivalent (Fe3+) ions (Fig. S8 ESI). However, we did observe binding of Mg2+ with an IC50 of 37.5 µM, which is about 3 times lower than that for Ca2+ (Fig. 1D and Fig. S7, ESI). In addition, we also tested whether Ca2+-dependent signal changes could be observed in the presence of other divalent metals both in MOPS buffer and human serum. Upon addition of 1 equivalent of [Mg2+]/[Zn2+]/[Cu2+], the absorbance peak amplitude decreased as compared to buffer and was further reduced by ~22% upon addition of Ca2+ (Fig. 1E and Fig. S9, ESI). Similarly, when EDTA was added to L mixed with human serum containing on average ~1.2 mM ionized Ca2+, a signal increase was observed indicating that metals were outcompeted from binding to L. Subsequent addition of 1 mM Ca2+ then lead to a ~40 % signal decrease that was reversible by further addition of EDTA (Fig. 1F and Fig. S10, ESI). We furthermore demonstrate that L can be used as a Ca2+sensing probe for PA Imaging. The probe was assessed in the absence and presence of different [Ca2+] concentration in 30 mM MOPS buffer [100 mM KCl, pH 7.20, 310K]. These experiments were conducted using a small animal PAI scanner (inVision 256-TF, iThera Medical GmbH, Munich, Germany). In brief, 40 µM of L was mixed with varying [Ca2+] concentrations [0 → 60 µM, (20 µM incremental points)] and the absorbance spectrum was measured. Each sample was then sequentially placed in a separate 3 mm diameter nonabsorbing plastic tube together with a reference sample containing no calcium. This was inserted at a defined position in a 20 mm diameter phantom made of 1.3% agar mimicking tissue

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scattering. Images were obtained with a tuneable laser set to different wavelengths (680-900 nm in 10 nm steps, with 50 averages per image). The images were reconstructed for each wavelength using a model-based reconstruction method and their intensity was normalized to the reference sample. As can be seen from the composite image in Figure 2A, there is a gradual decrease in the signal intensity of the sample (imaged at 760 nm) as a function of increasing [Ca2+] concentrations up to 60 µM. Addition of EDTA (80 µM) restores the signal to that of the metal-free probe by outcompeting Ca2+. Figure 2B shows the photoacoustic spectra plotted from the average pixel intensities obtained from a region of interest analysis of the photoacoustic images (solid lines). Corresponding absorbance spectra of the identical samples are displayed as broken lines (Fig. 2B insert); the inset shows the correlation of the peak signals from the absorbance and PA spectra (r = 0.99). We also imaged L in human serum and confirmed that the sensor’s response could be reversed by addition of EDTA (Fig. S11, ESI).

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX General and synthetic description of compounds with twelve figures showing HPLC chromatogram and detailed characterizations of L.



In summary, we have devised and characterised the first reversible NIR Ca2+ sensing probe for PA imaging. The engineered probe strongly absorbed light in the NIR range which is ideal for PAI methods since the background absorption and signal changes due to hemodynamics are relatively low in this wavelength range. The compound showed a robust reduction in absorbance upon addition of Ca2+ that translated into a strong decrease in the PA signal. The sensor showed a three-times higher affinity for Ca2+ over Mg2 which can be further tuned by changing the chelating moiety and/or altering its positioning on the chromophore e.g. via cyclohexene-oxygen or directly on the cyclohexene. [22] The IR-780 scaffold we introduced here could thus serve as a platform to generate a series of NIR metallochromic sensors for molecular imaging of specific divalent cations in deep tissue via photoacoustic tomography.



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AUTHOR INFORMATION

Corresponding Authors * Email: [email protected] (AM) * Email: [email protected] (GGW) Notes: The authors declare no competing financial interest.



ACKNOWLEDGMENT

We thank the European Research Council under grant agreements ERC-StG: 311552 (GGW), the Helmholtz Alliance ICEMED (AM, GGW), and the HMGU ERC recognition award (AM). We thank Panagiotis Symvoulidis for helpful comments on technical aspects of the manuscript.



Figure 2. Photoacoustic signal changes of L for varying [Ca2+] and its subsequent reversibility upon addition of EDTA. (A) 40 µM L in aqueous solution (30 mM MOPS, 100 mM KCl, pH 7.2, 310 K) was mixed with 0 → 60 µM of [Ca2+] and 60 µM [Ca2+] + 80 µM [EDTA] and filled into 3 mm diameter tubes that were inserted in a 20 mm diameter phantom made of 1.3% agar. Photoacoustic images were acquired for different wavelengths (680 to 900 nm in 10 nm steps, 9 ns laser pulses at a repetition rate of 10Hz, 50 averages per image). (B) Corresponding photoacoustic spectra (solid lines) extracted from the average pixel intensities of circular region of interests (ROIs) covering the samples on the photoacoustic images are plotted together with absorbance spectra (broken lines) obtained for each of the [Ca2+]. Color coding of the conditions is used as in (A). The inset shows the correlation between the absorbance and photoacoustic peak values.

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We have synthesised the first reversible near-infrared calciumsensing probe for photoacoustic imaging that showed a strong reduction in absorbance upon addition of Ca2+ that translated into robust contrast changes in photoacoustic images.

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