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. Author manuscript; available in PMC: 2020 Sep 27.
Published in final edited form as: ACS Sens. 2019 Aug 22;4(9):2412–2419. doi: 10.1021/acssensors.9b01010

Peroxidase Sensitive Amplifiable Probe for Molecular Magnetic Resonance Imaging of Pulmonary Inflammation

Aurora Rodríguez-Rodríguez 1, Sergey Shuvaev 1, Nicholas Rotile 1, Chloe M Jones 1, Clemens K Probst 2, Diego Dos Santos Ferreira 1, Katherine Graham-O´Regan 1, Eszter Boros 1, Rachel S Knipe 2, Jason W Griffith 2, Andrew M Tager 2,‡, Alexei Bogdanov Jr 3, Peter Caravan 1,*
PMCID: PMC6924005  NIHMSID: NIHMS1045747  PMID: 31397156

Abstract

An amplifiable MRI probe that combines the stability of the macrocyclic Gd-DOTAGA core with a peroxidase-reactive 5-hydroxytryptamide (5-HT) moiety is reported. The incubation of the complex under enzymatic oxidative conditions led to a 1.7-fold increase in r1 at 1.4 T that was attributed to an oligomerization of the probe upon oxidation. This probe, Gd-5-HT-DOTAGA, provided specific detection of lung inflammation by MRI in bleomycin injured mice.

Keywords: Gadolinium, MRI, contrast agent, pulmonary fibrosis, 5-hydroxytryptamide

Graphical Abstract

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Magnetic Resonance Imaging (MRI) is one of the most powerful diagnostic modalities in medicine. In order to improve imaging contrast and specificity for particular pathologies, responsive contrast agents, whose signal is modulated in the presence of an external stimuli, such as a specific biomarker or a biological event, are highly desirable. A number of responsive agents have been described that potentially can report on ion flux, pH, enzymatic activity, chemical potential (redox), and temperature in the body in a noninvasive manner.1 For contrast agents that shorten T1 of bulk water, the relaxivity enhancement is modulated by either a change in the hydration number, water residence time, and/or in the rotational dynamics of the complex.2 For a successful clinical translation, MRI contrast agents should have high solubility in water and exhibit sufficiently high kinetic stability to prevent gadolinium release in vivo. Gadolinium release has been associated with nephrogenic systemic fibrosis in patients with acute or chronic kidney disease,3 and there is concern about gadolinium retention in the body after injection, especially when more labile complexes are used.4–5

Inflammation is a common response to tissue injury and is a characteristic of many acute and chronic diseases such as heart attack, stroke, and many cancers. In the lungs, inflammation can arise from infection, cancer, and lung injury such as acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. In ARDS, patients with severe inflammation were shown to have worse outcomes.6–8 In pulmonary fibrosis, severe inflammation is associated with acute exacerbations which can portend rapid decline and death.9–11 The ability to noninvasively determine the presence, the distribution, and the degree of lung inflammation would be valuable for prognosis to select these patients for more aggressive therapies to treat their acute condition.

During the onset of the inflammatory process, plasma proteins and leukocytes leak into the extravascular space of the injured areas. In the lungs, leukocytes (neutrophils, eosinophils, and alveolar macrophages) are activated and release oxidative enzymes, e.g. myeloperoxidase (MPO) and eosinophil peroxidase (EOP), that can generate reactive oxygen species (ROS).12 MPO and EPO are heme-containing enzymes that stimulate migration of fibroblasts and promote biosynthesis of collagen extracellular matrix at sites of normal tissue repair and fibrosis.13 In the presence of H2O2, the peroxidase enzyme can be oxidized to generate hypochlorous acid (HOCl) or to catalyze the oxidation of aromatic fragments through one electron oxidation.14 It has been previously reported by Bogdanov and coworkers that gadolinium complexes bearing a 5-hydroxytryptamide or related moieties can be oxidized by oxidases in the presence of H2O2 to form oligomers or to form adducts with plasma and matrix proteins.15–20 As a result, the relaxivity is increased through an increased rotational correlation time and the complex is retained at the site of injury because of its increased size. This technology has been broadly applied to different models of oxidative stress.

The increased size and/or protein binding also results in retention at the site of injury and enabled such probes to detect inflammation in the heart,21 liver,22 brain,23 and blood vessels.24 However, the potential for human translation of these probes is decreased, due to their poor solubility in aqueous media and because of the risk of releasing Gd in vivo from acyclic DTPA-amide based chelates. Recent regulatory actions have limited or withdrawn the use of Gd-DTPA-bis(amide) chelates in clinical practice.25

Here we report a new peroxidase sensing MR probe termed Gd-5-HT-DOTAGA. To improve solubility and stability, we based the probe on the monoanionic Gd-DOTA core which is the most inert chelate with respect to Gd release among the clinically used agents and which there are no unconfounded cases of nephrogenic systemic fibrosis despite 10s of millions of administrations worldwide.26 We describe the synthesis, in vitro characterization, and demonstrate the specificity of Gd-5-HT-DOTAGA to image lung inflammation in a mouse model.

EXPERIMENTAL SECTION

1H and 13C NMR spectra were recorded on a JEOL 11.7 T NMR system equipped with a 5 mm broadband probe. Relaxivity measurements were performed on a Bruker mq60 Minispec at 1.41 T and 37 °C. UV-vis spectra were recorded on a SpectraMax M2 spectrophotometer at 37°C using quartz cuvettes with a 1 cm path length. Dynamic light-scattering measurements were performed using a Zetasizer Nano ZS (Malvern Instruments, MA) in triplicate. Intensity weighted values were used to determine the average particle diameter. HPLC purity analyses (both UV and MS detection) were carried out on an Agilent 1260 system (Phenomenex Luna C18(2) column: 100 mm × 2 mm, 0.8 mL/min flow rate) with UV detection at 220, 254, and 280 nm and +ESI using the following methods. Method A: 0.1% TFA in water, solvent B = 0.1% TFA in MeCN; 5–95% B over 10 min. Method B: 10 mM ammonium acetate in water, solvent B = 10% 10 mM ammonium acetate in 90% MeCN; 5–95% B over 10 min. Flash chromatography (Teledyne ISCO CombiFlash) was performed with UV detection at 220 and 254 nm using Method C and Method D (see ESI for details). High resolution mass spectra were measured with a high-resolution time-of-flight mass spectrometer (AccuTOF-DART, JEOL USA).27 Oxidations of Gd-5-HT-DOTAGA and Gd-5-T-DOTAGA were performed by adding horseradish peroxidase (1 U/mL) to a solution containing gadolinium complex and hydrogen peroxide solution (10 mM) in 0.1 M Tris buffer (pH = 7.4). The peroxidase activity was verified using a guaiacol assay.28 Chemicals were supplied by Sigma Aldrich, Alfa Aesar, Ambiopharm, Fluka, Acros Organics. Solvents (HPLC grade) were used as received. (R)-5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoic acid (tBu-DOTAGA) was prepared as described.29 All experiments and procedures with animals were performed in accordance with the National Institutes of Health’s “Guide for the Care and Use of Laboratory Animals” and were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee. C57Bl/6 adult male mice at 6 weeks of age (Charles River Laboratories, Wilmington MA) received a single 50 μL intratracheal dose of 1 U/kg bleomycin (Fresenius Kabi, Lake Zurich, Il) in sterile PBS. MR imaging experiments with Gd-5-HT-DOTAGA or the control Gd-T-DOTAGA were carried out at 3 weeks after bleomycin instillation.

The MR imaging protocol was as follows: Coronal and axial RARE (rapid acquisition with relaxation enhancement) sequences, matrix = 192 × 192; TR/TE = 1500 ms/8.11 ms; field of view (FOV) 32.763 × 32.763 mm; 2 averages; RARE factor 4. T1-weighted 3D FLASH (fast low angle shot), matrix = 192 × 112 × 80 mm; TR/TE/FA = 12.0 ms/2.463 ms/10°; FOV = 48 × 28 × 20 mm; 1 average. T1-weighted UTE (Ultrashort time to Echo), matrix = 192 × 192; TR/TE/FA = 5.00 ms/0.008 ms/12°; FOV 58.0 × 58.0 × 58.0 mm, 1 average. The probe, 100 μmol/kg, was then injected through an indwelling tail vein catheter and the FLASH sequence was repeated 5 times to estimate blood clearance. Then the UTE sequence was repeated. Data were analyzed using Horos software package. The RARE and post-probe FLASH images were used to segment out the large airways and blood vessels (see Figure S1). Regions of interest (ROIs) were then drawn over the upper, middle, and lower region of the lungs and copied to the UTE images for quantitative analysis. Mean signal intensity (SI) pre- and post-injection of the probe was measured in the lung and in the skeletal muscle, and we measured the standard deviation (SD) of the signal in the air outside the image to estimate noise. Contrast to noise ratio (CNR) is defined as (SIlung – SImuscle)/(SDair). We calculated ΔCNR (post injection image – pre injection) for each animal and then averaged for each group.

Mouse Lung Flow Cytometry

C57BL/6 WT mice were injected with intratracheal (IT) bleomycin at a standard dose (1.0 U/kg) per standard protocol.30 On day 20 after IT bleomycin, lungs were harvested and digested to generate a single cell suspension. Flow cytometry was performed to identify populations of inflammatory cells present in the two groups of mice, naïve and bleomycin treated. Alveolar macrophages were identified as CD45+ CD11c+ F4/80+ SiglecF+ cells. Interstitial macro-phages were identified as CD45+ CD11c- F4/80+ SiglecF- cells. Eosinophils were identified as CD45+ CD11c- F4/80+ SiglecF+ cells. Dendritic Cells were identified as CD45+ CD11c+ F4/80- Sig-lecF- MHCII+ cells. Neutrophils were identified as CD45+ CD11c- F4/80- SiglecF- CD11b+ Ly6G+ Ly6C- cells.

Syntheses

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Tri-tert-butyl 2,2’,2’’-(10-(1-(tert-butoxy)-5-((2-(5-hydroxy-1H-indol-3-yl)ethyl)amino)-1,5-dioxopentan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)(R)-triacetate (A). tBu-DOTAGA (1.17 g, 1.67 mmol, 1.0 eq), 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU, 97%, 0.74 g, 1.89 mmol, 1.13 eq) and N,N-diisopropylethylamine (DIPEA, 590 μL, 4.17 mmol, 2.5 eq) were dissolved in N,N-dimethylformamide (DMF, 5 mL) and stirred at rt for 1 h. Then 3-(2-aminoethyl)-5-hydroxyindole hydrochloride (5-HT, serotonin hydrochloride, 0.41 g, 1.87 mmol, 1.12 eq) was slowly added and the reaction mixture was stirred at rt for 16 h. The crude product was purified by flash chromatography with Method C, product elutes at 39 % MeCN. Fractions containing the desired product were combined, acetonitrile was removed under reduced pressure and remaining aqueous solution was and lyophilized to give a white solid (1.08 g, 75 % yield). LC-MS retention time (Method A): 3.65 min; calcd. Mass (C45H74N6O10+H+): 859.55, found: 859.5, calcd. mass (C45H74N6O10+2H+): 430.28, found: 430.3. 1H NMR (500 MHz, D2O) δ 7.19 (H12, d, 3JH-H = 7.5 Hz, 1H), 7.00 (H9, H15, br s, 2H), 6.68 (H13, d, 3JH-H = 7.5 Hz, 1H), 3.58–1.74 (H1, H2, H3, H6, H7, CH2COOtBu, cyclen, m, 31H), 1.39 (tBu, m, 36H); 13C NMR (126 MHz, D2O) δ 173.1–170.6, (C4, COOtBu), 150.7 (C14), 131.5 (C11), 128.8 (C16), 128.5 (C9), 123.2 (C15), 112.4 (C13), 112.0 (C12), 103.4 (C9), 81.5–81.3 (COOC(CH3)3), 63.7–33.3 (cyclen, C1, C3, C6, CH2COOtBu), 28.3–25.4 (C2, C7, COOC(CH3)3).

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(R)-2,2’,2’’-(10-(1-carboxy-4-((2-(5-hydroxy-1H-indol-3-yl)ethyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B). Compound (A) (1.02 g, 1.19 mmol) was dissolved in a formic acid/water mixture (1:1, 200 mL) and the reaction mixture was heated at 80 °C for 2 h. Solvent was removed under reduced pressure and lyophilized to give a beige solid. The solid was dissolved in HCl (0.3 M, 100 mL) and stirred under argon at rt for 48 h. The crude product was purified by flash chromatography, Method D, product elutes at 9 % MeCN, to give (B) as a white solid (0.6501 g, 86 % yield). LC-MS retention time (Method A): 1.85 min; calcd. mass (C29H42N6O10+H+): 635.30, found: 635.3, calcd. Mass (C45H74N6O10+Na+): 657.29, found: 657.2. 1H NMR (500 MHz, D2O) δ 7.20 (H12, d, J = 8.5 Hz, 1H), 7.03 (H9, s, 1H), 6.90 (H15, d, J = 2.0 Hz, 1H), 6.64 (H13, dd, J = 8.5 Hz, 2.0 Hz, 1H), 3.60–2.54 (CH2COOtBu, cyclen, H6, H7, m, 27H), 2.16 (H3, m, 2H), 1.52 (H2, m, 2H); 13C NMR (126 MHz, D2O) δ 175.1(CONH), 166.9 (COOH), 148.7 (C14), 131.4 (C11), 128.0 (C16), 124.7 (C9), 112.7 (C8), 111.5 (C12, C13), 102.9 (С15), 59.2–43.9 (С1, cyclen, CH2COOH), 40.1 (C6), 33.5 (C3), 24.0 (C2, C7).

Gadolinium(III) sodium (R)-2,2’,2’’-(10-(1-carboxylato-4-((2-(5-hydroxy-1H-indol-3-yl)ethyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (Gd-5-HT-DOTAGA). Compound (B) (0.63 g, 0.99 mmol, 1 eq) was dissolved in sodium acetate buffer (1 M, pH = 6, 50 mL) under argon. Gadolinium chloride hexahydrate (0.37 g, 0.99 mmol, 1 eq) in sodium acetate buffer (1 M, pH = 6, 10 mL) was slowly added and the reaction mixture was stirred under argon at rt for 16 h. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography with Method D, product elutes at 9 % MeCN, to give the desired product (Gd-5-HT-DOTAGA) as a white solid (0.8049 g, 98 % yield) after removal of acetonitrile under reduced pressure and subsequent lyophilization. LC-MS retention time (Method A): 1.85 min; m/z (HRMS-) 788.1841 (C29H38O10N6Gd requires 788.1897).

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Tri-tert-butyl 2,2’,2’’-(10-(5-((2-(1H-indol-3-yl)ethyl)amino)-1-(tert-butoxy)-1,5-dioxopentan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)(R)-triacetate (C). tBu-DOTAGA (1.3042 g, 1.86 mmol, 1 eq), HATU (0.83 g, 2.19 mmol, 1.14 eq) and DIPEA (657 μL, 4.65 mmol, 2.5 eq) were dissolved in DMF (5 mL) and the reaction mixture was stirred at rt for 1 h. 2-(1H-indol-3-yl)ethan-1-amine (T, tryptamine, 98 %, 0.35 g, 2.16 mmol, 1.14 eq) was slowly added, and the reaction mixture was stirred at rt for 16 h. The crude product was purified by flash chromatography, Method C, product elutes at 39 % MeCN. Fractions containing the desired product were combined and lyophilized to give the desired product (C) as a white solid (1.57 g, 85 % yield). LC-MS retention time (Method B): 3.13 min; calcd. mass (C45H74N6O9+H+): 843.56, found: 843.5, calcd. Mass (C45H74N6O9+2H+): 422.28, found: 422.4. 1H NMR (500 MHz, CDCl3) δ 8.93 (H10, s, 1H), 7.52 (H15, d, 3JH-H = 8.0 Hz, 1H), 7.36 (H12, d, 3JH-H = 8.0 Hz, 1H), 7.06 (H13, dd, 3JH-H = 7.5 Hz, 1H), 7.04 (H9, s, 1H), 6.98 (H14, dd, 3JH-H = 7.5 Hz, 1H), 6.12 (H5, m, 1H), 3.55–3.20 (CH2COOtBu, 1H, 6H m, 7H), 2.86 (CH2COOtBu, m, 4H), 2.42–2.38 (cyclen, H3, m, 8H), 2.27–1.86 (cyclen, H3, H7, m, 12H), 1.76–1.59 (H2, m, 2H), 1.36 (tBu, m, 36H); 13C NMR (126 MHz, CDCl3) δ 175.1 (C4), 172.9–172.7 (COOtBu), 136.7 (C11), 127.5 (C16), 123.0 (C9), 121.7 (C13), 119.0 (C14), 118.6 (C15), 112.3 (C8), 111.8 (C12), 82.0–81.9 (COOC(CH3)3), 60.2 (C1), 55.8–55.5 (CH2COOtBu), 53.6–47.0 (cyclen), 39.8 (C6), 35.2 (C3), 27.9–27.8 (COOC(CH3)3), 25.1 (C7), 21.0 (C2).

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(R)-2,2’,2’’-(10-(4-((2-(1H-indol-3-yl)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (D). Compound (C) (2.34 g, 2.78 mmol, 1 eq) was dissolved in a formic acid/water mixture (1:1, 500 mL) and stirred at 80 °C for 2 h. The solvent was removed under reduced pressure and the crude was lyophilized to give brown oil. The oil was dissolved in HCl (0.3 M, 100 mL) and stirred under argon at rt for 48 h. The crude product was purified by flash chromatography using Method D, product elutes at 9 % MeCN to give a white solid (1.56 g, 91 % yield) after removal of acetonitrile under reduced pressure and subsequent lyophilization. LC-MS retention time (Method A): 2.84 min; calcd. (C29H43N6O9+H+): 619.31, found: 619.3. 1H NMR (500 MHz, D2O) δ 7.51 (H15, d, J = 8.0 Hz, 1H), 7.33 (H12, d, J = 8.0 Hz, 1H), 7.07 (H9, H13, m, 2H), 6.99 (H14, m, 1H), 3.62–2.46 (CH2COOtBu, cyclen, H1, H7, H8, m, 27H), 2.19 (2H, m, H2), 1.54 (2H, m, H3); 13C NMR (126 MHz, D2O) δ 175.0 (CONH), 166.9 (COOH), 136.1 (C11), 127.2 (C16), 123.6 (C9), 121.8 (C13), 119.2 (C14), 118.7 (C15), 112.0 (C8), 111.8 (C12), 59.2–43.2 (С1, cyclen, CH2COOH), 40.1 (C6), 33.4 (C3), 24.0 (C2, C7).

Gadolinium(III) sodium (R)-2,2’,2’’-(10-(4-((2-(1H-indol-3-yl)ethyl)amino)-1-carboxylato-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (Gd-T-DOTAGA). Compound (D) (0.63 g, 1.02 mmol, 1 eq) was dissolved in sodium acetate buffer (1 M, pH = 8, 50 mL) under argon. Gadolinium chloride hexahydrate (0.38 g, 1.02 mmol, 1.0 eq) solution in sodium acetate buffer (1 M, pH = 6, 10 mL) was slowly added and the reaction mixture was stirred under argon at rt for 16 h. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography with Method D, product elutes at 9 % MeCN, to give the desired product (Gd-5-T-DOTAGA) as a white solid (0.79 g, 98 % yield) after removal of acetonitrile under reduced pressure and subsequent lyophilization. LC-MS retention time (Method B): 2.11 min; m/z (HRMS-) 772.1930 (C29H38O9N6Gd requires 772.1948).

RESULTS AND DISCUSSION

Gd-5-HT-DOTAGA was synthesized in three steps (Scheme 2). First, tert-butyl protected DOTAGA was coupled with serotonin hydrochloride (5-HT, 75% yield) using HATU as a coupling reagent. The tert-butyl protected ligand was hydrolyzed with TFA (86% yield) and complexed with GdCl3⋅6H2O in sodium acetate buffer (pH 6) under argon giving the desired complex Gd- 5- HT-DOTAGA in high yield (98%). A control compound, Gd-T-DOTAGA, in which the hydroxyl group is missing was prepared in an analogous manner using tryptamine instead of serotonin. Gd-T-DOTAGA is expected to have a similar relaxivity and pharmacokinetics to Gd-5-HT-DOTAGA, but is much less sensitive to oxidation and therefore acts as a negative control probe.

Scheme 2.

Scheme 2.

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Synthesis of the Gd-complexes used as probe (Gd-5-HT-DOTAGA) and control probe (Gd-T-DOTAGA)

The negatively charged Gd-DOTAGA chelate core imparts high solubility to these complexes (> 130 mM in phosphate buffered saline (PBS))

Reagents and conditions: (i) HATU, DIPEA, DMF; (ii) (a) HCOOH/H2O, Δ, (b) 0.2 M HCl; (iii) GdCl3·6H2O, 1.0 M NaOAc buffer (pH ~ 6).

The ability of Gd-5-HT-DOTAGA to act as an amplifiable probe for peroxidase activity was tested by challenging a solution of the complex with horseradish peroxidase enzyme (HRP, 1 U of activity) in the presence of hydrogen peroxide (10 mM). That reaction was followed by UV-vis spectroscopy, 1H relaxometry (60 MHz), and dynamic light scattering (DLS) (Fig. 1). Under these conditions, the absorbance at 350 nm increases with time for Gd-5-HT-DOTAGA and reaches a plateau after 20 min (Fig.1c), while absorbance doubles. However, there are no changes in the absorbance spectrum, when Gd-T-DOTAGA was incubated under the same conditions. These results are in accordance with the observed 1.7-fold increase in relaxivity r1 (60 MHz, 37°C) with added HRP/H2O2 for Gd-5-HT-DOTAGA, while no changes in relaxivity are observed for a control compound Gd-T-DOTAGA (Fig.1b and Fig.1c). We also observed a 40% increase in r1 (p < 0.01) when Gd-5-HT-DOTAGA was incubated with 0.3U MPO in lung tissue homogenate but no significant increase in r1 was observed under the same conditions with Gd-5-T-DOTAGA.

Figure 1.

Figure 1.

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The incubation of gadolinium complexes under enzymatic oxidative conditions leads to (a, b) an increase of r1 for Gd-5-HT-DOTAGA (1.7-fold after 20 min) and no change for Gd-T-DOTAGA (0.5 mM, 0.1M Tris buffer, pH = 7.4, 37°C), as well as (c) an increase in the intensity of the UV band at 350 nm for Gd-5-HT-DOTAGA and no changes for Gd-T-DOTAGA (0.5 mM, 0.1M Tris buffer, pH = 7.4, 37°C), due to the oligomerization of the active probe; (d) DLS measurements demonstrated an increase in size upon oxidation.

The observed increase in r1 is ascribed to an increased rotational correlation time of the molecule as a result of oligomerization. Indeed, an increase of the mean particle size from 0.6 nm to 2.0 nm by dynamic light scattering was detected for Gd-5-HT-DOTAGA with added HRP/H2O2 (Fig. 1d). Electrospray mass spectral analysis detected the presence of dimers and trimers.

In order to assess the kinetic inertness of Gd-5-HT-DOTAGA with respect to Gd dissociation, the complex was challenged with a 4-fold excess of a DTPA-based chelator MS-325 (pH = 3.0, 52°C), and the observed ligand exchange rate was monitored by RP-HPLC and compared with that of commercially available MRI contrast agents (see Supporting Information for details). Under these conditions, we observed a steady and very slow ligand exchange with less than 15% of the Gd from Gd-5-HT-DOTAGA undergoing transchelation after 3 days, consistent with similar results from Gd-DOTAGA based complexes.31–32 We previously observed almost identical results for Gd-DOTA.32 On the other extreme, the linear chelates Gd-DTPA and Gd-BOPTA reached equilibrium before the first time point was measured (30 min). The macrocyclic complex Gd-DO3A-butrol was also prone to ligand exchange under these conditions with 75% transchelation after 30 min and equilibrium was reached after 4 hours of incubation with >96% transchelation.

To illustrate the potential of Gd-5-HT-DOTAGA in detecting lung inflammation, a mouse model of bleomycin-induced lung injury was employed. Bleomycin is a chemotherapeutic agent known to cause side effects including pulmonary inflammation and fibrosis. Intratracheal instillation of bleomycin into the mouse lung results in a strong inflammatory response causing fibrosis. Figure 2 shows flow cytometry data on the leukocyte population in the lungs of a bleomycin-injured mouse taken 20 days after bleomycin instillation, which are compared with naïve (uninjured) mice. At this time point, the alveolar macrophage, dendritic cell, and eosinophil populations are greatly increased. Histology with hematoxylin and eosin staining shows increased cellular density and infiltration of inflammatory cells at day 21 post bleomycin compared to controls (Fig S2A), while trichrome staining reveals significant fibrosis in the bleomycin injured mice (Fig S2B) which was confirmed by 2-fold higher (p<0.01) hydroxyproline levels, a biochemical marker of total collagen, Fig. S2C.

Figure 2.

Figure 2.

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A. Flow cytometry was performed on naïve C57BL/6 WT mouse lungs to identify myeloid cell populations using a sequential gating strategy (Am = alveolar macrophages, Im = interstitial macrophages, E = eosinophils, D = dendritic cells, N = neutrophils). B. Flow cytometry on C57BL/6 WT mouse lungs at day 20 after intratracheal (IT) bleomycin challenge demonstrated expansion of alveolar macrophages, dendritic cells, and eosinophils.

The ability of Gd-5-HT-DOTAGA enhanced MRI to detect inflammation was tested in this model by imaging four groups of mice (n = 4 mice per group) and comparing bleomycin-injured animals with naïve mice. Figure 3 shows representative T1-weighted ultrashort echo time coronal images from 4 different mice imaged before and 1 h post-injection. It is apparent that administering Gd-5-HT-DOTAGA results in a strong and obvious signal enhancement in the lungs of bleomycin-injured mice (Fig. 3A) but not in naïve animals (Fig. 3C).

Figure 3.

Figure 3.

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Coronal T1-weighted UTE MR images before and 1h post-injection for Gd-5-HT-DOTAGA (A, C) or Gd-T-DOTAGA (B, D) of naïve mice (C, D) or bleomycin injured mice (A, B). Lungs outlined in green. T1-weighted images show a higher lung uptake of the probe in bleomycin-treated mice and minor enhancement in a control animal.

To demonstrate that the signal increase in the injured mice was specific to Gd-5-HT-DOTAGA and not just a result of nonspecific enhancement due to the pathology in the lung we performed similar experiments with the control probe Gd-T-DOTAGA which does not undergo oxidation and activation (Fig. 3, right). As expected, Gd-T-DOTAGA administration resulted in little lung enhancement in both bleomycin injured and naïve mice. Apart from lung uptake, both probes showed a similar biodistribution. After intravenous injection, the blood pool is transiently enhanced until the probe is cleared from the blood and passes through the kidneys into the bladder (Fig. S3A). The blood half-lives of the probes were estimated from the dynamic MR data and found to be 27 ± 6 min for Gd-5-HT-DOTAGA and 36 ± 4 min for Gd-T-DOTAGA. We also observed a significant uptake in the liver (this is seen in all animals, Fig. 3), followed by transport and excretion through the bile (Fig. S3B).

Figure 4a shows ΔCNR (post injection image – pre injection) for each group. ΔCNR in animals injected with Gd-5-HT-DOTAGA is twofold higher in bleomycin-injured mice compared to naïve animals. On the other hand, ΔCNR was the same in bleomycin-injured mice compared to naïve animals when Gd-T-DOTAGA was used. Ex vivo analysis of the lungs and blood samples showed significantly higher concentrations of Gd in the lungs of bleomycin-injured mice injected with Gd-5-HT-DOTAGA compared to Gd-T-DOTAGA or to naïve mice, Figure 4b.

Figure 4.

Figure 4.

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(left) Change in contrast-to-noise ratio after i.v. probe injection for bleomycin injured- and naïve mice 1h post-injection for Gd-5-HT-DOTAGA and Gd-T-DOTAGA showing specific enhancement of the injured lung with Gd-5-HT-DOTAGA. (right) ex vivo Gd quantification in the lungs of naive and bleomycin injured mice shows a higher lung uptake of the active probe in bleomycin-injured mice.

The ex vivo biodistribution of Gd-5-HT-DOTAGA and Gd-T-DOTAGA are consistent with the imaging results and show that both compounds are excreted through kidneys and liver (where the hepatobiliary excretion leads to an increase in the amount of gadolinium in the gallbladder and the intestines) (see Figure S3 in ESI).

CONCLUSION

We synthesized a highly soluble, kinetically inert, amplifiable molecular probe Gd-5HT-DOTAGA and demonstrated its potential to assess pulmonary inflammation by in a bleomycin lung injury model. Competitive transchelation experiments revealed comparable kinetic inertness with respect to Gd release to Gd-DOTA and much higher inertness than several FDA-approved gadolinium contrast agents. Gd-5HT-DOTAGA administration resulted in a specific signal enhancement in inflamed lung tissue, as a result of an increased rotational correlation time upon oligomerization and/or binding with extracellular matrix proteins.

Supplementary Material

Supporting Information

Scheme 1.

Scheme 1.

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Enzymatic oxidation of a serotonin fragment in the presence of hydrogen peroxide, followed by oligomerization or binding to a tyrosine moiety of a protein

Acknowledgments

Funding Sources

Research support from the National Heart Lung and Blood Institute (R01HL116315, R01HL131907 to P.C.), the National Institute for Neurodegenerative Diseases and Stroke (R01NS091552 to A.B.), National Institute of Biomedical Imaging and Bioengineering (RO1 EB000858 for A.B.) and the NIH Office of the Director (S10OD010650, S10OD025234 to P.C.) is gratefully acknowledged.

Footnotes

The Supporting Information is available free of charge on the ACS Publications website. ESI.pdf contains descriptions of how the ROIs were drawn, histology, hydroxyproline analysis, dynamic MR images, further data on the kinetic inertness of the compounds, biodistribution data, and details of the chromatographic methods.

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Associated Data

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Supplementary Materials

Supporting Information
NIHMS1045747-supplement-Supporting_Information.pdf (11.8MB, pdf)

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