Lanthanide-Based Probes for Imaging Detection of Enzyme Activities by NIR Luminescence, T1- and ParaCEST MRI.

Applying a single molecular probe to monitor enzymatic activities in multiple, complementary imaging modalities is highly desirable to ascertain detection and to avoid the complexity associated with the use of agents of different chemical entities. We demonstrate here the versatility of lanthanide (Ln3+) complexes with respect to their optical and magnetic properties and their potential for enzymatic detection in NIR luminescence, CEST and T1 MR imaging, controlled by the nature of the Ln3+ ion, while using a unique chelator. Based on X-ray structural, photophysical, and solution NMR investigations of a family of Ln3+ DO3A-pyridine model complexes, we could rationalize the luminescence (Eu3+, Yb3+), CEST (Yb3+) and relaxation (Gd3+) properties and their variations between carbamate and amine derivatives. This allowed the design of L n L G a l 5 ${{{\bf L n L}}_{{\bf G a l}}^{5}}$ probes which undergo enzyme-mediated changes detectable in NIR luminescence, CEST and T1-weighted MRI, respectively governed by variations in their absorption energy, in their exchanging proton pool and in their size, thus relaxation efficacy. We demonstrate that these properties can be exploited for the visualization of ß-galactosidase activity in phantom samples by different imaging modalities: NIR optical imaging, CEST and T1-weighted MRI.

Gd3+ Complexes for MRI Detection of Zn2+ in the Presence of Human Serum Albumin: Structure-Activity Relationships.

Zn2+-responsive magnetic resonance imaging (MRI) contrast agents are typically composed of a Gd chelate conjugated to a Zn2+-binding moiety via a linker. They allow for Zn2+ detection in the presence of human serum albumin (HSA). In order to decipher the key parameters that drive their Zn2+-dependent MRI response, we designed a pyridine-based ligand, PyAmC2mDPA, and compared the properties of GdPyAmC2mDPA to those of analogue complexes with varying Gd core, Zn-binding moiety, or linker sizes. The stability constants determined by pH potentiometry showed the good selectivity of PyAmC2mDPA for Gd3+ (log KGd = 16.27) versus Zn2+ (log KZn = 13.58), proving that our modified Zn2+-binding DPA moiety prevents the formation of previously observed dimeric species. Paramagnetic relaxation enhancement measurements indicated at least three sites that are available for GdPyAmC2mDPA binding on HSA, as well as a 2-fold affinity increase when Zn2+ is present (KD = 170 µM versus KDZn = 60 µM). Fluorescence competition experiments provided evidence of the higher affinity for site II vs site I, as well as the importance of both the Zn-binding part and the Gd core in generating enhanced HSA affinity in the presence of Zn2+. Finally, an analysis of nuclear magnetic relaxation dispersion (NMRD) data suggested a significantly increased rigidity for the Zn2+-bound system, which is responsible for the Zn2+-dependent relaxivity response.

Insights into the Responding Modes of Highly Potent Gadolinium-Based Magnetic Resonance Imaging Probes Sensitive to Zinc Ions.

Zn ions (Zn2+) play an important biological role in many diseases; hence, an imaging method for monitoring the Zn2+ distribution in tissues could provide important clinical insights. Recently, we reported a potent Zn-sensitive probe based on the Gd-DO3A (DO3A = 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid), modified tyrosine. and di(2-picolyl)amine chelator for this metal cation, which generates an outstanding magnetic resonance imaging (MRI) response. Here we further explored the origin of this unprecedented response and expanded the choice of potential MRI probes by preparing the free acid version of the initial MRI sensor. We report a detailed investigation of the 1H NMR dispersion, 17O NMR, and isothermal titration calorimetry properties of these two MRI probes upon interaction with Zn2+. The performed experiments confirm selective interaction of the MRI probes and target metal cation, which causes substantial changes in the coordination sphere of the paramagnetic center. It also evidenced some aggregation, which enhances the relaxivity response. Interestingly, conversion of the methyl ester to the free carboxylic acid of the tyrosine moiety changes the nature of the aggregates and leads to a smaller relaxivity response. The probes interact with human serum albumin (HSA) in the absence of Zn2+, which leads to a possible modification of the coordination sphere of Gd3+ or a substantial change in the exchange rate of second-sphere water molecules. In the presence of Zn2+, the interaction with HSA is very weak, demonstrating the importance of the Zn2+ coordination sphere in the behavior of these systems.

Lanthanide DO3A-Complexes Bearing Peptide Substrates: The Effect of Peptidic Side Chains on Metal Coordination and Relaxivity.

Two DO3A-type ligands conjugated to substrates of urokinase (L3) and caspase-3 (L4) via a propyl-amide linker were synthesized and their lanthanide(III) (Ln3+) complexes studied. A model compound without peptide substrate (L2) and an amine derivative ligand mimicking the state after enzymatic cleavage (L1) were also prepared. Proton Nuclear Magnetic Relaxation Dispersion (NMRD) profiles recorded on the gadolinium(III) (Gd3+) complexes, complemented with the assessment of hydration numbers via luminescence lifetime measurements on the Eu3+ analogues, allowed us to characterize the lanthanide coordination sphere in the chelates. These data suggest that the potential donor groups of the peptide side chains (carboxylate, amine) interfere in metal coordination, leading to non-hydrated LnL3 and LnL4 complexes. Nevertheless, GdL3 and GdL4 retain a relatively high relaxivity due to an important second-sphere contribution generated by the strongly hydrophilic peptide chain. Weak PARACEST effects are detected for the amine-derivative EuL1 and NdL1 chelates. Unfortunately, the GdL3 and GdL4 complexes are not significantly converted by the enzymes. The lack of enzymatic recognition of these complexes can likely be explained by the participation of donor groups from the peptide side chain in metal coordination.

Doxorubicin-Sensitized Luminescence of NIR-Emitting Ytterbium Liposomes: Towards Direct Monitoring of Drug Release.

Drug-loaded liposomes are typical examples of nanomedicines. We show here that doxorubicin, the anti-cancer agent in the liposomal drug Doxil, can sensitize Ytterbium (Yb3+ ) and generate its near-infrared (NIR) emission. When doxorubicin and amphiphilic Yb3+ chelates are incorporated into liposomes, the sensitized emission of Yb3+ is dependent on the integrity of the particles, which can be used to monitor drug release. We also established the first demonstration that the NIR Yb3+ emission signal is observable in living mice following intratumoral injection of the Yb3+ -doxorubicin-liposomes, using a commercial macroscopic setup equipped with a NIR camera.

Synthesis and In Vitro Studies of a Gd(DOTA)-Porphyrin Conjugate for Combined MRI and Photodynamic Treatment.

With the aim of developing new molecular theranostic agents, a π-extended Zn(II) porphyrin as photosensitizer for photodynamic therapy (PDT) linked to two GdDOTA-type complexes for magnetic resonance imaging (MRI) detection was synthesized. The relaxivity studies revealed a much higher relaxivity value per Gd ion for this medium sized molecule (19.32 mM-1 s-1 at 20 MHz and 298 K) compared to clinical contrast agents-a value which strongly increases in the presence of bovine serum albumin, reaching 25.22 mM-1 s-1. Moreover, the photophysical studies showed the strong ability of the molecule to absorb light in the deep red (670 nm, ε ≈ 60000 M-1 cm-1) and in the near-infrared following two-photon excitation (920 nm, σ2 ≈ 650 GM). The conjugate is also able to generate singlet oxygen, with a quantum yield of 0.58 in DMSO. Promising results were obtained in cellular studies, demonstrating that the conjugate is internalized in HeLa cells at micromolar concentration and leads to 70% of cell death following 30 min irradiation at 660 nm. These results confirm the potential of the designed molecule as an imaging and therapeutic agent.

Aza-BODIPY Platform: Toward an Efficient Water-Soluble Bimodal Imaging Probe for MRI and Near-Infrared Fluorescence.

In this study, an original aza-BODIPY system comprising two Gd3+ complexes has been designed and synthesized for magnetic resonance imaging/optical imaging applications, by functionalization of the boron center. This strategy enabled the obtainment of a positively charged bimodal probe, which displays an increased water solubility, optimized photophysical properties in the near-infrared region, and very promising relaxometric properties. The absorption and emission wavelengths are 705 and 741 nm, respectively, with a quantum yield of around 10% in aqueous media. Moreover, the system does not produce singlet oxygen upon excitation, which would be toxic for tissues. The relaxivity obtained is high at intermediate fields (16.1 mM-1 s-1 at 20 MHz and 310 K) and competes with that of bigger or more rigid systems. A full relaxometric and 17O NMR study and fitting of the data using the Lipari-Szabo approach showed that this high relaxivity can be explained by the size of the system and the presence of some small aggregates. These optimized photophysical and relaxometric properties highlight the potential use of such systems for future bimodal imaging studies.

High-Field Detection of Biomarkers with Fast Field-Cycling MRI: The Example of Zinc Sensing.

Many smart magnetic resonance imaging (MRI) probes provide response to a biomarker based on modulation of their rotational correlation time. The magnitude of such MRI signal changes is highly dependent on the magnetic field and the response decreases dramatically at high fields (>2 T). To overcome the loss of efficiency of responsive probes at high field, with fast-field cycling magnetic resonance imaging (FFC-MRI) we exploit field-dependent information rather than the absolute difference in the relaxation rate measured in the absence and in the presence of the biomarker at a given imaging field. We report here the application of fast field-cycling techniques combined with the use of a molecular probe for the detection of Zn2+ to achieve 166 % MRI signal enhancement at 3 T, whereas the same agent provides no detectable response using conventional MRI. This approach can be generalized to any biomarker provided the detection is based on variation of the rotational motion of the probe.

Toward MRI and Optical Detection of Zwitterionic Neurotransmitters: Near-Infrared Luminescent and Magnetic Properties of Macrocyclic Lanthanide(III) Complexes Appended with a Crown Ether and a Benzophenone Chromophore.

Thanks to their versatile magnetic and luminescence features, lanthanide complexes have gained a central position in biomedical imaging as magnetic resonance imaging (MRI) contrast agents and optical imaging probes. In addition, appropriate chemical design allows modification of the magnetic relaxation properties of GdIII complexes and the optical properties of visible- or near-infrared (NIR)-emitting lanthanide chelates upon interaction with various biomarkers, which makes them ideal candidates for the creation of responsive agents. In this Forum Article, we demonstrate such design principles as well as the difficulties encountered in the context of neurotransmitter (NT) detection. Lanthanide(III) complexes of a macrocyclic ligand incorporating a benzophenone chromophore and a monoazacrown ether (LnL3) have been synthesized as responsive probes to monitor amino acid NTs either in MRI (Ln = Gd) or in NIR optical detection (Ln = Nd or Yb). The parameters characterizing the water exchange and rotational dynamics of the gadolinium(III) complex were assessed by 17O NMR and 1H NMRD. In the presence of zwitterionic NTs, the inner-sphere water molecule is replaced by the carboxylate function of the NTs in the gadolinium(III) complex, leading to a decrease of the longitudinal relaxivity from 6.7 to 2-2.5 mM-1 s-1 (300 MHz and 37 °C). The apparent affinity constants range from Ka = 35 for γ-aminobutyric acid (GABA) to 80 M-1 for glycine and glutamate, and there is no selectivity with respect to hydrogen carbonate (Ka = 232; pH 7.4). The gadolinium(III) complex interacts with human serum albumin (HSA), resulting in a 60% increase in the relaxivity (20 MHz, 37 °C) in the absence of NTs. The HSA-bound complex, however, was revealed to be less responsive to NTs because of displacement of the GdIII-bound water by HSA, which was confirmed by the hydration number calculated from luminescence lifetimes of the HSA-bound europium(III) complex. The creation of an imaging agent suitable for NIR detection of NTs for an enhanced sensitivity in biological systems using the benzophenone (BP) moiety as the sensitizer of lanthanide luminescence was also attempted. Upon excitation at 300 nm of the BP chromophore in aqueous solutions of NdL3 and YbL3, characteristic NIR emissions of NdIII and YbIII were observed because of 4F3/2 → 4IJ (J = 9/2-13/2) and 2F5/2 → 2F7/2 transitions, respectively, indicating that this chromophore is a suitable antenna. Despite these promising results, luminescence titrations of NdIII and YbIII complexes with NTs were not conclusive because of chemical conversion of the ligand triggered by light, preventing quantitative analysis. The observed photochemical reaction of the ligand is strongly dependent on the nature of the lanthanide chelated; it is considerably slowed down in the presence of NdIII and EuIII.

A Porphyrin Dimer-GdDOTA Conjugate as a Theranostic Agent for One- and Two-Photon Photodynamic Therapy and MRI.

A molecular theranostic agent designed for photodynamic therapy (PDT) treatment in the near-infrared and for imaging tissue tumors with magnetic resonance imaging (MRI) is reported. It consists of a linear π-conjugated Zn(II) porphyrin dimer linked at each extremity to a GdDOTA-type complex. This agent has shown very promising potential for PDT applications with good singlet oxygen generation in DMSO and high linear absorption in the near-infrared (λmax = 746 nm, ε ≈ 105 M-1 cm-1). Moreover, this molecule has a propensity for two-photon excited PDT with high two-photon cross sections (∼8000 GM in 880-930 nm range), which should allow for deeper tumor treatments and higher spatial precision as compared to conventional one-photon PDT. Regarding the MRI contrast agent properties, the molecule has shown superior relaxivity (14.4 mM-1 s-1 at 40 MHz, 298 K) in comparison to clinical contrast agents and the ability to be internalized in cells, thanks to its amphiphilic character. Irradiation of HeLa cells using either one-photon (740 nm) or two-photon excitation (910 nm) has led in both cases to important cell death.

Proton Exchange in a Paramagnetic Chemical Exchange Saturation Transfer Agent from Experimental Studies and ab Initio Metadynamics Simulation.

The proton-exchange process between water and a carbamate has been studied experimentally and theoretically in a lanthanide-based paramagnetic chemical exchange saturation transfer agent endowed with potential multimodality detection capabilities (optical imaging, or T1 MRI for the Gd(III) analogue). In addition to an in-depth structural analysis by a combined approach (using X-ray crystallography, NMR, and molecular dynamics), our ab initio simulation in aqueous solution sheds light on the reaction mechanism for this proton exchange, which involves structural Grotthuss diffusion.

Synthetic water soluble di-/tritopic molecular receptors exhibiting Ca2+/Mg2+ exchange.

Structural integration of two synthetic water soluble receptors for Ca2+ and Mg2+, namely 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and o-aminophenol-N,N,O-triacetic acid (APTRA), respectively, gave novel di- and tritopic ionophores (1 and 2). As Mg2+ and Ca2+ cannot be simultaneously complexed by the receptors, allosteric control of complexation results. Potentiometric measurements established stepwise protonation constants and showed high affinity for Ca2+ (log K = 6.08 and 8.70 for 1 and 2, respectively) and an excellent selectivity over Mg2+ (log K = 3.70 and 5.60 for 1 and 2, respectively), which is compatible with magnesium-calcium ion exchange. While ion-exchange of a single Mg2+ for a single Ca2+ is possible in both 1 and 2, the simultaneous binding of two Mg2+ by 2 appears prohibitive for replacement of these two ions by a single Ca2+. Ion-binding and exchange was further rationalized by DFT calculations.

Prototypes of Lanthanide(III) Agents Responsive to Enzymatic Activities in Three Complementary Imaging Modalities: Visible/Near-Infrared Luminescence, PARACEST-, and T1-MRI.

We report first prototypes of responsive lanthanide(III) complexes that can be monitored independently in three complementary imaging modalities. Through the appropriate choice of lanthanide(III) cations, the same reactive ligand can be used to form complexes providing detection by (i) visible (Tb(3+)) and near-infrared (Yb(3+)) luminescence, (ii) PARACEST- (Tb(3+), Yb(3+)), or (iii) T1-weighted (Gd(3+)) MRI. The use of lanthanide(III) ions of different natures for these imaging modalities induces only a minor change in the structure of complexes that are therefore expected to have a single biodistribution and cytotoxicity.

A Theranostic Agent Combining a Two-Photon-Absorbing Photosensitizer for Photodynamic Therapy and a Gadolinium(III) Complex for MRI Detection.

The convergent synthesis and characterization of a potential theranostic agent, [DPP-ZnP-GdDOTA](-), which combines a diketopyrrolopyrrole-porphyrin component DPP-ZnP as a two-photon photosensitizer for photodynamic therapy (PDT) with a gadolinium(III) DOTA complex as a magnetic resonance imaging probe, is presented. [DPP-ZnP-GdDOTA](-) has a remarkably high longitudinal water proton relaxivity (19.94 mm(-1) s(-1) at 20 MHz and 25 °C) for a monohydrated molecular system of this size. The Nuclear Magnetic Relaxation Dispersion (NMRD) profile is characteristic of slow rotation, related to the extended and rigid aromatic units integrated in the molecule and to self-aggregation occurring in aqueous solution. The two-photon properties were examined and large two-photon absorption cross-sections around 1000 GM were determined between 910 and 940 nm in DCM with 1 % pyridine and in DMSO. Furthermore, the new conjugate was able to generate singlet oxygen, with quantum yield of 0.42 and 0.68 in DCM with 1 % pyridine and DMSO, respectively. Cellular studies were also performed. The [DPP-ZnP-GdDOTA](-) conjugate demonstrated low dark toxicity and was able to induce high one-photon and moderate two-photon phototoxicity on cancer cells.

pH-Responsive Relaxometric Behaviour of Coordination Polymer Nanoparticles Made of a Stable Macrocyclic Gadolinium Chelate.

Lanthanide-containing nanoscale particles have been widely explored for various biomedical purposes, however, they are often prone to metal leaching. Here we have created a new coordination polymer (CP) by applying, for the first time, a stable Gd(III) chelate as building block in order to prevent any fortuitous release of free lanthanide(III) ion. The use of the Gd-DOTA-4AmP complex as a design element in the CP allows not only for enhanced relaxometric properties (maximum r1 =16.4 mm(-1) s(-1) at 10 MHz), but also for a pH responsiveness (Δr1 =108 % between pH 4 and 6.5), beyond the values obtained for the low molecular weight Gd-DOTA-4AmP itself. The CP can be miniaturised to the nanoscale to form colloids that are stable in physiological saline solution and in cell culture media and does not show cytotoxicity.

Four Gadolinium(III) Complexes Appended to a Porphyrin: A Water-Soluble Molecular Theranostic Agent with Remarkable Relaxivity Suited for MRI Tracking of the Photosensitizer.

A molecular theranostic agent for magnetic resonance imaging (MRI) and photodynamic therapy (PDT) consisting of four [GdDTTA](-) complexes (DTTA(4-) = diethylenetriamine-N,N,N″,N″-tetraacetate) linked to a meso-tetraphenylporphyrin core, as well as its yttrium(III) analogue, was synthesized. A variety of physicochemical methods were used to characterize the gadolinium(III) conjugate 1 both as an MRI contrast agent and as a photosensitizer. The proton relaxivity measured in H2O at 20 MHz and 25 °C, r1 = 43.7 mmol(-1) s(-1) per gadolinium center, is the highest reported for a bishydrated gadolinium(III)-based contrast agent of medium size and can be related to the rigidity of the molecule. The complex displays also a remarkable singlet oxygen quantum yield of Ï•Δ = 0.45 in H2O, similar to that of a meso-tetrasulfonated porphyrin. We also evidenced the ability of the gadolinium(III) conjugate to penetrate in cancer cells with low cytotoxicity. Its phototoxicity on Hela cells was evaluated following incubation at low micromolar concentration and moderate light irradiation (21 J cm(-2)) induced 50% of cell death. Altogether, these results demonstrate the high potential of this conjugate as a theranostic agent for MRI and PDT.

Smart Contrast Agents for Magnetic Resonance Imaging.

By visualizing bioactive molecules or biological parameters in vivo, molecular imaging is searching for information at the molecular level in living organisms. In addition to contributing to earlier and more personalized diagnosis in medicine, it also helps understand and rationalize the molecular factors underlying physiological and pathological processes. In magnetic resonance imaging (MRI), complexes of paramagnetic metal ions, mostly lanthanides, are commonly used to enhance the intrinsic image contrast. They rely either on the relaxation effect of these metal chelates (T(1) agents), or on the phenomenon of paramagnetic chemical exchange saturation transfer (PARACEST agents). In both cases, responsive molecular magnetic resonance imaging probes can be designed to report on various biomarkers of biological interest. In this context, we review recent work in the literature and from our group on responsive T(1) and PARACEST MRI agents for the detection of biogenic metal ions (such as calcium or zinc), enzymatic activities, or neurotransmitter release. These examples illustrate the general strategies that can be applied to create molecular imaging agents with an MRI detectable response to biologically relevant parameters.

A Pyridine-Based Ligand with Two Hydrazine Functions for Lanthanide Chelation: Remarkable Kinetic Inertness for a Linear, Bishydrated Complex.

To study the influence of hydrazine functions in the ligand skeleton, we designed the heptadentate HYD ligand (2,2',2″,2‴-(2,2'-(pyridine-2,6-diyl)bis(2-methylhydrazine-2,1,1-triyl)) tetraacetic acid) and compared the thermodynamic, kinetic, and relaxation properties of its Ln(3+) complexes to those of the parent pyridine (Py) analogues without hydrazine (Py = 2,6-pyridinebis(methanamine)-N,N,N',N'-tetraacetic acid). The protonation constants of HYD were determined by pH-potentiometric measurements, and assigned by a combination of UV-visible and NMR spectroscopies. The protonation sequence is rather unusual and illustrates that small structural changes can strongly influence ligand basicity. The first protonation step occurs on the pyridine nitrogen in the basic region, followed by two hydrazine nitrogens and the carboxylate groups at acidic pH. Contrary to Py, HYD self-aggregates through a pH-dependent process (from pH ca. 4). Thermodynamic stability constants have been obtained by pH-potentiometry and UV-visible spectrophotometry for various Ln(3+) and physiological cations (Zn(2+), Ca(2+), Cu(2+)). LnHYD stability constants show the same trend as those of LnDTPA complexes along the Ln(3+) series, with log K = 18.33 for Gd(3+), comparable to the Py analogue. CuHYD has a particularly high stability (log K > 19) preventing its determination from pH-potentiometric measurements. The stability constant of CuPy was also revisited and found to be underestimated in previous studies, highlighting that UV-visible spectrophotometry is often indispensable to obtain reliable stability constants for Cu(2+) chelates. The dissociation of GdL, assessed by studying the Cu(2+)-exchange reaction, occurs mainly via an acid-catalyzed process, with limited contribution from direct Cu(2+) attack. The kinetic inertness of GdHYD is remarkable for a linear bishydrated chelate; the 25-fold increase in the dissociation half-life with respect to the monohydrated commercial contrast agent GdDTPA (t1/2 = 5298 h for GdHYD vs 202 h for GdDTPA) is related to the rigidity of the HYD ligand due to the pyridine and methylated hydrazine functions of the backbone. A combined analysis of variable-temperature (17)O NMR and NMRD data on GdHYD yielded the microscopic parameters influencing relaxation properties. The high relaxivity (r1 = 7.7 mM(-1) s(-1) at 20 MHz, 25 °C) results from the bishydrated character of the complex combined with an optimized water exchange rate (kex(298) = 7.8 × 10(6) s(-1)). The two inner-sphere water molecules are not replaced through interaction with biological cations such as carbonate, citrate, and phosphate as monitored by (1)H relaxivity and luminescence lifetime measurements.

Mechanistic studies of Gd3+-based MRI contrast agents for Zn2+ detection: towards rational design.

A series of novel pyridine-based Gd(3+) complexes have been prepared and studied as potential MRI contrast agents for Zn(2+) detection. By independent assessment of molecular parameters affecting relaxivity, we could interpret the relaxivity changes observed upon Zn(2+) binding in terms of variations of the rotational motion.

Nanozeolite-LTL with Gd(III) deposited in the large and Eu(III) in the small cavities as a magnetic resonance optical imaging probe.

The immense structural diversity of more than 200 known zeolites is the basis for the wide variety of applications of these fascinating materials ranging from catalysis and molecular filtration to agricultural uses. Despite this versatility, the potential of zeolites in medical imaging has not yet been much exploited. In this work a novel strategy is presented to selectively deposit different ions into distinct framework locations of zeolite-LTL (Linde type L) and it is demonstrated that the carefully ion-exchanged Gd/Eu-containing nanocrystals acquire exceptional magnetic properties in combination with enhanced luminescence. This smart exploitation of the framework structure yields the highest relaxivity density (13.7 s(-1) L g(-1) at 60 MHz and 25 °C) reported so far for alumosilicates, rendering these materials promising candidates for the design of dual magnetic resonance/optical imaging probes, as demonstrated in preliminary phantom studies.