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.     

Target Visualization by MRI Using the Avidin/Biotin Amplification Route: Synthesis and Testing of a Biotin–Gd‐DOTA Monoamide Trimer

To design efficient targeting strategies in magnetic resonance (MR) molecular imaging applications, the formation of supramolecular adducts between (strept)avidin ((S)Av) and tribiotinylated Gd‐DOTA‐monoamide complexes (DOTA=1,4,7,10‐tetraazacyclododecane‐N,N′,N′′,N′′′‐tetraacetic acid) was explored. Two compounds based on the trivalent core of tris(2‐aminoethyl)amine each containing three biotin molecules and one ( L1 ) or three ( L2 ) DOTA‐monoamide (DOTAMA) ligands were synthesized. In these tribiotinylated derivatives the biotins are spaced far enough apart to allow the formation of the supramolecular adduct with the protein and to host the chelating units in between the (S)Av layers. Size exclusion HPLC analyses indicated complete formation of very high molecular weight polymers (>2 MDa) with (S)Av in solution. A ¹H NMR spectroscopy relaxometric study on the obtained polymeric adducts showed a marked increase of the relaxivity at 35–40 MHz as a consequence of the lengthening of the tumbling time due to the formation of Gd‐chelates/(S)Av polymers. The most efficient Gd₃ L2 /(S)Av polymeric system was used for a test in cell cultures. The target is represented by a neural cell adhesion molecule (NCAM), which is overexpressed in Kaposi’s sarcoma cells and tumor endothelial cells (TEC) and that is efficiently recognized by a biotinylated tetrameric peptide (C3d‐Bio). In vitro experiments showed that only cells incubated with both C3d‐Bio and Gd₃ L2 /SAv polymer were hyperintense with respect to the control. Relaxation rates of cell pellets incubated with Gd₃ L2 /SAv alone were not significantly different from the untreated cells demonstrating the absence of a specific binding.     

Tetraphenylporphyrin‐based dual‐functional medical agent for magnetic resonance and fluorescence imaging

A bimodal magnetic resonance imaging contrast agent, TPP‐M‐Gd, was developed by modifying tetraphenylporphyrin (TPP) with a small dendritic molecule as a ligand (M) to chelate gadolinium (Gd) ions. The ligand featured four carboxylate groups, which contributed to good water solubility and a strong combination with metal ions. The longitudinal relaxivity (R₁) of the resulting agent was calculated to be 12.45 mM^?1 s^?1, which is much higher than that of DTPA‐Gd (4.49 mM^?1 s^?1). The magnetic resonance imaging experiments showed that the newly synthesized contrast agent could enhance T₁‐weighted magnetic resonance imaging quality both in vitro and in vivo. In addition, TPP‐M‐Gd exhibited good fluorescent property as shown in cell imaging experiments. The cytotoxicity of TPP‐M‐Gd was even better than that of clinically approved DTPA‐Gd, which makes it a promising dual‐functional medical imaging agent to provide more detailed information about biological and disease‐related events.     

A Carborane‐Derivative “Click” Reaction under Heterogeneous Conditions for the Synthesis of a Promising Lipophilic MRI/GdBNCT Agent

In this study, the Huisgen reaction has been used to functionalise a carborane cage with a lipophilic moiety and a 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) ligand to obtain a new Gd boron neutron‐capture therapy (BNCT)/magnetic resonance imaging (MRI) agent. The introduction of the triazole units has been accomplished under both heterogeneous conditions, by the use of a Cu‐supported ionic‐liquid catalyst, and homogeneous conditions. The ability of the Gd complex of the synthesised ligand to form stable adducts with low‐density lipoproteins (LDLs) has been evaluated and then MRI has been performed on tumour melanoma cells incubated in the presence of a Gd‐complex/LDL imaging probe. It has been concluded that the high amount of intracellular boron necessary to perform BNCT can be reached even in the presence of a relatively low‐boron‐containing LDL concentration.     

Near‐Infrared‐Emitting BODIPY‐trisDOTA111In as a Monomolecular Multifunctional Imaging Probe: From Synthesis to In Vivo Investigations

A new generation of monomolecular imaging probes (MOMIP) based on a distyryl‐BODIPY (BODIPY=boron‐dipyrromethene) coupled with three DOTA macrocycles has been prepared (DOTA=1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid). The MOMIP presents good fluorescence properties and is very stable in serum. The bimodal probe was conjugated to trastuzumab, and an optical in vivo study showed high accumulation of the imaging agent at the tumor site. ¹¹¹In radiometallation of the bioconjugate was performed in high radiochemical yield, highlighting the potential of this new BODIPY‐chelators derivative as a bimodal imaging probe.     

Synthesis and Characterization of α-Hexadecyl-DOTA and its Gd（Ⅲ） Chelate

Synthesis and characterization of the ligand, 10-(α-hexadecylcarboxymethyl)- 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (H4L), and its Gd(Ⅲ) chelate are described. Protonation constants for H4L ( lg Ki^H = 10.52, 9.45,4.74, 4.10) and the stability constant for GdL^-(lg KGdL^-=24.50) were determined by potentiometric titrations.The results obtained show that the ligand still maintains the strong chelating properties of the parent DOTA(1,4,7,10-tetraazacyclododecane-N,N‘,N“N′“-tetraacetic acid) after introduction of a linear chain hexadecyl group at the acetic side chain of DOTA, and its basicity is not significantly altered.     

Highly Water‐Dispersible Surface‐Modified Gd2O3 Nanoparticles for Potential Dual‐Modal Bioimaging

Water‐dispersible and luminescent gadolinium oxide (GO) nanoparticles (NPs) were designed and synthesized for potential dual‐modal biological imaging. They were obtained by capping gadolinium oxide nanoparticles with a fluorescent glycol‐based conjugated carboxylate (H L ). The obtained nanoparticles (GO‐ L ) show long‐term colloidal stability and intense blue fluorescence. In addition, L can sensitize the luminescence of europium(III) through the so‐called antenna effect. Thus, to extend the spectral ranges of emission, europium was introduced into L‐ modified gadolinium oxide nanoparticles. The obtained Eu^III‐doped particles (Eu:GO‐ L ) can provide visible red emission, which is more intensive than that without L capping. The average diameter of the monodisperse modified oxide cores is about 4 nm. The average hydrodynamic diameter of the L ‐modified nanoparticles was estimated to be about 13 nm. The nanoparticles show effective longitudinal water proton relaxivity. The relaxivity values obtained for GO‐ L and Eu:GO‐ L were r₁=6.4 and 6.3 s^?1 mM ^?1 with r₂/r₁ ratios close to unity at 1.4 T. Longitudinal proton relaxivities of these nanoparticles are higher than those of positive contrast agents based on gadolinium complexes such as Gd‐DOTA, which are commonly used for clinical magnetic resonance imaging. Moreover, these particles are suitable for cellular imaging and show good biocompatibility.     

Synthesis and Characterization of a Hypoxia‐Sensitive MRI Probe

Tissue hypoxia occurs in pathologic conditions, such as cancer, ischemic heart disease and stroke when oxygen demand is greater than oxygen supply. An imaging method that can differentiate hypoxic versus normoxic tissue could have an immediate impact on therapy choices. In this work, the gadolinium(III) complex of 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) with a 2‐nitroimidazole attached to one carboxyl group via an amide linkage was prepared, characterized and tested as a hypoxia‐sensitive MRI agent. A control complex, Gd(DO3A‐monobutylamide), was also prepared in order to test whether the nitroimidazole side‐chain alters either the water proton T₁ relaxivity or the thermodynamic stability of the complex. The stabilities of these complexes were lower than that of Gd(DOTA)^? as expected for mono‐amide derivatives. The water proton T₁ relaxivity (r₁), bound water residence lifetime (τ_M) and rotational correlation time (τ_R) of both complexes was determined by relaxivity measurements, variable temperature ¹⁷O NMR spectroscopy and proton nuclear magnetic relaxation dispersion (NMRD) studies. The resulting parameters (r₁=6.38 mM ^?1 s^?1 at 20 MHz , τ_M=0.71 μs, τ_R=141 ps) determined for the nitroimidazole derivative closely parallel to those of other Gd(DO3A‐monoamide) complexes of similar molecular size. In vitro MR imaging experiments with 9L rat glioma cells maintained under nitrogen (hypoxic) versus oxygen (normoxic) gas showed that both agents enter cells but only the nitroimidazole derivative was trapped in cells maintained under N₂ as evidenced by an approximately twofold decrease in T₁ measured for hypoxic cells versus normoxic cells exposed to this agent. These results suggest that the nitroimidazole derivative might serve as a molecular reporter for discriminating hypoxic versus normoxic tissues by MRI.     

Hyaluronic Acid‐Gadolinium Complex Nanospheres as Lymphatic System‐Specific Contrast Agent for Magnetic Resonance Imaging

A novel MRI contrast agent, hyaluronic acid gadolinium complex (HA‐Gd‐DTPA) nanospheres, is prepared by the synthesis of hyaluronic acid gadolinium complexes and their assembly. The physicochemical properties are characterized, and the lymphatic targeting in vitro and in vivo are also evaluated. The results show that the HA‐Gd‐DTPA nanospheres with suitable and stable physicochemical properties could be used for in vivo lymphatic targeting studies. Furthermore, the HA‐Gd‐DTPA nanospheres have obviously higher relaxation efficiency and MRI contrast between blood vessel and lymph vessel in rabbit than that of Magnevist. Thus, the novel MRI contrast agent can be taken up selectively by lymphatic system and used as a potential MRI contrast agents in lymphatic system.     

A Phthalocyanine–Peptide Conjugate with High In Vitro Photodynamic Activity and Enhanced In Vivo Tumor‐Retention Property

A novel zinc(II) phthalocyanine conjugated with a short peptide with a nuclear localization sequence, Gly‐Gly‐Pro‐Lys‐Lys‐Lys‐Arg‐Lys‐Val, was synthesized by click chemistry and a standard Fmoc solid‐phase peptide synthesis protocol. The conjugate was purified by HPLC and characterized with UV/Vis and high‐resolution mass spectroscopic methods. Both this compound and its non‐peptide‐conjugated analogue are essentially non‐aggregated in N,N‐dimethylformamide and can generate singlet oxygen effectively with quantum yields (Φ_Δ) of 0.84 and 0.81, respectively, relative to unsubstituted zinc(II) phthalocyanine (Φ_Δ=0.56). Conjugation of the peptide sequence, however, can enhance the cellular uptake, efficiency in generating intracellular reactive oxygen species, and photocytotoxicity of the phthalocyanine‐based photosensitizer against HT29 human colorectal carcinoma cells. The IC₅₀ value of the conjugate is as low as 0.21 μM . In addition, the conjugate shows an enhanced tumor‐retention property in tumor‐bearing nude mice. After 72 h post‐injection, the dye concentration in the tumor was significantly higher than that in other organs. The results suggest that this phthalocyanine–peptide conjugate is a highly promising photosensitizer for photodynamic therapy.     

Synthesis and characterization of an MRI Gd‐based probe designed to target the translocator protein

DPA‐713 is the lead compound of a recently reported pyrazolo[1,5‐a]pyrimidineacetamide series, targeting the translocator protein (TSPO 18 kDa), and as such, this structure, as well as closely related derivatives, have been already successfully used as positron emission tomography radioligands. On the basis of the pharmacological core of this ligands series, a new magnetic resonance imaging probe, coded DPA‐C₆‐(Gd)DOTAMA was designed and successfully synthesized in six steps and 13% overall yield from DPA‐713. The Gd‐DOTA monoamide cage (DOTA = 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid) represents the magnetic resonance imaging reporter, which is spaced from the phenylpyrazolo[1,5‐a]pyrimidineacetamide moiety (DPA‐713 motif) by a six carbon‐atom chain. DPA‐C₆‐(Gd)DOTAMA relaxometric characterization showed the typical behavior of a small‐sized molecule (relaxivity value: 6.02 mM^?1 s^?1 at 20 MHz). The good hydrophilicity of the metal chelate makes DPA‐C₆‐(Gd)DOTAMA soluble in water, affecting thus its biodistribution with respect to the parent lipophilic DPA‐713 molecule. For this reason, it was deemed of interest to load the probe to a large carrier in order to increase its residence lifetime in blood. Whereas DPA‐C₆‐(Gd)DOTAMA binds to serum albumin with a low affinity constant, it can be entrapped into liposomes (both in the membrane and in the inner aqueous cavity). The stability of the supramolecular adduct formed by the Gd‐complex and liposomes was assessed by a competition test with albumin. Copyright © 2013 John Wiley & Sons, Ltd.     

Tuning Glutamine Binding Modes in Gd‐DOTA‐Based Probes for an Improved MRI Visualization of Tumor Cells

Three new magnetic resonance imaging probes that target glutamine transporters have been synthesized. They consist of a Gd‐DOTA‐monoamide moiety (DOTA=1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid) linked through a six carbon atom chain to a vector represented by a glutamine residue bound through α‐carboxylic, γ‐carboxamidic, or α‐amino functionalities. Their uptake by HTC (rat hepatocarcinoma) and healthy rat hepatocytes has shown that the system containing the glutamine vector bound through the α‐carboxylic group displays a markedly higher affinity for tumor cells. The observed behavior is rationalized in terms of the exploitation of an additional glutamine transporter active in hepatic tumor cells.     

A Panchromatic Supramolecular Fullerene‐Based Donor–Acceptor Assembly Derived from a Peripherally Substituted Bodipy–Zinc Phthalocyanine Dyad

A panchromatic 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene –zinc phthalocyanine conjugate (Bodipy–ZnPc) 1 was synthesized starting from phthalocyanine aldehyde 4 , via dipyrromethane 3 and dipyrromethene 2 . Conjugate 1 represents the first example in which a Bodipy unit is tethered to the peripheral position of a phthalocyanine core. Electrochemical and optical measurements provided evidence for strong electronic interactions between the Bodipy and ZnPc constituents in the ground state of 1 . When conjugate 1 is subjected to photoexcitation in the spectral region corresponding to the Bodipy absorption, the strong fluorescence characteristic of the latter subunit is effectively quenched (i.e., ≥97 %). Excitation spectral analysis confirmed that the photoexcited Bodipy and the tethered ZnPc subunits interact and that intraconjugate singlet energy transfer occurs with an efficiency of ca. 25 %. Treatment of conjugate 1 with N‐pyridylfulleropyrrolidine ( 8 ), an electron‐acceptor system containing a nitrogen ligand, gives rise to the novel electron donor–acceptor hybrid 1 ? 8 through ligation to the ZnPc center. Irradiation of the resulting supramolecular ensemble within the visible range leads to a charge‐separated Bodipy–ZnPc^.+–C₆₀^.? radical‐ion‐pair state, through a sequence of excited‐state and charge transfers, characterized by a remarkably long lifetime of 39.9 ns in toluene.     

Multiple‐Frequency and Variable‐Temperature EPR Study of Gadolinium(III) Complexes with Polyaminocarboxylates: Analysis and Comparison of the Magnetically Dilute Powder and the Frozen‐Solution Spectra

An electron paramagnetic resonance (EPR) study of glasses and magnetically dilute powders of [Gd(DTPA)(H₂O)]^2?, [Gd(DOTA)(H₂O)]^?, and macromolecular gadolinate(1?) complexes P792 was carried out at the X‐ and Q‐bands and at 240 GHz (DTPA=diethylenetriaminepentaacetato; DOTA=1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetato). The results show that the zero‐field splitting (ZFS) parameters for these complexes are quite different in a powder as compared to the frozen aqueous solution. In several complexes, an inversion of the sign of the axial component D of the zero field splitting is observed, indicating a significant structural change. In contrary to what was expected, powder samples obtained by lyophilization do not allow a more precise determination of the static ZFS parameters. The results obtained in glasses are more relevant to the problem of electron spin relaxation in aqueous solution than those obtained from powders.     

Relative Ligand Exchange Rates in Gd‐based MRI Contrast Agent Formation as Probed by Gd–XO Complex

A novel methodology involving the use of Gd³⁺–Xylenol Orange (Gd–XO), a solution colorimetric probe, was used to determine the conditional relative rates of complex formation between the Gd³⁺ ion and polyamino carboxylate ligands used in MRI contrast agent development. Among the ligands tested, the order of rate of Gd‐complex formation was found to be DTPA ≈ DOTP > DOTA > DO3A > DOTAM. The observed rates are an effect of ligand denticity, backbone structure, and nature of the ligand donor groups.     

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.     

New insights into water‐soluble and water‐coordinated copper 15‐metallacrown‐5 gadolinium complexes designed for high‐field magnetic resonance imaging applications

The development of contrast agents specifically designed for high‐field magnetic resonance imaging (MRI) is required because the relaxation efficiency of classic Gd(III) contrast agents significantly decreases with increasing magnetic field strengths. With an idea of exploring the unique structure of lanthanide (Ln) 15‐MC‐5 metallacrowns, we developed a series of water‐soluble Gd(III) aqua‐complexes, bearing aminohydroxamate (glycine, α‐alanine, α‐phenylalanine and α‐tyrosine) ligands, with increasing number of water molecules directly coordinated to the Gd(III) ion: Gd(H₂O)₄[15‐MC_Cu(II)Glyha‐5](Cl)₃ ( 1 (Gd)), Gd(H₂O)₄[15‐MC_Cu(II)Alaha‐5](Cl)₃ ( 2 (Gd)), Gd(H₂O)₃[15‐MC_{Cu(II)Phalaha}‐5](Cl)₃ ( 3 (Gd)) and Gd(H₂O)₃[15‐MC_Cu(II)Tyrha‐5](Cl)₃ ( 4 (Gd)). In these systems, the Ln(III) central ion is coordinated by five oxygen donor atoms of the ligands and three or four inner‐sphere water molecules. The X‐ray crystal structure of metallacrown Ln(H₂O)_3,4[15‐MC_Cu(II)Rha‐5]³⁺ agrees with density functional theory predictions. The calculations demonstrate that the exchange of coordinated water molecules can proceed easily, resulting in increased relaxivity parameters. The longitudinal relaxivities (r₁) of 1 (Gd)– 4 (Gd) in water at ultrahigh magnetic field of 9.4 T were determined to be 11.5, 14.8, 13.9 and 12.2 mM^?1 s^?1, respectively. The ability to increase the number of Ln(III) inner‐sphere water molecules up to four, the planar metallacrown structure and the rich hydration shell due to strong hydrogen bonds between the [15‐MC‐5] moiety and bulk water molecules provide new opportunities for potential MRI applications.     

Synthesis,Relaxivity and MRI Enhancement of Linear Oligo‐Gd(III) Complexes with Poly (DTPA‐ester) Ligands Derived from Amino Acids

Six linear oligo‐DTPA‐ester Gd(III) complexes being used for potential MRI contrast agents were synthesized from amino adds and characterized. Their longitudinal relaxation rates were measured. One of them, die phenylalanine derivative, with high relaxivity, was chosen for the acute toxicity and T₁,‐weighted imaging test. The results indicated that there was no obvious toxicity for this new oligomeric Gd(III) complex, and it exhibits the highly enhanced MRI signal intensity and the increasing signal duration in the liver tissue compared to Gd‐DTPA.     

New 3‐(Heteroaryl)‐2‐iminocoumarin‐based Borate Complexes: Synthesis,Photophysical Properties,and Rational Functionalization for Biosensing/Biolabeling Applications

Members of a series of boron difluoride complexes with 3‐(heteroaryl)‐2‐iminocoumarin ligands bearing both a phenolic hydroxyl group (acting as a fluorogenic center) and an N‐aryl substituent (acting as a stabilizing moiety) have been synthesized in good yields by applying a straightforward two‐step method. These novel fluorogenic dyes belong to the family of “Boricos” (D. Frath et al., Chem. Commun.­ 2013 , 49, 4908–4910) and are the first examples of phenol‐based fluorophores of which the photophysical properties in the green‐yellow spectral range are dramatically improved by N,N‐chelation of a boron atom. Modulation of their fluorescence properties through reversible chemical modification of their phenol moieties has been demonstrated by the preparation of the corresponding 2,4‐dinitrophenyl (DNP) ethers, which led to a dramatic “OFF‐ON” fluorescence response upon reaction with thiols. Additionally, to expand the scope of these “7‐hydroxy‐Borico” derivatives, particularly in biolabeling, amine or carboxylic acid functionalities amenable to (bio)conjugation have been introduced within their scaffold. Their utility has been demonstrated in the preparation of fluorescent bovine serum albumin (BSA) conjugates and “Borico”‐DOTA‐like scaffolds in an effort to design novel monomolecular multimodal fluorescence‐ radioisotope imaging agents.