Development of hypoxia-sensitive Gd3+-based MRI contrast agents

Hypoxia occurs in various diseases, including cancer, ischemia, and acute and chronic vascular diseases. Here we describe the design and synthesis of the first hypoxia-sensitive MRI contrast agents, SAGds. SAGds showed a pH-dependent r(1) relaxivity change associated with intramolecular chelation of the nitrogen atom of the sulfonamide moiety to the Gd(3+) center. There was a correlation between the pK(a) of the r(1) relaxivity change and the sum of the Hammett σ constants of substituents on the aromatic ring. Among the synthesized compounds, 4NO(2)2MeOSAGd was selectively reduced to the amine by rat liver microsomes under hypoxic conditions, resulting in a 1.8-fold increment of the r(1) relaxivity owing to the change in pK(a) of the arylsulfonamide moiety. This enhancement of the r(1) relaxivity could be clearly detected in T(1)-weighted MR images. Thus, 4NO(2)2MeOSAGd is a 'smart' MRI contrast agent for the detection of hypoxia under physiological conditions.     

Cyclen-based Gd3+ complexes as MRI contrast agents: Relaxivity enhancement and ligand design

Magnetic Resonance Imaging (MRI) is a noninvasive radiology technique used to examine the internal organs of human body. It is useful for the diagnosis of structural abnormalities in the body. Contrast agents are used to increase the sensitivity of this technique. 1,4,7,10-Tetraazacyclododecane (cyclen) is a macrocyclic tetraamine. Its derivatives act as useful ligands to produce stable complexes with Gd³⁺ ion. Such chelates are investigated as MRI contrast agents. Free Gd³⁺ ion is extremely toxic for in vivo use. Upon complexation with a cyclen-based ligand, it is trapped in the preformed central cavity of the ligand resulting in the formation of a highly stable Gd³⁺-chelate. Better kinetic and thermodynamic stability of cyclen-based MRI contrast agents decrease their potential toxicity for in vivo use. Consequently, such agents have proved to be safest for clinical applications. Relaxivity is the most important parameter used to measure the effectiveness of a contrast agent. A number of factors influence this parameter. This article elucidates detailed strategies to increase relaxivity of cyclen-based MRI contrast agents. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) are two key ligands derived from cyclen. They also act as building blocks for the synthesis of novel ligands. A few important methodologies for the synthesis of DOTA and DO3A derivatives are described. Moreover, the coordination geometry of chelates formed by these ligands and their derivatives is discussed as well. Novel ligands can be developed by the appropriate derivatization of DOTA and DO3A. Gd³⁺-chelates of such ligands prove to be useful MRI contrast agents of enhanced relaxivity, greater stability, better clearance, lesser toxicity and higher water solubility.     

Lipari-Szabo approach as a tool for the analysis of macromolecular gadolinium(III)-based MRI contrast agents illustrated by the [Gd(EGTA-BA-(CH2)12)] n n + polymer

The parameters governing the water proton relaxivity of the [Gd(EGTA-BA-(CH2)12)]nn+ polymeric complex were determined through global analysis of 17O NMR, EPR and nuclear magnetic relaxation dispersion (NMRD) data [EGTA-BA2- = 3,12-bis(carbamoylmethyl)- 6,9-dioxa-3,12-diazatetradecanedioate(2-)]. The Lipari-Szabo approach that distinguishes the global motion of the polymer (tau g) from the local motion of the Gd(III)-water vector (tau l) was necessary to describe the 1H and 17O longitudinal relaxation rates; therefore for the first time it was included in the global simultaneous analysis of the EPR, 17O NMR and NMRD data. The polymer consists on average of only five monomeric units, which limits the intramolecular hydrophobic interactions operating between the (CH2)12 groups. Hence the global rotational correlation time is not very high (tau g298 = 3880 +/- 750 ps) compared to the corresponding DTPA-BA-based polymer (about 15 monomeric units), where tau g298 = 6500 ps. As a consequence, the relaxivity is limited by the rotation, which precludes the advantage obtained from the fast exchanging chelating unit (kex298 = 2.2 +/- 0.1 x 10(6) s-1).     

Analogs of Eu3+ DOTAM-Gly-Phe-OH and Tm3+ DOTAM-Gly-Lys-OH: synthesis and magnetic properties of potential PARACEST MRI contrast agents

Chelated lanthanide ions, especially gadolinium, have found wide use as contrast agents in magnetic resonance imaging. A new paradigm for generating contrast, termed PARACEST, was recently described that requires the slow exchange of water or other exchangeable protons present in the ligand framework. In previous work, we have described a synthetic method for the preparation of dipeptide conjugates of DOTAM for use as PARACEST agents. Two compounds possessed interesting magnetic properties: the Eu(3+) complex of DOTAM-Gly-Phe-OH and the Tm(3+) complex of DOTAM-Gly-Lys-OH. To understand the relationship between the structure of these complexes and their magnetic properties, we have expanded our synthetic methodology and prepared several new complexes. Ligands have been prepared in which the terminal phenylalanine moieties have been replaced with tryptophan or tyrosine, the distance to the amino acid residue possessing an alpha-substituent has been changed, or phenylalanine and lysine have been combined in the peptide sequence. The preparation of lanthanide(III) complexes of these ligands has been achieved and their PARACEST properties have been determined.     

Identification of cells with lowered sensitivity to cytostatic agents by the use of fluorescent dyes

With a help of stepwise increase of vincristine concentrations in culture medium several lines of mouse myeloma X63 Ag 8.863 cells resistant to low concentrations of vincristine (6-35-fold) were selected. Rhodamine 123 stained resistant cells and wild-type cells with an equal intensity. However, resistant cells differ significantly from the sensitive ones by the rate of rhodamine efflux. The rate of the efflux was in proportion to the degree of resistance. The efflux of the dye could be blocked by the addition to reserpine, the inhibitor of multidrug resistance. Thus, fluorescent dyes can be used for the detection of cells with low levels of multidrug resistance.     

Strategies for the covalent conjugation of a bifunctional chelating agent to albumin: synthesis and characterization of potential MRI contrast agents

With the purpose to develop macromolecular magnetic resonance imaging contrast agents, we herein report three different synthetic approaches to the covalent attachment of bifunctional chelating agents to human serum albumin followed by coordination to contrast enhancing gadolinium(III). Applied methods cover active ester-mediated conjugation, linkage through glutaryl spacer, as well as the connection by the employment of glutaraldehyde. The content of gadolinium(III) was evaluated by inductively-coupled-plasma mass-spectrometry (ICP-MS) measurements and indicated reproducible amounts of conjugated contrast enhancing material. Small angle X-ray scattering (SAXS) experiments provided the size and altered shape of the gadolinium loaded proteins in comparison to unmodified albumin. Finally, the magnetic resonance properties of the protein conjugates were evaluated. The results indicated suitability of the gadolinium(III) loaded protein conjugates for use as macromolecular contrast agents in magnetic resonance imaging (MRI).     

MRI contrast agents for functional molecular imaging of brain activity

Functional imaging with MRI contrast agents is an emerging experimental approach that can combine the specificity of cellular neural recording techniques with noninvasive whole-brain coverage. A variety of contrast agents sensitive to aspects of brain activity have recently been introduced. These include new probes for calcium and other metal ions that offer high sensitivity and membrane permeability, as well as imaging agents for high-resolution pH and metabolic mapping in living animals. Genetically encoded MRI contrast agents have also been described. Several of the new probes have been validated in the brain; in vivo use of other agents remains a challenge. This review outlines advantages and disadvantages of specific molecular imaging approaches and discusses current or potential applications in neurobiology.     

Study of different coupling agents in the conjugation of a V3-based synthetic MAP to carrier proteins.

The conjugation of synthetic peptides to carrier proteins is a widely used method for immunological studies. Different coupling agents have been described to form the conjugate with carrier proteins. In this paper, we demonstrate that the antibody response toward V3-based synthetic MAPs derived from HIV-1, JY1 isolate, conjugated to two different carrier proteins using either m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or beta-maleimidopropionic acid N-hydroxysuccinimide ester (MPS), or succinic anhydride (SA) show different behaviors. An excellent anti-JY1 response without a strong response to the coupling agent is observed in the case of succinic anhydride spacer. In contrast, MBS produces total abrogation of the antibody response with a high response toward the coupling agent.     

Two new fluorescent dyes applicable for visualization of fungal cell walls

Two fluorophores, Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B, not heretofore reported upon are described as useful dyes of fungal cell walls, septa and bud scars examined microscopically. The dyes, depending on the filter sets used, yield fluorescently stained material generally in the blue to green and yellow to red wavelengths for Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B, respectively. They provide an excellent alternative to the more commonly used fluorophore, Calcofluor White M2R. The two fluorophores, in addition to being used at various spectral wavelengths from mercury arc sources, can be used with laser sources providing 488 nm and 543 nm line wavelengths, common to most scanning confocal microscopes. Unlike Calcofluor, Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B do not fade quickly when exposed to selected light wavelengths; however, like Calcofluors they are compatible with living fungal cells.     

Calmodulin activation of the Ca2+ pump revealed by fluorescent chelator dyes in human red blood cell ghosts

Ca2+ transport in red blood cell ghosts was monitored with fura2 or quin2 incorporated as the free acid during resealing. This is the first report of active transport monitored by the fluorescent intensity of the chelator dyes fura2 (5-50 microM) or quin2 (250 microM) in hemoglobin-depleted ghosts. Since there are no intracellular compartments in ghosts and the intracellular concentrations of all assay chelator substances including calmodulin (CaM), the dyes, and ATP could be set, the intracellular concentrations of free and total Ca [( Cafree]i and [Catotal]i) could be calculated during the transport. Ghosts prepared with or without CaM rapidly extruded Ca2+ to a steady-state concentration of 60-100 nM. A 10(4)-fold gradient for Ca2+ was routinely produced in medium containing 1 mM Ca2+. During active Ca2+ extrusion, d[Cafree]i/dt was a second order function of [Cafree]i and was independent of the dye concentration, whereas d[Catotal]i/dt increased as a first order function of both the [Cafree]i and the concentration of the Ca:dye complex. CaM (5 microM) increased d[Catotal]i/dt by 400% at 1 microM [Cafree]i, while d[Cafree]i/dt increased by only 25%. From a series of experiments we conclude that chelated forms of Ca2+ serve as substrates for the pump under permissive control of the [Cafree]i, and this dual effect may explain cooperativity. Free Ca2+ is extruded, and probably also Ca2+ bound to CaM or other chelators, while CaM and the chelators are retained in the cell.     

Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties

Molecular optical imaging has shown promise in visualizing molecular biomarkers with subcellular resolution both noninvasively and in real-time. Here, we use gold nanoparticles as optical probes to provide meaningful signal in the presence of targeted biomarkers. We present a novel conjugation technique to control the binding orientation of antibodies on the surface of gold nanoparticles to maximize antibody functionality. Briefly, a heterobifunctional linker, hydrazide-polyethylene glycol-dithiol, is used to directionally attach the Fc, or nonbinding region of the antibody, to the gold nanoparticle surface. The conjugation strategy allows for multiplexing various glycosylated antibodies on a single nanoparticle. We present a method to prepare multifunctional nanoparticles by incorporating targeting and delivery moieties on the same nanoparticle that addresses the challenge of imaging intracellular biomarkers. The time estimate for the entire protocol is approximately 6 h.     

High-affinity near-infrared fluorescent small-molecule contrast agents for in vivo imaging of prostate-specific membrane antigen

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.     

Ternary Gd(III)L-HSA adducts: evidence for the replacement of inner-sphere water molecules by coordinating groups of the protein. Implications for the design of contrast agents for MRI

Two novel gadolinium(III) chelates based on the structure of the heptadentate macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) ligand have been synthesized and their relaxometric and luminescent properties thoroughly investigated. They contain two water molecules in the inner coordination sphere in fast exchange with the bulk solvent and bear either a p-bromobenzyl or a p-phosphonatomethylbenzanilido substituent for promoting further interaction with macromolecular substrates. Upon interaction with human serum albumin the expected relaxation enhancement is not observed owing to displacement of the two inner-sphere water molecules of the complexes by a donor atom (likely from a carboxylate group) on the protein and possibly the phosphate anion of the buffered solution, respectively. We modeled the observed behavior by measuring the decrease of the relaxation rate of the water protons upon addition of malonate anion to aqueous solutions of the complexes. Conversely, no change in the hydratation state of the Gd(III) center for both complexes has been observed when the substrate for the formation of the macromolecular adduct is represented by poly-beta-cyclodextrin.     

Molecular imaging of EGFR/HER2 cancer biomarkers by protein MRI contrast agents

Epidermal growth factor receptor (EGFR) and HER2 are major prognosis biomarkers and drug targets overexpressed in various types of cancer cells. There is a pressing need to develop MRI contrast agents capable of enhancing the contrast between normal tissues and tumors with high relaxivity, capable of targeting tumors, and with high intratumoral distribution and minimal toxicity. In this review, we first discuss EGFR signaling and its role in tumor progression as a major drug target. We then report our progress in the development of protein contrast agents with significant improvement of both r ₁ and r ₂ relaxivities, pharmacokinetics, in vivo retention time, and in vivo dose efficiency. Finally, we report our effort in the development of EGFR-targeted protein contrast agents with the capability to cross the endothelial boundary and with good tissue distribution across the entire tumor mass. The noninvasive capability of MRI to visualize spatially and temporally the intratumoral distribution as well as quantify the levels of EGFR and HER2 would greatly improve our ability to track changes of the biomarkers during tumor progression, monitor treatment efficacy, aid in patient selection, and further develop novel targeted therapies for clinical application.     

Calpastatin exon 1B-derived peptide,a selective inhibitor of calpain: enhancing cell permeability by conjugation with penetratin

The ubiquitous calpains, mu- and m-calpain, have been implicated in essential physiological processes and various pathologies. Cell-permeable specific inhibitors are important tools to elucidate the roles of calpains in cultivated cells and animal models. The synthetic N-acetylated 27-mer peptide derived from exon B of the inhibitory domain 1 of human calpastatin (CP1B) is unique as a potent and highly selective reversible calpain inhibitor, but is poorly cell-permeant. By addition of N-terminal cysteine residues we have generated a disulfide-conjugated CP1B with the cell-penetrating 16-mer peptide penetratin derived from the third helix of the Antennapedia homeodomain protein. The inhibitory potency and selectivity of CP1B for calpain versus cathepsin B and L, caspase 3 and the proteasome was not affected by the conjugation with penetratin. The conjugate was shown to efficiently penetrate into living LCLC 103H cells, since it prevents ionomycin-induced calpain activation at 200-fold lower concentration than the non-conjugated inhibitor and is able to reduce calpain-triggered apoptosis of these cells. Penetratin-conjugated CP1B seems to be a promising alternative to the widely used cell-permeable peptide aldehydes (e.g. calpain inhibitor 1) which inhibit the lysosomal cathepsins and partially the proteasome as well or even better than the calpains.     

Comprehensive investigation of the non-covalent binding of MRI contrast agents with human serum albumin

Three techniques, electrospray mass spectrometry, ultrafiltration, and proton relaxometry, are compared in the context of the quantitative analysis of non-covalent binding between human serum albumin (HSA) and MRI contrast agents. The study of the affinity by proton relaxometry reveals the association constant and the number of interaction sites assuming that all sites are identical and independent. Ultrafiltration was adapted for the study of paramagnetic complexes. This technique confirmed the results obtained by relaxometry. Electrospray mass spectrometry, an original method able to study non-covalent binding because of its soft ionization process that allows for the survival of weak binding, provides qualitative and quantitative results. Electrospray mass spectrometry confirmed the affinity measured by proton relaxometry and ultrafiltration. This technique requires very small amounts of products and directly gives the stoichiometry of the association, information not easily obtained by classic techniques. Nevertheless, proton relaxometry remains a useful and mandatory technique for determining the enhancement of the relaxation subsequent to the binding although it demands large amounts of compounds. It is to be pointed out that even if the three techniques lead to a similar ranking of the affinity of the contrast agents for HSA, the absolute values of the association constants disagree as a result of the difference in the experimental conditions (presence of salt, native protein or desalted one, approximations in the fitting of the data, liquid or gas phases).     

Development of contrast agents targeted to macrophage scavenger receptors for MRI of vascular inflammation

Atherosclerosis is a leading cause of death in the U.S. Because there is a potential to prevent coronary and arterial disease through early diagnosis, there is a need for methods to image arteries in the subclinical stage as well as clinical stage using various noninvasive techniques, including magnetic resonance imaging (MRI). We describe a development of a novel MRI contrast agent targeted to plaques that will allow imaging of lesion formation. The contrast agent is directed to macrophages, one of the earliest components of developing plaques. Macrophages are labeled through the macrophage scavenger receptor A, a macrophage specific cell surface protein, using an MRI contrast agent derived from scavenger receptor ligands. We have synthesized and characterized these contrast agents with a range of relaxivities. In vitro studies show that the targeted contrast agent accumulates in macrophages, and solution studies indicate that micromolar concentrations are sufficient to produce contrast in an MR image. Cell toxicity and initial biodistribution studies indicate low toxicity, no detectable retention in normal blood vessels, and rapid clearance from blood. The promising performance of this contrast agent targeted toward vascular inflammation opens doors to tracking of other inflammatory diseases such as tumor immunotherapy and transplant acceptance using MRI.     

Synthesis and characterization of multifunctional hyperbranched polyesters as prospective contrast agents for targeted MRI

Based on a commercially available hyperbranched aliphatic polyester, novel multifunctional gadolinium complexes were prepared bearing protective PEG chains, a folate targeting ligand and EDTA or DTPA chelate moieties. Their relatively high water relaxivity values coupled with biodegradability of the hyperbranched scaffold, folate receptor specificity render these non-toxic dendritic polymers promising candidates for MRI applications.