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.     

Molecular probes for fluorescence image-guided cancer surgery

In cancer surgery, complete surgical resection of tumor lesions is critical to optimize the outcome. However, it is sometimes difficult to distinguish the boundary between tumor and normal tissues, and residual tumor tissue can result in cancer recurrence. Intraoperative imaging with fluorescent molecular probes can assist surgeons to visualize tumor lesions and their boundaries during surgery. Here, we review molecular probes for fluorescence image-guided cancer surgery, focusing especially on recent developments in high-performance tumor-imaging probes and the strategies used for their design.     

Inorganic nanoparticle-based contrast agents for molecular imaging

Inorganic nanoparticles (NPs) including semiconductor quantum dots (QDs), iron oxide NPs and gold NPs have been developed as contrast agents for diagnostics by molecular imaging. Compared with traditional contrast agents, NPs offer several advantages: their optical and magnetic properties can be tailored by engineering the composition, structure, size and shape; their surfaces can be modified with ligands to target specific biomarkers of disease; the contrast enhancement provided can be equivalent to millions of molecular counterparts; and they can be integrated with a combination of different functions for multimodal imaging. Here, we review recent advances in the development of contrast agents based on inorganic NPs for molecular imaging, and also touch on contrast enhancement, surface modification, tissue targeting, clearance and toxicity. As research efforts intensify, contrast agents based on inorganic NPs that are highly sensitive, target-specific and safe to use are expected to enter clinical applications in the near future.     

Safe common agents for improved NMR contrast

As nuclear magnetic resonance imaging techniques have developed, a need for agents which can enhance and improve the natural tissue relaxation time differences has become apparent. Especially valuable would be agents that differentially alter NMR images in a manner related to tissue physiology and disease processes. Sophisticated para-magnetic and free radical contrast agents will be discussed in other papers in this issue. However, in this report, some common agents which are currently used in research and in human clinical studies for other purposes, but which can alter NMR contrast will be discussed. These agents include olive oil, estrogen hormones, diuretics, ethanol, glycerin, and dimethyl sulfoxide. Measurements of their relative effects on T1 and T2 of normal and cancerous breast tissues, a variety of body organs, and brain are presented. Some of these agents may have immediate practical applications in human NMR imaging studies.     

Method for enhancing cell penetration of Gd3+-based MRI contrast agents by conjugation with hydrophobic fluorescent dyes

Gadolinium ion (Gd(3+)) complexes are commonly used as magnetic resonance imaging (MRI) contrast agents to enhance signals in T(1)-weighted MR images. Recently, several methods to achieve cell-permeation of Gd(3+) complexes have been reported, but more general and efficient methodology is needed. In this report, we describe a novel method to achieve cell permeation of Gd(3+) complexes by using hydrophobic fluorescent dyes as a cell-permeability-enhancing unit. We synthesized Gd(3+) complexes conjugated with boron dipyrromethene (BDP-Gd) and Cy7 dye (Cy7-Gd), and showed that these conjugates can be introduced efficiently into cells. To examine the relationship between cell permeability and dye structure, we further synthesized a series of Cy7-Gd derivatives. On the basis of MR imaging, flow cytometry, and ICP-MS analysis of cells loaded with Cy7-Gd derivatives, highly hydrophobic and nonanionic dyes were effective for enhancing cell permeation of Gd(3+) complexes. Furthermore, the behavior of these Cy7-Gd derivatives was examined in mice. Thus, conjugation of hydrophobic fluorescent dyes appears to be an effective approach to improve the cell permeability of Gd(3+) complexes, and should be applicable for further development of Gd(3+)-based MRI contrast agents.     

Liposomal mr contrast agents

Abstract

Liposomes are spheres composed of relatively non-toxic and biodegradable lipids which are useful for entrapping a variety of drugs, decreasing drug toxicity and targeting. For a number of years we have evaluated the use of liposomes as MR contrast agents. We have prepared and tested contrast agents entrapped within the internal aqueous space of liposomes as well as liposomes incorporating lipophilic contrast agents in the lipid bilayer. When chelates such as Gd-DTPA are entrapped within the internal aqueous space of lipid vesicles, delivery is primarily to the Kupffer cells and clearance is slow. Manganese ions entrapped within lipid vesicles cause more enhancement per micromole of paramagnetic ion than gadolinium. Lipophilic derivatives of manganese EDTA chelates when incorporated into liposomes confer the greatest hepatic enhancement per micromole of metal ion and have favorable clearance kinetics. An apparently hepatocyte specific liposomal MR contrast agent has been prepared based upon a lipophilic derivative of manganese EDTA, which enhances the liver and increases liver/tumor contrast to noise more than most other contrast agents per micromole of metal ion. The agent has very high relaxivity, Rl over 30 and R2 over 40 per micromole of manganese. Cardiac imaging shows pronounced blood pool enhancement with potential for myocardial perfusion imaging. Membrane bound lipophilic paramagnetic chelates hold promise as improved liposomal contrast agents for MR.     

Metal-based environment-sensitive MRI contrast agents

Interactions of paramagnetic metal complexes with their biological environment can modulate their magnetic resonance imaging (MRI) contrast–enhancing properties in different ways, and this has been widely exploited to create responsive probes that can provide biochemical information. We survey progress in two rapidly growing areas: the MRI detection of biologically important metal ions, such as calcium, zinc, and copper, and the use of transition metal complexes as smart MRI agents. In both fields, new imaging technologies, which take advantage of other nuclei (¹⁹F) and/or paramagnetic contact shift effects, emerge beyond classical, relaxation-based applications. Most importantly, in vivo imaging is gaining ground, and the promise of molecular MRI is becoming reality, at least for preclinical research.     

Targeted contrast agents for magnetic resonance imaging and ultrasound

The development of contrast agents that can be localized to a particular tissue or cellular epitope will potentially allow the noninvasive visualization and characterization of a variety of disease states. Recent advances have been made in the field of molecular imaging with magnetic resonance imaging and ultrasound and varied approaches have been devised to overcome the high background tissue signal. The types of agents and applications developed include gadolinium-conjugated targeting molecules for imaging of fibrin, superparamagnetic iron oxide particles for stem-cell tracking, multimodal perfluorocarbon nanoparticles for visualization of angiogenesis, liposomes for targeting atheroma components, and microbubbles for imaging transplant rejection.     

On the unimportance of constitutive models in computing brain deformation for image-guided surgery

Imaging modalities that can be used intra-operatively do not provide sufficient details to confidently locate the abnormalities and critical healthy areas that have been identified from high-resolution pre-operative scans. However, as we have shown in our previous work, high quality pre-operative images can be warped to the intra-operative position of the brain. This can be achieved by computing deformations within the brain using a biomechanical model. In this paper, using a previously developed patient-specific model of brain undergoing craniotomy-induced shift, we conduct a parametric analysis to investigate in detail the influences of constitutive models of the brain tissue. We conclude that the choice of the brain tissue constitutive model, when used with an appropriate finite deformation solution, does not affect the accuracy of computed displacements, and therefore a simple linear elastic model for the brain tissue is sufficient.     

Promising strategies for Gd-based responsive magnetic resonance imaging contrast agents

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Highlights► Responsive MRI contrast agents enable the study of biochemical events. ► Recent Gd-based contrast agents are reviewed here. ► Responsive agents act by modulating hydration state, molecular tumbling or number of metal centres. ► Promising strategies for future probe design are identified.     

Conformation and structure of polymeric contrast agents for medical imaging

The structure of Gd-DTPA-polylysine, Gd-DOTA-polylysine, Gd-SCN-Bz-DOTA-polylysine, and Gd-DTPA-poly(glu:lys) was investigated with circular dichroism, gel permeation chromatography, low angle light scattering, and proton longitudinal relaxivity. Molecular modeling calculations were performed and predicted helical secondary structure for charged Gd-chelator residues, i.e., Gd-DTPA, when the DTPA conjugation levels reached 90% and higher. This helical secondary structure was observed with circular dichroism. The conformational transition from coiled to extended linear was observed also by gel permeation chromatography and by proton relaxivity measurements. The helical secondary structure was not observed when the chelator was changed to DOTA. The residue charge interactions were eliminated in this case since the Gd-DOTA complex had no net charge. For this construct, the gel permeation and relaxivity measurements indicated a coiled conformation. An extended linear conformation was regained when the chelator complex was changed to Gd-SCN-Bz-DOTA, which had a net negative charge. The functional aspects of these structures were investigated by MR imaging of an animal tumor model. The linear extended polymer constructs gave 10-fold higher tumor signals then the coiled-collapsed constructs, indicating a much higher degree of trans-endothelial transport in the tumors.     

Semiconductor quantum dots as contrast agents for whole animal imaging

Recent developments in quantum dot (QD) technology have paved the way for using QDs as optical contrast agents for in vivo imaging. Pioneering papers showed the use of QDs as luminescent contrast agents for imaging cancer and guiding cancer surgery. The possible future use of QDs for clinical applications is expected to have a significant impact, however many challenges in this field have yet to be overcome.     

Aptamer-targeted gold nanoparticles as molecular-specific contrast agents for reflectance imaging

Targeted metallic nanoparticles have shown potential as a platform for development of molecular-specific contrast agents. Aptamers have recently been demonstrated as ideal candidates for molecular targeting applications. In this study, we investigated the development of aptamer-based gold nanoparticles as contrast agents, using aptamers as targeting agents and gold nanoparticles as imaging agents. We devised a novel conjugation approach using an extended aptamer design where the extension is complementary to an oligonucleotide sequence attached to the surface of the gold nanoparticles. The chemical and optical properties of the aptamer-gold conjugates were characterized using size measurements and oligonucleotide quantitation assays. We demonstrate this conjugation approach to create a contrast agent designed for detection of prostate-specific membrane antigen (PSMA), obtaining reflectance images of PSMA(+) and PSMA(-) cell lines treated with the anti-PSMA aptamer-gold conjugates. This design strategy can easily be modified to incorporate multifunctional agents as part of a multimodal platform for reflectance imaging applications.