Zongren Zhang

Design, synthesis and evaluation of near-infrared fluorescent pH indicators in a physiologically relevant range

Removal of a hydroxylsulfonylbutyl arm from indocyanine green dye produced a pH-sensitive near-infrared (NIR) fluorescent indicator that is useful at physiological range.

Activatable molecular systems using homologous near-infrared fluorescent probes for monitoring enzyme activities in vitro, in cellulo, and in vivo.

We have developed a generic approach to determine enzyme activities in vitro and monitor their functional status in vivo. Specifically, a method to generate donor (CbOH)-acceptor (Me2NCp) near-infrared (NIR) fluorescent dye pairs for preparing enzyme activatable molecular systems were developed based on the structural template of heptamethine cyanine dyes. Using caspase-3 as a model enzyme, we prepared two new caspase-3 sensitive compounds with high fluorescence quenching efficiency: Me2NCp-DEVD-K(CbOH)-OH (4) and AcGK(Me2NCp)-DEVD-APK(CbOH)-NH2 (5). The mechanism of quenching was based on combined effects of direct (classical) and reverse fluorescence resonance energy transfer (FRET). Caspase-3 cleavage of the scissile DEVD amide bond regenerated the NIR fluorescence of both donor and acceptor dyes. While both compounds were cleaved by caspase-3, substrate 5 was cleaved more readily than 4, yielding k(cat) and K(M), values of 1.02 +/- 0.06 s(-1) and 15 +/- 3 microM, respectively. Treatment of A549 tumor cells with paclitaxel resulted in > 2-fold increase in the fluorescence intensity by NIR confocal microscopy, suggesting the activation of pro-caspase-3 to caspase-3. A similar trend was observed in a mouse model, where the fluorescence intensity was nearly twice the value in caspase-3-rich tissue relative to the control. These results demonstrate the use of the same NIR activatable molecular systems for monitoring the activities of enzymes across a wide spatial scale ranging from in vitro kinetics measurements to in cellulo and in vivo localization of caspase-3 activation. The NIR activatable molecular probes provide an effective strategy to screen new drugs in vitro and monitor treatment response in living organisms.

Targeting of ανβ3-Integrins Expressed on Tumor Tissue and Neovasculature Using Fluorescent Small Molecules and Nanoparticles

AIM Receptor-specific small molecules and nanoparticles are widely used in molecular imaging of tumors. Although some studies have described the relative strengths and weaknesses of the two approaches, reports of a direct comparison and analysis of the two strategies are lacking. Herein, we compared the tumor-targeting characteristics of a small near-infrared fluorescent compound (cypate-peptide conjugate) and relatively large perfluorocarbon-based nanoparticles (250 nm diameter) for imaging alpha(nu)beta(3)-integrin receptor expression in tumors. MATERIALS & METHODS Near-infrared fluorescent small molecules and nanoparticles were administered to living mice bearing subcutaneous or intradermal syngeneic tumors and imaged with whole-body and high-resolution optical imaging systems. RESULTS The nanoparticles, designed for vascular constraint, remained within the tumor vasculature while the small integrin-avid ligands diffused into the tissue to target integrin expression on tumor and endothelial cells. Targeted small-molecule and nanoparticle contrast agents preferentially accumulated in tumor tissue with tumor-to-muscle ratios of 8 and 7, respectively, compared with 3 for nontargeted nanoparticles. CONCLUSION Fluorescent small molecular probes demonstrate greater overall early tumor contrast and rapid visualization of tumors, but the vascular-constrained nanoparticles are more selective for detecting cancer-induced angiogenesis. A combination of both imaging agents provides a strategy to image and quantify integrin expression in tumor tissue and tumor-induced neovascular systems.

Near-Infrared Dichromic Fluorescent Carbocyanine Molecules†

Central to major advances in biochemical assays, molecularsensor technologies, and molecular optical imaging arefluorescent materials that provide high detection sensitivityformolecularprocesses.Inbiologicalopticalimaging,thelowtissueautofluorescenceandthedeeppenetrationoflightintothetissuesobservedatwavelengthsbetween650 and900nmallow the use of near-infrared (NIR) fluorescent dyes ascontrast agents in heterogeneous systems.

Spectral Properties of Pro-multimodal Imaging Agents Derived from a NIR Dye and a Metal Chelator

Abstract Monomolecular multimodal imaging agents (MOMIAs) are able to provide complementary diagnostic information of a target diseased tissue. We developed a convenient solid-phase approach to construct two pro-MOMIAs (before incorporating radiometal) derived from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and cypate, a near-infrared (NIR) fluorescent dye analogous to indocyanine green (ICG). The possible interaction between d orbitals of transition metal DOTA complexes or free metals and the p orbitals of cypate chromophore could quench the fluorescence of pro-MOMIAs. However, we did not observe significant changes in the spectral properties of cypate upon conjugation with DOTA and subsequent chelation with metals. The fluorescence intensity of the chelated and nonmetal-chelated PRO-MOMIAs remained fairly the same in dilute 20% aqueous dimethylsulfoxide (DMSO) solution (1 × 10−6 M). Significant reduction in the fluorescence intensity of pro-MOMIAs occurred in the presence of a large excess of metal ions (>1 molar ratio for indium and 20-fold for a copper relative to pro-MOMIA). This study suggests the feasibility of using MOMIAs for combined optical and radioisotope imaging.

Understanding dichromic fluorescence manifested in certain indocyanine green (ICG) analogs

Fluorescence has advanced our understanding in various aspects of biological processes. Fluorescence in the near infrared (NIR) region avoids background autofluorescence from biological samples, leading to improved image quality. In searching for indo-cyanine green (ICG) analogs that can be attached to biomolecules, we observed that dichromic fluorescence manifested in some mono reactive-group-functionalized ICG analogs. The two emission bands are distinctively separate from each other, making it a unique feature of fluorescent probes found in biological studies. We further demonstrated that the dichromism comes from the structure and is transferable from dye to its bioconjugates. In this paper, we used resonance theory and molecular orbital theory to explain the fluorophore photochemistry in an effort to understand the general fluorescence feature of ICG analogs and provide understanding of the secondary emission band.

Synthesis and evaluation of novel galactose-carbocyanine fluorescent contrast agents with enhanced hydrophilicity and rigid molecular constraint

A new carbocyanine optical molecular probe with enhanced water solubility and constrained structural conformations was designed and synthesized. The near infrared (NIR) fluorescent probe contains a nonionic D-galactopyranose, which could improve water solubility of the probe and enhance uptake in tumors mediated by glucose transporter. The possibility of multiple attachment points provides the potential to conjugate diverse bioactive molecules to the probe. We developed an efficient synthetic method that is optimized for large-scale synthesis. Preliminary in vivo biodistribution studies show that the probe is rapidly cleared from blood and localize in the liver as early as 5 minutes post-injection of the probe in nude mice. Additional studies to evaluate the tumor uptake of the probe and its bioactive peptide conjugates are in progress.

Multimodal optical-nuclear molecular imaging of tumors

Accurate diagnosis and treatment of various pathologic conditions can be achieved by integrating multimodal imaging systems that furnish complementary information to improve patient management. The availability of high-resolution tomography systems, fast computers, and image reconstruction algorithms has improved disease diagnosis and treatment management. As medical practice moves into the molecular era, the realization that no single imaging method can provide solutions to the complex information derived from molecular imaging has heightened interest in the use of multimodal imaging to harness the strengths of different imaging methods. For example, co-registration of pathologic tissues with computed tomography (CT) and radionuclear platform provides complementary anatomical and functional diagnostic information, respectively. Optical imaging (OI) promises to complement established imaging methods by reporting molecular events with high detection sensitivity. Although combining molecular optical contrasts with MRI or CT provides co-registered reference anatomy, the disparate contrast agent concentrations needed for OI and MRI or CT present a barrier to integration. An alternative approach is to combine OI with an established imaging modality possessing similar detection sensitivity but complementary reporting strategies. Because of the high sensitivity of both nuclear and OI methods and the compatibility of their imaging agents, incorporating nuclear to OI will provide a unique opportunity to fuse the imaging datasets with identical pharmacokinetics but different contrast mechanisms. To appreciate the potential benefits of combining optical and SPECT/PET, we have tabulated the similarities and differences between the two imaging methods in Table 1 (1). Based on these properties, several potential opportunities to combine the two platforms become apparent.

Design, synthesis, and evaluation of near-infrared fluorescent pH indicators at physiological range

Biochemical processes frequently involve protonation and deprotonation, resulting in pH changes that can be monitored with pH indicators. In heterogenous media such as tissue where indicator concentration or visual observation is limited, highly sensitive pH indicators with reduced tissue autofluorescence are needed. Because of the reduced tissue autofluorescence in the near infrared (NIR) region, NIR fluorescence dyes such as indocyanine green (ICG) and its derivatives have been used to image molecular processes. Removing one of the N-subsitituent of ICG led to pHsensitive dyes operating in the NIR region and at physiologically relevant range. Further modification of the compound and synthetic procedure allowed their conjugation to peptides for specific delivery to target cells and tissues. Synthetic strategy and preliminary results on the spectral and biological properties of such dyes and their bioconjugates are described.