Maged Henary

Targeted zwitterionic near-infrared fluorophores for improved optical imaging

The signal-to-background ratio (SBR) is the key determinant of sensitivity, detectability and linearity in optical imaging. As signal strength is often constrained by fundamental limits, background reduction becomes an important approach for improving the SBR. We recently reported that a zwitterionic near-infrared (NIR) fluorophore, ZW800-1, exhibits low background. Here we show that this fluorophore provides a much-improved SBR when targeted to cancer cells or proteins by conjugation with a cyclic RGD peptide, fibrinogen or antibodies. ZW800-1 outperforms the commercially available NIR fluorophores IRDye800-CW and Cy5.5 in vitro for immunocytometry, histopathology and immunoblotting and in vivo for image-guided surgery. In tumor model systems, a tumor-to-background ratio of 17.2 is achieved at 4 h after injection of ZW800-1 conjugated to cRGD compared to ratios of 5.1 with IRDye800-CW and 2.7 with Cy5.5. Our results suggest that introducing zwitterionic properties into targeted fluorophores may be a general strategy for improving the SBR in diagnostic and therapeutic applications.

Synthesis and in vivo fate of zwitterionic near-infrared fluorophores.

A longstanding problem in the field of image-guided surgery is the development of ideal near-infrared (NIR) fluorophores. The heptamethine NIR fluorophore indocyanine green (ICG) has been used extensively for image-guided surgery because of clinical availability and safety.[1-3] However, ICG is far from ideal because it exhibits high uptake in the liver, contaminates the gastrointestinal (GI) tract, provides moderate optical properties,[4] is unstable in aqueous media,[3,5] and is unable to conjugate covalently to targeting ligands.[2] Although several classes of novel molecules have been described,[6-13] none to date exhibit simultaneous low background binding, bifunctionality, excellent optical properties, low protein binding, and high serum stability. Although it is intuitive that physicochemical properties, i.e., positive/negative charge density, hydrophilicity/lipophilicity, and charge distribution, will impact in vivo performance, chemical structures that exhibit ideal characteristics have not yet been defined.

Near IR Heptamethine Cyanine Dye–Mediated Cancer Imaging

Purpose: Near-IR fluorescence imaging has great potential for noninvasive in vivo imaging of tumors. In this study, we show the preferential uptake and retention of two hepatamethine cyanine dyes, IR-783 and MHI-148, in tumor cells and tissues. Experimental Design: IR-783 and MHI-148 were investigated for their ability to accumulate in human cancer cells, tumor xenografts, and spontaneous mouse tumors in transgenic animals. Time- and concentration-dependent dye uptake and retention in normal and cancer cells and tissues were compared, and subcellular localization of the dyes and mechanisms of the dye uptake and retention in tumor cells were evaluated using organelle-specific tracking dyes and bromosulfophthalein, a competitive inhibitor of organic anion transporting peptides. These dyes were used to detect human cancer metastases in a mouse model and differentiate cancer cells from normal cells in blood. Results: These near-IR hepatamethine cyanine dyes were retained in cancer cells but not normal cells, in tumor xenografts, and in spontaneous tumors in transgenic mice. They can be used to detect cancer metastasis and cancer cells in blood with a high degree of sensitivity. The dyes were found to concentrate in the mitochondria and lysosomes of cancer cells, probably through organic anion transporting peptides, because the dye uptake and retention in cancer cells can be blocked completely by bromosulfophthalein. These dyes, when injected to mice, did not cause systemic toxicity. Conclusions: These two heptamethine cyanine dyes are promising imaging agents for human cancers and can be further exploited to improve cancer detection, prognosis, and treatment. Clin Cancer Res; 16(10); 2833–44. ©2010 AACR.

Structure-inherent targeting of near-infrared fluorophores for parathyroid and thyroid gland imaging

The typical method for creating targeted contrast agents requires covalent conjugation of separate targeting and fluorophore domains. In this study, we demonstrate that it is possible to create near-infrared (NIR) fluorophores with different tissue specificities driven by their inherent chemical structures. Thus, a single compact molecule performs both targeting and imaging. We use this strategy to solve a major problem in head and neck surgery: the identification and preservation of parathyroid and thyroid glands. We synthesized 700-nm and 800-nm halogenated fluorophores that show high uptake into these glands after a single intravenous (IV) injection of 0.06 mg kg−1 in a pig. By using a dual-channel NIR imaging system, we observed—in real time and with high sensitivity—the unambiguous distinction of parathyroid and thyroid glands simultaneously in the context of blood and surrounding soft tissue. This novel technology lays a foundation for performing head and neck surgery with increased precision and efficiency along with potentially lower morbidity, and it provides a general strategy for developing targeted NIR fluorophores.

Kinetically Controlled Photoinduced Electron Transfer Switching in Cu(I)-Responsive Fluorescent Probes

Copper(I)-responsive fluorescent probes based on photoinduced electron transfer (PET) switching consistently display incomplete recovery of emission upon Cu(I) binding compared to the corresponding isolated fluorophores, raising the question of whether Cu(I) might engage in adverse quenching pathways. To address this question, we performed detailed photophysical studies on a series of Cu(I)-responsive fluorescent probes that are based on a 16-membered thiazacrown receptor ([16]aneNS(3)) tethered to 1,3,5-triarylpyrazoline-fluorophores. The fluorescence enhancement upon Cu(I) binding, which is mainly governed by changes in the photoinduced electron transfer (PET) driving force between the ligand and fluorophore, was systematically optimized by increasing the electron withdrawing character of the 1-aryl-ring, yielding a maximum 29-fold fluorescence enhancement upon saturation with Cu(I) in methanol and a greater than 500-fold enhancement upon protonation with trifluoroacetic acid. Time-resolved fluorescence decay data for the Cu(I)-saturated probe indicated the presence of three distinct emissive species in methanol. Contrary to the notion that Cu(I) might engage in reductive electron transfer quenching, femtosecond time-resolved pump-probe experiments provided no evidence for formation of a transient Cu(II) species upon photoexcitation. Variable temperature (1)H NMR experiments revealed a dynamic equilibrium between the tetradentate NS(3)-coordinated Cu(I) complex and a ternary complex involving coordination of a solvent molecule, an observation that was further supported by quantum chemical calculations. The combined photophysical, electrochemical, and solution chemistry experiments demonstrate that electron transfer from Cu(I) does not compete with radiative deactivation of the excited fluorophore, and, hence, that the Cu(I)-induced fluorescence switching is kinetically controlled.

Evaluation of Polymethine Dyes as Potential Probes for Near Infrared Fluorescence Imaging of Tumors: Part - 1

Near-infrared (NIR) organic dyes have become important for many biomedical applications, including in vivo optical imaging. Conjugation of NIR fluorescent dyes to photosensitizing molecules (photosensitizers) holds strong potential for NIR fluorescence image guided photodynamic therapy (PDT) of cancer. Therefore, we were interested in investigating the photophysical properties, in vivo tumor-affinity and fluorescence imaging potential of a series of heterocyclic polymethine dyes, which could then be conjugated to certain PDT agents. For our present study, we selected a series of symmetrical polymethine dyes containing a variety of bis-N-substituted indole or benzindole moieties linked by linear conjugation with and without a fused substituted cyclohexene ring. The N-alkyl side chain at the C-terminal position was functionalized with sulfonic, carboxylic acid, methyl ester or hydroxyl groups. Although, among the parent cyanine dyes investigated, the commercially available, cyanine dye IR783 (3) (bis-indole-N-butylsulfonate)-polymethine dye with a cyclic chloro-cyclohexene moiety showed best fluorescence-imaging ability, based on its spectral properties (λAbs=782 nm, λFl=810 nm, ε = 261,000 M-1cm-1, ΦFl≈0.08) and tumor affinity. In addition to 3, parent dyes IR820 and Cypate (6) were also selected and subjected to further modifications by introducing desired functional groups, which could enable further conjugation of the cyanine dyes to an effective photosensitizer HPPH developed in our laboratory. The synthesis and biological studies (tumor-imaging and PDT) of the resulting bifunctional conjugates are discussed in succeeding paper (Part-2 of this study).

Phosphonated Near-Infrared Fluorophores for Biomedical Imaging of Bone†

The conventional method for creating targeted contrast agents is to conjugate separate targeting and fluorophore domains. A new strategy is based on the incorporation of targeting moieties into the non-delocalized structure of pentamethine and heptamethine indocyanines. Using the known affinity of phosphonates for bone minerals in a model system, two families of bifunctional molecules that target bone without requiring a traditional bisphosphonate are synthesized. With peak fluorescence emissions at approximately 700 or 800 nm, these molecules can be used for fluorescence-assisted resection and exploration (FLARE) dual-channel imaging. Longitudinal FLARE studies in mice demonstrate that phosphonated near-infrared fluorophores remain stable in bone for over five weeks, and histological analysis confirms their incorporation into the bone matrix. Taken together, a new strategy for creating ultra-compact, targeted near-infrared fluorophores for various bioimaging applications is described.

Cartilage-Specific Near-Infrared Fluorophores for Biomedical Imaging†

A novel class of near-infrared fluorescent contrast agents was developed. These agents target cartilage with high specificity and this property is inherent to the chemical structure of the fluorophore. After a single low-dose intravenous injection and a clearance time of approximately 4 h, these agents bind to all three major types of cartilage (hyaline, elastic, and fibrocartilage) and perform equally well across species. Analysis of the chemical structure similarities revealed a potential pharmacophore for cartilage targeting. Our results lay the foundation for future improvements in tissue engineering, joint surgery, and cartilage-specific drug development.

Structure-activity relationship analysis of substituted 4-quinolinamines, antagonists of immunostimulatory CpG-oligodeoxynucleotides

On the basis of a systematic SAR analysis of substituted quinolines, a derivative 32 was synthesized that shows half-maximal inhibition of the immunostimulatory effect of CpG-oligodeoxynucleotides in vitro at the concentration of 0.24 nM.

Percutaneous penetration enhancement activity of aromatic S,S-dimethyliminosulfuranes

The activity of three series of iminosulfuranes (classes I-III) as potential transdermal penetration enhancers was investigated. These dimethyl sulfoxide (DMSO) related compounds were synthesized from activated DMSO by trifluoroacetic anhydride. Structure confirmation was accomplished by 1H NMR, and 13C NMR spectroscopy and elemental analysis prior to in vitro testing. Hydrocortisone (HC) was used as a model drug, and the effect of the iminosulfuranes on the penetration of HC through hairless mouse skin was evaluated. All enhancers tested were applied to the skin as saturated suspensions in propylene glycol to ensure their maximum thermodynamic activity. Three compounds, S,S-dimethyl-N-(4-bromobenzoyl)iminosulfurane (9), S,S-dimethyl-N-(5-nitro-2-pyridyl)iminosulfurane (13), and S, S-dimethyl-N-(4-phenylazaphenyl)iminosulfurane (16) showed statistically significant activity quantitated by amounts of model drug permeated through the skin in 24 h (Q(24)), and flux values, compared to control (propylene glycol without enhancer). Highest Q(24) and flux values were obtained for 9: 996.2+/-192.5 microg/cm(2) and 42.9+/-7.5 microg/cm(2) per h, respectively. All arylsulfonyl substituted compounds showed lower or similar enhancement activity when compared to control. S, S-dimethyl-N-(benzenesulfonyl)iminosulfurane (1), S, S-dimethyl-N-(2-methoxycarbonylbenzenesulfonyl)iminosulfurane++ + ( 7) and S,S-dimethyl-N-(4-chlorobenzenesulfonyl)iminosulfurane (8) decreased the permeation of HC significantly (P<0.05). It is possible that these agents work as retardants under these experimental conditions. None of the enhancers tested showed significant skin model drug retention, suggesting that these compounds could be useful for increasing systemic rather than local drug delivery.