Human NQO1 as a Selective Target for Anticancer Therapeutics and Tumor Imaging.

Human NAD(P)H-quinone oxidoreductase1 (HNQO1) is a two-electron reductase antioxidant enzyme whose expression is driven by the NRF2 transcription factor highly active in the prooxidant milieu found in human malignancies. The resulting abundance of NQO1 expression (up to 200-fold) in cancers and a barely detectable expression in body tissues makes it a selective marker of neoplasms. NQO1 can catalyze the repeated futile redox cycling of certain natural and synthetic quinones to their hydroxyquinones, consuming NADPH and generating rapid bursts of cytotoxic reactive oxygen species (ROS) and H2O2. A greater level of this quinone bioactivation due to elevated NQO1 content has been recognized as a tumor-specific therapeutic strategy, which, however, has not been clinically exploited. We review here the natural and new quinones activated by NQO1, the catalytic inhibitors, and the ensuing cell death mechanisms. Further, the cancer-selective expression of NQO1 has opened excellent opportunities for distinguishing cancer cells/tissues from their normal counterparts. Given this diagnostic, prognostic, and therapeutic importance, we and others have engineered a large number of specific NQO1 turn-on small molecule probes that remain latent but release intense fluorescence groups at near-infrared and other wavelengths, following enzymatic cleavage in cancer cells and tumor masses. This sensitive visualization/quantitation and powerful imaging technology based on NQO1 expression offers promise for guided cancer surgery, and the reagents suggest a theranostic potential for NQO1-targeted chemotherapy.

Rational Design of a Small Molecular Near-Infrared Fluorophore for Improved In Vivo Fluorescence Imaging.

The near-infrared (NIR) fluorescence imaging modality has great potential for application in biomedical imaging research owing to its unique characteristics, such as low tissue autofluorescence and noninvasive visualization with high spatial resolution. Although a variety of NIR fluorophores are continuously reported, the commercially available NIR fluorophores are still limited, owing to complex synthetic processes and poor physicochemical properties. To address this issue, a small molecular NIR fluorophore (SMF800) was designed and developed in the present work to improve in vivo target-specific fluorescence imaging. After conjugation with pamidronate (PAM) and bovine serum albumin (BSA), the SMF800 conjugates exhibited successful in vivo targeting in bone and tumor tissues with low background uptake, respectively. The improved in vivo performance of the SMF800 conjugate demonstrated that the small molecular NIR fluorophore SMF800 can be widely used in a much broader range of imaging applications. The structure of SMF800, which was developed by considering two important physicochemical properties, water solubility and conjugatability, is first introduced. Therefore, this work suggests a simple and rational approach to design small, hydrophilic, and conjugatable NIR fluorophores for targeted bioimaging.

3D cell-printing of gradient multi-tissue interfaces for rotator cuff regeneration.

Owing to the prevalence of rotator cuff (RC) injuries and suboptimal healing outcome, rapid and functional regeneration of the tendon-bone interface (TBI) after RC repair continues to be a major clinical challenge. Given the essential role of the RC in shoulder movement, the engineering of biomimetic multi-tissue constructs presents an opportunity for complex TBI reconstruction after RC repair. Here, we propose a gradient cell-laden multi-tissue construct combined with compositional gradient TBI-specific bioinks via 3D cell-printing technology. In vitro studies demonstrated the capability of a gradient scaffold system in zone-specific inducibility and multi-tissue formation mimicking TBI. The regenerative performance of the gradient scaffold on RC regeneration was determined using a rat RC repair model. In particular, we adopted nondestructive, consecutive, and tissue-targeted near-infrared fluorescence imaging to visualize the direct anatomical change and the intricate RC regeneration progression in real time in vivo. Furthermore, the 3D cell-printed implant promotes effective restoration of shoulder locomotion function and accelerates TBI healing in vivo. In summary, this study identifies the therapeutic contribution of cell-printed constructs towards functional RC regeneration, demonstrating the translational potential of biomimetic gradient constructs for the clinical repair of multi-tissue interfaces.

Molecular Tuning of IR-786 for Improved Tumor Imaging and Photothermal Therapy.

A tumor-targeted near-infrared (NIR) fluorophore CA800Cl was developed based on commercially available IR-786 by modulating its physicochemical properties. IR-786, a hydrophobic cationic heptamethine cyanine fluorophore, was previously recognized as a mitochondria-targeting NIR agent with excellent optical properties. Owing to the poor tumor specificity of IR-786 itself, in vivo studies on tumor-targeted imaging have not yet been investigated. A chloro-cyclohexene ring and indolium side groups on the heptamethine chain are key structural features that improve tumor targetability, owing to better biodistribution and clearance. Thus, IR-786 should be designed to be more soluble in aqueous solutions so that it can preferentially accumulate in the tumor based on the structure-inherent targeting strategy. In this study, we developed a bifunctional NIR fluorophore CA800Cl by incorporating carboxylate moieties in the basic structure of IR-786. This improved its tumor targetability and water solubility, thereby enabling the use of CA800Cl for enhanced photothermal cancer therapy.

Overcoming Spectral Dependence: A General Strategy for Developing Far-Red and Near-Infrared Ultra-Fluorogenic Tetrazine Bioorthogonal Probes.

Bioorthogonal fluorogenic dyes are indispensable tools in wash-free bioimaging of specific biological targets. However, the fluorogenicity of existing tetrazine-based bioorthogonal probes deteriorates as the emission wavelength shifts towards the NIR window, greatly limiting their applications in live cells and tissues. Herein, we report a generalizable molecular design strategy to construct ultra-fluorogenic dyes via a simple substitution at the meso-positions of various far-red and NIR fluorophores. Our probes demonstrate significant fluorescence turn-on ratios (102 -103 -fold) in the range 586-806 nm. These results will greatly expand the applications of bioorthogonal chemistry in NIR bioimaging and biosensing.

NIR-II Fluorophore with Dithienylethene as an Electron Donor for Fluorescence/Photoacoustic Dual-Model Imaging and Photothermal Therapy.

Well-designed second near-infrared (NIR-II) fluorophores are promising in optical diagnosis and therapy of tumors. In this work, we synthesized a donor-acceptor-donor (D-A-D) NIR-II fluorophore named BBTD-BET with dithienylethene as an electron donor and benzobisthiadiazole as an electron acceptor. To the best of our knowledge, this is the first report of using dithienylethene, a typical photochromic molecule, as a building block for NIR-II fluorophores. We studied the geometrical configuration, electronic state, and optical properties of BBTD-BET by both theoretical and experimental means. BBTD-BET had absorption and emission in the NIR-I and NIR-II spectral ranges, respectively. Using PEGylated BBTD-BET as a theranostic agent, we achieved NIR-II fluorescence/photoacoustic (PA) dual-modal imaging and attained high imaging resolution, desired signal-to-noise ratio, and excellent photothermal therapy (PTT) efficacy. After one PTT treatment, the tumors established in mice were eradicated. This work provides a novel organic conjugated molecule integrating NIR-II/PA dual-modal imaging and PTT functionalities that is very promising in the theranostic of tumors.

Radiolabeled Silicon-Rhodamines as Bimodal PET/SPECT-NIR Imaging Agents.

Radiolabeled fluorescent dyes are decisive for bimodal imaging as well as highly in demand for nuclear- and optical imaging. Silicon-rhodamines (SiRs) show unique near-infrared (NIR) optical properties, large quantum yields and extinction coefficients as well as high photostability. Here, we describe the synthesis, characterization and radiolabeling of novel NIR absorbing and emitting fluorophores from the silicon-rhodamine family for use in optical imaging (OI) combined with positron emission tomography (PET) or single photon emission computed tomography (SPECT), respectively. The presented photostable SiRs were characterized using NMR-, UV-Vis-NIR-spectroscopy and mass spectrometry. Moreover, the radiolabeling conditions using fluorine-18 or iodine-123 were extensively explored. After optimization, the radiofluorinated NIR imaging agents were obtained with radiochemical conversions (RCC) up to 70% and isolated radiochemical yields (RCY) up to 54% at molar activities of g.t. 70 GBq/µmol. Radioiodination delivered RCCs over 92% and allowed to isolate the 123I-labeled product in RCY of 54% at a molar activity of g.t. 7.6 TBq/µmol. The radiofluorinated SiRs exhibit in vitro stabilities g.t. 70% after two hours in human serum. The first described radiolabeled SiRs are a promising step toward their further development as multimodal PET/SPECT-NIR imaging agents for planning and subsequent imaging-guided oncological surgery.

Near-infrared Fluorophores for Thrombosis Diagnosis and Therapy.

Thrombosis is an adverse physiological event wherein the resulting thrombus and thrombus-induced diseases collectively result in high morbidity and mortality rates. Currently, nano-medicines that incorporate fluorophores emitting in the near-infrared-I (NIR-I, 700-900 nm) spectral region into their systems have been adopted to afford thrombosis theranostics. However, several unsolved problems such as limited penetration depth and image quality severely impede further applications of such nano-medicine systems. Fortunately, the ability to incorporate fluorophores emitting in the NIR-II (1000-1700 nm) window into nano-medicine systems can unambiguously identify biological processes with high signal-to-noise, deep tissue penetration depth, and high image resolution. Considering the inherently favorable properties of NIR-II fluorophores, we believe such have enormous potential to quickly become incorporated into nano-medicine systems for thrombosis theranostics. In this review, we i) discuss the development of NIR fluorescence as an imaging modality and fluorescent agents; ii) comprehensively summarize the recent development of NIR-I fluorophore-based nano-medicine systems for thrombosis theranostics; iii) highlight the state-of-the-art NIR-II fluorophores that have been designed for the specific purpose of affording thrombotic diagnosis; iv) speculate on possible forward avenues for the use of NIR-II fluorophores towards thrombosis diagnosis and therapy; and v) discuss the potential for their clinical translation.

Near-InfraRed PhotoImmunoTherapy (NIR-PIT) for the local control of solid cancers: Challenges and potentials for human applications.

Near-InfraRed PhotoImmunoTherapy (NIR-PIT) is a novel cancer-targeted treatment effected by a chemical conjugation between a photosensitiser (e.g. the NIR phthalocyanine dye IRDye700DX) and a cancer-targeting moiety (e.g. a monoclonal antibody, moAb). Delivery of a conjugate in vivo leads to accumulation at the tumour cell surface by binding to cell surface receptors or antigens. Upon deployment of focal NIR-light, irradiation of the conjugate results in a rapid, targeted cell death. However, the mechanisms of action to produce the cytotoxic effects have yet to be fully understood. Herein, we bring together the current knowledge of NIR-PIT from preclinical and clinical studies in a variety of cancers highlighting the key unanswered research questions. Furthermore, we discuss how to enhance the local control of solid cancers using this novel treatment regimen.

Structurally symmetric near-infrared fluorophore IRDye78-protein complex enables multimodal cancer imaging.

Rationale: Most contemporary cancer therapeutic paradigms involve initial imaging as a treatment roadmap, followed by the active engagement of surgical operations. Current approved intraoperative contrast agents exemplified by indocyanine green (ICG) have a few drawbacks including the inability of pre-surgical localization. Alternative near-infrared (NIR) dyes including IRDye800cw are being explored in advanced clinical trials but often encounter low chemical yields and complex purifications owing to the asymmetric synthesis. A single contrast agent with ease of synthesis that works in multiple cancer types and simultaneously allows presurgical imaging, intraoperative deep-tissue three-dimensional visualization, and high-speed microscopic visualization of tumor margins via spatiotemporally complementary modalities would be beneficial. Methods: Due to the lack of commercial availability and the absence of detailed synthesis and characterization, we proposed a facile and scalable synthesis pathway for the symmetric NIR water-soluble heptamethine sulfoindocyanine IRDye78. The synthesis can be accomplished in four steps from commercially-available building blocks. Its symmetric resonant structure avoided asymmetric synthesis problems while still preserving the benefits of analogous IRDye800cw with commensurable optical properties. Next, we introduced a low-molecular-weight protein alpha-lactalbumin (α-LA) as the carrier that effectively modulates the hepatic clearance of IRDye78 into the preferred renal excretion pathway. We further implemented 89Zr radiolabeling onto the protein scaffold for positron emission tomography (PET). The multimodal imaging capability of the fluorophore-protein complex was validated in breast cancer and glioblastoma. Results: The scalable synthesis resulted in high chemical yields, typically 95% yield in the final step of the chloro dye. Chemical structures of intermediates and the final fluorophore were confirmed. Asymmetric IRDye78 exhibited comparable optical features as symmetric IRDye800cw. Its well-balanced quantum yield affords concurrent dual fluorescence and optoacoustic contrast without self-quenching nor concentration-dependent absorption. The NHS ester functionality modulates efficient covalent coupling to reactive side-chain amines to the protein carrier, along with desferrioxamine (DFO) for stable radiolabeling of 89Zr. The fluorophore-protein complex advantageously shifted the biodistribution and can be effectively cleared through the urinary pathway. The agent accumulates in tumors and enables triple-modal visualization in mouse xenograft models of both breast and brain cancers. Conclusion: This study described in detail a generalized strategic modulation of clearance routes towards the favorable renal clearance, via the introduction of α-LA. IRDye78 as a feasible alternative of IRDye800cw currently in clinical phases was proposed with a facile synthesis and fully characterized for the first time. This fluorophore-protein complex with stable radiolabeling should have great potential for clinical translation where it could enable an elegant workflow from preoperative planning to intraoperative deep tissue and high-resolution image-guided resection.

Near-infrared fluorescence imaging in immunotherapy.

Near-infrared (NIR) light possesses many suitable optophysical properties for medical imaging including low autofluorescence, deep tissue penetration, and minimal light scattering, which together allow for high-resolution imaging of biological tissue. NIR imaging has proven to be a noninvasive and effective real-time imaging methodology that provides a high signal-to-background ratio compared to other potential optical imaging modalities. In response to this, the use of NIR imaging has been extensively explored in the field of immunotherapy. To date, NIR fluorescence imaging has successfully offered reliable monitoring of the localization, dynamics, and function of immune responses, which are vital in assessing not only the efficacy but also the safety of treatments to design immunotherapies optimally. This review aims to provide an overview of the current research on NIR imaging of the immune response. We expect that the use of NIR imaging will expand further in response to the recent success in cancer immunotherapy. We will also offer our insights on how this technology will meet rapidly growing expectations in the future.

A New Phenolate-Ion-Type Two-Photon Near Infrared Fluorophore-Based Biosensor for High-Performance Detection of HNO.

Although (E)-4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)phenolate anion (DCPO- ) has recently emerged as a potential near infrared (NIR) biosensor signaling unit, the pKa value of its conjugate acid is relatively high (∼9); this will lead to relatively low concentrations of DCPO- under physiological conditions and, hence, unsatisfactory sensitivity of DCPO- -based bio-probes. By difluoro-substitution on DCPO- , we have exploited a new fluorophore of o-FDCPO- whose conjugate acid has a much lower pKa value of 7.42. Meanwhile, o-FDCPO- is NIR emissive with λem =693 nm and has a 0.76-fold higher fluorescence efficiency than DCPO- . The significant superiority of o-FDCPO- over DCPO- in sensitivity for NIR biosensor applications was confirmed by comparative studies on two HNO probes, namely o-FDCPO-P and DCPO-P, which bear signaling units of o-FDCPO- and DCPO- , respectively. Moreover, o-FDCPO-P has been demonstrated to be a high-performance HNO probe with high selectivity, high sensitivity (detection limit: 50 nm), and a rapid response, together with a two-photon NIR-excitation imaging capability.

Bioluminescence and near-infrared fluorescence imaging for detection of metastatic bone tumors.

Bioluminescence imaging is being increasingly utilized in biological research. However, since the most commonly used firefly luciferase generates relatively weak bioluminescent signals, detection of low numbers of luciferase-expressing cells in vivo is challenging. The weak signal makes it difficult to detect cells located in deep tissues, which is problematic for preclinical research in tumor metastasis. In this study, three different types of fluorophores such as D-luciferin, AkaLumine-HCl, and P800SO3 were compared to evaluate the progression of bone metastasis induced by MDA-MB-231 breast cancer cells in vivo. The fluorescent signals for D-luciferin, AkaLumine-HCl, and P800SO3 were differently detected in the chest and knee joint. In particular, the fluorescence signal of P800SO3 was clearly observed in a section of the ribs, where it pointed out fractured bone fragments by tumor mass. Moreover, the P800SO3 signal from the left knee joint also showed a small bone fragment in the distal femur and was highlighted in the proximal tibia. Using targeted NIR fluorophores, metastatic bone tumors were monitored under the NIR fluorescence imaging system in real time, which enabled the in vivo diagnosis of bone metastasis by providing the location of the metastatic bone tumors.

Bridge-Caging Strategy in Phosphorus-Substituted Rhodamine for Modular Development of Near-Infrared Fluorescent Probes.

Replacement of the bridging oxygen atom in rhodamine with phosphorus is one of the most efficient ways for bright near-infrared (NIR) fluorophores with wavelengths over 700 nm. However, the organophosphorus bridge is more versatile than just being a spectrum tuner, it is also a profound solubility booster and photostability enhancer, as proved by a series of phosphorus-substituted rhodamines (PRBs). A unique bridge-caging strategy for efficiently manipulating fluorescence has further been innovated in example PRB2. Consistent with theoretical calculations, the formation of organophosphinate by a caging group as a fluorescence-controller locks the spirolactone into a colorless and nonfluorescent form, whereas decaging, a process induced by a specific stimulus, results in a ring-opened form, which yields strong fluorescence. The bridge-caging strategy is feasible for the modular development of NIR probes. Efficient in vivo imaging of photoillumination, hydrogen peroxide, and enzyme have been achieved on the PRB2 scaffold as a photoactivatable fluorophore, PRB2-hν; fluorescent indicator, PRB2-H2 O2 ; and fluorogenic enzyme substrate, PRB2-NTR, respectively.

Real-Time Tracking of Ex Vivo-Expanded Natural Killer Cells Toward Human Triple-Negative Breast Cancers.

Introduction: Ex vivo-expanded natural killer (NK) cells are a potential candidate for cancer immunotherapy based on high cytotoxicity against malignant tumor cells. However, a limited understanding of the migration of activated NK cells toward solid tumors is a critical dilemma in the development of effective and adoptive NK cell-based immunotherapy. Methods: Ex vivo-expanded NK cells from healthy donors were stained with near-infrared fluorophores at different concentrations. NK cell proliferation and cytotoxicity were assessed using a WST-8 assay, while the expression levels of surface molecules were analyzed by flow cytometry. To investigate the biodistribution of NK cells in both normal and tumor-bearing NSG mice, NK cells labeled with ESNF13 were subjected to NIR fluorescence imaging using the Mini-FLARE imaging system. Finally, mice were sacrificed and histopathological tests were performed in resected organs. Results: The signal intensity of ESNF-stained NK cells was long-lasting at 72 h using concentrations as low as 0.04 µM. At a low dose range, ESNF13 did not affect NK cell purity, expression levels of surface receptors, or cytotoxic functions against MDA-MB-231 cancer cells. Ex vivo-expanded NK cells labeled with ESNF13 had a 4-h biodistribution in non-tumor-bearing NSG mice that mainly localized to the lungs immediately after injection and then fully migrated to the kidney after 4 h. In an MDA-MB-231 tumor-bearing NSG mice with extensive metastasis in both lungs, the fluorescence signal was dominant in both lungs and steady at 1, 2, and 4 h post-injection. In a early phase of tumor progression, administered NK cell migrated to the lungs and tumor sites within 30 min post-injection, the signal dominated the tumor site after 1 h, and remained steady at 4 h. Conclusion: Optical imaging with NIR fluorophore ESNF13 is a highly sensitive, applicable, and inexpensive method for the real-time tracking of ex vivo-expanded NK cells both in vitro and in vivo. Administered NK cells had different patterns of NK cell distribution and accumulation to the tumor site according to tumor progression in triple-negative breast cancer xenograft models.

Selective Conversion of P=O-Bridged Rhodamines into P=O-Rhodols: Solvatochromic Near-Infrared Fluorophores.

The substitution of an oxygen atom in rhodols with a phosphine oxide (P=O) moiety affords P=O-bridged rhodols as a new type of near-infrared (NIR) fluorophore. This compound class can be readily accessed upon exposure of the corresponding rhodamines to aqueous basic conditions. The electron-withdrawing effect of the P=O group facilitates the hydrolytic deamination, and, moreover, prolonged exposure to aqueous basic conditions generates P=O-bridged fluoresceins, that is, a series of three P=O-bridged xanthene dyes is available in one simple operation. The P=O-bridged rhodols show significant bathochromic shifts of the longest-wavelength absorption maximum (Δλ=125 nm; >3600 cm-1 ) upon changing the solvent from toluene to water, whereas the emission is shifted less drastically (Δλ=70 nm; 1600 cm-1 ). The hydrogen bonding between the P=O and C=O groups with protic solvents results in substantial stabilization of the LUMO level, which is responsible for the solvatochromism.

Synthesis and Characterization of Water-Soluble Conjugated Oligoelectrolytes for Near-Infrared Fluorescence Biological Imaging.

Near-infrared (NIR) fluorophores attract increasing attention as a molecular marker (or probe) for in vivo and in vitro biological fluorescence imaging. Three types of new NIR fluorescent conjugated oligoelectrolytes (COEs: Q-FlTBTTFl, Q-FlBBTFl, and Q-FlTBBTTFl) are synthesized with quaternized ammonium ionic groups in their side-chains for water solubility. The emission wavelength is modulated in the range 600-1300 nm, by adjusting the intramolecular charge transfer in the molecular backbone based on the electron-rich fluorene (and/or thiophene) and electron-deficient benzo[2,1,3]thiadiazole (or benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole) moieties. The COEs show a remarkably larger Stokes shift (147-276 nm) compared to commercial rhodamine and cyanine dyes in water, avoiding self-quenching and interference from the excitation backscattered light. The photoluminescence (PL) quantum efficiency is improved substantially by up to 27.8% in water by fabricating a vesicular complex, COE/v, with a block ionomer, poly[(ethylene oxide)-block-(sodium 2-acrylamido-2-methyl-1-propanesulfonate)]. In vitro cellular uptake images with the COEs are obtained with good biocompatibility by confocal single-photon and two-photon microscopy. The ex vivo and in vivo images of a mouse xenograft model treated with the Q-FlBBTFl/v exhibit a substantially stronger fluorescence signal at the tumor site than at the other organs, highlighting the potential of the COE/v as an NIR fluorescent imaging agent for the diagnosis of cancer.

A Highly Selective Sensor for Cyanide in Organic Media and on Solid Surfaces.

The application of IR 786 perchlorate (IR-786) as a selective optical sensor for cyanide anion in both organic solution (acetonitrile (MeCN), 100%) and solvent-free solid surfaces was demonstrated. In MeCN, IR-786 was selective to two anions in the following order: CN(-) > OH(-). A significant change in the characteristic dark green color of IR-786 in MeCN to yellow was observed as a result of nucleophilic addition of CN(-) to the fluorophore, i.e., formation of IR 786-(CN), which was also verified by a blue shift in the 775 nm absorbance peak to 430 nm. A distinct green fluorescence emission from the IR-786-(CN) in MeCN was also observed, which demonstrated the selectivity of IR-786 towards CN(-) in MeCN. Fluorescence emission studies of IR-786 showed that the lower detection limit and the sensitivity of IR-786 for CN(-) in MeCN was 0.5 µM and 0.5 to 8 µM, respectively. The potential use of IR-786 as a solvent-free solid state sensor for the selective sensing and monitoring of CN(-) in the environment was also demonstrated. On solvent-free solid state surfaces, the sensitivity of the IR-786 to CN(-) in water samples was in the range of 50-300 µM with minimal interference by OH(-).

Charge and hydrophobicity effects of NIR fluorophores on bone-specific imaging.

Recent advances in near-infrared (NIR) fluorescence imaging enabled real-time intraoperative detection of bone metastases, bone growth, and tissue microcalcification. Pamidronate (PAM) has been widely used for this purpose because of its high binding affinity toward bone and remarkable therapeutic effects. Herein we describe the development of a series of PAM-conjugated NIR fluorophores that varied in net charges and hydrophobicity, and compared their bone targeting efficiency, biodistribution, and blood clearance. Since the targeting moiety, PAM, is highly negatively charged but small, the overall in vivo bone targeting and biodistribution were mediated by the physicochemical properties of conjugated fluorophores.

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(-1)cm(-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).