Recent advances in light sources, detectors and other optical imaging technologies coupled with the development of novel optical contrast agents have enabled real-time, high resolution, in vivo monitoring of molecular targets. Noninvasive monitoring of molecular targets is particularly relevant to photodynamic therapy (PDT), including the delivery of photosensitizer in the treatment site and monitoring of molecular and physiological changes following treatment. Our lab has developed optical imaging technologies to investigate these various aspects of photodynamic therapy (PDT). We used a laser scanning confocal microscope to monitor the pharmacokinetics of various photosensitizers in in vitro as well as ex vivo samples, and developed an intravital fluorescence microscope to monitor photosensitizer delivery in vivo in small animals. A molecular specific contrast agent that targets the vascular endothelial growth factor (VEGF) was developed to monitor the changes in the protein expression following PDT. We were then able to study the physiological changes due to post-treatment VEGF upregulation by quantifying vascular permeability with in vivo imaging.

In vivo imaging of the nanoparticle-tissue interaction reveals processes which aid in the improvement of disease-specific markers. Magnetomotive optical coherence tomography (MM-OCT) may fill this role by imaging magnetic nanoparticles (Fe₃O₄, 20-30nm diameter) similar to those currently used for MRI contrast. This is performed by modulating a small (<20mm) electromagnet during conventional OCT imaging and detecting the induced displacement (magnetomotion) of the nanoparticles. In a recent advance, increased specificity was achieved using a 3-pulse sequence to measure the intrinsic background fluctuation to normalize the magnetomotive signal. In this way ghosting due to physiological and Brownian motion are eliminated. Silicone tissue phantoms which are both optically and mechanically similar to soft human tissue were used to measure the scaling of the magnetomotive signal with magnetic field strength, local optical scattering efficiency, and magnetic nanoparticle concentration. MM-OCT is sensitive to magnetite nanoparticles at a concentration of 220μg/g (P>.975), with the possibility of detecting even lower concentrations (63μg/g) with minor improvements. The MM-OCT signal exhibits a gentler falloff in depth (~4dB over 0.5mm) than conventional OCT imaging, limited ultimately by shot noise. The performance of MM-OCT was evaluated in vivo in a Xenopus laevis tadpole exposed to magnetic nanoparticles for 24 hours prior to imaging. Corresponding histology demonstrates the ability to correctly identify regions of high nanoparticle concentration with in vivo MM-OCT.

A new multimodal imaging agent for imaging the somatostatin receptor has been synthesized and evaluated in vitro and in vivo. A somatostatin analog, conjugated to both 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTA) and cypate (BS-296), was synthesized entirely on the solid phase (Fmoc) and purified by RP-HPLC. DOTA was added as a ligand for radiometals such as ⁶⁴Cu or ¹⁷⁷Lu for either radio-imaging or radiotherapy respectively. Cytate, a cypatesomatostatin analog conjugate, has previously demonstrated the ability to visualize somatostatin receptor rich tumor xenografts and natural organs by optical imaging techniques. BS-296 exhibited low nanomolar inhibitory capacity toward the binding of radiolabeled somatostatin analogs in cell membranes enriched in the somatostatin receptor, demonstrating the high affinity of this multimodal imaging peptide and indicating its potential as a molecular imaging agent. ⁶⁴Cu, an isotope for diagnostic imaging and radiotherapy, was selected as the isotope for radiolabeling BS-296. BS-296 was radiolabeled with ⁶⁴Cu in high specific activity (200 μCi/μg) in 90% radiochemical yield. Addition of 2,5-dihydroxybenzoic acid (gentisic acid) prevented radiolysis of the sample, allowing for study of the ⁶⁴Cu -BS-296 the day following radiolabeling. Furthermore, inclusion of DMSO at a level of 20% was found not to interfere with radiolabeling yields and prevented the adherence of ⁶⁴Cu -BS-296 to the walls of the reaction vessel.

Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.

The molecular first hyperpolarizability β of di-2-ANEPEQ and di-8-ANEPPS have been measured using hyper-Rayleigh scattering. Because of the strong fluorescence produced by the ANEP dyes, time correlated single photon counting Hyper-Rayleigh scattering measurements and spectral decomposition have been used to obtain accurate β values that correct for the competing fluorescent signal. The molecular hyperpolarizability β is measured for various wavelengths and compared with a two level model. Solvent effects have also been measured; the experimental results are consistent with a two valence bond state model.

In this work we applied core-shell CdS/Cd(OH)2 quantum dots (QDs) as fluorescent labels in the Leishmania amazonensis protozoarium. The nanocrystals (8-9 nm) are obtained via colloidal synthesis in aqueous medium, with final pH=7 using sodium polyphosphate as the stabilizing agent. The surface of the particles is passivated with a cadmium hydroxide shell and the particle surface is functionalized with glutaraldehyde. The functionalized and non-functionalized particles were conjugated to Leishmania organisms in the promastigote form. The marked live organisms were visualized using confocal microscopy. The systems exhibit a differentiation of the emission color for the functionalized and non-functionalized particles suggesting different chemical interactions with the promastigote moieties. Two photon emision spectra (λ_exc=795nm) were obtained for the promastigotes labeled with the functionalized QDs showing a significant spectral change compared to the original QDs suspension. These spectral changes are discussed in terms of the possible energy deactivation processes.

Interest in novel RGD peptides has been increasingly growing as the interactions between RGD peptides and integrins are the basis for a variety of cellular functions and medical applications such as modulation of cell adhesion, invasion, tumor angiogenesis, and metastasis. In particular, we have been interested in novel NIR fluorescent RGD peptides as potential optical contrast agents for in vivo tumor optical imaging. Therefore, two cyclic RGD penta-peptides conjugated with a NIR fluorescent carbocyanine (Cypate), i.e. lactam-based cyclo[RGDfK(Cypate)] (1) and disulfide-containing Cypate-cyclo(CRGDC)-NH₂ (2), were designed and synthesized. The competitive binding assay between the purified α_vβ₃ integrin and the peptide ligands using ¹²⁵I-echistatin as a tracer showed that 1 had a higher receptor binding affinity (IC₅₀~10^-7 M) than 2 (IC₅₀~10^-6 M). Furthermore, the internalization of 1 in A549 cells in vitro was less than 2, as revealed by fluorescence microscopy. These results suggest that both the lactam- and disulfide-based cyclic RGD penta-peptides should be further studied structurally and functionally to elucidate the advantages of each class of compounds.

Cyclodextrins (CD) are often projected as potential vehicles in targeted drug delivery. However, if the membrane structure is disrupted by CD, then it cannot be considered to be a good drug delivery vehicle. When an extrinsic fluorescence probe is used to monitor such interactions, there are no less than three possible equilibria that can operate simultaneously: surfactant-cyclodextrin, surfactant-fluorophore and cyclodextrin-fluorophore. The fluorescence intensity / lifetime might be affected by all these and so, the results depend strongly on the fluorophore used as well as the nature of the surfactant. This aspect highlights the importance of the suitability of the fluorescence probe to be used to study complicated systems and interaction. In the present work, chlorin p₆, prepared from chlorophyll from spinach leaves, has been used as the fluorescence probe to investigate the interaction between α-CD and β-CD with the neutral surfactants TX 100 and CTAB. The fluorophore is found to be a sensitive one for the study of the interaction of α- and β-CD with the surfactants Tritron X 100 (TX 100) and cetyl trimethyl ammonium bromide (CTAB). It is found that contrary to earlier reports, a complex between α-CD and TX 100 is formed, even though the binding constant is not very high. This observation can be obtained with chlorin p₆, which does not bind to the CDs, but not with a fluorophore like TNS, which binds to the CD as well and thus complicates the situation as the binding with CD is stronger than that between TX 100 and α-CD as compared to that between TNS and CD.

Near-Infrared (NIR) absorbing chromophores have been valuable in analytical and bioanalytical chemistry. NIR probes and labels have been used for several applications, including solvent polarity, hydrophobicity, DNA sequencing, immunoassays, CE separations, etc. The NIR region (700-1100 nm) is more advantageous for the bioanalytical chemist due to the inherently lower background interference and the high molar absorptivities. NIR dyes can be used as simple probes to investigate biomolecule properties or just simply to detect the presence of biomolecules. Another typical application is the use of NIR fluorophores as labels. In these applications covalent labeling is the preferred method but it requires NIR dyes with appropriate reactive moieties. Due to the hydrophobic nature of NIR chromophores non-covalent labeling may be a viable alternative. For this purpose novel bis(carbocynines) have been developed in our laboratories. These dyes form intramolecular H-aggregates in polar solvents, even at very low concentrations. Spectral properties of this intramolecular dimer greatly depend on the properties of heterocyclic moieties and the length, the location and/or flexibility of the connecting chain. This form of the dye can be described as a clamshell complex with two interacting hydrophobic carbocyanine moieties. This intramolecular H-aggregate has a low extinction coefficient and fluorescence quantum yield. Upon opening the clamshell that can be facilitated by changing microhydrophobicity (i.e., binding to biomolecules) the H-and D- bands are decreased and the monomeric band is increased, with concomitant increase in fluorescence intensity. The main analytical utility of these bis(carbocyanines) is that the free dye (i.e., not complexed to an analyte) has negligible fluorescence in a typical aqueous buffer environment. Examples of different applications of these bis(carbocyanines) are given including forensic applications.

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.

We present the results of our investigation of new derivatives of cyclohexanone and piperidone compounds that have exhibited in the past anticancer effect due to biochemical destruction of cancer cells. In this study we focus on using these compounds as markers for malignant cells because of their strong two-photon excited fluorescence. Their molecular cross-section of two-photon absorption can be as high as 3000x10-50cm4s/photon and compares well with Rhodamine B, well-known fluorescent molecular probe. This provides an option for monitoring the biochemical destruction of cancer cells by means of two-photon excited fluorescence spectroscopy. Initially we studied the two-photon fluorescence of the solutions of pure compounds. Then we mixed the compounds with amino acids (Glycine and Alanine), the major building blocks of proteins in cells, hoping that the fluorescence will give some insight in the interaction between the compounds and bio substances. We discuss the solubility issues, the cross-section of twophoton absorption of the compounds, and also the features of the spectrum of the two-photon excited fluorescence. Compounds combining both properties (cytotoxicity and two-photon excited fluorescence), which are now carried by different chemical agents, are expected to improve the efficiency of cancer treatment and lower the cost.

Although magnesium ions play a key role in many fundamental biological processes, information about its intracellular regulation is still scarce, due to the lack of appropriate detection methods. Here, we report the spectroscopic characterization of two diaza-18-crown-6 hydroxyquinoline derivatives (DCHQ) and we propose their application for the determination of total Mg²⁺ concentration and in confocal imaging as effective Mg²⁺ indicators. DCHQ derivatives 1 and 2 bind Mg²⁺ with much higher affinity than other available probes (Kd = 44 and 73 mM, respectively) with a concomitant strong fluorescence increase. On the other hand, the fluorescence intensity is not significantly affected by other divalent cations, most importantly Ca²⁺, or by pH changes within the physiological range. Evidence is provided on the use of fluorometric data to derive totalcellular Mg²⁺content, which is in agreement with atomic absorption data. Furthermore, we show that DCHQ compounds can be effectively employed to map intracellular ion distribution and movements in live cells by confocal microscopy. These findings suggest that DCHQ derivatives may serve as new probes for the study of Mg²⁺ regulation, allowing sensitive and straightforward detection of both static and dynamic signals.

The tetracycline-europium (Tc-Eu) complex is known to show emission at 615 nm. On addition of hydrogen peroxide (HP), a strongly fluorescent complex is formed. In this paper it is reported that the europium fluorescence intensity is increased when urea hydrogen peroxide is added to the tetracycline-europium aqueous solution. It was conceived that this enhancement could be used to determine urea hydrogen peroxide (UHP) levels. This method is simple, practical, and relatively free of interference from coexisting substances, and it can be applied successfully to assess urea peroxide in biological samples, for example, on human whole blood. The values obtained for whole blood agree with the urea concentration variation verified in 50 patients, including 25 pre-dialysis, 15 dialysis subjects and 10 controls. This method is non invasive and can help in the identification of renal and cardiac diseases.

Current organic fluorophores used as labeling reagents for biomolecule conjugation have significant limitations in photostability. This compromises their performance in applications that require a photostable fluorescent reporting group. For example, in molecular imaging and single molecule microscopy, photostable fluorescent labels are important for observing and tracking individual molecular events over extended period of time. We report in this paper an extremely photostable and highly fluorescent phthalocyanine dye, IRDye^TM 700DX, as a near-infrared fluorescence labeling reagent to conjugate with biomolecules. This novel water-soluble silicon phthalocyanine dye has an isomericly pure chemical structure. The dye is about 45 to 128 times more photostable than current near-IR fluorophores, e.g. Alexa Fluor"R"680, Cy^TM 5.5, Cy^TM 7 and IRDye^TM 800CW dyes; and about 27 times more photostable than tetramethylrhodamine (TMR), one of the most photostable organic dyes. This dye also meets all the other stringent requirements as an ideal fluorophore for biomolecules labeling such as excellent water solubility, no aggregation in high ionic strength buffer, large extinction coefficient and high fluorescent quantum yield. Antibodies conjugated with IRDye^TM 700DX at high D/P ratio exist as monomeric species in high ionic buffer and have bright fluorescence. The IRDye^TM 700DX conjugated antibodies generate sensitive, highly specific detection with very low background in Western blot and cytoblot assays.

The HIV-1 protease enzyme is an excellent target for drug therapy of HIV infection/AIDS. To measure the protease activity and screen for potent protease inhibitors, homogeneous protease assays based on quenched fluorescent peptide substrates have been widely used as a high-throughput screening methods. The major problem in these assays is the compound interference or assay artifacts from colored or insoluble materials in the assay, e.g. assay components, screening library compounds, etc. We report in this paper a near-infrared fluorescence resonance energy transfer (NIRFRET) based HIV-1 protease assay that can dramatically reduce or completely eliminate these assay artifacts by using a novel near-IR donor-quencher pair and long wavelength excitation (780 nm) and detection (820±10 nm). In this assay, a HIV-1 protease peptide substrate is conjugated with a near-IR fluorescent donor (IRDye^TM 800CW), and a novel near-IR non-fluorescent quencher (QC1) on opposite sides of the proteolytic cleavage site. The quencher, QC1, has extremely good spectral overlap of its absorption spectrum with the donor emission spectrum to ensure the efficient quenching of the donor's fluorescence. In the HIV-1 protease assay, this NIR-FRET system shows a large dynamic range, high signal to noise ratio, excellent Z'-factors, a wide range of DMSO tolerance, and no compound interference. This system provides a sensitive, robust assay for high-throughput screening (HTS) and can be readily adapted to other therapeutically significant protease targets.

In order to explore novel NIR fluorescent probes for optical imaging in biomedicines, one desferrioxamine (DFO)-bearing NIR fluorescent probe was designed and synthesized based on a dicarboxylic acid-containing carbocyanine (Cypate). Similar to the free DFO, the resulting conjugate Cypate-DFO showed high binding affinity with Fe(III) and Ga(III) as identified by ES-MS. Nevertheless, the iron binding was found to quench its fluorescent emission significantly, suggesting that the siderophore moiety might perturb the spectroscopic properties of the attached carbocyanine fluorophore through metal binding. As observed by fluorescence microscopy, Cypate-DFO showed significant cellular internalization in A549 cells in vitro. Further studies on novel Cypate-DFO derivatives of this type may reveal some exciting properties and biological activities.

Expression of integrin α_vβ₃ is upregulated in a number of cancers including colon, pancreas, lung and breast. Previous studies demonstrated that near infrared (NIR) fluorescent probes designed to target α_vβ₃ accumulated both in vitro and in vivo in α_vβ₃-positive tumor cells. To evaluate the selectivity of some NIR-labeled RGD peptides for α_vβ₃, the molecular probes were incubated in different cells, including the α_vβ₃-positive U87 and A549 cells, and α_vβ₃-negative HT29 cells. Whereas the RGD compounds tested internalized in the A549 cells, their uptake by the HT29 cell line, which is positive for α_vβ₅ and α_vβ₆, was low. The uptake of these probes in U87 depended on the structural features of the compounds. Further studies with functional blocking antibodies showed that the internalization in the α_vβ₃-positive cells may be mediated by different integrin receptor subtypes. The preliminary results suggest that the internalization of linear RGD peptides is mediated by the α_vβ₃ heterodimer but rearrangement of the peptide sequence could alter the selectivity of the molecular probes for different integrin subunits in the dimeric α and β proteins. Thus, a careful choice of RGD peptides can be used to monitor the functional status of different integrins in cells and tissues.