Novel near-infrared cyanine fluorochromes: synthesis,properties, and bioconjugation

Recently, near-infrared (NIR) fluorescence light has been applied to image various biological events in vivo, because it penetrates tissue more efficiently than light in the visible spectrum. Compounds exhibiting fluorescent properties in the NIR range are key elements for this upcoming optical imaging technology. In this paper, we report the synthesis of four new, water-soluble NIR cyanine fluorochromes which have superior chemical stability and optical properties. Each fluorochrome was designed with a monoreactive carboxyl group for labeling purposes. When multiple fluorochromes were attached to a single macromolecule, fluorescence quenching was observed. On the basis of this property, a novel autoquenched enzyme sensitive NIR fluorescence probe was prepared.     

Miniaturized multichannel near infrared endoscope for mouse imaging

We describe the design and construction of a miniaturized multichannel near infrared (NIR) endoscopic imaging system developed for high-resolution imaging of mice. The device allows for simultaneous real-time video images in white light and two independent NIR channels. Testing demonstrated independent acquisition of nanomolar concentrations of fluorochromes Cy5.5 and Cy7. Cross-talk between the NIR channels, partially a result of broad tails in the spectra of commonly used organic fluorochromes, was assessed, modeled for the linear range of the concentration/signal intensity function, and compensated. The calculated compensation was 5.5% and 22% of the total signal intensity in the two channels NIR700 and NIR780, respectively, at equal concentrations of the two fluorochromes. Using a mouse model of colonic adenomatosis, we show that both perfusion and protease activity can be detected simultaneously, independently, and repeatedly in live mice. The developed device should be useful for in vivo imaging of diverse molecular targets.     

Developing a peptide-based near-infrared molecular probe for protease sensing

Recently near-infrared (NIR) molecular probes have become important reporter molecules for a number of types of in vivo biomedical imaging. A peptide-based NIR fluorescence probe consisting of a NIR fluorescence emitter (Cy5.5), a NIR fluorescence absorber (NIRQ820), and a protease selective peptide sequence was designed to sense protease activity. Using a MMP-7 model, we showed that NIRQ820 efficiently absorbs the emission energy of Cy5.5 resulting in a low initial signal. Upon reacting with its target, MMP-7, the fluorescence signal of the designed probe was increased by 7-fold with a K(cat)/K(m) of 100 000 M(-)(1) s(-)(1). The described synthetic strategy should have wide application for other NIR probe preparations.     

Near-infrared emissive polymer-coated IR-820 nanoparticles assisted photothermal therapy for cervical cancer cells

Photothermal therapy (PTT) has attracted wide attention due to its noninvasiveness and its thermal ablation ability. As photothermal agents are crucial factor in PTT, those with the characteristics of biocompatibility, non-toxicity and high photothermal stability have attracted great interest. In this work, new indocyanine green (IR-820) was utilized as a photothermal agent and near-infrared (NIR) fluorescence imaging nanoprobe. To improve the biocompatibility, poly(styrene-co-maleic anhydride) (PSMA) was utilized to encapsulate the IR-820 molecules to form novel IR-820@PSMA nanoparticles (NPs). Then, the optical and thermal properties of IR-820@PSMA NPs were studied in detail. The IR-820@PSMA NPs showed excellent photothermal stability and biocompatibility. The cellular uptaking ability of the IR-820@PSMA NPs was further confirmed in HeLa cells by the NIR fluorescent confocal microscopic imaging technique. The IR-820@PSMA NPs assisted PTT of living HeLa cells was conducted under 793 nm laser excitation, and a high PTT efficiency of 73.3% was obtained.     

A novel near-infrared indocyanine dye-polyethylenimine conjugate allows DNA delivery imaging in vivo

Near-infrared (NIR) fluorescence light has been applied to monitor several biological events in vivo since it penetrates tissues more efficiently than visible light. Dyes exhibiting NIR fluorescence and having large Stokes shift are key elements for this promising optical imaging technology. Here, we report the synthesis of a novel conjugate between a near-infrared indocyanine dye and an organic polyamine polymer (polyethylenimine, PEI) (IR820-PEI) with high chemical stability and good optical properties. IR820-PEI absorbs at 665 nm, emits at 780 nm, and displays a large Stokes shift (115 nm). Moreover, the reported conjugate is able to bind DNA, and the delivery process can be monitored in vivo with noninvasive optical imaging techniques. These characteristics make IR820-PEI one of the most effective and versatile indocyanine dye polymeric-conjugate reported so far.     

Synthesis and properties of sulfhydryl-reactive near-infrared cyanine fluorochromes for fluorescence imaging

Near-infrared fluorochromes (NIRF) are useful compounds for diverse biotechnology applications and for in vivo biomedical imaging. Such NIRF must have high quantum yield, be biocompatible, and be conjugatable to a wide variety of proteins, peptides, and other affinity ligands. Here, we describe the synthesis of four new nonsymmetrical sulfhydryl-reactive cyanine NIRF with excellent optical and chemical properties. Each fluorochrome was designed to contain an iodoacetamido group that reacts specifically with sulfhydryl-containing molecules. The synthesized fluorochromes were used to label model peptides and sulfhydryl-containing biomolecules.     

A novel thymidine phosphoramidite synthon for incorporation of internucleoside phosphate linkers during automated oligodeoxynucleotide synthesis

A novel thymidine phosphoramidite synthon was synthesized and successfully used for incorporation of primary amino groups, attached through a triethylene glycol linker to the internucleoside phosphates, at desired locations during automated oligodeoxynucleotide synthesis. The synthesized amino-linker bearing oligonucleotides are stable under deprotection conditions and exhibit Watson-Crick base-pairing properties. Covalent labeling of oligonucleotides with carbocyanine near-infrared fluorochromes resulted in 2.5 times higher labeling yields when compared with oligonucleotides containing base-attached aminolinkers. We anticipate that the developed synthetic approach will be useful for nucleotide sequence-specific attachment of single or multiple ligands or reporter molecules.     

A quantitative continuous enzyme assay of intramolecularly quenched fluorogenic phospholipase substrates for molecular imaging

There has been recent growth in the development of activatable near-infrared (NIR) fluorescent probes for molecular imaging, generally designed by placing fluorochromes on a cleavable substrate in close proximity to one another, such that they self-quench, but fluoresce on separation via enzymatic cleavage of the substrate. Although these probes offer excellent contrast, the detection of enzyme activity has largely only been described qualitatively. In order to assess the effectiveness of a probe, it is useful to have a quantitative measure, such as the enzyme-substrate kinetic parameters. We have developed an assay to determine kinetic parameters and applied it to an intramolecularly quenched molecule, Pyro-PtdEtn-BHQ, a NIR fluorescent probe specific to phosphatidylcholine-specific phospholipase C. The development of this assay includes corrections for intermolecular quenching, calibration, optimization of reaction mixtures, and determination of kinetic and inhibition parameters. This assay can easily be extended to analyze and compare the efficiency of other fluorescent activatable phospholipase probes as suitable molecular imaging agents.     

Single-Molecule Spectroscopy Reveals that Individual Low-Light LH2 Complexes from Rhodopseudomonas palustris 2.1.6. Have a Heterogeneous Polypeptide Composition

Rhodopseudomonas palustris belongs to the group of purple bacteria that have the ability to produce LH2 complexes with unusual absorption spectra when they are grown at low-light intensity. This ability is often related to the presence of multiple genes encoding the antenna apoproteins. Here we report, for the first time to our knowledge, direct evidence that individual low-light LH2 complexes have a heterogeneous αβ-apoprotein composition that modulates the site energies of Bchl a molecules, producing absorption bands at 800, 820, and 850 nm. The arrangement of the Bchl a molecules in the “tightly coupled ring” can be modeled by nine αβ-Bchls dimers, such that the Bchls bound to six αβ-pairs have B820-like site energies and the remaining Bchl a molecules have B850-like site energies. Furthermore, the experimental data can only be satisfactorily modeled when these six αβ-pairs with B820 Bchl a molecules are distributed such that the symmetry of the assembly is reduced to C₃. It is also clear from the measured single-molecule spectra that the energies of the electronically excited states in the mixed B820/850 ring are mainly influenced by diagonal disorder.     

Biochemical characterization of the dissociated forms from the core antenna proteins from purple bacteria

The core light-harvesting protein from Rhodospirillum rubrum is of particular interest for studying membrane polypeptide association, as it can be reversibly dissociated in the presence of n-octyl-beta-D-glucopyranoside (betaOG) into smaller subunit forms, which exhibit dramatically blue-shifted absorption properties (Miller et al. (1987) Biochemistry 26, 5055-5062). During this dissociation/reassociation process, two main spectroscopic forms are observed, absorbing at 820 (B820) and 777 (B777) nm, respectively. By using polyacrylamide gel electrophoresis in the presence of betaOG, these forms were characterized from a biochemical point of view. B777 consist of a mixture of alpha or beta polypeptide chains, retaining their bound bacteriochlorophyll (BChl) molecules. The absorption properties of the BChl molecules bound to the monomeric polypeptides do not depend on the chemical nature of the polypeptides they are bound to. B820 is more complex and consist of equilibrium between alphabeta-containing oligomers and beta only containing dimers, all exhibiting very similar electronic absorption properties. Resonance Raman spectroscopy indicates that the binding site provided by the beta-only B820 to the BChl molecules is very similar to that provided by the alphabeta B820. This, together with the observation that the alpha polypeptide alone is unable to form B820, suggests that the local organization of the BChl molecules tightly depends on BChl-protein interactions. On the other hand, our results suggest that the affinity of the beta-BChl complexes for itself and for the alpha-BChl ones are of the same order of magnitude, the formation of heterodimeric complexes being mainly driven by the inability of alpha-BChl complexes to self-associate.     

Determination of the B820 subunit size of a bacterial core light-harvesting complex by small-angle neutron scattering

The B820 subunit is an integral pigment-membrane protein complex and can be obtained by both dissociation of the core light-harvesting complex (LH1) in photosynthetic bacteria and reconstitution from its component parts in the presence of n-octyl beta-D-glucopyranoside (OG). Intrinsic size of the B820 subunit from Rhodospirillum rubrum LH1 complex was measured by small-angle neutron scattering in perdeuterated OG solution and evaluated by Guinier analysis. Both the B820 subunits prepared by dissociation of LH1 and reconstitution from apopolypeptides and pigments were shown to have a molecular weight of 11,400 +/- 500 and radius of gyration of 11.0 +/- 1.0 A, corresponding to a heterodimer consisting of one pair of alphabeta-polypeptides and two bacteriochlorophyll a molecules. Molecular weights of micelles formed by OG alone in solutions were determined in a range from 30,000 to 50,000 over concentrations of 1-5% (w/v), and thus are much larger than that of the B820 subunit. Similar measurement on the pigment-depleted apopolypeptides revealed highly heterogeneous behavior in the OG solutions, indicating that aggregates with various sizes were formed. The result provides evidence that bacteriochlorophyll a molecules play a crucial role in stabilizing and maintaining the B820 subunits in the dimeric state in solution. Further measurements on individual alpha- and beta-polypeptides exhibited a marked difference in aggregation property between the two polypeptides. The alpha-polypeptides appear to be uniformly dissolved in OG solution in a monomeric form, whereas the beta-polypeptides favor a self-associated form and tend to form large aggregates even in the presence of detergent. The difference in aggregation tendency was discussed in relation to the different behavior between alpha- and beta-polypeptides in reconstitution with bacteriochlorophyll a molecules.     

A universally applicable method of isolating somatic cell hybrids by two-colour flow sorting

A generally applicable technique is described that permits easy identification and isolation of heterokaryons a few hours after fusion. It is based on the labelling of living cells with different fluorochromes, which, at appropriate concentrations do not affect viability or gene expression. Both fluorochromes are relatively stable and do not cross-contaminate unlabelled cells. The technique has a powerful potential in studies on gene regulation in somatic cell hybrids since heterofluorescent hybrids between any type of cells can be isolated directly from large populations of monofluorescent parental cells by using a cell sorter equipped with a single laser. Thus the technique avoids the need for genetically marked parental cells for selection.     

Determination of restriction enzyme activity when cutting DNA labeled with the TOTO dye family

Optical mapping, a single DNA molecule genome analysis platform that can determine methylation profiles, uses fluorescently labeled DNA molecules that are elongated on the surface and digested with a restriction enzyme to produce a barcode of that molecule. Understanding how the cyanine fluorochromes affect enzyme activity can lead to other fluorochromes used in the optical mapping system. The effects of restriction digestion on fluorochrome labeled DNA (Ethidium Bromide, DAPI, H33258, EthD-1, TOTO-1) have been analyzed previously. However, TOTO-1 is a part of a family of cyanine fluorochromes (YOYO-1, TOTO-1, BOBO-1, POPO-1, YOYO-3, TOTO-3, BOBO-3, and POPO-3) and the rest of the fluorochromes have not been examined in terms of their effects on restriction digestion. In order to determine if the other dyes in the TOTO-1 family inhibit restriction enzymes in the same way as TOTO-1, lambda DNA was stained with a dye from the TOTO family and digested. The restriction enzyme activity in regards to each dye, as well as each restriction enzyme, was compared to determine the extent of digestion. YOYO-1, TOTO-1, and POPO-1 fluorochromes inhibited ScaI-HF, PmlI, and EcoRI restriction enzymes. Additionally, the mobility of labeled DNA fragments in an agarose gel changed depending on which dye was intercalated.     

IRDye78 conjugates for near-infrared fluorescence imaging

The detection of human malignancies by near-infrared (NIR) fluorescence will require the conjugation of cancer-specific ligands to NIR fluorophores that have optimal photoproperties and pharmacokinetics. IRDye78, a tetra-sulfonated heptamethine indocyanine NIR fluorophore, meets most of the criteria for an in vivo imaging agent, and is available as an N-hydroxysuccinimide ester for conjugation to low-molecular-weight ligands. However, IRDye78 has a high charge-to-mass ratio, complicating purification of conjugates. It also has a potentially labile linkage between fluorophore and ligand. We have developed an ion-pairing purification strategy for IRDye78 that can be performed with a standard C18 column under neutral conditions, thus preserving the stability of fluorophore, ligand, and conjugate. By employing parallel evaporative light scatter and absorbance detectors, all reactants and products are identified, and conjugate purity is maximized. We describe reversible and irreversible conversions of IRDye78 that can occur during sample purification, and describe methods for preserving conjugate stability. Using seven ligands, spanning several classes of small molecules and peptides (neutral, charged, and/or hydrophobic), we illustrate the robustness of these methods, and confirm that IRDye78 conjugates so purified retain bioactivity and permit NIR fluorescence imaging of specific targets.     

Applications of Fluorochromes to Pollen Biology. I. Mithramycin and 4',6-Diamidino-2-Phenylindole (Dapi) as Vital Stains and for Quantitation of Nuclear Dna

The two DNA-specific fluorochromes DAPI and mithramycin have been found to be extremely useful dyes in studies of pollen development and growth. Both fluorochromes stain nuclei brilliantly either in fixed or in living tricellular and bicellular angiosperm pollen, thereby permitting rapid scanning for pollen abnormalities and easy observation of nuclear details. These water soluble dyes can be incorporated into the germination medium for studies of pollen germination in vitro, facilitating observation of the movement of generative, sperm and tube nuclei during pollen growth. In fixed pollen, the fluorochromes bind quantitatively with DNA and thus may be used to quantitate ploidy changes and to study cell cycles during pollen development, germination and fertilization.     

Preparation of a cathepsin D sensitive near-infrared fluorescence probe for imaging.

A variety of proteases are overexpressed or activated during pathogenesis and represent important targets for therapeutic drugs. We have previously shown that optical imaging probes sensitive in the near-infrared fluorescence (NIRF) spectrum can be used for in vivo imaging of enzyme activity. In the current study, we show that these probes can be designed with specificity for specific enzymes, for example, cathepsin D which is known to be overexpressed in many tumors. A NIR cyanine fluorochrome served as the optical reporter and was attached to the amino terminal of an 11 amino acid peptide sequence with specificity for cathepsin D. The peptides were subsequently attached to a synthetic graft copolymer for efficient tumoral delivery. The close spatial proximity of the multiple fluorochromes resulted in quenching of fluorescence in the bound state. A 350-fold signal amplification was observed post cleavage during in vitro testing. Cell culture experiments using a rodent tumor cell line stably transfected with human cathepsin D confirmed enzyme specific activation within cells. This sequence but not a scrambled control sequence showed enzyme specificity in vitro. We conclude that activatable NIRF optical probes can be synthesized to potentially probe for specific enzymes in living organisms.     

Fluorescent indicator displacement assay of ligands targeting 10 microRNA precursors

Fluorescent indicator displacement (FID) assay is a rapid and convenient assay for identifying new ligands that bind to the target molecules. In our previous studies, we have shown that a series of 2,7-diaminoalkoxy xanthone and thioxanthone derivatives can be used as fluorescent indicators for detecting the interaction between RNA and a ligand. The xanthone and thioxanthone fluorochromes showed efficient fluorescence quenching upon binding to target RNA. Subsequent displacement of the bound-fluorochrome with a ligand that binds more strongly to the target RNA led to the recovery of the fluorescence by releasing the fluorochrome from RNA. Here we report a pilot screening of a chemical library that contains 9600 structurally diverse compounds for molecules that bind to a specific miRNA precursor using the FID assay.     

Light-Induced Dark States of Organic Fluochromes Enable 30 nm Resolution Imaging in Standard Media

We show that high quantum efficiency fluorophores can exhibit reversible photobleaching. This observation provides the basis for an imaging technique we call reversible photobleaching microscopy. We demonstrate applicability of this technique using antibody labeled biological samples in standard aqueous (or glycerol based) media to produce far-field images at ∼30 nm resolution. Our novel method relies on intense illumination to reversibly induce a very long-lived (>10 s) dark state from which single fluorochromes slowly return stochastically. As in other localization microscopy methods, reversible photobleaching microscopy localizes single fluorochromes, but has the advantage that specialized photoactivatible and photoswitchable molecules or special immersion/embedding media are not required.     

The crystallographic structure of the B800-820 LH3 light-harvesting complex from the purple bacteria Rhodopseudomonas acidophila strain 7050

The B800-820, or LH3, complex is a spectroscopic variant of the B800-850 LH2 peripheral light-harvesting complex. LH3 is synthesized by some species and strains of purple bacteria when growing under what are generally classed as "stressed" conditions, such as low intensity illumination and/or low temperature (<30 degrees C). The apoproteins in these complexes modify the absorption properties of the chromophores to ensure that the photosynthetic process is highly efficient. The crystal structure of the B800-820 light-harvesting complex, an integral membrane pigment-protein complex, from the purple bacteria Rhodopseudomonas (Rps.) acidophila strain 7050 has been determined to a resolution of 3.0 A by molecular replacement. The overall structure of the LH3 complex is analogous to that of the LH2 complex from Rps. acidophila strain 10050. LH3 has a nonameric quaternary structure where two concentric cylinders of alpha-helices enclose the pigment molecules bacteriochlorophyll a and carotenoid. The observed spectroscopic differences between LH2 and LH3 can be attributed to differences in the primary structure of the apoproteins. There are changes in hydrogen bonding patterns between the coupled Bchla molecules and the protein that have an effect on the conformation of the C3-acetyl groups of the B820 molecules. The structure of LH3 shows the important role that the protein plays in modulating the characteristics of the light-harvesting system and indicates the mechanisms by which the absorption properties of the complex are altered to produce a more efficient light-harvesting component.     

Comparative study of the optical and heat generation properties of IR820 and indocyanine green

AbstractNear-infrared (NIR) fluorophores are the focus of extensive research for combined molecular imaging and hyperthermia. In this study, we showed that the cyanine dye IR820 has optical and thermal generation properties similar to those of indocyanine green (ICG) but with improved in vitro and in vivo stability. The fluorescent emission of IR820 has a lower quantum yield than ICG but less dependence of the emission peak location on concentration. IR820 demonstrated degradation half-times approximately double those of ICG under all temperature and light conditions in aqueous solution. In hyperthermia applications, IR820 generated lower peak temperatures than ICG (4-9%) after 3-minute laser exposure. However, there was no significant difference in hyperthermia cytotoxicity, with both dyes causing significant cell growth inhibition at concentrations ≥ 5 μM. Fluorescent images of cells with 10 μM IR820 were similar to ICG images. In rats, IR820 resulted in a significantly more intense fluorescence signal and significantly higher organ dye content than for ICG 24 hours after intravenous dye administration (p < .05). Our study shows that IR820 is a feasible agent in experimental models of imaging and hyperthermia and could be an alternative to ICG when greater stability, longer image collection times, or more predictable peak locations are desirable.