Fluorescence Imaging Using Deep-Red Indocyanine Blue, a Complementary Partner for Near-Infrared Indocyanine Green.

Indocyanine Blue (ICB) is the deep-red pentamethine analogue of the widely used clinical near-infrared heptamethine cyanine dye Indocyanine Green (ICG). The two fluorophores have the same number of functional groups and molecular charge and vary only by a single vinylene unit in the polymethine chain, which produces a predictable difference in spectral and physicochemical properties. We find that the two dyes can be employed as a complementary pair in diverse types of fundamental and applied fluorescence imaging experiments. A fundamental fluorescence spectroscopy study used ICB and ICG to test a recently proposed Förster Resonance Energy Transfer (FRET) mechanism for enhanced fluorescence brightness in heavy water (D2O). The results support two important corollaries of the proposal: (a) the strategy of using heavy water to increase the brightness of fluorescent dyes for microscopy or imaging is most effective when the dye emission band is above 650 nm, and (b) the magnitude of the heavy water florescence enhancement effect for near-infrared ICG is substantially diminished when the ICG surface is dehydrated due to binding by albumin protein. Two applied fluorescence imaging studies demonstrated how deep-red ICB can be combined with a near-infrared fluorophore for paired agent imaging in the same living subject. One study used dual-channel mouse imaging to visualize increased blood flow in a model of inflamed tissue, and a second mouse tumor imaging study simultaneously visualized the vasculature and cancerous tissue in separate fluorescence channels. The results suggest that ICB and ICG can be incorporated within multicolor fluorescence imaging methods for perfusion imaging and hemodynamic characterization of a wide range of diseases.

Lipophilic Anchors that Embed Bioconjugates in Bilayer Membranes: A Review.

A wide range of biomaterials and engineered cell surfaces are composed of bioconjugates embedded in liposome membranes, surface-immobilized bilayers, or the plasma membranes of living cells. This review article summarizes the various ways that Nature anchors integral and peripheral proteins in a cell membrane and describes the strategies devised by chemical biologists to label a membrane protein in living cells. Also discussed are modern synthetic and semisynthetic methods to produce lipidated proteins. Subsequent sections describe methods to anchor a three-component synthetic construct that is composed of a lipophilic membrane anchor, hydrophilic linker, and exposed functional component. The surface exposed payload can be a fluorophore, aptamer, oligonucleotide, polypeptide, peptide nucleic acid, polysaccharide, branched dendrimer, or linear polymer. Hydrocarbon chains are commonly used as the membrane anchor, and a general experimental trend is that a two chain lipid anchor has higher membrane affinity than a cholesteryl or single chain lipid anchor. Amphiphilic fluorescent dyes are effective molecular probes for cell membrane imaging and a zwitterionic linker between the fluorophore and the lipid anchor promotes high persistence in the plasma membrane of living cells. A relatively new advance is the development of switchable membrane anchors as molecular tools for fundamental studies or as technology platforms for applied biomaterials.

Doubly Strapped Zwitterionic NIR-I and NIR-II Heptamethine Cyanine Dyes for Bioconjugation and Fluorescence Imaging.

Heptamethine cyanine dyes enable deep tissue fluorescence imaging in the near infrared (NIR) window. Small molecule conjugates of the benchmark dye ZW800-1 have been tested in humans. However, long-term imaging protocols using ZW800-1 conjugates are limited by their instability, primarily because the chemically labile C4'-O-aryl linker is susceptible to cleavage by biological nucleophiles. Here, we report a modular synthetic method that produces novel doubly strapped zwitterionic heptamethine cyanine dyes, including a structural analogue of ZW800-1, with greatly enhanced dye stability. NIR-I and NIR-II versions of these doubly strapped dyes can be conjugated to proteins, including monoclonal antibodies, without causing undesired fluorophore degradation or dye stacking on the protein surface. The fluorescent antibody conjugates show excellent tumor-targeting specificity in a xenograft mouse tumor model. The enhanced stability provided by doubly strapped molecular design will enable new classes of in vivo NIR fluorescence imaging experiments with possible translation to humans.

Single-molecule characterization of a bright and photostable deep-red fluorescent squaraine-figure-eight (SF8) dye.

Squaraine Figure Eight (SF8) dyes are a unique class of deep-red fluorescent dyes with self-threaded molecular architecture that provides structural rigidity while simultaneously encapsulating and protecting the emissive fluorochrome. Previous cell microscopy and bulk phase studies of SF8 dyes indicated order of magnitude enhancements in photostability over conventional pentamethine cyanine dyes such as Cy5. Studies conducted at the single molecule level now reveal that these ensemble level enhancements carry over to the single molecule level in terms of enhanced emission quantum yields, longer times to photobleaching, and enhanced total photon yields. When compared to Cy5, the SF8-based dye SF8(D4)2 possesses a three-fold larger single molecule emission quantum yield, exhibits order of magnitude longer average times before photobleaching, and exhibits twenty times larger photon yields. Additional features such as water solubility, fluorochrome encapsulation to protect it against nucleophilic attack, and selective biomarker targeting capability make SF8-based dyes promising candidates for biological labeling and microscopy applications and single molecule tracking.

Spontaneous Transfer of Indocyanine Green from Liposomes to Albumin Is Inhibited by the Antioxidant α-Tocopherol.

Indocyanine Green (ICG) is a clinically approved organic dye with near-infrared absorption and fluorescence. Over the years, many efforts to improve the photophysical and pharmacokinetic properties of ICG have investigated numerous nanoparticle formulations, especially liposomes with membrane-embedded ICG. A series of systematic absorption and fluorescence experiments, including FRET experiments using ICG as a fluorescence energy acceptor, found that ICG transfers spontaneously from liposomes to albumin protein residing in the external solution with a half-life of ∼10 min at 37 °C. Moreover, transfer of ICG from liposome membranes to external albumin reduces light-activated leakage from thermosensitive liposomes with membrane-embedded ICG. A survey of lipophilic liposome additives discovered that the presence of clinically approved antioxidant, α-tocopherol, greatly increases ICG retention in the liposomes (presumably by forming favorable aromatic stacking interactions), inhibits ICG photobleaching and prevents albumin-induced reduction of light-triggered liposome leakage. This new insight will help researchers with the specific task of optimizing ICG-containing liposomes for fluorescence imaging or phototherapeutics. More broadly, the results suggest a broader design concept concerning light triggered liposome leakage, that is, proximity of the light absorbing dye to the bilayer membrane is a critical design feature that impacts the extent of liposome leakage.

Sterically Shielded Hydrophilic Analogs of Indocyanine Green.

A modular synthetic process enables two or four shielding arms to be appended strategically over the fluorochromes of near-infrared cyanine heptamethine dyes to create hydrophilic analogs of clinically approved indocyanine green. A key synthetic step is the facile substitution of a heptamethine 4'-Cl atom by a phenol bearing two triethylene glycol chains. The lead compound is a heptamethine dye with four shielding arms, and a series of comparative spectroscopy studies showed that the shielding arms (a) increased dye photostability and chemical stability and (b) inhibited dye self-aggregation and association with albumin protein. In mice, the dye cleared from the blood primarily through the renal pathway rather than the biliary pathway for ICG. This change in biodistribution reflects the much smaller hydrodynamic diameter of the shielded hydrophilic ICG analog compared to the 67 kDa size of the ICG/albumin complex. An attractive feature of versatile synthetic chemistry is the capability to systematically alter the dye's hydrodynamic diameter. The sterically shielded hydrophilic ICG dye platform is well-suited for immediate incorporation into dynamic contrast-enhanced (DCE) spectroscopy or imaging protocols using the same cameras and detectors that have been optimized for ICG.

Comparison of cRGDfK Peptide Probes with Appended Shielded Heptamethine Cyanine Dye (s775z) for Near Infrared Fluorescence Imaging of Cancer.

Previous work has shown that the sterically shielded near-infrared (NIR) fluorescent heptamethine cyanine dye, s775z, with a reactive carboxyl group produces fluorescent bioconjugates with an unsurpassed combination of high photostability and fluorescence brightness. This present contribution reports two new reactive homologues of s775z with either a maleimide group for reaction with a thiol or a strained alkyne group for reaction with an azide. Three cancer-targeting NIR fluorescent probes were synthesized, each with an appended cRGDfK peptide to provide selective affinity for integrin receptors that are overexpressed on the surface of many cancer cells including the A549 lung adenocarcinoma cells used in this study. A set of cancer cell microscopy and mouse tumor imaging experiments showed that all three probes were very effective at targeting cancer cells and tumors; however, the change in the linker structure produced a statistically significant difference in some aspects of the mouse biodistribution. The mouse studies included a mock surgical procedure that excised the subcutaneous tumors. A paired-agent fluorescence imaging experiment co-injected a binary mixture of targeted probe with 850 nm emission, an untargeted probe with 710 nm emission and determined the targeted probe's binding potential in the tumor tissue. A comparison of pixelated maps of binding potential for each excised tumor indicated a tumor-to-tumor variation of integrin expression levels, and a heterogeneous spatial distribution of integrin receptors within each tumor.