Structural Engineering of Fluorescent Self-Threaded Peptide Probes for Targeted Cell Imaging.

Squaraine figure-eight (SF8) molecules are a new class of deep-red fluorescent probes that are well suited for fluorescence cell microscopy due to their very high fluorescence brightness and excellent stability. Three homologous SF8 probes, with peptidyl loops that differ by very minor changes in the peptide sequence, were synthesized and assessed for probe uptake by cancer cells. One of probes included the RGD motif that is recognized by many classes of integrin receptors that reside on the surface of the cancer cells, and it permeated the cells by receptor-mediated endocytosis. In contrast, cell microscopy showed that there was negligible cell uptake of the two homologous SF8 probes indicating differences in probe targeting capability. The synthetic method allows for easy alteration of the peptide sequence; thus, it is straightforward to develop new classes of peptidyl SF8 probes with loop sequences that target other cancer biomarkers.

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.

Chiral figure-eight molecular scaffold for fluorescent probe development.

Targeted fluorescent molecular probes are useful for cell microscopy, diagnostics, and biological imaging. An emerging discovery paradigm is to screen libraries of fluorescent molecules and identify hit compounds with interesting targeting properties. However, a current limitation with this approach is the lack of fluorescent molecular scaffolds that can produce libraries of probe candidates with three dimensional globular shape, chiral centers, and constrained conformation. This study evaluated a new probe scaffold called squaraine figure-eight (SF8), a self-threaded molecular architecture that is comprised of an encapsulated deep-red fluorescent squaraine dye, surrounding tetralactam macrocycle, and peripheral loops. Easy synthetic variation of the loops produced four chiral isomeric SF8 probes, with the same log P values. Cell microscopy showed that subtle changes in the loop structure led to significant differences in intracellular targeting. Most notably, a comparison of enantiomeric probes revealed a large difference in mitochondrial accumulation, very likely due to differences in affinity for a chiral biomarker within the organelle. A tangible outcome of the research is a probe candidate that can be: (a) developed further as a bright and photostable, deep-red fluorescent probe for mitochondrial imaging, and (b) used as a molecular tool to identify the mitochondrial biomarker for selective targeting. It will be straightforward to expand the SF8 probe chemical space and produce structurally diverse probe libraries with high potential for selective targeting of a wide range of biomarkers.

High-Performance Near-Infrared Fluorescent Secondary Antibodies for Immunofluorescence.

A broad array of imaging and diagnostic technologies employs fluorophore-labeled antibodies for biomarker visualization, an experimental technique known as immunofluorescence. Significant performance advantages, such as higher signal-to-noise ratio, are gained if the appended fluorophore emits near-infrared (NIR) light with a wavelength >700 nm. However, the currently available NIR fluorophore antibody conjugates are known to exhibit significant limitations, including low chemical stability and photostability, weakened target specificity, and low fluorescence brightness. These fluorophore limitations are resolved by employing a NIR heptamethine cyanine dye named s775z whose chemical structure is very stable, charge-balanced, and sterically shielded. Using indirect immunofluorescence for imaging and visualization, a secondary IgG antibody labeled with s775z outperformed IgG analogues labeled with the commercially available NIR fluorophores, IRDye 800CW and DyLight800. Comparison experiments include three common techniques: immunocytochemistry, immunohistochemistry, and western blotting. Specifically, the secondary IgG labeled with s775z was 3-8 times brighter, 3-6 times more photostable, and still retained excellent target specificity when the degree of antibody labeling was high. The results demonstrate that antibodies labeled with s775z can emit total photon counts that are 1-2 orders of magnitude higher than those currently possible, and thus enable unsurpassed performance for NIR fluorescence imaging and diagnostics. They are especially well suited for analytical applications that require sensitive NIR fluorescence detection or use modern photon-intense methods that require high photostability.

Fluorescent Self-Threaded Peptide Probes for Biological Imaging.

A general synthetic method creates a new class of covalently connected, self-threaded, fluorescent molecular probes with figure-eight topology, an encapsulated deep-red fluorophore, and two peripheral peptide loops. The globular molecular shape and rigidified peptide loops enhance imaging performance by promoting water solubility, eliminating probe self-aggregation, and increasing probe stability. Moreover, the peptide loops determine the affinity and selectivity for targets within complex biological samples such as cell culture, tissue histology slices, or living subjects. For example, a probe with cell-penetrating peptide loops targets the surface of cell plasma membranes, whereas, a probe with bone-targeting peptide loops selectively stains the skeleton within a living mouse. The unique combination of bright deep-red fluorescence, high stability, and predictable peptide-based targeting is ideal for photon intense fluorescence microscopy and biological imaging.

Cell organelle targeting of near-infrared croconaine dye controls photothermal outcome.

Near-infrared croconaine-peptide conjugates that target the cell nucleus promote photothermal induced cell death. In contrast, a croconaine-morpholine conjugate that targets the cell lysosomes promotes lysosome permeabilization without measurable cell phototoxicity.

Sterically Shielded Heptamethine Cyanine Dyes for Bioconjugation and High Performance Near-Infrared Fluorescence Imaging.

The near-infrared window of fluorescent heptamethine cyanine dyes greatly facilitates biological imaging because there is deep penetration of the light and negligible background fluorescence. However, dye instability, aggregation, and poor pharmacokinetics are current drawbacks that limit performance and the scope of possible applications. All these limitations are simultaneously overcome with a new molecular design strategy that produces a charge balanced and sterically shielded fluorochrome. The key design feature is a meso-aryl group that simultaneously projects two shielding arms directly over each face of a linear heptamethine polyene. Cell and mouse imaging experiments compared a shielded heptamethine cyanine dye (and several peptide and antibody bioconjugates) to benchmark heptamethine dyes and found that the shielded systems possess an unsurpassed combination of photophysical, physiochemical, and biodistribution properties that greatly enhance bioimaging performance.

Dual-Targeted Phototherapeutic Agents as Magic Bullets for Cancer.

Imagine the ideal cancer drug that only kills cancer cells and does not affect nearby noncancerous cells. In the words of Paul Ehrlich, the drug acts like a magic bullet. This Topical Review summarizes an emerging new strategy to achieve this audacious goal. The central concept is a dual-targeted phototherapeutic agent for photodynamic or photothermal therapy. The dual-targeted phototherapeutic agent promotes cancer cell specificity by leveraging three levels of selectivity. Cell death will only occur in the anatomical location that is illuminated with light (Selectivity Level 1) and in cancer cells within the illumination area that have selectively accumulated the agent (Selectivity Level 2). The cancer cell killing effect is highly localized if the agent accumulates in hypersensitive intracellular organelles (Selectivity Level 3). The common targeting units for cancer cells and organelles are described, along with recent examples of dual-targeted phototherapeutic agents that incorporate these two classes of targeting units.

Paired Agent Fluorescence Imaging of Cancer in a Living Mouse Using Preassembled Squaraine Molecular Probes with Emission Wavelengths of 690 and 830 nm.

New methods are described for the construction of targeted fluorescence probes for imaging cancer and the assessment of tumor targeting performance in a living mouse model. A novel noncovalent assembly process was used to fabricate a set of structurally related targeted fluorescent probes with modular differences in three critical assembly components: the emission wavelength of the squaraine fluorochrome, the number of cRGDfK peptide units that target the cancer cells, and the length of the polyethylene glycol chains as pharmacokinetic controllers. Selective targeting of cancer cells was proven by a series of cell microscopy experiments followed by in vivo imaging of subcutaneous tumors in living mice. The mouse imaging studies included a mock surgery that completely removed a fluorescently labeled tumor. Enhanced tumor accumulation due to probe targeting was first evaluated by conducting Single Agent Imaging (SAI) experiments that compared tumor imaging performance of a targeted probe and untargeted probe in separate mouse cohorts. Although there was imaging evidence for enhanced tumor accumulation of the targeted probe, there was moderate scatter in the data due to tumor-to-tumor variability of the vasculature structure and interstitial pressure. A subsequent Paired Agent Imaging (PAI) study coinjected a binary mixture of targeted probe (with emission at 690 nm) and untargeted probe (with emission at 830 nm) into the same tumor-burdened animal. The conclusion of the PAI experiment also indicated enhanced tumor accumulation of the targeted probe, but the statistical significance was much higher, even though the experiment required a much smaller cohort of mice. The imaging data from the PAI experiment was analyzed to determine the targeted probe's Binding Potential (BP) for available integrin receptors within the tumor tissue. In addition, pixelated maps of BP within each tumor indicated a heterogeneous spatial distribution of BP values. The results of this study show that the combination of fluorescent probe preassembly and PAI is a promising new way to rapidly develop targeted fluorescent probes for tumors with high BP and eventual use in clinical applications such as targeted therapy, image guided surgery, and personalized medicine.

High affinity threading of a new tetralactam macrocycle in water by fluorescent deep-red and near-infrared squaraine dyes.

A new tetralactam macrocycle was prepared and found to encapsulate deep-red and near-infrared squaraine and croconaine dyes in water with tunable threading kinetics. The new supramolecular paradigm of guest back-folding was used to increase macrocycle/squaraine affinity by 370-fold and achieve an association constant of 2.8 × 109 M-1.

Chemoenzymatic Dynamic Kinetic Resolution of Primary Benzylic Amines using Pd0 -CalB CLEA as a Biohybrid Catalyst.

Herein, we report on the use a biohybrid catalyst consisting of palladium nanoparticles immobilized on cross-linked enzyme aggregates of lipase B of Candida antarctica (CalB CLEA) for the dynamic kinetic resolution (DKR) of benzylic amines. A set of amines were demonstrated to undergo an efficient DKR and the recyclability of the catalysts was studied. Extensive efforts to further elucidate the structure of the catalyst are presented.

Molecular conjugation using non-covalent click chemistry.

Molecular conjugation refers to methods used in biomedicine, advanced materials and nanotechnology to link two partners - from small molecules to large and sometimes functionally complex biopolymers. The methods ideally have a broad structural scope, proceed under very mild conditions (including in H2O), occur at a rapid rate and in quantitative yield with no by-products, enable bioorthogonal reactivity and have zero toxicity. Over the past two decades, the field of click chemistry has emerged to afford us new and efficient methods of molecular conjugation. These methods are based on chemical reactions that produce permanently linked conjugates, and we refer to this field here as covalent click chemistry. Alternatively, if molecular conjugation is undertaken using a pair of complementary molecular recognition partners that associate strongly and selectively to form a thermodynamically stable non-covalent complex, then we refer to this strategy as non-covalent click chemistry. This Perspective is concerned with this latter approach and highlights two distinct applications of non-covalent click chemistry in molecular conjugation: the pre-assembly of molecular conjugates or surface-coated nanoparticles and the in situ capture of tagged biomolecular targets for imaging or analysis.

Molecular Imaging of Aminopeptidase N in Cancer and Angiogenesis.

This review focuses on recent advances in the molecular imaging of aminopeptidase N (APN, also known as CD13), a zinc metalloenzyme that cleaves N-terminal neutral amino acids. It is overexpressed in multiple cancer types and also on the surface of vasculature undergoing angiogenesis, making it a promising target for molecular imaging and targeted therapy. Molecular imaging probes for APN are divided into two large subgroups: reactive and nonreactive. The structures of the reactive probes (substrates) contain a reporter group that is cleaved and released by the APN enzyme. The nonreactive probes are not cleaved by the enzyme and contain an antibody, peptide, or nonpeptide for targeting the enzyme exterior or active site. Multivalent homotopic probes utilize multiple copies of the same targeting unit, whereas multivalent heterotopic molecular probes are equipped with different targeting units for different receptors. Several recent preclinical cancer imaging studies have shown that multivalent APN probes exhibit enhanced tumor specificity and accumulation compared to monovalent analogues. The few studies that have evaluated APN-specific probes for imaging angiogenesis have focused on cardiac regeneration. These promising results suggest that APN imaging can be expanded to detect and monitor other diseases that are associated with angiogenesis.

Non-Covalently Pre-Assembled High-Performance Near-Infrared Fluorescent Molecular Probes for Cancer Imaging.

New fluorescent molecular probes, which can selectively target specific cell surface receptors, are needed for microscopy, in vivo imaging, and image guided surgery. The preparation of multivalent probes using standard synthetic chemistry can be a laborious process due to low reaction yields caused by steric effects. In this study, fluorescent molecular probes were prepared by a programmed non-covalent pre-assembly process that used a near-infrared fluorescent squaraine dye to thread a macrocycle bearing a cyclic arginine-glycine-aspartate peptide antagonist (cRGDfK) as a cancer targeting unit. Cell microscopy studies using OVCAR-4 (ovarian cancer) and A549 (lung cancer) cells that express high levels of the integrin αvß3 or αvß5 receptors, respectively, revealed a multivalent cell targeting effect. That is, there was comparatively more cell uptake of a pre-assembled probe equipped with two copies of the cRGDfK antagonist than a pre-assembled probe with only one appended cRGDfK antagonist. The remarkably high photostability and low phototoxicity of these near-infrared probes allowed for acquisition of long-term fluorescence movies showing endosome trafficking in living cells. In vivo near-infrared fluorescence imaging experiments compared the biodistribution of a targeted and untargeted probe in a xenograft mouse tumor model. The average tumor-to-muscle ratio for the pre-assembled targeted probe was 3.6 which matches the tumor targeting performance reported for analogous cRGDfK-based probes that were prepared entirely by covalent synthesis. The capability to excite these pre-assembled near-infrared fluorescent probes with blue or deep-red excitation light makes it possible to determine if a target site is located superficially or buried in tissue, a probe performance feature that is likely to be very helpful for eventual applications such as fluorescence guided surgery.

High expression of integrin αvß3 enables uptake of targeted fluorescent probes into ovarian cancer cells and tumors.

The cell line OVCAR-4 was recently ranked as one of the most representative cell lines for high grade serous ovarian cancer (HGSOC). However, little work has been done to assess the susceptibility of OVCAR-4 cells and tumors to the more common types of molecular targeting. Proteome profiles suggest OVCAR-4 express high levels of integrin αvß3 receptors. Using flow cytometry with fluorescent antibodies we determined that OVCAR-4 cells have high number of integrin αvß3 receptors ([9.8 ±â€¯2.5] × 104/cell) compared to the well-characterized cell line U87-MG ([5.2 ±â€¯1.4] × 104/cell). However, OVCAR-4 cells also have roughly three times the surface area of U87-MG cells, so the average αvß3 receptor density is actually lower (11 ±â€¯3 versus 18 ±â€¯6 receptors/µm2). A series of new fluorescent molecular probes was prepared with structures comprised of a deep-red squaraine fluorophore (∼680 nm emission) covalently attached to zero, one, or two cyclic pentapeptide cRGD sequences for integrin targeting. Microscopy studies showed that uptake of the divalent probe into cultured OVCAR-4 cells was 2.2 ±â€¯0.4 higher than the monovalent probe, which in turn was 2.2 ±â€¯0.4 higher than the untargeted probe. This probe targeting trend was also seen in OVCAR-4 mouse tumor models. The results suggest that clinically relevant OVCAR-4 cells can be targeted using molecular probes based on αvß3 integrin receptor antagonists such as the cRGD peptide. Furthermore, deep-red fluorescent cRGD-squaraine probes have potential as targeted stains of cancerous tissue associated with HGSOC in surgery and pathology settings.

Inhibitor of p52 NF-κB subunit and androgen receptor (AR) interaction reduces growth of human prostate cancer cells by abrogating nuclear translocation of p52 and phosphorylated AR(ser81).

Accumulating evidence shows that androgen receptor (AR) activation and signaling plays a key role in growth and progression in all stages of prostate cancer, even under low androgen levels or in the absence of androgen in the castration-resistant prostate cancer. Sustained activation of AR under androgen-deprived conditions may be due to its interaction with co-activators, such as p52 NF-κB subunit, and/or an increase in its stability by phosphorylation that delays its degradation. Here we identified a specific inhibitor of AR/p52 interaction, AR/p52-02, via a high throughput screen based on the reconstitution of Gaussia Luciferase. We found that AR/p52-02 markedly inhibited growth of both castration-resistant C4-2 (IC50 ∼6 µM) and parental androgen-dependent LNCaP (IC50 ∼4 µM) human prostate cancer cells under low androgen conditions. Growth inhibition was associated with significantly reduced nuclear p52 levels and DNA binding activity, as well as decreased phosphorylation of AR at serine 81, increased AR ubiquitination, and decreased AR transcriptional activity as indicated by decreased prostate-specific antigen (PSA) mRNA levels in both cell lines. AR/p52-02 also caused a reduction in levels of p21(WAF/CIP1), which is a direct AR targeted gene in that its expression correlates with androgen stimulation and mitogenic proliferation in prostate cancer under physiologic levels of androgen, likely by disrupting the AR signaling axis. The reduced level of cyclinD1 reported previously for this compound may be due to the reduction in nuclear presence and activity of p52, which directly regulates cyclinD1 expression, as well as the reduction in p21(WAF/CIP1), since p21(WAF/CIP1) is reported to stabilize nuclear cyclinD1 in prostate cancer. Overall, the data suggest that specifically inhibiting the interaction of AR with p52 and blocking activity of p52 and pARser81 may be an effective means of reducing castration-resistant prostate cancer cell growth.