Method To Diversify Cyanine Chromophore Functionality Enables Improved Biomolecule Tracking and Intracellular Imaging.

Heptamethine indocyanines are invaluable probes for near-infrared (NIR) imaging. Despite broad use, there are only a few synthetic methods to assemble these molecules, and each has significant limitations. Here, we report the use of pyridinium benzoxazole (PyBox) salts as heptamethine indocyanine precursors. This method is high yielding, simple to implement, and provides access to previously unknown chromophore functionality. We applied this method to create molecules to address two outstanding objectives in NIR fluorescence imaging. First, we used an iterative approach to develop molecules for protein-targeted tumor imaging. When compared to common NIR fluorophores, the optimized probe increases the tumor specificity of monoclonal antibody (mAb) and nanobody conjugates. Second, we developed cyclizing heptamethine indocyanines with the goal of improving cellular uptake and fluorogenic properties. By modifying both the electrophilic and nucleophilic components, we demonstrate that the solvent sensitivity of the ring-open/ring-closed equilibrium can be modified over a wide range. We then show that a chloroalkane derivative of a compound with tuned cyclization properties undergoes particularly efficient no-wash live cell imaging using organelle-targeted HaloTag self-labeling proteins. Overall, the chemistry reported here broadens the scope of accessible chromophore functionality, and, in turn, enables the discovery of NIR probes with promising properties for advanced imaging applications.

Palladium responsive liposomes for triggered release of aqueous contents.

Palladium (Pd) is a promising metal catalyst for novel bioorthogonal chemistry and prodrug activation. This report describes the first example of palladium responsive liposomes. The key molecule is a new caged phospholipid called Alloc-PE that forms stable liposomes (large unilamellar vesicles, ∼220 nm diameter). Liposome treatment with PdCl2 removes the chemical cage, liberates membrane destabilizing dioleoylphosphoethanolamine (DOPE), and triggers liposome leakage of encapsulated aqueous contents. The results indicate a path towards liposomal drug delivery technologies that exploit transition metal triggered leakage.

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.

Supramolecular Mitigation of the Cyanine Limit Problem.

Currently, there is a substantial research effort to develop near-infrared fluorescent polymethine cyanine dyes for biological imaging and sensing. In water, cyanine dyes with extended conjugation are known to cross over the "cyanine limit" and undergo a symmetry breaking Peierls transition that favors an unsymmetric distribution of π-electron density and produces a broad absorption profile and low fluorescence brightness. This study shows how supramolecular encapsulation of a newly designed series of cationic, cyanine dyes by cucurbit[7]uril (CB7) can be used to alter the π-electron distribution within the cyanine chromophore. For two sets of dyes, supramolecular location of the surrounding CB7 over the center of the dye favors a nonpolar ground state, with a symmetric π-electron distribution that produces a sharpened absorption band with enhanced fluorescence brightness. The opposite supramolecular effect (i.e., broadened absorption and partially quenched fluorescence) is observed with a third set of dyes because the surrounding CB7 is located at one end of the encapsulated cyanine chromophore. From the perspective of enhanced near-infrared bioimaging and sensing in water, the results show how that the principles of host/guest chemistry can be employed to mitigate the "cyanine limit" problem.

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.

Conformational Effects on the Threading Kinetics of Dumbbell-Shaped Guests into the Cavity of Oxatub[4]arene.

Unprecedented threading kinetics were revealed between viologen-based guests and conformationally adaptive oxatub[4]arene. Three representative conformations of oxatub[4]arene are involved in the kinetic and thermodynamic products which follow the opposite orders in their rankings. Consequently, error correction was involved and a complex kinetic process was observed in a simple two-component system. Moreover, it was found that some viologen-based guests have much faster threading kinetics than those of DABCO-based with the same stoppers. This was enabled by an unprecedented threading mechanism in which a tilted conformation of the guests is adopted by involving one linear alkyl group on the 3,5-dialkoxybenzyl stoppers, the viologen core, and the methylene spacers in the transition states. This new mechanism even allows the viologen-based guests with the 3,5-dicetyloxybenzyl stoppers to form a pseudorotaxane with oxatub[4]arene.

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.

Fecal Fusobacterium nucleatum harbored virulence gene fadA are associated with ulcerative colitis and clinical outcomes.

OBJECT: Fusobacterium nucleatum (F.nucleatum), a gram-negative, obligately anaerobe of oral commensal，has been regarded as culprit of periodontal diseases previously and is being unveiled as possible pathogen of gastrointestinal disorders. The key virulence factor of F.nucleatum is FadA adhesin for binding and invading of the host's epithelial cells. Here, we detected fecal F.nucleatum and virulence gene fadA in patients with ulcerative colitis(UC) and evaluated the clinical relevance with UC. METHODS AND SUBJECTS: A total of 310 subjects were enrolled including 100 patients with UC, 70 healthy controls (HC), 70 patients with irritable bowel syndrome subtype diarrhea(IBS-D), and 70 colorectal cancer patients(CRC). Stool samples of UC patients compared with healthy controls as well as IBS-D and CRC patients were collected for Polymerase Chain Reaction(PCR) detection of F.nucleatum (based on 16s rRNA) and virulence gene fadA. RESULTS: The detection rate of 16s rRNA based PCR for F.nucleatum of UC patients(39/100, 39.00%) and CRC(26/70, 37.14%) patients are significantly higher than HC (12/70, 17.14%, P < 0.01) and IBS-D patients (14/70, 20.00%, P < 0.01). Moreover, 19 samples were detected fadA positive from 39 F.nucleatum positive samples of UC patients (19/39, 48.72%), which is significantly higher than HC(2/12, 16.66%, P < 0.05). There were 3 samples detected fadA positive from 14 F.nucleatum positive samples of IBS-D patients(3/14, 21.43%) and 13 out of 26(50.00%) of CRC patients, which were both no significant differences compared with UC patients(21.4% vs 48.72%, P > 0.05; 50.00% vs 48.72%, P > 0.05). For both F.nucleatum and fadA gene positive patients, there were no statistical significances between erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), white blood cells(WBC), and hemoglobin compared with negative patients(defined by either F.nucleatum or fadA negative, or both negative). However, it is worth noting that detection rate of F.nucleatum with virulence gene fadA in patients of severe ulcerative colitis was significantly higher than patients with mild and moderate colitis(28.89% vs 10.91%, P < 0.05). In addition, the fecal F.nucleatum and fadA gene positive patients were more likely to have pancolitis other than left-sided colitis(pancolitis/left-sided colitis: 26.92% vs 10.42%, P < 0.05). CONCLUSIONS: The presence of F.nucleatum and fadA gene increased in UC patients, especially in patients with severe colitis and pancolitis. Strains of F.nucleatum harbored virulence gene fadA are suggested to play a role in the pathogenesis of UC.

Deuterated Indocyanine Green (ICG) with Extended Aqueous Storage Shelf-Life: Chemical and Clinical Implications.

Indocyanine Green (ICG) is a clinically approved near-infrared fluorescent dye that is used extensively for various imaging and diagnostic procedures. One drawback with ICG is its instability in water, which means that reconstituted clinical doses have to be used very shortly after preparation. Two deuterated versions of ICG were prepared with deuterium atoms on the heptamethine chain, and the spectral, physiochemical, and photostability properties were quantified. A notable mechanistic finding is that self-aggregation of ICG in water strongly favors dye degradation by a photochemical oxidative dimerization reaction that gives a nonfluorescent product. Storage stability studies showed that replacement of C-H with C-D decreased the dimerization rate constant by a factor of 3.1, and it is likely that many medical and preclinical procedures will benefit from the longer shelf-lives of these two deuterated ICG dyes. The discovery that ICG self-aggregation promotes photoinduced electron transfer can be exploited as a new paradigm for next-generation photodynamic therapies.

NMR Relaxation Dispersion Reveals Macrocycle Breathing Dynamics in a Cyclodextrin-based Rotaxane.

A distinctive feature of mechanically interlocked molecules (MIMs) is the relative motion between the mechanically bonded components, and often it is the functional basis for artificial molecular machines and new functional materials. Optimization of machine or materials performance requires knowledge of the underlying atomic-level mechanisms that control the motion. The field of biomolecular NMR spectroscopy has developed a diverse set of pulse schemes that can characterize molecular dynamics over a broad time scale, but these techniques have not yet been used to characterize the motion within MIMs. This study reports the first observation of NMR relaxation dispersion related to MIM motion. The rotary (pirouette) motion of α-cyclodextrin (αCD) wheels was characterized in a complementary pair of rotaxanes with pirouetting switched ON or OFF. 13C and 1H NMR relaxation dispersion measurements reveal previously unknown exchange dynamics for the αCD wheels in the pirouette-ON rotaxane with a rate constant of 2200 s-1 at 298 K and an activation barrier of ΔF‡ = 43 ± 3 kJ/mol. The exchange dynamics disappear in the pirouette-OFF rotaxane, demonstrating their switchable nature. The 13C and 1H sites exhibiting relaxation dispersion suggest that the exchange involves "macrocycle breathing", in which the αCD wheel fluctuates between a contracted or expanded state, the latter enabling diffusive rotary motion about the axle. The substantial insight from these NMR relaxation dispersion methods suggests similar dynamic NMR methods can illuminate the fast time scale (microsecond to millisecond) mechanisms of intercomponent motion in a wide range of MIMs.

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.

Naphthocage: A Flexible yet Extremely Strong Binder for Singly Charged Organic Cations.

We report a quite flexible naphthol-based cage (so-called "naphthocage") which adopts a self-inclusion conformation in its free state and is able to bind singly charged organic cations extremely strongly ( Ka > 107 M-1). Ion-selective electrodes prepared with this naphthocage show a super-Nernstian response to acetylcholine. In addition, the highly stable complex (1010 M-1) between ferrocenium and the naphthocage can be switched electrochemically, which lays a basis for its application in stimuli-responsive materials.

Shape-Selective Recognition of Quaternary Ammonium Chloride Ion Pairs.

Synthetic receptors that recognize ion pairs are potentially useful for many technical applications, but to date there has been little work on selective recognition of quaternary ammonium (Q+) ion pairs. This study measured the affinity of a tetralactam macrocycle for 11 different Q+·Cl- salts in chloroform solution. In each case, NMR spectroscopy was used to determine the association constant ( Ka) and the structure of the associated complex. Ka was found to depend strongly on the molecular shape of Q+ and was enhanced when Q+ could penetrate the macrocycle cavity and engage in attractive noncovalent interactions with the macrocycle's NH residues and aromatic sidewalls. The highest measured Ka of 7.9 × 103 M-1 was obtained when Q+ was a p-CN-substituted benzylic trimethylammonium. This high-affinity Q+·Cl- ion pair was used as a template to enhance the synthetic yield of macrocyclization reactions that produce the tetralactam receptor or structurally related derivatives. In addition, a permanently interlocked rotaxane was prepared by capping the end of a noncovalent complex composed of the tetralactam macrocycle threaded by a reactive benzylic cation. The synthetic method provides access to a new family of rotaxanated ion pairs that can likely act as anion sensors, molecular shuttles, or transport molecules.

Molecular recognition using tetralactam macrocycles with parallel aromatic sidewalls.

This review summarizes the supramolecular properties of tetralactam macrocycles that have parallel aromatic sidewalls and four NH residues directed into the macrocyclic cavity. These macrocycles are versatile hosts for a large number of different guest structures in water and organic solvents, and they are well-suited for a range of supramolecular applications. The macrocyclic cavity contains a mixture of polar functional groups and non-polar surfaces which is reminiscent of the amphiphilic binding pockets within many proteins. In water, the aromatic surfaces in the tetralactam cavity drive high affinity due the hydrophobic effect and the NH groups provide secondary interactions that induce binding selectivity. In organic solvents, the supramolecular factors are reversed; the polar NH groups drive high affinity and the aromatic surfaces provide the secondary interactions. In addition to an amphiphilic cavity, macrocyclic tetralactams exhibit conformational flexibility, and the combination of properties enables them to be effective hosts for a wide range of guest molecules including organic biscarbonyl derivatives, near-infrared dyes, acenes, precious metal halide complexes, trimethylammonium ion-pairs, and saccharides.

Electronic Substituent Effects of Guests on the Conformational Network and Binding Behavior of Oxatub[4]arene.

A series of quaternary ammonium guests have been synthesized, and their binding behavior with oxatub[4]arene have been studied. In particular, remote electronic substituents of the guests can significantly affect the binding affinities mainly through a field/inductive effect by following a linear free energy relationship. More surprisingly, oxatub[4]arene, with a complex conformational network, shows a large amplitude of conformational change in response to the remote electronic substituents on the guests. This novel mode of synthetic molecular recognition may also have biological relevance.

A algorithm for prediction of exudative retinal detachment risk of patients with pregnancy-induced hypertension.

AIM: To investigate the risk of exudative retinal detachment (ERD) morbidity in patients with pregnancy-induced hypertension (PIH) by using the logistic regression combined with the receiver operating characteristic (ROC) curve. METHODS: A total of 46 patients with ERD and 142 patients with non-ERD were diagnosed as PIH from January 2017 to February 2020. A retrospective comparison of the clinical manifestations and laboratory tests were conducted. The risk of ERD morbidity with PIH was predicted by using logistic regression combined with an ROC curve model. RESULTS: There was no significant difference in age and body mass index between the two groups before pregnancy (P>0.05). However, significant differences were found in gestational weeks, duration of hypertension, maximum and minimum systolic and diastolic blood pressure (BP), and plasma total protein (PTP) concentration between the two groups (P<0.05). Binary logistic regression analysis showed that the maximum systolic BP (OR=1.050, 95%CI: 1.016-1.085) and PTP concentration (OR=0.764, 95%CI: 0.702-0.832) were independent prediction risks of ERD in PIH. The sensitivities of maximum systolic BP, PTP concentration and combined diagnosis were 0.717, 0.870, and 0.870, respectively; the specificities were 0.617, 0.837, and 0.908, respectively; the area under the curve (AUC) was 0.707 (95%CI: 0.622-0.792), 0.917 (95%CI: 0.868-0.967), and 0.933 (95%CI: 0.890-0.975), respectively; the AUC of combined diagnosis was higher than that of single diagnosis (P<0.01). CONCLUSION: Logistic regression and ROC curve model combined with maximum systolic BP and PTP can improve the early identification of high-risk PIH patients in the hospital.

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.

[Determination of trace elements in traditional Chinese medicine from Changbai Mountain by ICP-MS].

The effects of different digestives for the fritillaria and atractylodes were compared. Many trace elements in the planted and wild fritillaria and atractylodes were determined by ICP-MS The results show that the RSD and recovery are better if the planted and wild fritillaria and atractylodes were digested with HNO3-H2O2. Among the many elements determined from the fritillaria and atractylodes, Cu, Zn, Fe, Mg and Mn are the dominant chemicals. The content of Fe was higher in the wild fritillaria and atractylodes than that in the planted fritillaria and atractylodes, while the contents of heavy metal Pb and Cd were lower in the wild fritillaria and atractylodes than those in the planted fritillaria and atractylodes. The wild fritillaria and atractylodes contain Co, which was not determined in the planted fritillaria and atractylodes. The experimental results showed that the detection limits were lower than 0.086 ng x g(-1) with low RSD(n = 7, 4.85%) for most metal chemicals determined, and the standard recoveries (n = 7) ranged from 96.8 to 103.4%.

A sensitive electrochemical sensor based on ZIF-8-acetylene black-chitosan nanocomposites for rutin detection.

Herein, we fabricated a sensitive rutin electrochemical sensor via modifying glassy carbon electrode (GCE) with zeolitic imidazolate framework-8 (ZIF-8) and acetylene black (AB) in the presence of chitosan (CS). The electrochemical activity and experimental parameters of the ZIF-8-AB-CS/GCE sensor were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under the optimal conditions, the sensor presented a reasonable linear response in the range of 0.1-10 µM with a limit of detection (LOD) as low as 0.004 µM (S/N = 3). The sensor possessed good reproducibility and high stability, and was successfully applied to detect rutin tablet samples with satisfactory results, which was attributed to the synergistic effect between ZIF-8 and AB. Meanwhile, the sensor displayed a potential application for detection of other analytes in real samples. Furthermore, a probable interaction mechanism was proposed to account for the interaction between rutin and the nanocomposite electrode, which was not discussed in previous reports.

[Interaction between carbaryl and calf thymus DNA enhanced via Cu2+].

The interaction between carbaryl and calf thymus DNA with Cu2+ was studied using fluorescence spectroscopy (FS) and UV spectrum. The quenching process was proved to be single static quenching and the quenching constant decreases with temperature increasing. The experimental results showed that the carbaryl can be intercalated into the twin-screw structure of calf thymus DNA, forming ctDNA-carbaryl adducts. The UV spectrum showed that ctDNA can also result in the increasing color and red shift. In the presence of Cu2+, it was discovered that the apparent association constant (Kalpha) increases and the binding sites number (n) of carbaryl molecules on ctDNA obviously increases. The interaction of the carbaryl and ctDNA was driven mainly by electrostatic force which was enhanced by Cu2+, thus the contribution of deltaH to deltaG increased in the presence of Cu2+ So the interaction between carbaryl and calf thymus DNA was enhanced via Cu+.