Pearson's Colocalization Coefficient Research Articles

Water-soluble red fluorescent protein dimers for hypoxic two-photon photodynamic therapy.

In this study, two water-soluble red fluorescent protein (RFP) dimers, FP2R' and FP2R'', were synthesized by linking two phenothiazine-based RFP chromophore analogues through alkyl chains or alkoxy chains for hypoxic two-photon photodynamic therapy. RFP dimers are heavy-atom-free two-photon photosensitizers in which the intersystem crossing process is boosted by S and N heteroatoms. In terms of the aqueous solubility, the saturation concentration of FP2R'' was 3.5 mM, the emission wavelength was 677 nm, the singlet oxygen yield was 18%, and the two-photon absorption coefficient (β) was 2.1 × 10-11 cm W-1. Further, the RFP dimer FP2R'' showed excellent biocompatibility, negligible dark toxicity, and could produce 1O2 and O2˙- simultaneously. Under 460 nm illumination, the photosensitizer FP2R'' showed high phototoxicity with an IC50 value of 4.08 μM in an hypoxia environment, indicating that the photosensitizer FP2R'' has an excellent anti-hypoxia ability. In addition, the photosensitizer FP2R'' demonstrated a precise localization ability to lysosomes and its Pearson's colocalization coefficient was 0.94, which could guide the aggregation of photosensitizers in the lysosomes of tumor cells to effectively improve its photodynamic therapy (PDT) effect. In particular, when exposed to 800 nm two-photon excitation, FP2R'' effectively produced 1O2 and O2˙- in zebrafish and exhibited a bright two-photon fluorescence imaging capability. At the same time, the efficacy of two-photon photodynamic therapy mediated by the photosensitizer FP2R'' was verified in the tumor zebrafish model, and the growth of tumor cells in zebrafish was significantly inhibited under a two-photon laser irradiation. The water-soluble two-photon photosensitizer FP2R'' that was reasonably constructed in this study can be used as a high-efficiency hypoxic two-photon photosensitizer to inhibit deep tumor tissues.

A hemicyanine-based near-infrared fluorescent probe with large Stokes shift for non-invasive bioimaging of brown adipose tissue.

Brown adipose tissue (BAT), characterized by the presence of numerous mitochondria, plays a key role in metabolism and energy expenditure. Accurately reporting the presence and activation of BAT is beneficial to study obesity, diabetes, and other metabolic disorders. Near-infrared (NIR) fluorescence imaging has the advantages of high sensitivity, non-radioactivity, and simple operation. However, most NIR probes for BAT imaging exhibit small Stokes shifts, which may lead to self-quenching, reducing the signal-to-noise ratio, and introducing cross-talk. Herein, we rationally designed and synthesized a series of hemicyanine-based NIR fluorescent probes HCYBAT-1-3. Among them, HCYBAT-1 demonstrated favorable properties such as near-infrared emission (776 nm), large Stokes shift (139 nm), good biocompatibility and specific mitochondrial targeting (Pearson's colocalization coefficient of 0.87). Meanwhile, HCYBAT-1 was successfully employed to differentiate BAT from white adipose tissue (WAT). Quantitative analysis of NIR fluorescent images showed that HCYBAT-1 could be used for real-time monitoring of BAT activation in mice stimulated by norepinephrine (NE) and cold exposure. Overall, probe HCYBAT-1 showcased its efficacy in non-invasive evaluation of BAT metabolism in vivo with high selectivity and sensitivity.

A terpyridyl functionalized rhodamine dye for viscosity sensing with fluorescence enhancement and mitochondria-targeting imaging

Exploring new fluorescent organic dyes with mitochondria-targeted and viscosity-sensitive functions attracts extensive attention because they may provide access to a better understanding of the microenvironments of the subcellular level via biological imaging. Herein a terpyridyl functionalized rhodamine dye (TRMe) has been facilely designed and prepared, which shows a highly selective fluorescence enhancement response to viscosity (1.0–1238.0 cp) in water-glycerol mixtures with the increased glycerol content. In addition, TRMe can respond to viscosity enhancement over a wide pH range (1.0–10.0) and presents low cell toxicity. Besides, a good linearity between the viscosity and fluorescence lifetime of TRMe can be observed. Significantly, TRMe shows excellent mitochondria-targeted performance with a Pearson's colocalization coefficient of 0.958 and can be employed as a tracer for viscosity imaging in Nystatin-stimulated living HeLa cells.

Development of a Facile Lysosome-Targeting Nitric Oxide Fluorescent Probe Based on a Novel Reaction Mechanism.

As a ubiquitous signal molecule in biosystems, nitric oxide (NO) plays an important role in many physiological and pathological processes. Therefore, it is of great significance to detect NO in organisms for the study of related diseases. Currently, a variety of NO fluorescent probes have been developed based on several types of reaction mechanisms. However, due to the inherent disadvantages of these reactions, like potential interference by biologically related species, there is a great need to develop NO probes based on the new reactions. Herein, we report our discovery of the unprecedented reaction between a widely used fluorophore of 4-(dicyanomethylene)-2-methyl-6-(p-(dimethylamino)styryl)-4H-pyran (DCM) and NO under mild conditions with fluorescence changes. By the analysis of the structure of the product, we proved that DCM undergoes a particular nitration process and proposed a mechanism for fluorescence changes due to the interruption of the intramolecular charge transfer (ICT) process of DCM by the nitrated product of DCM-NO2. Based on the understanding of this specific reaction, we then easily constructed our lysosomal-localized NO fluorescent probe LysoNO-DCM by linking DCM and a morpholine group, a lysosomal-targeting functional group. LysoNO-DCM exhibits excellent selectivity, sensitivity, pH stability, and outstanding lysosome localization ability with Pearson's colocalization coefficient of up to 0.92 and is successfully applied to the imaging of exogenous and endogenous NO in cells and zebrafish. Our studies expand design methods for NO fluorescence probes based on the novel reaction mechanism and will benefit the studies of this signaling molecule.

Mitochondrial targeting half-sandwich iridium(III) and ruthenium(II) dppf complexes and in vitro anticancer assay

Considering the potential application of half-sandwich and ferrocenyl-containing organometallic complexes in the area of anticancer, four half-sandwich iridium(III) (IrIII) and ruthenium(II) (RuII) diphenylphosphino ferrocene (dppf) complexes were prepared in this study. Complexes showed favorable anti-proliferation activity towards A549 cell lines compared to cisplatin, meanwhile, which could effectively inhibit cell migration. These complexes followed an energy dependence uptake mechanism, effectively accumulated in mitochondria with a Pearson's Colocalization Coefficient (PCC) of 0.77, decreased the mitochondrial membrane potential, induced a surge of reactive oxygen species, disturbed cell cycle, and eventually led to apoptosis. Western blot assay further confirmed that these complexes induced apoptosis following a mitochondrial pathway. Above all, half-sandwich IrIII and RuII dppf complexes show the prospect of becoming a new multifunctional therapeutic platform for mitochondrial targeted imaging and anticancer drugs.

Cyclooxygenase-2-targeting fluorescent carbon dots for the selective imaging of Golgi apparatus

Golgi apparatus is an important organelle responsible for transporting proteins in cells. Accurate monitoring of Golgi apparatus status by targeting fluorescence imaging technology is of great significance for visualization of Golgi apparatus' physiological status. Cyclooxygenase-2 (COX-2) is a protease on the Golgi apparatus surface of cells. By means of hydrogen bonding between COX-2 and sulfonamide groups, orange-emitting Golgi apparatus-targeting carbon dots (GTCDs) were developed for the efficient imaging monitoring of Golgi apparatus. GTCDs were synthesized from p-phenylenediamine and benzene sulfonamide by one-step solvothermal methods. GTCDs had uniform size (∼3.36 nm), graphitized structure, maximum emission wavelength of 612 nm, low cytotoxicity, 24 h metabolic time, and good photostability. In addition, GTCDs had an excellent Golgi apparatus-targeting effect (average Pearson's co-localization coefficient = 0.92) and ultrafast imaging capability of Golgi apparatus (imaging immediately after the addition of GTCDs). The selective targeting of GTCDs to Golgi apparatus was attributed to the binding of the sulfonamide moiety on the surface of GTCDs to the active pocket site of COX-2 in Golgi apparatus through hydrogen bonds, which led to the localization of GTCDs to Golgi apparatus. It is believed that GTCDs have great potential in the fields of precise organelle imaging and targeting drug delivery as Golgi apparatus-targeting biological fluorescent probes.

Sensitive monitoring mitochondrial peroxynitrite based on a new reaction site and cell imaging by anthracycline-based red emitting fluorescence probe

The change in the level of ONOO− is usually indicative of an abnormality in bodily function. Therefore, there is a need to develop highly reliable ONOO− assays to determine its role in the biological environment. Thus, a long-wavelength anthracycline-based fluorescent probe (LAP) with a new reaction site was presented to detect peroxynitrite (ONOO−) by one step synthesize between 6-hydroxy-1-tetralone and salicylaldehyde. LAP responded to ONOO− about 8-fold fluorescence changes at 628 nm and the detection limit was 3.7 nM. The ONOO− sensing of LAP with the disruption of the conjugated skeleton was highly specific and could identify ONOO− by naked eyes. LAP was specifically localized to the mitochondria and co-localization results of LAP and MitoTrancker Green showed that the merged fluorescent images were observed with high Pearson's co-localization coefficient 0.92 and 0.90 in SMMC-7721 and RAW264.7 cells, respectively. As a specific and sensitive fluorescence probe, LAP with mitochondria-targetable moiety was ability to detect exogenous ONOO− in SMMC-7721 and RAW264.7 cells.

Imidazole and Benzimidazole Modified Half-Sandwich IridiumIII N-Heterocyclic Carbene Complexes: Synthesis, Anticancer Application, and Organelle Targeting.

Herein, we report the synthesis, characterization and anticancer activity of a series of half-sandwich iridiumIII imidazole and benzimidazole N-heterocyclic carbene (NHC) anticancer complexes, and the general formula of which can be expressed as [(η5-Cpx)Ir(C∧N)Cl]Cl (Cpx: pentamethylcyclopentadienyl (Cp*) or biphenyl derivatives (Cpxbiph); C∧N: imidazole and benzimidazole NHC chelating ligands). Compared with cis-platin, these complexes showed interesting antitumor activity against A549 cells. Complexes could bind to bovine serum albumin (BSA) by means of static quenching mode, catalyze the oxidation of nicotinamide adenine dinucleotide (NADH) and increase the levels of reactive oxygen species (ROS). Meanwhile, these complexes could arrest the cell cycles of A549 cells and influence the mitochondrial membrane potential significantly. Due to the inherent luminescence property, laser confocal test show that complexes could enter cells followed an energy-dependent mechanism and effectively accumulate in lysosome (the value of Pearson's co-localization coefficient is 0.70 after 1 h), further destroy lysosome integrity and induce apoptosis.

Dual functions of iridium(III) 2-phenylpyridine complexes: Metastasis inhibition and lysosomal damage

Six N-phenylcarbazole/triphenylamine-appended half-sandwich iridium(III) 2-phenylpyridine complexes ([(η5-Cp*)Ir(C^N)Cl]) were prepared and characterized. Compared with cisplatin, these complexes exhibited potential antitumor activity against A549 and HeLa tumor cells, with IC50 values (half-maximum inhibitory concentration) that changed from 2.8 ± 0.8 μM to 39.5 ± 2.7 μM, and could block the migration of tumor cells. These complexes also effectively bound to protein (binding constant: ~104 M−1) and were transported through serum proteins, catalyzed the oxidation of coenzyme nicotinamide-adenine dinucleotide. Additionally, laser confocal microscopy and flow cytometry confirmed that these complexes possessed a non-energy-dependent cellular uptake mechanism, effectively accumulated in lysosomes (Pearson colocalization coefficient: ~0.74), damaged the integrity of acidic lysosomes, led to a change in the mitochondrial membrane potential, disrupted the cell cycle (G0/G1 phase), and eventually induced apoptosis. Above all, these complexes are potential antitumor agents with dual functions: metastasis inhibition and lysosomal damage.

Fluorescent iridium(iii) coumarin-salicylaldehyde Schiff base compounds as lysosome-targeted antitumor agents.

Six fluorescent half-sandwich iridium(iii) coumarin-salicylaldehyde Schiff base (O^N) compounds ([(η5-Cp*)Ir(O^N)Cl]) were prepared and characterized. The introduction of a coumarin unit increased the antitumor activity (IC50: 9.9 ± 0.1 μM-40.7 ± 12.9 μM) of these compounds, the best of which was nearly two times that of clinical cisplatin. The results of laser confocal microscopy demonstrated that these compounds possessed an energy-dependent cellular uptake mechanism, accumulated in the lysosomes (Pearson co-localization coefficient: ∼0.7), damaged the integrity of the lysosomes, and induced apoptosis. The compounds could also decrease the mitochondrial membrane potential, catalyze the oxidation of the coenzyme (nicotinamide-adenine dinucleotide) and improve the levels of the intracellular reactive oxygen species, following an antitumor mechanism of oxidation. Additionally, these compounds could block the metastasis of tumor cells. Above all, these iridium(iii) compounds show potential as antitumor agents with dual functions: lysosomal damage and anti-metastasis.

Preparation and the anticancer mechanism of configuration-controlled Fe(II)-Ir(III) heteronuclear metal complexes.

A series of configuration-controlled Fe(ii)-Ir(iii) heteronuclear metal complexes, including ferrocene and half-sandwich like iridium(iii) complex units, have been designed and prepared. These complexes show better anticancer activity than cisplatin under the same conditions, especially cis-configurational ones. Laser confocal microscopy analysis confirms that the complexes follow a non-energy-dependent cellular uptake mechanism, accumulate in lysosomes (pearson co-localization coefficient: ∼0.7), lead to lysosomal damage, and eventually induce apoptosis. These complexes can reduce the mitochondrial membrane potential, disturb the cell circle, catalyze the oxidation of nicotinamide-adenine dinucleotide (NADH) and increase the levels of intracellular reactive oxygen species (ROS), following an anticancer mechanism of oxidation. In addition, the complexes could bind to serum protein, and transport through it. Above all, the Fe(ii)-Ir(iii) heteronuclear metal complexes hold promise as potential anticancer agents for further study.

New Organometallic Tetraphenylethylene⋅Iridium(III) Complexes with Antineoplastic Activity.

Iridium(III) complexes have attracted more and more attention in the past few years because of their potential antineoplastic activity. In this study, four IrIII complexes of the types [(η5 -Cpx )Ir(N^N)Cl]PF6 (complexes 1 and 2) and [Ir(Phpy)2 (N^N)]PF6 (complexes 3 and 4) have been synthesized and characterized. They exhibit potential antineoplastic activity towards A549 cells, especially in the case of complex 1 [IC50 =(3.56±0.5) μm], which was nearly six times as effective as cisplatin [(21.31±1.7) μm]. Additionally, these complexes show some selectivity towards cancer cells over normal cells. They could be transported by serum albumin (binding constants were changed from 0.37×105 to 81.71×105 m-1 ). IrIII complexes 1 and 2 could catalyze the transformation of nicotinamide adenine dinucleotide reduced form (NADH) into NAD+ (turnover numbers 43.2, 11.9] and induce the accumulation of reactive oxygen species, thus confirming their antineoplastic mechanism of oxidation, whereas the cyclometalated complexes 3 and 4 were able to target the lysosome [Pearson co-localization coefficient (PCC)=0.73], cause lysosomal damage, and induce apoptosis. Understanding the mechanism of action would help further structure-activity optimization on these IrIII complexes as emerging cancer therapeutics.

Ferrocene-Appended Iridium(III) Complexes: Configuration Regulation, Anticancer Application, and Mechanism Research.

A series of ferrocene-appended half-sandwiched iridium(III) phenylpyridine complexes have been designed and synthesized. These complexes show better anticancer activity than cisplatin widely used in clinic under the same conditions. Meanwhile, complexes could effectively inhibit cell migration and colony formation. Complexes could interact with protein and transport through serum protein, effectively catalyzing the oxidation of nicotinamide-adenine dinucleotid and inducing the accumulation of reactive oxygen species (ROS, 1O2), which confirmed the anticancer mechanism of oxidation. Furthermore, laser scanning confocal detection indicates that these complexes can enter cells followed by a non-energy-dependent cellular uptake mechanism, effectively accumulating in the lysosome (Pearson's colocalization coefficient: ∼0.90), leading to lysosome damage, and reducing the mitochondrial membrane potential (MMP). Taken together, ferrocene-appended iridium(III) complexes possess the prospect of becoming a new multifunctional therapeutic platform, including lysosome-targeted imaging and anticancer drugs.

Half‐sandwich Ruthenium (II) complexes with triphenylamine modified dipyridine skeleton and application in biology/luminescence imaging

Four half‐sandwich rutheniumII (RuII) complexes with triphenylamine‐modifed dipyridine frameworks were synthesized and characterized. The cytotoxicity of target complexes toward A549 (lung cancer cells), HeLa (cervical cancer cells) and HepG2 (hepatoma cells) were obtained by the MTT assay, which were superior to cisplatin with the IC50 values changed from 2.4 ± 0.1 μM to 9.2 ± 2.7 μM. Meanwhile, complexes possess the ability of antimetastasis to cancer cells. RuII complexes could be transported by serum albumin, catalyze the conversion of NADH (the reduced state of nicotinamide‐adenine dinucleotide) to NAD+ and induce the accumulation of reactive oxygen species, which confirmed the antineoplastic mechanism of oxidation. RuII complexes could enter A549 cells followed by a non‐energy dependent cellular uptake mechanism, target lysosomes with the Pearson's colocalization coefficient of 0.75, lead to lysosomal damage, disturb the cell cycle (S phase), and eventually induce apoptosis. The results demonstrate that these RuII complexes are potential anticancer agents with dual functions, including metastasis inhibition and lysosomal damage.

Half-sandwich iridium(III) complexes with α-picolinic acid frameworks and antitumor applications

Eight half-sandwich iridiumIII (IrIII) complexes of the general formula [(η5-Cpxbiph)Ir(O^N)Cl] (Cpxbiph is tetramethyl(biphenyl)cyclopentadienyl, and the O^N is α-picolinic acid chelating ligand and its derivatives) were synthesized and characterized. Compared with cis-platin widely used in clinic, target IrIII complexes showed at most five times more potent antitumor activity against A549 cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. IrIII complexes could be transported by serum albumin, bind with DNA, catalyze the oxidation of nicotinamide-adenine dinucleotid (NADH) and induce the production of reactive oxygen species, which confirmed the antitumor mechanism of oxidation. IrIII complexes could enter A549 cells followed by an energy-dependent cellular uptake mechanism, meanwhile, target the mitochondria and lysosomes with the Pearson's colocalization coefficient of 0.33 and 0.74, respectively, lead to the lysosomal destruction and the change of mitochondrial membrane potential (ΔΨm), and eventually induce apoptosis.

Half-sandwich Iridium(III) Benzimidazole-Appended Imidazolium-Based N-heterocyclic Carbene Complexes and Antitumor Application.

A series of half-sandwich iridium(III) benzimidazole-appended imidazolium-based N-heterocyclic carbene (NHC) antitumor complexes [(η5 -Cpx )Ir(C^N)Cl]Cl, where Cpx is pentamethylcyclopentadienyl (Cp*) or its biphenyl derivative (Cpxbiph ) and C^N is a NHC chelating ligand, were successfully synthesized and characterized. The IrIII complexes showed potential antitumor activity against A549 cells, at most three times more potent than cis-platin under the same conditions. Complexes could bind to BSA by a static quenching mode, catalyzing the change of NADH to NAD+ and inducing the production of reactive oxygen species (maximum turnover number, 9.8), which play an important role in regulating cell apoptosis. Confocal microscopy showed that the complexes could specifically target lysosomes in cells with a Pearson's co-localization coefficient 0.76 and 0.72 after 1 h and 6 h, respectively, followed an energy-dependent cellular uptake mechanism and damaged the integrity of lysosomes. At the same time, complexes caused a marked loss of mitochondrial membrane potential.

Half-sandwich IridiumIIIN-heterocyclic carbene antitumor complexes and biological applications

Series of half-sandwich IrIIIN-heterocyclic carbene (NHC) antitumor complexes [(η5-Cp*)Ir(C^C)Cl] have been synthesized and characterized (Cp* is pentamethyl cyclopentadienyl, and C^C are four NHC chelating ligands containing phenyl rings at different positions). IrIII complexes showed potent antitumor activity with IC50 values ranged from 3.9 to 11.8 μM against A549 cells by the MTT assay. Complexes can catalyze the conversion of the coenzyme NADH to NAD+ and induce the production of reactive oxygen species (ROS), and bonding to BSA by static quenching mode. Complexes can arrest the cell cycle in G1 or S phase and reduce the mitochondrial membrane potential. Confocal microscopy test show complexes could target the lysosome and mitochondria in cells with the Pearson's colocalization coefficient of 0.82 and 0.21 after 12 h, respectively, and followed by an energy-dependent cellular uptake mechanism.

Half-sandwich Iridium(III) complexes with triphenylamine-substituted dipyridine frameworks and bioactivity applications

Nine half-sandwich IrIII complexes [(η5-Cpx)Ir(NˆN)Cl]PF6, where Cpx is Cp* or its phenyl Cpxph or biphenyl Cpxbiph derivative and NˆN is a triphenylamine (TPA)-substituted bipyridyl ligand, were synthesized and characterized. MTT assay revealed that the complexes show potent antitumor activity with IC50 values of 1.2–36.3 μM against A549 lung cancer cells. The IrIII complexes bound to BSA and oxidized NADH to NAD+, leading to production of H2O2 (maximum turnover number, 37.9), which plays an important role in regulating cell apoptosis. Confocal microscopy showed that the complexes could target lysosomes effectively in cells with a high Pearson's colocalization coefficient (∼0.5) and followed an energy-dependent cellular uptake mechanism.

Endoplasmic reticulum-targeted two-photon turn-on fluorescent probe for nitroreductase in tumor cells and tissues

Hypoxia conditions could increase the activity of intracellular nitroreductase (NTR) and lead to many malignant diseases. Therefore, monitoring the activity of NTR is of great significance to study the related diseases. Organelles play crucial roles in the metabolism of living cells. In these organelles, the endoplasmic reticulum (ER) possesses single membrane structure, and it is the largest organelle in the cell. ER performs the synthesis, processing and modification of proteins and lipid, stabilizing the intracellular Ca2+ concentration and other physiological functions in living cells. Therefore, it is of great significance to develop ER-target probes in living system. Toward this goal, a new endoplasmic reticulum-targeted two-photon fluorescence turn-on NTR probe Na-NTR-ER is designed and synthesized. Probe Na-NTR-ER has been proved to display high sensitivity (36 ng/mL) and selectivity to NTR. Particularly, probe Na-NTR-ER has been successfully applied for the monitoring of NTR in ER with a high the Pearson's colocalization coefficient as 0.90 in HeLa cells and cancerous mouse tissues up to the depth of 100 μm with significant fluorescence signals.