Pt(II) Bis(pyrrole-imine) complexes: Luminescent probes and cytotoxicity in MCF-7 cells†.

Four Pt(II) bis(pyrrole-imine) Schiff base chelates (1-4) were synthesised by previously reported methods, through a condensation reaction, and the novel crystal structure of 2,2'-{propane-1,3-diylbis[nitrilo(E)methylylidene]}bis(pyrrol-1-ido)platinum(II) (1) was obtained. Pt(II) complexes 1-4 exhibited phosphorescence, with increased luminescence in anaerobic solvents or when bound to human serum albumin (HSA). One of the complexes shows a 15.6-fold increase in quantum yield when bound to HSA and could be used to detect HSA concentrations as low as 5 nM. Pt(II) complexes 1-3 was investigated as potential theranostic agents in MCF-7 breast cancer cells, but only complex 3 exhibited cytotoxicity when irradiated with UV light (λ355nmExcitation). Interestingly, the cytotoxicity of complex 1 was unresponsive to UV light irradiation. This indicates that only complex 3 can be considered a potential photosensitising agent.

A Dinuclear Osmium(II) Complex Near-Infrared Nanoscopy Probe for Nuclear DNA.

With the aim of developing photostable near-infrared cell imaging probes, a convenient route to the synthesis of heteroleptic OsII complexes containing the Os(TAP)2 fragment is reported. This method was used to synthesize the dinuclear OsII complex, [{Os(TAP)2}2tpphz]4+ (where tpphz = tetrapyrido[3,2-a:2',3'-c:3″,2''-h:2‴,3'''-j]phenazine and TAP = 1,4,5,8- tetraazaphenanthrene). Using a combination of resonance Raman and time-resolved absorption spectroscopy, as well as computational studies, the excited state dynamics of the new complex were dissected. These studies revealed that, although the complex has several close lying excited states, its near-infrared, NIR, emission (λmax = 780 nm) is due to a low-lying Os → TAP based 3MCLT state. Cell-based studies revealed that unlike its RuII analogue, the new complex is neither cytotoxic nor photocytotoxic. However, as it is highly photostable as well as live-cell permeant and displays NIR luminescence within the biological optical window, its properties make it an ideal probe for optical microscopy, demonstrated by its use as a super-resolution NIR STED probe for nuclear DNA.

Covalent immobilization of luminescent oxygen indicators reduces cytotoxicity.

Luminescence-based oxygen sensing is a widely used tool in cell culture applications. In a typical configuration, the luminescent oxygen indicators are embedded in a solid, oxygen-permeable matrix in contact with the culture medium. However, in sensitive cell cultures even minimal leaching of the potentially cytotoxic indicators can become an issue. One way to prevent the leaching is to immobilize the indicators covalently into the supporting matrix. In this paper, we report on a method where platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin (PtTFPP) oxygen indicators are covalently immobilized into a polymer matrix consisting of polystyrene and poly(pentafluorostyrene). We study how the covalent immobilization influences the sensing material's cytotoxicity to human induced pluripotent stem cell-derived (hiPSC-derived) neurons and cardiomyocytes (CMs) through 7-13 days culturing experiments and various viability analyses. Furthermore, we study the effect of the covalent immobilization on the indicator leaching and the oxygen sensing properties of the material. In addition, we demonstrate the use of the covalently linked oxygen sensing material in real time oxygen tension monitoring in functional hypoxia studies of the hiPSC-derived CMs. The results show that the covalently immobilized indicators substantially reduce indicator leaching and the cytotoxicity of the oxygen sensing material, while the influence on the oxygen sensing properties remains small or nonexistent.

A near-infrared phosphorescent iridium(iii) complex for fast and time-resolved detection of cysteine and homocysteine.

Thiol-containing amino acids, cysteine (Cys) and homocysteine (Hcy), play crucial roles in the biosystem; their abnormal contents in the cells are linked to many diseases. Herein, we designed and synthesized a novel near-infrared (NIR) phosphorescent iridium(iii) complex-based probe (FNO1) that can detect Cys and Hcy in real-time in the biosystem. Due to the advantages of the iridium complex, the FNO1 probe had excellent chemical stability and photostability, high luminescence efficiency, and long luminescence lifetime. In addition, the probe showed a fast response, high sensitivity, and low cytotoxicity. As verified by high resolution mass spectra (HR-MS) and density functional theory (DFT) calculations, the detection was achieved through the addition of the α,ß-unsaturated ketone group in FNO1 by the nucleophilic thiol group in Cys and Hcy. Through time-resolved emission spectroscopy (TRES) and in the presence of a strongly fluorescent dye rhodamine B, the FNO1 probe could detect Cys and Hcy due to its long luminescence lifetime (260/197 ns). Finally, owing to its NIR-emitting properties, the FNO1 probe was successfully applied in the imaging of Cys and Hcy in living cells, zebrafish, and mice.

A novel mitochondria-targeted phosphorescence probe for hypochlorite ions detection in living cells.

Monitoring hypochlorite anion (ClO-) in living cells is particularly meaningful and valuable, because over-exposure of the ClO- may cause a potential health hazard towards animals and humans. Considering the special structure and properties of the gemini surfactant, a novel amphiphilic gemini-iridium complex Ir[(ppy-iso)2(bpy-tma2Br2)] (Ir-iso) with isoniazide as a recognition site for ClO- was designed. The Ir-iso possessed an excellent water-solubility as well as a strong ClO- binding capacity, as revealed from the rapid response of emission signal towards ClO-. It was worth noting that such probe had a highly-specific selectivity with a low detection limit (20.5 nM) and was suitable in physiological environment. The cell viability assay, cell imaging, and co-location studies further proved that the Ir-iso had little cytotoxicity and was specifically localized in the mitochondria of breast cancer cells, being a promising candidate of chemo-sensor to detect the endogenous ClO- in living cells.

Dispersion stability and biocompatibility of four ligand-exchanged NaYF4: Yb, Er upconversion nanoparticles.

Surface modification to obtain high dispersion stability and biocompatibility is a key factor for bio-application of upconversion nanoparticles (UCNPs). A systematic study of UCNPs modified with four hydrophilic molecules separately, comparing their dispersion stability in biological buffers and cellular biocompatibility is reported here. The results show that carboxyl-functionalized UCNPs (modified by 3,4-dihydrocinnamic acid (DHCA) or poly(monoacryloxyethyl phosphate (MAEP)) with negative surface charge have superior even-distribution in biological buffers compared to amino-functionalized UCNPs (modified by (aminomethyl)phosphonic (AMPA) or (3-Aminopropyl)triethoxysilane (APTES)) with positive surface charge. Subsequent investigation of cellular interactions revealed high levels of non-targeted cellular uptake of the particles modified with either of the three small molecules (AMPA, APTES, DHCA) and high levels of cytotoxicity when used at high concentrations. The particles were seen to be trapped as particle-aggregates within the cellular cytoplasm, leading to reduced cell viability and cell proliferation, along with dysregulation of the cell cycle as assessed by DNA content measurements. The dramatically reduced proportion of cells in G1 phase and the slightly increased proportion in G2 phase indicates inhibition of M phase, and the appearance of sub-G1 phase reflects cell necrosis. In contrast, MAEP-modified UCNPs are bio-friendly with increased dispersion stability in biological buffers, are non-cytotoxic, with negligible levels of non-specific cellular uptake and no effect on the cell cycle at both low and high concentrations. MAEP-modified UCNPs were further functionalized with streptavidin for intracellular microtubule imaging, and showed clear cytoskeletal structures via their upconversion luminescence. STATEMENT OF SIGNIFICANCE: Upconversion nanoparticles (UCNP) are an exciting potential nanomaterial for bio-applications. Their anti-Stokes luminescence makes them especially attractive to be used as imaging probes and thermal therapeutic reagents. Surface modification is the key to achieving stable and compatible hydrophilic-UCNPs. However, the lack of criteria to assess molecular ligands used for ligand exchange of nanoparticles has hampered the development of surface modification, and further limits UCNP's bio-application. Herein, we report a systematic comparative study of modified-UCNPs with four distinct hydrophilic molecules, assessing each particles' colloidal stability in biological buffers and their cellular biocompatibility. The protocol established here can serve as a potential guide for the surface modification of UCNPs in bio-applications.

Bioluminescence imaging of carbon monoxide in living cells based on a selective deiodination reaction.

d-Luciferin is a popular bioluminescent substrate of luciferase in the presence of ATP. It is used in luciferase-based bioluminescence imaging and cell-based high-throughput screening applications. Herein, the iodination of d-luciferin was undertaken and explored as a bioluminescence probe without the need for light excitation to sensitively trace and image carbon monoxide (CO) in liver cancer cells. The bioluminescent probe (7'-iodo-luciferin) exhibited excellent selectivity for CO detection in vitro. This new probe could image exogenous and endogenous CO in the luciferase-transfected cancer cells. This new probe might be used for evaluating the roles of CO in various biological processes.

Bioluminescence Imaging of Selenocysteine in Vivo with a Highly Sensitive Probe.

Selenocysteine (Sec), a vital member of reactive selenium species, is closely implicated in diverse pathophysiological states, including cancer, cardiovascular diseases, diabetes, neurodegenerative diseases, and male infertility. Monitoring Sec in vivo is of significant interest for understanding the physiological roles of Sec and the mechanisms of human diseases associated with abnormal levels of Sec. However, no bioluminescence probe for real-time monitoring of Sec in vivo has been reported. Herein, we present a novel bioluminescent probe BF-1 as an effective tool for the determination of Sec in living cells and in vivo for the first time. BF-1 has advantages of high sensitivity (a detection limit of 8 nM), remarkable bioluminescence enhancement (580-fold), reasonable selectivity, low cytotoxicity, and high signal-to-noise ratio imaging feasibility of Sec in living cells and mice. More importantly, BF-1 affords high sensitivity for monitoring Sec stimulated by Na2SeO3 in tumor-bearing mice. These results demonstrate that our new probe could serve as a powerful tool to selectively monitor Sec in vivo, thus providing a valuable approach for exploring the physiological and pathological functions and anticancer mechanisms of selenium.

Highly Stable and Luminescent Oxygen Nanosensor Based on Ruthenium-Containing Metallopolymer for Real-Time Imaging of Intracellular Oxygenation.

Metal complex-based luminescent oxygen nanosensors have been intensively studied for biomedical applications. In terms of monitoring dynamics of intracellular oxygen, however, high-quality nanosensors are still badly needed, because of stringent requirements on stability, biocompatibility and luminescence intensity, aside from oxygen sensitivity. In this paper, we reported a type of highly luminescent and stable oxygen nanosensors prepared from metallopolymer. First, a novel ruthenium(II)-containing metallopolymer was synthesized by chelating the oxygen probe [Ru(bpy)3]2+ with a bipyridine-branched hydrophobic copolymer, which was then doped into polymeric nanoparticles (NPs) by a reprecipitation method, followed by further conjugation to selectively target mitochondria (Mito-NPs). The resultant Mtio-NPs possessed a small hydrodynamic size of ∼85 nm, good biocompatibility and high stability resulting from PEGylation and stable nature of Ru-complex. Because the complexed [Ru(bpy)3]2+ homogeneously resided on particle surface, Mito-NPs exhibited strong luminescence at 608 nm that was free of aggregation-caused-quenching, the utmost oxygen sensitivity of free [Ru(bpy)3]2+ probe ( Q = 75%), and linear Stern-Volmer oxygen luminescence quenching plots. Taking advantage of the mitochondria-specific nanosensors, intracellular oxygenation and deoxygenation processes were real-time monitored for 10 min by confocal luminescence imaging, visualized by the gradual weakening (by more than 90%) and enhancing (by 50%) of the red emission, respectively.

Luminescent Rhenium(I)-Polypyridine Complexes Appended with a Perylene Diimide or Benzoperylene Monoimide Moiety: Photophysics, Intracellular Sensing, and Photocytotoxic Activity.

This communication reports novel luminescent rhenium(I)-polypyridine complexes appended with a perylene diimide (PDI) or benzoperylene monoimide (BPMI) moiety through a non-conjugated linker. The photophysical and photochemical properties originating from the interactions of the metal polypyridine and perylene units were exploited to afford new cellular reagents with thiol-sensing capability and excellent photocytotoxic activity.

Design of ruthenium-albumin hydrogel for cancer therapeutics and luminescent imaging.

Improving cell uptake of metal compounds has became an important goal in the field of metal-based anticancer agents. This may combat platinum resistance and side effects seen commonly in current anticancer chemotherapy regimes. Here, we explore a novel degradable ruthenium-albumin hydrogel, which shows strong luminescence for cell imaging and high selectivity for cancer cells versus non-cancer cells. This is an early indication of the possibility of reducing unwanted side effects of metals by using bovine serum albumin hydrogel as a delivery strategy. This work provides a strong basis for development of a new class of metal-based cancer therapeutic agents.

Host-guest supramolecular assembly directing beta-cyclodextrin based nanocrystals towards their robust performances.

Fluorescent CdTe nanocrystals (NCs) capped with beta-cyclodextrin (ß-CD) are successfully synthesized by host-guest supramolecular assembly of the hydrophobic alkyl chains of N-acetyl-l-cysteine (NAC) on the surface of CdTe NCs and eco-friendly ß-CD via the promising simple hydrothermal method in our experiments. The as-prepared NCs display better stability and lower toxicity compared with traditional those only capped with NAC. Specially, cytotoxicity experiments to human umbilical vein endothelial cells in vitro and zebrafish embryo toxicological tests in vivo are performed to determine the toxicity of CdTe NCs. For their practical applications, the promising red-luminescent NCs are employed as stable and low poison red phosphors to fabricate white light-emitting diodes (WLEDs) with remarkable color-rendering index (CRI) being 91.6. This research offers significance for solving the difficulty in toxicity and instability of heavy metal based NCs, which has potential applications in future optoelectronic devices and biomarkers.

A Novel Chemiluminescent Probe Based on 1,2-Dioxetane Scaffold for Imaging Cysteine in Living Mice.

A novel chemiluminescent probe for detection of cysteine (Cys) from other biothiols has been reported by utilizing the excellent chemiluminescent Schaap's adamantylidene-dioxetane scaffold. After careful assessment, the probe CL-Cys could detect Cys with high sensitivity and total light photons increased by 28-fold after the probe was treated with Cys, with the detection limit of 7.5 × 10-8 M. Finally, CL-Cys was further utilized for the chemiluminescent imaging of endogenous Cys in a living mouse. In general, this probe has a remarkable ability to detect Cys, which provides a valuable method for interrogation of the Cys roles in more biological and pathological processes.

A ratiometric Raman probe for live-cell imaging of hydrogen sulfide in mitochondria by stimulated Raman scattering.

Stimulated Raman Scattering (SRS) coupled with alkyne tags has been an emerging imaging technique to visualize small-molecule species with high sensitivity and specificity. Here we describe the development of a ratiometric Raman probe for visualizing hydrogen sulfide (H2S) species in living cells as the first alkyne-based sensor for SRS microscopy. This probe uses an azide unit as a selective reactive site, and it targets mitochondria with high specificity. The SRS ratiometric images show a strong response to H2S level changes in living cells.

Robust and Biocompatible Functionalization of ZnS Nanoparticles by Catechol-Bearing Poly(2-methyl-2-oxazoline)s.

Zinc sulfide (ZnS) nanoparticles (NPs) are particularly interesting materials for their electronic and luminescent properties. Unfortunately, their robust and stable functionalization and stabilization, especially in aqueous media, has represented a challenging and not yet completely accomplished task. In this work, we report the synthesis of colloidally stable, photoluminescent and biocompatible core-polymer shell ZnS and ZnS:Tb NPs by employing a water-in-oil miniemulsion (ME) process combined with surface functionalization via catechol-bearing poly-2-methyl-2-oxazoline (PMOXA) of various molar masses. The strong binding of catechol anchors to the metal cations of the ZnS surface, coupled with the high stability of PMOXA against chemical degradation, enable the formation of suspensions presenting excellent colloidal stability. This feature, combined with the assessed photoluminescence and biocompatibility, make these hybrid NPs suitable for optical bioimaging.

Visualization of mercury(ii) accumulation in vivo using bioluminescence imaging with a highly selective probe.

Mercury is a highly toxic environmental pollutant that negatively affects human health. Thus, an in vivo method for noninvasive imaging of mercury(ii) and visualization of its accumulation within living systems would be advantageous. Herein, we describe a reaction-based bioluminescent probe for detection of mercury(ii) in vitro and accumulation in vivo. The application of this probe would help to shed light on the intricate contributions of mercury(ii) to various physiological and pathological processes.

Tracking mitochondrial pH fluctuation during cell apoptosis with two-photon phosphorescent iridium(iii) complexes.

Two iridium(iii) complexes with ligands containing morpholine groups were used to track mitochondrial pH fluctuation during cell apoptosis as their emissive intensities linearly increased with increase in pH values.

Bioluminescent probe for detecting endogenous hypochlorite in living mice.

As a kind of biologically important reactive oxygen species (ROS), hypochlorite (ClO-) plays a crucial role in many physiological processes. As such, endogenous ClO- is a powerful antibacterial agent during pathogen invasion. Nonetheless, excessive endogenous ClO- could pose a health threat to mammalian animals including humans. However, the detection of endogenous ClO- by bioluminescence probes in vivo remains a considerable challenge. Herein, based on a caged strategy, we developed a turn-on bioluminescent probe 1 for the highly selective detection of ClO-in vitro and imaging endogenous ClO- in a mouse inflammation model. We anticipate that such a probe could help us understand the role of endogenous ClO- in a variety of physiological and pathological processes.

Protein staining agents from low toxic platinum(ii) complexes with bidentate ligands.

Three new luminescent platinum(ii) complexes with bidentate C^N and O^O ligands have been designed and synthesized in this work. Along with the changing of C^N ligands, the emission peaks of these complexes range from 489 to 629 nm and the photoluminescence quantum efficiencies are up to 55% at room temperature. DFT and TD-DFT calculations have also been employed to investigate the ground and excited states of these platinum(ii) complexes. Most importantly, these platinum(ii) complexes with bidentate ligands have almost no cytotoxicity towards HeLa cells and their applications in living cell imaging and protein staining are focused on in this work.

Perspectives and challenges of photon-upconversion nanoparticles - Part II: bioanalytical applications.

In Part II of this review series on lanthanide-doped photon-upconversion nanoparticles (UCNPs), we present and critically discuss the performance and suitability of UCNPs as background-free luminescent reporters in bioimaging and bioanalytical applications. The preparation of a biocompatible nanoparticle surface is an integral step for all life - science-related applications. UCNPs have found their way into a large number of diagnostic platforms, homogeneous and heterogeneous assay formats, and sensor applications. Many bioanalytical detection schemes involve Förster resonance energy transfer (FRET), which is still debated for UCNPs and needs to be much improved. The need for dedicated and standardized instruments as well as recent studies on the dissolution and potential toxicity of UCNPs are addressed. Finally we outline future trends and challenges in the field of upconversion. Graphical Abstract Both synthesis / spectroscopy as well bioanalytical applications of UCNPs are driven by the COST Action CM1403 "The European Upconversion Network".