Formation of Highly Emissive Anthracene Excimers for Aggregation-Induced Emission/Self-Assembly Directed (Bio)imaging.

AIEgens have emerged as a promising alternative to molecular rotors in bioimaging applications. However, transferring the concept of aggregation-induced emission (AIE) from solution to living systems remains a challenge. Given the highly heterogeneous nature and the compartmentalization of the cell, different approaches are needed to control the self-assembly within the crowded intricate cellular environment. Herein, we report for the first time the self-assembly mechanism of an anthracene-guanidine derivative (AG) forming the rare and highly emissive T-shaped dimer in breast cancer cell lines as a proof of concept. This process is highly sensitive to the local environment in terms of polarity, viscosity, and/or water quantity that should enable the use of the AG as a fluorescence lifetime imaging biosensor for intracellular imaging of cellular structures and the monitoring of intracellular state parameters. Different populations of the monomer and T-shaped and π-π dimers were observed in the cell membrane, cytoplasm, and nucleoplasm, related to the local viscosity and presence of water. The T-shaped dimer is formed preferentially in the nucleus because of the higher density and viscosity compared to the cytoplasm. The present results should serve as a precursor for the development of new design strategies for molecular systems for a wide range of applications such as cell viscosity, density, or temperature sensing and imaging.

Unusually High Affinity of the PLK Inhibitors RO3280 and GSK461364 to HSA and Its Possible Pharmacokinetic Implications.

The binding processes of two Polo-like kinase inhibitors, RO3280 and GSK461364, to the human serum albumin (HSA) protein as well as the protonation equilibria of both compounds have been studied combining absorbance and fluorescence spectroscopy experiments together with density functional theory calculations. We found that the charge states of RO3280 and GSK461364 are +2 and +1, respectively, at the physiological pH. Nevertheless, RO3280 binds to HSA in the charge state +1 prior to a deprotonation pre-equilibrium. Binding constants to site I of HSA of 2.23 × 106 and 8.80 × 104 M-1 were determined for RO3280 and GSK461364, respectively, at 310 K. The binding processes of RO3280 and GSK461364 to HSA are entropy- and enthalpy-driven, respectively. The positive enthalpy found for the RO3280-HSA complex formation could be related to a proton pre-equilibrium of RO3280.

Shedding light on the binding mechanism of kinase inhibitors BI-2536, Volasetib and Ro-3280 with their pharmacological target PLK1.

In the present work, the interactions of the novel kinase inhibitors BI-2536, Volasetib (BI-6727) and Ro-3280 with the pharmacological target PLK1 have been studied by fluorescence spectroscopy and molecular dynamics calculations. High Stern-Volmer constants were found in fluorescence experiments suggesting the formation of stable protein-ligand complexes. In addition, it was observed that the binding constant between BI-2536 and PLK1 increases about 100-fold in presence of the phosphopeptide Cdc25C-p that docks to the polo box domain of the protein and releases the kinase domain. All the determined binding constants are higher for the kinase inhibitors than for their competitor for the active center (ATP) being BI-2536 and Volasertib the inhibitors that showed more affinity for PLK1. Calculated binding free energies confirmed the higher affinity of PLK1 for BI-2536 and Volasertib than for ATP. The higher affinity of the inhibitors to PLK1 compared to ATP was mainly attributed to stronger van der Waals interactions. Results may help with the challenge of designing and developing new kinase inhibitors more effective in clinical cancer therapy.

A Novel Quantum Dot-Based pH Probe for Long-Term Fluorescence Lifetime Imaging Microscopy Experiments in Living Cells.

The use of two nanoparticles for quantitative pH measurements in live cells by means of fluorescence lifetime imaging microscopy (FLIM) is investigated here. These nanoparticles are based on CdSe/ZnS quantum dots (QDs), functionalized with N-acetylcysteine (CdSe/ZnS-A) and with a small peptide containing D-penicillamine and histidine (CdSe/ZnS-PH). CdSe/ZnS-A has tendency to aggregate and nonlinear pH sensitivity in a complex medium containing salts and macromolecules. On the contrary, CdSe/ZnS-PH shows chemical stability, low toxicity, efficient uptake in C3H10T1/2 cells, and good performance as an FLIM probe. CdSe/ZnS-PH also has key advantages over a recently reported probe based on a CdSe/ZnS QD functionalized with D-penicillamine (longer lifetimes and higher pH-sensitivity). A pH(±2σ) of 6.97 ± 0.14 was determined for C3H10T1/2 cells by FLIM employing this nanoprobe. In addition, the fluorescence lifetime signal remains nearly constant for C3H10T1/2 cells treated with CdSe/ZnS-PH for 24 h. These results show the promising applications of this nanoprobe to monitor the intracellular pH and cell state employing the FLIM technique.

New titanocene derivative with improved stability and binding ability to albumin exhibits high anticancer activity.

Titanium-based therapies have emerged as a promising alternative for the treatment of cancer patients, particularly those with cisplatin resistant tumors. Unfortunately, some titanium compounds show stability and solubility problems that have hindered their use in clinical practice. Here, we designed and synthesized a new titanium complex containing a titanocene fragment, a tridentate ligand to improve its stability in water, and a long aliphatic chain, designed to facilitate a non-covalent interaction with albumin, the most abundant protein in human serum. The stability and human serum albumin affinity of the resulting titanium complex was investigated by UV-Vis absorption and fluorescence spectroscopy techniques. Complex [TiCp2{(OOC)2py-O-myr}] (3) (myr = C14H29, py = pyridine) and its analogous [TiCp2{(OOC)2py-OH}] (4), lacking the aliphatic chain, showed improved stability in phosphate saline buffer compared with [TiCp2Cl2] (1). 3 showed a strong interaction with human serum albumin in a 1:1 stoichiometry. The cytotoxic effect of 3 was higher compared to [TiCp2Cl2] in tumor cell lines and showed potential tumor selectivity when assayed in non-tumor human epithelial cells. Finally, 3 showed an antiproliferative effect on cancer cells, decreasing the population in the S phase, and increasing apoptotic cells in a significant manner. All this makes the novel Ti(IV) compound 3 a firm candidate to continue further studies of its therapeutic potential in vitro and in vivo.

Novel Fluorescence Guanidine Molecules for Selective Sulfate Anion Detection in Water Complex Samples over a Wide pH Range.

Quantitative analysis of sulfate anions in water still remains an important challenge for the society. Among all the methodologies, the most successful one is based on optical supramolecular receptors because the presence of small concentrations of sulfate anion modifies the photophysical properties of the receptor. In this case, fluorescence anion sensors have been designed by the incorporation of guanidine motifs into fluorenyl cores. The photophysical behaviors of the new mono- (M) and bis-guanidine (B) derivatives were studied through pH dependence, solvent effects, and ion sensing on steady-state spectra and time-resolved fluorescence spectroscopy. In more detail, the results demonstrate that M is a highly selective and sensitive sulfate ion receptor in real water samples and, even more importantly, its function remains unchanged at different ranges of pH. The reason behind this resides on the fluorescence quenching produced by an internal charge-transfer process when the sulfate anion is complexed with M. It is worth noting that the global and partial affinity constants (1010 M-2 and 105 M-1, respectively) of complex formation are far above from the current sulfate sensors in water (104 M-1) which give an LOD of 0.10 µM in water with an analytical range of 2.5-10 µM. On the other hand, although it would seem, at first sight, that the B derivate will be the most promising one, the possibility of having two simultaneous protonation states reduces the complex formation and, therefore, its sensitivity to sulfate anions. The results presented here offer the possibility of using a new molecule in water environments, which opens the door to infinite applications such as the detection of trace amounts of sulfate ions in food or water.

Functionalized CdSe/ZnS Quantum Dots for Intracellular pH Measurements by Fluorescence Lifetime Imaging Microscopy.

pH is an important biomarker for many human diseases and great efforts are being made to develop new pH probes for bioimaging and biomedical applications. Here, the use of three different CdSe/ZnS QDs, functionalized with d-penicillamine and small peptides, as pH probes for fluorescence lifetime imaging microscopy (FLIM) is investigated. The fluorescence pH sensitivity of the nanoparticles is analyzed in different experimental media: aqueous solution, synthetic intracellular medium, and mesenchymal C3H10T1/2 and tumoral SK-MEL-2 cell lines. Different experiments along with theoretical calculations are conducted to unravel the mechanisms causing pH sensitivity of the nanoparticles and the effect of the length and composition of the peripheral branches on their photophysical properties. Absolute intracellular pH values measured in live cells with FLIM using a fluorescent probe based on a QD are reported here for the first time (intracellular pH values of 7.0 and 7.1 for C3H10T1/2 and SK-MEL-2 cells, respectively). These fluorescent nanoprobes can also be used to distinguish between different types of cells in cocultures on the basis of their different fluorescence lifetimes in dissimilar intracellular environments.

The role of water and influence of hydrogen bonding on the self-assembly aggregation induced emission of an anthracene-guanidine-derivative.

We report a luminescent anthracene-guanidine derivative that forms rare T-shape dimers, resulting in an excimer with a quantum yield approaching one. Water plays a fundamental role through H-bonding guiding the self-assembly. These results establish a new framework for environmentally friendly aggregation-induced emission luminogens.

Screening and Preliminary Biochemical and Biological Studies of [RuCl(p-cymene)(N,N-bis(diphenylphosphino)-isopropylamine)][BF4] in Breast Cancer Models.

Breast cancer is the second leading cause of cancer death worldwide. Despite progress in drug discovery, identification of the correct population is the limiting factor to develop new compounds in the clinical setting. Therefore, the aim of this study is to evaluate the effects of a new metallodrug, [RuCl(p-cymene)(N,N-bis(diphenylphosphino)-isopropylamine)][BF4] (pnpRu-14), as a lead pnp-Ru compound by screening and preliminary biochemical and biological studies in different breast cancer subtypes. The results show that complex pnpRu-14 is much more effective in promoting in vitro cytotoxic effects on HER2+ and RH+/HER2- breast cancer than the reference metallodrugs cisplatin, carboplatin, or RAPTA-C. It is important to highlight that pnpRu-14 shows an impressive cytotoxicity against BT474 cells. Caspase-dependent apoptosis is the mechanism of action for these compounds. In addition, treatment of SKBR3, BT474, T47D, and MCF7 cancer cells with pnpRu-14 caused an accumulation of cells in the G0/G1 phase cells. The human serum albumin, DNA, and H1 histones binding properties of the lead compound are reported. Pharmacokinetic and biodistribution studies show a quick absorption of pnpRu-14 in serum with no significant accumulation in any of the tested organs. This work provides evidence to support the preclinical and clinical development of pnpRu-14 in breast cancer.

Binding of the anticancer drug BI-2536 to human serum albumin. A spectroscopic and theoretical study.

BI-2536 is a potent Polo-like kinase inhibitor which induces apoptosis in diverse human cancer cell lines. The binding affinity of BI-2536 for human serum albumin (HSA) protein may define its pharmacokinetic and pharmacodynamic profile. We have studied the binding of BI-2536 to HSA by means of different spectroscopic techniques and docking calculations. We have experimentally observed that the affinity of BI-2536 for HSA is higher than that of other common HSA binding drugs. Therefore, it can be postulated that the drug dose should be increased to achieve a certain concentration of free drug in plasma, although BI-2536 could also reach tumour tissues by uptaking HSA/BI-2536 complex. Only a single binding site on HSA has been observed for BI-2536 which seems to correspond to the subdomain IIA pocket. The formation of the HSA/BI-2536 complex is a spontaneous and entropy-driven process that does not cause a significant change of the secondary structure of the protein. Its endothermic character could be related to proton release. Thermodynamic analysis showed that the main protein-drug interactions are of the van der Waals type although the presence of amide and ether groups in BI-2536 could also allow H-bonding with some residues in the subdomain IIA pocket.