Efficient two-step excitation energy transfer in artificial light-harvesting antenna based on bacteriochlorophyll aggregates.

Chlorosomes of green photosynthetic bacteria are large light-harvesting complexes enabling these organisms to survive at extremely low-light conditions. Bacteriochlorophylls found in chlorosomes self-organize and are ideal candidates for use in biomimetic light-harvesting in artificial photosynthesis and other applications for solar energy utilization. Here we report on the construction and characterization of an artificial antenna consisting of bacteriochlorophyll c co-aggregated with ß-carotene, which is used to extend the light-harvesting spectral range, and bacteriochlorophyll a, which acts as a final acceptor for excitation energy. Efficient energy transfer between all three components was observed by means of fluorescence spectroscopy. The efficiency varies with the ß-carotene content, which increases the average distance between the donor and acceptor in both energy transfer steps. The efficiency ranges from 89 to 37% for the transfer from ß-carotene to bacteriochlorophyll c, and from 93 to 69% for the bacteriochlorophyll c to bacteriochlorophyll a step. A significant part of this study was dedicated to a development of methods for determination of energy transfer efficiency. These methods may be applied also for study of chlorosomes and other pigment complexes.

Anticancer Potential of Diruthenium Complexes with Bridging Hydrocarbyl Ligands from Bioactive Alkynols.

Diruthenacyclopentenone complexes of the general composition [Ru2Cp2(CO)2{µ-η1:η3-CH═C(C(OH)(R))C(═O)}] (2a-c; Cp = η5-C5H5) were synthesized in 94-96% yields from the reactions of [Ru2Cp2(CO)2{µ-η1:η3-C(Ph)═C(Ph)C(═O)}] (1) with 1-ethynylcyclopentanol, 17α-ethynylestradiol, and 17-ethynyltestosterone, respectively, in toluene at reflux. Protonation of 2a-c by HBF4 afforded the corresponding allenyl derivatives [Ru2Cp2(CO)3{µ-η1:η2-CH═C═R}]BF4 (3a-c) in 85-93% yields. All products were thoroughly characterized by elemental analysis, mass spectrometry, and IR, UV-vis, and nuclear magnetic resonance spectroscopy. Additionally, 2a and 3a were investigated by cyclic voltammetry, and the single-crystal diffraction method was employed to establish the X-ray structures of 2b and 3a. The cytotoxicity in vitro of 2b and 3a-c was evaluated against nine human cancer cell lines (A2780, A2780R, MCF-7, HOS, A549, PANC-1, Caco-2, PC-3, and HeLa), while the selectivity was assessed on normal human lung fibroblast (MRC-5). Overall, complexes exert stronger cytotoxicity than cisplatin, and 3b (comprising 17α-estradiol derived ligand) emerged as the best-performing complex. Inductively coupled plasma mass spectrometry cellular uptake studies in A2780 cells revealed a higher level of internalization for 3b and 3c compared to 2b, 3a, and the reference compound RAPTA-C. Experiments conducted on A2780 cells demonstrated a noteworthy impact of 3a and 3b on the cell cycle, leading to the majority of the cells being arrested in the G0/G1 phase. Moreover, 3a moderately induced apoptosis and oxidative stress, while 3b triggered autophagy and mitochondrial membrane potential depletion.

Heteroleptic Copper(II) Complexes Containing 2'-Hydroxy-4-(Dimethylamino)Chalcone Show Strong Antiproliferative Activity.

A series of six heteroleptic copper(II) complexes with 2'-hydroxy-4-(dimethylamino)chalcone (HL) with the composition [Cu(N-N)(L)]NO3 (1-6), where N-N stands for dmbpy = 5,5'-dimethyl-2,2'-bipyridine (1), bphen = 4,7-diphenyl-1,10-phenanthroline (2), dbbpy = 4,4'-di-tert-butyl-2,2'-bipyridine (3), nphen = 5-nitro-1,10-phenanthroline (4), bpy = 2,2'-bipyridine, (5), and dpa = 2,2'-dipyridylamine (6), was prepared and thoroughly characterized. The in vitro cytotoxicity screening on eight human cancer cell lines identified complex 2, containing the bulkiest N-donor ligands (bphen) as highly cytotoxic against cancer cells, with IC50 values ranking from 1.0 to 2.3 µM, with good selectivity and low toxicity against healthy human fetal lung fibroblasts MRC-5. The cell-based assays, involving the most effective complex 2 in A2780 cancer cells, revealed its strong pro-apoptotic effects based on the effective activation of caspases 3/7, ROS overproduction, and autophagy in the A2780 cells while not impeding the cell cycle and mitochondrial membrane functions. The cellular uptake studies in A2780 and 22Rv1 cells uncovered no intracellular transport of the cationic complex 2, supporting the hypothesis that the in vitro anticancer effects of complex 2 are based on the combined extrinsic activation of apoptosis and autophagy induction.

New glycoconjugation strategies for Ruthenium(II) arene complexes via phosphane ligands and assessment of their antiproliferative activity.

Glycoconjugation is a powerful tool to improve the anticancer activity of metal complexes. Herein, we modified commercial arylphosphanes with carbohydrate-derived fragments for the preparation of novel glycoconjugated ruthenium(II) p-cymene complexes. Specifically, d-galactal and d-allal-derived vinyl epoxides (VEß and VEα) were coupled with (2-hydroxyphenyl)diphenylphosphane, affording the 2,3-unsaturated glycophosphanes 1ß and 1α. Ligand exchange with [Ru(C2O4)(η6-p-cymene)(H2O)] gave the glycoconjugated complexes Ru1ß and Ru1α which were subsequently dihydroxylated with OsO4/N-methylmorpholine N-oxide to Ru2ß and Ru2α containing O-benzyl d-mannose and d-gulose units respectively. Besides, aminoethyl tetra-O-acetyl-ß-d-glucopyranoside was condensed with borane-protected (4-diphenylphosphanyl)benzoic acid by HATU/DIPEA under MW heating, to afford the amide 3∙BH3. Zemplén deacylation with MeONa/MeOH gave the deprotected d-glucopyranoside derivative 4∙BH3. The glycoconjugated phosphane complexes Ru3 and Ru4 were obtained by reaction of the phosphane-boranes 3∙BH3 and 4∙BH3 with [Ru(C2O4)(η6-p-cymene)(H2O)]. The employed synthetic strategies were devised to circumvent unwanted phosphine oxidation. The compounds were purified by silica chromatography, isolated in high yield and purity and characterized by analytical and spectroscopic (IR and multinuclear NMR) techniques. The behaviour of the six glycoconjugated Ru complexes in aqueous solutions was assessed by NMR and MS measurements. All compounds were screened for their in vitro cytotoxicity against A2780/A2780R human ovarian and MCF7 breast cancer cell lines, revealing a significant cytotoxicity for complexes containing the 2,3-unsaturated glycosyl unit (Ru1ß, Ru1α). Additional studies on five other human cancer cells, as well as time-dependent toxicity and cell-uptake analyses on ovarian cancer cells, confirmed the prominent activity of these two compounds - higher than cisplatin - and the better performance of the ß anomer. However, Ru1ß, Ru1α did not show preferential activity against cancer cells with respect to fetal lung fibroblast and human embryonic kidney cells as models of normal cells. The effects of the two ruthenium glycoconjugated compounds in A2780 ovarian cancer cells were further investigated by cell cycle analysis, induction of apoptosis, intracellular ROS production, activation of caspases 3/7 and disruption of mitochondrial membrane potential. The latter is a relevant factor in the mechanism of action of the highly cytotoxic Ru1ß, inducing cell death by apoptosis.

Graphene oxide interaction with Lemna minor: Root barrier strong enough to prevent nanoblade-morphology-induced toxicity.

The production of graphene oxide (GO) along with its applications in various aquatic environments is vastly increasing thanks to its rapidly expanding range of new GO-based environmental technologies. Therefore, the fate of GO in aquatic environments is an important issue, as it could become an environmental challenge if its potential toxic mechanism is not addressed properly. Number of studies reporting the toxicity of GO to various aquatic organisms is still increasing. However, research data on the possible toxic mechanism of GO towards aquatic plants have yet to be collected, especially regarding GO's surface chemistry. Here, we studied the interaction of three differently oxidized GO systems with model aquatic plant Lemna minor. We found that although none of the three GOs caused lethal phytotoxicity to Lemna after 7 days, the mechanism of action was dependent on the GO's surface oxidation. Based on the amount of functional surface groups, the GO was able to directly interact with the Lemna's root through its edges. However, in this case in contrast to algae and crustaceans, the interaction did not lead to a mechanical damage. Therefore, our results showed that GO is not hazardous to Lemna minor even at very high concentrations (up to 25 mg/L), because the root barrier proved to be strong enough to prevent GO's penetration and its consequent toxicity.

Identification of potent anticancer copper(ii) complexes containing tripodal bis[2-ethyl-di(3,5-dialkyl-1H-pyrazol-1-yl)]amine moiety.

A series of heteroleptic copper(ii) complexes of the composition [Cu(L1-5)Cl]X, where X = ClO4 and/or PF6 and [bis(2-ethyl-di(3,5-dimethyl-1H-pyrazol-1-yl))-(6-methyl-(2-pyridylmethyl))]amine (L1), [bis(2-ethyl-di(3,5-dimethyl-1H-pyrazol-1-yl))-(3,4-dimethoxy-(2-pyridylmethyl))]amine (L2), [bis(2-ethyl-di(3,5-dimethyl-1H-pyrazol-1-yl)-(2-quinolymethyl)]amine (L3), [bis(2-ethyl-di(3,5-dimethyl-1H-pyrazolyl)-(di(3,5-dimethyl-1H-pyrazol-1-yl-methyl))]amine (L4) and [bis(2-ethyl-di(3,5-dimethyl-1H-pyrazol-1-yl)-(5-methyl-3-phenyl-1H-pyrazol-1-yl-methyl)]amine (L5), were prepared and thoroughly characterized including single-crystal X-ray diffraction technique. The in vitro cytotoxicity of complexes against A2780, A2780R, HOS and MCF-7 human cancer cell lines was evaluated using the MTT test. The results revealed that complexes [Cu(L1)Cl]PF6 (1-PF6), [Cu(L2)Cl]ClO4 (2-ClO4) and [Cu(L3)Cl]PF6 (3-PF6) are the most effective, with IC50 values ranging from 1.4 to 6.3 µM, thus exceeding the cytotoxic potential of metallodrug cisplatin (IC50 values ranging from 29.9 to 82.0 µM). The complexes [Cu(L4)Cl]PF6 (4-PF6) and [Cu(L5)Cl]PF6 (5-PF6) showed only moderate cytotoxicity against A2780, with IC50 = 53.6 µM, and 33.8 µM, respectively. The cell cycle profile, time-resolved cellular uptake, interactions with small sulfur-containing biomolecules (cysteine and glutathione), intracellular ROS production, induction of apoptosis and activation of caspases 3/7 were also evaluated in the case of the selected complexes. It has been found that the best performing complexes 1 and 2 cause cell arrest in the G2/M phase and induce apoptosis via the increase in production of ROS, dominantly due to the overproduction of superoxide.

Copper(II) Complexes Containing Natural Flavonoid Pomiferin Show Considerable In Vitro Cytotoxicity and Anti-inflammatory Effects.

A series of new heteroleptic copper(II) complexes of the composition [Cu(L)(bpy)]NO3·2MeOH (1), [Cu(L)(dimebpy)]NO3·2H2O (2), [Cu(L)(phen)]NO3·2MeOH (3), [Cu(L)(bphen)]NO3·MeOH (4), [Cu(L)(dppz)]NO3·MeOH (5) was prepared, where HL = 3-(3,4-dihydroxyphenyl)-5-hydroxy-8,8-dimethyl-6-(3-methylbut-2-ene-1-yl)-4H,8H-benzo[1,2-b:3,4-b']dipyran-4-one, (pomiferin) and bpy = 2,2'-bipyridine, dimebpy = 4,4'-dimethyl-2,2'-bipyridine, phen = 1,10-phenanthroline, bphen = 4,7-diphenyl-1,10-phenanthroline, and dppz = dipyrido[3,2-a:2',3'-c]phenazine. The complexes were characterized using elemental analysis, infrared and UV/Vis spectroscopies, mass spectrometry, thermal analysis and conductivity measurements. The in vitro cytotoxicity, screened against eight human cancer cell lines (breast adenocarcinoma (MCF-7), osteosarcoma (HOS), lung adenocarcinoma (A549), prostate adenocarcinoma (PC-3), ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R), colorectal adenocarcinoma (Caco-2) and monocytic leukemia (THP-1), revealed the complexes as effective antiproliferative agents, with the IC50 values of 2.2-13.0 µM for the best performing complexes 3 and 5. All the complexes 1-5 showed the best activity against the A2780R cells (IC50 = 2.2-6.6 µM), and moreover, the complexes demonstrated relatively low toxicity on healthy human hepatocytes, with IC50 > 100 µM. The complexes were evaluated by the Annexin V/propidium iodide apoptosis assay, induction of cell cycle modifications in A2780 cells, production of reactive oxygen species (ROS), perturbation of mitochondrial membrane potential, inhibition of apoptosis and inflammation-related signaling pathways (NF-κB/AP-1 activity, NF-κB translocation, TNF-α secretion), and tested for nuclease mimicking activity. The obtained results revealed the corresponding complexes to be effective antiproliferative and anti-inflammatory agents.

Toxicity of Carbon Nanomaterials-Towards Reliable Viability Assessment via New Approach in Flow Cytometry.

The scope of application of carbon nanomaterials in biomedical, environmental and industrial fields is recently substantially increasing. Since in vitro toxicity testing is the first essential step for any commercial usage, it is crucial to have a reliable method to analyze the potentially harmful effects of carbon nanomaterials. Even though researchers already reported the interference of carbon nanomaterials with common toxicity assays, there is still, unfortunately, a large number of studies that neglect this fact. In this study, we investigated interference of four bio-promising carbon nanomaterials (graphene acid (GA), cyanographene (GCN), graphitic carbon nitride (g-C3N4) and carbon dots (QCDs)) in commonly used LIVE/DEAD assay. When a standard procedure was applied, materials caused various types of interference. While positively charged g-C3N4 and QCDs induced false results through the creation of free agglomerates and intrinsic fluorescence properties, negatively charged GA and GCN led to false signals due to the complex quenching effect of the fluorescent dye of a LIVE/DEAD kit. Thus, we developed a new approach using a specific gating strategy based on additional controls that successfully overcame all types of interference and lead to reliable results in LIVE/DEAD assay. We suggest that the newly developed procedure should be a mandatory tool for all in vitro flow cytometry assays of any class of carbon nanomaterials.

Silver Covalently Bound to Cyanographene Overcomes Bacterial Resistance to Silver Nanoparticles and Antibiotics.

The ability of bacteria to develop resistance to antibiotics is threatening one of the pillars of modern medicine. It was recently understood that bacteria can develop resistance even to silver nanoparticles by starting to produce flagellin, a protein which induces their aggregation and deactivation. This study shows that silver covalently bound to cyanographene (GCN/Ag) kills silver-nanoparticle-resistant bacteria at concentrations 30 times lower than silver nanoparticles, a challenge which has been so far unmet. Tested also against multidrug resistant strains, the antibacterial activity of GCN/Ag is systematically found as potent as that of free ionic silver or 10 nm colloidal silver nanoparticles. Owing to the strong and multiple dative bonds between the nitrile groups of cyanographene and silver, as theory and experiments confirm, there is marginal silver ion leaching, even after six months of storage, and thus very high cytocompatibility to human cells. Molecular dynamics simulations suggest strong interaction of GCN/Ag with the bacterial membrane, and as corroborated by experiments, the antibacterial activity does not rely on the release of silver nanoparticles or ions. Endowed with these properties, GCN/Ag shows that rigid supports selectively and densely functionalized with potent silver-binding ligands, such as cyanographene, may open new avenues against microbial resistance.

Superradiance of bacteriochlorophyll c aggregates in chlorosomes of green photosynthetic bacteria.

Chlorosomes are the main light-harvesting complexes of green photosynthetic bacteria that are adapted to a phototrophic life at low-light conditions. They contain a large number of bacteriochlorophyll c, d, or e molecules organized in self-assembling aggregates. Tight packing of the pigments results in strong excitonic interactions between the monomers, which leads to a redshift of the absorption spectra and excitation delocalization. Due to the large amount of disorder present in chlorosomes, the extent of delocalization is limited and further decreases in time after excitation. In this work we address the question whether the excitonic interactions between the bacteriochlorophyll c molecules are strong enough to maintain some extent of delocalization even after exciton relaxation. That would manifest itself by collective spontaneous emission, so-called superradiance. We show that despite a very low fluorescence quantum yield and short excited state lifetime, both caused by the aggregation, chlorosomes indeed exhibit superradiance. The emission occurs from states delocalized over at least two molecules. In other words, the dipole strength of the emissive states is larger than for a bacteriochlorophyll c monomer. This represents an important functional mechanism increasing the probability of excitation energy transfer that is vital at low-light conditions. Similar behaviour was observed also in one type of artificial aggregates, and this may be beneficial for their potential use in artificial photosynthesis.

Crucial cytotoxic and antimicrobial activity changes driven by amount of doped silver in biocompatible carbon nitride nanosheets.

The use of Ag-modified nanomaterials continues to attract attention in biological contamination control, their potential cytotoxicity is often overlooked. Herein, biocompatible carbon nitride is modified with 1 and 5 wt.% Ag and effects of different nanomaterial dose and Ag content on antimicrobial activity and cytotoxicity is studied. Pure Ag nanoparticles and AgNO3 is tested for comparison, together with ten bacterial strains including pan-resistant Pseudomonas aeruginosa. Cytotoxicity is then investigated in three adherent and two suspension human cell lines, and results confirm that cancer adherent cell lines are the most immune lines and human cervical adenocarcinoma cells (HeLa) are more resilient than human lung adenocarcinoma cells (A549). The HeLa remains over 90 % viable even after 24 -h treatment with the highest concentration of 5%Ag/g-C3N4 (300 mg L-1) while A549 sustained viability only up to 100 mg L-1. Higher concentrations then induce cytotoxicity and A549 cell viability decreases. Our results show the importance of complementary testing of cytotoxicity by LIVE/DEAD assay using flow cytometry with more different human cell lines, which might be less immune to tested nanomaterials than HeLa and A549. Combined controls of new antibacterial agent activity tests then provide increased knowledge of their biocompatibility.

The environmental fate of graphene oxide in aquatic environment-Complete mitigation of its acute toxicity to planktonic and benthic crustaceans by algae.

Graphene oxide (GO) as the most studied hydrophilic graphene derivative can be deployed in a broad spectrum of environmental technologies opening the issue of its ecotoxicity. Nevertheless, the information about its behavior in complex aquatic environment is still not sufficient. Here, we studied the interaction of three differently oxidized GO systems with planktonic and benthic crustaceans. By standard toxicity tests, we observed the importance of feeding strategy as well as the surface oxidation of GO with respect to GO's ecotoxicity. However, to gain a clearer insight into GO's environmental fate, we introduced a pre-treatment with algae as the most common source of food for crustaceans. Such an adjustment mimicking the conditions in real aquatic ecosystems resulted in complete mitigation of acute toxicity of GOs to all organisms and, more importantly, to the eradication of oxidative stress caused by GOs. We argue, that the pre-exposition of food is a crucial factor in GO's overall environmental fate, even though this fact has been completely neglected in recent studies. These experiments proved that GO is not a hazardous material in complex aquatic environments because its acute toxicity can be successfully mitigated through the interaction with algae even at very high concentrations (25 mg/L).

The Differential Effect of Carbon Dots on Gene Expression and DNA Methylation of Human Embryonic Lung Fibroblasts as a Function of Surface Charge and Dose.

This study presents a toxicological evaluation of two types of carbon dots (CD), similar in size (<10 nm) but differing in surface charge. Whole-genome mRNA and miRNA expression (RNAseq), as well as gene-specific DNA methylation changes, were analyzed in human embryonic lung fibroblasts (HEL 12469) after 4 h and 24 h exposure to concentrations of 10 and 50 µg/mL (for positive charged CD; pCD) or 10 and 100 µg/mL (for negative charged CD, nCD). The results showed a distinct response for the tested nanomaterials (NMs). The exposure to pCD induced the expression of a substantially lower number of mRNAs than those to nCD, with few commonly differentially expressed genes between the two CDs. For both CDs, the number of deregulated mRNAs increased with the dose and exposure time. The pathway analysis revealed a deregulation of processes associated with immune response, tumorigenesis and cell cycle regulation, after exposure to pCD. For nCD treatment, pathways relating to cell proliferation, apoptosis, oxidative stress, gene expression, and cycle regulation were detected. The expression of miRNAs followed a similar pattern: more pronounced changes after nCD exposure and few commonly differentially expressed miRNAs between the two CDs. For both CDs the pathway analysis based on miRNA-mRNA interactions, showed a deregulation of cancer-related pathways, immune processes and processes involved in extracellular matrix interactions. DNA methylation was not affected by exposure to any of the two CDs. In summary, although the tested CDs induced distinct responses on the level of mRNA and miRNA expression, pathway analyses revealed a potential common biological impact of both NMs independent of their surface charge.