Hybrid FePt/SiO2/Au nanoparticles as a theranostic tool: in vitro photo-thermal treatment and MRI imaging.

We have produced an innovative, theranostic material based on FePt/SiO2/Au hybrid nanoparticles (NPs) for both, photo-thermal therapy and magnetic resonance imaging (MRI). Furthermore, a new synthesis approach, i.e., Au double seeding, for the preparation of Au nanoshells around the FePt/SiO2 cores, is proposed. The photo-thermal and the MRI response were first demonstrated on an aqueous suspension of hybrid FePt/SiO2/Au NPs. The cytotoxicity together with the internalization mechanism and the intracellular fate of the hybrid NPs were evaluated in vitro on a normal (NPU) and a half-differentiated cancerous cell line (RT4). The control samples as well as the normal cell line incubated with the NPs showed no significant temperature increase during the in vitro photo-thermal treatment (ΔT < 0.8 °C) and thus the cell viability remained high (∼90%). In contrast, due to the high NP uptake by the cancerous RT4 cell line, significant heating of the sample was observed (ΔT = 4 °C) and, consequently, after laser irradiation the cell viability dropped significantly to ∼60%. These results further confirm that the hybrid FePt/SiO2/Au NPs developed in the scope of this work were not only efficient but also highly selective photo-thermal agents. Furthermore, the improvement in the contrast and the easier distinction between the healthy and the cancerous tissues were clearly demonstrated with in vitro MRI experiments, proving that hybrid NPs have an excellent potential to be used as contrast agents.

Magnetic interactions and in vitro study of biocompatible hydrocaffeic acid-stabilized Fe-Pt clusters as MRI contrast agents.

A detailed magnetic study of separated Fe-Pt NPs and Fe-Pt clusters was performed to predict their optimal size and morphology for the maximum saturation magnetization, a factor that is known to influence the performance of a magnetic-resonance-imaging (MRI) contrast agent. Excellent stability and biocompatibility of the nanoparticle suspension was achieved using a novel coating based on hydrocaffeic acid (HCA), which was confirmed with a detailed Fourier-transform infrared spectroscopy (FTIR) study. An in vitro study on a human-bladder papillary urothelial neoplasm RT4 cell line confirmed that HCA-Fe-Pt nanoparticles showed no cytotoxicity, even at a very high concentration (550 µg Fe-Pt per mL), with no delayed cytotoxic effect being detected. This indicates that the HCA coating provides excellent biocompatibility of the nanoparticles, which is a prerequisite for the material to be used as a safe contrast agent for MRI. The cellular uptake and internalization mechanism were studied using ICP-MS and TEM analyses. Furthermore, it was shown that even a very low concentration of Fe-Pt nanoparticles (<10 µg mL-1) in the cells is enough to decrease the T 2 relaxation times by 70%. In terms of the MRI imaging, this means a large improvement in the contrast, even at a low nanoparticle concentration and an easier visualization of the tissues containing nanoparticles, proving that HCA-coated Fe-Pt nanoparticles have the potential to be used as an efficient and safe MRI contrast agent.

Electrochemotherapy of mouse sarcoma tumors using electric pulse trains with repetition frequencies of 1 Hz and 5 kHz.

Electrochemotherapy is an efficient local treatment of tumors that combines administration of a chemotherapeutic drug with the subsequent application of electric pulses to the tumor. Although no difference in clinical response of the treated tumors to the electrochemotherapy when using 1 Hz or 5 kHz repetition frequency was observed, it is mandatory to be aware of possible differences in the effectiveness of electrochemotherapy when using suboptimal doses of the drugs. Therefore, this study compares the antitumor effectiveness of electrochemotherapy using electric pulse trains with repetition frequencies of 1 Hz and 5 kHz at suboptimal drug doses of bleomycin or cisplatin. Electrochemotherapy of fibrosarcoma SA-1 subcutaneous tumors transplanted in A/J mice resulted in good antitumor effectiveness, but antitumor effectiveness was significantly better at 1 Hz repetition frequency than at 5 kHz. The platinum content was higher in tumors treated with a 1 Hz repetition frequency. The application of electric pulses to the tumors at a 5 kHz repetition frequency induced an immediate reduction in tumor perfusion, comparable to the reduction at 1 Hz but with faster reperfusion. The greater effectiveness of electrochemotherapy using electric pulse trains of 1 Hz compared to 5 kHz is due to the greater electroporative effect and longer time in which electroporated tumors are exposed to the two chemotherapeutic drugs. These differences are observed at suboptimal drug doses, whereas at optimal drug doses of bleomycin or cisplatin the antitumor effectiveness is the same, as demonstrated in clinical trials.

Metallothionein-like proteins and zinc--copper interaction in the hindgut of Porcellio scaber (Crustacea: Isopoda) exposed to zinc.

Metallothioneins (MTs) are ubiquitous low-molecular-weight metal-binding proteins, with a variety of functions in metal metabolism ascribed to them. Among terrestrial invertebrates, MTs have been studied in nematodes, insects, snails, and earthworms. The aim of this study was the characterization of MT-like proteins in the terrestrial isopod crustacean Porcellio scaber in order to analyze their probable role in the metabolism of copper (Cu) and zinc (Zn). Dietary Zn supplementation (793 microg Zn/g dry food, 6 d) was applied to stimulate MT synthesis. After separation of the hindgut post-microsomic supernatant (cytosol) of Zn-exposed animals by gel filtration on a Sephadex G-75 column, a Cu- and Zn-containing peak was detected in the position of Ve/Vo approximately 2, where MTs are expected to elute. Rechromatography of these fractions by size-exclusion chromatography-high-performance liquid chromatography revealed that the 215-nm absorbance peak coincided with the absorbance peak of the rabbit MT II standard. These low-molecular-weight Cu- and Zn-binding compounds, detected in the cytosol of the hindgut cells in Zn-exposed P. scaber, are considered to be Cu, Zn-MT-like proteins. To our knowledge, this is the first report on the characterization of MT-like proteins in isopod crustaceans. These results also indicate that both Zn and Cu dynamics in P. scaber hindgut are affected at the given dietary Zn supplementation and that MT-like proteins are involved in this Zn-Cu interaction.

Simultaneous sample preparation and species-specific isotope dilution mass spectrometry analysis of monomethylmercury and tributyltin in a certified oyster tissue.

A rapid, accurate, sensitive, and simple method for simultaneous speciation analysis of mercury and tin in biological samples has been developed. Integrated simultaneous sample preparation for tin and mercury species includes open focused microwave extraction and derivatization via ethylation. Capillary gas chromatography-inductively plasma mass spectrometry (CGC-ICPMS) conditions and parameters affecting the analytical performance were carefully optimized both for species-specific isotope dilution analysis of MMHg and TBT and for conventional analysis of MBT and DBT201Hg-enriched monomethylmercury and 117Sn-enriched tributyltin were used for species-specific isotope dilution mass spectrometry (SIDMS) analysis. As important, accurate isotope dilution analysis requires equilibration between the spike and the analyte to achieve successful analytical procedures. Since the spike stabilization and solubilization are the most critical and time-consuming steps in isotope dilution analysis, different spiking procedures were tested. Simultaneous microwave-assisted spike stabilization and solubilization can be achieved within less than 5 min. This study originally introduces a method for the simultaneous speciation and isotope dilution of mercury and tin in biological tissues. The sample throughput of the procedure was drastically reduced by fastening sample preparation and GC separation steps. The accuracy of the method was tested by both external calibration analysis and species-specific isotope dilution analysis using the first biological reference material certified for multielemental speciation (oyster tissue, CRM 710, IRMM). The results obtained demonstrate that isotope dilution analysis is a powerful method allowing the simultaneous speciation of TBT and MMHg with high precision and excellent accuracy. Analytical problems related to low recovery during sample preparation are thus minimized by SIDMS. In addition, a rapid procedure allows us to establish a performant routine method using CGC-ICPMS technique.

Hepatic metallothioneins in two neotenic salamanders, Proteus anguinus and Necturus maculosus (Amphibia, Caudata).

The presence of metallothionein (MT) and the subcellular distribution of copper, zinc and cadmium were investigated in livers of two neotenic salamanders, Proteus anguinus and Necturus maculosus. In P. anguinus, caught in the wild, hepatic MTs were present as a single isoform of (Zn, Cu, Cd)-thioneins, whose molecular weight was estimated to be approximately 12000 by size exclusion chromatography. The percentage of zinc and cadmium was higher in the cytosol and of copper in the pellet. Cytosolic cadmium was almost exclusively associated with MTs (80%), while zinc and copper were also present in the regions of higher-molecular weight proteins. In laboratory bred N. maculosus, MTs were isolated from the liver cytosol and extract of the pellet as (Cu, Zn)- and (Zn, Cu)-thioneins, respectively. According to the low amount of copper extracting from liver pellets of N. maculosus, the presence of water insoluble aggregated forms of Cu-thioneins should be checked in further investigations.

DNA damage and metallothionein synthesis in human hepatoma cells (HepG2) exposed to cadmium.

Cadmium is an important heavy metal environmental toxicant, which is classified as a human carcinogen. The comet assay was used to evaluate the levels of DNA damage in a metabolically competent HepG2 cell line after treatment with low, non-cytotoxic and physiologically relevant concentrations of cadmium, alone and in combination with the dietary mutagen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and with the environmental mutagen benzo[a]pyrene (B(a)P). After exposure of the cells to 10, 100 and 1000 nM CdCl(2), a dose- and time-dependent increase of DNA damage was detected. Maximal damage was found after 12 h of treatment, but declined with further incubation with CdCl(2). The increased synthesis of metallothioneins on exposure to CdCl(2) up to 12 h suggests that they are responsible for the adaptation of HepG2 cells to the DNA damaging effects of CdCl(2). Co-treatment of the cells with CdCl(2) (10-1000 nM) and IQ (300 microM) induced a dose-dependent increase of DNA damage compared to cells treated with IQ alone. Co-genotoxic activity was also observed by increased formation of micronuclei in cells exposed to IQ and 1000 nM CdCl(2); at this concentration, CdCl(2) alone also induced micronuclei in HepG2 cells. Our results support the hypothesis that direct and indirect mechanisms are involved in cadmium-induced DNA damage.

Environmentally safe sewage sludge disposal: the impact of liming on the behaviour of Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn.

Dewatered sewage sludge containing relatively high total concentrations of Cr (945 micrograms ml-1), Cu (523 micrograms ml-1), Ni (1186 micrograms ml-1) and Zn (2950 micrograms ml-1) was treated with quicklime and sawdust for sludge disinfection and post-stabilisation. The mobility of the heavy metals in the sludge samples was assessed by applying a modified five-step Tessier sequential extraction procedure. Water was added as a first step for estimation of the proportion of the easily soluble metal fractions. To check the precision of the analytical work the concentrations of heavy metals in steps 1-6 of the extraction procedure were summed and compared to the total metal concentrations. The mass balance agreed within +/- 3% for Cd, Cu, Cr, and Zn and within +/- 5% for Ni, Pb, Fe and Mn. Data from the partitioning study indicate that in the lime-treated sludge at a pH of 12 the mobility of Cu and Ni notably increased with the solubilisation of these metals from their organic and/or carbonate and Fe and Mn oxide and hydroxide fractions, respectively. Liming slightly decreased the proportion of other heavy metals in the easily soluble fractions while its impact on the partitioning between other sludge phases was almost insignificant. Due to the increased solubility of Ni and Cu as well as potential Cr oxidation at high pH, liming cannot be recommended for sludge disinfection. Addition of sawdust did not change the heavy metal partitioning.

Use of nitric acid in sample pretreatment for determination of trace elements in various biological samples by ETAAS.

Trace elements in liquid biological samples may be determined by direct electrothermal atomic absorption spectrometry (ETAAS). In our previous work it was found that samples containing proteins or DNA may leak out of the graphite tube before the drying step, despite the addition of various modifiers. In order to keep the sample to the graphite tube, samples were diluted before analysis 1 + 1 with 32% v/v nitric acid, or 5 microl of 32% v/v nitric acid was added to the graphite tube before ETAAS determination. Applying the proposed procedure, the concentrations of lead in eluted fractions after gel chromatographic separation of human cerebellar nucleus dentatus supernatant and platinum in isolated DNA samples were determined. The use of nitric acid in sample pretreatment prevent sample leakage out of the graphite tube, provided for even drying and considerably reduced nonspecific absorption in lead determination. The repeatability of measurements was better than + 6%. The accuracy of the procedure was checked by spiking samples. The recoveries for both elements lay between 93--104%. Nitric acid was found to be a better modifier than TRITON X-100.

Total metal concentrations and partitioning of Cd, Cr, Cu, Fe, Ni and Zn in sewage sludge.

Application of the BCR three-step sequential extraction procedure to sewage sludge samples collected at an urban wastewater treatment plant (Domzale, Slovenia) is reported. The total concentrations of Cd, Cr, Cu, Fe, Ni and Zn and their concentrations in fractions after extraction were determined by flame or electrothermal atomic absorption spectrometry (FAAS, ETAAS) under optimised measurement conditions. Total acid digestion including hydrofluoric acid (HF) treatment and aqua regia extraction were compared in order to estimate the efficiency of aqua regia extraction for determination of total metal concentrations in sewage sludge. It was found experimentally that aqua regia quantitatively leached these heavy metals from the sewage sludge and could therefore be applied in analysis of total heavy metal concentrations. The total concentrations of 856 mg kg(-1) Cr, 621 mg kg(-1) Ni and 2032 mg kg(-1) Zn were higher than those set by Slovenian legislation for sludge to be used in agriculture. Total concentrations of 2.78 mg kg(-1) Cd, 433 mg kg(-1) Cu and 126 mg kg(-1) Pb were below those permitted in the relevant legislation. CRM 146R reference material was used to follow the quality of the analytical process. The results of the BCR three-step sequential extraction procedure indicate high Ni and Zn mobility in the sludge analysed. The other heavy metals were primarily in sparingly soluble fractions and hence poorly mobile. Due to the high total Ni concentration and its high mobility the investigated sewage sludge could not be used in agriculture.

Determination of trace elements and calcium in bone of the human iliac crest by atomic absorption spectrometry.

A rapid and reliable analytical method for the determination of trace elements in human bone by atomic absorption spectrometry is reported. Calcium was determined to estimate the homogeneity of samples. Human bone from the iliac crest was obtained at autopsy of adult subjects. Before analysis samples were decomposed by microwave digestion and acid digestion in a Parr bomb. Zinc, rubidium, strontium, calcium and iron were determined by flame atomic absorption spectrometry (FAAS) and aluminium, copper and lead by electrothermal atomic absorption spectrometry (ETAAS) at optimum measurement conditions. The results for the two digestion procedures agreed for zinc, rubidium and calcium within +/-5%, for copper within +/-7% and for strontium, iron, aluminium and lead within +/-10%. The repeatability of measurement (R.S.D.) for determination of calcium and trace elements after microwave digestion and acid digestion in a Parr bomb was tested in one representative autopsy bone sample by six parallel determinations. It was found to be better than +/-5% either for microwave digested samples or samples digested in a Parr bomb, for all elements determined by FAAS and ETAAS techniques. The accuracy of the applied digestion procedures was checked by analysis of trace elements in NIST SRM 1486 Bone Meal reference material. Good agreement of the results with certified values was obtained for both digestion procedures. The microwave procedure developed for digestion of small amounts of sample was applied in trace elements analysis of bone biopsy samples from dialysis patients.

Effect of arsenic trioxide on metallothionein and its conversion to different arsenic metabolites in hen liver.

The metabolism of arsenic, its affinity to metallothionein (MT), its influence on selenium levels, and its biotransformation to different metabolites in the liver tissue of laying hens exposed to arsenic trioxide (As2O3) was investigated. The experiment was performed with two groups of hens fed for 19 d with either a standard diet or with the same diet enriched in arsenic (30 microg/g). The major findings were as follows: 1. After 19 d exposure, about 65% of the total liver As was found in the water-soluble phase (100,000g centrifuged supernatant). In liver supernatant, As binding was found mostly in the range of very low-molecular-weight proteins (Mr < 10,000). Although after exposure the amount of MT-like proteins increased, the As bound to it was only in trace amounts. The protein was identified by convential procedures as Zn,Cu-thionein with traces of selenium and arsenic. 2. Arsenic exposure resulted in almost unchanged Se levels regarding its tissue concentrations and distribution between supernatant and pellet, where about 10% of total Se was found in the supernatant. On the contrary, As exposure did affect Cd levels. Tissue Cd concentration was slightly diminished, but the percentage of tissue Cd found in the water-soluble phase was increased from 20% to 40%. 3. In methanol extracts of tissue and supernatant of the As-exposed group, only two arsenic compounds were detected, As(III) and dimethylarsinic acid (DMA), the latter prevailing.

Problems related to determination of trace elements in spent continuous ambulatory peritoneal dialysis fluids by electrothermal atomic absorption spectrometry.

Critical parameters which influence the direct determination of trace elements in spent continuous ambulatory peritoneal dialysis (CAPD) fluids by electrothermal atomic absorption spectrometry (ETAAS) were investigated. Samples were collected after CAPD fluid exchange in 5.0 ml polyethylene cups and stored at -20 degrees C. Daily spent CAPD fluids were frozen immediately, while nightly spent CAPD fluids were frozen 2-3 h after the morning exchange. Before analysis samples were equilibrated to room temperature and analysed within 8 h. It was found experimentally that some samples which were not frozen immediately leaked out of the pyrolytically coated graphite tube before the drying step. In order to keep the sample in the graphite tube and to obtain reproducible measurements of copper, aluminium and iron, samples were diluted before analysis 1 + 1 with 32% v/v nitric acid. The standard addition method was used in the calibration procedure. The results of direct ETAAS determinations for copper agreed well (+/-5-10%) with those obtained after acid digestion of CAPD fluids in Parr bomb. The procedure for direct determination of all three elements was validated with spiked samples.

Increased platinum accumulation in SA-1 tumour cells after in vivo electrochemotherapy with cisplatin.

Electrochemotherapy is an anti-tumour treatment that utilizes locally delivered electric pulses to increase cytotoxicity of chemotherapeutic drugs. The aim of our study was to determine whether anti-tumour effectiveness of electrochemotherapy with cisplatin is a consequence of increased plasma membrane permeability caused by electroporation that enables cisplatin binding to DNA. For this purpose, anti-tumour effectiveness of electrochemotherapy was evaluated on SA-1 tumours treated with electric pulses 3 min after intravenous injection of cisplatin (4 mg kg(-1)). Anti-tumour effectiveness was correlated with platinum accumulation in tumours and the amount of platinum bound to DNA, as determined by atomic absorption spectrometry. In tumours treated with electrochemotherapy, cell kill was increased by a factor of 20 compared with treatment with cisplatin only, as determined from tumour growth curves. The amount of platinum bound to DNA and platinum content in the tumours treated by electrochemotherapy was approximately two times higher than in cisplatin-treated tumours. Based on our results, we conclude that in vivo application of electric pulses potentiates anti-tumour effectiveness of cisplatin by electroporation that consequently results in cisplatin increased delivery into the cells. In addition, besides electroporation, immune system and tumour blood flow changes could be involved in the observed anti-tumour effectiveness of electrochemotherapy.

Optimisation of parameters for determination of rubidium in spent CAPD fluids by flame and electrothermal atomic absorption spectrometry.

An analytical procedure is reported for the determination of rubidium in spent continuous ambulatory peritoneal dialysis (CAPD) fluids by flame and electrothermal atomic absorption spectrometry (FAAS, ETAAS). Samples of spent CAPD fluids were collected as 5 ml aliquots in polyethylene tubes and stored in a freezer at -20 degrees C. Before analysis, samples were equilibrated to room temperature and analysed within 8 h. A total of 2 mg ml(-1) of caesium was added to each sample and standard solution to overcome interferences from ionisation. An air-acetylene flame was applied in FAAS determinations. Analysis was performed against aqueous standards. The calibration graph was linear from 30.0 up to 5000 microg l(-1) Rb, while the limit of detection (3 s) was found to be 20.0 microg l(-1) rubidium. Good repeatability of measurement (RSD 1%) was obtained. Parameters were also optimised for determination of rubidium in spent CAPD fluids by ETAAS. Ten-fold diluted samples (3.5% nitric acid) were analysed applying standard addition calibration. The calibration graph was linear from 2.0 up to 30.0 microg l(-1) rubidium, while the limit of detection (3 s) was found to be 1.0 microg l(-1) rubidium (sample volume 10 microl). Good repeatability of measurement (RSD 5%) was obtained. The results of direct determination by FAAS and ETAAS were compared to those obtained after acid digestion of samples in Parr bombs. The accuracy of the procedure for direct determination was checked by spiking samples. In 73% of samples analysed, the differences between the results obtained by the two techniques, either for direct determinations of samples or for samples digested in a Parr bomb did not exceed +/-10%.