Bovine sperm chromatin is not protected from the effects of ultrasmall gold nanoparticles

The response of ejaculated bovine spermatozoa to gold nanoparticles was studied by the standard method of nuclear chromatin decondensation in vitro. After the treatment of semen samples with a hydrosol containing gold nanoparticles with an average diameter of ～3.0 nm and a concentration of 1 × 10¹⁵ particles/mL, the ability of sperm nuclei to decondense in the presence of sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) dramatically changed compared to the control. The frequencies of gametes with nondecondensed (“intact”), partially decondensed, and completely decondensed nuclei correlated as 40: 32: 28% and 0: 36: 64% in the experiment and the control, respectively. Moreover, the appearance of a sufficiently large number of gametes with destructed and almost completely destroyed nuclei was noticed in the spermatozoa treated with gold nanoparticles. This article suggests the putative mechanisms of action of ultrasmall gold nanoparticles on the structural and functional integrity of the deoxyribonucleoprotein (DNP) complex of mature male gametes.     

In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg‐jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø_o (sperm‐specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca²⁺, pH, and this activity could be a serine‐proteinase. Thermal denaturalization of the oocyte extracts (80°C, 10–15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø_o) will be a first condition in the process of sperm chromatin remodeling.     

Utility of gold nanoparticles in luminescence determination of trovafloxacin: comparison of chemiluminescence and fluorescence detection

Two novel sensitive sequential injection chemiluminescence analysis and fluorescence methods for trovafloxacin mesylate detection have been developed. The methods were based on the enhancement effect of gold nanoparticles on luminol–ferricyanide–trovafloxacin and europium(III)–trovafloxacin complex systems. The optimum conditions for both detection methods were investigated. The chemiluminescence signal was emitted due to the enhanced effect of gold nanoparticles on the reaction of luminol–ferricyanide–trovafloxacin in an alkaline medium. The response was linear over a concentration range of 1.0 × 10^–9 to 1.0 × 10^–2 mol/L (%RSD = 1.3), (n = 9, r = 0.9991) with a detection limit of 1.7 × 10^–10 mol/L (S/N = 3). The weak fluorescence intensity signal of the oxidation complex of europium(III)–trovafloxacin was strongly enhanced by gold nanoparticles and detected at λ_ex = 330 and λ_em = 540 nm. Fluorescence detection enabled the determination of trovafloxacin mesylate over a linear range of 1.0 × 10^–8 to 1.0 × 10^–3 mol/L (%RSD = 1.2), (n = 6, r = 0.9993) with a detection limit of 3.3 × 10^–9 mol/L. The proposed methods were successfully applied to the determination of the studied drug in its bulk form and in pharmaceutical preparations. The results were treated statistically and compared with those obtained from other reported methods. Copyright © 2015 John Wiley & Sons, Ltd.     

Sperm nuclear chromatin transformations in somatic cell-free extracts

HeLa cell extracts induced decondensation of lysolecithin permeabilized Xenopus, pig, and human sperm chromatin; decondensation began almost immediately on incubation in the extract and was completed within 10–20 min. The average enlargements of human and pig sperm nuclei were 15-fold and 3-fold, respectively. The structural organization of pig and human sperm chromatin was significantly differnt. Decondensation was differentially inhibited by Mg⁺⁺ and polyamines; inhibition was least for Xenopus and most for pig sperm nuclei. The nuclear membrane was disintegrated on chromatin dispersion, whereas the nuclei which failed to decondense exhibited distinct nuclear envelopes. The decondensing factors were stable at 65°C for 15 min. The dispersed chromatin was remodelled to somatic nucleosomal structures within 60 min. The remodelled chromatin could be recondensed to chromosome-like structures, when incubated further in extracts from mitosis arrested HeLa cells. © 1994 Wiley-Liss, Inc.     

Eco‐friendly synthesis of gelatin‐capped bimetallic Au–Ag nanoparticles for chemiluminescence detection of anticancer raloxifene hydrochloride

This study described the utility of green analytical chemistry in the synthesis of gelatin‐capped silver, gold and bimetallic gold–silver nanoparticles (NPs). The preparation of nanoparticles was based on the reaction of silver nitrate or chlorauric acid with a 1.0 wt% aqueous gelatin solution at 50°C. The gelatin‐capped silver, gold and bimetallic NPs were characterized using transmission electron microscopy, UV–vis, X‐ray diffraction and Fourier transform infrared spectroscopy, and were used to enhance a sensitive sequential injection chemiluminescence luminol–potassium ferricyanide system for determination of the anticancer drug raloxifene hydrochloride. The developed method is eco‐friendly and sensitive for chemiluminescence detection of the selected drug in its bulk powder, pharmaceutical injections and biosamples. After optimizing the conditions, a linear relationship in the range of 1.0 × 10^–9 to 1.0 × 10^–1 mol/L was obtained with a limit of detection of 5.0 × 10^–10 mol/L and a limit of quantification of 1.0 × 10^‐9 mol/L. Statistical treatment and method validation were performed based on ICH guidelines. Copyright © 2016 John Wiley & Sons, Ltd.     

Stopped‐flow chemiluminescence determination of the anticancer drug capecitabine: Application in pharmaceutical analysis and drug‐delivery systems

Capecitabine is a chemotherapeutic agent used for the treatment of patients with metastatic cancers. This study aimed at determining the drug capecitabine in a simple chemiluminescence (CL) system of acidic potassium permanganate using the stopped‐flow injection technique. Statistical methods were used to detect optimum conditions. The method showed two linear calibration ranges from 6.7 × 10^?6 to 6.7 × 10^?5 mol L^?1 and from 6.7 × 10^?5 to 2.7 × 10^?3 mol L^?1 with a detection limit of 1.5 × 10^?6 mol L^?1. Chitosan‐modified magnetic nanoparticles were studied in the drug‐delivery experiments. According to the pH sensitivity of chitosan and low pH values in tumour cells, the chitosan‐coated magnetic nanoparticles could provide a good targeting drug‐delivery system to tumour sites. To evaluate the applicability of the method, the capecitabine‐loaded magnetic chitosan nanoparticles were synthesized with two different cross‐linkers; loading and releasing rates of the drug were investigated using the proposed CL method and an ultraviolet–visible light spectrophotometric method (absorption at 305 nm). The results showed a good correlation between the two methods, and it was found that the synthesized chitosan‐modified magnetic nanoparticles could be used for pH‐dependent release of capecitabine in cancer cells. Moreover, determination of capecitabine in tablets and synthetic samples was performed.     

Gold nanoparticles‐based fluorescence enhancement of the terbium–levofloxacin system and its application in pharmaceutical preparations

A sensitive fluorescence (FL) technique is proposed for the determination of levofloxacin (LVX). The method is based on the fact that the weak FL signal of the Tb(III)–LVX system is strongly enhanced in the presence of gold nanoparticles. Gold nanoparticles were prepared by the citrate reduction of HAuCl₄ and characterized by transmission electron microscopy (TEM). Levofloxacin and Tb(III) ion form a fluorescence complex in aqueous solution, and its maximum emission wavelength was found at 545 nm. Optimal conditions for the formation of the levofloxacin–Tb(III) complexes were studied. Levofloxacin was detected by measuring the FL intensity, which increases linearly with the concentration of LVX in the range 6.2 × 10⁻¹⁰–2.6 × 10⁻⁸ mol/L. Recovery of the target analytes was > 96% with good quality parameters: linearity (r² > 0.996), limit of detection (LOD) and limit of quantification (LOQ) values 2.1 × 10⁻¹⁰ mol/L and 7.2 × 10⁻¹⁰ mol/L, and run‐to‐run and day‐to‐day precisions with relative standard deviations (RSDs) around 3%. Thus, the proposed method can be successfully applied to the routine determination of levofloxacin in pharmaceutical preparations. Copyright © 2011 John Wiley & Sons, Ltd.     

A Visualized Assay for Quercetin Based on the Formation of Silver–Gold Alloy Nanoparticles

A visualized assay for quercetin (QU) was first developed based on the formation of silver–gold alloy nanoparticles in this contribution. With the ability to reduce metal ions to metal substances, QU could reduce Ag⁺ absorbed on the surface of gold nanoparticles to metallic silver. The thickness of the formed Ag shell and the color change of the solution were proportional to the concentration of QU. Therefore, visualized detection of QU could be realized by studying the surface resonance plasmon absorption spectra of the analytical systems after addition of different concentration of QU. Under optimum conditions, trace amount of QU could be detected in the linear range 9.0?×?10^?7–1.0?×?10^?4 mol L^?1 with a detection limit of 6.5?×?10^?7 mol L^?1. The present assay was applied in the determination of QU in human serum and satisfactory results were obtained. This assay is simple, rapid, and cost-effective, and it is a powerful complement for the spectroscopy assays for QU. Also, it is the first visualized spectroscopic assay of QU until now.     

Electron microscopy of human interphase nuclei

Quantitative electron microscopy was used to analyze surface-spread, critical-point-dried human interphase nuclei and chromatin. The following information is presented: (1) Unstimulated interphase nuclei of lymphocytes from peripheral blood have a mean dry mass of 50.30×10^?12 g. The mean dry mass of stimulated nuclei of lymphocytes was determined to be 59.34×10^?12 g, a significant statistical difference from the unstimulated ones. (2) Mean diameter of chromatin fibers and mean fiber mass per micron were 199ű15% coefficient of variation (C.V.) and 5.95×10^?16g×29% C.V., respectively. (3) A line of regression of fiber mass on fiber diameter for 83 fibers indicated that a 200-Å fiber has a mass of 5.86×10^?16g/μ, or almost the same as the mean fiber mass of 5.95× 10^?16g/μ. (4) With the value 7×10^?12g for the DNA content of an unstimulated lymphocyte nucleus, a total length of 215 cm is calculated for the DNA double helix. When this length is compared to the mean length of chromatin fiber per nucleus (7.59 cm), a ratio of 28.3 to 1 results, which is called the DNA-packing ratio. (5) This DNA-packing ratio of 28.3 is reasonably close to the packing ratio of 26.9 suggested from model calculations for the second DNA supercoil in a 200-Å chromatin fiber.     

Toxicity of gold nanoparticles for plants in experimental aquatic system

Increased production and use of nanomaterials can lead to new types of pollution of the environment, including aquatic ecosystems. Pollution of the aqueous environment with nanoparticles can be a new type of pollution of the environment. This requires a more detailed study of the biological effects during exposure of nanoparticles on aquatic organisms. The interactions of gold nanoparticles (Au) with aquatic macrophytes Ceratophyllum demersum have been studied. Aquatic microcosms with these plants were used. Gold nanoparticles (Au) were added to the aqueous medium of C. demersum macrophyte containing microcosms. The state of the plants was then analyzed. Phytotoxicity of Au nanoparticles for aquatic macrophytes was shown for the first time. A new method of phytotoxicity detection was suggested and successfully approved. Phytotoxicity at a concentration of Au (in the form of nanoparticles) of 6 × 10^?6 M-1.8 × 10^?5 M was shown.     

Surface-functionalized gold nanoparticles mediate bacterial transformation: a nanobiotechnological approach

Transformation of bacteria is an important step in molecular biology. Viral and non-virus-based gene delivery techniques, including chemical/biological and physical approaches, have been applied to bacterial, mammalian and plant cells. E. coli is not competent to take up DNA; hence, different methods are used to incorporate plasmid DNA. A novel method has been developed using glutathione-functionalized gold nanoparticles to mediate transformation of plasmid DNA (pUC19) into E. coli DH5α that does not require the preparation of competent cells. The glutathione-functionalized gold nanoparticles acted as a vector and facilitated the entry of DNA into the host cell. The method also gave a higher transformation efficiency (4.2 × 10⁷/μg DNA) compared to 2.3 × 10⁵/μg DNA using the conventional CaCl₂-mediated method. It was also non-toxic to the bacterium making it suitable for biotechnological applications.     

Micronucleus assay in bovine lymphocytes after exposure to bisphenol A in vitro

Bisphenol A (BPA) [2,2-bis-(4-hydroxyphenyl) propane] is an important industrial agent, made by combining acetone and phenol, that is used extensively as a monomer in the production of polycarbonate plastics and as a precursor of epoxy resins. Micronucleus assays have served as an index of cytogenetic damage in in vivo and in vitro studies. We studied the genotoxic and cytotoxic effects of BPA on bovine peripheral lymphocytes in vitro. Lymphocyte cultures from two donors were exposed to four different concentrations of BPA (1?×?10^?4, 1?×?10^?5, 1?×?10^?6, and 1?×?10^?7 mol.L^?1) for 48 h. The highest concentration of BPA (1?×?10^?4 mol.L^?1) resulted in a significant increase in the number of micronuclei in comparison with the negative control (67.50?±?2.121/1,000 binucleated cells versus 36.0?±?5.657/1,000 binucleated cells in the DMSO control, P??=??0.018). BPA did not affect the nuclear division index at any treatment concentrations. The present results thus demonstrate a significant genotoxic effect by BPA on bovine peripheral lymphocytes in vitro, only at the highest concentration.     

Effect of gold nanoparticles on mouse spermatogenesis

The response of the mouse male germ cells exposed to gold nanoparticles (??2.5 nm) was studied. Our investigation demonstrates that treatment with Au nanoparticles for four days does not impair the architecture of the spermatogenic epithelium. Cytogenetic evaluation using micronucleus assay showed that gold nanoparticles can affect the chromosomes of early primary spermatocytes. However, gold nanoparticles did not induce chromosome abnormalities in spermatogonial stem cells. Further, the cauda epididymal sperm was isolated on the 14th day after treatment and was incubated in SDS solution (Na dodecyl sulphate) and then in a solution containing DTT (dithiothreitol) to induce nuclear chromatin decondensation. Observations showed that after four days of treatment of spermiogenic (postmeiotic) cells with gold nanoparticles the decondensation process had no differences from the control. On the contrary, in the experiment with the same cells and period of fixation but with a single exposure to gold nanoparticles, the number of mature gametes with totally decondensed nuclei reached 100% as opposed to 44% in the controls.     

A new spectrofluorimetric method for the determination of some tetracyclines based on their interfering effect on resonance fluorescence energy transfer

A rapid, simple, inexpensive and highly sensitive spectrofluorimetric method was developed for the determination of trace amounts of some tetracyclines (TCs), namely tetracycline hydrochloride (TCH), oxytetracycline hydrochloride (OTCH) and minocycline hydrochloride (MCH). Binding rhodamine B (RhB) to gold nanoparticles (Au NPs) resulted in quenching of the fluorescence of RhB by a resonance energy transfer (FRET) mechanism, with Au NPs as the energy acceptors. The presence of TCs caused the release of RhB molecules and recovered their fluorescence, and this was used as a basis for the quantitative determination of TCs. The reaction was monitored spectrofluorimetrically by measuring the increase in fluorescence of RhB at 572 nm starting 5 min after mixing the reagents in Tris buffer solution (pH 6.5). The effect of various experimental factors such as buffer type, pH, concentrations of the involved reagents and reaction time were studied to optimize the reaction conditions. Under optimum conditions, the calibration graphs were linear within the ranges 2.08 × 10^?9–1.04 × 10^?6 mol/L, 2.01 × 10^?9–1.00 × 10^?6 mol/L and 2.02 × 10^?9–1.01 × 10^?6 mol/L and detection limits (LODs) of 0.61 × 10^?9, 0.32 × 10^?9 and 0.66 × 10^?9 mol/L were calculated for TCH, OTCH and MCH, respectively, with corresponding percent relative standard deviations (%RSDs) of 1.18, 1.21 and 1.54 (n = 5). The method was successfully applied to the determination of TCs in drinking water, human urine, bovine milk and breast milk samples. Copyright © 2014 John Wiley & Sons, Ltd.     

A novel method for sensing of methimazole using gold nanoparticle‐catalyzed chemiluminescent reaction

Based on the inhibition effect of methimazole (MMI) on the reaction of luminol–H₂O₂ catalyzed by gold nanoparticles, a novel chemiluminescence (CL) method was developed for the determination of MMI. Under the optimum conditions, the relative CL intensity was linearly related to MMI concentration in the range from 5.0 × 10^?8 to 5.0 × 10^?5 mol L^?1. The detection limit was 1.6 × 10^?8 mol L^?1 (S/N = 3), and the RSD for 6.0 × 10^?6 mol L^?1 MMI was 4.83 (n = 11). This method has high sensitivity, wide linear range, inexpensive instrumentation and has been applied to detect MMI in pharmaceutical tablets and pig serum samples. Furthermore, a possible reaction mechanism is discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

Speckled SiO2@Au Core–Shell Particles as Surface Enhanced Raman Scattering Probes

Silica particles of ~800 nm size were functionalized using 3-amino propyl triethoxysilane molecules on which gold particles (~20 nm size) were deposited. The resulting particles appeared to form speckled SiO₂@Au core–shell particles. The surface roughness, along with hot spots, due to nanogaps between the gold nanoparticles was responsible for the enhancement of the Raman signal of crystal violet molecules by ~3.2?×?10⁷ and by ~1.42?×?10⁸ of single-wall carbon nanotubes. It has also been observed that the electromagnetic excitation near surface plasmon resonance (SPR) of core–shell particles is more effective than off resonance SPR excitation.     

Flow-injection chemiluminescence method for the determination of moxifloxacin in pharmaceutical tablets and human urine using silver nanoparticles sensitized calcein–KMnO<Subscript>4</Subscript> system

Silver nanoparticles have been synthesized and were utilized for the enhanced luminometric estimation of moxifloxacin antibiotic. During the experimental procedure, it was clearly found that the addition of silver nanoparticles intensifies the weak chemiluminescence signal intensity of calcein–KMnO₄ system by several folds. It was also obvious that the intensity enhancement was linearly proportional to the moxifloxacin concentration and this phenomenon was further utilized for the quantitative determination of target analyte. Effects of the different chemical variables during the experiment were studied to achieve best chemiluminescence signal. Under the optimized experimental parameters, the linear calibration graph was established over the moxifloxacin concentration range of 6.0 × 10^?8 M to 2.5 × 10^?6 M with coefficient of correlation (r ²) value 0.9998. The lower detection limit was found to be 5.6 × 10^?9 M. The percentage relative standard deviation calculated from five replicate chemiluminescence measurements was found to be 2.63 %. The developed chemiluminescence technique was successfully applied to the determination of moxifloxacin in tablet formulation and spiked human urine sample.     

A highly efficient and selective turn‐on fluorescent sensor for Hg2+, Ag+ and Ag nanoparticles based on a coumarin dithioate derivative

Based on chelation‐enhanced fluorescence, a new fluorescent coumarin derivative probe 3(1‐(7‐hydroxy‐4‐methylcoumarin)ethylidene)hydrazinecarbodithioate for Hg²⁺, Ag⁺ and Ag nanoparticles is reported. Fluorescent probe acts as a rapid and highly selective “off–on” fluorescent probe and fluorescence enhancement by factors 5 to12 times was observed upon selective complexation with Hg²⁺, Ag⁺ and Ag nanoparticles. The molar ratio plots indicated the formation of 1:1 complexes between Hg²⁺ and Ag⁺ with the probe. The linear response range covers a concentration range 0.1 × 10^–5–1.9 × 10^–5 mol/L, 0.1 × 10^–5–2.3 × 10^–5 mol/L and 0.146 × 10^–12–2.63 × 10^–12 mol/L for Hg²⁺, Ag⁺ and Ag nanoparticles, respectively. The interference effect of some anions and cations was also tested. Copyright © 2013 John Wiley & Sons, Ltd.     

Folate receptor-targeted multimodal fluorescence mesosilica nanoparticles for imaging,delivery palladium complex and in vitro G-quadruplex DNA interaction

New folic acid-conjugated mesoporous silica nanoparticles were synthesized. The effect of calcination at 400°C on the fluorescence characteristics of mesoporous silica nanoparticles were studied in this work. The formed carbon dots (CDs) from calcination were used as the source of fluorescence. 3-Aminopropyltriethoxysilane was then used to amine-functionalized the fluorescent surface of mesoporous silica nanoparticles. The amine fluorescence mesoporous silica nanoparticles (amine-FMSNs) were coupled with folic acid (FA) as the target ligand (FA-amine-FMSNs). A palladium complex was also synthesized and encapsulated in the FA-amine-FMSNs yielded fluorescent property with therapeutic effect. The in vitro release of an entrapped palladium complex from FA-amine-FMSNs was studied under physiological conditions. According to the cell viability assay on HeLa (positive FR) and Hep-G2 (negative FR) cells, the targeted delivery system inhibited the growth of positive FR with higher selectivity compared with negative FR cells. Also, the emission CDs were used for fluorescence microscopic imaging. To confirm anti-cancer activity of the palladium complex, the interaction between palladium complex and G-quadruplex DNA were investigated with multi-spectroscopic methods and molecular modeling. The molecular docking studies showed a partial intercalation mode with a 4.27 × 10⁵ M^?1 binding constant.     

Cell density dependent response of E. Coli cells to weak ELF magnetic fields

The effects of weak magnetic fields of extremely low frequency (ELF) on E. coli K12 AB1157 cells were studied by the method of anomalous viscosity time dependencies (AVTD). E. coli cells at different densities within a range of 5 × 10⁵–10⁹ cell/ml were exposed to ELF (sinusoidal, 30 μT peak, 15 min) at a frequency of 9 Hz. A transient effect with maximum 40–120 min after exposure was observed. Kinetics of the per-cell-normalised ELF effects fitted well to a Gaussian distribution for all densities during exposure. A maximum value of these kinetics and a time for this maximum were strongly dependent on the cell density during exposure. These data suggest a cell-to-cell interaction during response to ELF. Both dependencies had three regions close to a plateau within the ranges of 3 × 10⁵ − 2 × 10⁷ cell/ml, 4 × 10⁷ − 2 × 10⁸ cell/ml and 4 × 10⁸–10⁹ cell/ml and two rather sharp transitions between these plateaus. The effect reached a maximum value at a density of 4 × 10⁸ cell/ml. Practically no effect was observed at the lowest density of 3 × 10⁵ cell/ml. The data suggested that the ELF effect was mainly caused by a secondary rather than a primary reaction. The filtrates from exposed cells neither induced significant AVTD changes in unexposed cells nor increased the ELF effect when were added to cells before exposure. The data did not provide evidence for significant contribution of stable chemical messengers, but some unstable compounds such as radicals could be involved in the mechanism of cell-to-cell interaction during response to ELF. The results obtained were also in accordance with a model based on an re-emission of secondary photons during resonance fluorescence. Bioelectromagnetics 19:300–309, 1998. © 1998 Wiley-Liss, Inc.