Effects induced by η6-p-cymene ruthenium(II) complexes on Langmuir monolayers mimicking cancer and healthy cell membranes do not correlate with their toxicity.

The mechanism of chemotherapeutic action of Ru-based drugs involves plasma membrane disruption and valuable insights into this process may be gained using cell membrane models. The interactions of a series of cytotoxic η6-p-cymene ruthenium(II) complexes, [Ru(η6-p-cymene)P(3,5-C(CH3)3-C6H3)3Cl2] (1), [Ru(η6-p-cymene)P(3,5-CH3-C6H3)3Cl2] (2), [Ru(η6-p-cymene)P(4-CH3O-3,5-CH3-C6H2)3Cl2] (3), and [Ru(η6-p-cymene)P(4-CH3O-C6H4)3Cl2] (4), were examined using Langmuir monolayers as simplified healthy and cancerous outer leaflet plasma membrane models. The cancerous membrane (CM1 and CM2) models contained either 40 % 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 30 % cholesterol (Chol), 20 % 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 10 % 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS). Meanwhile, the healthy membrane (HM1 and HM2) models were composed of 60 % DPPC or DOPC, 30 % Chol and 10 % DPPE. The complexes affected surface pressure isotherms and decreased compressional moduli of cancerous and healthy membrane models, interacting with the monolayers headgroup and tails according to data from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). However, the effects did not correlate with the toxicity of the complexes to cancerous and healthy cells. Multidimensional projection technique showed that the complex (1) induced significant changes in the CM1 and HM1 monolayers, though it had the lowest cytotoxicity against cancer cells and is not toxic to healthy cells. Moreover, the most toxic complexes (2) and (4) were those that least affected CM2 and HM2 monolayers. The findings here support that the ruthenium complexes interact with lipids and cholesterol in cell membrane models, and their cytotoxic activities involve a multifaceted mode of action beyond membrane disruption.

Nanoconjugates based on a novel organic-inorganic hybrid silsesquioxane and gold nanoparticles as hemocompatible nanomaterials for promising biosensing applications.

A new hybrid organic-inorganic silsesquioxane material, 3-n-propyl(2-amino-4-methyl)pyridium chloride (SiAMPy+Cl-), was synthesized and successfully applied for the synthesis of stable nanoconjugates with gold nanoparticles (AuNPs-SiAMPy+). SiAMPy+Cl- was obtained through a simple sol-gel procedure by using chloropropyltrimetoxysilane and tetraethylorthosilicate as precursors and 2-amino-4-methylpyridine as the functionalizing agent. The resulting material was characterized by employing FTIR, XRD, and 1H-, 13C-, and 29Si-NMR spectroscopy. The synthesis of AuNPs-SiAMPy+ nanoconjugates was optimized through a 23 full factorial design. UV-VIS, FTIR, TEM, DLS, and ζ-potential measurements were used to characterize the nanoconjugates, which presented a spherical morphology with an average diameter of 5.8 nm. To investigate the existence of toxic effects of AuNPs-SiAMPy+ on blood cells, which is essential for their future biomedical applications, toxicity assays on human erythrocytes and leukocytes were performed. Interestingly, no cytotoxic effects were observed for both types of cells. The nanoconjugates were further applied in the construction of electrochemical immunosensing devices, aiming the detection of anti-Trypanosoma cruzi antibodies in serum as biomarkers of Chagas disease. The AuNPs-SiAMPy+ significantly enhanced the sensitivity of the biodevice, which was able to discriminate between anti-T. cruzi positive and negative serum samples. Thus, the AuNPs-SiAMPy+-based biosensor showed great potential to be used as a new tool to perform fast and accurate diagnosis of Chagas disease. The promising findings described herein strongly confirm the remarkable potential of SiAMPy+Cl- to obtain nanomaterials, which can present notable biomedical properties and applications.

Porphyran-capped silver nanoparticles as a promising antibacterial agent and electrode modifier for 5-fluorouracil electroanalysis.

In the present work, the green synthesis of silver nanoparticles (AgNPs) using the sulfated polysaccharide porphyran (PFR) as capping agent and d-glucose as reducing agent is described. PFR was extracted from red seaweed and characterized by employing 13C NMR and determination of total sugar, protein, and sulfate contents. The obtained AgNPs-PFR were characterized by using UV-VIS spectroscopy, zeta potential determination, FESEM, and TEM, which demonstrated that PFR was effective at capping the AgNPs, yielding stable suspensions. The AgNPs-PFR presented good antimicrobial properties against Gram-positive and Gram-negative bacterial strains (Staphylococcus aureus and Escherichia coli, respectively). The AgNPs-PFR were also employed as the modifier of carbon paste electrodes, which were efficiently applied as electrochemical sensors for the determination of 5-fluorouracil (5-FU), an important anticancer drug, through square wave voltammetry (SWV). The AgNPs-PFR improved the electrochemical properties of the electrodes, and enhanced their electroanalytical performance. The developed sensing device presented detection and quantification limits equal to 10.7 and 35.8 µmol L-1, respectively, towards 5-FU determination. The proposed electrochemical sensor successfully quantified 5-FU in a real pharmaceutical formulation, confirming its potential as a new promising analytical detection tool for 5-FU quality control purposes.

Electrochemical, mechanistic, and DFT studies of amine derived diphosphines containing Ru(II)-cymene complexes with potent in vitro cytotoxic activity against HeLa and triple-negative breast cancer cells MDA-MB-231.

Complexes with general formula [RuCl(η6-p-cymene)(P-NR-P)]X (R = CH2Py (Py = pyridine) - [1a]+, CH2Ph (Ph = phenyl) - [1b]+, Ph - [1c] and p-tol (p-tol = p-tolyl) - [1d]+; X = PF6- or BF4-) were evaluated as cytotoxic agents against two cancer cell lines (HeLa and MDA-MB-231). All metal complexes are active in the range of concentrations tested (up to 100 µmol L-1). The IC50 (µmol L-1) values for the metal complexes are lower than that found for cisplatin. The activities are up to 6- and 15-fold higher than cisplatin for HeLa and MDA-MB-231 cancer cell lines, respectively. Studies of DNA binding and DNA cleavage were performed. DNA binding studies revealed a modest hypochromic shift in the metal complexes electronic spectra, indicating a weak interaction with Kb values in the range of 1.7 × 103-1.6 × 104. Although the cleavage tests revealed that in the dark DNA is not a biological target for these metal complexes, upon blue light irradiation they are activated causing DNA cleavage. Electrochemical studies showed the presence of two independent redox processes, one attributed to the oxidation process of Ru2+ → Ru3+ (EC process) and the other one to the reduction of Ru2+ → Ru1+, which is further reduced to Ru0 (ECE mechanism). In both processes, coupled chemical reactions were observed. DFT calculations were performed to support the electrochemical/chemical behavior of the complexes. The reactivity of complex [1b]BF4 with CH3CN was evaluated and two complexes were isolated [2b]BF4 and [3b]BF4. The complex mer-[RuCl(CH3CN)3(P-NCH2Ph-P)]BF4 ([2b]BF4) was isolated after refluxing the precursor [1b]BF4 in CH3CN. Isomerization of [2b]BF4 in CH3CN resulted in the formation of fac-[RuCl(CH3CN)3(P-NCH2Ph-P)]BF4. An attempt to isolate the fac-isomer by adding diethyl ether was unsuccessful, and the complex [3b]BF4 was observed as the major component. The complex [Ru2(µ-Cl3)(CH3CN)2(P-NCH2Ph-P)2]BF4 ([3b]BF4) proved to be very stable and can be obtained from both the mer- and the fac-isomers. The molecular structures of [1b]BF4 and [3b]BF4 were solved by single-crystal X-ray diffraction.

Distinguishing Activities in the Photodynamic Arsenals of the Pigmented Ciliates Blepharisma sinuosum Sawaya, 1940 and Blepharisma japonicum Suzuki, 1954 (Ciliophora: Heterotrichea).

Blepharismins are photodynamic hypericin-like dianthrones produced as a variable pigment blend in Blepharisma ciliates and mostly studied in the Afro-Asiatic Blepharisma japonicum. The present work describes the bioactivity of pigments from the Brazilian Blepharisma sinuosum. Comparative analyses showed that the pigments from both species can trigger photo-induced modifications in phospholipids, but different redox properties and biological activities were assigned for each pigment blend. Stronger activities were detected for B. sinuosum pigments, with the lethal concentration LC50 10 × lower than B. japonicum pigments in light-irradiated tests against Bacillus cereus and less than half for treatments on the human HeLa tumor cells. HPLC showed B. sinuosum producing a simpler pigment blend, mostly with the blepharismin-C (~ 70%) and blepharismin-E (~ 30%) types. Each blepharismin engaged a specific dose-response profile on sensitive cells. The blepharismin-B and blepharismin-C were the most toxic pigments, showing LC50  ~ 2.5-3.0 µm and ~ 100 µm on B. cereus and HeLa cells, respectively, after illumination. Similarity clustering analysis compiling the bioactivity data revealed two groups of blepharismins: the most active, B and C, and the less active, A, D and E. The B. sinuosum pigment blend includes one representative of each clade. Functional and medical implications are discussed.

Bulky 2,6-disubstituted aryl siloxanes and a disilanamine.

The crystal structures of 5-bromo-1,3-di-tert-butyl-2-[(tri-methyl-sil-yl)-oxy]benzene, C17H29BrOSi, (I), 1,3-di-tert-butyl-2-[(tri-methyl-sil-yl)-oxy]benzene, C17H30OSi, (II), and N-(2,6-diiso-propyl-phen-yl)-1,1,1-trimethyl-N-(tri-methyl-sil-yl)silanamine, C18H35NSi2, (III), are reported. Compound (I) crystallizes in space group P21/c with Z' = 1, (II) in Pnma with Z' = 0.5 and (III) in Cmcm with Z' = 0.25. Consequently, the mol-ecules of (II) are constrained by m and those of (III) by m2m site symmetries. Despite this, both (I) and (II) are distorted towards mild boat conformations, as is typical of 2,6-di-tert-butyl-substituted phenyl compounds, reflecting the high local steric pressure of the flanking alkyl groups. Compound (III) by contrast is planar and symmetric, and this lack of distortion is compatible with the lower steric pressure of the flanking 2,6-diisopropyl substituents.

The antibacterial activity of p-tert-butylcalix[6]arene and its effect on a membrane model: molecular dynamics and Langmuir film studies.

The antibacterial activity of a calixarene derivative, p-tert-butylcalix[6]arene (Calix6), was assessed and was shown not to inhibit the growth of E. coli, S. aureus and B. subtilis bacteria. With the aim of gaining more insights into the absence of antibacterial activity of Calix6, the interaction of this derivative with DPPG, a bacterial cell membrane lipid, was studied. Langmuir monolayers were used as the model membrane. Pure DPPG and pure Calix6 monolayers, as well as binary DPPG:Calix6 mixtures were studied using surface pressure measurements, compressional modulus, Brewster angle and fluorescence microscopies, ellipsometry, polarization-modulation infrared reflection absorption spectroscopy and molecular dynamics simulations. Thermodynamic properties of the mixed monolayers were additionally calculated using thermodynamic parameters. The analysis of isotherms showed that Calix6 significantly affects the DPPG monolayers, modifying the isotherm profile and increasing the molecular area, in agreement with the molecular dynamics simulations. The presence of Calix6 in the mixed monolayers decreased the interfacial elasticity, indicating that calixarene disrupts the strong intermolecular interactions of DPPG hindering its organization into a compact arrangement. At low molar ratios of Calix6, the DPPG:Calix6 interactions are preferentially attractive, due to the interactions between the hydrophobic tails of DPPG and the tert-butyl groups of Calix6. Increasing the proportion of calixarene generates repulsive interactions. Calix6 significantly affects the hydrophobic tail organization, which was confirmed by PM-IRRAS measurements. Calix6 appears to be expelled from the mixed films at a biologically relevant surface pressure, π = 30 mN m-1, indicating a low interaction with the cell membrane model related to the absence of antibacterial activity.

A sensitive label-free impedimetric DNA biosensor based on silsesquioxane-functionalized gold nanoparticles for Zika Virus detection.

Zika virus (ZIKV) has recently become a global health challenge due to its rapid geographical expansion, since it is associated with serious neurological anomalies such as Guillain-Barré syndrome and microcephaly. Currently, the techniques for ZIKV diagnosis require labor-intensive, expensive and lengthy tests using sophisticated equipment. Moreover, false-positive or false-negative results can occur. In the present work, a DNA biosensor to detect ZIKV in real human serum samples was developed using an oxidized glassy carbon electrode (ox-GCE) modified with silsesquioxane-functionalized gold nanoparticles (AuNPs-SiPy). This nanohybrid was characterized by UV-Vis, FTIR and Raman spectroscopies, DLS, and XRD. The conditions for the immobilization of a ZIKV ssDNA probe on the electrode surface (ox-GCE-[AuNPs-SiPy]) were optimized by univariate and multivariate analysis. The optimized biosensor was characterized by CV, EIS and AFM experiments. The ZIKV target recognition was based on the variation of the charge transfer resistance (ΔRct) of the redox marker ([Fe(CN)6]3-/4-) used and the roughness (Rq) of the electrode surface. The proposed biosensor presented a LOD of 0.82 pmol L-1, with a linear range of 1.0 x10-12 - 1.0 x10-6 mol L-1. Moreover, the reported device showed a suitable stability and satisfactory sensitivity and selectivity to quantify ZIKV in human serum samples, which suggests its promising clinical applications for the early diagnosis of ZIKV-associated pathologies.

Label-free impedimetric immunosensor based on arginine-functionalized gold nanoparticles for detection of DHEAS, a biomarker of pediatric adrenocortical carcinoma.

Pediatric adrenocortical carcinoma (pACC) is a rare and aggressive malignancy of high occurrence in Southern Brazil. pACC is characterized by the usual overproduction of dehydroepiandrosterone sulfate (DHEAS), whose detection in serum or plasma can be effective to the early diagnosis of the disease. Therefore, the present paper reports, for the first time, the construction and application of a label-free impedimetric immunosensor to detect DHEAS, which was based on the modification of an oxidized glassy carbon electrode with arginine-functionalized gold nanoparticles (AuNPs-ARG) and anti-DHEA IgM antibodies (ox-GCE/AuNPs-ARG/IgM). AuNPs-ARG was synthesized by a green route, and characterized by UV-VIS spectroscopy, FTIR, TEM, DLS, and XRD. The construction of ox-GCE/AuNPs-ARG/IgM was optimized through factorial design and response surface methodology. Cyclic voltammetry and electrochemical impedance spectroscopy measurements were employed to characterize the optimized immunosensor. The DHEAS detection principle was based on the variation of charge transfer resistance (∆Rct) relative to the Fe(CN)64-/3- electrochemical probe after immunoassays in the presence of the biomarker. A linear relationship between ∆Rct and DHEAS concentration was verified in the range from 10.0 to 110.0 µg dL-1, with a LOD of 7.4 µg dL-1. Besides the good sensitivity, the immunosensor displayed accuracy, stability, and specificity to detect DHEAS. The promising analytical performance of ox-GCE/AuNPs-ARG/IgM was confirmed by quantifying DHEAS in real patient plasma samples, with results that were comparable to the reference chemiluminescence assay. Our results suggest that the presented immunosensor can find clinical applications in the early diagnosis of pACC and to monitor DHEAS levels in other adrenal pathologies.

Characterization and Cytotoxic Evaluation of Silver and Gold Nanoparticles Produced with Aqueous Extract of Lavandula dentata L. in Relation to K-562 Cell Line

Abstract Metallic nanoparticles have great potential as a chemotherapeutic agent. The aim of this study was to develop and characterize silver and gold nanoparticles using a simple method, as well as evaluating the potential cytotoxic activity in relation to the K-562 cell line. For the synthesis, a solution containing the metallic ions was subjected to magnetic stirring with the aqueous extract of Lavandula dentata L. and a change of colour was observed. With the data obtained from the analyses we concluded that the nanoparticles were successfully obtained by a simple and green method using the aqueous extract of L. dentata. The obtained nanoparticles presented a reduced size, a low level of polydispersion, and a homogenous spherical shape. The nanoparticles presented intense and characteristic diffraction peaks, which could be correlated to the planes of the centred cubic structure of the silver and gold. The two formulations presented predominantly crystalline characteristics. The infrared analysis suggested that the amides and alcohols present in the samples may have been responsible for the reduction and limitation of the size and dispersion of the silver and gold nanoparticles. The cytotoxic assay showed that the nanoparticles demonstrated great potential to reduce the cell viability of the K-562 cell line, especially the gold nanoparticles.

An experimental and theoretical study of the aggregate structure of calix[6]arenes in Langmuir films at the water/air interface.

In this paper, the aggregate formation of para-tert-butylcalix[6]arene molecules (Calix6) in dimeric structures was investigated at the water/air interface using experimental and theoretical studies. A specific orientation for such Calix6 molecules was observed with an average area of 133 Å(2), which corresponds to a flat-on orientation with the OH groups parallel to the interface. By varying the pressure on the Calix6 monolayer, the molecules tend to organize at the water/air interface and subsequently, at higher pressures, aggregates were formed atop the monolayer as cluster structures. Morphological characterization by the Brewster Angle Microscopy technique showed the formation of larger domains at lower pressures. Based on such experimental evidence, molecular dynamics (MD) simulations were performed to investigate possible dimeric structures for aggregated Calix6 molecules, which are localized at the water/air interface, where one molecule remains in the water phase and the other remains in the air phase. By increasing surface pressure, experimental and theoretical results corroborate the intermolecular interactions among Calix6 molecules. These results are relevant because a dimeric structure has a molecular cavity, which is a candidate for host-guest chemistry, an ion receptor or a drug-delivery system.

Interaction of para-tert-butylcalix[6]arene molecules in Langmuir films with cadmium ions and their effects on molecular conformation and surface potential.

In this paper, we employ the surface-specific polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) and sum-frequency generation (SFG) methods with surface pressure and surface potential isotherms to determine the organization of p-tert-butylcalix[6]arene molecules and their interaction with Cd(2+) ions in Langmuir monolayers. The area per molecule was estimated to be 135 Å(2), which corresponds to the Calix6 axis perpendicular to the air-water interface with most OH groups parallel to the interface. This area is larger than predicted by molecular modeling with quantum chemical calculations with a PM3 Hamiltonian (109 Å(2)), which is ascribed to the repulsion between Calix6 molecules. The incorporation of Cd(2+) ions in the subphase leads to drastic changes in the dipole moment contribution of the monolayer surface potential. Rather than increasing with incorporation of Cd(2+) ions owing to a decrease in the negative double-layer potential, the measured surface potential decreased monotonically with increasing ion concentration. This unexpected result was ascribed to a strong interaction with Cd(2+) ions that induced the calyx of the molecule to adopt a more open conformation at the air/water interface and affected the orientation of hydration water molecules, according to the SFG data. This finding allows us to understand the reason why the Gouy-Chapman model fails to explain surface potential results for subphases containing divalent or trivalent ions, and may be relevant for the application of calixarenes in sensing.

Silsesquioxane as a new building block material for modified electrodes fabrication and application as neurotransmitters sensors.

Nanostructured films comprising a 3-n-propylpyridinium silsesquioxane polymer (designated as SiPy+Cl-) and copper (II) tetrasulfophthalocyanine (CuTsPc) were produced using the Layer-by-Layer technique (LbL). To our knowledge this is the first report on the use of silsesquioxane derivative polymers as building blocks for nanostructured thin films fabrication. Deposition of the multilayers were monitored by UV-Vis spectroscopy revealing the linear increment in the absorbance of the Q-band from CuTsPc at 617 nm with the number of SiPy+Cl-/CuTsPc or CuTsPc/SiPy+Cl-bilayers. FTIR analyses showed that specific interactions between SiPy+Cl- and CuTsPc occurred between SO3- groups of tetrasulfophthalocyanine and the pyridinium groups of the polycation. Morphological studies were carried out using the AFM technique, which showed that the roughness and thickness of the films increase with the number of bilayers. The films displayed electroactivity and were employed to detection of dopamine (DA) and ascorbic acid (AA) using cyclic voltammetry, at concentrations ranging from 1.96 x 10(-4) to 1.31 x 10(-3) molL(-1). The number and the sequence of bilayers deposition influenced the electrochemical response in presence of DA and AA. Using differential pulse technique, films comprising SiPy+/-/CuTsPc were able to distinguish between DA and ascorbic acid (AA), with a potential difference of approximately with 500 mV, in the concentration range of 9.0 x 10(-5) to 2.0 x 10(-4) molL(-1), in pH 3.0.

Langmuir and Langmuir-Blodgett films containing a porphyrin-ruthenium complex.

The fabrication of supramolecular structures from the tetraruthenated porphyrin-containing phosphines, {TPyP[RuCl3(dppb)]4}, RuTPyP, is demonstrated with Langmuir and Langmuir-Blodgett films. The surface pressure-molecular area isotherms (pi-A) point to an edge-on arrangement for the RuTPyP molecules in the condensed state. Weak aggregation in the Langmuir films was indicated by non-zero surface potentials at large areas per molecule and a slight red shift in the ultraviolet-visible absorption spectrum in comparison to the spectrum in solution. Further aggregation occurs in the Z-type Langmuir-Blodgett films, which was confirmed with ultraviolet-visible spectroscopy of the deposited films. Fourier transform infrared and Raman spectroscopic data for powder and Langmuir-Blodgett films indicate that the RuTPyP molecules are chemically stable in Langmuir-Blodgett films regardless of the contact with water during film fabrication. The nanostructured nature of the Langmuir-Blodgett films was manifested in cyclic voltammetry due to the high sensitivity of the metallic centers in RuTPyP. Electrodes modified with Langmuir-Blodgett films exhibit an anodic peak at 100 mV and a cathodic peak at 7 mV, which is assigned to RuIII/RuII redox processes. Furthermore, Langmuir-Blodgett films from RuTPyP showed electrocatalytic activity for oxidation of benzyl alcohol, illustrated by a large shift of 100 mV in the anodic peak at 400 mV, while electropolymerized and cast films of the same compound displayed smaller and no activities, respectively.

High-performance taste sensor made from Langmuir-Blodgett films of conducting polymers and a ruthenium complex.

A sensor array made up of nanostructured Langmuir-Blodgett (LB) films is used as an electronic tongue capable of identifying sucrose, quinine, NaCl, and HCl at the parts-per-billion (ppb) level, being in some cases 3 orders of magnitude below the human threshold. The sensing units comprise LB films from conducting polymers and a ruthenium complex transferred onto gold interdigitated electrodes. Impedance spectroscopy is used as the principle of detection, and the importance of using nanostructured films is confirmed by comparing results from LB films with those obtained from cast films.