Shape-Driven Response of Gold Nanoparticles to X-rays.

Radiotherapy (RT) involves delivering X-ray beams to the tumor site to trigger DNA damage. In this approach, it is fundamental to preserve healthy cells and to confine the X-ray beam only to the malignant cells. The integration of gold nanoparticles (AuNPs) in the X-ray methodology could be considered a powerful tool to improve the efficacy of RT. Indeed, AuNPs have proven to be excellent allies in contrasting tumor pathology upon RT due to their high photoelectric absorption coefficient and unique physiochemical properties. However, an analysis of their physical and morphological reaction to X-ray exposure is necessary to fully understand the AuNPs' behavior upon irradiation before treating the cells, since there are currently no studies on the evaluation of potential NP morphological changes upon specific irradiations. In this work, we synthesized two differently shaped AuNPs adopting two different techniques to achieve either spherical or star-shaped AuNPs. The spherical AuNPs were obtained with the Turkevich-Frens method, while the star-shaped AuNPs (AuNSs) involved a seed-mediated approach. We then characterized all AuNPs with Transmission Electron Microscopy (TEM), Uv-Vis spectroscopy, Dynamic Light Scattering (DLS), zeta potential and Fourier Transform Infrared (FTIR) spectroscopy. The next step involved the treatment of AuNPs with two different doses of X-radiation commonly used in RT, namely 1.8 Gy and 2 Gy, respectively. Following the X-rays' exposure, the AuNPs were further characterized to investigate their possible physicochemical and morphological alterations induced with the X-rays. We found that AuNPs do not undergo any alteration, concluding that they can be safely used in RT treatments. Lastly, the actin rearrangements of THP-1 monocytes treated with AuNPs were also assessed in terms of coherency. This is a key proof to evaluate the possible activation of an immune response, which still represents a big limitation for the clinical translation of NPs.

A simple strategy based on ATR-FTIR difference spectroscopy to monitor substrate intake and metabolite release by growing bacteria.

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) difference spectroscopy has been employed for a variety of applications spanning from reaction mechanisms analysis to interface phenomena assessment. This technique is based on the detection of spectral changes induced by the chemical modification of the original sample. In the present study, we highlight the potential of the ATR-FTIR difference approach in the field of microbial biochemistry and biotechnology, reporting on the identification of main soluble species consumed and released by growing bacteria during the biohydrogen production process. Specifically, the mid-infrared spectrum of a model culture broth, composed of glucose, malt extract and yeast extract, was used as background to acquire the FTIR difference spectrum of the same broth as modified by Enterobacter aerogenes metabolism. The analysis of difference signals revealed that only glucose is degraded during hydrogen evolution in anaerobic conditions, while ethanol and 2,3-butanediol are the main soluble metabolites released with H2. This fast and easy analytical approach can therefore represent a sustainable strategy to screen different bacterial strains and to select raw and waste materials to be employed in the field of biofuel production.

Polymer Encapsulated Liposomes for Oral Co-Delivery of Curcumin and Hydroxytyrosol.

Curcumin (Cur) is a hydrophobic polyphenol from the rhizome of Curcuma spp., while hydroxytyrosol (HT) is a water-soluble polyphenol from Olea europaea. Both show outstanding antioxidant properties but suffer from scarce bioavailability and low stability in biological fluids. In this work, the co-encapsulation of Cur and HT into liposomes was realized, and the liposomal formulation was improved using polymers to increase their survival in the gastrointestinal tract. Liposomes with different compositions were formulated: Type 1, composed of phospholipids and cholesterol; Type 2, also with a PEG coating; and Type 3 providing an additional shell of Eudragit® S100, a gastro-resistant polymer. Samples were characterized in terms of size, morphology, ζ-potential, encapsulation efficiency, and loading capacity. All samples were subjected to a simulated in vitro digestion and their stability was investigated. The Eudragit®S100 coating demonstrated prevention of early releases of HT in the mouth and gastric phases, while the PEG shell reduced bile salts and pancreatin effects during the intestinal digestion. In vitro antioxidant activity showed a cumulative effect for Cur and HT loaded in vesicles. Finally, liposomes with HT concentrations up to 40 µM and Cur up to 4.7 µM, alone or in combination, did not show cytotoxicity against Caco-2 cells.

Liposomes containing nanoparticles: preparation and applications.

The impetuous development of nanotechnology over the past two decades has enabled the production of a plethora of nanomaterials with outstanding optical, magnetic, electrical, catalytic and mechanical properties. The versatility of these materials attracted attention from the very beginning in the most disparate sectors of science and technology. The application of nanomaterials in the biological and biomedical fields soon benefited from the interaction with liposomes, which increased their biocompatibility and biostability. Liposomes indeed are versatile self-assembling supramolecular (nano)structures constituted of an aqueous core enclosed by a lipid bilayer, able to host hydrophobic and hydrophilic cargo, and with superior biocompatibility and great similarity with the biological membranes. The result is the construction of hybrid nanoscale architectures, in which nanoparticles (NPs) are allocated either in the aqueous core, in the palisade of the lipid bilayer or on the outer surface of the vesicles. In the first part of this review, the principal methods for the preparation of NP-loaded liposomes are carefully illustrated in a tutorial manner. In the second part, an overview of the great potentialities deriving from the conjugation of liposomes with NPs is presented. In each paragraph, the main characteristics of the most notable classes of NPs, the related issues, and the advantages arising from their association with liposomes are shown. Here, the most significant research works in literature for each kind of system are presented.

Chemical and morphological effects of the contraceptive hormone 17 α-ethynylestradiol on fluid lipid membranes.

The lack of studies involving the effects in human health associated with the chronic ingestion of pollutants lead to the path of investigating the action of these compounds in cell membrane models. We demonstrated the interaction (causes and consequences) of the hormone 17 α-ethinylestradiol (EE2) with lipid monolayers (prepared as Langmuir films) and bilayers prepared as small unilamellar vesicles (SUVs) and giant unilamellar vesicles (GUVs). Both fluidity and majority chemical composition of real plasma cell membrane were guaranteed using the phospholipid 1-palmitoil-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Surface pressure-mean molecular area (π-A) isotherms and PM-IRRAS measurements highlighted the strong interaction of EE2 with POPC monolayers, leading the hormone to remain at the air/water interface and promoting its penetration into the phospholipid hydrophobic chains. In the case of bilayers, the entrance of the hormone inside the SUV is likely facilitated by their high curvature. In GUVs, EE2 was responsible for changes in the spherical shape, forming structures like buds and lipid protrusions. The set of results indicates the strong effects of EE2 on fluid membranes, which is an important feature to predict its damage in human cells.

Nanocellulose/Fullerene Hybrid Films Assembled at the Air/Water Interface as Promising Functional Materials for Photo-electrocatalysis.

Cellulose nanomaterials have been widely investigated in the last decade, unveiling attractive properties for emerging applications. The ability of sulfated cellulose nanocrystals (CNCs) to guide the supramolecular organization of amphiphilic fullerene derivatives at the air/water interface has been recently highlighted. Here, we further investigated the assembly of Langmuir hybrid films that are based on the electrostatic interaction between cationic fulleropyrrolidines deposited at the air/water interface and anionic CNCs dispersed in the subphase, assessing the influence of additional negatively charged species that are dissolved in the water phase. By means of isotherm acquisition and spectroscopic measurements, we demonstrated that a tetra-sulfonated porphyrin, which was introduced in the subphase as anionic competitor, strongly inhibited the binding of CNCs to the floating fullerene layer. Nevertheless, despite the strong inhibition by anionic molecules, the mutual interaction between fulleropyrrolidines at the interface and the CNCs led to the assembly of robust hybrid films, which could be efficiently transferred onto solid substrates. Interestingly, ITO-electrodes that were modified with five-layer hybrid films exhibited enhanced electrical capacitance and produced anodic photocurrents at 0.4 V vs Ag/AgCl, whose intensity (230 nA/cm2) proved to be four times higher than the one that was observed with the sole fullerene derivative (60 nA/cm2).

Tomato Oil Encapsulation by α-, ß-, and γ-Cyclodextrins: A Comparative Study on the Formation of Supramolecular Structures, Antioxidant Activity, and Carotenoid Stability.

Cyclodextrins (CDs) are oligosaccharides, comprising 6 (α), 7 (ß), or 8 (γ) glucose residues, used to prepare oil-in-water emulsions and improve oil stability towards degradation. In this research, the aptitude of α-, ß-, and γ-CDs to form complexes with a supercritical CO2 extracted lycopene-rich tomato oil (TO) was comparatively assessed. TO/CD emulsions and the resulting freeze-dried powders were characterized by microscopy, Fourier transform infrared-attenuated total reflection (FTIR-ATR), and differential scanning calorimetry (DSC), as well as for their antioxidant activity. Furthermore, carotenoid stability was monitored for 90 days at 25 and 4 °C. Confocal and SEM microscopy revealed morphological differences among samples. α- and ß-CDs spontaneously associated into microcrystals assembling in thin spherical shells (cyclodextrinosomes, Ø ≈ 27 µm) at the oil/water interface. Much smaller (Ø ≈ 9 µm) aggregates were occasionally observed with γ-CDs, but most TO droplets appeared "naked". FTIR and DSC spectra indicated that most CDs did not participate in TO complex formation, nevertheless structurally different interfacial complexes were formed. The trolox equivalent antioxidant capacity (TEAC) activity of emulsions and powders highlighted better performances of α- and ß-CDs as hydrophobic antioxidants-dispersing agents across aqueous media. Regardless of CDs type, low temperature slowed down carotenoid degradation in all samples, except all-[E]-lycopene, which does not appear efficiently protected by any CD type in the long storage period.

Phosphate Modified Screen Printed Electrodes by LIFT Treatment for Glucose Detection.

The design of new materials as active layers is important for electrochemical sensor and biosensor development. Among the techniques for the modification and functionalization of electrodes, the laser induced forward transfer (LIFT) has emerged as a powerful physisorption method for the deposition of various materials (even labile materials like enzymes) that results in intimate and stable contact with target surface. In this work, Pt, Au, and glassy carbon screen printed electrodes (SPEs) treated by LIFT with phosphate buffer have been characterized by scanning electron microscopy and atomic force microscopy to reveal a flattening effect of all surfaces. The electrochemical characterization by cyclic voltammetry shows significant differences depending on the electrode material. The electroactivity of Au is reduced while that of glassy carbon and Pt is greatly enhanced. In particular, the electrochemical behavior of a phosphate LIFT treated Pt showed a marked enrichment of hydrogen adsorbed layer, suggesting an elevated electrocatalytic activity towards glucose oxidation. When Pt electrodes modified in this way were used as an effective glucose sensor, a 1⁻10 mM linear response and a 10 µM detection limit were obtained. A possible role of phosphate that was securely immobilized on a Pt surface, as evidenced by XPS analysis, enhancing the glucose electrooxidation is discussed.

Selective Targeting of Proteins by Hybrid Polyoxometalates: Interaction Between a Bis-Biotinylated Hybrid Conjugate and Avidin.

The Keggin-type polyoxometalate [γ-SiW10O36]8- was covalently modified to obtain a bis-biotinylated conjugate able to bind avidin. Spectroscopic studies such as UV-vis, fluorimetry, circular dichroism, coupled to surface plasmon resonance technique were used to highlight the unique interplay of supramolecular interactions between the homotetrameric protein and the bis-functionalized polyanion. In particular, the dual recognition mechanism of the avidin encompasses (i) a complementary electrostatic association between the anionic surface of the polyoxotungstate and each positively charged avidin subunit and (ii) specific host-guest interactions between each biotinylated arm and a corresponding pocket on the tetramer subunits. The assembly exhibits peroxidase-like reactivity and it was used in aqueous solution for L-methionine methyl ester oxidation by H2O2. The recognition phenomenon was then exploited for the preparation of layer-by-layer films, whose structural evolution was monitored in situ by ATR-FTIR spectroscopy. Finally, cell tracking studies were performed by exploiting the specific interactions with a labeled streptavidin.

An SPR based immunoassay for the sensitive detection of the soluble epithelial marker E-cadherin.

Protein biomarkers are important diagnostic tools for cancer and several other diseases. To be validated in a clinical context, a biomarker should satisfy some requirements including the ability to provide reliable information on a pathological state by measuring its expression levels. In parallel, the development of an approach capable of detecting biomarkers with high sensitivity and specificity would be ideally suited for clinical applications. Here, we performed an immune-based label free assay using Surface Plasmon Resonance (SPR)-based detection of the soluble form of E-cadherin, a cell-cell contact protein that is involved in the maintaining of tissue integrity. With this approach, we obtained a specific and quantitative detection of E-cadherin from a few hundred microliters of serum of breast cancer patients by obtaining a 10-fold enhancement in the detection limit over a traditional colorimetric ELISA.

Encapsulation of Curcumin-Loaded Liposomes for Colonic Drug Delivery in a pH-Responsive Polymer Cluster Using a pH-Driven and Organic Solvent-Free Process.

The present study aimed to develop and optimize liposome formulation for the colonic delivery of biologically active compounds. A strategy to facilitate such targeting is to formulate liposomes with a polymer coating sensitive to the pH shifts in the gastrointestinal tract. To this end, liposomes encapsulating curcumin-chosen as the biologically active compound model-and coated with the pH-responsive polymer Eudragit S100 were prepared and characterized. Curcumin was encapsulated into small unilamellar vesicles (SUVs) by the micelle-to-vesicle transition method (MVT) in a simple and organic solvent-free way. Curcumin-loaded liposomes were coated with Eudragit S100 by a fast and easily scalable pH-driven method. The prepared liposomes were evaluated for size, surface morphology, entrapment efficiency, stability, in vitro drug release, and curcumin antioxidant activity. In particular, curcumin-loaded liposomes displayed size lower than 100 nm, encapsulation efficiency of 98%, high stability at both 4 °C and 25 °C, high in vitro antioxidant activity, and a cumulative release that was completed within 200 min. A good Eudragit S100 coating which did not alter the properties of the curcumin-loaded liposomes was obtained. The present work therefore provides a fast and solvent-free method to prepare pH-responsive polymer-coated liposomes for the colonic delivery of biologically active compounds.

Modeling the microscopic electrical properties of thrombin binding aptamer (TBA) for label-free biosensors.

Aptamers are chemically produced oligonucleotides, able to bind a variety of targets such as drugs, proteins and pathogens with high sensitivity and selectivity. Therefore, aptamers are largely employed for producing label-free biosensors (aptasensors), with significant applications in diagnostics and drug delivery. In particular, the anti-thrombin aptamers are biomolecules of high interest for clinical use, because of their ability to recognize and bind the thrombin enzyme. Among them, the DNA 15-mer aptamer (TBA), has been widely explored around the possibility of using it in aptasensors. This paper proposes a microscopic model of the electrical properties of TBA and of the aptamer-thrombin complex, combining information from both structure and function, following the issues addressed in an emerging branch of electronics known as proteotronics. The theoretical results are compared and validated with measurements reported in the literature. Finally, the model suggests resistance measurements as a novel tool for testing aptamer-target affinity.

Luminescent CdSe@ZnS nanocrystals embedded in liposomes: a cytotoxicity study in HeLa cells.

The use of fluorescent nanocrystals (NCs) as probes for bioimaging applications has emerged as an advantageous alternative to conventional organic fluorescent dyes. Therefore their toxicological evaluation and intracellular delivery are currently a primary field of research. In this work, hydrophobic and highly fluorescent CdSe@ZnS NCs were encapsulated into the lipid bilayer of liposomes by the micelle-to-vesicle transition (MVT) method. The obtained aqueous NC-liposome suspensions preserved the spectroscopic characteristics of the native NCs. A systematic study of the in vitro toxicological effect on HeLa cells of these red emitting NC-liposomes was then carried out and compared to that of empty liposomes. By using liposomes of different phospholipid composition, we evaluated the effect of the lipid carrier on the cytotoxicity towards HeLa cells. Surprisingly, a cell proliferation and death study along with the MTT test on HeLa cells treated with NC-liposomes have shown that the toxic effects of NCs, at concentrations up to 20 nM, are negligible compared to those of the lipid carrier, especially when this is constituted by the cationic phospholipid DOTAP. In particular, obtained data suggest that DOTAP has a dose- and time-dependent toxic effect on HeLa cells. In contrast, the addition of PEG to the liposomes does not alter significantly the viability of the cells. In addition, the ability of NC-liposomes to penetrate the HeLa cells was assessed by fluorescence and confocal microscopy investigation. Captured images show that NC-liposomes are internalized into cells through the endocytic pathway, enter early endosomes and reach lysosomes in 1 h. Interestingly, red emitting NCs co-localized with endosomes and were positioned at the limiting membrane of the organelles. The overall results suggest that the fluorescent system as a whole, NCs and their carrier, should be considered for the development of fully safe biological applications of CdSe@ZnS NCs, and provide essential indications to define the optimal experimental conditions to use the proposed system as an optical probe for future in vivo experiments.

Effect of ultrasound on the function and structure of a membrane protein: The case study of photosynthetic Reaction Center from Rhodobacter sphaeroides.

Ultrasounds are used in many industrial, medical and research applications. Properties and function of proteins are strongly influenced by the interaction with the ultrasonic waves and their bioactivity can be lost because of alteration of protein structure. Surprisingly, to the best of our knowledge no study was carried out on Integral Membrane Proteins (IMPs), which are responsible for a variety of fundamental biological functions. In this work, the photosynthetic Reaction Center (RC) of the bacterium Rhodobacter sphaeroides has been used as a model for the study of the ultrasound-induced IMP denaturation. Purified RCs were suspended in i) detergent micelles, in ii) detergent-free buffer and iii) reconstituted in liposomes, and then treated with ultrasound at 30W and 20kHz at increasing times. The optical absorption spectra showed a progressive and irreversible denaturation in all cases, resulting from the perturbation of the protein scaffold structure, as confirmed by circular dichroism spectra that showed progressive alterations of the RC secondary structure. Charge recombination kinetics were studied to assess the protein photoactivity. The lifetime for the loss of RC photoactivity was 32min in detergent micelles, ranged from 3.8 to 6.5min in the different proteoliposomes formulations, and 5.5min in detergent-free buffer. Atomic force microscopy revealed the formation of large RC aggregates related to the sonication-induced denaturation, in agreement with the scattering increase observed in solution.

Semiquinone oscillations as a tool for investigating the ubiquinone binding to photosynthetic reaction centers.

Semiquinone oscillations induced by light pulses in the presence of exogenous electron donors are a valuable source of information on the kinetics and thermodynamics of ubiquinone chemistry relevant to the QB site of the photosynthetic reaction center (RC). In previous attempts to achieve the quantitative interpretation of data, the ubiquinone concentration was considered constant during the experiment since it was much bigger than that of RC. In this work, we extended existing models to low ubiquinone concentrations revealing several hidden processes taking place during the ubiquinone photoreduction and enabling the evaluation of the ubiquinone binding constant K Q at the QB site. The proposed approach provides for the first time the evaluation of K Q without any preliminary treatment of ubiquinone extraction from the binding site, thereby better preserving its native structure.

The binding of quinone to the photosynthetic reaction centers: kinetics and thermodynamics of reactions occurring at the QB-site in zwitterionic and anionic liposomes.

Liposomes represent a versatile biomimetic environment for studying the interaction between integral membrane proteins and hydrophobic ligands. In this paper, the quinone binding to the QB-site of the photosynthetic reaction centers (RC) from Rhodobacter sphaeroides has been investigated in liposomes prepared with either the zwitterionic phosphatidylcholine (PC) or the negatively charged phosphatidylglycerol (PG) to highlight the role of the different phospholipid polar heads. Quinone binding (K Q) and interquinone electron transfer (L AB) equilibrium constants in the two type of liposomes were obtained by charge recombination reaction of QB-depleted RC in the presence of increasing amounts of ubiquinone-10 over the temperature interval 6-35 °C. The kinetic of the charge recombination reactions has been fitted by numerically solving the ordinary differential equations set associated with a detailed kinetic scheme involving electron transfer reactions coupled with quinone release and uptake. The entire set of traces at each temperature was accurately fitted using the sole quinone release constants (both in a neutral and a charge separated state) as adjustable parameters. The temperature dependence of the quinone exchange rate at the QB-site was, hence, obtained. It was found that the quinone exchange regime was always fast for PC while it switched from slow to fast in PG as the temperature rose above 20 °C. A new method was introduced in this paper for the evaluation of constant K Q using the area underneath the charge recombination traces as the indicator of the amount of quinone bound to the QB-site.

Photosynthetic machineries in nano-systems.

Photosynthetic reaction centres are membrane-spanning proteins, found in several classes of autotroph organisms, where a photoinduced charge separation and stabilization takes place with a quantum efficiency close to unity. The protein remains stable and fully functional also when extracted and purified in detergents thereby biotechnological applications are possible, for example, assembling it in nano-structures or in optoelectronic systems. Several types of bionanocomposite materials have been assembled by using reaction centres and different carrier matrices for different purposes in the field of light energy conversion (e.g., photovoltaics) or biosensing (e.g., for specific detection of pesticides). In this review we will summarize the current status of knowledge, the kinds of applications available and the difficulties to be overcome in the different applications. We will also show possible research directions for the close future in this specific field.

Development and characterization of a novel bioactive polymer with antibacterial and lysozyme-like activity.

The development and characterization of a novel bioactive polymer based on the immobilization of glucose oxidase enzyme (GOx) in a polyvinyl alcohol (PVA) film showing antibacterial activity is presented. The PVA-GOx composite material was extensively characterized by UV-vis, X-ray Photoelectron (XPS) spectroscopy and by Fourier Transform Infrared (FTIR) spectroscopy to verify the preservation of enzyme structural integrity and activity. The antimicrobial activity of this composite material against Escherichia coli and Vibrio alginolyticus was assessed. Furthermore the lysozyme-like activity of PVA-GOx was highlighted by a standard assay on Petri dishes employing Micrococcus lysodeikticus cell walls. The findings from this study have implications for future investigations related to the employment of PVA-GOx system as a composite material of pharmaceutical and technological interest.

Oxidoreductase activity of chromatophores and purified cytochrome bc1 complex from Rhodobacter sphaeroides: a possible role of cardiolipin.

Osmotic shock was used as a tool to obtain cardiolipin (CL) enriched chromatophores of Rhodobacter sphaeroides. After incubation of cells in iso- and hyper-osmotic buffers both chromatophores with a physiological lipid profile (Control) and with an almost doubled amount of CL (CL enriched) were isolated. Spectroscopic properties, reaction centre (RC) and reducible cytochrome (cyt) contents in Control and CL enriched chromatophores were the same. The oxidoreductase activity was found higher for CL enriched than for Control chromatophores, raising from 60 ± 2 to 93 ± 3 mol cyt c s(-1) (mol total cyt c)(-1). Antymicin and myxothiazol were tested to prove that oxidoreductase activity thus measured was mainly attributable to the cyt bc ( 1 ) complex. The enzyme was then purified from BH6 strain yielding a partially delipidated and almost inactive cyt bc ( 1 ) complex, although the protein was found to maintain its structural integrity in terms of subunit composition. The ability of CL in restoring the activity of the partially delipidated cyt bc ( 1 ) complex was proved in micellar systems by addition of exogenous CL. Results here reported indicate that CL affects oxidoreductase activity in the bacterium Rhodobacter sphaeroides both in chromatophore and in purified cyt bc ( 1 ) complex.

Light induced transmembrane proton gradient in artificial lipid vesicles reconstituted with photosynthetic reaction centers.

Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c2 and K4[Fe(CN)6]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.