The melting curves of calf thymus-DNA are buffer specific.

The specific effects of salts (strong electrolytes) on biomolecular properties have been investigated for more than a century. By contrast, the specific role of pH buffers (weak electrolytes and their salts) has usually been ignored. Here, specific buffer effects on DNA thermal stability were evaluated by measuring the melting curve of calf thymus DNA through UV-vis spectroscopy. The study was carried out using phosphate, Tris, citrate and cacodylate buffers at fixed pH 7.4 at concentrations varying systematically in the range 1-600 mM. DNA stability increases with buffer concentration and is influenced specifically by buffer type. To interpret empirical data, a theoretical model was applied with parameters quantifying the impact of buffer on the DNA backbone charge. Comparing the buffer effects via buffer ionic strength rather than buffer concentration, we find that the buffers stabilize DNA in the order Tris > cacodylate > phosphate > citrate.

BSA fragmentation specifically induced by added electrolytes: An electrospray ionization mass spectrometry investigation.

Biointerfaces are significantly affected by electrolytes according to the Hofmeister series. This work reports a systematic investigation on the effect of different metal chlorides, sodium and potassium bromides, iodides and thiocyanates, on the ESI/MS spectra of bovine serum albumin (BSA) in aqueous solution at pH = 2.7. The concentration of each salt was varied to maximize the quality of the ESI/MS spectrum, in terms of peak intensity and bell-shaped profile. The ESI/MS spectra of BSA in the absence and in the presence of salts showed a main protein pattern characterized by the expected mass of 66.5 kDa, except the case of BSA/RbCl (mass 65.3 kDa). In all systems we observed an additional pattern, characterized by at least three peaks with low intensity, whose deconvolution led to suggest the formation of a BSA fragment with a mass of 19.2 kDa. Only NaCl increased the intensity of the peaks of the main BSA pattern, while minimizing that of the fragment. NaCl addition seems to play a crucial role in stabilizing the BSA ionized interface against hydrolysis of peptide bonds, through different synergistic mechanisms. To quantify the observed specific electrolyte effects, two "Hofmeister" parameters (Hs and Ps) are proposed. They are obtained using the ratio of (BSA-Salt)/BSA peak intensities for both the BSA main pattern and for its fragment. SYNOPSIS: NaCl stabilizes BSA ion and almost prevents fragmentation due to denaturing pH.

Specific electrolyte effects on hemoglobin in denaturing medium investigated through electro spray ionization mass spectrometry.

We examine Hofmeister specific ion effects of electrolytes added to protein solution under conditions minimizing electrostatic attraction between cations and positively charged protein. Hemoglobin (Hb) in aqueous solution at the denaturing pH = 2.7 is investigated in the presence of several metal chlorides, along with sodium and potassium bromides, iodides and thiocyanates, using electrospray ionization mass spectrometry (ESI-MS). Salt concentration was varied to maximize peak intensity and bell-shaped profile in the ESI-MS spectrum. The α-chain of myoglobin is identified as the main pattern of the ESI-MS spectra in all Hb-salt systems. Both peak intensity and quality of the bell-shaped profile of the protein spectrum decrease in the cation order: K+ > > Mg2+ > Li+ > > Na+ > Ca2+ ≈ Cs+ > Rb+ for Hb-Metal Chloride systems, and decrease in the anion order: Cl- > Br- > I- > SCN- for systems of both Hb-NaX and Hb-KX salts. To quantify salt addition effects two Hofmeister specific electrolyte parameters HS, and PS are proposed. HS is the mean (Hb-salt)/Hb peak intensity ratio, measured for the nine peaks used for ESI-MS spectra deconvolution, taken at the same m/z values of the Hb profile. PS is the ratio between HS standard deviation and HS, and provides a specific perturbation parameter measuring the loss of protein structure. These two Hofmeister parameters give clear evidence of the effects induced either by KCl, MgCl2 and LiCl that enhance protein peak intensity, or by NaBr, NaI, NaSCN and KSCN that induce the protein fragmentation, due to electrolyte-mediated dissociation.

Adsorption and Release of Sulfamethizole from Mesoporous Silica Nanoparticles Functionalised with Triethylenetetramine.

Mesoporous silica nanoparticles (MSN) were synthesised and functionalised with triethylenetetramine (MSN-TETA). The samples were fully characterised (transmission electron microscopy, small angle X-ray scattering, Fourier transform infrared spectroscopy, thermogravimetric analysis, zeta potential and nitrogen adsorption/desorption isotherms) and used as carriers for the adsorption of the antimicrobial drug sulphamethizole (SMZ). SMZ loading, quantified by UV-Vis spectroscopy, was higher on MSN-TETA (345.8 mg g-1) compared with bare MSN (215.4 mg g-1) even in the presence of a lower surface area (671 vs. 942 m2 g-1). The kinetics of SMZ adsorption on MSN and MSN-TETA followed a pseudo-second-order model. The adsorption isotherm is described better by a Langmuir model rather than a Temkin or Freundlich model. Release kinetics showed a burst release of SMZ from bare MSN samples (k1 = 136 h-1) in contrast to a slower release found with MSN-TETA (k1 = 3.04 h-1), suggesting attractive intermolecular interactions slow down SMZ release from MSN-TETA. In summary, the MSN surface area did not influence SMZ adsorption and release. On the contrary, the design of an effective drug delivery system must consider the intermolecular interactions between the adsorbent and the adsorbate.

Specific Buffer Effects on the Intermolecular Interactions among Protein Molecules at Physiological pH.

BSA and lysozyme molecular motion at pH 7.15 is buffer-specific. Adsorption of buffer ions on protein surfaces modulates the protein surface charge and thus protein-protein interactions. Interactions were estimated by means of the interaction parameter kD obtained from plots of diffusion coefficients at different protein concentrations (Dapp = D0[1 + kDCprotein]) via dynamic light scattering and nuclear magnetic resonance. The obtained results agree with recent findings confirming doubts regarding the validity of the Henderson-Hasselbalch equation, which has traditionally provided a basis for understanding pH buffers of primary importance in solution chemistry, electrochemistry, and biochemistry.

Assembly of Multicomponent Nano-Bioconjugates Composed of Mesoporous Silica Nanoparticles, Proteins, and Gold Nanoparticles.

The purpose of this work was the assembly of multicomponent nano-bioconjugates based on mesoporous silica nanoparticles (MSNs), proteins (bovine serum albumin, BSA, or lysozyme, LYZ), and gold nanoparticles (GNPs). These nano-bioconjugates may find applications in nanomedicine as theranostic devices. Indeed, MSNs can act as drug carriers, proteins stabilize MSNs within the bloodstream, or may have therapeutic or targeting functions. Finally, GNPs can either be used as contrast agents for imaging or for photothermal therapy. Here, amino-functionalized MSNs (MSN-NH2) were synthesized and characterized through various techniques (small angle X-rays scattering TEM, N2 adsorption/desorption isotherms, and thermogravimetric analysis (TGA)). BSA or lysozyme were then grafted on the external surface of MSN-NH2 to obtain MSN-BSA and MSN-LYZ bioconjugates, respectively. Protein immobilization on MSNs surface was confirmed by Fourier transform infrared spectroscopy, ζ-potential measurements, and TGA, which also allowed the estimation of protein loading. The MSN-protein samples were then dispersed in a GNP solution to obtain MSN-protein-GNPs nano-bioconjugates. Transmission electron microscopy (TEM) analysis showed the occurrence of GNPs on the MSN-protein surface, whereas almost no GNPs occurred in the protein-free control samples. Fluorescence and Raman spectroscopies suggested that proteins-GNP interactions involve tryptophan residues.

Gold Nanoparticles: A Powerful Tool to Visualize Proteins on Ordered Mesoporous Silica and for the Realization of Theranostic Nanobioconjugates.

Ordered mesoporous silica (OMS) is a very interesting nanostructured material for the design and engineering of new target and controlled drug-delivery systems. Particularly relevant is the interaction between OMS and proteins. Large pores (6­9 nm) micrometric particles can be used for the realization of a drug depot system where therapeutic proteins are adsorbed either inside the mesopores or on the external surface. Small pores (1­2 nm) mesoporous silica nanoparticles (MSNs), can be injected in the blood stream. In the latter case, therapeutic proteins are mainly adsorbed on the MSNs' external surface. Whenever a protein-OMS conjugate is prepared, a diagnostic method to locate the protein either on the internal or the external silica surface is of utmost importance. To visualize the fine localization of proteins adsorbed in mesoporous silica micro- and nanoparticles, we have employed specific transmission electron microscopy (TEM) analytical strategies based on the use of gold nanoparticles (GNPs) conjugates. GNPs are gaining in popularity, representing a fundamental tool to design future applications of MSNs in nanomedicine by realizing theranostic nanobioconjugates. It may be pointed out that we are at the very beginning of a new age of the nanomaterial science: the "mesoporous golden age".

Lipid based liquid-crystalline stabilized formulations for the sustained release of bioactive hydrophilic molecules.

Lipid based formulations, endowed of long term stability as a result of the formation of lamellar liquid crystals, were prepared using the natural lipids lecithin and glycerol trioleate in water, and characterized using optical microscopy, SAXRD and NMR. The formulations, designed as possible carriers for lysozyme and caffeine, were evaluated for structural features and stability after the loading of the guest molecules. Release experiments were performed at 37 °C using the PBS medium. No burst release was observed either for lysozyme or caffeine. Although lysozyme released from the lipid formulations does not fully retain its biological activity, the investigated liquid crystal stabilized formulations display a promising potential as drug and cosmetic carriers for topical applications, due to their high biocompatibility.

Structural, rheological and dynamics insights of hydroxypropyl guar gel-like systems.

A dynamic, rheological, and structural characterization of aqueous gel-like systems containing hydroxypropyl guar gum (HPG), borax and glycerol is presented in this paper. The role of glycerol, which is introduced as a plasticizer in the formulation, is investigated by means of 11B NMR and 1H NMR PGSTE measurements in order to clarify its contribution to the gel network formation and its interaction with borax, with whom it forms a complex. The effect of gels components on the rheological behaviour and on the activation energy related to the relaxation process of the system was assessed by means of rheology. The results obtained suggest that the mechanical properties of these gels can be tuned and controlled by modulating the formulation in a wide range of compositions. Moreover, a structural characterisation has been also carried out by means of Small Angle X-ray Scattering (SAXS) to highlight the role of the various components on the mesh size of the network. The structural and mechanical characteristics of these systems suggest their potential use for applicative purposes. In this regard, one of the gel set up has been successfully tested as cleaning agent on the surface of a XIX stucco fragment coming from the La Fenice theatre (Venice, Italy) for the removal of a dirt layer composed by dust and particulated matter originated during a fire in 1996.

Mesoporous silica nanoparticles functionalized with hyaluronic acid. Effect of the biopolymer chain length on cell internalization.

Mesoporous silica nanoparticles (MSNs) were functionalized with amino groups (MSN-NH2) and then with hyaluronic acid, a biocompatible biopolymer which can be recognized by CD44 receptors in tumor cells, to obtain a targeting drug delivery system. To this purpose, three hyaluronic acid samples differing for the molecular weight, namely HAS (8-15 kDa), HAM (30-50 kDa) and HAL (90-130 kDa), were used. The MSN-HAS, MSN-HAM, and MSN-HAL materials were characterized through zeta potential and dynamic light scattering measurements at pH = 7.4 and T = 37 °C to simulate physiological conditions. While zeta potential showed an increasing negative value with the increase of the HA chain length, an anomalous value of the hydrodynamic diameter was observed for MSN-HAL, which was smaller than that of MSN-HAS and MSN-HAM samples. The cellular uptake of MSN-HA samples on HeLa cells at 37 °C was studied by optical and electron microscopy. HA chain length affected significantly the cellular uptake that occurred at a higher extent for MSN-NH2 and MSN-HAS than for MSN-HAM and MSN-HAL samples. Cellular uptake experiments carried out at 4 °C showed that the internalization process was inhibited for MSN-HA samples but not for MSN-NH2. This suggests the occurrence of two different mechanisms of internalization. For MSN-NH2 the uptake is mainly driven by the attractive electrostatic interaction with membrane phospholipids, while MSN-HA internalization involves CD44 receptors overexpressed in HeLa cells.

Phase behavior in the biologically important oleic acid/sodium oleate/water system.

The phase behavior in the oleic acid/sodium oleate/normal saline (0.15M NaCl aqueous solution) system has been determined. For this purpose visual inspection of samples between crossed polarizers, and Small Angle X-ray diffraction was used to identify the various phases and their unit cell dimensions. A rich phase behavior was observed for the ternary system, featuring reverse micellar, micellar cubic, hexagonal, and cubic phases, and large regions with lamellar phases. As expected the ratio the 'oleic acid/sodium oleate' determines the pH and as a consequence the phase behavior. The results could be modeled by an extended Henderson-Hasselbalch (HH) equation, which takes into account the electrostatic potential at the aqueous lipid interface. The knowledge obtained is important for understanding the lipolysis of triglycerides, as the phase behavior of the end-product of the reaction regulates how well the insoluble product can be dispersed and consequently the kinetics of the process.

Adsorption and release of ampicillin antibiotic from ordered mesoporous silica.

In this work the adsorption and the release of ampicillin - a ß-lactam penicillin-like antibiotic - from MCM-41, SBA-15, and (amino functionalized) SBA-15-NH2 ordered mesoporous silica (OMS) materials were investigated. The silica matrices differ for their pore size (SBA-15 vs. MCM-41) mainly, and also for surface charge (SBA-15 and MCM-41, vs. SBA-15-NH2). OMS samples were characterized through small-angle X-rays scattering (SAXS), transmission electron microscopy (TEM), N2 adsorption-desorption isotherms, Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and potentiometric titrations. The quantification of immobilized and released ampicillin was monitored by mean of UV-Vis spectroscopy. Experimental adsorption isotherms evidenced that ampicillin's loading is not related to the pore size (dBJH) of the adsorbent. Indeed the maximal loadings were 237mg/g for SBA-15 (dBJH=6.5nm), 278mg/g for MCM-41 (dBJH=2.2nm), and 333mg/g for SBA-15-NH2 (dBJH=5.6nm). Loading seems, instead, to be related to the surface charge density (σ) of the sorbent surface. Indeed, at pH 7.4 ampicillin drug is negatively charged and likely prefers to interact with SBA-15-NH2 (σSBA-15-NH2=+0.223Cm-2) rather than the slightly negatively charged silicas (σSBA-15=-0.044Cm-2 and σMCM-41=-0.033Cm-2). Similarly, ampicillin release is affected by interfacial interactions. Indeed, we found a burst release from pure silica samples (SBA-15 and MCM-41), whereas a sustained one from SBA-15-NH2 sample. We explain this behavior as a result of an attractive interaction between the protonated amino group of SBA-15-NH2 and the negatively charged carboxylate group of ampicillin. In summary, in order to obtain a sustained drug release, the chemical nature of the matrix's surface plays a role which is more important than its textural features. SBA-15-NH2 matrix is hence a suitable candidate for local sustained release of antibiotic drugs.

Cubosomes for in vivo fluorescence lifetime imaging.

Herein we provided the first proof of principle for in vivo fluorescence optical imaging application using monoolein-based cubosomes in a healthy mouse animal model. This formulation, administered at a non-cytotoxic concentration, was capable of providing both exogenous contrast for NIR fluorescence imaging with very high efficiency and chemospecific information upon lifetime analysis. Time-resolved measurements of fluorescence after the intravenous injection of cubosomes revealed that the dye rapidly accumulated mainly in the liver, while lifetimes profiles obtained in vivo allowed for discriminating between free dye or dye embedded within the cubosome nanostructure after injection.

Effect of electrolytes on proteins physisorption on ordered mesoporous silica materials.

This short review highlights the effect of electrolytes on the performance of proteins-mesoporous silica conjugates which can open interesting perspectives in biotechnological fields, particularly nanomedicine and biocatalysis. Indeed therapeutic proteins and peptides represent a challenging innovation for several kinds of diseases, but since their self-life in biological fluids is very short, they need a stealth protective carrier. Similarly, enzymes need a solid support to improve thermal stability and to allow for recycling. Ordered mesoporous silica materials represent a valid choice as widely demonstrated. Both proteins and silica mesoporous materials possess charged surfaces, and here, the crucial role of pH, buffer, ionic strength and electrolyte type is posed in relation with loading/release of proteins onto/from the silica support through the analysis of adsorption and release processes. A delicate interplay of electrostatic and van der Waals interactions arises from considering electrolytes' effects on the two different charged surfaces. Clear outcomes concern the effect of pH and ionic strength. Protein loading onto the silica matrix is favored by an adsorbing solution having a pH close to the protein pI, and by a high ionic strength that reduces the Debye length. Release is instead favored by an adsorbing solution characterized by an intermediate ionic strength, close to the physiological values. Significant specific ions effects are shown to affect both proteins and silica matrices, as well as protein adsorption onto silica matrices. Further work is needed to quantify specific ion effects on the preservation of the biological activity, and on the release performance.

Mesoporous Silica Nanoparticles Functionalized with Hyaluronic Acid and Chitosan Biopolymers. Effect of Functionalization on Cell Internalization.

Mesoporous silica nanoparticles (MSNs), based on the MCM-41 matrix, were functionalized with amino groups, and then with hyaluronic acid (HA) or chitosan (CHIT) to fabricate bioactive conjugates. The role of the functional groups toward cytotoxicity and cellular uptake was investigated using 3T3 mouse fibroblast cells. A very high biocompatibility of MSN-NH2, MSN-HA and MSN-CHIT matrices was assessed through the MTS biological assay and Coulter counter evaluation. No significant differences in cytotoxicity data arise from the presence of different functional groups in the investigated MSNs. Fluorescence microscopy experiments performed using fluorescein isothiocyanate-conjugated MSN-NH2, MSN-HA, and MSN-CHIT, and transmission electron microscopy experiments performed on slices of the investigated systems embedded in epoxy resins give evidence of significant differences due to type of functionalization in terms of cellular uptake and stability of the particles in the biological medium. MSN-NH2 and MSN-HA conjugates are easily internalized, the uptake of the HA-functionalized MSNs being much higher than that of the -NH2-functionalized MSNs. Differently, MSN-CHIT conjugates tend to give large aggregates dispersed in the medium or localized at the external surface of the cell membranes. Both fluorescence microscopy and TEM images show that the MSNs are distributed in the cytoplasm of the cells in the case of MSN-NH2 and MSN-HA, whereas only a few particles are internalized in the case of MSN-CHIT. Flow cytometry experiments confirmed quantitatively the selectively high cellular uptake of MSN-HA particles.

Silver Enhancement for Transmission Electron Microscopy Imaging of Antibody Fragment-Gold Nanoparticles Conjugates Immobilized on Ordered Mesoporous Silica.

Ordered mesoporous silica (OMS) materials are receiving great attention as possible carriers for valuable but unstable drugs as, for example, therapeutic proteins. A key issue is to prove that the therapeutic protein is effectively able to penetrate the pores of OMS during the adsorption step. Here, we immobilized an antibody fragment [F(ab')GAMIgG] conjugated with ultrasmall gold nanoparticles (GNPs) onto amino-functionalized SBA-15 (SBA-NH2) mesoporous silica. The aim of this work is the visualization of the location of the conjugates adsorbed onto SBA-NH2 with transmission electron microscopy (TEM). Because of the ultrasmall size of GNPs (<1 nm), we use the silver enhancement procedure to amplify their size. In this procedure, ultrathin sections of conjugate-loaded SBA-NH2 particles are prepared by a ultramicrotome sectioning technique. The ultrasmall GNPs located on the top side of the 70-90 nm thick slices act as microcrystallization nucleation sites for the deposition of reduced metallic silver. Consequently, the ultrasmall GNPs increase their size. This allows for the direct imaging of the conjugates adsorbed. We clearly localize the F(ab')GAMIgG-GNPs conjugates either on the external surface of the particles or inside the mesopores of SBA-NH2 through TEM.

Docetaxel-Loaded Fluorescent Liquid-Crystalline Nanoparticles for Cancer Theranostics.

Here, we describe a novel monoolein-based cubosome formulation engineered for possible theranostic applications in oncology. The Docetaxel-loaded nanoparticles were stabilized in water by a mixture of commercial Pluronic (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer) F108 (PF108) and rhodamine- and folate-conjugated PF108 so that the nanoparticles possess targeting, therapeutic, and imaging properties. Nanoparticles were investigated by DLS, cryo-TEM, and SAXS to confirm their structural features. The fluorescent emission characterization of the proposed formulation indicated that the rhodamine conjugated to the PF108 experiences an environment less polar than water (similar to chloroform), suggesting that the fluorescent fragment is buried within the poly(ethylene oxide) corona surrounding the nanoparticle. Furthermore, these nanoparticles were successfully used to image living HeLa cells and demonstrated a significant short-term (4 h incubation) cytotoxicity effect against these cancer cells. Furthermore, given their analogy as nanocarriers for molecules of pharmaceutical interest and to better stress the singularities of these bicontinuous cubic nanoparticles, we also quantitatively evaluated the differences between cubosomes and multilamellar liposomes in terms of surface area and hydrophobic volume.

Cubosome formulations stabilized by a dansyl-conjugated block copolymer for possible nanomedicine applications.

We present here an innovative, fluorescent, monoolein-based cubosome dispersion. Rather than embedded within the monoolein palisade, the fluorescent imaging agent, namely dansyl, was conjugated to the terminal ethylene oxide moieties of the block copolymer Pluronic F108. We discuss the physicochemical and photophysical properties of this fluorescent Pluronic and of a cubosome formulation stabilized by a mixture of dansyl-conjugated and non-conjugated Pluronic, also including an anticancer drug (quercetin). Furthermore, we performed biocompatibility tests against HeLa cells to assess internalization and cytotoxicity features of this nanoparticles aqueous dispersion. Cryo-TEM, SAXS, and DLS analysis, proved the bicontinuous cubic inner nanostructure and the morphology of this fluorescent cubosome dispersion, while photophysical measurements and biocompatibility results basically validate their potential use for theranostic nanomedicine applications.

The molecular motion of bovine serum albumin under physiological conditions is ion specific.

Specific ion effects on the Brownian molecular motion of BSA protein under physiological conditions are investigated. New useful insights into Hofmeister phenomena related to electrolyte-protein interactions are presented.

Penetration Enhancer-Containing Vesicles: Does the Penetration Enhancer Structure Affect Topical Drug Delivery?

The aim of this study was to elucidate the influence of the edge activator structure on the properties of novel deformable liposomes, Penetration Enhancer-containing Vesicles (PEVs), capable of delivering drugs to the skin. The PEVs were prepared by testing five different amphiphilic penetration enhancers as edge activators in the bilayer composition, together with soy phosphatidylcholine and oleic acid. The penetration enhancers contained the same lipophilic tail (one or more C8-C10 carbon chains) and different hydrophilic heads. Conventional phospholipid liposomes were prepared and used as a control. Lidocaine was chosen as a model drug. Liquid and gelified PEVs were obtained, depending on the penetration enhancer used. The vesicular systems were characterized by measuring size distribution, zeta potential, incorporation efficiency, and monitoring these parameters over 90 days. Accelerated ageing tests were also performed to check the stability of the dispersions. The effects of the different nature of the edge activator on the features of the obtained PEVs were assessed by TEM, SAXS and WAXS, rheological and deformability studies. Higher interactions of the most lipophilic penetration enhancers with the lipid bilayers and a consequent higher stability and elasticity of the obtained PEVs were observed. In vitro experiments through pig skin confirmed the superior potential as carriers for lidocaine of the PEVs prepared with the most lipophilic penetration enhancers, even in comparison with commercial EMLA cream.