Mechanisms of fibrinogen-acebutolol interactions: Insights from DSC, CD and LS

The complex formed due to the interaction of the amphiphilic betablocker acebutolol with fibrinogen in a buffer solution (50mN glycine, pH of 8.5) has been investigated using a multipronged physicochemical approach. Differential scanning calorimetry measurements of the complexes have shown no reversibility of thermal denaturation as indicated by the three observed peaks and the opposite role that acebutolol plays in the folding different domains of the fibrinogen molecule and the stability of such domains. While circular dichroism measurements have revealed that interaction of acebutolol with fibrinogen affects the protein secondary structure to a different extent depending on the temperature and drug concentration, dynamic light scattering analysis showed evidence for protein aggregation mainly to tetramers and dimers.     

In situ conformational analysis of fibrinogen adsorbed on Si surfaces

Fibrinogen is a major plasma protein. Previous investigations of structural changes of fibrinogen due to adsorption are mostly based on indirect evidence after its desorption, whereas our measurements were performed on fibrinogen in its adsorbed state. Specific enzyme-linked immunosorption experiments showed that the amount of adsorbed fibrinogen increased as the surface became more hydrophobic. Atomic force microscopy (AFM) investigations revealed the trinodular shape of fibrinogen molecules adsorbed on hydrophilic surfaces, whereas all of the molecules appeared globular on hydrophobic surfaces. The distribution of secondary structures in adsorbed fibrinogen was quantified by in situ Fourier-transform infrared (FTIR) analysis. Substrates of identical chemical bulk composition but different surface hydrophobicity permit direct comparison among them. Adsorption properties of fibrinogen are different for each degree of hydrophobicity. Although there is some increase of turn structure and decrease of β-sheet structure, the secondary structure of adsorbed fibrinogen on hydrophilic surface turned out to be rather similar to that of the protein in solution phase with a major -helix content. Hydrophilic surfaces exhibit superior blood compatibility as required for medical applications.     

Competitive adsorption of fibrinogen and dipalmitoylphosphatidylcholine at the air/aqueous interface

The competitive adsorption of fibrinogen (FB) and DPPC at the air/aqueous interface, in phosphate buffer saline at 25 degrees C, was studied with tensiometry, infrared reflection absorption spectroscopy (IRRAS), and ellipsometry. For FB/DPPC mixtures with 750 ppm (0.075 wt%) FB and 1000 ppm (0.10 wt%) DPPC, the tension behavior was found to be similar to that of FB when alone, even with DPPC and FB being at the interface. Thus, FB interferes with adsorption of DPPC and inhibits its surface tension lowering ability. When FB protein is introduced in the solution after a DPPC monolayer has formed, the adsorption of FB is inhibited by the DPPC monolayer. When a DPPC monolayer is spread onto a solution with a preadsorbed FB layer, the DPPC monolayer excludes FB from the surface and controls the tension behavior with little inhibition by FB. When a DPPC dispersion is introduced with the Trurnit method, or sprayed dropwise, onto an aqueous FB/DPPC surfaces, the DPPC layer formed on the surface prevents the adsorption of FB and dominates the surface tension behavior. These results have implications in controlling the inhibition of lung surfactant tension behavior by serum proteins, when they leak at the alveolar lining layer, and in developing surfactant replacement therapies for alveolar respiratory diseases.     

Investigation of early stages of fibrin association

Interactions between fibrinogen molecules proteolytically cleaved with thrombin were investigated using atomic force microscopy (AFM) and dynamic light scattering (DLS). Gradually decreased fibrinogen concentrations were used to study the fibrin network, large separated fibrils, small fibrils in the initial association stages, and protofibrils. In addition, a new type of structure was found in AFM experiments at a low fibrinogen concentration (20 nM): the molecules in these single-stranded associates are arranged in a row, one after the other. The height, diameter, and distance between domains in these single-stranded associates were the same as those in the original fibrinogen molecules. DLS data assumed formation of extended associates in bulk solution at fibrinogen concentration as low as 20 nM.     

Vesicle-micelle transition in aqueous mixtures of the cationic dioctadecyldimethylammonium and octadecyltrimethylammonium bromide surfactants

The vesicle-micelle transition in aqueous mixtures of dioctadecyldimethylammonium and octadecyltrimethylammonium bromide (DODAB and C(18)TAB) cationic surfactants, having respectively double and single chain, was investigated by differential scanning calorimetry (DSC), steady-state fluorescence, dynamic light scattering (DLS) and surface tension. The experiments performed at constant total surfactant concentration, up to 1.0 mM, reveal that these homologous surfactants mix together to form mixed vesicles and/or micelles, depending on the relative amount of the surfactants. The melting temperature T(m) of the mixed DODAB-C(18)TAB vesicles is larger than that for the neat DODAB in water owing to the incorporation of C(18)TAB in the vesicle bilayer. The surface tension decreases sigmoidally with C(18)TAB concentration and the inflection point lies around x(DODAB) approximately 0.4, indicating the onset of micelle formation owing to saturation of DODAB vesicles by C(18)TAB molecules. When x(DODAB)>0.5 C(18)TAB molecules are mainly solubilised by the vesicles, but when x(DODAB)<0.25 micelles are dominant. Fluorescence data of the Nile Red probe incorporated in the system at different surfactant molar fractions indicate the formation of micelle and vesicle structures. These structures have apparent hydrodynamic radius R(H) of about 180 and 500-800 nm, respectively, as obtained by DLS measurements.     

Creation of highly stable selenium nanoparticles capped with hyperbranched polysaccharide in water

Water-dispersible selenium nanoparticles (SeNPs) were created by using natural hyperbranched polysaccharide (HBP) as the stabilizer and capping agent under extremely safe conditions. The structure, morphology, size, and stability of the nanocomposites were investigated by transmission electron microscopy (TEM), atomic force microscopy (AFM), and static and dynamic light scattering (DLS) measurements. The results revealed that the spherical selenium nanoparticles (mean particle size of about 24 nm) were ligated with HBP to form nanocomposites (Se-HBP) in aqueous solution and were stable for over one month. In our findings, supported by the results of FTIR, TEM, AFM, and DLS, SeNPs were capped with the HBP macromolecules, as a result of strong physical adsorption of OH groups on Se surfaces, leading to a highly stable structure of Se nanoparticles in water. This work provided reaction sites for the complexation between HBP and Se to fabricate well-dispersed Se nanoparticles in aqueous system with potential bioapplications.     

Long circulating 10-hydroxycamptothecin-loaded nanoparticles fabricated from poly(ethylene glycol)/poly(L-lactic acid) multi-block copolymers

A series of poly(ethylene glycol) (PEG)/poly(L-lactic acid) (PLLA) multi-block copolymers were facilely synthesized using triphosgene as coupling agent. With the resulting multi-block copolymers, 10-hydroxycamptothecin (HCPT)-loaded nanoparticles were successfully prepared by dialysis method. The results obtained from dynamic light scattering (DLS) measurements confirmed that HCPT-loaded nanoparticles had the size of less than 200 nm and the average diameter decreased with increasing PLLA content. TEM images demonstrated that most of the drug-loaded nanoparticles had a distinct spherical shape and smooth surface without any aggregation. Atomic force microscopy (AFM) images further indicated that the nanoparticles were in spherical shape with smooth surface, no drug crystal was visualized on their surface. To investigate the drug state in HCPT-loaded nanoparticles, differential scanning calorimetry (DSC) and X-ray powder diffraction (XRD) measurements were carried out. The results from these tests suggested that HCPT was molecularly dispersed in the amorphous polymer matrix. Drug loading content and in vitro drug release behavior of HCPT-loaded nanoparticles showed dependence on polymer composition. Cytotoxicity test indicated that HCPT-loaded nanoparticles exhibited greatly superior cytotoxicity compared to free HCPT due to its molecular dispersion in the polymer matrix. Furthermore, the nanoparticles significantly increased the duration of the drug in circulation. All these results demonstrated that PEG/PLLA nanoparticles have great potential as promising delivery system for poorly soluble antitumor drugs.     

Long circulating 10-hydroxycamptothecin-loaded nanoparticles fabricated from poly(ethylene glycol)/poly(L-lactic acid) multiblock copolymers

A series of poly(ethylene glycol) (PEG)/poly(L-lactic acid) (PLLA) multiblock copolymers were facilely synthesized using triphosgene as coupling agent. With the resulting multiblock copolymers, 10-hydroxycamptothecin (HCPT)-loaded nanoparticles were successfully prepared by dialysis method. The results obtained from dynamic light scattering (DLS) measurements confirmed that HCPT-loaded nanoparticles had the size of less than 200 nm and the average diameter decreased with increasing PLLA content. TEM images demonstrated that most of the drug-loaded nanoparticles had a distinct spherical shape and smooth surface without any aggregation. Atomic force microscopy (AFM) images further indicated that the nanoparticles were in spherical shape with smooth surface, no drug crystal was visualized on their surface. To investigate the drug state in HCPT-loaded nanoparticles, differential scanning calorimetry (DSC) and X-ray powder diffraction (XRD) measurements were carried out. The results from these tests suggested that HCPT was molecularly dispersed in the amorphous polymer matrix. Drug loading content and in vitro drug release behavior of HCPT-loaded nanoparticles showed dependence on polymer composition. Cytotoxicity test indicated that HCPT-loaded nanoparticles exhibited greatly superior cytotoxicity compared to free HCPT due to its molecular dispersion in the polymer matrix. Furthermore, the nanoparticles significantly increased the duration of the drug in circulation. All these results demonstrated that PEG/PLLA nanoparticles have great potential as promising delivery system for poorly soluble antitumor drugs.     

Characterization of synthetic hematite (α-Fe2O3) nanoparticles using a multi-technique approach

The aim of this work was to investigate the surface structure of aqueous hematite dispersions characterized by a large variability of morphology and particle size combining structural investigations obtained from Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM) techniques with in vitro particle size distributions and zeta potential measurements from Dynamic Light Scattering (DLS) technique, and we achieved a self-consistent and detailed characterization of hematite particles whose sizes and morphologies could be correlated to the synthesis conditions (type of added anion, Al substitution and pH). Surface AFM characterization provided an accurate analysis of particle microstructure and also indicated that the growth of microcrystals followed different surface roughness. DLS, AFM, and TEM techniques furnished complementary information on the average particle dimensions, whose variation could be attributed to the morphological difference of hematites, ranging from platy to regular or irregular hexagonal or ellipsoidal shape. Finally, a correlation between the average particle dimensions and the measured zeta potential was also been found in aqueous dilute suspensions characterized by neither pH nor-ionic-strength-control, for which a drop of zeta potential from positive to negative values was detected for hematite particle dimensions larger than a threshold size of ~150 nm.     

Quantitative analysis of protein adsorption via atomic force microscopy and surface plasmon resonance

Surface properties have a significant influence on the performance of biomedical devices. The influence of surface chemistry on the amount and distribution of adsorbed proteins has been evaluated by a combination of atomic force microscopy (AFM) and surface plasmon resonance (SPR). Adsorption of albumin, fibrinogen, and fibronectin was analyzed under static and dynamic conditions, employing self-assembled monolayers (SAMs) as model surfaces. AFM was performed in tapping mode with antibody-modified tips. Phase-contrast images showed protein distribution on SAMs and phase-shift entity provided information on protein conformation. SPR analysis revealed substrate-specific dynamics in each system investigated. When multi-protein solutions and diluted human plasma interacted with SAMs, SPR data suggested that surface chemistry governs the equilibrium composition of the protein layer.     

Surface and bulk properties of two anionic amphiphilic penicillins in a selective solvent

The surface physicochemical properties of two anionic penicillins-cloxacillin and dicloxacillin-in mixed ethanol-water solvent were investigated by surface tension and dynamic light scattering (DLS). The data were analyzed according to the treatment of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory to study the stability of the systems. The aim of the study is to obtain information about the effects of ethanol on the surface activity, bulk properties, and aggregate stability of these amphiphilic drugs, keeping in mind that both penicillins have the same counterion, and the difference in their structures is only a Cl atom in the phenyl ring that makes dicloxacillin more hydrophobic. The surface tension data show a minimum area per molecule increment with ethanol concentration that is related to the variation of the dielectric constant with the alcohol. Dicloxacillin has lower values of the standard Gibbs energies of adsorption than does cloxacillin, which gives this drug a more marked escaping tendency from the aqueous environment to the air-water monolayer. DLS data was fitted to an exponential function for cloxacillin at any drug or alcohol concentration in the range of concentrations studied that indicates that the system can be modeled as an ergodic system of dilute diffusing monodisperse particles. Dicloxacillin DLS data at an ethanol concentration of 5% (v/v) had to be fitted at a sum of an exponential and a stretched exponential function, which indicates that, besides the drug aggregates, a small population of penicillin clusters with longer relaxation times is present. The stability curves predicted by the DLVO theory, for both penicillins, indicate the predominance of electrostatic repulsion, leading to a stable system over the drug-ethanol concentration range studied, but the height of the reduced pair interaction potential energy barrier decreases with ethanol concentration, thus it is expected to undergo a transition from a stable dispersion to a coagulated one.     

Adsorption-induced conformational changes of proteins onto ceramic particles: differential scanning calorimetry and FTIR analysis

Three model proteins, bovine serum albumin, hen's egg lysozyme and bovine serum fibrinogen, were adsorbed from aqueous solution onto finely dispersed ceramic particles, namely different kinds of alumina and hydroxyapatite particles. The influence of adsorption on protein secondary structure was investigated. The FTIR spectroscopic findings were compared with the results of DSC measurements. In almost all cases it was found that adsorption results in destabilisation and structural loss of the bound protein. A decrease in transition enthalpy is correlated with a loss in alpha-helical structure, which seems to be the most sensitive structure on adsorption-induced rearrangements. A total collapse of structure in the adsorbed proteins was not determined on any ceramic surface. Some residual structure is always retained. Structural changes in the D- or E-domains of fibrinogen could be independently observed by two different calorimetric signals. The two techniques applied in the present study -- micro-DSC and FTIR spectroscopy -- can be concluded to provide complementary information on adsorption-induced structural changes on both the molecular (thermal stability, overall structure) and the sub-molecular level (secondary structure).     

Structure, stability, and activity of myoglobin adsorbed onto phosphate-grafted zirconia nanoparticles

The adsorption of myoglobin (Mb) onto phosphate grafted-zirconia (ZrO2-P) nanoparticles was studied in terms of conformational studies and thermal stability, determined by circular dichroism (CD), differential scanning calorimetry (DSC), and atomic force microscopy (AFM). The changes in protein structure have been correlated with the catalytic activity of free and adsorbed Mb. CD and DSC studies indicate marked rearrangements in Mb structure upon adsorption onto phosphate-grafted zirconia nanoparticles. These structural rearrangements of Mb could be responsible for the loss of catalytic activity observed for the adsorbed Mb. In particular, the conformational changes due to the adsorption process induced a reduction of kcat and KM. AFM measurements indicate that the interaction with the grafted-zirconia nanoparticles also affects the morphology of the bound protein, inducing the nucleation of prefibrillar-like aggregates.     

Humidity-dependent surface tension measurements of individual inorganic and organic submicrometre liquid particles

Surface tension, an important property of liquids, is easily measured for bulk samples. However, for droplets smaller than one micron in size, there are currently no reported measurements. In this study, atomic force microscopy (AFM) and force spectroscopy have been utilized to measure surface tension of individual submicron sized droplets at ambient pressure and controlled relative humidity (RH). Since the surface tension of atmospheric aerosols is a key factor in understanding aerosol climate effects, three atmospherically relevant systems (NaCl, malonic and glutaric acids) were studied. Single particle AFM measurements were successfully implemented in measuring the surface tension of deliquesced particles on the order of 200 to 500 nm in diameter. Deliquesced particles continuously uptake water at high RH, which changes the concentration and surface tension of the droplets. Therefore, surface tension as a function of RH was measured. AFM based surface tension measurements are close to predicted values based on bulk measurements and activities of these three chemical systems. Non-ideal behaviour in concentrated organic acid droplets is thought to be important and the reason for differences observed between bulk solution predictions and AFM data. Consequently, these measurements are crucial in order to improve atmospheric climate models as direct measurements hitherto have been previously inaccessible due to instrument limitations.     

Interaction of sodium N-lauroylsarcosinate with N-alkylpyridinium chloride surfactants: spontaneous formation of pH-responsive, stable vesicles in aqueous mixtures

The interaction of sodium N-lauroylsarcosinate (SLS) with N-cetylpyridinium chloride (CPC) and N-dodecylpyridinium chloride (DPC) was investigated in aqueous mixtures. A strong interaction between the anionic and cationic surfactants was observed. The interaction parameter, β was determined for a wide composition range and was found to be negative. The mixed systems were found to have much lower critical micelle concentration (cmc) and surface tension at cmc. The surfactant mixtures exhibit synergism in the range of molar fractions investigated. The self-assembly formation in the mixtures of different compositions and total concentrations were studied using a number of techniques, including surface tension, fluorescence spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), confocal fluorescence microscopy (CFM). Thermodynamically stable unilamellar vesicles were observed to form upon mixing of the anionic and cationic surfactants in a wide range of composition and concentrations in buffered aqueous media. TEM as well as DLS measurements were performed to obtain shape and size of the vesicular structures, respectively. These unilamellar vesicles are stable for periods as long as 3 months and appear to be the equilibrium form of aggregation. Effect of pH, and temperature on the stability was investigated. The vesicular structures were observed to be stable at pH as low as 2.0 and at biological temperature (37°C). In presence of 10 mol% of cholesterol the mixed surfactant vesicles exhibited leakage of the encapsulated calcein dye, showing potential application in pH-triggered drug release.     

Surface modification of polyethylene for improving the adhesion of a highly fluorinated UV-cured coating

The surface of low density polyethylene (LDPE) was modified by grafting a photoinitiator on it, after an Ar plasma treatment. The functionalisation was characterized by contact angle measurements, XPS analyses and AFM. The grafted LDPE was then coated with a UV-curable formulation based on highly fluorinated oligomers. Although the surface tension of the coating is very low, a good adhesion onto the substrate was obtained due to the surface treatment which was applied.     

Interaction between bovine serum albumin and equimolarly mixed cationic-anionic surfactants decyltriethylammonium bromide-sodium decyl sulfonate

The interactions of bovine serum albumin (BSA) with the anionic surfactant sodium decylsulfonate (C10SO3), the cationic surfactant decyltriethylammonium bromide (C10NE) and equimolarly mixed cationic-anionic surfactants C10NE-C10SO3 were investigated by surface tension, viscosity, dynamic light scattering (DLS) and circular dichroism (CD). It was shown that the single ionic surfactant C10SO3 or C10NE has obvious interaction with BSA. The presence of C10SO3 or C10NE modified BSA structure. However, the equimolarly mixed cationic-anionic surfactants C10NE-C10SO3 showed very weak interactions with BSA. The surface tension-log concentration (gamma-logC) plot for the aqueous solutions of C10NE-C10SO3/BSA mixtures coincided with that of C10NE-C10SO3 solutions. Viscometry showed that there is no significant change in the rheological properties for the C10NE-C10SO3/BSA mixed solutions. DLS showed that BSA monomers and mixed aggregates of C10NE-C10SO3 existed in the C10NE-C10SO3/BSA mixed solutions. From CD spectra no obvious modification of BSA structure in the presence of C10NE-C10SO3 mixtures was observed. The weak interactions between BSA and C10NE-C10SO3 might be explained in terms of the very low critical micelle concentration (cmc) of C10NE-C10SO3 mixtures that made the concentration of ionic surfactant monomers much lower than that needed for inducing the modification of BSA structure. In other words, the very strong synergism between oppositely charged cationic and anionic surfactants makes the formation of cationic-anionic surfactant mixed aggregates in the bulk solution a more favorable process than binding to proteins.     

Interaction between β‐Cyclodextrin and Mixed Cationic‐Anionic Surfactants (1): Thermodynamic Study

The interactions between β‐cyclodextrin (β‐CD) and the mixtures of cationic‐anionic surfactants in aqueous solution were investigated by surface tension and ¹H NMR measurements. It was shown that the critical micelle concentration (cmc) increased linearly with the increase of β‐CD concentration. Furthermore, β‐CD formed 1∶1 inclusion complex with both cationic and anionic surfactants in the mixed surfactant systems, and no significant selective inclusion was observed. The thermodynamic parameters of the inclusion process of β‐CD to mixed cationic‐anionic surfactants were calculated by a numerical method based on the surface tension measurements, and it was found that the inclusion process was both enthalpy and entropy favorable.     

Thermal and surface study of phenolic resin from cashew nut shell liquid cured by plasma treatment

In this study, phenolic resins from cashew nut shell liquid (CNSL) were applied as coating on Carbon Steel 1020 samples and successfully cured by plasma treatment or with hexamethylenetetramine (HMTA). The crosslinked samples were characterized by thermal analysis using thermogravimetry (TG), derivative thermogravimetry (DTG), differential scanning calorimetry (DSC), and atomic force microscopy (AFM) techniques in order to evaluate the thermal stability of these samples, as well as understand and study the curing process. TG/DTG curves showed that the thermal stability of the HMTA-cured resin was slightly higher than the resin treated by plasma. According to the DSC curves, HMTA-cured resin and plasma-treated resin exhibited only transition temperatures, so both resins were predominantly amorphous. Images generated by AFM provided qualitative evaluation of the resin surfaces, demonstrating that the coating surface with best homogeneity was cured by plasma treatment.