Alteplase and DNase for the treatment of pleural empyema in rats.

BACKGROUND AND OBJECTIVES: Adjunctive intrapleural fibrinolytic is an option to treat empyema at fibrinopurulent stage, but there is controversy about which should be use. Our objective is to evaluate the action of alteplase and/or desoxyribonuclease at physical and chemical properties in vitro pus derived from an experimental induced empyema in rats. METHODS: Streptococcus pneumoniae was introduced into the pleural cavity by thoracentesis through pleural pressure monitor. Animals were euthanized after 24 h, with macroscopic thoracic evaluation and measurement of amount of intrapleural liquid that was posteriorly stored at -80 °C. Selected samples were randomly distributed into four groups, then thawed at room temperature before exposure to one of the following: G1 = alteplase (n = 12), G2 = DNase (n = 12), G3 = alteplase + DNase (n = 12), or G4 = saline (n = 6). The mean molecular size in the fluid portion of the empyema was evaluated using dynamic light scattering; viscosity of the empyema fluid was measured using the drip method. RESULTS: Macroscopic showed purulent liquid, with fibrin and septation, with mean volume of 4.16 ml (0.5-8 ml). All samples were culture-positive for Streptococcus pneumoniae. Comparing with control, all experimental groups presented reduction of larger than 135 nm molecular size, but there was only significant difference with alteplase (p = 0,02). Viscosity reduced at all experimental groups, but increased at control. DNase group presented negative median (-5 mPa/s) of viscosity, and differed significantly from that observed in the control group (p = 0.04). CONCLUSIONS: Alteplase, DNase and alteplase + DNase changed significantly physical and chemical properties of experimental empyema at fibrinopurulent phase: alteplase reduced molecular size larger than 135 nm and DNase reduced viscosity.

Smart Starch-Poly( N-isopropylacrylamide) Hybrid Microgels: Synthesis, Structure, and Swelling Behavior.

In this study, we present hybrid microgels made of starch nanoparticles (SNPs) and poly( N-isopropylacrylamide) [p(NIPAM)]. SNPs were formed through nanoprecipitation. Hybrid microgels were prepared by surfactant-free precipitation polymerization (SFPP) or in the presence of surfactant precipitation polymerization (PP) at different NIPAM/SNP ratios. Dynamic light scattering results of hybrid microgels synthesized by SFPP revealed changes in volume phase transition temperature according to SNP amount, where the increase in the hydrophilic content caused small shifts in the lower critical solution temperature (LCST), reaching nearly 35 °C. Colloidal stability was improved with the SNP content, leading to increased stability because of the hydroxyl groups. Small-angle X-ray scattering indicates a core-shell structure above the LCST, where SNPs chains cover a p(NIPAM) core. Swelling curves experimentally obtained were analyzed using the Flory-Rehner model, where the interaction parameter (χ) has been modeled either by a series expansion of the swelling ratio or by a Hill-like equation for a cooperative thermotropic transition.

Synthesis and characterization of acetylated amylose and development of inclusion complexes with rifampicin.

Amylose (AM) tends to form single helical inclusion complexes with suitable agents. These complexes are considered promising biomaterial carrier since the guest molecules can be released later, leading to many applications, especially in the pharmaceutical industry. Rifampicin (RIF) has long been recognized as an active drug against Mycobacterium tuberculosis, however, the administration of RIF in high dosages can originate unwanted side-effects. Due to the fact that the use of native amylose (AM) in the formation of complexes is limited by their low water solubility, it was acetylated with a medium degree of substitution (DS), allowing solubilizing (0.5gL-1) acetylated amylose (AMA) in water at neutral pH, in opposition to that observed with native amylose (trace solubility). The resulting acetylated amylose was characterized by means of Fourier Transform Infrared (FT-IR) spectroscopy and Scanning Electron Microscopy (SEM). FT-IR results indicated that the acetylation of anhydroglucose units of amylose corresponds to a low DS, whereas SEM results suggested that the smooth surfaces of amylose granules were changed into rougher surfaces after acetylation. Ultraviolet absorption spectroscopy (UV-vis) analysis confirmed the formation and allowed the quantification of both native (AM-RIF) and acetylated (AMA-RIF) amylose inclusion complexes. Their characterization in solution was performed by dynamic light scattering (DLS) and zeta potential (ZP) measurements. The average size of inclusion complexes as determined by DLS, ranged between 70 and 100nm. Besides, ZP analysis showed that both complexes are more stable in the presence of RIF. This study may lead to the development of an effective method for the preparation of amylose inclusion complexes, which is beneficial to their further application in drug delivery systems.

Films based on neutralized chitosan citrate as innovative composition for cosmetic application.

In this work, citrate and acetate buffers, were investigated as neutralizers to chitosan salts in order to provide biocompatible and stable films. To choose the appropriate film composition for this study, neutralized chitosan citrate and acetate films, with and without the plasticizer glycerol, were prepared and characterized by thickness, moisture content, degree of swelling, total soluble matter in acid medium, simultaneous thermal analysis and differential scanning calorimetry. Chitosan films neutralized in citrate buffer showed greater physical integrity resulted from greater thicknesses, lower moisture absorbance, lower tendency to solubility in the acid medium, and better swelling capacities. According to thermal analyses, these films had higher interaction with water which is considered an important feature for cosmetic application. Since the composition prepared in citrate buffer without glycerol was considered to present better physical integrity, it was applied to investigate hyaluronic acid release in a skin model. Skins treated with those films, with or without hyaluronic acid, show stratum corneum desquamation and hydration within 10min. The results suggest that the neutralized chitosan citrate film prepared without glycerol promotes a cosmetic effect for skin exfoliation in the presence or absence of hyaluronic acid.

Silver nanoparticle-ionic silsesquioxane: a new system proposed as an antibacterial agent.

Spherical silver nanoparticles with an average size of ca. 5 nm were synthesized in aqueous medium using a charged silsesquioxane containing a quaternary ammonium group, the bridged 1,4-diazoniabicyclo[2.2.2]octane nitrate, as a stabilizer and size controller. For the first time this system was synthesized and applied as an antibacterial agent and its activity was confirmed with excellent results. The new system shows high stability, which can be confirmed by the unchanged UV-Vis band even one year later. The magnitude of the zeta-potential (ζ) (+24.7 mV) indicated electrostatic contribution for the silver nanoparticles stability and the signal showed that the nanoparticles have a positively charged surface. In vitro antibacterial tests were performed against E. coli, P. aeruginosa and S. aureus bacteria, and the minimum concentrations of silver in the nanoparticle form for complete inhibition of bacteria were 0.60, 1.1 and 2.0 µg mL-1, respectively. These values are very low when compared to the previous reports, making this system very promising. The cytotoxicity assay showed that these silver nanoparticles are safe for mammalian cells at the studied concentrations.

Sustained antioxidant activity of quercetin-loaded lipid-core nanocapsules.

Lipid-core nanocapsules (LNC) are vesicular nanocarriers prepared by solvent displacement. LNC have been previously prepared using medium-chain triglyceride and sorbitan monostearate as liquid and solid lipophilic components dispersed in the core, surrounded by poly(epsilon-caprolactone) (PCL). Our objective was to investigate the antioxidant activity of LNC containing quercetin (QUE), a radical scavenger, prepared with octyl methoxycinnamate and sorbitan monostearate as lipophilic core components and PCL as the polymer wall. We selected Saccharomyces cerevisae cells as the proposed biological model. QUE-LNC presented z-average diameter of 212 nm, pH of 5.51 and zeta potential of -11 mV. Multiple light scattering analysis (TurbiscanLab) showed a photon path length of 172 microm. Furthermore, a validated turbidimetric study determined that the density of particles in suspension was 1.66 x 10(13). DSC analysis showed that the melting temperature of PCL shifted to lower values when in contact with octyl methoxycinnamate indicating a molecular interaction. After 1 h (7 h), the QUE-LNC formulation and QUE solution incubated with H2O2 showed cell survival of 84.4% (87.7%) and 65.6% (7.3%), respectively. After 35 h of incubation, cell survival was 31.7% and 0.9%, respectively. The QUE-LNC showed sustained antioxidant activity and potential as a nanostructured material to formulate final products.

Dynamic rheological properties of native and cross-linked gliadin proteins.

A comparison of cross-linked and native gliadin suspensions, with respect to the state of protein globular structure was carried out using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS) and rheological analysis. Gliadin suspensions were also analyzed in the presence and absence of glycerol. DLS analysis showed that R(h) increased only with gliadin/EDC/NHS suspensions. However, Kratky plots revealed that gliadin and gliadin/L-cysteine maintained their globular shape even in absence or presence of glycerol. Rheological experiments revealed that gliadin and gliadin/L-cysteine suspension exhibited a similar profile with three main domains, and a sol-gel transition. Gliadin/EDC/NHS did not present any sol-gel transition, and this fact corroborates with DLS results and the hypothesis of lower protein-protein interaction, which are in agreement with G″ > G'.

Chitosan coated liposomes as an innovative nanocarrier for drugs.

Chitosomes are chitosan coated liposomes that represent an alternative to conventional liposomes since they present better stability and bioadhesivity. The aim of this work was to develop and evaluate the physico-chemical stability of melatonin (MEL)-loaded chitosomes as well as to compare their properties with that of MEL loaded liposomes. Structural characteristics of nanovesicles were also studied by dynamic light scattering and small angle X-ray scattering. The liposome and chitosome suspensions presented mean diameters between 150 nm and 254 nm, polydispersity indexes around 0.4, zeta potential values between -38 mV and -28 mV, pH values close to 4.0, MEL content close to 100% and encapsulation efficiency between 34.4% and 60.8%. Small angle X-rays scattering showed the presence of unilamelar structures, which were also observed by transmission electronic microscopy. Stability studies focusing on the particle diameter indicated that, within 90 days, the liposome suspensions had a decrease in mean diameter values and in polydispersity indexes, but no alterations were detected in zeta potentials and MEL content. The chitosome suspensions remained stable in relation to these parameters during 90 days. Multiple light scattering analysis (Turbiscan LAb) corroborated the the findings in the stability studies. The result sets pointed out the physico-chemical stability of chitosomes and the chitosan influence in their supramolecular structure.

Effects of the composite nanovesicles on the physical properties and cellular adhesion of chitosan films.

Chitosan films were prepared by the casting of a chitosan gel in absence and presence of composite nanovesicles. The microscopy images showed the occurrence of agglomerates on the surface and internal pores when the nanovesicles were added to the films, differently from the smooth surface of the pure chitosan films. Despite the hydrophobic character that composite nanovesicles gave to the chitosan films, as showed by the reduction of the water permeation at prolonged times, there was a reduction on the contact angle values for these samples related to the roughness of the surface. The peak of water desorption observed on calorimetric analysis of chitosan was shifted to higher values when the nanovesicles were added to the films. Furthermore, the desappearance of Tg on the films containing nanovesicles denoted their plastifier effect in the chitosan film. The swelling results showed higher water diffusion at the first times for the films containing nanovesicles because of the pores observed by microscopy. However, at prolonged times, there was a reduction on the swelling because of the lipofilic composition of the nanovesicles. Furthermore, the presence of nanovesicles led to a reduction on the water content in the chitosan films. Due to the effect on the physical properties of the chitosan films, the addition of nanovesicles on discrete concentrations contributed to the cell adhesion.

Echinococcus granulosus antigen B structure: subunit composition and oligomeric states.

BACKGROUND: Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. METHODOLOGY/PRINCIPAL FINDINGS: The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. CONCLUSIONS/SIGNIFICANCE: For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

Internal structural characterization of triblock copolymer micelles with looped corona chains.

We report the characterization through SAXS measurements of micelles produced from a new series of block copolymers: one diblock and four triblock copolymers bearing short poly[5-(N,N-diethylamino)isoprene] and long polystyrene blocks. Micellar aggregates produced in DMF (selective solvent for polystyrene) from the same set of samples were previously successfully characterized through light scattering measurements. The X-ray scattering profiles of starlike (from the diblock copolymer sample) and flowerlike micelles (from the triblock copolymers samples) could be fitted using the spherical copolymer micelle model proposed by Pedersen and Gerstenberg (Macromolecules 1996, 29, 1363.) where in the case of flowerlike micelles, the particles were understood as formed by hypothetical diblock copolymers having half of the true polymeric molar mass. Using the spherical copolymer micelle model, it could be possible to attest the unswollen nature of the micellar cores. The total micellar size suggested thus that the chains forming the corona are extended which is mainly related to a small core surface area per corona chain entering the core (Ac/n), which also induced a small number of aggregation (N(agg)) of all self-assembled particles. The total micellar size fits well with our previous light scattering measurements.

Electroformation of giant vesicles from an inverse phase precursor.

We discuss a simple modification of the well-known method of giant vesicle electroformation that allows for a direct addition of water-soluble species to the phospholipid bilayers. Using this modified method, we prepare phospholipid vesicles decorated with chitosan, a water-soluble polysaccharide currently investigated for potential pharmacological applications. We find that the method allows this polysaccharide with primary amino groups on every glucose subunit to be tightly bound to the membrane, rather than simply being encapsulated.

Aggregation behavior of a new series of ABA triblock copolymers bearing short outer A blocks in B-selective solvent: from free chains to bridged micelles.

A combination of dynamic (DLS) and static (SLS) light scattering measurements was employed to study the self-assembly behavior of a new series of triblock copolymers bearing poly[5-(N,N-diethylamino isoprene)] (PAI) short outer blocks and polystyrene (PS) as the major middle block. Previously, it was verified that PAI outer blocks can be quaternized leading the formation of crew-cut aggregates in water (Riegel, I. C.; Eisenberg, A.; Petzhold, C. L.; Samios, D. Langmuir 2002, 18, 3358). Herein, we focus on the copolymer's ability in the nonquaternized version to undergo self-aggregation in dimethylformamide (DMF), a selective solvent for the middle block. Light scattering measurements showed that formation of well-defined flowerlike micelles is likely to occur. Aggregates with a relatively narrow distribution, small average size, and number of aggregation ranging from 21 to 39 chains/micelle were experimentally observed. The results also suggested that approximately 5-6 polymeric units per each short outer block are needed to induce aggregation. The middle block length governs the size of the micelles and influences the number of aggregation of the resultant particles as well. Furthermore, when the polystyrene middle block was particularly long (degree of polymerization DP > 600), dynamic and static light scattering measurements suggested the formation of bridged micelles in an open structure in concentrations as low as 15 mg mL-1.

Effect of the alkaline treatment on the ultrastructure of C-type starch granules.

The effect of alkaline treatment on the ultrastructure of C-type starch granules was investigated during the alkaline extraction of Araucaria angustifolia (pinhao) starch. The efficiency in protein removal was evaluated using intrinsic fluorescence and Kjeldahl's method. In parallel, morphological changes of starch granules were observed using scanning electron microscopy and atomic force microscopy. The starch crystallinity was monitored by wide-angle X-ray scattering and the lamellar structure was studied by small-angle X-ray scattering (SAXS). The paracrystalline model was employed to interpret the SAXS curves. It was found that the granular organization was significantly altered when alkaline solutions were used during the extraction. A partial degradation of B-type allomorph of starch and a significant compression of semicrystalline growth rings were observed.

Single crystals of V-amylose complexed with alpha-naphthol.

Lamellar square single crystals of V-amylose were obtained by adding alpha-naphthol to metastable dilute aqueous solutions of synthetic amylose chains with an average degree of polymerization of 100. The morphology and structure of the crystals were studied using low-dose transmission electron microscopy including high-resolution imaging, as well as electron and X-ray diffraction. The crystals are crystallized in a tetragonal P4(1)2(1)2 or P4(3)2(1)2 space group with unit cell parameters, calculated from X-ray diffraction data, a = b = 2.2844 nm (+/-0.0005) and c = 0.7806 nm (+/-0.001), implying the presence of two amylose chains per unit cell. High-resolution lattice images of the crystals confirmed that the amylose chains were crystallized as 8-fold helices corresponding to the repeat of four maltosyl units.

Self-assembly and structural characterization of Echinococcus granulosus antigen B recombinant subunit oligomers.

Echinococcus granulosus antigen B is an oligomeric protein of 120-160 kDa composed by 8-kDa (AgB8) subunits. Here, we demonstrated that the AgB8 recombinant subunits AgB8/1, AgB8/2 and AgB8/3 are able to self-associate into high order homo-oligomers, showing similar properties to that of parasite-produced AgB, making them valuable tools to study AgB structure. Dynamic light scattering, size exclusion chromatography and cross-linking assays revealed approximately 120- to 160-kDa recombinant oligomers, with a tendency to form populations with different aggregation states. Recombinant oligomers showed helical circular dichroism spectra and thermostability similar to those of purified AgB. Cross-linking and limited proteolysis experiments indicated different degrees of stability and compactness between the recombinant oligomers, with the AgB8/3 one showing a more stable and compact structure. We have also built AgB8 subunit structural models in order to predict the surfaces possibly involved in electrostatic and hydrophobic interactions during oligomerization.

Physico-chemical characterization and in vivo evaluation of indomethacin ethyl ester-loaded nanocapsules by PCS, TEM, SAXS, interfacial alkaline hydrolysis and antiedematogenic activity.

Nanocapsules are vesicular drug carriers constituted of an oil core, a polymeric wall, and surfactants. A general understanding about the influence of the polymeric wall of nanocapsules on the release profiles of drugs is not known. So, this work was devoted to characterize formulations prepared without polymer or containing it at different concentrations. The indomethacin ethyl ester was used as model and the strategy was based on its interfacial alkaline hydrolysis simulating a sink condition for the release. The antiedematogenic activity in rats for ester-loaded-nanocarriers was also evaluated. The nanocapsules (NC) and nanoemulsion (NE) presented particle sizes below 300 nm, polydispersity lower than 1.2 and pH around 5. SAXS analyses showed that the sorbitan monostearate is dissolved in the oil and the polymer presents regions of crystallinity independently on the PCL concentration. TEM analyses showed droplets (NE) and spherical particles (NC). The time for the total disappearance of the ester varied from 12 h to 24 h depending on the polymer concentration. The biexponential model showed that the indomethacin ester was essentially entrapped within the nanocarriers in an extension of 85 to 95%. The half-lives varied from 147 to 289 min for the sustained phases and from 3 to 6 min for the burst phases. The ester-loaded-NC showed significant antiedematogenic activity, while the ester-loaded-NE did not inhibit the carrageenin-induced paw edema. The nanocapsules promoted the absorption of the indomethacin ethyl ester and the presence of the polymer is important to achieve the pharmacological effect.

Diffusion and mathematical modeling of release profiles from nanocarriers.

The aim of this work was to establish models and to differentiate the kinetic release behavior of drug models from nanocapsules, nanoemulsion and nanospheres by physico-chemical characterization and release experiments. SAXS analysis showed that the polymer is organized in the nanocapsules, while in the nanospheres the sorbitan monostearate is organized and acts as an impurity of the poly(epsilon-caprolactone) suggesting that constituents in these nanocarriers are differently organized. Formulations presented particle sizes ranging from 178 to 297 nm, probe content from 0.981 to 0.997 mg/mL, pH values from 4.90 to 5.10 and zeta potential from -37.9 to -51.9 mV. The kinetic experiments showed that the nanostructures present similar behaviors when the probe is adsorbed on the nanocarriers (indomethacin-loaded formulations). However, when the probe is entrapped within the nanocarriers (indomethacin ethyl ester-loaded formulations), nanocapsules, nanospheres and nanoemulsion presented different kinetic behaviors. Mathematical modeling of the release profiles was conducted, showing that the presence of the polymer increases the half-lives of the burst phases (5.9, 4.4 and 2.7 min) while the presence of the oil increases the half-lives of the sustained phases (288.8, 87.7 and 147.5 min) for nanocapsules, nanospheres and nanoemulsion, respectively.

Time-resolved optical Kerr-effect investigation on CS2/polystyrene mixtures.

The relaxation dynamics of carbon disulfide are investigated in mixtures with polystyrene (PS) using the time-resolved optical heterodyne-detected optical Kerr effect (OHD-OKE). The data are analyzed using both the model-dependent approach, which assumes four distinct temporal responses, and the model-independent Fourier transform approach, which generates a spectral response that can be compared with results obtained by depolarized Rayleigh scattering. A slow dynamics is observed for the OHD-OKE transient decaying exponentially with a time constant that varies from 1.68 ps for neat CS2 to 3.76 ps for the most concentrated CS2PS mixture. The increase of this time constant accompanies an increase in the viscosity of the mixture, so we can associate this component with the diffusive reorientation process of the induced polarizability anisotropy of the carbon disulfide in the mixture. The short-time nuclear response is characterized in the frequency domain by a broad band that peaks around 30 cm(-1) for neat carbon disulfide, and is associated with a complex relaxation pattern. The vibrational distribution shifts to higher frequencies when the PS concentration is increased in the mixture. This result is discussed in terms of an increase in the interaction strength between the PS phenyl rings and the carbon disulfide molecules.

Jack bean urease (EC 3.5.1.5) aggregation monitored by dynamic and static light scattering.

Aggregation of jack bean urease (JBU) is involved in many alterations of its biological properties, notably the ureolytic and entomotoxic activities. In order to investigate this phenomenon, protein aggregates were characterized by dynamic (DLS) and static light scattering (SLS) spectroscopies through determination of apparent hydrodynamic radii, the average molecular masses, radii of gyration and second virial coefficients. No effect of disulfide reducing agents on protein association was observed contrasting with previous reports implicating their function in the prevention of JBU aggregation. The influence of freeze-thawing cycles on protein aggregation was also investigated. Our results showed that after freeze-thawing cycles the native form of JBU with apparent hydrodynamic radius of 7 nm and radius of gyration of 12 nm is replaced by high-order oligomers and this aggregation is not reverted neither by dithiothreitol (DTT) treatment nor by high concentration of salts. Altogether the data help to understand the complex behavior of JBU in solution and may correlate with the diversity of biological properties of this enzyme.