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'.

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.

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.