Preparation and characterization of NaF/Chitosan supramolecular complex and their effects on prevention of enamel demineralization.

Fluoridated mouthrinse is indicated for individuals with high risk of caries. Chitosan (Chit) exhibits antibacterial properties, but little is known about its effects on enamel when combined with sodium fluoride (NaF) to form NaF/Chit supramolecular complexes. In our study, NaF/Chit supramolecular complexes structured as microparticles were synthetized and characterized, and their effects on human enamel were evaluated after cariogenic challenge simulating the daily mouthrinse use. Initially, NaF/Chit complex formation was investigated based on several titrations by measuring the zeta potential, electrical conductivity (κ), hydrodynamic diameter (Dh), viscosity (η) and heat flow (by isothermal titration calorimetry) against the molar ratio [NaF]/[Chitmonomer], which allowed us to identify the interactions between Chit-NaF with a stoichiometry of approximately 0.68. Spontaneous microparticle formation was observed. Samples of enamel blocks were prepared and divided into eight groups (n = 10/group): (i) 0.2% Chit; (ii) 0.2% NaF; (iii) 0.2% NaF/Chit suspension; (iv) 0.2% acetic acid; (v) 0.05% Chit; (vi) 0.05% NaF; (vii) 0.05% NaF/Chit suspension; and (viii) 0.05% acetic acid. Cariogenic challenge was performed in each sample by cycling in demineralization and remineralization solutions for 7 days. Before each demineralization cycle, the corresponding substances were passively applied daily for 90 s, even in groups with 0.02% concentration. After 7 days, samples were examined for Knoop hardness (KHN) measurements. The data were analyzed by repeated-measures ANOVA and Tukey tests (α = 0.05). The 0.2% NaF and 0.2% NaF/Chit groups showed higher KHNpost-challenge values than the other groups. The 0.2% NaF/Chit microparticle suspension reduced the enamel hardness loss after cariogenic challenge as effectively as the 0.2% NaF solution and demonstrated potential for use in a formulation with anti-caries effects.

Synthesis of a chitosan nanoparticle suspension and its protective effects against enamel demineralization after an in vitro cariogenic challenge.

OBJECTIVE: Our study aims to synthesize, characterize, and determine the effects of a ChNPs suspension on human enamel after cariogenic challenge via pH-cycling. METHODOLOGY: ChNPs were synthesized by ion gelation and characterized by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering. Forty enamel blocks were divided into four groups (n=10/group): (i) ChNPs suspension; (ii) chitosan solution; (iii) 0.05% sodium fluoride (NaF) solution; and (iv) distilled water. Specimens were exposed to cariogenic challenge by cycling in demineralization solution (3 h) and then remineralized (21h) for 7 days. Before each demineralization cycle, the corresponding solutions were passively applied for 90 s. After 7 days, specimens were examined for surface roughness (Ra) and Knoop hardness (KHN) before and after the cariogenic challenge; % KHN change (variation between initial and final hardness), and surface topography by an optical profilometer. The data were analyzed by repeated-measures ANOVA, One-way ANOVA, and Tukey tests (α=0.05). RESULTS: TEM images showed small spherical particles with diameter and zeta potential values of 79.3 nm and +47.9 mV, respectively. After the challenge, all groups showed an increase in Ra and a decrease in KHN values. Optical profilometry indicated that ChNPs- and NaF-treated specimens showed uneven roughness interspersed with smooth areas and the lowest %KHN values. CONCLUSION: The ChNPs suspension was successfully synthesized and minimized human enamel demineralization after a cariogenic challenge, showing an interesting potential for use as an oral formulation for caries prevention.

Evaluation of biological activities, structural and conformational properties of bovine beta- and alpha-trypsin isoforms in aqueous-organic media.

The study of the biological activity of trypsin isoforms in aqueous-organic media is of great interest to various fields of knowledge and biochemistry applications. Thus enzymatic, structural, and energetic properties of bovine ß- and α-trypsin isoforms were compared in aqueous-organic media using 30 mg of each isoform. The results showed that the changes induced on the structure and activity of the same trypsin isoform occur at different concentrations. Better results for activity (ionic strength of 0.11 mol·L-1, at 37 °C and pH 8.0) were found in 0-40% of ethanolic media in which the activity for ß-trypsin was about 60% higher than ɑ-trypsin. The ethanolic system does not cause significant changes in the level of secondary structure but the ß-trypsin isoform undergoes a major rearrangement. The use of until 60% (v/v) ethanol showed that ß-trypsin presents a denaturation process 17% more cooperative. The organic solvent causes redistribution in the supramolecular arrangement of both isoforms: all concentrations used induced the ß-trypsin molecules to rearrange into agglomerates. The ɑ-trypsin rearranges into agglomerates up to 60% (v/v) of ethanol and aggregates at 80% (v/v) of ethanol. Both isoforms keep the enzymatic activity up to 60% (v/v) of ethanol.

Synthesis of a chitosan nanoparticle suspension and its protective effects against enamel demineralization after an in vitro cariogenic challenge

Abstract Objective Our study aims to synthesize, characterize, and determine the effects of a ChNPs suspension on human enamel after cariogenic challenge via pH-cycling. Methodology ChNPs were synthesized by ion gelation and characterized by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering. Forty enamel blocks were divided into four groups (n=10/group): (i) ChNPs suspension; (ii) chitosan solution; (iii) 0.05% sodium fluoride (NaF) solution; and (iv) distilled water. Specimens were exposed to cariogenic challenge by cycling in demineralization solution (3 h) and then remineralized (21h) for 7 days. Before each demineralization cycle, the corresponding solutions were passively applied for 90 s. After 7 days, specimens were examined for surface roughness (Ra) and Knoop hardness (KHN) before and after the cariogenic challenge; % KHN change (variation between initial and final hardness), and surface topography by an optical profilometer. The data were analyzed by repeated-measures ANOVA, One-way ANOVA, and Tukey tests (α=0.05). Results TEM images showed small spherical particles with diameter and zeta potential values of 79.3 nm and +47.9 mV, respectively. After the challenge, all groups showed an increase in Ra and a decrease in KHN values. Optical profilometry indicated that ChNPs- and NaF-treated specimens showed uneven roughness interspersed with smooth areas and the lowest %KHN values. Conclusion The ChNPs suspension was successfully synthesized and minimized human enamel demineralization after a cariogenic challenge, showing an interesting potential for use as an oral formulation for caries prevention.

Development and in vivo evaluation of chitosan-gel containing Mitracarpus frigidus methanolic extract for vulvovaginal candidiasis treatment.

Vulvovaginal candidiasis (VVC) is characterized by inflammatory changes in the vaginal mucosa caused by abnormal colonization of Candida species. Traditional topical therapies using reference antifungal drugs usually present several issues and limitations for VVC treatment. Thus, the interest in new vaginal formulations, mainly those based on compounds from natural origin, has been growing over the last years. Methanolic extract from the plant species Mitracarpus frigidus (Willd. Ex Reem Schult.) K. Schum (MFM) has presented potential antifungal activity against C. albicans vaginal infection. Here, we aimed to develop and characterize a gynecological gel formulation based on chitosan containing MFM and to evaluate its anti-C. albicans effectiveness in the treatment of VVC. First, MFM was incorporated into a gel formulation based on chitosan in three final concentrations: 2.5 %, 5.0 %, and 10.0 %. Next, these gel formulations were subjected to stationary and oscillatory rheological tests. Finally, the gel was tested in an experimental VVC model. The rheological tests indicated pseudoplastic fluids, becoming more viscous and elastic with the increase of the extract concentration, indicating intermolecular interactions. Our in vivo analyses demonstrated a great reduction of vulvovaginal fungal burden and infection accompanied with the reduction of mucosal inflammation after MFM chitosan-gel treatment. The present findings open perspectives for the further use of the MFM-chitosan-gel formulation as a therapeutic alternative for VVC treatment.

Synthesis and characterization of TPP/chitosan nanoparticles: Colloidal mechanism of reaction and antifungal effect on C. albicans biofilm formation.

In the present study chitosan (Chit) nanoparticles were synthetized by the ionic gelation process, using tripolyphosphate (TPP) as crosslinking agent. The TPP/Chit nanoparticle formation was evaluated by titrations, measuring electrical conductivity (k), zeta potential (ZP), hydrodynamic diameter (Dh), viscosity (η) and heat by isothermal calorimetry (ITC). The antifungal effects were evaluated by C. albicans time-kill assays, inhibition of C. albicans initial adhesion and biofilm formation in comparison with nystatin and chitosan. Conductometric titration exhibited a typical precipitation profile, with an inflection at molar ratio of [TPP]/[Chitmon] ≈ 0.3, suggesting a 1:3.3 stoichiometry. The highest Dh, ZP and η values were shown at the beginning of titrations, due to the intramolecular repulsion between Chit-Chit. With addition of TPP, the values showed gradual reduction, with an intermediary transition at [TPP]/[Chitmon] ≈ 0.16, which was attributed to the partial breakdown of interchain crosslinking and formation of discrete charged aggregates. After this point, reaction should occur by neutralization of these assemblies, causing new reduction in values of Dh, ZP and η until [TPP]/[Chitmon] ≈ 0.3, when they reached their lowest values. ITC experiment also showed the occurrence of two bindings (K1 = 3.6 × 103 and K2 = 7.7 × 104), which were entropy driven. Biological results showed lower C. albicans viability for TPP/Chit over 24 h compared with chitosan and nystatin at MIC and 2 MIC. Moreover, TPP/Chit showed 25-50% inhibition of C. albicans adhesion and biofilm formation. The results showed that TPP/Chit nanoparticles reduced the initial adhesion and biofilm formation of C. albicans and demonstrated potential for use in a formulation for the treatment of oral candidiasis.

Hydrophobic nanoprecipitates formed by benzoylphenylureas and ß-cyclodextrin inclusion compounds: synthesis, characterization and toxicity against aedes aegypti larvae.

The aim of this work was to synthesize and characterize the inclusion compounds formed by the complexation of ß-cyclodextrin (ßCD) with insecticides from the class of benzoylphenylureas (BPUs), named novaluron (NOV) and diflubenzuron (DIF), beyond evaluate their larvicidal activity against Aedes aegypti larvae. Solid state characterization by FTIR showed changes in the main peaks of BPUs and ßCD, suggesting the formation of inclusion compounds in solid phase. DTA and TGA thermal analysis showed changes in temperatures of BPUs decomposition as result of molecular interactions. 1H NMR experiments allowed to observe the occurrence of interactions in solution through changes in chemical shifts of BPUs aromatic hydrogens. However, the presence of H-H intermolecular correlations in 2D ROESY was found only for the DIF/ßCD complex, suggesting different topology for each complex. Such hypothesis was corroborated by thermodynamic analysis using ITC, which showed different profile of titration curves, beyond endothermic and exothermic interactions for NOV/ßCD and DIF/ßCD complexes, respectively. DLS titrations of BPUs or BPUs/ßCD DMSO solutions in aqueous solution demonstrated that the spontaneously formed hydrophobic nanoprecipitates (HNPs) have different profile of sizes depending on the BPU/ßCD system, corroborating also with the hypothesis about the existence of different topologies for each complex. Finally, the HNPs of inclusion compounds showed to be more efficient than free BPUs, allowing proposing a new insecticide formulation.

Evaluation of the interaction between polymyxin B and Pseudomonas aeruginosa biofilm and planktonic cells: reactive oxygen species induction and zeta potential.

BACKGROUND: Although the most widely accepted mechanism of action for polymyxins is related to bacterial lysis via disruption, we hypothesized that this antimicrobial drug class could have other effects on Pseudomonas aeruginosa planktonic and sessile cells. Little is known regarding oxidative burst and zeta potential (ZP) data associated with the interaction between polymyxin B and P. aeruginosa cells. The present study evaluated endogenous reactive oxygen species (ROS) production and changes in the net charges of biofilm and planktonic cells in response to polymyxin B. RESULTS: Polymyxin B induced concentration-dependent killing at all concentrations tested in planktonic and sessile cells from P. aeruginosa strains. Sublethal concentrations of polymyxin B induced oxidative burst. ROS production was higher in resistant planktonic cells than in biofilm cells but this was not observed for susceptible cells. Moreover, no net surface charge alterations were observed in planktonic cells from a susceptible strain treated with polymyxin B, but a significant increase of ZP was noted in planktonic cells from a resistant strain. CONCLUSION: Oxidative burst generated by planktonic and sessile cells from P. aeruginosa strains against polymyxin B indicates that ROS may have an important role in the mechanism of action of this drug. ZP data revealed that electrostatic interactions of the cationic peptide with the anionic surface of the cells are strain-dependent. Therefore, we suggested that the intracellular effects of polymyxin B should be further investigated to understand polymyxin B-induced stress in P. aeruginosa.

Biophysical Effects of a Polymeric Biosurfactant in Candida krusei and Candida albicans Cells.

This study evaluated the effects of a polymeric biosurfactant produced by Trichosporon montevideense CLOA72 in the adhesion of Candida albicans and Candida krusei cells to human buccal epithelial cells and its interference in biofilm formation by these strains. The biofilm inhibition by biosurfactant (25 mg/mL) in C. krusei and C. albicans in polystyrene was reduced up to 79.5 and 85 %, respectively. In addition, the zeta potential and hydrodynamic diameter of the yeasts altered as a function of the biosurfactant concentration added to the cell suspension. The changes in the cell surface characteristics and the interface modification can contribute to the inhibition of the initial adherence of yeasts cells to the surface. In addition, the analyses of the biofilm matrix and planktonic cell surfaces demonstrated differences in carbohydrate and protein concentrations for the two studied strains, which may contribute to the modulation of cell adhesion or consolidation of biofilms, especially in C. krusei. This study suggests a possible application of the of CLOA72 biosurfactant in inhibiting the adhesion and formation of biofilms on biological surfaces by yeasts of the Candida genus.

Heteroresistance to Itraconazole Alters the Morphology and Increases the Virulence of Cryptococcus gattii.

Cryptococcus gattii is the main etiological agent of cryptococcosis in immunocompetent individuals. The triazole drug itraconazole is one of the antifungals used to treat patients with cryptococcosis. Heteroresistance is an adaptive mechanism to counteract the stress of increasing drug concentrations, and it can enhance the ability of a microorganism to survive under antifungal pressure. In this study, we evaluated the ability of 11 C. gattii strains to develop itraconazole heteroresistance. Heteroresistant clones were analyzed for drug susceptibility, alterations in cell diameter, capsule properties, and virulence in a murine model. Heteroresistance to itraconazole was intrinsic in all of the strains analyzed, reduced both the capsule size and the cell diameter, induced molecular heterogeneity at the chromosomal level, changed the negatively charged cells, reduced ergosterol content, and improved the antioxidant system. A positive correlation between surface/volume ratio of original cells and the level of heteroresistance to itraconazole (LHI) was observed in addition to a negative correlation between capsule size of heteroresistant clones and LHI. Moreover, heteroresistance to itraconazole increased the engulfment of C. gattii by macrophages and augmented fungal proliferation inside these cells, which probably accounted for the reduced survival of the mice infected with the heteroresistant clones and the higher fungal burden in lungs and brain. Our results indicate that heteroresistance to itraconazole is intrinsic and increases the virulence of C. gattii. This phenomenon may represent an additional mechanism that contributes to relapses of cryptococcosis in patients during itraconazole therapy.

Development of sulfadiazine-decorated PLGA nanoparticles loaded with 5-fluorouracil and cell viability.

The aim of this work was to synthesize sulfadiazine-poly(lactide-co-glycolide) (SUL-PLGA) nanoparticles (NPs) for the efficient delivery of 5-fluorouracil to cancer cells. The SUL-PLGA conjugation was assessed using FTIR, 1H-NMR, 13C-NMR, elemental analysis and TG and DTA analysis. The SUL-PLGA NPs were characterized using transmission and scanning electron microscopy and dynamic light scattering. Additionally, the zeta potential, drug content, and in vitro 5-FU release were evaluated. We found that for the SUL-PLGA NPs, Dh = 114.0 nm, ZP = -32.1 mV and the encapsulation efficiency was 49%. The 5-FU was released for up to 7 days from the NPs. Cytotoxicity evaluations of 5-FU-loaded NPs (5-FU-SUL-PLGA and 5-FU-PLGA) on two cancer cell lines (Caco-2, A431) and two normal cell lines (fibroblast, osteoblast) were compared. Higher cytotoxicity of 5-FU-SUL-PLGA NPs were found to both cancer cell lines when compared to normal cell lines, demonstrating that the presence of SUL could significantly enhance the cytotoxicity of the 5-FU-SUL-PLGA NPs when compared with 5-FU-PLGA NPs. Thus, the development of 5-FU-SUL-PLGA NPs to cancer cells is a promising strategy for the 5-FU antitumor formulation in the future.

Pharmaceutical composition of valsartan: beta-cyclodextrin: physico-chemical characterization and anti-hypertensive evaluation.

Valsartan, a water-insoluble drug, is mainly used in the treatment of hypertension albeit with reduced oral bioavailability. The aim of work was to develop a valsartan:beta-cyclodextrin (VAL:beta-CD) pharmaceutical composition in order to improve its water solubility and bioavailability. The VAL:beta-CD complexes were prepared by the kneading, solid dispersion and freeze-drying methods, of which the freeze-drying method (FDY) was found to be the best to prepare an inclusion complex. A physical mixture PM was also prepared. Complexes were characterized by thermal analysis, Fourier transformed-infrared (FTIR) spectroscopy, Powder X-ray diffractometry, intrinsic dissolution and NMR (2D-ROESY). Phase-solubility analysis showed A(L)-type diagrams with beta-cyclodextrin (beta-CD). Microcalorimetric titrations suggested the formation of 1:1 inclusion complex between VAL and beta-CD. The apparent stability constants K(1:1) calculated from phase-solubility plots were 165.4 M(-1) (298 K), 145.0 M(-1) (303 K) and 111.3 M(-1) (310 K). In vivo experiments in rats showed that reduction in arterial pressure for the FDY complex is better than with valsartan used alone. The better activity of FDY can be attributed to the higher solubility of valsartan after inclusion in the cyclodextrin cavity, as suggest by the intrinsic dissolution studies.

Caracterização físico-química de complexos de insulina: dimetil-beta-ciclodextrina e insulina: hidroxipropil-beta-ciclodextrina e avaliação da influência do tipo de complexo na produção de microesferas biodegradáveis / Physico-chemical caracterization of insulin: dimethyl-beta-cyclodextrin and insulin:hydroxypropil-beta-cyclodextrin complexes and evaluation of the kind of complexes on the biodegradable microspheres preparation

A etapa principal na ativação e ligação da insulina ao seu receptor é a dissociação dos hexâmeros do hormônio, normalmente presente nas preparações farmacêuticas, para a forma monomérica bioativa. A utilização de diferentes ciclodextrinas (CDs) como adjuvantes em formulações contendo insulina vem sendo explorada e os estudos realizados demonstram que estas substâncias podem aumentar a absorção da insulina principalmente por diminuírem sua capacidade de formar dímeros e hexâmeros em meio aquoso. No presente trabalho, complexos de insulina:hidroxipropil-beta-ciclodextrina (INS:HP-beta-CD) e insulina:dimetil-beta-ciclodextrina (INS:DM-beta-CD) foram caracterizados utilizando técnicas de titulação calorimétrica isotérmica e espalhamento dinâmico de luz. Por meio da titulação calorimétrica foram determinados os parâmetros termodinâmicos de interação entre a insulina e as CDs utilizadas, sugerindo que o mecanismo de complexação ocorre com aumento de entropia para ambos os sistemas. Os experimentos de espalhamento dinâmico de luz não indicaram diminuição do diâmetro hidrodinâmico das espécies moleculares de insulina após a complexação com as CDs. Os complexos INS:HP-beta-CD e INS:DM-beta-CD foram encapsulados em microesferas (MEs) de PLGA 50:50. A caracterização das MEs obtidas revelou aumento considerável na taxa de encapsulamento de insulina quando complexada com as CDs sem que ocorresse diferença significativa no diâmetro das partículas em função da complexação.

The main stage in the linking and activation of the specific receptors by the insulin is the dissociation of this peptide hexamers, normally present in pharmaceutical formulations, in the monomeric active form. Because of this, the use of different cyclodextrins as adjuvants in the formulations containing insulin has been explored and the realized studies have demonstrated that the cyclodextrins can increase the absorption of the insulin mainly by reducing the ability of insulin oligomerization in aqueous media. In this work, complexes of INS:HP-beta-CD and INS:DM-beta-CD have been characterized by the use of isothermal calorimetry titration (ICT) and dynamic scattering of light. By means of ICT, the thermodynamic parameters of interaction between insulin and the cyclodextrins have been determined, and it was observed that the complexation occurs with an increase of entropy for both systems. The experiments of dynamic scattering of light have not showed reduction in the size of insulin particles, which could indicate the dissociation of insulin hexamers after the complexation with cyclodextrins. Then, the INS: HP-beta-CD and INS:DM-beta-CD complexes were encapsulated in PLGA microspheres. These systems were characterized and it was not observed any significant difference in the microspheres diameter, but a considerable increase in the hormone loading after the complexation with HP-beta-CD and DM-beta-CD was shown.