Diethylaminoethyl- chitosan as an efficient carrier for siRNA delivery: Improving the condensation process and the nanoparticles properties.

Chitosan has been indicated as a promising carrier for the preparation of small interfering RNA (siRNA) delivery systems due to its remarkable properties. However, its weak interactions with siRNA molecules makes the condensation of siRNA molecules into nanoparticles difficult. In this work, a non-viral gene delivery system based on diethylaminoethyl chitosan (DEAE-CH) derivatives of varied Mw (25-230 kDa) having a low degree of substitution of 15% was investigated. The presence of secondary and tertiary amino groups strengthened the interaction of siRNA and DEAE-CH derivatives of higher Mw (130 kDa to 230 kDa) and provided the preparation of spherical nanoparticles at low charge ratios (N/P 2 to 3) with low polydispersities (0.15 to 0.2) in physiological ionic strength. Nanoparticles prepared with all derivatives exhibited remarkable silencing efficiencies (80% to 90%) on different cell lines (HeLa, MG-63, OV-3) by adjusting the charge ratios. A selected PEG-folic acid labeled derivative (FA-PEG-DEAE15-CH230) was synthesized and its nanoparticles completely inhibited the mRNA expression level of TNF-α in RAW 264.7 macrophages. The study demonstrates that the insertion of DEAE groups provides improved physical properties to chitosan-siRNA nanoparticles and holds potential for in vivo applications.

Chitosan derivatives for gene transfer: effect of phosphorylcholine and diethylaminoethyl grafts on the in vitro transfection efficiency.

The purpose of this work was to improve the functional properties of chitosan for gene transfer by inserting phosphorylcholine (PC) and diethylaminoethyl (DEAE) groups into the main chain. A series of derivatives containing increasing contents of DEAE and a fixed content of PC groups were synthesized and characterized, aiming to evaluate the effect of these groups on the nanoparticles' properties and the in vitro transfection efficiency. The derivatives were soluble at physiological pH levels and all derivatives were less cytotoxic than the control, the lipid lipofectamine. The obtained derivatives complexed pDNA into nanoparticles with smaller sizes and higher zeta potentials than plain chitosan. The in vitro transfection was performed with nanoparticles prepared at pH 6.3 and 7.4 and the results showed that nanoparticles prepared with derivatives containing 20% of PC groups (PC18-CH) and high degrees of substitution by DEAE (PC20-CH-DEAE100, CH-DEAE80, CH-DEAE100) displayed the better transfection efficiencies in HeLa cells, reaching relative values comparable to lipofectamine. The most effective derivative, PC18CH, was selected for complexation with siRNA and its complexes demonstrated an in vitro knockdown efficiency highly dependent on the N/P ratio. Our combined results indicated that, by means of controlled modifications, the limitations of chitosan can be overcome to obtain more effective carriers based on chitosan, and the derivatives here studied hold potential for in vivo studies.

Toxicity and Loss of Mitochondrial Membrane Potential Induced by Alkyl Gallates in Trypanosoma cruzi.

American trypanosomiasis or Chagas disease is a debilitating disease representing an important social problem that affects, approximately, 10 million people in the world. The main aggravating factor of this situation is the lack of an effective drug to treat the different stages of this disease. In this context, the search for trypanocidal substances isolated from plants, synthetic or semi synthetic molecules, is an important strategy. Here, the trypanocidal potential of gallates was assayed in epimastigotes forms of T. cruzi and also, the interference of these substances on the mitochondrial membrane potential of the parasites was assessed, allowing the study of the mechanism of action of the gallates in the T. cruzi organisms. Regarding the preliminary structure-activity relationships, the side chain length of gallates plays crucial role for activity. Nonyl, decyl, undecyl, and dodecyl gallates showed potent antitrypanosomal effect (IC50 from 1.46 to 2.90 µM) in contrast with benznidazole (IC50 = 34.0 µM). Heptyl gallate showed a strong synergistic activity with benznidazole, reducing by 10(5)-fold the IC50 of benznidazole. Loss of mitochondrial membrane potential induced by these esters was revealed. Tetradecyl gallate induced a loss of 53% of the mitochondrial membrane potential, at IC50 value.

Antifungal Activity of Decyl Gallate against Several Species of Pathogenic Fungi.

This work aims to demonstrate that the gallic acid structure modification to the decyl gallate (G14) compound contributed to increase the antifungal activity against several species of pathogenic fungi, mainly, Candida spp., Cryptococcus spp., Paracoccidioides spp., and Histoplasma capsulatum, according to standardized microdilution method described by Clinical Laboratory Standard Institute (CLSI) documents. Moreover this compound has a particularly good selectivity index value, which makes it an excellent candidate for broad-spectrum antifungal prototype and encourages the continuation of subsequent studies for the discovery of its mechanism of action.

Anti-trichophyton activity of protocatechuates and their synergism with fluconazole.

Dermatophytosis and superficial mycosis are a major global public health problem that affects 20-25% of the world's population. The increase in fungal resistance to the commercially available antifungal agents, in conjunction with the limited spectrum of action of such drugs, emphasises the need to develop new antifungal agents. Natural products are attractive prototypes for antifungal agents due to their broad spectrum of biological activities. This study aimed to verify the antifungal activity of protocatechuic acid, 3,4-diacetoxybenzoic, and fourteen alkyl protocatechuates (3,4-dihydroxybenzoates) against Trichophyton rubrum and Trichophyton mentagrophytes and to further assess their activities when combined with fluconazole. Susceptibility and synergism assays were conducted as described in M38-A2 (CLSI), with modifications. Three strains of Trichophyton rubrum and three strains of Trichophyton mentagrophytes were used in this work. The pentyl, hexyl, heptyl, octyl, nonyl, and decyl protocatechuates showed great fungicidal effects, with minimum inhibitory concentration (MIC) values ranging from 0.97 to 7.8 mg/L. Heptyl showed a synergistic activity (FIC index = 0.49), reducing the MIC of fluconazole by fourfold. All substances tested were safe, especially the hexyl, heptyl, octyl, and nonyl compounds, all of which showed a high selectivity index, particularly in combination with fluconazole. These ester associations with fluconazole may represent a promising source of prototypes in the search for anti-Trichophyton therapeutic agents.

Alkyl caffeates as anti-Helicobacter pylori and scavenger of oxidants produced by neutrophils.

Helicobacter pylori pathogenic action involves the colonization of the gastrointestinal tract and a large production of reactive oxygen species (ROS) by the neutrophils attracted to the site of infection. The aim of this study was to evaluate caffeic acid and its alkyl esters as inhibitors of the release of ROS by Helicobacter pylori activated neutrophils and their bactericidal effect. The increased hydrophobicity caused by esterification had direct consequence in their efficiency as bactericidal agents against H. pylori and inhibitors of the production of ROS by neutrophils. The minimum inhibitory concentration (MIC) decreased from higher than 1000 µg/mL (caffeic acid) to 250 µg/mL to butyl and heptyl caffeate. The release of total ROS, superoxide anion and hypochlorous acid by activated neutrophils was also significantly decreased and the esters were more efficient than the acid precursor. In conclusion, the alkyl esters of caffeic acid have two properties that are complementary for the treatment of H. pylori infections: bactericidal activity and inhibitory effect upon generation of ROS by neutrophils. Hence, we propose that these easily synthesized and non-expensive substances should be applied to in vivo experimental models of H. pylori induced gastric infections.

Light emission from tryptophan oxidation by hypobromous acid.

The emission of ultraweak light from cells is a phenomenon associated with the oxidation of biomolecules by reactive oxygen species. The indole moiety present in tryptophan, serotonin and melatonin is frequently associated with the emission of light during the oxidation of these metabolites. This study presents results for hypobromous acid (HOBr) oxidation of tryptophan as a putative endogenous source of ultraweak light emission. We found that chemiluminescence elicited by the oxidation of tryptophan by HOBr was significantly higher than by hypochlorous acid (HOCl). This difference was related to secondary oxidation reactions, which were more intense using HOBr. The products identified during oxidation by HOCl, but depleted by using HOBr, were N-formylkynurenine, kynurenine, 1,2,3,3a,8,8a-hexahydro-3a-hydroxypyrrolo[2,3-b]-indole-2-carboxylic acid, oxindolylalanine and dioxindolylalanine. The emission of light is dependent on the free α-amino group of tryptophan, and hence, the indole of serotonin and melatonin, although efficiently oxidized, did not produce chemiluminescence. The emission of light was even greater using taurine monobromamine and dibromamine as the oxidant compared to HOBr. A mechanism based on bromine radical intermediates is suggested for the higher efficiency in light emission. Altogether, the experimental evidence described in the present study indicates that the oxidation of free tryptophan or tryptophan residues in proteins is an important source of ultraweak cellular emission of light. This light emission is increased in the presence of taurine, an amino acid present in large amounts in leukocytes, where this putative source of ultraweak light emission is even more relevant.

Inhibitory effect of gallic acid and its esters on 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion of intracellular glutathione in erythrocytes.

The protective effect of gallic acid and its esters, methyl, propyl, and lauryl gallate, against 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion of intracellular glutathione (GSH) in erythrocytes was studied. The inhibition of hemolysis was dose-dependent, and the esters were significantly more effective than gallic acid. Gallic acid and its esters were compared with regard to their reactivity to free radicals, using the DPPH and AAPH/pyranine free-cell assays, and no significant difference was obtained. Gallic acid and its esters not only failed to inhibit the depletion of intracellular GSH in erythrocytes induced by AAPH but exacerbated it. Similarly, the oxidation of GSH by AAPH or horseradish peroxidase/H(2)O(2) in cell-free systems was exacerbated by gallic acid or gallates. This property could be involved in the recent findings on pro-apoptotic and pro-oxidant activities of gallates in tumor cells. We provide evidence that lipophilicity and not only radical scavenger potency is an important factor regarding the efficiency of antihemolytic substances.