RESUMO
Edible films and coatings can enhance the quality of food products, protecting them from biological deterioration, especially against fungal diseases and pathogenic microorganisms. In this study, films from chitosan, diethylaminoethyl-chitosan (DEAE-CH) and its hydrophobicized derivative DEAE-CH-DD were prepared by casting and their physicochemical and antimicrobial properties evaluated. The grafting with DEAE and dodecyl groups resulted in films with an elasticity modulus up to five times higher than commercial chitosan and increased water vapor permeability. Field emission gun - scanning electron microscopy and atomic force microscopy techniques showed films with smooth surfaces and the contact angle measurements revealed a correlation between the grafted group and hydrophilic/hydrophobic nature of the surface of the film. The amphiphilic derivatives exhibited better antimicrobial activity than unmodified chitosan against Penecillium expansum, Alternaria alternata and Alternaria solani. The amphiphilics DEAE-CH and DEAE-CH-DD showed no toxicity and delayed rotting and loss of water in strawberries and bananas, suggesting that this kind of film has great potential for increasing the shelf-life of different fruits.
Assuntos
Quitosana/química , Filmes Comestíveis , Peso Molecular , Animais , Sobrevivência Celular , Fenômenos Químicos , Concentração de Íons de Hidrogênio , Fenômenos Mecânicos , Camundongos , Células NIH 3T3 , Análise Espectral , Termogravimetria , Difração de Raios XRESUMO
Chitosan has received a lot of attention as a carrier for small interfering RNA (siRNA), due to its capacity for complexation and intracellular release of these molecules. However, one of its limitations is its insolubility at neutral pH and the tendency towards aggregation of its nanoparticles in isotonic ionic strength. In this study, a series of amphipathic chitosans were synthesized by varying the degree of acetylation (DA) from Ë2 to Ë30 mol% and the degree of substitution (DS) from 5 to 25%. by tertiary amino groups (DEAE) The results showed that the adjustment of these parameters decreases the interparticle interactions mediated by hydrogen bonding to obtain nanoparticles with improved colloidal stability. siRNA-containing nanoparticles of 100 to 150 nm with low polydispersities (0.15-0.2) and slightly positive zeta potentials (Ë+ 5 mV) were resistant to aggregation at pH 7.4 and ionic strength of 150 mM. This resistance to aggregation is provided by changes on the nanoparticle surface and highlights the importance of more organized self-assembly in providing colloidal stability at physiological conditions. Additionally, the PEGylation of the most promising vectors conferred favorable physicochemical properties to nanoparticles. The chitosans and their nanoparticles exhibited low toxicity and an efficient cell uptake, as probed by confocal microscopy of rhodamine labeled vectors. The results provide a new approach to overcome the limited stability of chitosan nanoparticles at physiological conditions and show the potential of these amphipathic chitosans as siRNA carriers.
Assuntos
Quitosana/análogos & derivados , Portadores de Fármacos/química , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Tensoativos/química , Anidridos Acéticos/química , Acetilação , Animais , Quitosana/síntese química , Quitosana/metabolismo , Quitosana/toxicidade , Dietilaminas/química , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/toxicidade , Fluorescência , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Camundongos , Nanopartículas/metabolismo , Nanopartículas/toxicidade , Tamanho da Partícula , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/toxicidade , Células RAW 264.7 , RNA Interferente Pequeno/química , Rodaminas/química , Tensoativos/síntese química , Tensoativos/metabolismo , Tensoativos/toxicidadeRESUMO
The antimicrobial activity of chitosan and derivatives to human and plant pathogens represents a high-valued prospective market. Presently, two low molecular weight derivatives, endowed with hydrophobic and cationic character at different ratios were synthesized and characterized. They exhibit antimicrobial activity and increased performance in relation to the intermediate and starting compounds. However, just the derivative with higher cationic character showed cytotoxicity towards human cervical carcinoma cells. Considering cell membranes as targets, the mode of action was investigated through the interaction with model lipid vesicles mimicking bacterial, tumoral and erythrocyte membranes. Intense lytic activity and binding are demonstrated for both derivatives in anionic bilayers. The less charged compound exhibits slightly improved selectivity towards bacterial model membranes, suggesting that balancing its hydrophobic/hydrophilic character may improve efficiency. Observing the aggregation of vesicles, we hypothesize that the "charge cluster mechanism", ascribed to some antimicrobial peptides, could be applied to these chitosan derivatives.
Assuntos
Quitosana/síntese química , Quitosana/farmacologia , Bicamadas Lipídicas/química , Membranas Artificiais , Bioensaio , Quitosana/química , Escherichia coli/efeitos dos fármacos , Fluorometria , Células HeLa , Humanos , Testes de Sensibilidade Microbiana , Permeabilidade , Espectroscopia de Prótons por Ressonância Magnética , Staphylococcus aureus/efeitos dos fármacos , Eletricidade Estática , Tensoativos/químicaRESUMO
In this study, the antifungal activity of chitosan derivatives against A. flavus was studied to understand the contribution of the molecular mass (Mw) and of the hydrophobic and electrostatic forces to the inhibition of fungal growth. The interaction of amphiphilics ranging from 8 to 130â¯kDa with model membranes of zwitterionic L-α-phosphatidylcholine (PC) and anionic L-α-phosphatidylcholine/L-α-phosphatidyl-DL-glycerol (PC:PG, 80:20â¯mol%) were exploited to obtain information on the inhibition mechanism. The results indicated that concurrent interactions control the antifungal activity. The decrease in the Mw weakens the self-association favoring the electrostatic and hydrophobic associations with the cell wall and anionic lipids of the lipid bilayer, indicating an increasing association of the amphiphilics with the fungal membrane. Laser confocal scanning microscopy of rhodamine labeled-derivatives and transmission electronic microscopy techniques showed that the amphiphilics affect the cell wall integrity by inducing the aggregation of hydrophobic constituents of the conidia.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Aspergillus flavus/efeitos dos fármacos , Quitosana/química , Quitosana/farmacologia , Antifúngicos/metabolismo , Membrana Celular/metabolismo , Quitosana/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Peso Molecular , Eletricidade EstáticaRESUMO
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 (25-230â¯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 (130â¯kDa to 230â¯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.
Assuntos
Quitosana/química , Etanolaminas/química , Técnicas de Transferência de Genes , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Animais , Sobrevivência Celular , Quitosana/análogos & derivados , Quitosana/síntese química , Células HeLa , Humanos , Camundongos , Peso Molecular , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Células RAW 264.7 , TransfecçãoRESUMO
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.
Assuntos
Quitosana/química , Portadores de Fármacos/química , Etanolaminas/química , Fosforilcolina/química , Transfecção , Quitosana/toxicidade , DNA/química , DNA/genética , Portadores de Fármacos/toxicidade , Inativação Gênica , Células HeLa , Humanos , Nanopartículas/química , Tamanho da Partícula , Plasmídeos/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genéticaRESUMO
Two series of new chitosan derivatives were synthesized by reaction of deacetylated chitosan (CH) with propyl (CH-Propyl) and pentyl (CH-Pentyl) trimethylammonium bromides to obtain derivatives with increasing degrees of substitution (DS). The derivatives were characterized by (1)H NMR and potentiometric titration techniques and their antifungal activities on the mycelial growth of Aspergillus flavus were investigated in vitro. The antifungal activities increase with DS and the more substituted derivatives of both series, CH-Propyl and CH-Pentyl, exhibited antifungal activities respectively three and six times higher than those obtained with commercial and deacetylated chitosan. The minimum inhibitory concentrations (MIC) were evaluated at 24, 48 and 72 h by varying the polymer concentration from 0.5 to 16 g/L and the results showed that the quaternary derivatives inhibited the fungus growth at polymer concentrations four times lower than that obtained with deacetylated chitosan (CH). The chitosans modified with pentyltrimethylammonium bromide exhibited higher activity and results are discussed taking into account the degree of substitution (DS).