Impact of Degree of Ionization and PEGylation on the Stability of Nanoparticles of Chitosan Derivatives at Physiological Conditions.

Nowadays, the therapeutic efficiency of small interfering RNAs (siRNA) is still limited by the efficiency of gene therapy vectors capable of carrying them inside the target cells. In this study, siRNA nanocarriers based on low molecular weight chitosan grafted with increasing proportions (5 to 55%) of diisopropylethylamine (DIPEA) groups were developed, which allowed precise control of the degree of ionization of the polycations at pH 7.4. This approach made obtaining siRNA nanocarriers with small sizes (100-200 nm), positive surface charge and enhanced colloidal stability (up to 24 h) at physiological conditions of pH (7.4) and ionic strength (150 mmol L-1) possible. Moreover, the PEGylation improved the stability of the nanoparticles, which maintained their colloidal stability and nanometric sizes even in an albumin-containing medium. The chitosan-derivatives displayed non-cytotoxic effects in both fibroblasts (NIH/3T3) and macrophages (RAW 264.7) at high N/P ratios and polymer concentrations (up to 0.5 g L-1). Confocal microscopy showed a successful uptake of nanocarriers by RAW 264.7 macrophages and a promising ability to silence green fluorescent protein (GFP) in HeLa cells. These results were confirmed by a high level of tumor necrosis factor-α (TNFα) knockdown (higher than 60%) in LPS-stimulated macrophages treated with the siRNA-loaded nanoparticles even in the FBS-containing medium, findings that reveal a good correlation between the degree of ionization of the polycations and the physicochemical properties of nanocarriers. Overall, this study provides an approach to enhance siRNA condensation by chitosan-based carriers and highlights the potential of these nanocarriers for in vivo studies.

Biopolymeric Films of Amphiphilic Derivatives of Chitosan: A Physicochemical Characterization and Antifungal Study.

The chemical modification of chitosan has been an active subject of research in order to improve the physicochemical and antifungal properties of chitosan-based films. The aim of this study was to evaluate the physiochemical and antifungal properties of films prepared with chitosan and its derivatives containing diethylaminoethyl (DEAE) and dodecyl groups (Dod). Chitosans and selected derivatives were synthesized and characterized, and their films blended with glycerol and sorbitol (5%, 10%, and 20%). They were studied by means of the evaluation of their mechanical, thermal, barrier, and antifungal properties. The collected data showed that molecular weight (Mw), degree of acetylation, and grafting with DEAE and Dod groups greatly affected the mechanical, thickness, color, and barrier properties, all of which could be tailored by the plasticizer percentage. The antifungal study against Aspergillus flavus, Alternaria alternata, Alternaria solani, and Penicillium expansum showed that the films containing DEAE and Dod groups exhibited higher antifungal activity than the non-modified chitosans. The mechanical properties of highly soluble films were improved by the plasticizers at percentages of 5% and 10%, indicating these derivatives as potential candidates for the coating of seeds, nuts and fruits of various crops.

Amphiphilic diethylaminoethyl chitosan of high molecular weight as an edible film.

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.

Insights on the antifungal activity of amphiphilic derivatives of diethylaminoethyl chitosan against Aspergillus flavus.

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 130 kDa with model membranes of zwitterionic L-α-phosphatidylcholine (PC) and anionic L-α-phosphatidylcholine/L-α-phosphatidyl-DL-glycerol (PC:PG, 80:20 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.

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.

Biochemical Characterization, Thermal Stability, and Partial Sequence of a Novel Exo-Polygalacturonase from the Thermophilic Fungus Rhizomucor pusillus A13.36 Obtained by Submerged Cultivation.

This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5-47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. Ea(d) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and ΔG(d) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot.

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.