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.

Synthesis, Characterization, and Antifungal Activities of Amphiphilic Derivatives of Diethylaminoethyl Chitosan against Aspergillus flavus.

Amphiphilic derivatives of diethylaminoethyl chitosan (DEAE-CH) were synthesized using a two-step process involving initial substitution with diethylaminoethyl (DEAE) groups followed by reductive amination with dodecylaldehyde. The synthesized derivatives were characterized by (1)H NMR, gel permeation, and FTIR. The associative behaviors of these compounds in aqueous solution were studied using fluorescence spectroscopy, whereas their antifungal activities against Aspergillus flavus were evaluated in terms of mycelial growth. The effects of deacetylated chitosans and their derivatives on the mycelial growth of A. flavus were evaluated at several polymer concentrations (0.05-1.0 g/L), and the results were compared. The inhibition indices of the deacetylated chitosans increased with increasing Mw (16.9 kDa < 176 kDa < 517.7 kDa); however, derivatives with a combination of either a high molecular weight (Mw) and low hydrophobicity or a low Mw and high hydrophobicity were the most effective in inhibiting the in vitro radial growth of A. flavus.

Synthesis and evaluation of diethylethylamine-chitosan for gene delivery: composition effects on the in vitro transfection efficiency.

Chitosan has been indicated as a safe and promising polycation vector for gene delivery. However its low transfection efficiency has been a challenging obstacle for its application. To address this limitation, we synthesized chitosan derivatives which had increasing amounts of diethylethylamine groups (DEAE) attached to the chitosan main chain. The plasmid DNA VR1412 (pDNA), encoding the ß-galactosidase (ß-gal) reporter gene was used to prepare nanoparticles with the chitosan derivatives, and the transfection studies were performed with HeLa cells. By means of dynamic light scattering and zeta potential measurements, it was shown that diethylethylamine-chitosan derivatives (DEAE(x)-CH) were able to condense DNA into small particles having a surface charge depending on the polymer/DNA ratio (N/P ratio). Nanoparticles prepared with derivatives containing 15 and 25% of DEAE groups (DEAE(15)-CH and DEAE(25)-CH) exhibited transfection efficiencies ten times higher than that observed with deacetylated chitosan (CH). For derivatives with higher degrees of substitution (DS), transfection efficiency decreased. The most effective carriers showed low cytotoxicity and good transfection activities at low charge ratios (N/P). Vectors with low DS were easily degraded in the presence of lysozyme at physiological conditions in vitro and the nontoxicity displayed by these vectors opens up new opportunities in the design of DEAE-chitosan-based nanoparticles for gene delivery.