Double-grafted chitosans as siRNA nanocarriers: effects of diisopropylethylamine substitution and labile-PEG coating

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

“…Overall, while nanoparticles formulated with PEGylated-only chitosan (PEG-Ch d ) formed complexes composed of large aggregates with a negative charge surface ( Figure S18 ), the derivatives have formed nanoparticles with promising sizes (100–200 nm) and zeta potentials (6–12 mV) to be applied as vectors for gene therapy. Notably, the importance of the degree of ionization ( α ) of these derivatives for obtaining nanovectors of adequate Dh and ζ was also observed in a previous study [ 16 ], in which it was shown that high MW DIPEA-chitosans with low DS DIPEA (15%) are more prone to formulate efficient siRNA nanocarriers (with small size, positive ζ, and improved stability) at pH 6.3 ( α ≥ 80%) than at pH 7.4 ( α < 35%), due to an increasing degree of ionization with decreasing pH.…”

“…Similarly, the molecular weights for derivatives tended to decrease upon DIPEA grafting (especially for the more substituted ones), a sign that main chain degradation took place during the chemical modification of Ch d , as earlier reported for DEAE-chitosan [ 27 ]. Another relevant effect of MW may be observed in the synthesis of derivatives when the data in Table 1 are compared to previous results reported by our research group [ 16 ] for DIPEA-chitosans of higher MW (200 kDa), i.e., under the same experimental conditions, chitosans of high MW need to be fed with more DIPEA-Cl (about 20–30%) to originate DIPEA-chitosans with the same DS DIPEA of derivatives listed in Table 1 , which have about 100 kDa.…”

“…RAW 264.7 [ 15 , 51 ] and HeLa-GFP [ 52 ] cell lines were obtained from BCRJ (Banco de Células do Rio de Janeiro, Brazil). NIH/3T3 fibroblasts [ 16 , 53 ] were obtained from ATCC ® (Virginia, USA). A CellTiter96 ® Aqueous One Solution kit composed of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and a phenazine ethosulfate (PES) was purchased from Promega Corporation (Fitchburg, MA, USA).…”

“…In order to overcome this limitation, some research groups have been modifying chitosan with additional amino groups of higher pKa to increase its applicability at neutral pH and the results have shown more compact, stable, and efficient siRNA nanocarriers [ 12 , 13 , 14 , 15 ]. Particularly, the grafting of hydrophobic-ionizable groups containing tertiary amines, such as diethylaminoethyl (DEAE) and diisopropylethylamine (DIPEA) groups that improve the ability of chitosan as a gene therapy agent [ 15 , 16 , 17 ], is highlighted.…”

“…Recently, our research team has shown that the combination of chitosan with DIPEA groups is a promising alternative for the development of siRNA nanocarriers. However, the best transfection efficiencies were still centered at low pH values due to the higher degrees of ionization of the derivatives [ 16 ]. Thus, this study proposed to better explore the effect of the degree of ionization of the polycations on the physicochemical and biological properties of the nanocarrier by studying a higher variation of DIPEA grafts (5–55%) on a highly deacetylated chitosan, as shown in Figure 1 .…”