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

Abstract

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-CH) 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.

<p>Evidence Supporting the Safety of Pegylated Diethylaminoethyl-Chitosan Polymer as a Nanovector for Gene Therapy Applications</p>

“…as well as their endosomal escape and the release of cargo in the cytoplasm, which enhances their transfection efficiency. 19,26 As previously shown, adding FA to our nanovector also improves the capacity to target cells expressing the folate receptor (FR). 28 Herein, we synthesized stable DEAE 12 -CH-PEG-FA 2 /siRNA nanoparticles at physiological pH (7.2), as demonstrated by the uniformity in size and PDI measurements over 24 h at a N/P ratio of 15:1.…”

“…Additional Information about DEAE 12 -CH-PEG-FA 2 characterization is described in our previous study. 19 …”

“…Additional Information about DEAE 12 -CH-PEG-FA 2 characterization is described in our previous study. 19 Nanoparticle Preparation in DPBS pH 7.2 DEAE 12 -CH-PEG-FA 2 was dissolved overnight in a 0.1 M HCL solution, then heated at 50°C for 30 min, adjusted with Dulbecco's Phosphate-Buffered Saline (DPBS) to the desired final concentration, and finally filtered with a 0.45 μm polyether sulfone membrane filter. To prepare DEAE 12 -CH-PEG-FA 2 /siRNA-SSB nanoparticles, siRNA-SSB and modified CH stock solutions were added to DPBS and vortexed immediately at moderate speed for 1 min.…”

“…The present study systematically evaluates the safety of a pegylated diethylaminoethyl CH (DEAE 12 -CH-PEG-FA 2 ), with or without siRNA-SSB complexation. This derivative showed a high in vitro transfection efficiency on varied cell lines 19 and its in vivo therapeutic efficacy was demonstrated in a murine collagen antibody-induced arthritis model. 2 Among the assays proposed by ISO 10,993–4:2009, 20 ISO/TR 16,197, 17 ASTM F1903, 21 ASTM E56.03 committee, 22 and NCL protocols, 23 the following were chosen: endotoxin contamination, physicochemical characterization, cytotoxicity assay, hemotoxicity (hemolysis, hemagglutination, complement activation, platelet aggregation and coagulation tests), inflammatory response (cytokines) and oxidative stress response (reactive oxygen species (ROS) and nitric oxide (NO)).…”

Hyaluronan/Diethylaminoethyl Chitosan Polyelectrolyte Complexes as Carriers for Improved Colistin Delivery

“…DEAECS [22][23][24][25] and HA [26][27][28][29] have attractive biomedical properties, as both are biodegradable, biocompatible, and non-toxic, water-soluble polymers. Chitosan and its water-soluble derivatives are enzymatically biodegraded in body fluids and tissues by lysozyme and are mainly distributed in the kidneys and urine, where they are further destructed by enzymes and excreted; this prevents their accumulation in the body [30][31][32].…”