Abstract
Chitosan (CS) nanoparticles have been extensively studied for siRNA delivery; however, their stability and efficacy are highly dependent on the types of cross-linker used. To address this issue, three common cross-linkers; tripolyphosphate (TPP), dextran sulphate (DS) and poly-D-glutamic acid (PGA) were used to prepare siRNA loaded CS-TPP/DS/PGA nanoparticles by ionic gelation method. The resulting nanoparticles were compared with regard to their physicochemical properties including particle size, zeta potential, morphology, binding and encapsulation efficiencies. Among all the formulations prepared with different cross linkers, CS-TPP-siRNA had the smallest particle size (ranged from 127 ± 9.7 to 455 ± 12.9 nm) with zeta potential ranged from +25.1 ± 1.5 to +39.4 ± 0.5 mV, and high entrapment (>95%) and binding efficiencies. Similarly, CS-TPP nanoparticles showed better siRNA protection during storage at 4˚C and as determined by serum protection assay. TEM micrographs revealed the assorted morphology of CS-TPP-siRNA nanoparticles in contrast to irregular morphology displayed by CS-DS-siRNA and CS-PGA-siRNA nanoparticles. All siRNA loaded CS-TPP/DS/PGA nanoparticles showed initial burst release followed by sustained release of siRNA. Moreover, all the formulations showed low and concentration-dependent cytotoxicity with human colorectal cancer cells (DLD-1), in vitro. The cellular uptake studies with CS-TPP-siRNA nanoparticles showed successful delivery of siRNA within cytoplasm of DLD-1 cells. The results demonstrate that ionically cross-linked CS-TPP nanoparticles are biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting.
Highlights
Small interfering RNAs have the potential for therapeutic application in many diseases, including cancer [1,2]
This was expected because of the lower viscosity observed at lower concentrations of CS, which resulted in better solubility and interaction between CS and crosslinkers, and smaller particle size[27]
Significant decrease in particle size was observed after loading of siRNA into CS-TPP/dextran sulphate (DS)/polyD-glutamic acid (PGA) nanoparticles in comparison to unloaded CS-TPP/DS/PGA nanoparticles as shown in (Table 2), which was in accordance with previous findings[28]
Summary
Small interfering RNAs (siRNAs) have the potential for therapeutic application in many diseases, including cancer [1,2]. Despite efficient and reliable gene silencing activity in vitro, only limited siRNA intracellular delivery has been achieved, owing to its rapid enzymatic degradation and poor cellular uptake [3,4]. Viral and non-viral vectors are used as carriers to deliver genes. Non-viral delivery systems using cationic liposomes and polymers, such as polyethylenimine (PEI), poly (L-lysine) (PLL), and their respective derivatives have been used to condense siRNAs to form nanoparticles[8,9]. CS is known to be biocompatible, minimally toxic, nonimmunogenic, and degradable by enzymes[16,17,18,19]. Despite these advantages, the stability of CS nanoparticles remains a crucial issue for researchers
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