Development and characterisation of chitosan nanoparticles for siRNA delivery
Introduction
Over the past few decades, antisense approaches including oligonucleotides, ribozymes and DNAzymes have been extensively investigated as tools for controlling cellular processes. Only recently, small interfering RNAs (siRNAs) have proven to be versatile agents for controlling gene expression in mammalian cells. They have also been shown more potent than conventional antisense strategies [1], [2], [3]. Overall, they appear to be a much more robust and efficient technology offering significant potential.
siRNA consisting of 21–23 nucleotides can regulate gene expression in mammalian cells through RNA interfering (RNAi). As the administration of siRNA could bypass non-specific inhibition of protein synthesis induced by long double-stranded RNA [4], [5], it has therefore been employed as a novel tool in blocking the expression of genes such as those expressed in infectious diseases and cancers. However, similar to hydrophilic and polyanion-mediated Gene Therapy, siRNA also suffers particular problems including poor cellular uptake, rapid degradation by ubiquitous nucleases as well as limited blood stability [6], [7]. As a result of these limitations, unassisted delivery of siRNA to the cells is frustrating. Although various chemical modifications of siRNA can be used to overcome these problems, these modifications posses disadvantages such as a decreased mRNA hybridization, higher cytotoxicity and increased unspecific effects [8]. Therefore, effective systems which can protect and transport siRNA to the cytoplasm of the target cells are needed to exploit the promising potential applications offered by successful delivery of siRNA.
From among the gene vectors that have been studied, non-viral vectors have attracted more and more attention in comparison to viral vectors, although viral vectors have been proven to yield higher transfection efficiency in most cell lines. This is due to the advantages of non-viral vectors such as ease of synthesis, low immune response against the vector and unrestricted gene materials size in addition to potential benefits in terms of safety [9]. In recent years, chitosan-based carriers are one of the non-viral vectors that have gained increasing interest as a safer and cost-effective delivery system for gene materials including plasmid DNA (pDNA), oligonucleotide (ODN) as well as proteins and peptides. Chitosan has beneficial qualities such as low toxicity, low immunogenicity, [10] excellent biodegradability, biocompatibility, [10], [11] as well as a high positive charge that can easily form polyelectrolyte complexes with negatively charged nucleotides by electrostatic interaction.
Although chitosan has been studied for more than a decade as a gene vector for pDNA and ODN [12], so far to our knowledge, there is no study that has been carried out to investigate the use of chitosan to deliver siRNA in vitro. In this report, we studied three methods of siRNA association; by simple complexation, ionic gelation (siRNA entrapment) and adsorption of siRNA onto the surface of preformed chitosan nanoparticles for preparation of chitosan-based nano-carriers. To address the lack of information regarding the behaviour of interaction between siRNA and chitosan – since siRNA's structure and size is quite different to that of pDNA, these systems were fully characterised with regards to their physical and biological features by exploiting commercial chitosan products. The ability of these nanoparticulate systems to mediate gene silencing was then assessed on CHO K1 and HEK 293 cells in vitro.
Section snippets
Materials
Four different types of medical grade chitosan with the degree of deacetylation (DD) of ∼ 86% were used: chitosan hydrochloride with molecular weight of 270 kDa (Cl213) and 110 kDa (Cl113) as well as chitosan glutamate with molecular weight of 470 kDa (G213) and 160 kDa (G113) (Protasan Ultra-pure, Pronova Biomedical, Norway). Pentasodium tripolyphosphate (TPP) was obtained from Fluka and sodium acetate from Sigma-Aldrich. siRNA targeting against pGL3 luciferase gene (sense:
Particle size
Mean particle size of chitosan–siRNA complexes prepared by simple complexation for both chitosan hydrochloride and glutamate were increased when the concentration of chitosan was increased from 25 to 300 μg/ml in distilled water (Fig. 1). However, a smaller mean particle size of chitosan nanoparticles was obtained when the lower molecular weight of chitosan was used compared to the higher molecular weight for the individual chitosan derivatives. In addition, chitosan glutamate (G213, G113)
Discussion
In this study, the simple complexation and ionic gelation methods were chosen to prepare chitosan–siRNA complexes or nanoparticles since both processes are simple and mild [11]. In addition, the use of TPP in ionic gelation as a polyanion to cross-link with the cationic chitosan through electrostatic interaction could avoid possible toxicity of reagents used in chemical cross-linking (e.g. glutaraldehyde). Modulation of size and surface charge of the particles could also be easily done by using
Conclusions
In conclusion, we have demonstrated that chitosan could be used as a delivery system for siRNA and from among four types of chitosan studied, chitosan glutamate with higher molecular weight, G213 is the best candidate as a vector for siRNA. Furthermore, in vitro study has revealed the transfection efficiency of siRNA depends on the method of siRNA association to the chitosan and entrapping siRNA using ionic gelation has shown to yield a better biological effect than simple complexation or siRNA
Acknowledgements
The authors would like to thank Ministry of Science, Technology and Environment of Malaysia for the funding of this work and also Dr. S. Somavarapu as well as Dr. Xiong Wei Li for useful discussions and technical assistance.
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