Chitosan-based formulations for delivery of DNA and siRNA

Abstract

Among non-viral vectors, chitosan and chitosan derivatives have been developed in vitro and in vivo for DNA and siRNA delivery systems because of their cationic charge, biodegradability and biocompatibility, as well as their mucoadhesive and permeability-enhancing properties. However, the transfection efficiency of chitosan is too low for clinical application. Studies indicated that the transfection efficiency depends on a series of chitosan-based formulation parameters, such as the Mw of chitosan, its degree of deacetylation, the charge ratio of chitosan to DNA/siRNA (N/P ratio), the chitosan salt form used, the DNA/siRNA concentration, pH, serum, additives, preparation techniques of chitosan/nucleic acid particles and routes of administration. In this paper, chitosan-based formulations for the delivery of DNA and siRNA were reviewed to facilitate the process of chitosan vector development for clinical application. In addition to formulation optimization, chitosan structure modification or additive incorporation is an effective way to improve the stability of the polyplex in biological fluids, enhance targeted cell delivery and facilitate endo-lysosomal release of the complex. In summary, the transfection efficiency of chitosan-based delivery systems can be adjusted by changing formulation-related parameters.

Introduction

In recent decades, gene delivery research has grown very rapidly due to its huge potential as a future therapeutic strategy for clinical applications to treat many inheritable or acquired diseases by replacing defective genes, substituting missing genes, or silencing unwanted gene expression. However, naked therapeutic genes are rapidly degraded by nucleases, showing poor cellular uptake, non-specificity to the target cells, and low transfection efficiency [1]. Therefore, the development of safe and efficient gene carriers is one of the prerequisites for the success of gene therapy [2]. At present, major gene delivery systems use either viral or non-viral vectors. Although viral systems are very efficient for in vivo transfection, as well as immunization, their major drawbacks include possible toxicity, immunogenicity, and inflammatory potential [3]. A recent, additional cause for concern over using viral vectors is the phenomenon known as insertional mutagenesis, in which the ectopic chromosomal integration of viral DNA either disrupts the expression of a tumor-suppressor gene or activates an oncogene, leading to the malignant transformation of cells. A murine leukemia virus (MLV)-based clinical trial for treating X-linked severe combined immunodeficiency (X-SCID) highlighted the high risk of insertional mutagenesis: 3 of 11 treated children developed a type of T-cell leukemia, subsequently resulting in the death of one patient [4]. Genetic analysis of the malignant cells showed that the retroviral vector had inserted into, and activated, an oncogene called LMO2 that is associated with childhood leukemia [4]. Therefore, viral-based vectors urgently need to be reassessed with regard to their safety for human gene therapy. Non-viral vectors have attracted increasing attention due to such advantages as ease of synthesis, low immune response against the vector and unrestricted gene materials size in addition to potential benefits in terms of safety [5]. Non-viral vectors include liposomes, complexes of the negatively charged plasmid with cationic polymers, and nanoparticles. Liposome vesicles have shown a relatively low encapsulation efficiency, poor storage stability, and rapid clearance from the blood. Although it is widely believed that the steric hindrance of PEG grafted on the liposome surface can prevent the opsonization by serum proteins and the subsequent interaction with cells of the MPS, resulting in increased retention time of the liposomes in circulating blood, the ‘accelerated blood clearance (ABC) phenomenon’ was observed for PEGylated liposomes when a second dose is administered after a several-day interval [6], limiting their application. Therefore, non-viral systems based on cationic polymers containing several amine groups in their backbone have been used extensively as gene carriers [7]. They can provide unlimited DNA packaging capacity, well-defined physicochemical properties, and a high degree of molecular diversity that allows extensive modifications to overcome extracellular and intracellular obstacles of gene delivery [8]. An ideal polymeric carrier should form a stable complex with nucleic acids to maintain its stability in a biological solution, hide from the host surveillance systems, and deliver the therapeutic nucleic acid to the desired cell population by recognizing a specific characteristic on the cell surface. After being localized within the cells, the carrier should provide appropriate functionalities to escape from the endocytotic pathways, deliver the complexes to the vicinity of the targets, perform decomplexation (or unpacking) in response to a certain intracellular environment, such as pH or redox potential, and actively transport the nucleic acids to the target [8].

In recent years, chitosan-based carriers have become one of the non-viral vectors that have gained increasing interest as a safe delivery system for gene materials including plasmid DNA (pDNA), oligonucleotides and siRNA. Chitosan has beneficial qualities such as low toxicity, low immunogenicity, excellent biocompatibility [9], [10] as well as a high positive charge density. Due to its positive charge, it can easily form polyelectrolyte complexes with negatively charged nucleotides by electrostatic interaction. However, gene delivery efficiency of chitosan is significantly influenced by formulation-related parameters. In this paper, chitosan-based formulations for the delivery of DNA and siRNA were reviewed to facilitate the process of chitosan vector development for clinical applications.