Convenient targeting of stealth siRNA-lipoplexes to cells with chelator lipid-anchored molecules

Abstract

A major obstacle for the use of siRNAs as novel therapeutics is the requirement for functional delivery to specific cells in vivo. siRNA delivery by cationic agents is generally non-specific and a convenient targeting strategy has been lacking. This work explored the potential for using the chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA₃-DTDA) with neutral stealth liposomes to target siRNA to cells. A novel method for incorporating siRNAs into lipoplexes was developed which utilised helper lipids and the ionisable lipid 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). This approach results in an efficient (> 50%) incorporation of siRNA into lipoplexes, which when incorporated with Ni-NTA₃-DTDA and engrafted with a His-tagged form of murine CD4 can target siRNA to murine A20 B cells, in vitro. Also, siRNA-lipoplexes engrafted with His-tagged peptides that target receptors on HEK-293 cells, or the receptor for tumour necrosis factor alpha expressed on the murine dendritic cell line DC2.4, could target siRNA and silence the expression of enhanced green fluorescence protein (EGFP). siRNA-lipoplexes produced by this method are ~ 240 nm dia, exhibit low zeta-potential (− 1 mV), and target cells in serum-containing media. The results show that NTA₃-DTDA can be used to target siRNA-lipoplexes to cells, and could provide a convenient approach for targeting siRNA to cells in vivo for therapeutic applications.

Introduction

RNA interference (RNAi) has been hailed as one of the most exciting discoveries in functional genomics of the past decade, having enormous potential as a tool for analysing gene function, and for developing novel therapeutics based on gene silencing [1], [2]. The finding that the introduction of small interfering RNA (siRNA) duplexes (19–23 nucleotides long) into cells can induce a sequence-specific degradation and inhibition in the expression of the targeted mRNA, seems set to revolutionise the potential for effective use of RNAi for both research and therapeutic applications [3], [4]. siRNAs for use in mammalian cells can be produced as synthetic molecules, and pre-designed synthetic siRNAs optimised for maximum potency and specificity using effective and extensively tested algorithms, have become commercially available for > 99% of all human, mouse and rat genes. siRNAs act catalytically to mediate cleavage of the targeted mRNA, and are very potent [5]. Systemic delivery of siRNAs can achieve effective knockdown of certain genes [6], [7], but this approach lacks efficiency and tissue specificity, and increases the likelihood of unwanted off-target effects. A convenient strategy for targeting siRNAs to specific cell types is required to better exploit their therapeutic potential [8], [9], [10], [11].

An attractive approach for delivery of siRNA to cells is the use of liposomes as targeted delivery vehicles [12]. Cationic lipids can form complexes with nucleic acids, and are widely used as components of liposomal reagents used for the transfection of cells with DNA and siRNAs in vitro [13], [14], [15]. Such liposomes often contain a large proportion of one or more cationic lipids, e.g. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and the zwitterionic helper lipid 1,2-dioleoyl-phosphatidylethanolamine (DOPE). Cationic lipids interact efficiently with nucleic acids, forming lipid/nucleic acid complexes (lipoplexes). Unfortunately, whilst cationic liposomes can facilitate transfection of cells in vitro, their propensity to aggregate and to interact non-specifically with negative charges on the surface of cells makes their targeted delivery to specific cells in vivo difficult [16], [17].

As an alternative to using cationic liposomes, the encapsulation of siRNA into neutral stealth liposomes seems an excellent strategy for delivering siRNAs to cells in vivo. Such liposomes are generally non-toxic, avoid non-specific interactions with blood components [18], [19], and the lipid barrier protects the encapsulated siRNA cargo from rapid degradation by serum nucleases. Despite progress towards the efficient encapsulation of drugs and nucleic acids into neutral stealth liposomes [12], [20], a convenient method of targeting has been lacking [4], [9], [21]. Using the metal chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA₃-DTDA) [22], we recently developed a method for engrafting targeting molecules onto liposomes for targeting to specific cells [23], [24]. Thus, the incorporation of NTA₃-DTDA into stealth liposomes containing antigen and cytokine, allowed stable engraftment (by Ni²⁺-chelating linkage) of His-tagged forms of B7.1, CD40 and ScFv, and enabled targeting of the liposomes to T cells and dendritic cells in vitro and in vivo [23], [24]. In the present work we explored the potential of the NTA₃-DTDA technology for targeting siRNA encapsulated within stealth liposomes. The results show that a formulation of NTA₃-DTDA-containing liposomes can be designed to enable convenient receptor-mediated delivery of lipoplex-incorporated siRNAs to cells for the induction of gene silencing. The potential of this approach for targeting siRNA delivery in therapeutic applications is discussed.