Carbonate apatite-facilitated intracellularly delivered siRNA for efficient knockdown of functional genes

Abstract

Gene therapy through intracellular delivery of a functional gene or a gene-silencing element is a promising approach to treat critical diseases. Elucidation of the genetic basis of human diseases with complete sequencing of human genome revealed many vital genes as possible targets in gene therapy programs. RNA interference (RNAi), a powerful tool in functional genomics to selectively silence messenger RNA (mRNA) expression, can be harnessed to rapidly develop novel drugs against any disease target. The ability of synthetic small interfering RNA (siRNA) to effectively silence genes in vitro and in vivo, has made them particularly well suited as a drug therapeutic. However, since naked siRNA is unable to passively diffuse through cellular membranes, delivery of siRNA remains the major hurdle to fully exploit the potential of siRNA technology. Here pH-sensitive carbonate apatite has been developed to efficiently deliver siRNA into the mammalian cells by virtue of its high affinity interactions with the siRNA and the desirable size of the resulting siRNA/apatite complex for effective cellular endocytosis. Moreover, following internalization by cells, siRNA was found to be escaped from the endosomes in a time-dependent manner and finally, more efficiently silenced reporter genes at a low dose than commercially available lipofectamine. Knockdown of cyclin B1 gene with only 10 nM of siRNA delivered by carbonate apatite resulted in the significant death of cancer cells, suggesting that the new method of siRNA delivery is highly promising for pre-clinical and clinical cancer therapy.

Introduction

Genes encode proteins through messenger RNA (mRNA) to carry out the major functions of a biological system and a disorder either acquired or genetic is usually associated with the suppression or the overexpression of certain genes. Regulation of the gene expression particularly through the delivery of exogenous gene(s) or gene-silencing element(s) could assist in restoring the regular physiological functions for treatment of a genetic or an acquired disease. RNA interference (RNAi) being one of the mechanisms to selectively silence mRNA expression can be harnessed to rapidly develop novel drugs against target genes [1], [2], [3], [4], [5], [6]. There are two basic ways of implementing RNAi for selective gene inhibition: 1) cytoplasmic delivery of short interfering RNA (siRNA) and 2) nuclear delivery of gene expression plasmid to express a short hairpin RNA (shRNA) [7]. Silencing by synthetic siRNA, an RNA duplex of 21–23 nucleotides, is more advantageous than shRNA partly due to the difficulty of constructing shRNA expression systems prior to the selection and verification of the active sequences [7] and the requirement of the expression system to cross the nuclear membrane for shRNA expression [8]. The ability of siRNA to potently, but reversibly, silence genes in vivo has made them a highly promising drug therapeutic with several different clinical trials ongoing and more poised to enter the clinic in the future [2], [5]. However, due to the strong anionic phosphate backbone with consequential electrostatic repulsion from the anionic cell membrane, siRNA is unable to passively diffuse across the membrane [9]. For the intracellular delivery of siRNA, both viral or non-viral vectors have been investigated. Although the viral vectors are highly efficient, they are limited to shRNA delivery and remain highly immunogenic and carcinogenic. On the other hand, the non-viral systems are promising alternatives for siRNA delivery since they are relatively safe and cost-effective. Being usually polycationic, they are able to form complexes with anionic nucleic acid, protecting it from nuclease attack and facilitating cellular uptake through electrostatic interactions with negatively charged plasma membrane or through specific interactions between the ligand attached to the complex and the receptor on cell membrane [8]. Among the non-viral vectors, both polyplexes as well as lipoplexes have been found efficient for siRNA delivery with significant gene-silencing effect both in vitro and in vivo [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38]. However, synthetic non-viral systems are inefficient and an increase in performance is often associated with an increase in cytotoxicity [39]. The major obstacle for siRNA delivery in the non-viral route is the degradation of a significant portion of the internalized siRNA by lysosomal nucleases [40]. The endosomal escape of siRNA is, therefore, a crucial step in successful gene silencing.

We have recently developed an efficient delivery system based on some fascinating properties of carbonate apatite-ability of preventing crystal growth for generation of nanoscale particles as needed for efficient endocytosis and fast dissolution kinetics in endosomal acidic compartments to facilitate the release of delivered therapeutics from the particles and endosomes [41], [42], [43], [44], [45], [46], [47], [48], [49], [50]. Here, we show that pH-sensitive carbonate apatite particles having high affinity interactions with siRNAs, mediate efficient endocytosis and subsequent endosomal escape of the siRNAs, leading to the silencing of reporter gene expression more effectively than commercially available lipofectamine. Moreover, nanoparticle-assisted delivery of validated siRNA against cyclin B1 results in significant inhibition of cancer cell growth.