Inspired by a natural cellular defence mechanism to protect own genome discovered in Petunia flowers in 1990, the idea of "post‐transcriptional gene silencing" grabbed a lot of attention in the field of biological research [1]. Following the demonstration of RNAi in mammalian cells in 2001, the scientific community quickly realized that this highly specific mechanism of sequence‐specific gene silencing might be harnessed to develop a novel class of drugs that interfere with disease‐
causing or disease‐promoting genes. RNAi therapeutics represent a fundamentally new way to treat human disease by addressing targets that are otherwise ‘undruggable’ with existing medicines [2].
In the first step of the RNAi process, the long, dsRNA (delivered to cells or expressed intracellularly from plasmids) are processed into short 21–25 base pair fragments by an RNase III‐type enzyme called Dicer. Then, each of these small fragments, so called "small interference RNA (siRNA)", is loaded into an RNA‐induced silencing complex (RISC). Depending on the directionality of siRNA loading, Ago2, an enzyme component of RISC, cleaves and discards one strand (passenger) and retains the other (guide) to activate the mature RISC complex. The siRNA guide strand then directs RISC to mRNA molecules containing a complementary sequence. Through Watson–Crick base pairing, the siRNA strand binds to the complementary portion of the mRNA molecule and the endonuclease region of RISC cleaves the mRNA in this region of homology. The cleaved mRNA, which is subsequently degraded by intracellular nucleases, is no longer available for translation into protein [3]. The RNAi mechanism is thoroughly reviewed by de Fougerolles, et al. [2].
The ability to use long dsRNAs to silence gene expression with a high specificity has been extended to a variety of organisms [4]. Unfortunately, it was found that in mammalian cells, dsRNAs longer
than about 30 bp initiated a harsh antiviral response called the interferon response, which ultimately results in non‐specific suppression of gene expression through activation of RNase L and general degradation of RNA molecules [5].
A major advance to the successful use of RNAi as a therapy in humans was the discovery that some smaller dsRNAs, less than 30 bp, were able to bypass the mammalian immune response to facilitate gene‐specific silencing [6]. This discovery revealed that synthetic siRNAs designed with a sequence complementary to a target gene could be delivered to cells instead of long dsRNA. The RNAi mechanism works the same way, except that the dicing step is no longer necessary. It should be noted that siRNAs containing specific nucleotide sequence motifs that are recognizable by toll‐like receptors in the endosomal pathway are capable of triggering immunostimulatory activity, as well.
Avoidance of these sequence motifs (e.g., GU) when designing siRNAs for in vivo use is one way to decrease the probability of inducing the innate mammalian immune response [3, 7].
However, for siRNA‐based therapeutics to hit the market, there are still quite a few challenges related to their efficient delivery to the targeted cell cytoplasm where they become effective in inducing silencing. Naked siRNAs have a half‐life time of less than an hour in human plasma, and circulating siRNAs are rapidly excreted by kidneys owing to their small size. Furthermore, naked RNAs cannot penetrate cellular lipid membranes and therefore, systemic application of unmodified siRNAs is unlikely to be successful [8]. Finally, since cellular uptake of siRNA mainly proceeds through the endosomal pathway, endosomal escape to the cytoplasm compartment is an additional requirement to efficiently deliver siRNA. Thus, specially designed delivery systems are needed.
Ideally, delivery systems for siRNA‐based therapeutics have to: protect siRNAs from rapid degradation by serum and tissue nucleases, prolong circulation time to have enough time to reach their target and perform their function, provide stealth properties to be undetectable by macrophages (unless organs of the mononuclear phagocyte system (MPS) such as spleen and liver are targeted), offer a targeting mechanism to lead the cargo to the desired cells and/or tissues (ligands such as galactose, transferrin, antibodies or peptides), enhance cellular uptake, and endosomal escape to the cytoplasm after cellular internalization [9] (summarized in Figure 1).
Figure 1: Required properties to form an efficient siRNA delivery system. An efficient delivery system protects siRNA against degradation, elimination, non‐specific distribution and tissue barricades. Stability and stealth properties of the delivery system can be achieved by surface modification. To deliver siRNA to a specific cell type or tissue, the delivery system can be recovered by ligands as galactose, transferrin, antibodies or peptides. Once the siRNA is free in the cytoplasm, it can act by the RNAi pathway. “Reprinted from reference [9]. Copyright 2010, with permission of Elsevier.”
Reported delivery systems in literature can be classified into two main groups: viral and non‐viral delivery systems. Viral delivery systems are mostly used to deliver DNA plasmids or precursor molecules to induce RNAi, whereas for synthetic siRNA‐delivery non‐viral delivery systems are applied [9]. Both groups of delivery systems have their advantages and disadvantages. High transduction efficacy, due to the inherent ability of viruses to transport genetic material into cells, is one of their advantageous properties [10]. But the potential of mutagenicity or oncogenesis, several host immune responses, and the high cost of production limit their application [11]. For these reasons, different kinds of non‐viral siRNA delivery systems have been tested to avoid viral vectors (for example, the injection of chemically stabilized or modified RNA, encapsulating siRNA in polymeric microparticles or nanoparticles, liposomes, binding siRNA to cationic or other particulate carriers).
siRNA delivery using polymeric particles has been reported extensively. One of the major limitations for the use of siRNA in vivo is the inability of naked siRNA to passively diffuse through cellular membranes due to the strong anionic charge of the phosphate backbone and resulting electrostatic repulsion from the anionic cell membrane surface [12]. Thus, polymers used are mainly polycations forming strong electrostatic interactions with the negatively charged polynucleotide molecules allowing for “self‐assembly”, which can substantially hinder or prevent enzymatic degradation of the incorporated polynucleotide in the bloodstream [13]. However, it is worth mentioning that although siRNA‐polymer complexes of overall positive charge are superior in cell culture studies, as they are binding to the negatively charged cell walls, for in vivo applications, a positive surface charge is rather a handicap. Systemically administered cationic particles are neutralized following interaction with blood components such as negatively charged serum albumin and other opsonins, resulting in aggregation, size increase reducing the internalisation efficacy, rapid clearance by reticuloendothelial system or phagocytes and short plasma circulation time, or even more seriously, aggregates can cause lung embolism [14, 15]. Therefore, certain techniques have to be adapted to solve these issues as we will discuss later.